Methods for treating multiple sclerosis

Abstract

To provide methods for treating multiple sclerosis (MS) in a patient.SOLUTION: Provided is a method comprising administering to a patient an effective amount of an anti-CD20 antibody, the method comprising a first exposure and second, third, and / or fourth additional exposures.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 238,103, filed October 6, 2015, U.S. Provisional Patent Application No. 62 / 238,674, filed October 7, 2015, U.S. Provisional Patent Application No. 62 / 271,985, filed December 28, 2015, U.S. Provisional Patent Application No. 62 / 296,049, filed February 16, 2016, U.S. Provisional Patent Application No. 62 / 322,734, filed April 14, 2016, U.S. Provisional Patent Application No. 62 / 342,633, filed May 27, 2016, and U.S. Provisional Patent Application No. 62 / 355,299, filed June 27, 2016, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 146392035245SEQLISTING.txt, Date Recorded: October 4, 2016, Size: 41 KB).

[0003] The present invention relates to methods for treating multiple sclerosis (MS) in a patient and to articles of manufacture containing instructions for such use. [Background technology]

[0004] Multiple sclerosis (MS) is an inflammatory and demyelinating degenerative disease of the human central nervous system (CNS). It is a worldwide disease affecting approximately 300,000 people in the United States; it is a disease of young adults, with 70% to 80% of cases occurring between the ages of 20 and 40 (Anderson et al. Ann Neurology 31(3):333-6 (1992); Noonan et al. Neurology 58:136-8 (2002)). MS is a heterogeneous disease based on clinical course, magnetic resonance imaging (MRI) scan evaluation, and pathological analysis of biopsy and autopsy material (Lucchinetti et al. Ann Neurol 47:707-17 (2000)). The disease manifests in numerous possible combinations of defects, including spinal cord, brainstem, cranial nerve, cerebellar, cerebral, and cognitive syndromes. Progressive disability is the fate of most patients with MS, especially over the 25-year period. Half of MS patients require a cane to walk within 15 years of disease onset. MS is a leading cause of neurological disability in young and middle-aged adults, and until the past decade, no useful treatments were known. MS is difficult to diagnose due to its nonspecific clinical findings, which have led to the development of highly structured diagnostic criteria, including several advanced techniques consisting of MRI scans, evoked potentials, and cerebrospinal fluid (CSF) studies. All diagnostic criteria rely on the general principle of scattered lesions throughout the central white matter occurring at different times and not explained by other etiologies such as infection, vascular disease, or autoimmune disease (McDonald et al. Ann Neurol 50:121-7 (2001)). MS has four disease patterns: relapsing-remitting MS (RRMS; 80%-85% of cases), primary progressive MS (PPMS; 10%-15% of cases), progressive relapsing MS (PRMS; 5% of cases), and secondary progressive MS (SPMS) (Kremenchutzky et al. Brain 122(Pt 10):1941-50 (1999); Confavreux et al. N Engl J Med 343(20):1430-8 (2000)).An estimated 50% of patients with RRMS will develop SPMS within 10 years, and up to 90% of patients with RRMS will eventually develop SPMS (Weinshenker et al. Brain 112(Pt 1):133-46 (1989)).

[0005] Several disease-modifying drugs from five classes are approved in the United States for the treatment of RRMS, but none are approved for PPMS. Treatments for RRMS include: the interferon class, IFN-β-1a (REBIF®, Extavia, AVONEX®, PLEGRIDY™, and IFN-β-1b (BETASERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®), alemtuzumab (LEMTRADA®), both monoclonal antibodies; dimethyl fumarate (TECFIDERA®), and furosemide. These include ingolimod (GILENYA®), both small molecules, and mitoxantrone (NOVANTRONE®), a cytotoxic agent; and teriflunomide (AUBAGIO®). Other disease-modifying drugs, including methotrexate, cyclophosphamide, azathioprine, and intravenous (IV) immunoglobulin, have been used with varying degrees of success. Current treatments lack sufficient efficacy to quell early disease or are not used in early MS due to associated side effects (Hartung et al. (2011) Expert Rev Neurother. 11, 351–62; Freedman et al., Mult. Scler. Relat. Disord. 3(2):147–55, 2014). Thus, there remains a need for highly effective treatments with acceptable benefit-risk profiles that can be administered early in the disease course to reduce the long-term impact of cumulative disability and improve patients' quality of life.

[0006] All references cited herein are incorporated by reference in their entirety. Summary of the Invention

[0007] Provided herein are methods for treating multiple sclerosis in a human patient, the methods comprising administering an effective amount of an anti-CD20 antibody. Any of the anti-CD20 antibodies described herein can be administered. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0008] In one aspect, provided herein is a method of improving functional capacity in a human patient with multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient has improved functional capacity after treatment; wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, functional capacity is improved in the patient after treatment. In some embodiments, the patient has confirmed improvement in disability 12 weeks after treatment. In some embodiments, the patient has confirmed improvement in disability 24 weeks after treatment. In some embodiments, the improvement in functional capacity in the patient is sustained for at least 12 weeks. In some embodiments, the improvement in functional capacity in the patient is sustained for at least 24 weeks. In some embodiments, the improvement in functional capacity is measured by the Timed 25-Foot Walk (T-25FW) test or EDSS score. In some embodiments, the improvement in functional capacity is measured by the Timed 25-Foot Walk (T-25FW) test and EDSS score.

[0009] In some embodiments, the patient has T1 gadolinium staining lesions at baseline. In some embodiments, the patient does not have T1 gadolinium staining lesions at baseline.

[0010] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0011] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0012] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0013] In some embodiments, the patient has improved functional capacity after one, two, three, and / or four exposures to anti-CD20 antibodies.

[0014] In another aspect, a method of inhibiting the progression of complex disorders in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a reduction in confirmed disorder progression events, and the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, administration results in a reduction in confirmed disorder progression for 12 weeks. In some embodiments, administration results in a reduction in confirmed disorder progression for 24 weeks. In some embodiments, confirmed disorder progression is determined by Expanded Disability Status Scale (EDSS) score, Timed 25-Foot Walk (T25-FW), or 9-Hole Peg Test (9-HPT). In some embodiments, confirmed complex disability progression is determined by Expanded Disability Status Scale (EDSS) score, Timed 25-Foot Walk (T25-FW), and 9-Hole Peg Test (9-HPT).

[0015] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0016] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0017] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0018] In some embodiments, complex disorder progression is inhibited in patients after exposure to one or two, three, and / or four doses of an anti-CD20 antibody. In some embodiments, patients have a delayed onset or reduced risk of confirmed disorder progression 12 weeks after exposure to one, two, three, and / or four doses of an anti-CD20 antibody. In some embodiments, the onset of confirmed disorder progression 24 weeks is delayed in patients after exposure to one, two, three, and / or four doses of an anti-CD20 antibody. In some embodiments, the risk of confirmed disorder progression 24 weeks is reduced in patients after exposure to one, two, three, and / or four doses of an anti-CD20 antibody. In some embodiments, confirmed disorder progression is determined by Expanded Disability Status Scale (EDSS) score.

[0019] In another aspect, provided herein is a method of inhibiting disability progression in a human patient with multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a reduction in confirmed disability progression events, and the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, administration results in a reduction in confirmed disability progression for 12 weeks. In some embodiments, administration results in a reduction in confirmed disability progression for 24 weeks. In some embodiments, confirmed disability progression is determined by Expanded Disability Status Scale (EDSS) score. In some embodiments, confirmed disability progression is determined by an increase in EDSS.

[0020] In some embodiments, confirmed disability progression is determined by change in Timed 25-foot walk (T25-FW). In some embodiments, confirmed disability progression is determined by rate of change in MRI total T2 lesion volume. In some embodiments, confirmed disability progression is determined by rate of change in MRI whole brain volume. In some embodiments, confirmed disability progression is determined by change in Short Form-36 (SF-36) physical component score.

[0021] In some embodiments, the patient has T1 gadolinium staining lesions at baseline. In some embodiments, the patient does not have T1 gadolinium staining lesions at baseline.

[0022] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0023] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0024] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0025] In some embodiments, the patient has suppressed progression of the complex disorder after one or two, three, or four exposures to the anti-CD20 antibody.

[0026] In another aspect, provided herein are methods of delaying the onset of or reducing the risk of confirmed disability progression in a human patient with multiple sclerosis, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, administration results in a delay of onset or a reduction in risk of confirmed disability progression for 12 weeks. In some embodiments, administration results in a delay of onset or a reduction in risk of confirmed disability progression for 24 weeks.

[0027] In some embodiments, the patient has T1 gadolinium staining lesions at baseline. In some embodiments, the patient does not have T1 gadolinium staining lesions at baseline.

[0028] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0029] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0030] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0031] In some embodiments, the patient has a delayed onset of confirmed disorder progression or a reduced risk of confirmed disorder progression after exposure to one, two, three, and / or four of the anti-CD20 antibodies.

[0032] A method for reducing T2 lesion volume in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the patient has T1 gadolinium-staining lesions at baseline. In certain embodiments, the patient does not have T1 gadolinium-staining lesions at baseline.

[0033] In another aspect, provided herein are methods of delaying or preventing brain volume loss in a human patient with multiple sclerosis, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein brain volume loss is delayed or prevented in the patient; and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, further loss of brain volume in patients who have experienced brain volume loss is delayed or prevented.

[0034] Also provided is a method of reducing brain atrophy in a human patient with multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein brain atrophy is delayed or prevented, the anti-CD20 antibody comprising: a) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, further brain atrophy in patients who have experienced brain atrophy is delayed or prevented.

[0035] Methods of treating a human patient with multiple sclerosis are provided, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in no observed disease activity (NEDA) for at least 12 weeks, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the treatment results in no observed disease activity (NEDA) for at least 24 weeks.

[0036] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0037] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0038] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations on one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0039] In some embodiments, the treatment results in NEDA for at least 12 weeks after one, two, three, and / or four exposures of the anti-CD20 antibody.

[0040] Also provided herein are methods of treating a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in the patient achieving a lesion-free state after 24, 48, or 96 weeks of treatment, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the treatment results in the patient achieving a lesion-free state after 48 weeks of treatment. In certain embodiments, the treatment results in the patient achieving a lesion-free state after 24 weeks of treatment. In certain embodiments, the treatment results in the patient achieving a lesion-free state after 48 weeks of treatment. In certain embodiments, the treatment results in the patient being free of gadolinium-staining lesions. In certain embodiments, the treatment results in the patient being free of T2 lesions.

[0041] Provided herein are methods of treating a human patient with a relapsing form of multiple sclerosis, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment comprises: a) Patients who are relapse-free at 96 weeks; b) Patients without a confirmed disability progression event at 96 weeks; c) patients without T1 gadolinium-enhancing lesions at 96 weeks; d) patients with no new and / or enlarging T2 lesions at 96 weeks; Here, the anti-CD20 antibody comprises: 1) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and 2) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0042] Provided herein is a method of treating a human patient with a relapsing form of multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in the patient having no confirmed events of disability progression at 96 weeks, wherein the anti-CD20 antibody comprises 1) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and 2) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0043] In one embodiment, the treatment comprises: a) Patients who are relapse-free at 96 weeks; b) patients without T1 gadolinium-enhancing lesions at 96 weeks; c) Patients with no new and / or enlarging T2 lesions at 96 weeks;

[0044] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0045] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0046] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0047] In some embodiments, the treatment results in the patient having no confirmed disease progression events at 96 weeks after one, two, three, and / or four exposures to the anti-CD20 antibody. a) Patients who are relapse-free at 96 weeks; b) Patients without T1 gadolinium-enhancing lesions at 96 weeks; and / or c) patients with no new and / or enlarging T2 lesions at 96 weeks after exposure to one, two, three, and / or four doses of anti-CD20 antibody.

[0048] Provided herein are methods of treating a human patient with a relapsing form of multiple sclerosis, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment comprises: a) Patients who are relapse-free; b) Patients without confirmed disability progression events; c) patients without T1 gadolinium-enhancing lesions; d) Patients without new and / or enlarging T2 lesions; wherein the anti-CD20 antibody comprises 1) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12, and 2) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6, and wherein the treatment results in any one or more of a) to d) after one, two, three, and / or four exposures to the anti-CD20 antibody.

[0049] In another aspect, provided herein is a method of treating a human patient with highly active multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the patient with highly active multiple sclerosis is an inadequate responder to other therapies for multiple sclerosis. In some embodiments, the patient with highly active multiple sclerosis has not previously been treated with other therapies for multiple sclerosis. In some embodiments, the other therapies for multiple sclerosis are interferon or glatiramer acetate. In some embodiments, administration of the anti-CD20 antibody is effective in one or more of: (1) reducing the number of lesions in the patient's brain; (2) reducing the annualized relapse rate; (3) reducing the progression of disability; and (4) improving functional ability. In some embodiments, the method further comprises performing an MRI scan to determine whether the patient has hyperactive multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

[0050] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0051] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0052] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0053] In some embodiments, patients with highly active multiple sclerosis have (1) a decrease in the number of lesions in the patient's brain; (2) a decrease in the annualized relapse rate; (3) a decrease in the progression of disability; and / or (4) an improvement in functional ability after exposure to one, two, three, and / or four of the anti-CD20 antibodies.

[0054] In another aspect, provided herein is a method of treating a human patient with early multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the method further comprises diagnosing the patient with early multiple sclerosis before administering the anti-CD20 antibody to the patient. In some embodiments, the patient has been diagnosed with multiple sclerosis but has not received treatment for at least two years before administering the anti-CD20 antibody. In certain embodiments, early multiple sclerosis is the first clinical presentation of multiple sclerosis in the patient. In some embodiments, the first clinical manifestation of multiple sclerosis is a first demyelinating event (FCDE) (also known as clinically isolated syndrome (CIS)), i.e., the first episode of neurological symptoms, which lasts at least 24 hours and is caused by inflammation or demyelination in the central nervous system. In some embodiments, FDCE affects the optic nerve, brainstem, subcortical white matter, or spinal cord. In some embodiments, early multiple sclerosis refers to a diagnosis of clinically evident multiple sclerosis (CDMS), in which a patient experiences a second clinical attack following FCDE.

[0055] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0056] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0057] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0058] In another aspect, provided herein is a method of treating a human patient with multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in no observed disease activity (NEDA) in the patient, wherein the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, NEDA is defined as no relapse, no CDP events, no new or enlarging T2 lesions, and no Gd-enhancing T1 lesions, as defined in the protocol.

[0059] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0060] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0061] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0062] In some embodiments, the treatment results in NEDA after one, two, three, and / or four exposures of anti-CD20 antibodies.

[0063] In certain embodiments, treatment or administration of an anti-CD20 antibody to a human patient with a relapsing form of multiple sclerosis comprises the following: a) any of about a 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% reduction in annualized relapse rate over time (e.g., over a period of at least about 1 year, 1.5 years, or 2 years) compared to patient(s) being treated with interferon beta-1a (e.g., REBIF®), including any ranges therebetween; b) any of about a 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% reduction in the risk of confirmed disability progression for at least 12 weeks compared to patient(s) receiving treatment with interferon beta-1a (e.g., REBIF®) (including any ranges therebetween); c) any of about a 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% reduction in the risk of confirmed disability progression for at least 24 weeks compared to patient(s) receiving treatment with interferon beta-1a (e.g., REBIF®); including any ranges therebetween; d) any of about a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction in the total number of T1 gadolinium lesions (including any ranges therebetween) compared to patient(s) receiving treatment with interferon beta-1a (e.g., REBIF®); e) any of about a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction in the mean number of T1 gadolinium lesions at 24, 48, and / or 96 weeks compared to patient(s) treated with interferon beta-1a (e.g., REBIF®); including any ranges therebetween; f) any of about a 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% reduction in the number of new and / or enlarging T2 hyperintense lesions (including any ranges therebetween) compared to patient(s) receiving treatment with interferon beta-1a (e.g., REBIF®); g) about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% of the mean number of new and / or enlarging T2 hyperintense lesions at 24 weeks, 48 ​​weeks, and / or 96 weeks compared to patient(s) receiving treatment with interferon beta-1a (e.g., REBIF®); Any of the following: a 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction (including any range between these values); h) any of the following (including any ranges between these values) - a decrease of approximately 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% in the rate of total brain volume loss compared to untreated patient(s); i) about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 1 Any of the following improvements (including any range between these values): 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%; j) any of about a 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% reduction in the number of new T1 hypointense lesions compared to patients receiving treatment with interferon beta-1a (e.g., REBIF®); including any ranges therebetween; k) results in one or more of about a 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% increase in the likelihood of reaching NEDA (No Evidence of Disease Activity); Here, NEDA is defined as no protocol-defined recurrence, no CDP events, no new or enlarging T2 lesions, and no Gd-enhancing T1 lesions compared to patients treated with interferon beta-1a (e.g., REBIF®), including any range between these values.

[0064] In certain embodiments, the treatment additionally or alternatively results in any of about a 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% reduction in brain atrophy, including any range between these values, compared to patients receiving treatment with interferon beta-1a (e.g., REBIF®).

[0065] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0066] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0067] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0068] In some embodiments, the treatment results in one or more of a) through k) after one, two, three, and / or four exposures of anti-CD20 antibodies.

[0069] In certain embodiments, treatment or administration of an anti-CD20 antibody to a patient with primary progressive multiple sclerosis comprises one of the following: a) Any of the following (including any ranges between these values) of approximately 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% reduction in the risk of progression of confirmed disability for at least 12 weeks compared to untreated patient(s); b) Any of the following (including any ranges between these values) reductions in the risk of confirmed disability progression for at least 24 weeks compared to untreated patient(s); c) Any of the following (including any ranges between these values) of approximately 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% decrease in the rate of increase in walking time as measured by the Timed 25-Foot Walk compared to untreated patient(s); d) Any of the following (including any ranges between these values) reductions in T2 lesion volume of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% compared to untreated patient(s); e) a decrease from baseline in hyperintense T2 lesion volume of about 1%, 1.5%, 2%, 2.5%, 3%, 3.4%, 3.5%, 4%, 4.5%, or 5% at any of about Weeks 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, and 120; and f) resulting in one or more of the following (including any ranges therebetween): approximately 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% decrease in the rate of whole brain volume loss compared to patient(s) not receiving treatment.

[0070] In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0071] In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0072] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0073] In some embodiments, the treatment results in one or more of a) through f) after one, two, three, and / or four exposures of an anti-CD20 antibody.

[0074] In certain embodiments, a method of reducing the risk of confirmed disability progression over at least 12 weeks in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody results in any of about a 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% reduction (including any ranges between these values) in the risk of confirmed disability progression over at least 12 weeks in the patient, compared to a patient(s) not receiving the treatment, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0075] In certain embodiments, a method of reducing the risk of confirmed disability progression over at least 24 weeks in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody results in any of about a 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% reduction (including any ranges between these values) in the risk of confirmed disability progression over at least 24 weeks in the patient, compared to patient(s) not receiving the treatment, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0076] In certain embodiments, a method of reducing the rate of increase in walking time, as measured by a Timed 25-Foot Walk, in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody results in any of about a 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% reduction (including any ranges between these values) in the rate of increase in walking time in the patient, compared to patient(s) not receiving the treatment, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0077] In certain embodiments, a method of reducing T2 lesion volume in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody results in any of about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% reduction in T2 lesion volume in the patient (including any ranges between these values) compared to a patient(s) not receiving the treatment, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0078] In certain embodiments, a method of reducing hyperintense T2 lesion volume in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody reduces hyperintense T2 lesion volume in the patient at about week 20, week 24, week 28, week 32, week 36, week 40, week 44, week 48, week 52, week 56, week 60, week 64, week 68, week 72, week 76, week 80, week 82, week 84, week 86, week 88, week 89, week 90, week 91, week 92, week 93, week 94, week 95, week 96, week 97, week 98, week 99, week 100, week 101, week 102, week 103, week 104, week 105, week 106, week 107, week 108, week 109, week 110, week 111, week 112, week 113, week 114, week 115, week 116, week 117, week 118, week 119, week 120, week 121, week 122, week 123, week 124, week 125, week 126, week 127, week 128, week 129, week 130, week 131, week 132, week 133, week 134, week 135, week 136, week 137, week 138, week 139, week 140, week 141, week 142, week 143, week 144, week 145, week 146, week 1 and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, wherein the antibody results in about a 1%, 1.5%, 2%, 2.5%, 3%, 3.4%, 3.5%, 4%, 4.5%, or 5% reduction from baseline in hyperintense T2 lesion volume at any of weeks 1, 2, 3, 4, 5, 6, 7, 8, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, and 120, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2.

[0079] In certain embodiments, a method of reducing the rate of total brain volume loss in a patient with primary progressive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration of the anti-CD20 antibody results in any of about a 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20% reduction (including any ranges between these values) in the rate of total brain volume loss in the patient, compared to patient(s) not receiving the treatment, wherein the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20-24 weeks or about 5-6 months. In some embodiments, an anti-CD20 antibody is administered to a patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0080] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0081] In certain embodiments, a) risk of confirmed disability progression for at least 12 weeks, b) risk of confirmed disability progression for at least 24 weeks, c) rate of increase in walking time as measured by Timed 25-Foot Walk, d) T2 lesion volume, e) hyperintense T2 lesion volume, and / or f) rate of whole brain volume loss is reduced after exposure to one, two, three, and / or four of the anti-CD20 antibodies. In certain embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0082] In certain embodiments of any of the methods, the patient maintains the ability to mount a humoral response to the antigen during treatment. In certain embodiments, the antigen is a mumps antigen, a rubella antigen, a varicella antigen, a Streptococcus pneumoniae antigen, a tetanus toxoid antigen, a pneumococcal antigen, or an influenza antigen.

[0083] In certain embodiments, the patient is premedicated prior to infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with methylprednisolone (or equivalent) approximately 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with 100 mg of IV methylprednisolone (or equivalent) approximately 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antihistamine (e.g., diphenhydramine) approximately 30-60 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antipyretic (e.g., acetaminophen / paracetamol).

[0084] In some embodiments of the methods described above and herein, the anti-CD20 antibody is the first medicament, and the second medicament is administered with the initial exposure or a subsequent exposure. In some embodiments, the second medicament is selected from the group consisting of interferon, glatiramer acetate, a cytotoxic agent, a chemotherapeutic agent, mitoxantrone, methotrexate, cyclophosphamide, chlorambucil, azathioprine, gamma globulin, Campath, anti-CD4, cladribine, corticosteroids, mycophenolate mofetil (MMF), cyclosporine, cholesterol-lowering drugs of the statin class, estradiol, testosterone, hormone replacement drugs, TNF inhibitors, disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), levothyroxine, cyclosporine A, somatostatin analogs, cytokine or cytokine receptor antagonists, antimetabolites, immunosuppressants, integrin antagonists or antibodies, LFA-1 antibodies, efalizumab, alpha 4 integrin antibodies, natalizumab, and other B cell surface marker antibodies.

[0085] In some embodiments of the methods described above and herein, the multiple sclerosis is a relapsing form of multiple sclerosis. In some embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the relapsing form of multiple sclerosis is secondary progressive multiple sclerosis with overlapping relapses (rSPMS). In some embodiments, the multiple sclerosis is progressive multiple sclerosis. In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis (PPMS).

[0086] In certain embodiments according to (or as applied to) any of the above embodiments, the patient is selected for treatment based on having a relapsing form of multiple sclerosis (e.g., RRMS or rSPMS). In certain embodiments according to (or as applied to) any of the above embodiments, the treatment is based on the patient having a relapsing form of multiple sclerosis (e.g., RRMS or rSPMS). In certain embodiments according to (or as applied to) any of the above embodiments, the patient has been diagnosed with a relapsing form of multiple sclerosis (e.g., RRMS or rSPMS) prior to treatment.

[0087] In certain embodiments according to (or as applied to) any of the above embodiments, the patient is selected for treatment based on having a progressive form of multiple sclerosis (e.g., PPMS). In certain embodiments according to (or as applied to) any of the above embodiments, the treatment is based on the patient having a progressive form of multiple sclerosis (e.g., PPMS). In certain embodiments according to (or as applied to) any of the above embodiments, the patient has been diagnosed with a progressive form of multiple sclerosis (e.g., PPMS) prior to treatment.

[0088] In some embodiments of the methods described above and herein, an anti-CD20 antibody is administered to a patient to provide an initial anti-CD20 antibody exposure, followed by one or more additional anti-CD20 antibody exposures, where each exposure is about 600 mg of antibody, where each exposure is provided to the patient as one or two doses of anti-CD20 antibody, and where the interval between each exposure is about 20-24 weeks or about 5-6 months. In some embodiments, "about 20-24 weeks" refers to a time point between 20 and 24 weeks. In some embodiments, "about 20-24 weeks" refers to a variant of 1 week or 7 days before or after the 24th week. In some embodiments, "about 5-6 months" refers to a time point between 5 and 6 months.

[0089] In some embodiments, the first exposure comprises a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In some embodiments, the first exposure and the second, third, and / or fourth additional exposures comprise a first and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about two weeks or about 14 days (e.g., 13 or 15 days).

[0090] In certain embodiments according to (or as applied to) any of the above embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments according to (or as applied to) any of the above embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 26 or SEQ ID NO: 27, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0091] In certain embodiments according to (or as applied to) any of the above embodiments, an anti-CD20 antibody is administered to a patient to provide a second anti-CD20 antibody exposure following a first anti-CD20 antibody exposure, where the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months. In certain embodiments according to (or as applied to) any of the above embodiments, an anti-CD20 antibody is administered to a patient to provide a third anti-CD20 antibody exposure, where the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months. In certain embodiments according to (or as applied to) any of the above embodiments, the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, where the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20-24 weeks or about 5-6 months.

[0092] In certain embodiments according to (or as applied to) any of the above embodiments, the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days (e.g., 13 days or 15 days). In some embodiments, "about 14 days" refers to variations on one day before or after the 14th day. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses are administered intravenously. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses each comprise 250 mL of anti-CD20 antibody at a concentration of about 1.2 mg / mL. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses are each infused at a rate of 30 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the infusion rate of the first and second doses can be increased in 30 mL / hour increments up to a maximum rate of 180 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the first dose and the second dose are each given over approximately 2.5 hours.

[0093] In some embodiments, the second, third, and / or fourth exposures comprise a single dose of about 600 mg. In certain embodiments according to (or as applied to) any of the above embodiments, the second, third, and / or fourth exposures are administered intravenously. In certain embodiments according to (or as applied to) any of the above embodiments, the second, third, and / or fourth exposures each comprise 500 mL of anti-CD20 antibody at a concentration of about 1.2 mg / mL. In certain embodiments according to (or as applied to) any of the above embodiments, the second, third, and / or fourth exposures are each infused at a rate of 40 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the infusion rate of the second, third, and / or fourth exposures can be increased in 40 mL / hour increments up to a maximum rate of 200 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the first dose and the second dose are each given over approximately 3.5 hours.

[0094] In some embodiments, the first exposure, and the second, third, and / or fourth additional exposures, comprise a first and second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days (e.g., 13 days or 15 days). In some embodiments, "about 14 days" refers to variations on one day before or after the 14th day. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses are administered intravenously. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses each comprise 250 mL of anti-CD20 antibody at a concentration of about 1.2 mg / mL. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses each are infused at a rate of 30 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the infusion rates of the first and second doses may be increased in 30 mL / hour increments up to a maximum rate of 180 mL / hour. In certain embodiments according to (or as applied to) any of the above embodiments, the first and second doses are each given over approximately 2.5 hours.

[0095] In certain embodiments, the patient receives at least 2, 3, 4, or more than 4 anti-CD20 antibody exposures.

[0096] In some embodiments, the anti-CD20 antibody is administered to the patient to provide one or more additional anti-CD20 antibody exposures after the fourth exposure, wherein the one or more additional exposures are about 600 mg of antibody, and the interval between the fourth and additional exposures is about 20-24 weeks or about 5-6 months. In some embodiments, "about 20-24 weeks" refers to a time point between 20 and 24 weeks. In some embodiments, "about 20-24 weeks" refers to a variation of 1 week or 7 days before or after the 24th week. In some embodiments, "about 5-6 months" refers to a time point between 5 and 6 months. In some embodiments, the interval between each additional exposure following the fourth exposure is 20-24 weeks or about 5-6 months. In some embodiments, the one or more exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 14 days (e.g., 13 days or 15 days). In some embodiments, "about 14 days" refers to variations on one day before or after day 14. In some embodiments, the one or more exposures include a first dose and a second dose of an anti-CD20 antibody, where each dose is about 300 mg, and the first and second doses are separated by about 14 days (e.g., 13 days or 15 days).

[0097] In some embodiments of the methods described above and herein, the anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:26 or SEQ ID NO:15 or SEQ ID NO:27 and a light chain comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, the anti-CD20 antibody is ocrelizumab. In some embodiments, the anti-CD20 antibody is present in a pharmaceutically acceptable composition. In some embodiments, the anti-CD20 antibody is in a formulation comprising 30 mg / mL antibody, 20 mM sodium acetate, 106 mM trehalose, 0.02% polysorbate 20, pH 5.3. In some embodiments, the antibody in the formulation is stored at about 2-8°C at 300 mg / vial. In some embodiments, the antibody is diluted in saline (0.9% sodium chloride) in an IV bag for administration by infusion. In some embodiments, the anti-CD20 antibody is an antigen-binding fragment thereof.

[0098] In some embodiments of the methods described above and herein, the anti-CD20 antibody is administered intravenously. In some embodiments, the anti-CD20 antibody is administered intravenously for each antibody exposure. In some embodiments of the methods described above and herein, the antibody is administered subcutaneously. In some embodiments, the anti-CD20 antibody is administered subcutaneously for each antibody exposure.

[0099] In some embodiments, a composition is provided for use in treating multiple sclerosis in a patient according to any of the above-described methods, the composition comprising an anti-CD20 antibody comprising: a) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0100] In some embodiments, an anti-CD20 antibody is provided for use in the manufacture of a medicament for the treatment of multiple sclerosis in a patient according to any of the above-described methods, the anti-CD20 antibody comprising: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0101] In certain embodiments of any of the methods described above and / or herein, the decrease or reduction or improvement following administration of an anti-CD20 antibody can be compared to baseline levels, levels in untreated patient(s), and / or levels in patient(s), e.g., the mean, average, or median levels of a group of patients receiving different treatments (e.g., interferon beta-1a or REBIF®, etc.).

[0102] In another aspect, provided herein is an article of manufacture comprising: (a) a container containing an anti-CD20 antibody (e.g., ocrelizumab); and (b) a package insert containing instructions for treating multiple sclerosis in a patient according to any of the methods described above and herein.

[0103] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. [Brief explanation of the drawings]

[0104] [Figure 1A]Figure 1 shows a sequence alignment comparing the amino acid sequences of the light chain variable domains (VL) of mouse 2H7 (SEQ ID NO: 1), the humanized 2H7.v16 variant (SEQ ID NO: 2), and the human kappa light chain subgroup I (SEQ ID NO: 3). The CDRs of the VLs of 2H7 and hu2H7 are as follows: CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6). CDR1, CDR2, and CDR3 in each chain are enclosed in square brackets and flanked by framework regions FR1 through FR4 as indicated. 2H7 refers to the mouse 2H7 antibody. Asterisks between the two sequences indicate positions that differ between the two sequences. Residue numbering is according to Kabat et al., Sequences of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions indicated as a, b, c, d, and e. [Figure 1B] Figure 1 is a sequence alignment comparing the amino acid sequences of the heavy chain variable domains (VH) of mouse 2H7 (SEQ ID NO: 7), the humanized 2H7.v16 variant (SEQ ID NO: 8), and the human consensus sequence for heavy chain subgroup III (SEQ ID NO: 9). The CDRs of the VHs of 2H7 and hu2H7 are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12). CDR1, CDR2, and CDR3 in each chain are enclosed in square brackets and flanked by framework regions FR1-FR4 as indicated. 2H7 refers to the mouse 2H7 antibody. Asterisks between the two rows of sequences indicate positions that differ between the two sequences. Residue numbering is according to Kabat et al. Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions indicated as a, b, c, d, and e. [Figure 2] The amino acid sequence of the mature 2H7.v16 heavy chain (SEQ ID NO: 13) is shown. [Figure 3]The amino acid sequence of the mature 2H7.v16 heavy chain (SEQ ID NO: 14) is shown. [Figure 4] The amino acid sequence of the mature 2H7.v31 heavy chain (SEQ ID NO: 15) is shown. The light chain of 2H7.v31 is the same as that of 2H7.v16. [Figure 5] 1 shows the sequence alignment of the mature 2H7.v16 and 2H7.v511 light chains (SEQ ID NOs: 13 and 16, respectively) with Kabat variable domain residue numbering and Eu constant domain residue numbering. [Figure 6] 1 shows the sequence alignment of the mature 2H7.v16 and 2H7.v511 heavy chains (SEQ ID NOs: 14 and 17, respectively) with Kabat variable domain residue numbering and Eu constant domain residue numbering. [Figure 7]

[0023] Figure 1 shows an overview of the study design for two identical Phase III studies (i.e., Study I and Study II) of ocrelizumab compared with interferon beta-1a (REBIF®) in patients with relapsing multiple sclerosis. [Figure 8] The hierarchical statistical analysis plan for Study I and Study II is shown. [Figure 9] The breakdown of patients in Study I and Study II is shown. [Figure 10] Figure 10A shows the annualized relapse rates (ARR) at week 96 for patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study I. Figure 10B shows the annualized relapse rates (ARR) at week 96 for patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study II. [Figure 11] Figure 1 shows time to onset of confirmed disability progression (CDP) of at least 12 weeks over 96 weeks in patients treated with ocrelizumab compared with patients treated with interferon beta-1a (pooled data from Study I and Study II). [Figure 12]Figure 12A shows the time to onset of confirmed disability progression of at least 12 weeks over 96 weeks in patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study I. Figure 12B shows the time to onset of confirmed disability progression of at least 12 weeks over 96 weeks in patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study II. [Figure 13] Figure 1 shows time to onset of confirmed disability progression of at least 24 weeks over 96 weeks in patients treated with ocrelizumab compared with patients treated with interferon beta-1a (pooled data from Study I and Study II). [Figure 14] Figure 14A shows the time to onset of confirmed disability progression of at least 24 weeks over 96 weeks in patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study I. Figure 14B shows the time to onset of confirmed disability progression of at least 24 weeks over 96 weeks in patients treated with ocrelizumab compared to patients treated with interferon beta-1a in Study II. [Figure 15] Figure 15A shows the proportion of patients with at least 12 weeks of confirmed disability improvement (i.e., CDI) (baseline EDSS score of 2.0 or greater) in patients receiving ocrelizumab compared to patients receiving interferon beta-1a (pooled data from Studies I and II). Figure 15B shows the proportion of patients with at least 24 weeks of confirmed disability improvement (i.e., CDI) (baseline EDSS score of 2.0 or greater) in patients receiving ocrelizumab compared to patients receiving interferon beta-1a (pooled data from Studies I and II). [Figure 16]Figure 16A shows the proportion of patients with at least 12 weeks of confirmed disability improvement (i.e., CDI) (baseline EDSS score of 2.0 or greater) in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 16B shows the proportion of patients with at least 12 weeks of confirmed disability improvement (i.e., CDI) (baseline EDSS score of 2.0 or greater) in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 16C] 1 shows the change in Multiple Sclerosis Functional Composite Score from baseline to Week 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. [Figure 16D] 1 shows the change in Multiple Sclerosis Functional Composite Score from baseline to Week 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 17] Figure 17A shows the total number of T1 gadolinium-enhancing lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 17B shows the total number of T1 gadolinium-enhancing lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 18] Figure 18A shows the mean total number of T1 gadolinium-enhancing lesions in patients receiving ocrelizumab at weeks 24, 48, and 96 compared to patients receiving IFN β-1a in Study I. Figure 18B shows the mean total number of T1 gadolinium-enhancing lesions in patients receiving ocrelizumab at weeks 24, 48, and 96 compared to patients receiving IFN β-1a in Study II. [Figure 19A-B]Figure 19A shows the total number of new and / or enlarging hyperintense T2 lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I and Study II. Figure 19B shows the total number of new and / or enlarging hyperintense T2 lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 19C-D] Figure 19C shows the mean number of new and / or enlarging hyperintense T2 lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 19D shows the mean number of new and / or enlarging hyperintense T2 lesions detected at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 20] Figure 20A shows the rate of brain volume loss from weeks 24 to 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 20B shows the rate of brain volume loss from weeks 24 to 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 21] Figure 21A shows the rate of brain volume loss from baseline to week 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 21B shows the rate of brain volume loss from baseline to week 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 22]Figure 22A shows the total number of new T1 hypointense lesions per MRI scan at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study I. Figure 22B shows the total number of new T1 hypointense lesions per MRI scan at weeks 24, 48, and 96 in patients receiving ocrelizumab compared to patients receiving interferon beta-1a in Study II. [Figure 23] Figure 23A shows infusion-related reactions in patients receiving IFN β-1a in Study I and Study II (pooled). Figure 23B shows infusion-related reactions in patients receiving ocrelizumab in Study I and Study II (pooled). [Figure 24] 1 shows the study design for a Phase III study of ocrelizumab in patients with primary progressive multiple sclerosis (PPMS). [Figure 25] To provide statistical stratification for a phase III study of ocrelizumab in patients with PPMS. [Figure 26] The breakdown of patients in the Phase III PPMS study at the clinical cutoff date is shown below. [Figure 27] Figure 1 shows the time to onset of confirmed disability progression of at least 12 weeks in patients treated with ocrelizumab compared to patients receiving placebo in a Phase III PPMS study. [Figure 28] Figure 1 shows the time to onset of confirmed disability progression of at least 24 weeks in patients treated with ocrelizumab compared to patients receiving placebo in a Phase III PPMS study. [Figure 29] Figure 1 shows the percent decline in walking speed, as measured by the Timed 25-Foot Walk, in patients receiving 600 mg of ocrelizumab compared to patients receiving placebo from baseline to week 120 in the Phase 3 PPMS study. [Figure 30]Figure 1 shows the percent decline in walking speed relative to baseline at week 120 in patients receiving 600 mg of ocrelizumab compared to patients receiving placebo in the Phase III PPMS study. [Figure 31] Figure 1 shows the percent change in whole brain volume from week 24 to week 96 in patients receiving 600 mg of ocrelizumab compared to patients receiving placebo in a Phase III PPMS study. [Figure 32] 1 shows the change in T2 lesion volume in patients receiving 600 mg of ocrelizumab compared to patients receiving placebo in a Phase III PPMS study. [Figure 33] Figure 1 shows infusion-related reactions (IRR) in patients receiving 600 mg of ocrelizumab compared with patients receiving placebo, by exposure and severity, through the clinical cutoff date in the Phase III PPMS study. [Figure 34] Figure 34A shows the proportion of patients receiving IFN β-1a with no observed disease activity (NEDA) compared to the proportion of patients receiving ocrelizumab with no observed disease activity (NEDA) in Study I. Figure 34B shows the proportion of patients receiving IFN β-1a with no observed disease activity (NEDA) compared to the proportion of patients receiving ocrelizumab with no observed disease activity (NEDA) in Study II. [Figure 35]Figure 35A shows the percent change reduction in walking time from baseline to week 120 in patients with Gd+ lesions at baseline receiving ocrelizumab relative to patients with Gd+ lesions at baseline receiving placebo in the Timed 25-Foot Walk study. Figure 35B shows the percent change reduction in walking time from baseline to week 120 in patients without Gd+ lesions at baseline receiving ocrelizumab relative to patients without Gd+ lesions at baseline receiving placebo in the Timed 25-Foot Walk study. Figure 35C shows the percent change reduction in walking time from baseline to week 120 in patients in the overall study population receiving ocrelizumab relative to patients in the overall study population receiving placebo in the Timed 25-Foot Walk study. [Figure 36] Figure 36A shows the difference in total brain volume loss from weeks 24 to 120 in patients with T1 Gd+ lesions at baseline who were treated with ocrelizumab compared to patients with T1 Gd+ lesions at baseline who received a placebo. Figure 36B shows the difference in total brain volume loss from weeks 24 to 120 in patients without T1 Gd+ lesions at baseline who were treated with ocrelizumab compared to patients without T1 Gd+ lesions at baseline who received a placebo. Figure 36C shows the rate of total brain volume loss from weeks 24 to 120 in patients in the overall study population who were treated with ocrelizumab compared to patients in the overall study population who received a placebo. [Figure 37]Figure 37A shows the difference in T2 lesion volume from baseline to week 120 in patients receiving ocrelizumab relative to patients receiving placebo. Figure 37B shows the difference in T2 lesion volume from baseline to week 120 in patients receiving ocrelizumab with T1 Gd+ lesions at baseline relative to patients receiving placebo with T1 Gd+ lesions at baseline. Figure 37C shows the difference in T2 lesion volume from baseline to week 120 in patients receiving ocrelizumab without T1 Gd+ lesions at baseline relative to patients receiving placebo without T1 Gd+ lesions at baseline. [Figure 38] Figure 1 shows the difference in reported improvement in quality of life change between patients receiving ocrelizumab and patients receiving IFN beta-1a, as measured by the Short Form-36 (SF-36) Physical Component Summary (PCS). [Figure 39A] 1 shows the difference between the change in EDSS score from baseline to week 96 in patients receiving ocrelizumab versus patients receiving IFN beta-1a in Study I. [Figure 39B] 1 shows the difference between the change in EDSS score from baseline to week 96 in patients receiving ocrelizumab versus patients receiving IFN beta-1a in Study II. [Figure 40]Figure 40A shows the change in SF-36 physical component summary score from baseline to week 120 in patients in the overall study population receiving 600 mg of ocrelizumab compared to patients in the overall study population receiving placebo. Figure 40B shows the change in SF-36 physical component summary score from baseline to week 120 in patients with T1 gadolinium-enhancing lesions at baseline receiving 600 mg of ocrelizumab compared to patients with T1 gadolinium-enhancing lesions at baseline receiving placebo. Figure 40C shows the change in SF-36 physical component summary score from baseline to week 120 in patients without T1 gadolinium-enhancing lesions at baseline receiving 600 mg of ocrelizumab compared to patients without T1 gadolinium-enhancing lesions at baseline receiving placebo. [Figure 41] Figure 1 shows the time to onset of confirmed composite disability progression of at least 12 weeks in patients treated with ocrelizumab compared to patients receiving placebo in a Phase III PPMS study. [Figure 42] Figure 1 shows the time to onset of confirmed composite disability progression of at least 24 weeks in patients treated with ocrelizumab compared with patients receiving placebo in a Phase III PPMS study. [Figure 43] Annualized protocol-defined relapse rates through week 96 are shown for the following subgroups: active inadequate responders, active treatment-naive patients, highly active inadequate responders, and highly active treatment-naive patients. [Figure 44] Time to onset of CDP of at least 12 weeks in the following subgroups: active inadequate responders, active treatment-naive patients, highly active inadequate responders, and highly active treatment-naive patients is shown. [Figure 45] Time to onset of CDP of at least 24 weeks in the following subgroups: active inadequate responders, active treatment-naive patients, highly active inadequate responders, and highly active treatment-naive patients is shown. [Figure 46]The proportion of patients with at least 12 weeks of CDI in the following subgroups: active inadequate responders, active treatment-naive patients, highly active inadequate responders, and highly active treatment-naive patients is shown. [Figure 47] The total number of T1 gadolinium-enhancing lesions detected by brain MRI in the following subgroups: active inadequate responders, active treatment-naive patients, highly active inadequate responders, and highly active treatment-naive patients is shown. DETAILED DESCRIPTION OF THE INVENTION

[0105] I. Definition "B cells" are lymphocytes that mature in the bone marrow and include naive B cells, memory B cells, or effector B cells (plasma cells). B cells herein can be normal or non-malignant B cells.

[0106] As used herein, a "B cell surface marker" or "B cell surface antigen" is an antigen expressed on the surface of a B cell that can be targeted by an antibody that binds to it. Exemplary B cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85, and CD86 leukocyte surface markers (for a description, see The Leukocyte Antigen Facts Book, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B cell surface markers include RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. B cell surface markers of particular interest herein are preferentially expressed on B cells relative to other non-B cell tissues in mammals and can be expressed on both precursor B cells and mature B cells. A preferred B cell surface marker herein is CD20.

[0107] The "CD20" antigen, or "CD20," is a non-glycosylated phosphoprotein of approximately 35 kDa found on the surface of more than 90% of B cells in peripheral blood or lymphoid organs. CD20 is present on both normal and malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include "B lymphocyte-restricted antigen" and "Bp35." The CD20 antigen is described, for example, in Clark et al. Proc. Natl. Acad. Sci. (USA) 82:1766 (1985).

[0108] An "antibody antagonist" herein is an antibody that, upon binding to a B cell surface marker on a B cell, destroys or depletes B cells in a mammal and / or interferes with one or more B cell functions, e.g., by reducing or preventing a humoral response induced by the B cell. Preferably, the antibody antagonist is capable of depleting B cells (i.e., reducing circulating B cell levels) in a mammal treated therewith. Such depletion may be achieved through a variety of mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), inhibition of B cell proliferation, and / or induction of B cell death (e.g., via apoptosis).

[0109] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibody on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 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 may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0110] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. In one embodiment, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and NK cells being preferred.

[0111] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. Furthermore, preferred FcRs bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (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 herein by the term "FcR." This term also includes the neonatal receptor, FcRn, involved in the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0112] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0113] A "growth inhibitory" antibody is one that inhibits or reduces the proliferation of cells expressing the antigen to which the antibody binds. For example, the antibody may inhibit or reduce the proliferation of B cells in vitro and / or in vivo.

[0114] An antibody that "induces apoptosis" is one that induces programmed cell death, e.g., of B cells, as measured by standard apoptosis assays, e.g., Annexin V binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum dilation, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies).

[0115] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0116] "Antibody fragments" include portions of intact antibodies, preferably including their antigen-binding regions. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0117] For purposes herein, an "intact antibody" is one comprising heavy and light chain variable domains, as well as an Fc region.

[0118] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) followed by a number of constant domains. H Each light chain has at one end a variable domain (V L ) at one end and a constant domain at the other end; the light-chain constant domain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0119] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FRs, largely in a β-sheet configuration, connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are held in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains 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).

[0120] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site; the remainder is designated the "Fc" fragment, reflecting its ability to be readily crystallized. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0121] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. The three hypervariable regions of each variable domain interact to form the V H -V LIt is in this configuration that defines an antigen-binding site on the surface of the dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0122] Fab fragments also contain the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0123] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0124] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0125] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L In one embodiment, an Fv polypeptide comprises V domains, and these domains are present in a single polypeptide chain.H and V L The scFv further comprises a polypeptide linker between the 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. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0126] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) to the heavy chain variable domain (V H ) linked together. A linker too short to allow pairing of the two domains on the same chain is used to force pairing of the domains with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in detail, for example, in European Patent Application Publication No. 404097; International Patent Application Publication No. WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0127] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible mutations that may arise during production of the monoclonal antibody, which may generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and does not imply that the antibody must be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be made, for example, by recombinant DNA methodologies (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0128] As used herein, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains corresponds to or resembles 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) corresponds to or resembles 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 (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies containing variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

[0129] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. By way of example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for the FR substitutions noted above. Humanized antibodies optionally also comprise at least a portion of an immunoglobulin constant region, 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).

[0130] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable regions may be comprised of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain, and 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0131] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0132] Purely for purposes herein, and unless otherwise specified, "humanized 2H7" means a humanized antibody, or antigen-binding fragment thereof, that binds to human CD20, wherein the antibody is effective in depleting primate B cells in vivo, and wherein the antibody is a humanized antibody comprising a heavy chain variable region (V H ) comprising at least the CDR H3 sequence of SEQ ID NO: 12 (FIG. 1B) derived from an anti-human CD20 antibody and a sequence substantially similar to that of the human heavy chain subgroup III (V HIn some embodiments, the antibody further comprises a heavy chain CDR H1 sequence of SEQ ID NO: 10 and a CDR H2 sequence of SEQ ID NO: 11, and in some embodiments, a light chain CDR L1 sequence of SEQ ID NO: 4, a light chain CDR L2 sequence of SEQ ID NO: 5, a light chain CDR L3 sequence of SEQ ID NO: 6, and a light chain CDR L4 sequence of SEQ ID NO: 7, and a light chain CDR L5 sequence of SEQ ID NO: 8, and a light chain CDR L6 sequence of SEQ ID NO: 9, and a light chain CDR L7 sequence of SEQ ID NO: 10, a light chain CDR L8 sequence of SEQ ID NO: 11, and a light chain CDR L9 sequence of SEQ ID NO: 12, and a light chain CDR L10 sequence of SEQ ID NO: 13, a light chain CDR L11 sequence of SEQ ID NO: 14, a light chain CDR L12 sequence of SEQ ID NO: 15, a light chain CDR L13 sequence of SEQ ID NO: 16, and a light chain CDR L14 sequence of SEQ ID NO: 17, and a light chain CDR L15 sequence of SEQ ID NO: 18, and a light chain CDR L16 sequence of SEQ ID NO: 19, and a light chain CDR L17 sequence of SEQ ID NO: 20, and a light chain CDR L18 sequence of SEQ ID NO: 21, and a light chain CDR L19 sequence of SEQ ID NO: 22, and a light chain CDR L19 sequence of SEQ ID NO: 23, and a light chain CDR L11 sequence of SEQ ID NO: 24, a light chain CDR L12 sequence of SEQ ID NO: 25, a light chain CDR L13 sequence of SEQ ID NO: 26, and a light chain CDR L14 sequence of SEQ ID NO: 27, and a light chain CDR L1 κ I), wherein V H The region may be linked to a human IgG chain constant region, which may be, for example, IgG1 or IgG3. In some embodiments, such antibodies comprise the V of SEQ ID NO:8. H 1B), and optionally V of SEQ ID NO: 2 L The antibody may comprise the sequence (v16 shown in FIG. 1A), which may have amino acid substitutions of D56A and N100A in the H chain and S92A in the L chain (v96). In some embodiments, the antibody is an intact antibody comprising the light and heavy chain amino acid sequences of SEQ ID NOs: 13 and 14, as shown in FIGS. 2 and 3, respectively. In some embodiments, the antibody is 2H7.v31, which comprises the light and heavy chain amino acid sequences of SEQ ID NOs: 13 and 15, as shown in FIGS. 2 and 4, respectively. The antibody herein may further comprise at least one amino acid substitution in the Fc region that improves ADCC and / or CDC activity, e.g., the amino acid substitutions are S298A / E333A / K334A, and in some embodiments, is 2H7.v31, which has the heavy chain amino acid sequence of SEQ ID NO: 15 (shown in FIG. 4). Any of these antibodies may further comprise at least one amino acid substitution in the Fc region that reduces CDC activity, e.g., including at least the substitution K322A. See U.S. Patent No. 6,528,624 B1 (Idusogie et al.).

[0133] As used herein, the term "ocrelizumab" (CAS Registry Number 637334-45-3) refers to a genetically engineered humanized monoclonal antibody raised against the CD20 antigen and comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13, and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14, including fragments thereof that retain the ability to bind to CD20. Ocrelizumab is available from Genentech.

[0134] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, as determined by the Lowry assay; (2) sufficiently 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 non-reducing or reducing conditions using Coomassie blue or, in some embodiments, silver staining. 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.

[0135] A "subject" or "patient" herein refers to a human subject or patient. Generally, the subject or patient is suitable for treatment of multiple sclerosis. For purposes herein, such eligible subjects or patients are those who are experiencing, have experienced, or are likely to experience one or more signs, symptoms, or other indicators of multiple sclerosis; those who have been diagnosed with multiple sclerosis, e.g., newly diagnosed ("new onset" MS), newly diagnosed with a relapse or exacerbation, or previously diagnosed and in remission; and / or those who are at risk for developing multiple sclerosis. Individuals with or at risk for multiple sclerosis may optionally be identified by screening for elevated levels of CD20-positive B cells in serum, cerebrospinal fluid (CSF), and / or MS lesions, and / or screened and assessed qualitatively and preferably quantitatively using assays that detect autoantibodies. Exemplary such autoantibodies associated with multiple sclerosis include anti-myelin basic protein (MBP), anti-myelin oligodendrocyte glycoprotein (MOG), anti-anti-ganglioside, and / or anti-neurofilament antibodies. Such autoantibodies may be detected in a subject's serum, cerebrospinal fluid (CSF), and / or MS lesions. "Elevated" antibody or B-cell levels herein refer to levels of such autoantibodies or B-cells that significantly exceed levels in individuals without MS.

[0136] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired results include, but are not limited to, one or more of the following: reducing one or more symptoms resulting from the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), slowing or slowing the progression of the disease, ameliorating the disease state, reducing the dose of one or more other medications needed to treat the disease, and / or increasing quality of life.

[0137] As used herein, "delaying" or "slowing" the progression of multiple sclerosis means preventing, postponing, hindering, slowing, retarding, stabilizing, and / or prolonging the development of the disease. This delay can be for varying lengths of time, depending on the history of the disease and / or the individual being treated.

[0138] As used herein, "at the time of treatment initiation" refers to the time at or before the first exposure to a multiple sclerosis medication, such as an anti-CD20 antibody. In some embodiments, "at the time of treatment initiation" refers to any of one year, nine months, six months, three months, two months, or one month prior to the administration of a multiple sclerosis medication, such as an anti-CD20 antibody. In some embodiments, "at the time of treatment initiation" refers to just prior to the first exposure to a multiple sclerosis medication, such as an anti-CD20 antibody.

[0139] As used herein, "based on" includes (1) assessing, determining, or measuring a patient characteristic described herein (preferably to select a patient suitable for treatment), and (2) administering a treatment described herein.

[0140] A "symptom" of MS is any morbid phenomenon or deviation from the normal in structure, function, or sensation experienced by a subject and indicative of MS.

[0141] "Multiple sclerosis" means a chronic, often disabling disease of the central nervous system characterized by the progressive destruction of myelin. There are four internationally recognized forms of MS: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS).

[0142] As used herein, "progressive multiple sclerosis" refers to primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS). In some embodiments, progressive multiple sclerosis is characterized by a documented irreversible loss of neurological function that persists for six months or more and cannot be attributed to a clinical relapse.

[0143] "Primary progressive multiple sclerosis" or "PPMS" is characterized by a gradual progression of disease from its onset, with relapses and remissions rarely overlapping. There may be periods of stable disease activity, with good days or weeks and bad days or weeks. PPMS differs from RRMS and SPMS in that onset typically occurs in the late 30s or 40s, men are equally susceptible to it, and initial disease activity is often in the spinal cord, not the brain. PPMS disease activity can be observed (or detected) in the brain. PPMS is the MS subtype least likely to show inflammatory (gadolinium-enhancing) lesions on MRI scans. The primary progressive form of the disease affects 10 to 15% of all people with multiple sclerosis. PPMS may be defined according to the criteria in Polman et al., Ann Neurol 69:292-392 (2010). PPMS patients treated here typically have a probable or definitive diagnosis of PPMS.

[0144] "Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by relapses (also known as exacerbations) during which new symptoms may appear and old ones return or worsen. Relapses are followed by periods of remission, during which the person fully or partially recovers from deficits acquired during the relapse. Relapses may last for days, weeks, or months, and recovery may be slow, gradual, or almost instantaneous. Most people with MS are initially diagnosed with RRMS. This typically occurs when people are in their 20s or 30s, although earlier and later diagnoses are known. Twice as many women as men present with this subtype of MS. During relapses, myelin, the protective shielding sheath around nerve fibers (neurons) in the white matter regions of the central nervous system (CNS), can be damaged by an inflammatory response by the body's own immune system. This causes a wide range of neurological symptoms that vary considerably depending on which areas of the CNS are damaged. Shortly after a relapse, the inflammatory response subsides, and a special type of glial cell in the CNS (called oligodendrocytes) assists in remyelination (the process by which the myelin sheath around axons can be repaired). It is this remyelination that can cause remission. Approximately 50% of patients with RRMS convert to SPMS within 10 years of disease onset. After 30 years, this number rises to 90%. At any one time, the relapsing-remitting form of the disease accounts for approximately 55% of all people with MS.

[0145] "Secondary progressive multiple sclerosis" or "SPMS" is characterized by a steady progression of clinical neurological damage, with or without overlapping relapses, minor remissions, and plateaus. People who develop SPMS will have previously experienced periods of RRMS, which can last from two to over 40 years. Any overlapping relapses and remissions tend to subside over time. With the onset of the secondary progressive phase of the disease, disability begins and progresses more rapidly than in RRMS, although progression can still be quite slow in some individuals. After 10 years, 50% of people with RRMS have developed SPMS. By 25 to 30 years, that number rises to 90%. SPMS tends to be associated with lower levels of inflammatory lesions than in RRMS, but the overall disease burden continues to progress. At any given time, SPMS accounts for approximately 30% of all people with multiple sclerosis.

[0146] "Progressive relapsing multiple sclerosis" refers to "PRMS," a disease characterized by a steady progression of clinical neurological damage, with overlapping relapses and remissions. Significant recovery may occur immediately after each relapse, but there is a gradual worsening of symptoms between relapses. PRMS affects approximately 5% of all people with multiple sclerosis. Some neurologists believe PRMS is a variant of PPMS. The phrase "effective amount" refers to an amount of an antibody (or other drug) that is effective in remission or treatment of multiple sclerosis. Such an effective amount generally results in a slowing or delay in disease progression (e.g., using the Expanded Disability Status Scale, EDSS), including improvements in the signs, symptoms, or other indicators of MS, such as a reduction in relapse rate, prevention of disability, a reduction in the number and / or volume of brain MRI lesions, improvement in a timed 25-foot walk, or an increase in the time to disease progression.

[0147] "Antibody exposure" refers to contact with or exposure to one or more doses of the antibodies herein administered over a period of about 1 to 20 days. The doses may be administered once or at regular or irregular times throughout this exposure period. The initial and subsequent (e.g., second or third) antibody exposures are separated in time from each other as detailed herein.

[0148] As used herein, the "interval" between antibody exposures refers to the period between a previous antibody exposure and a subsequent antibody exposure. The antibody exposures of the present disclosure can include one or two doses. When an antibody exposure includes one dose, the interval between two antibody exposures refers to the amount of time that elapses between the dose of one antibody exposure (e.g., on day 1) and the dose of the next antibody exposure. When one antibody exposure includes two doses and the next antibody exposure includes one dose, the interval between two antibody exposures refers to the amount of time that elapses between the first of the two doses of the first antibody exposure (e.g., on day 1) and the dose of the next antibody exposure. When each of the two antibody exposures includes two doses, the interval between antibody exposures refers to the amount of time that elapses between the first of the two doses of the first antibody exposure (e.g., on day 1) and the first of the two doses of the second antibody exposure. For example, if the methods of the present disclosure include a first antibody exposure having two doses and a second antibody exposure having two doses, and the second antibody exposure is not administered until about 24 weeks or 6 months after the first antibody exposure, the interval between the first dose of the first antibody exposure and the first dose of the second antibody exposure is about 24 weeks or 6 months. In certain embodiments, the second antibody exposure is not administered until about 20-24 weeks or about 5-6 months after the first antibody exposure. In some embodiments, the interval between the first dose of the first antibody exposure and the first dose of the second antibody exposure is about 20-24 weeks or about 5-6 months. In some embodiments, "about 20-24 weeks" refers to a time point between 20 and 24 weeks. In some embodiments, "about 20-24 weeks" refers to a variation of 1 week or 7 days before or after the 24th week. In some embodiments, "about 5-6 months" refers to a time point between 5 and 6 months.

[0149] The term "immunosuppressant" as used herein with respect to adjunctive therapy means a substance that acts to suppress or mask the immune system of the mammal being treated herein. This includes substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticosteroids such as cortisol or aldosterone, anti-inflammatory agents such as cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, or leukotriene receptor antagonists; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies against MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs such as prednisone, methylprednisone, and decongestants. dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); hydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antagonists, including anti-interferon-α, -β, or -γ antibodies, anti-tumor necrosis factor-α antibodies (infliximab or adalimumab); anti-TNF-α immunoadhesin (etanercept), anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies, and anti-IL-2 receptor anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; xenogeneic antilymphocyte globulins; Pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptides containing the LFA-3 binding domain (WO 90 / 08187 published July 26, 1990); streptokinase; TGF-β; streptodornase; host-derived RNA or DNA; FK506; RS-61443; deoxyspergualin; rapamycin;These include T cell receptors (Cohen et al., U.S. Pat. No. 5,114,721); T cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90 / 11294; Janeway, Nature, 341:482 (1989)); and WO 91 / 01133); and T cell receptor antibodies such as T10B9 (European Patent Application Publication No. 340,109).

[0150] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as enzymatically active or small molecule toxins of bacterial, fungal, plant, or animal origin, or fragments thereof.

[0151] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocalma Isin (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine (p nitrogen mustards such as rednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994));Dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein (enediyne antibiotic chromophore), aclacinomycins, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin n), chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as florinic acid acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; Hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridine A, and anguidine); urethane;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylolnithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0152] Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON toremifene; such as 4(5)-imidazoles, aminoglutethimide, MEGASE® megestol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastomoses. aromatase inhibitors, such as Trozole, which inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell growth, such as PKC-α, Ralf, and H-Ras; vaccines, such as gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of the above.

[0153] The term "cytokine" is a general term for proteins released by one cell population and acting as intercellular mediators on other cells. Examples of such cytokines are lymphokines, monokines; interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, and IL-15; tumor necrosis factors, e.g., TNF-α or TNF-β; and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines, such as synthetically produced small molecule entities and pharmaceutically acceptable derivatives and salts thereof.

[0154] The term "hormone" refers to a polypeptide hormone secreted by a glandular organ, usually having a duct. Hormones include, for example, growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); prolactin, placental lactogen, mouse gonadotropin-related peptide; inhibin; activin; Müllerian inhibitory substance; and thrombopoietin. As used herein, the term "hormone" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native-sequence hormones, e.g., synthetically produced small molecule forms and pharmaceutically acceptable derivatives and salts thereof.

[0155] The term "growth factor" refers to a protein that promotes growth, such as hepatic growth factor; fibroblast growth factor; vascular endothelial growth factor; nerve growth factor, e.g., NGF-β; platelet-derived growth factor; transforming growth factors (TGFs), e.g., TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteogenic factor; interferons, e.g., interferon-α, -β, and -γ; and colony-stimulating factors (CSFs), e.g., macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF). As used herein, the term growth factor includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native-sequence growth factors, e.g., synthetically produced small molecule forms and pharmaceutically acceptable derivatives and salts thereof.

[0156] The term "integrin" refers to receptor proteins that allow cells to both bind to and respond to the extracellular matrix and are involved in various cellular functions, such as wound healing, cell differentiation, tumor cell homing, and apoptosis. They are part of a large family of cell adhesion receptors involved in cell-extracellular matrix and cell-cell interactions. Functional integrins consist of two noncovalently linked transmembrane glycoprotein subunits, termed α and β. As in the β subunit, all α subunits share some homology with each other. Receptors always contain one α chain and one β chain. Examples are α6β1, α3β1, α7β1, LFA-1, α4 integrin, etc. As used herein, the term integrin includes proteins from natural sources or recombinant cell culture and biologically active equivalents of native-sequence integrins, such as synthetically produced small molecule forms and pharmaceutically acceptable derivatives and salts thereof.

[0157] Examples of "integrin antagonists or antibodies" herein include LFA-1 antibodies; alpha 4 integrin antibodies such as natalizumab (TYSABRI®) available from Biogen Idec / Elan Pharmaceuticals, Inc.; diazacyclic phenylalanine derivatives (WO 2003 / 89410); phenylalanine derivatives (WO 2003 / 70709, WO 2002 / 28830, WO 2002 / 16329, and WO 2003 / 53926); phenylpropionic acid derivatives (WO 2003 / 10135); enamine derivatives (WO 2001 / 79173); protease inhibitors (WO 2001 / 79174); These include panic acid derivatives (WO 2000 / 37444); alkanoic acid derivatives (WO 2000 / 32575); substituted phenyl derivatives (U.S. Pat. Nos. 6,677,339 and 6,348,463); aromatic amine derivatives (U.S. Pat. No. 6,369,229); and ADAM disintegrin domain polypeptides (U.S. Pat. App. Pub. No. 2002 / 0042368), antibodies against αβ3 integrin (European Pat. App. Pub. No. 633945); aza-bridged bicyclic amino acid derivatives (WO 2002 / 02556), and the like.

[0158] For purposes herein, "tumor necrosis factor alpha (TNF-α)" refers to the human TNFα molecule containing the amino acid sequence set forth in Pennica et al., Nature, 312:721 (1984) or Aggarwal et al., JBC, 260:2345 (1985).

[0159] As used herein, a "TNFα inhibitor" is an agent that inhibits to some extent the biological activity of TNFα, generally through binding to and neutralizing the activity of TNFα. Examples of TNF inhibitors contemplated herein are etanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA™).

[0160] Examples of "disease-modifying antirheumatic drugs" or "DMARDs" include hydroxychloroquinone, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate), azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, and Staphylococcal protein A immunoadsorbent, including salts and derivatives thereof.

[0161] Examples of "nonsteroidal anti-inflammatory drugs" or "NSAIDs" include acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, tolmetin, and salts and derivatives thereof.

[0162] "Corticosteroid" refers to any one of several synthetic or naturally occurring substances with the general chemical structure of a steroid that mimics or enhances the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, glucocorticoids, and betamethasone.

[0163] "Package insert" is used to refer to instructions customarily included in commercial packaging for therapeutic products, including indications, directions for use, dosage, administration, contraindications, other therapeutic products to be used in conjunction with the packaged product, and / or warnings regarding the use of such therapeutic products.

[0164] "Label" is used herein to refer to commercial packaging of pharmaceutical preparations, including containers such as vials and package inserts, as well as information conventionally included on other types of packaging.

[0165] Reference herein to "approximately" a value or parameter includes (describes) a variation on the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X."

[0166] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0167] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art.

[0168] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

[0169] II. Treatment Methods In certain embodiments, methods of improving functional capacity in patients with multiple sclerosis are provided, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient has improved functional capacity following treatment. In some embodiments, the methods further comprise measuring the patient's functional capacity (e.g., using a method described elsewhere herein, e.g., measuring EDSS score and / or Timed 25-Foot Walk (T25-FW)) after one, two, three, four, or more exposures to the anti-CD20 antibody. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0170] In some embodiments, patients have a confirmed improvement in disability for at least about 12 weeks after treatment. In some embodiments, patients have a confirmed improvement in disability for at least about 24 weeks after treatment. In some embodiments, the confirmed improvement in disability is determined by Expanded Disability Status Scale (EDSS) score. In some embodiments, the patient's EDSS score is reduced by at least about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, or more than about 1.0 point (such as about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 points).

[0171] In certain embodiments, the improvement in functional capacity persists for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks (including any ranges therebetween). In certain embodiments, the improvement in functional capacity persists for at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, or more than about 75 weeks (including any ranges therebetween).

[0172] In some embodiments, the improvement in functional ability is measured by the Timed 25-Foot Walk (T25-FW) test. In some embodiments, the time taken to walk 25 feet after initiating treatment is reduced by about 5 seconds, about 10 seconds, about 30 seconds, about 60 seconds, about 90 seconds, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes, about 8.5 minutes, about 9 minutes, about 9.5 minutes, or about 10 minutes relative to the time taken to walk 25 feet immediately before initiating treatment.

[0173] In some embodiments, improvement in functional activity is demonstrated by no evidence of disease activity (NEDA). In some embodiments, NEDA is demonstrated by the absence of new or enlarging T2 lesions or T1 gadolinium-enhancing lesions on magnetic resonance imaging. In some embodiments, NEDA is demonstrated by the absence of recurrence. In some embodiments, NEDA is demonstrated by the absence of progression. In some embodiments, NEDA is demonstrated by the absence of worsening EDSS. In some embodiments, NEDA is defined as no protocol-defined recurrence, no CDP events, no new or enlarging T2 lesions, and no gadolinium-enhancing T1 lesions. In some embodiments, the NEDA lasts for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks (including any ranges therebetween). In certain embodiments, the improvement in functional capacity persists for at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 35 weeks, at least about 40 weeks, at least about 45 weeks, at least about 50 weeks, at least about 55 weeks, at least about 60 weeks, at least about 65 weeks, at least about 70 weeks, at least about 75 weeks, or more than about 75 weeks (including any ranges therebetween).

[0174] In some embodiments, the patient has T1 gadolinium-staining lesions at baseline (i.e., before treatment begins). In some embodiments, the patient does not have T1 gadolinium-staining lesions at baseline (i.e., before treatment begins).

[0175] In certain embodiments, a method of inhibiting complex disability progression in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a reduction in confirmed disability progression events, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, administration results in a reduction in progression of confirmed complex disorders for 12 weeks. In certain embodiments, administration results in a reduction in progression of confirmed complex disorders for 24 weeks.

[0176] In certain embodiments, a method of delaying the onset of complex disability progression in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a reduction in confirmed disability progression events, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, administration results in a reduction in progression of confirmed complex disorders for 12 weeks. In certain embodiments, administration results in a reduction in progression of confirmed complex disorders for 24 weeks.

[0177] In some embodiments, confirmed combined disability progression is determined by Expanded Disability Status Scale (EDSS) score. In some embodiments, confirmed combined disability progression is defined as EDSS progression (i.e., an increase in EDSS score). In some embodiments, confirmed combined disability progression is determined by Timed 25-foot Walk (T25-FW). In some embodiments, confirmed combined disability progression is defined as at least a 20% increase in T25-FW. In some embodiments, confirmed combined disability progression is determined by 9-Hole Peg Test (9-HPT). In some embodiments, confirmed combined disability progression is defined as at least a 20% increase in 9-hole peg test (9-HPT) time. In some embodiments, confirmed combined disability progression is determined by EDSS progression, Timed 25-Foot Walk, and 9-Hole Peg Test.

[0178] In certain embodiments, a method of inhibiting disorder progression in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a reduction in confirmed disorder progression events. In certain embodiments, the reduction in confirmed disorder progression events is observed after 1, 2, 3, 4, or more than 4 exposures to the anti-CD20 antibody. In certain embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In certain embodiments, administration results in a reduction in progression of confirmed disorders at 12 weeks. In certain embodiments, administration results in a reduction in progression of confirmed disorders at 24 weeks. In certain embodiments, administration results in a reduction in the risk of progression of confirmed disorders at 12 weeks. In certain embodiments, administration results in a reduction in the risk of progression of confirmed disorders at 24 weeks.

[0179] In some embodiments, the patient has T1 gadolinium-staining lesions at baseline (i.e., before treatment begins). In some embodiments, the patient does not have T1 gadolinium-staining lesions at baseline (i.e., before treatment begins).

[0180] In certain embodiments, a method of delaying the onset of confirmed disability progression in a human patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0181] In some embodiments, the confirmed disability progression is determined by Expanded Disability Status Scale (EDSS) score. In some embodiments, the patient's EDSS score increases by at least about 1.0 point from a baseline EDSS score of about 5.5 or less. In some embodiments, the patient's EDSS score increases by about 0.5 point from a baseline EDSS score of more than 5.5. In some embodiments, the EDSS increase is confirmed at least 12 weeks after the initial neurological deterioration.

[0182] In some embodiments, the patient has T1 gadolinium-staining lesions at baseline (i.e., before treatment begins). In some embodiments, the patient does not have T1 gadolinium-staining lesions at baseline (i.e., before treatment begins).

[0183] Methods of reducing T2 lesion volume in a human patient with multiple sclerosis are provided, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the patient has T1 gadolinium-staining lesions at baseline. In certain embodiments, the patient has no T1 gadolinium-staining lesions at baseline.

[0184] In certain embodiments, a method of delaying or preventing brain volume loss in a patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein brain volume loss is delayed or prevented in the patient; and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, brain volume loss is delayed. In certain embodiments, brain volume loss is delayed or prevented in patients who have not experienced brain volume loss, hi certain embodiments, further loss of brain volume is delayed or prevented in patients who have experienced brain volume loss.

[0185] In certain embodiments, a method of delaying or preventing brain atrophy in a patient with multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein brain atrophy is delayed or prevented in the patient; and the anti-CD20 antibody comprises a) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, brain atrophy is delayed. In certain embodiments, brain atrophy is delayed or prevented in patients who have not experienced brain atrophy, hi certain embodiments, further brain atrophy is delayed or prevented in patients who have experienced brain atrophy.

[0186] In certain embodiments, methods of treating a human patient with multiple sclerosis are provided, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in no observed disease activity (NEDA) for at least 12 weeks, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the treatment results in no observed disease activity (NEDA) for at least 24 weeks.

[0187] A method of treating a human patient with multiple sclerosis, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in the patient achieving a lesion-free state after 96 weeks of treatment, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the treatment results in the patient achieving a lesion-free state after 48 weeks of treatment. In some embodiments, the treatment results in a patient achieving a lesion-free state after 24 weeks of treatment. In some embodiments, the treatment results in a patient free of gadolinium-staining lesions. In some embodiments, the treatment results in a patient free of T2 lesions.

[0188] In one embodiment, a method of treating a human patient with a relapsing form of multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in one or more of: a) patients who are relapse-free at 96 weeks; b) patients who have no confirmed disability progression events at 96 weeks; c) patients who have no T1 gadolinium-enhancing lesions at 96 weeks; or d) patients who have no new and / or enlarging T2 lesions at 96 weeks; wherein the anti-CD20 antibody comprises 1) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and 2) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0189] In some embodiments, the patient has not been previously treated with other therapies for multiple sclerosis (i.e., a "naive patient"). In some embodiments, the naive patient has experienced at least two relapses in the two years prior to starting treatment. In some embodiments, the naive patient has experienced at least one relapse in the last year prior to starting treatment.

[0190] In some embodiments, the patient is an inadequate responder to other therapies for multiple sclerosis.In some embodiments, the inadequate responder patient has previously been treated with interferon beta-1a or glatiramer acetate for at least 1 year.In some embodiments, the inadequate responder patient has experienced at least one relapse or at least one baseline gadolinium-enhancing lesion while being treated with another therapy for multiple sclerosis.

[0191] In certain embodiments, a method of treating a human patient with hyperactive multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0192] In some embodiments, patients with highly active multiple sclerosis have not been previously treated with other therapies for multiple sclerosis (i.e., "naive patients"). In some embodiments, naive patients with highly active multiple sclerosis have experienced at least two relapses in the last year before randomization and either (a) at least one baseline gadolinium lesion or (b) an increase in T2 lesions (a definitive change from 0-5 to 6-9 lesions or from 6-9 lesions to >9 lesions) at the baseline visit compared to a previous MRI.

[0193] In some embodiments, patients with highly active multiple sclerosis are inadequate responders to other therapies for multiple sclerosis.In some embodiments, patients with highly active multiple sclerosis who are inadequate responders have previously been treated with interferon beta-1a or glatiramer acetate for at least one year.In some embodiments, patients with highly active multiple sclerosis who are inadequate responders have experienced at least one relapse while being treated with another therapy for multiple sclerosis, and at baseline, either (a) have at least 9 T2 lesions or (b) have at least 1 gadolinium lesion.

[0194] In certain embodiments, administration of an anti-CD20 antibody to a patient with highly active multiple sclerosis is effective in one or more of the following: (1) reducing the number of lesions in the patient's brain; (2) reducing the annualized relapse rate; (3) reducing the progression of disability; and (4) improving functional ability. In certain embodiments, the method of treating a patient with highly active multiple sclerosis further comprises performing an MRI scan to determine whether the patient has highly active multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

[0195] In certain embodiments, a method of treating a human patient with early stage multiple sclerosis is provided, the method comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises: a) a heavy chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 12; and b) a light chain variable region comprising: CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the method further comprises diagnosing the patient with early stage multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

[0196] In certain embodiments, methods of treating a human patient with multiple sclerosis are provided, the methods comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in a state of no disease activity in the patient, and the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody comprises a) a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 11, and a CDR3 having the amino acid sequence of SEQ ID NO: 12, and b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0197] In some embodiments, the patient has a relapsing form of multiple sclerosis. In some embodiments, relapsing forms of MS (RMS) refer to a patient population that typically consists of both RRMS and SPMS with overlapping relapses (commonly referred to as "relapsing SPMS"). In some embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the relapsing form of multiple sclerosis is secondary progressive multiple sclerosis with overlapping relapses (rSPMS). In some embodiments, the patient is under 18 years of age. In some embodiments, the patient is between 18 and 55 years of age. In some embodiments, the patient is over 55 years of age. In some embodiments, the patient has a diagnosis of multiple sclerosis according to the 2010 revised McDonald criteria (Polman et al. (2011) "Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria." Ann Neurol 69, 292-302). Additionally or alternatively, in some embodiments, the patient has an Expanded Disability Status Scale (EDSS, see world-wide-web.neurostatus.org) score of 0 to 5.5 at screening. Additionally or alternatively, in some embodiments, the patient has had at least two documented clinical attacks within the previous two years or one clinical attack occurring within the previous year. Additionally or alternatively, in some embodiments, the patient has a documented MRI of the brain with abnormalities consistent with multiple sclerosis.

[0198] In some embodiments, patients with relapsing forms of multiple sclerosis do not have a diagnosis of primary progressive multiple sclerosis. Additionally or alternatively, in some embodiments, patients have not previously been treated with any B cell targeted therapy, systemic corticosteroids, and / or immunosuppressants. Additionally or alternatively, in some embodiments, patients do not have a history of primary or secondary immunodeficiency, active infection, or the presence of recurrent or chronic infection (e.g., hepatitis B or C, HIV, syphilis, tuberculosis), or a history of progressive multifocal leukoencephalopathy.

[0199] In some embodiments, the patient has progressive multiple sclerosis. In some embodiments, the progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, the patient is under 18 years old. In some embodiments, the patient is between 18 and 55 years old. In some embodiments, the patient is over 55 years old. In some embodiments, the patient has a diagnosis of primary progressive multiple sclerosis according to the 2005 revised McDonald criteria (Polman et al. (2011) "Diagnostic criteria for multiple sclerosis: 2005 revisions to the McDonald criteria." Ann Neurol 58, 840-846). Additionally or alternatively, in some embodiments, the patient has an Expanded Disability Status Scale (EDSS) score of 3 to 6.5 at screening. Additionally or alternatively, in some embodiments, the patient has a score of at least 2.0 on the pyramidal functioning component of the Functional Systems Scale (FSS). Additionally or alternatively, in certain embodiments, the patient has a documented history or presence of an elevated IgG index in a cerebrospinal fluid (CSF) sample and / or one or more IgG oligoclonal bands detected by isoelectric focusing in a cerebrospinal fluid (CSF) sample at screening. Additionally or alternatively, in certain embodiments, the patient does not have a history of relapsing-remitting multiple sclerosis (RRMS). Additionally or alternatively, in certain embodiments, the patient does not have a history of secondary progressive multiple sclerosis (SPMS). Additionally or alternatively, in certain embodiments, the patient has a history of progressive relapsing multiple sclerosis (PRMS).

[0200] In certain embodiments, the patient has had prior treatment with a B-cell targeted therapy (e.g., rituximab, ocrelizumab, abatacept, belimumab, or ofatumumab). In certain embodiments, the patient has not had prior treatment with a B-cell targeted therapy (e.g., rituximab, ocrelizumab, abatacept, belimumab, or ofatumumab).

[0201] In some embodiments, "confirmed disability progression" or "CDP" refers to an increase of at least 1.0 point from baseline EDSS score in patients with a baseline score of 5.5 or less, or an increase of 0.5 point in patients with a baseline score of greater than 5.5, over a 96-week period, where the increase in EDSS is confirmed at a regularly scheduled clinic visit at least 12 weeks after initial neurological deterioration.

[0202] In some embodiments, "confirmed disability improvement" or "CDI" refers to a reduction in EDSS score of at least 1.0 compared to baseline in patients with a baseline EDSS score of 5.5 or less, or a reduction of 0.5 points in patients with a baseline EDSS score of greater than 5.5.

[0203] In some embodiments, brain atrophy refers to one or more of the following: axonal loss in the brain, tissue loss in gray matter or white matter lesions, lesions, or Wallerian degeneration in pathways related to lesion burden. In some embodiments, brain atrophy refers to a decrease in total brain volume. In some embodiments, brain atrophy refers to a decrease in the volume of one or more brain structures (including but not limited to the cerebrum, cerebellum, thalamus, frontotemporal neocortex, brainstem, hippocampus, parietal lobe, and / or hypothalamus). In some embodiments, brain atrophy refers to thinning of the cortex of the precentral gyrus, superior frontal gyrus, thalamus, and / or putamen. In some embodiments, brain atrophy refers to a loss of brain volume of at least about 0.4%, at least about 0.5%, at least about 0.6%, or at least about 0.7% per year. Further details regarding brain atrophy are described, for example, in Riley et al. (2012) Expert Rev Neurother 12(3), 323-333.

[0204] In some embodiments, the patient or subject has highly active multiple sclerosis. In some embodiments, "highly active multiple sclerosis" in the treatment of naive patients refers to a patient who has not previously been treated with other therapies for multiple sclerosis, who has experienced at least two relapses in the last year before randomization, and who has experienced either (a) at least one baseline gadolinium lesion or (b) an increase in T2 lesions (a definitive change from 0-5 to 6-9 lesions or from 6-9 lesions to >9 lesions) at the baseline visit compared to a previous MRI. In some embodiments, "highly active multiple sclerosis" refers to a patient who has previously been treated with other therapies for multiple sclerosis, who has experienced at least one relapse in the last year, and who has either (a) at least 9 T2 lesions or (b) at least one gadolinium lesion at baseline.

[0205] In certain embodiments, baseline levels in a patient refer to levels prior to administration of an anti-CD20 antibody to the patient or treatment with an anti-CD20 antibody, e.g., about 2 months, about 1.5 months, about 1 month, about 30 days, about 25 days, about 21 days, about 14 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day prior to administration of an anti-CD20 antibody to the patient or treatment with an anti-CD20 antibody.

[0206] In certain embodiments, the patient maintains the ability to mount a humoral immune response to the antigen during treatment. In certain embodiments, the antigen is a mumps antigen, a rubella antigen, a varicella antigen, a Streptococcus pneumoniae antigen, a tetanus toxoid antigen, a pneumococcal antigen, or an influenza antigen.

[0207] The methods described herein may include any combination of the embodiments described herein.

[0208] III. Dose According to some embodiments of any of the methods or articles of manufacture described herein, the methods or instructions comprise administering an effective amount of an anti-CD20 antibody to a patient with multiple sclerosis, resulting in an initial antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 grams), followed by a second antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 grams), where the second antibody exposure does not occur until about 16 to about 60 weeks after the initial antibody exposure. For purposes of this invention, the second antibody exposure is the next opportunity to treat the patient with an anti-CD20 antibody after the first antibody exposure, with no intervening anti-CD20 antibody treatment or exposure between the first and second exposures. In some embodiments, the initial antibody exposure and / or the second antibody exposure is about any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, or 1.0 grams.

[0209] The interval between the initial and second or subsequent antibody exposures can be measured from the first dose of the initial antibody exposure.

[0210] In some embodiments, antibody exposure is approximately 24 weeks or 6 months apart, or approximately 48 weeks or 12 months apart. In some embodiments, antibody exposure is approximately about 20-24 weeks or about 5-6 months. In some embodiments, "about 20-24 weeks" refers to a time point between 20 and 24 weeks. In some embodiments, "about 20-24 weeks" refers to a variation of 1 week or 7 days before or after the 24th week. In some embodiments, "about 5-6 months" refers to a time point between 5 and 6 months.

[0211] In one embodiment, the second antibody exposure is not provided until about 20 to about 30 weeks after the first exposure, optionally followed by a third antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams), which is not administered until about 46 to 60 weeks (preferably about 46 to 54 weeks) after the first exposure, and then, in some embodiments, no further antibody exposure is provided until at least about 70-75 weeks after the first exposure. In some embodiments, the third antibody exposure is any of about 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, or 1.0 grams.

[0212] In other embodiments, the second antibody exposure does not occur until about 46 to 60 weeks after the first exposure, and the subsequent antibody exposure, if any, does not occur until about 46 to 60 weeks after the previous antibody exposure.

[0213] According to some embodiments of any of the methods or articles of manufacture described herein, the methods or instructions comprise administering an effective amount of an anti-CD20 antibody to a patient with multiple sclerosis, comprising an initial antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 grams), followed by a second antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 grams), the second antibody exposure occurring about 20 to 30 days after the initial antibody exposure. and about 30 weeks after the first exposure), followed by a third antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 gram), where the third antibody exposure does not occur until about 46 to about 54 weeks after the first exposure, followed by a fourth antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 gram), where the fourth antibody exposure does not occur until about 70 to about 75 weeks after the first exposure.

[0214] In certain embodiments, the fourth antibody exposure is followed by one or more antibody exposures of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, e.g., about 0.6 grams or about 1.0 grams). In certain embodiments, each subsequent antibody exposure is about 20 to about 30 weeks after the previous exposure.

[0215] For purposes of this invention, each subsequent exposure is the next opportunity to treat the patient with an anti-CD20 antibody after the first antibody exposure, e.g., there is no intervening anti-CD20 antibody treatment or exposure between the first and second exposures, the second and third exposures, or the third and fourth exposures, etc. In some embodiments, the first, second, third, fourth, and / or subsequent antibody exposures are about any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 grams, or 1.0 grams.

[0216] Any one or more of the antibody exposures herein can be administered to a patient as a single dose of antibody or as two separate doses of antibody (i.e., comprising a first and a second dose). The specific number of doses (one or two) used for each antibody exposure depends, for example, on the type of MS being treated, the type of antibody used, whether a second medication is used, and the method and frequency of administration. When two separate doses are administered, the second dose is preferably administered about 3 to 17 days, more preferably about 6 to 16 days, and most preferably about 13 to 16 days, from the time the first dose was administered. In some embodiments, when two separate doses are administered, the second dose is administered about 14 days (e.g., 13 or 15 days). In some embodiments, "about 14 days" refers to variations of one day before or after the 14th day. When two separate doses are administered, the first and second doses of antibody are preferably about 0.3 to 1.5 grams, more preferably about 0.3 to about 1.0 gram. In some embodiments, when two separate doses are administered, the first and second doses of antibody are about 0.3 grams, 0.4 grams, 0.5 grams, or 0.6 grams. In some embodiments, the first ocrelizumab exposure comprises a first dose and a second dose of ocrelizumab, wherein the first and second doses of ocrelizumab are about 0.3 grams. In some embodiments, the second ocrelizumab exposure comprises a single dose of ocrelizumab, wherein the single dose of ocrelizumab is 0.6 grams.

[0217] In one embodiment, the patient is provided with at least about 3, at least about 4, or at least about 5 exposures of antibody, e.g., about 3 to 60 exposures, more particularly about 3 to 40 exposures, and most particularly about 3 to 20 exposures. In some embodiments of any of the methods, the method further comprises providing about 1 to about 3 subsequent ocrelizumab exposures. In some embodiments, such exposures are administered at intervals of approximately 24 weeks or 6 months, or 48 weeks or 12 months, respectively. In certain embodiments, the interval is shortened by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, one or more intervals are shortened by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, the intervals are extended by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, one or more intervals are extended by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In certain embodiments, one or more intervals are shortened by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day, or are lengthened by about 4 weeks, about 3.5 weeks, about 3 weeks, about 2.5 weeks, about 2 weeks, about 1.5 weeks, about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day.

[0218] In one embodiment, each antibody exposure is provided as a single dose of antibody. In other embodiments, each antibody exposure is provided as two separate doses of antibody. In some embodiments, several exposures are provided as a single dose or as two separate doses.

[0219] The antibody can be a naked antibody or can be conjugated to another molecule, such as a cytotoxic agent, such as a radioactive compound. In some embodiments, the antibody is rituximab, humanized 2H7 (e.g., comprising the variable domain sequences of SEQ ID NOs: 2 and 8), or humanized 2H7 comprising the variable domain sequences of SEQ ID NOs: 23 and 24, or huMax-CD20 (Genmab). In some embodiments, the antibody is ocrelizumab (e.g., having (a) a light chain comprising the amino acid sequence of SEQ ID NO: 13 and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 14).

[0220] In one embodiment, the patient has never been previously treated with an agent such as an immunosuppressant to treat multiple sclerosis and / or has never been previously treated with an antibody against a B cell surface marker (e.g., has never been previously treated with a CD20 antibody).

[0221] The antibody may be administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and / or intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Intrathecal administration is also contemplated (see, e.g., U.S. Patent Application Publication No. 2002 / 0009444, Grillo-Lopez, A., regarding intrathecal delivery of CD20 antibodies). The antibody may also be suitably administered by pulse infusion, e.g., with declining doses of antibody. In some embodiments, the dosage is administered intravenously, subcutaneously, or intrathecally. In some embodiments, the dosage is given by intravenous infusion.

[0222] In certain embodiments, the patient is premedicated prior to infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with methylprednisolone (or equivalent) approximately 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with 100 mg of IV methylprednisolone (or equivalent) approximately 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antihistamine (e.g., diphenhydramine) approximately 30-60 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antipyretic (e.g., acetaminophen / paracetamol).

[0223] Although the CD20 antibody can be the only agent administered to a patient to treat multiple sclerosis, optionally a second medication, such as a cytotoxic agent, chemotherapeutic agent, immunosuppressant, cytokine, cytokine antagonist or antibody, growth factor, hormone, integrin, integrin antagonist or antibody (e.g., LFA-1 antibody, or an alpha 4 integrin antibody such as natalizumab (TYSABRI®) available from Biogen Idec / Elan Pharmaceuticals, Inc.), can be administered along with the antibody that binds to a B cell surface marker (e.g., a CD20 antibody).

[0224] In some embodiments of the combination therapy, the antibody is administered in combination with an interferon class agent, such as IFN-β-1a (REBIF® and AVONEX®) or IFN-β-1b (BETASERON®); an oligopeptide such as glatiramer acetate (COPAXONE®); a cytotoxic agent such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenous immunoglobulin (gamma globulin); lymphodepleting therapy (e.g., mitoxantrone, cyclophosphamide, alemtuzumab (Campath® LEMTRADA™), anti-CD4, cladribine, total body irradiation, bone marrow transplantation, or the like). marrow transplantation); corticosteroids, including systemic corticosteroid therapy (e.g., methylprednisolone, prednisone, dexamethasone, or glucocorticoids); non-lymphocyte-depleting immunosuppressive therapy (e.g., mycophenolate mofetil (MMF) or cyclosporine); cholesterol-lowering agents in the "statin" class, such as cerivastatin (BAYCOL®), fluvastatin (LESCOL®), atorvastatin (LIPITOR®), lovastatin (MEVACOR®), pravastatin (PRAVACHOL®), and simvastatin (ZOCOR®); estradiol; testosterone (sometimes at high doses; Stuve et al. Neurology 8:290-301(2002)); hormone replacement therapy; treatment of secondary or associated symptoms of MS (e.g., spasticity, ataxia, pain, fatigue), combined with TNF inhibitors; disease-modifying antirheumatic drugs (DMARDs); nonsteroidal anti-inflammatory drugs (NSAIDs); plasmapheresis; levothyroxine; cyclosporine A; somatastatin analogs; cytokine or cytokine receptor antagonists; antimetabolites; immunosuppressants; rehabilitative surgery; radioactive iodine; thyroid ablation; other B-cell surface antagonists / antibodies, etc.

[0225] The second medicament is administered with the initial and / or subsequent exposure to the CD20 antibody, and such combined administration includes simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in either order, preferably with a period during which both (or all) active agents exert their biological activities simultaneously.

[0226] Aside from administering the antibody to a patient, the present application contemplates administering the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the phrase administering an "effective amount" of an antibody. See, e.g., WO 96 / 07321, published March 14, 1996, concerning the use of gene therapy to generate intracellular antibodies.

[0227] There are two major approaches to introducing nucleic acids (optionally contained in a vector) into a patient's cells: in vivo and ex vivo. In in vivo delivery, nucleic acids are injected directly into the patient, usually at the site where the nucleic acid is needed. In ex vivo therapy, the patient's cells are removed, nucleic acids are introduced into these isolated cells, and the modified cells are administered to the patient either directly or encapsulated in a porous membrane, for example, that is implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are various techniques available for introducing nucleic acids into living cells. The techniques vary depending on whether the nucleic acid is transferred into cultured cells in vitro or into the cells of the intended host in vivo. Techniques suitable for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, etc. A commonly used vector for ex vivo gene delivery is a retrovirus.

[0228] In some embodiments, in vivo nucleic acid transfer techniques involve transfection using viral vectors (e.g., adenovirus, herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated gene transfer include DOTMA, DOPE, and DC-Chol, for example). In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cell, such as an antibody specific for a cell surface membrane protein or target cell, a ligand for a receptor on the target cell, etc. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting and / or to facilitate uptake, such as capsid proteins or fragments thereof with tropism for specific cell types, antibodies for proteins that undergo internalization during cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, in Wu et al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990). For a review of currently known gene marking and gene therapy protocols, see Anderson et al., Science 256:808-813 (1992). See also WO 93 / 25673 and the references cited therein.

[0229] IV. Antibodies and Their Production The methods and articles of manufacture of the present invention use or incorporate antibodies that bind to B cell surface markers, particularly those that bind to CD20. Accordingly, methods for producing such antibodies are described herein.

[0230] In some embodiments, the anti-CD20 antibody used in the methods described herein is produced by a method comprising expressing in a host cell a nucleic acid encoding a humanized antibody comprising the heavy and light chain amino acid sequences of SEQ ID NO: 14 or 13, respectively, and recovering the humanized antibody or antigen-binding fragment thereof expressed in the host cell. In some embodiments, the host cell is a mammalian cell (e.g., a CHO cell), an insect cell, or a plant cell. In some embodiments, the host cell is a bacterial cell. Methods for producing anti-CD20 are described in further detail, for example, in U.S. Patent No. 7,799,900.

[0231] The B cell surface marker used to produce or screen for antibodies can be a soluble form of the marker or a portion thereof containing, for example, a desired epitope. Alternatively, or in addition, cells expressing the marker on their cell surface can be used to produce or screen for antibodies. Other forms of B cell surface markers useful for producing antibodies will be apparent to those skilled in the art.

[0232] Exemplary techniques for the production of antibodies to be used in accordance with the present invention are described below.

[0233] (i) Polyclonal antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. A protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, is coupled to the relevant antigen via a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (for coupling through cysteine ​​residues), N-hydroxysuccinimide (for coupling through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R. 1 N=C=NR, where R and R 1 are different alkyl groups).

[0234] Animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. In some embodiments, animals are boosted with a conjugate of the same antigen but conjugated to a different protein and / or through a different cross-linking agent. Conjugates can also be made in recombinant cell culture as protein fusions. Additionally, aggregating agents such as alum are preferably used to enhance the immune response.

[0235] (ii) Monoclonal antibody Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants which arise during the production of the monoclonal antibody and which are generally present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.

[0236] For example, monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0237] In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster, is immunized as described herein to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0238] The hybridoma cells thus prepared are plated and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells are deficient in the enzyme hypoxanthine guanidine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine, substances that prevent the growth of HGPRT-deficient cells (HAT medium).

[0239] In some embodiments, the 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 medium such as HAT medium. Among these, in some embodiments, the myeloma cell line is derived from a mouse myeloma line, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0240] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen, hi some embodiments, 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).

[0241] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0242] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Additionally, the hybridoma cells can be grown in vivo as ascites tumors in animals.

[0243] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0244] DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody). In some embodiments, hybridoma cells serve as the source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be 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 immunoglobulin proteins, to achieve synthesis of the monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0245] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. The following publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0246] The DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0247] Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0248] (iii) humanized antibody Methods for humanizing non-human antibodies are well known in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically from an "import" variable domain. Humanization can be performed using the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by substituting the relevant hypervariable region sequences of essentially a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0249] To reduce antigenicity, the selection of human variable domains, both light and heavy, to be used in generating humanized antibodies is crucial. In the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) of the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Other methods use specific framework regions derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0250] It is also important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in some method embodiments, 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. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to 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, is achieved. In general, hypervariable region residues are directly and most substantially influencing antigen binding.

[0251] In some embodiments, the humanized anti-CD20 antibody is a humanized 2H7 antibody. In some embodiments, the humanized 2H7 antibody preferably comprises one, two, three, four, five, or six of the CDR sequences shown in Figures 1A and 1B. In some embodiments, the humanized 2H7 antibody preferably comprises one, two, three, four, five, or six of the following CDR sequences: CDR L1 sequence RASSSVSYXH, where X is M or L (SEQ ID NO: 18), e.g., RASSSVSYMH (SEQ ID NO: 4) (Figure 1A); CDR L2 sequence APSNLAS (SEQ ID NO: 5) (Figure 1A), CDR L3 sequence QQWXFNPPT, where X is S or A (SEQ ID NO: 19), e.g., QQWSFNPPT (SEQ ID NO: 6) (Figure 1A); CDR H1 sequence GYTFTSYNMH (SEQ ID NO: 10) (Figure 1B), CDR H2 sequence AIYPGNGXTSYNQKFKG, where X is D or A (SEQ ID NO: 20), e.g., AIYPGNGDTSYNQKFKG (SEQ ID NO: 11) (Figure 1B), and CDR H3 sequence VVYYSXXYWYFDV, where X at position 6 is N, A, Y, W, or D and X at position 7 is S or R (SEQ ID NO: 21), e.g., VVYYSNSYWYFDV (SEQ ID NO: 12) (Figure 1B).

[0252] The above CDR sequences are generally found within human variable light and variable heavy chain framework sequences, e.g., substantially human light chain kappa subgroup I (V L 6I) and substantially the human consensus FR residues of human heavy chain subgroup III (V H III) are present in the human consensus FR residues. See also WO 2004 / 056312 (Lowman et al.).

[0253] In some embodiments, the variable heavy chain region may be joined to a human IgG chain constant region, where the region comprises a native sequence and a variable constant region, for example, IgG1 or IgG3.

[0254] In some embodiments, such antibodies comprise the variable heavy chain domain sequence of SEQ ID NO:8 (v16 shown in FIG. 1B), and optionally also comprise the variable light chain domain sequence of SEQ ID NO:2 (v16 shown in FIG. 1A), optionally comprising one or more amino acid substitutions at positions 56, 100, and / or 100a in the variable heavy chain domain, e.g., D56A, N100A or N100Y, and / or S100aR, and one or more amino acid substitutions at positions 32 and / or 92 in the variable light chain domain, e.g., M32L and / or S92A. In some embodiments, the antibody is an intact antibody comprising the light chain amino acid sequence of SEQ ID NO:13 or 16, and the heavy chain amino acid sequence of SEQ ID NO:14, 15, 17, 22, or 25. In some embodiments, the humanized 2H7 antibody is ocrelizumab (Genentech).

[0255] In one embodiment, humanized 2H7 has the variable light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 2); and the variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 8).

[0256] In some embodiments, the humanized 2H7 is an intact antibody, and in some embodiments, has the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13); and heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) or the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26) or the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15) or the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 27).

[0257] In some embodiments, the amino acid K at the C-terminus of the heavy chain is removed.

[0258] In some embodiments, the humanized 2H7 antibody has the 2H7.v511 variable light chain domain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 23) and 2H7.v511 variable heavy chain domain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS (SEQ ID NO: 24).

[0259] In some embodiments, the humanized 2H7.v511 antibody is an intact antibody and has the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16) and the heavy chain amino acid sequence of SEQ ID NO: 17 or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 25).

[0260] In some embodiments, the antibodies herein may further comprise at least one amino acid substitution in the Fc region that improves ADCC activity, e.g., amino acid substitutions at positions 298, 333, and 334, preferably S298A, E333A, and K334A, using the EU numbering of heavy chain residues. See also U.S. Patent No. 6,737,056 B1, Presta. Any of these antibodies may comprise at least one substitution in the Fc region that improves FcRn binding or serum half-life, e.g., a substitution at heavy chain position 434, such as N434W. See also U.S. Patent No. 6,737,056 B1, Presta. Any of these antibodies may comprise at least one amino acid substitution in the Fc region that enhances CDC activity, preferably at least a substitution at position 326, such as K326A or K326W. See also U.S. Patent No. 6,528,624 B1 (Idusogie et al.).

[0261] In some embodiments, the humanized 2H7 variant comprises a variable light chain domain of SEQ ID NO: 2 and a variable heavy chain domain of SEQ ID NO: 8, with or without substitutions in the Fc region (if present), and a variable heavy chain with the modifications N100A; or D56A and N100A; or D56A, N100Y, and S100aR in SEQ ID NO: 8; and a variable light chain domain with the modifications M32L; or S92A; or M32L and S92A in SEQ ID NO: 2. M34 in the variable heavy chain domain of 2H7.v16 has been identified as a potential source of antibody stability and is another potential candidate for substitution.

[0262] In some embodiments of the invention, the variable region of a variant based on 2H7.v16 comprises the amino acid sequence of v16 except for the positions of the amino acid substitutions shown in Table 1 below. Unless otherwise stated, the 2H7 variant will have the same light chain as v16. TIFF2025170299000002.tif166170

[0263] (iv) human antibody As an alternative to humanization, human antibodies can be produced. For example, it is now 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. For example, the antibody heavy chain joining region (J) in chimeric and germ-line mutant mice can be expressed in a variety of ways. H It has been described that homozygous deletion of the IgG1 gene results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807.

[0264] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technology, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats; for a review, see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes obtained from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed, and antibodies against a diverse array of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0265] Human antibodies may also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0266] (v)Antibody fragment 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 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, 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 directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0267] (vi) Bispecific antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a B cell surface marker. Other such antibodies can bind to a B cell surface marker and also to a second, different B cell surface marker. Alternatively, an anti-B cell surface marker-binding arm can be combined with an arm that binds to an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), or a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2 or CD3), to focus cellular defense mechanisms on B cells. Bispecific antibodies can also be used to localize cytotoxic agents to B cells. These antibodies have a B cell surface marker-binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or a radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).

[0268] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy and light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (tetrahybrids) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography steps, is quite cumbersome and results in low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0269] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1), containing the site necessary for light chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows for great flexibility in adjusting the relative proportions of the three polypeptide fragments, in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, when expression of equal ratios of at least two polypeptide chains results in high yields, or when the ratio is not particularly critical, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector.

[0270] In some embodiments of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a facile separation method. This approach is disclosed in WO 94 / 04690. For further details on producing bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0271] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. In some embodiments, the interface is located between the C and C regions of the antibody constant domains. HThe antibody comprises at least a portion of the three domains. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chains are created on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0272] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be conjugated to avidin, while the other is conjugated to biotin. Such antibodies have been proposed for use, for example, in targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and in treating HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be made by any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0273] Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0274] Various methods for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were attached to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. The fragments consist of a light-chain variable domain (V) connected by a linker that is too short to allow pairing between the two domains on the same chain. L ) to the heavy chain variable domain (V H ) are bonded together. Therefore, the V H and V L The domain is complementary to the V L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0275] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).

[0276] V. Conjugates and Other Modifications of Antibodies The antibodies used in the methods or included in the articles of manufacture herein are optionally conjugated to a cytotoxic agent. For example, the antibodies can be conjugated to an agent as described in WO 2004 / 032828.

[0277] Chemotherapeutic agents useful in the production of such antibody-cytotoxic agent conjugates are described above.

[0278] Conjugates of an antibody and one or more small molecule toxins, such as calicheamicin, maytansine (U.S. Pat. No. 5,208,020), trichothene, and CC1065, are also contemplated herein. In one embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (e.g., about 1 to about 10 maytansine molecules per antibody molecule). Maytansine can be converted, for example, to May-SS-Me, which can be reduced to May-SH3 and reacted with the modified antibody (Chari et al. Cancer Research 52:127-131 (1992)), to generate the maytansinoid-antibody conjugate.

[0279] Alternatively, the antibody is conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that can be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl γ1 I , PSAG, and θ I 1 (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al. Cancer Research 58:2925-2928 (1998)).

[0280] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. See, for example, WO 93 / 21232, published October 28, 1993.

[0281] The present invention further contemplates antibodies conjugated to a compound with nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0282] A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , and radioactive isotopes of Lu.

[0283] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl 1-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene-2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene-triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radionucleotides to antibodies. See International Publication No. WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic agent within the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker, or disulfide-containing linker (Chari et al. Cancer Research 52:127-131 (1992)) can be used.

[0284] Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, eg by recombinant techniques or peptide synthesis.

[0285] In yet another embodiment, antibodies can be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting, where the antibody-receptor conjugate is administered to a patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation and the administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0286] The antibodies of the invention may also be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active anti-cancer drug. See, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278.

[0287] The enzyme component of such conjugates includes any enzyme capable of acting on a prodrug to convert it into its more active, cytotoxic form.

[0288] Enzymes useful in the methods of the invention include, but are not limited to, alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil; proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs. Examples of suitable enzymes include those useful for converting prodrugs containing D-amino acid substituents; D-alanylcarboxypeptidases, useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes, such as neuraminidase and β-galactosidase, useful for converting glycosylated prodrugs into free drugs; β-lactamases, useful for converting β-lactam-derivatized drugs into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert the prodrugs of the present invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivering abzymes to tumor cell populations.

[0289] The enzymes of the invention can be covalently bound to the antibodies by techniques well known in the art, such as the heterobifunctional cross-linking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least the functionally active site of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312:604-608 (1984)).

[0290] Other modifications of antibodies are contemplated herein. For example, the antibody can be conjugated to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, antibody fragments, such as Fab', are conjugated to one or more PEG molecules.

[0291] The antibodies disclosed herein can also be formulated as liposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545; and International Publication No. WO 97 / 38731, published October 23, 1997. Liposomes with long circulation times are disclosed in U.S. Patent No. 5,013,556.

[0292] Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising 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 the antibody of the present invention can be conjugated to liposomes via a disulfide-interchange reaction as described by Martin et al. J. Biol. Chem. 257:286-288 (1982). Optionally, a chemotherapeutic agent is incorporated into the liposomes. See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

[0293] Amino acid sequence modifications of antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processing of the antibody, such as changing the number or position of glycosylation sites.

[0294] A useful method for identifying certain residues or regions of an antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science, 244:1081-1085 (1989). In this method, target residues or groups are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions are then refined by introducing further or other substitutions at or for the substitution sites. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.

[0295] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 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 or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0296] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. The sites of greatest interest for substitutional mutagenesis of antibodies include the hypervariable regions, although FR modifications are also considered. Conservative substitutions are shown in Table 2 under the heading "preferred substitutions." If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table 2, can be introduced and the products screened. TIFF2025170299000003.tif182169

[0297] Substantial modifications in the biological properties of antibodies can be achieved by selecting substitutions that significantly differ in their effect on (a) the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical configuration, (b) the charge or hydrophobicity of the target site molecule, or (c) maintaining the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polarity: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0298] Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu (4) basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0299] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0300] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0301] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and neighboring residues are candidates for substitution using the techniques described herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0302] Another type of antibody amino acid mutation alters the original glycosylation pattern of the antibody, such as deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody.

[0303] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are the recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0304] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0305] If the antibody contains an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody are described in U.S. Patent Application Publication No. 2003 / 0157108A1 (Presta, L.); see also U.S. Patent Application Publication No. 2004 / 0093621A1 (Kyowa Hakko Kogyo Co., Ltd.) regarding CD20 antibody compositions. Antibodies with bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate chain attached to the Fc region of the antibody are referenced in WO 03 / 011878 (Jean-Mairet et al.) and U.S. Patent No. 6,602,684 (Umana et al.). Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody are reported in WO 97 / 30087 (Patel et al.). See also WO 98 / 58964 (Raju, S.) and WO 99 / 22764 (Raju, S.) regarding antibodies having engineered carbohydrate chains attached to their Fc region.

[0306] In some embodiments, the glycosylation variants herein comprise an Fc region, and the carbohydrate structure attached to the Fc region lacks fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333, and / or 334 (Eu residue numbering) of the Fc region. Examples of "defucosylated" or "fucose-deficient" antibodies in the literature include: U.S. Patent Application Publication No. 2003 / 0157108A1, Presta, L; WO 00 / 61739A1; WO 01 / 29246A1; U.S. Patent Application Publication No. 2003 / 0115614A1; U.S. Patent Application Publication No. 2002 / 0164328A1; U.S. Patent Application Publication No. 2004 / 0093621A1; U.S. Patent Application Publication No. 2005 / 0093621A1; U.S. Patent Application Publication No. 2006 / 0093621A1; U.S. Patent Application Publication No. 2007 / 0093621A1; U.S. Patent Application Publication No. 2009 ... 04 / 0132140A1; U.S. Patent Application Publication No. 2004 / 0110704A1; U.S. Patent Application Publication No. 2004 / 0110282A1; U.S. Patent Application Publication No. 2004 / 0109865A1; WO 2003 / 085119A1; WO 03 / 084570A1; WO 2005 / 035778; WO 2005 / 035586 (describing RNA inhibition (RNAi) of fucosylation); Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108A1, Presta, L; and WO 2004 / 056312, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)).

[0307] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared antibody variants or non-variants.

[0308] It may be desirable to modify the antibody of the invention with respect to effector function, for example to improve antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid modifications into the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody may be engineered which has dual Fc regions and may thereby possess enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

[0309] WO 00 / 42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies contain amino acid substitutions in their Fc region. In some embodiments, the antibodies with improved ADCC contain substitutions at positions 298, 333, and / or 334 in the Fc region. In some embodiments, the modified Fc region is a human IgG1 Fc region containing or consisting of substitutions at one, two, or three of these positions.

[0310] Antibodies with altered C1q binding and / or complement dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Pat. No. 6,194,551 B1, U.S. Pat. No. 6,242,195 B1, U.S. Pat. No. 6,528,624 B1, and U.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of their Fc region.

[0311] To increase the serum half-life of an antibody, a salvage receptor binding epitope can be introduced into the antibody (e.g., antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for extending the in vivo serum half-life of the IgG molecule. Antibodies with substitutions in their Fc region and extended serum half-lives are also described in WO 00 / 42072 (Presta, L.).

[0312] Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Patent Application Publication No. 2002 / 0004587A1, Miller et al.).

[0313] VI. Pharmaceutical Formulations Therapeutic formulations of antibodies used according to the present invention are prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the antibody having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium 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); and 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0314] An exemplary anti-CD20 antibody formulation is described in WO 98 / 56418, which describes a liquid multi-dose formulation containing 40 mg / mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, and 0.02% polysorbate 20, pH 5.0, with a minimum shelf life of 2 years at 2-8° C. Another anti-CD20 formulation of interest is one containing 10 mg / mL rituximab, 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection, pH 6.5.

[0315] Lyophilized formulations adapted for subcutaneous administration are described in U.S. Patent No. 6,267,958 (Andya et al.). Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the mammal being treated herein.

[0316] Crystallized forms of antibodies or antibodies are also contemplated. See, e.g., U.S. Patent Application Publication No. 2002 / 0136719A1 (Shenoy et al.).

[0317] The formulations herein may also contain more than one active compound as needed for the particular indication being treated, in some embodiments those with complementary activities that do not adversely affect each other. For example, cytotoxic agents, chemotherapeutic agents; immunosuppressants; cytokines; cytokine antagonists or antibodies; growth factors; hormones; integrins; integrin antagonists or antibodies (e.g., LFA-1 antibodies, or Biogen Idec / Elan antibodies). alpha-4 integrin antibodies, such as natalizumab (TYSABRI®) available from Eppendorf Pharmaceuticals, Inc.; interferon class agents, such as IFN-beta-1a (REBIF® and AVONEX®) and IFN-beta-1b (BETASERON®); oligopeptides, such as glatiramer acetate (COPAXONE®); cytotoxic agents, such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, or azathioprine; intravenous immunoglobulin (gamma globulin); lymphodepleting agents (e.g., mitoxantrone, cyclophosphamide, Campath, anti-CD4, or cladribine); non-lymphocyte-depleting immunosuppressants (e.g., mycophenolate mofetil (MMF) or cyclosporin). It may be desirable to further provide in the formulation: anti-inflammatory drugs such as steroids; cholesterol-lowering agents of the "statin" class; estradiol; testosterone; hormone replacement therapy; agents treating secondary or associated symptoms of MS (e.g., spasticity, ataxia, pain, fatigue); TNF inhibitors; disease-modifying antirheumatic drugs (DMARDs); nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids (e.g., methylprednisolone, prednisone, dexamethasone, or glucorticoids); levothyroxine; cyclosporine A; somatastatin analogs; cytokine antagonists; antimetabolites; immunosuppressants; integrin antagonists or antibodies (e.g., LFA-1 antibodies, e.g., efalizumab or alpha-4 integrin antibodies such as natalizumab); or other B-cell surface antagonists / antibodies.The type and effective amount of such other agents will depend, for example, on the amount of antibody present in the formulation, the type of multiple sclerosis being treated, and the patient's clinical parameters, and they will generally be used in the same dosages and by the same route of administration as previously used, or at approximately 1 to 99% of the dosage previously used.

[0318] The active ingredient can also be entrapped in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules of colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0319] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0320] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0321] In some embodiments, the formulation contains one or more of the group consisting of histidine buffer, trehalose, sucrose, and polysorbate 20. In some embodiments, the histidine buffer is a histidine-acetate buffer, pH 6.0. Examples of formulations suitable for administration of anti-CD20 antibodies are found in Andya et al., U.S. Patent Application Publication No. 2006 / 0088523, which is incorporated by reference in its entirety with respect to formulations.

[0322] Exemplary anti-CD20 antibody formulations are described in U.S. Patent Application Publication No. 2006 / 0088523 and WO 98 / 56418 to Andya et al., which are incorporated by reference in their entireties. In some embodiments, the formulation is a liquid multi-dose formulation comprising 40 mg / mL anti-CD20 antibody, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 (pH 5.0) with a minimum shelf life of 2 years stored at 2-8° C. In some embodiments, a subject anti-CD20 formulation comprises 10 mg / mL antibody, 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection (pH 6.5). In some embodiments, the anti-CD20 antibody is contained in an aqueous pharmaceutical formulation containing 10-30 mM sodium acetate at about pH 4.8 to about pH 5.5, preferably pH 5.5, polysorbate as a surfactant in an amount of about 0.01-0.1% v / v, trehalose in an amount of about 2-10% w / v, and benzyl alcohol as a preservative (U.S. Pat. No. 6,171,586, incorporated by reference in its entirety). Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801, incorporated by reference in its entirety. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the mammal being treated herein.

[0323] In some embodiments, the humanized 2H7 variant formulation is antibody at 12-14 mg / mL in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8. In a particular embodiment, 2H7 variants, and particularly 2H7.v16, are formulated at 20 mg / mL in 10 mM histidine sulfate, 60 mg / mL sucrose, 0.2 mg / mL polysorbate 20, and sterile water for injection, pH 5.8. In a particular embodiment, one IV formulation of humanized 2H7 v16 is antibody at 30 mg / mL in 20 mM sodium acetate, 4% trehalose dihydrate, 0.02% polysorbate 20 (Tween 20™), pH 5.3. In some embodiments, the humanized 2H7.v511 variant formulation is 15-30 mg / mL of antibody, preferably 20 mg / mL of antibody, in 10 mM histidine sulfate, 60 mg / mL sucrose (6%), 0.2 mg / mL polysorbate 20 (0.02%), and sterile water for injection (pH 5.8). In yet another embodiment, the formulation of a 2H7 variant, particularly 2H7.v511, is 20 mg / mL 2H7, 20 mM sodium acetate, 4% trehalose dihydrate, 0.02% polysorbate 20, pH 5.5, for intravenous administration. In some embodiments, the 2H7.v114 formulation is 15-25 mg / ml, preferably 20 mg / ml, of antibody in 20 mM sodium acetate, 240 mM (8%) trehalose dihydrate, 0.02% polysorbate 20, pH 5.3. In some embodiments, an anti-CD20 antibody (e.g., 2H7.v16) is formulated at 30 mg / ml of antibody, 20 mM sodium acetate, 106 mM trehalose, 0.02% polysorbate 20, pH 5.3. Liquid formulations containing the antibody may be in 300 mg / vial and may be stored at 2-8°C, protected from light. In some embodiments, prior to administration, the antibody is diluted with saline (0.9% sodium chloride) in an IV bag for administration by infusion.

[0324] VII. Manufactured Articles and Kits The present invention provides articles of manufacture or kits (e.g., kits of parts) containing materials useful for treating progressive multiple sclerosis as described herein. In some embodiments, the articles of manufacture include a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with multiple sclerosis and provides improvement in functional ability in patients with multiple sclerosis.

[0325] In some embodiments, the article of manufacture or kit comprises a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with multiple sclerosis and inhibits the progression of disability in patients with multiple sclerosis.

[0326] In some embodiments, the article of manufacture or kit includes a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with multiple sclerosis and delays the onset of confirmed disability progression in patients with multiple sclerosis. In some embodiments, confirmed disease progression is a 12-week sustained increase in EDSS. In some embodiments, confirmed disease progression is a 24-week sustained increase in EDSS.

[0327] In some embodiments, the article of manufacture or kit comprises a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with multiple sclerosis and slows or prevents brain volume loss in patients with multiple sclerosis.

[0328] In some embodiments, the article of manufacture or kit comprises a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with highly active multiple sclerosis. In some embodiments, the label further indicates that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with highly active multiple sclerosis who have not previously been treated with other therapies for multiple sclerosis. In some embodiments, the label further indicates that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with highly active multiple sclerosis who have previously been treated with other therapies for multiple sclerosis. In some embodiments, the label further indicates that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with highly active multiple sclerosis who are poor responders to other therapies for multiple sclerosis.

[0329] In some embodiments, the article of manufacture or kit comprises a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier, packaged together with a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for treating patients with multiple sclerosis, and that the anti-CD20 antibody or pharmaceutical composition is effective in one or more of the following: (1) reducing the number of lesions in the patient's brain; (2) reducing the annualized relapse rate; (3) reducing disability progression; and (4) improving functional ability.

[0330] In some embodiments of any of the articles of manufacture or kits, the multiple sclerosis is progressive multiple sclerosis. In some embodiments, the progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, the multiple sclerosis is a relapsing form of multiple sclerosis. In some embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis. In some embodiments, the relapsing form of multiple sclerosis is secondary progressive multiple sclerosis with overlapping relapses (rSPMS).

[0331] In some embodiments of any of the articles of manufacture or kits, the anti-CD20 antibody comprises a) a heavy chain variable region comprising SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and b) a light chain variable region comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the anti-CD20 antibody is ocrelizumab.

[0332] In some embodiments, the package insert comprises instructions representing (i.e., indicating) that an amount of ocrelizumab is administered to a patient effective to provide an initial ocrelizumab exposure of between about 0.3 and about 0.6 grams, followed by a second ocrelizumab exposure of between about 0.3 and about 0.6 grams, where the second exposure is not provided until about 16 to 60 weeks after the initial exposure, and each exposure of ocrelizumab is provided to the patient as one or two doses of ocrelizumab. In some embodiments, the initial ocrelizumab exposure is about 0.6 grams. In some embodiments, the second ocrelizumab exposure is about 0.6 grams. In some embodiments, the second exposure is administered starting about 20 to 24 weeks after the initial exposure. In some embodiments, "about 20 to 24 weeks" refers to a time point between 20 and 24 weeks. In some embodiments, "about 20 to 24 weeks" refers to variations of 1 week or 7 days before or after the 24th week. In some embodiments, one or more of the ocrelizumab exposures are provided to the patient as one dose of ocrelizumab. In some embodiments, one or more of the ocrelizumab exposures are provided to the patient as two doses of ocrelizumab. In some embodiments, two doses of ocrelizumab comprise about 0.3 grams of ocrelizumab.

[0333] In some embodiments, the article of manufacture or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container may house or contain a composition effective for treating multiple sclerosis and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody. In some embodiments, the container contains about 0.3 to about 4.0 grams of anti-CD20 antibody. In some embodiments, the container contains about 0.3 to about 1.5 grams of anti-CD20 antibody.

[0334] The label or package insert indicates that the composition is used for treating multiple sclerosis in patients with the disease and includes specific guidance regarding dosage amounts and intervals of the antibody and any other medications provided. The article of manufacture may further include a second container containing a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0335] In some cases, the articles of manufacture or kits provided herein further comprise a container that includes an agent other than an antibody for treatment and further comprises instructions for treating a patient with such an agent, preferably a chemotherapeutic or immunosuppressive agent, an interferon class agent, e.g., IFN-β-1a (REBIF® and AVONEX®) or IFN-β-1b (BETASERON®); an oligopeptide such as glatiramer acetate (COPAXONE®); a cytotoxic agent such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenous immunoglobulin (gamma globulin); lymphodepleting therapy (e.g., mitoxantrone, cyclophosphamide, Campath, anti-CD4, or cladribine); a non-lymphodepleting immunosuppressant. (e.g., mycophenolate mofetil (MMF) or cyclosporine); cholesterol-lowering agents of the "statin" class; estradiol; hormone replacement therapy; drugs treating secondary or associated symptoms of MS (e.g., spasticity, ataxia, pain, fatigue); TNF inhibitors; disease-modifying antirheumatic drugs (DMARDs); nonsteroidal anti-inflammatory drugs (NSAIDs); corticosteroids (e.g., methylprednisolone, prednisone, dexamethasone, or glucocorticoids); levothyroxine; cyclosporine A; somatastatin analogs; cytokine or cytokine receptor antagonists; antimetabolites; immunosuppressants; integrin antagonists or antibodies (e.g., LFA-1 antibodies, e.g., efalizumab, or alpha-4 integrin antibodies such as natalizumab); and other B-cell surface marker antibodies. [Example]

[0336] Example 1: Phase III study of ocrelizumab compared with interferon beta-1a (Rebif) in patients with relapsing multiple sclerosis Multiple sclerosis is a heterogeneous disease with an unpredictable disease course and no cure (Scalfari et al. (2013) JAMA Neurol. 70, 214-22; Tremlett et al. (2006) Neurology. 66, 172-7; Markowitz (2010) Am J Manag Care. 16, S211-8; Hauser et al. (2013) Ann Neurol. 74, 317-27). Despite the various treatments available in recent years for relapsing forms of multiple sclerosis, many patients continue to develop neurological impairment, and therefore there is a significant unmet need for more effective and well-tolerated treatments (Markowitz (2010) Am J Manag Care. 16, S211-8; Rotstein et al. (2015) JAMA Neurol. 72, 152-8; Sorensen (2007) J Neurol Sci. 259, 128-32). In addition, the risk profile of more effective treatments has previously prevented their use earlier in the disease course (Markowitz (2010) Am J Manag Care. 16, S211-8; Hartung et al. (2011) Expert Rev Neurother. 11, 351-62; Hauser SL. (2015) Mult Scler. 21, 8-21).

[0337] B cells are an important contributor to the pathogenesis of multiple sclerosis (Monson (2005) J Neuroimmunol. 158, 170-81; Hauser SL. (2015) Mult Scler. 21, 8-21). While rarely observed in the cerebrospinal fluid of healthy controls, B cells are frequently found at low percentages in the cerebrospinal fluid of patients with multiple sclerosis (Cepok (2005) Brain. 128, 1667-76; Cross et al. (2011) Biochim Biophys Acta. 1812, 231-8), and elevated levels in the cerebrospinal fluid correlate with faster disease progression in relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis (Cepok (2005) Brain. 128, 1667-76). B cells are involved in multiple functions: antigen presentation (Constant (1999) J Immunol. 162, 5695-703; Crawford et al. (2006) J Immunol. 176, 3498-506), autoantibody production (Bar-Or A (2010) Ann Neurol. 67, 452-61; Duddy (2007) J Immunol. 178, 6092-9), cytokine regulation (Genain et al. (1999) Nat Med. 5, 170-5; Storch et al. (1998) Ann Neurol. 43, 465-71), and the formation of ectopic lymphoid follicle-like aggregates (Magliozzi et al. (2010) Ann Neurol. 68, 477-93; Serafini et al. (2004) Brain Pathol. 14:164-74, which impacts the underlying pathogenesis of multiple sclerosis. Interest in B cells has increased with proof-of-concept and observational studies, and interest in their utility in multiple sclerosis has developed over time (Hauser et al. (2008) N Engl J Med. 358, 676-88; Kappos et al. (2011) Lancet. 378, 1779-87; Lehmann-Horn et al. (2013) Ther Adv Neurol Disord. 6, 161-73).

[0338] CD20 is a cell surface antigen found on pre-B cells, mature B cells, and memory B cells, but is not expressed on lymphoid stem cells or plasma cells (Stashenko et al. (1980) J Immunol. 125, 1678-85; Loken et al. (1987) Blood. 70, 1316-24; Tedder et al. (1994) Immunol Today. 15, 450-4). In the HERMES study, rituximab, an anti-CD20 chimeric monoclonal antibody, significantly reduced inflammatory brain lesions and clinical relapses compared with placebo in patients with relapsing-remitting multiple sclerosis; thus, selective depletion of CD20+ B cells provides evidence that it is a potentially effective therapeutic approach in multiple sclerosis (Kappos et al. (2011) Lancet. 378, 1779-87).

[0339] Ocrelizumab is a recombinant humanized monoclonal antibody that selectively depletes CD20-expressing B cells (Klein et al. (2013) MAbs. 5, 337-8; Genovese et al. (2008) Arthritis Rheum. 58, 2652-61), while preserving the capacity for B cell reconstitution and pre-existing humoral immunity (Martin et al. (2006) Annu Rev Immunol. 24, 467-96; DiLillo et al. (2008) J Immunol. 180, 361-71). Ocrelizumab binds with high affinity to the large extracellular loop of CD20 and selectively depletes B cells through several mechanisms, including antibody-dependent cell-mediated phagocytosis, antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and induction of apoptosis (Klein et al. (2013) MAbs. 5, 22-33).

[0340] Two identical phase 3, multicenter, randomized, double-blind, double-dummy, parallel-group studies (Study I and Study II) were conducted to investigate the efficacy and safety of ocrelizumab compared with interferon (IFN) beta-1a in patients with relapsing forms of multiple sclerosis. Results from these two studies are reported here.

[0341] method Eligibility and Exclusion Criteria Key eligibility criteria included: age 18 to 55 years; the 2010 revised McDonald criteria (Polman et al. (2011) "Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria." Ann Neurol 69, 292-302); Expanded Disability Status Scale (EDSS, see world-wide-web.neurostatus.org) score of 0 to 5.5 at screening; at least two confirmed clinical episodes within the previous 2 years or one clinical episode within the year prior to screening (but not within 30 days prior to screening); a confirmed MRI of the brain with abnormalities consistent with multiple sclerosis; and neurological stability for at least 30 days prior to both screening and baseline.

[0342] Key exclusion criteria included: a diagnosis of primary progressive multiple sclerosis; patients with disease duration of 10 years or more combined with an EDSS score of 2.0 or less at screening; known presence of other neurological disorders that could mimic multiple sclerosis; pregnancy or lactation; previous treatment with any B-cell targeted therapy or other contraindications (i.e., requirement of long-term treatment with systemic corticosteroids or immunosuppressants during the course of the study, history or currently active primary or secondary immunodeficiency, history or known presence of active infection, or recurrent or chronic infection (e.g., hepatitis B or C, HIV, syphilis, tuberculosis), history of progressive multifocal leukoencephalopathy, contraindication or intolerance to oral or intravenous corticosteroids, or contraindication to or incompatibility with the use of Rebif).

[0343] Research Plan Patients were randomized (1:1) to receive either ocrelizumab 600 mg by intravenous infusion every 24 weeks (administered as two 300 mg infusions on days 1 and 15 as the first dose, followed by a single 600 mg infusion on day 1 of each 24-week treatment course) or IFNβ-1a subcutaneously at a dose of 44 μg three times per week throughout the 96-week treatment period (see Figure 7). Patients in the ocrelizumab and IFNβ-1a groups also received subcutaneous and intravenous placebo, respectively. All patients received intravenous methylprednisolone 100 mg (as well as any analgesic / antipyretic and antihistamine) prior to the infusion. Randomization was performed centrally by an independent medical provider. Patients were stratified by region (USA / rest of the world) and baseline EDSS score (<4 / ≥4).

[0344] To maintain confidentiality of study group assignment, each study center had a separate treating investigator (a neurologist experienced in treating multiple sclerosis) and testing investigator (a neurologist or other healthcare professional), all of whom were blinded throughout the course of the study. The treating investigators had access to safety and blinded efficacy data and made treatment decisions based on the patient's clinical response and laboratory findings. The researchers included EDSS scores (Kurtze (1983) "Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)." Neurology. 33, 1444-52), functional system scores (Kurtze (1983) "Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)." Neurology. 33, 1444-52; Haber and LaRocca, eds. Minimal Record of Disability for Multiple Sclerosis. New York: National Multiple Sclerosis Society; 1985), multiple sclerosis functional composite (MSFC) (Rudick et al. (2002) "The multiple sclerosis functional composite: a new clinical outcome measure for multiple sclerosis trials." Multiple sclerosis (Houndmills, Basingstoke, England) 8, 359-65), and low-contrast visual acuity (LCVA) tests (Wieder et al. al.(2013) “Low contrast visual acuity testing is associated with cognitive performance in multiple sclerosis: a pilot study.” BMC Neurology.13,167), Symbol Digit Modality Test (SDMT) (Smith A.(1982).Neurological evaluations were performed, including the Symbol digit modalities test (Manual. Los Angeles: Western Psychological Services) and the Karnofsky Performance Status Scale (Mor et al. "The Karnofsky Performance Status Scale. An examination of its reliability and validity in a research setting." Cancer. 53. 2002-2007). MRI evaluations were analyzed independently by a central MRI reader who was blinded to treatment assignment.

[0345] Patients who completed the 96-week treatment period were eligible to enter the open-label extension phase of the study. Patients who discontinued or did not wish to participate in the open-label extension were entered into a 48-week safety follow-up phase that included B-cell monitoring.

[0346] Study Procedures EDSS scores were determined at screening, baseline, and every 12 weeks; MSFC, LCVA, and SDMT scores were determined at baseline and every 12 weeks; and Karnofsky Performance Status Scale was determined at baseline and every 24 weeks. MRI was performed at baseline and at weeks 24, 48, and 96. Adverse events were monitored throughout the study.

[0347] The primary endpoint was the annualized protocol-defined relapse rate at week 96, where a relapse was defined as a new or worsening neurological symptom lasting 24 hours or more and attributed solely to multiple sclerosis. New or worsening neurological symptoms must be accompanied by objective neurological deterioration consistent with an increase of at least half a step on the EDSS scale, a 2-point increase in one EDSS functional system score, or a 1-point increase in each of two or more EDSS functional system scores. Relapses were reclassified as protocol-defined relapses by an automated algorithm according to the rules described above. The algorithm was written before database closure and data unblinding.

[0348] Key secondary endpoints include: time to onset of 12-week confirmed disability progression (i.e., CDP), defined in the study as an increase of at least 1.0 points from baseline EDSS score in patients with a baseline score of 5.5 or less, or an increase of 0.5 points in patients with a baseline score of greater than 5.5, at 96 weeks, where the EDSS increase is confirmed at a regularly scheduled clinic visit at least 12 weeks after initial neurological deterioration; and at weeks 24, 48, and 96. The total number of T1 gadolinium-enhancing lesions; the total number of new and / or enlarging T2 hyperintense lesions detected at weeks 24, 48, and 96; the proportion of patients with 12-week confirmed disability improvement (i.e., CDI) during week 96 (only analyzed for the subgroup of patients with a baseline EDSS score of at least 2.0); the time to onset of 24-week CDP confirmed at week 24 after initial neurological deterioration at week 96; the pharmacokinetics, immunogenicity, and pharmacodynamics of ocrelizumab; and the safety and tolerability of ocrelizumab. CDI in this study was defined as a reduction in EDSS score of at least 1.0 compared to baseline in patients with a baseline EDSS score of 5.5 or less, or a reduction of 0.5 points in patients with a baseline EDSS score of greater than 5.5.

[0349] The proportion of patients with an EDSS score ≥ 2.0 with no evidence of disease activity (NEDA) by week 96 was an exploratory endpoint; the NEDA analysis in all patients was an exploratory endpoint. The percentage change in brain volume as detected by brain MRI from week 24 to week 96 from baseline to week 96 was also performed as an exploratory analysis.

[0350] statistical analysis The statistical hierarchy is provided in Figure 8. To achieve sufficient statistical power to test the effects of ocrelizumab on CDP and CDI, it was prespecified that these endpoints be pooled for both Phase III studies. All other endpoints were analyzed separately for each study. Primary and secondary efficacy analyses were performed on the intention-to-treat population.

[0351] All efficacy analyses were performed in the intention-to-treat population. Annualized relapse rate (ARR), the primary efficacy endpoint, was analyzed for each patient using a negative binomial distribution model, including onset between the date of randomization and early treatment discontinuation / week 96, in statistical analyses that considered length of exposure, treatment group, region (US / rest of world), and baseline EDSS score (<4.0 / ≥4.0) as covariates. Significant results at a two-sided alpha of less than 0.05 demonstrate the superior effect of ocrelizumab in reducing the ARR compared with IFN beta-1a.

[0352] The sample size for each study was based on an estimated annualized relapse rate of 0.165 for the ocrelizumab group and 0.33 for the IFNβ-1a group. Using a two-tailed t-test, it was calculated that a sample of 400 patients per treatment group would provide 84% statistical power to detect a 50% relative reduction in ocrelizumab compared with IFNβ-1a (assuming a dropout rate of approximately 20%), while maintaining a type I error rate of 0.05. A per-protocol sensitivity analysis assessed the effect of major protocol violations on the primary endpoint.

[0353] Ten secondary efficacy endpoints were tested in a hierarchical order of decreasing clinical importance with a two-sided alpha of 0.05 (see Figure 8; ARR = annualized relapse rate; CDI = confirmed disability improvement; CDP = confirmed disability progression; Gd = gadolinium; MSFC = multiple sclerosis functional composite; NEDA = no evidence of disease activity; SF-36 PCS = Short-Form 36, Physical Component Summary. NEDA is defined as no protocol-defined relapses, no CDP events, no new or enlarging T2 lesions, and no Gd-enhancing T1 lesions. Analysis of secondary efficacy endpoints at the individual study level is as follows:

[0354] For the first secondary efficacy endpoint (time to onset of confirmed disability progression of at least 12 weeks), the study-level p-value is interpreted as unconfirmed due to insufficient statistical power at the study level to detect a relevant treatment difference.

[0355] The second secondary efficacy endpoint (total number of T1 Gd-enhancing lesions at weeks 24, 48, and 96) will be tested in a confirmatory manner if and only if the first secondary efficacy endpoint reaches a significance level of 0.05 (i.e., pooled p≦0.05) in the analysis of both combined studies. If the pooled p>0.05 for the first secondary efficacy endpoint in the analysis of the combined studies, the p-values ​​for the second and subsequent secondary efficacy endpoints within the hierarchy will be interpreted as unconfirmed.

[0356] The third secondary efficacy endpoint (total number of new and / or enlarging T2 hyperintense lesions at Weeks 24, 48, and 96) will be tested in a confirmatory manner if, and only if, the second secondary efficacy endpoint (total number of T1 Gd-enhancing lesions at Weeks 24, 48, and 96) reaches a significance level of 0.05 (i.e., p≦0.05). If the second secondary efficacy endpoint has p>0.05, the p-values ​​for the third and subsequent secondary efficacy endpoints within the stratum will be interpreted as unconfirmed.

[0357] For the fourth (proportion of patients with confirmed disability improvement for at least 12 weeks) and fifth (time to onset of confirmed disability progression for at least 24 weeks) secondary efficacy endpoints, study-level p-values ​​are interpreted as unconfirmed due to insufficient statistical power at the study level to detect relevant treatment differences.

[0358] The sixth secondary efficacy endpoint (total number of new T1 hypointense lesions (chronic black holes) at Weeks 24, 48, and 96) will be tested in a confirmatory manner if, and only if, the fifth secondary efficacy endpoint (time to onset of confirmed disability progression of at least 12 weeks) reaches a significance level of 0.05 (i.e., pooled p<0.05) in the analysis of the combined study. If the pooled p>0.05 for the fifth secondary efficacy endpoint in the analysis of both combined studies, the p-values ​​for the sixth and subsequent secondary efficacy endpoints within the hierarchy will be interpreted as unconfirmed.

[0359] The seventh (and subsequent) secondary efficacy endpoint will be tested in a confirmatory manner if and only if the sixth (or immediately preceding) secondary efficacy endpoint reaches a significance level of 0.05 (i.e., p≦0.05). If p>0.05 for the sixth (and subsequent) secondary efficacy endpoint, the p-values ​​for the seventh (or current) and all subsequent secondary efficacy endpoints in the hierarchy will be interpreted as unconfirmed.

[0360] Additionally, for the analysis of secondary efficacy endpoints, data from both studies will be combined (thus providing sufficient statistical power for comparison of all primary and secondary efficacy endpoints):

[0361] The first secondary efficacy endpoint (time to onset of confirmed disability progression of at least 12 weeks) will be tested in a confirmatory manner if and only if the primary efficacy endpoint (2-year annualized protocol-defined relapse rate) reaches a significance level of 0.05 (i.e., pooled p≦0.05). If the pooled p for the primary efficacy endpoint is >0.05, the pooled p values ​​for all secondary efficacy endpoints within the stratum will be interpreted as unconfirmed.

[0362] The second (and subsequent) secondary efficacy endpoint will be tested in a confirmatory manner if and only if the first (or immediately preceding) secondary efficacy endpoint reaches a significance level of 0.05 (i.e., pooled p ≤ 0.05). If the pooled p for the first (and subsequent) secondary efficacy endpoint is > 0.05, the p values ​​for the pooled analyses of the second (or current) and all subsequent secondary efficacy endpoints within the stratum will be interpreted as unconfirmed.

[0363] The safety population was used for all analyses of safety data and included all patients who received any study treatment.

[0364] result patient Overall, 1656 patients were enrolled in the first (N=821) and second (N=835) Phase III studies (intent-to-treat populations). Baseline demographic and disease characteristics were similar between each study and between the two study populations (see Table 3). TIFF2025170299000004.tif251170TIFF2025170299000005.tif49170

[0365] A total of 366 (89%) and 340 (83%) patients in the ocrelizumab and IFN β-1a treatment groups, respectively, completed the first study (see Figure 9), and 360 (86%) and 320 (77%) in the ocrelizumab and IFN β-1a treatment groups, respectively, completed the second study (see Figure 9). More than 85% of patients in the ocrelizumab treatment group completed Studies I and II. All randomized patients were included in the intention-to-treat (ITT) population. Patients who withdrew from the study early for any reason and patients whose evaluations were not performed for any reason were still included in the intention-to-treat (ITT) analysis.

[0366] Effectiveness Clinical and MRI results from the first and second studies are summarized in Table 4. The frequency of multiple sclerosis relapses was reduced by ocrelizumab in both studies, with the adjusted annualized relapse rate (ARR) at 96 weeks (primary endpoint) for ocrelizumab in Study I being 0.156 (vs. 0.292 for IFN β-1a) and for ocrelizumab in Study II being 0.155 (vs. 0.290 for IFN β-1a) (see Table 4 and Figures 10A and 10B). The annualized relapse rate (based on protocol-defined relapses) in patients treated with ocrelizumab was reduced by 46% and 47% in Studies I and II, respectively, relative to IFN β-1 (p<0.0001 for both comparisons). The annualized relapse rate (based on protocol-defined relapses) in patients treated with ocrelizumab was reduced by 46% versus IFN β-1 (Study I and Study II, pooled data; p=0.0001). Adjusted ARR was calculated by binomial regression and adjusted for baseline EDSS score (<4.0 vs. ≥4.0) and geographic location (United States vs. rest of the world). The annualized relapse rate (based on all clinical relapses) in patients treated with ocrelizumab was reduced by 42% (p=0.001) and 47% (p<0.0001) versus IFN β-1 in Study I and Study II, respectively. TIFF2025170299000006.tif255170TIFF2025170299000007.tif250170TIFF2025170299000008.tif255170TIFF2025170299000009.tif212170*± Values ​​are mean ± standard deviation. †Bold indicates pre-specified pooled endpoints. ‡In patients with a baseline EDSS score of at least 2.0 §NEDA is defined as no protocol-defined recurrence, no CDP events, no new or enlarging T2 lesions, and no gadolinium-enhancing lesions. Adjusted for baseline lesion count, EDSS (<4.0 / ≥4.0), and geographic region (US / ROW).

[0367] hindrance When compared with IFN β-1a, ocrelizumab reduced the risk of 12-week sustained confirmed disability progression (i.e., CDP) by 40% over the 96-week study period (Studies I and II, pooled data; p=0.0006; Figure 11 , CI = confidence interval; HR = hazard ratio). Non-pooled data from Studies I and II showing that ocrelizumab reduced the risk of 12-week sustained confirmed disability progression (i.e., CDP) compared with IFN β-1a are shown in Figure 12A (Study I) and Figure 12B (Study II). Ocrelizumab also reduced the risk of 24-week sustained CDP by 40% over the 96-week study period relative to IFN β-1a (Studies I and II, pooled data; p=0.0025; Figure 13 ). Unpooled data from Studies I and II showing that ocrelizumab, compared with IFN β-1a, reduced the risk of confirmed disability progression (i.e., CDP) sustained for 24 weeks are shown in Figures 14A and 14B. The proportion of patients (with a baseline EDSS score of 2.0 or greater) with at least 12 weeks of confirmed disability improvement (i.e., CDI) was 20.7% for patients receiving ocrelizumab (n=628) versus 15.6% for patients receiving IFN β-1a (n=614), representing a 33% risk improvement with ocrelizumab (Study I and Study II, pooled data; p=0.0194). See Figure 15A. The proportion of patients (with a baseline EDSS score of 2.0 or greater) with at least 12 weeks of confirmed disability improvement (i.e., CDI) was 15.6% for patients receiving ocrelizumab (n=628) compared with 11.6% for patients receiving IFNβ-1a (n=614), representing a 36% risk improvement with ocrelizumab (Study I and Study II, pooled data; p=0.0343). See Figure 15B. For patients with a baseline EDSS score of ≥2.0 and ≤5.5, disability improvement was defined as a decrease in EDSS score of ≥1.0 points compared to the baseline EDSS score.For patients with a baseline EDSS score of >5.5, disability improvement was defined as a decrease in EDSS score of ≥0.5 points; p values ​​for relative improvement are from Cochran-Mantel-Haenszel chi-square tests stratified by study, baseline EDSS score (<4.0 vs. ≥4.0), and geographic region (United States vs. rest of the world), including stratification factors. Patients with missing EDSS or without post-onset confirmation of disability improvement were counted as not having CDI.

[0368] Unpooled data for the proportion of patients with at least 12 weeks of confirmed disabilit...

Claims

1. A method of improving functional capacity in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient has improved functional capacity after treatment; wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

2. 10. The method of claim 1, wherein the patient has a confirmed improvement in the disorder 12 weeks after treatment.

3. 10. The method of claim 1, wherein the patient has confirmed improvement in the disorder 24 weeks after treatment.

4. 4. The method of any one of claims 1 to 3, wherein the improvement in functional ability in the patient is sustained for at least 12 weeks.

5. 4. The method of any one of claims 1 to 3, wherein the improvement in functional ability in the patient is sustained for at least 24 weeks.

6. 6. The method of any one of claims 1 to 5, wherein the improvement in functional capacity is measured by the Timed 25-Foot Walk (T-25FW) test or EDSS score.

7. 6. The method of any one of claims 1 to 5, wherein the improvement in functional capacity is measured by the Timed 25-Foot Walk (T-25FW) test and EDSS score.

8. 8. The method of any one of claims 1 to 7, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

9. 10. The method of claim 8, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

10. 10. The method of claim 9, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

11. 11. The method of any one of claims 8 to 10, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

12. 12. The method of claim 11, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

13. 11. The method of any one of claims 8 to 10, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

14. 14. The method of any one of claims 8 to 13, wherein the patient has improved functional capacity after exposure to one, two, three, and / or four of the anti-CD20 antibodies.

15. 15. The method of any one of claims 1 to 14, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

16. A method for inhibiting the progression of complex disorders in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration results in a reduction in confirmed disorder progression events, and wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

17. 17. The method of claim 16, wherein administration results in a reduction in progression of confirmed complex disorders for 12 weeks.

18. 17. The method of claim 16, wherein administration results in a reduction in progression of confirmed complex disorders for 24 weeks.

19. 18. The method of any one of claims 16 to 17, wherein the progression of confirmed complex disability is determined by Expanded Disability Status Scale (EDSS) score, Timed 25-Foot Walk (T25-FW), and 9-Hole Peg Test (9-HPT).

20. 20. The method of any one of claims 16 to 19, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

21. 21. The method of claim 20, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

22. 22. The method of claim 21, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

23. 23. The method of any one of claims 20-22, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

24. 24. The method of claim 23, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

25. 23. The method of any one of claims 20-22, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

26. 26. The method of any one of claims 20 to 25, wherein progression of the complex disorder is inhibited in the patient after exposure to one or two, three, or four of the anti-CD20 antibodies.

27. 27. The method of any one of claims 16 to 26, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

28. A method of inhibiting disability progression in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein administration results in a reduction in confirmed disability progression events, and wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

29. 29. The method of claim 28, wherein administration results in a reduction in progression of the confirmed disorder for 12 weeks.

30. 29. The method of claim 28, wherein administration results in a reduction in progression of the confirmed disorder for 24 weeks.

31. 31. The method of any one of claims 28 to 30, wherein confirmed disability progression is determined by Expanded Disability Status Scale (EDSS) score.

32. 32. The method of any one of claims 28 to 31, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

33. 33. The method of claim 32, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

34. 34. The method of claim 33, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

35. 35. The method of any one of claims 32-34, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and wherein the first and second doses are separated by about 2 weeks or about 14 days.

36. 36. The method of claim 35, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

37. 35. The method of any one of claims 32 to 34, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

38. 38. The method of any one of claims 32 to 37, wherein progression of the complex disorder is inhibited in the patient after exposure to one or two, three, or four of the anti-CD20 antibodies.

39. 39. The method of any one of claims 28 to 38, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

40. A method for delaying the onset of or reducing the risk of confirmed disability progression in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

41. 41. The method of claim 40, wherein administration results in a delay in the onset of or a reduction in the risk of confirmed progression of the disorder for 12 weeks.

42. 41. The method of claim 40, wherein administration results in a delay in the onset of or a reduction in the risk of confirmed progression of the disorder for 24 weeks.

43. 43. The method of any one of claims 40 to 42, wherein the patient has T1 gadolinium staining lesions at baseline.

44. 43. The method of any one of claims 40 to 42, wherein the patient has no T1 gadolinium staining lesions at baseline.

45. 45. The method of any one of claims 40 to 44, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

46. 46. ​​The method of claim 45, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

47. 47. The method of claim 46, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

48. 48. The method of any one of claims 45-47, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

49. 49. The method of claim 48, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

50. 48. The method of any one of claims 45 to 47, wherein the initial exposure, and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

51. 51. The method of any one of claims 40 to 50, wherein the patient has a delayed onset of confirmed disorder progression or a reduced risk of confirmed disorder progression after exposure to one, two, three, and / or four of the anti-CD20 antibodies.

52. 52. The method of claim 51, wherein the patient has a delayed onset or reduced risk of confirmed disease progression 12 weeks after exposure to one, two, three, and / or four of the anti-CD20 antibodies.

53. 52. The method of claim 51, wherein the patient has a delayed onset or reduced risk of confirmed disease progression 24 weeks after exposure to one, two, three, and / or four doses of the anti-CD20 antibody.

54. 54. The method of any one of claims 51 to 53, wherein confirmed disability progression is determined by Expanded Disability Status Scale (EDSS) score.

55. 55. The method of any one of claims 40 to 54, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

56. A method of treating a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in no observed disease activity (NEDA) for at least 12 weeks, and wherein the anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

57. 57. The method of claim 56, wherein the treatment results in no observed disease activity (NEDA) for at least 24 weeks.

58. 58. The method of any one of claims 56-57, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months.

59. 59. The method of claim 58, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

60. 60. The method of claim 59, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

61. 60. The method of any one of claims 58-59, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and wherein the first and second doses are separated by about 2 weeks or about 14 days.

62. 62. The method of claim 61 , wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

63. 60. The method of any one of claims 58-59, wherein the initial exposure, and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

64. 64. The method of any one of claims 58-63, wherein the treatment results in NEDA for at least 12 weeks after one, two, three, and / or four exposures to anti-CD20 antibodies.

65. 65. The method of any one of claims 56 to 64, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

66. A method of treating a human patient with a relapsing form of multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in the patient having no confirmed events of disability progression at 96 weeks, and wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

67. Treatment, a) Patients who are relapse-free at 96 weeks; b) Patients without T1 gadolinium-enhancing lesions at 96 weeks; c) patients with no new and / or enlarging T2 lesions at 96 weeks.

68. 68. The method of claim 66 or 67, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

69. 69. The method of claim 68, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

70. 70. The method of claim 69, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

71. 71. The method of any one of claims 68-70, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and wherein the first and second doses are separated by about 2 weeks or about 14 days.

72. 72. The method of claim 71, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

73. 71. The method of any one of claims 68-70, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

74. 74. The method of any one of claims 68-73, wherein the patient has no confirmed disease progression events at 96 weeks after exposure to one, two, three, and / or four doses of anti-CD20 antibodies.

75. 75. The method of any one of claims 66 to 74, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

76. A method of treating a human patient with highly active multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

77. 77. The method of claim 76, wherein the patient with hyperactive multiple sclerosis is a poor responder to other therapies for multiple sclerosis.

78. 78. The method of claim 77, wherein the other treatment for multiple sclerosis is interferon or glatiramer acetate.

79. 77. The method of claim 76, wherein the patient with hyperactive multiple sclerosis has not been previously treated with other therapies for multiple sclerosis.

80. 80. The method of any one of claims 76 to 79, wherein administration of an anti-CD20 antibody is effective in one or more of: (1) reducing the number of lesions in the patient's brain; (2) reducing the annualized relapse rate; (3) reducing the progression of disability; and (4) improving functional ability.

81. 81. The method of any one of claims 76 to 80, further comprising performing an MRI scan to determine whether the patient has hyperactive multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

82. 82. The method of any one of claims 76 to 81, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

83. 83. The method of claim 82, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

84. 84. The method of claim 83, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

85. 85. The method of any one of claims 82-84, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and wherein the first and second doses are separated by about 2 weeks or about 14 days.

86. 86. The method of claim 85, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

87. 85. The method of any one of claims 82 to 84, wherein the initial exposure, and the second, third, and / or fourth additional exposures, comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

88. 88. The method of any one of claims 82 to 87, wherein the patient has (1) a reduction in the number of lesions in the patient's brain; (2) a reduction in the annualized relapse rate; (3) a reduction in the progression of disability; and / or (4) an improvement in functional ability after exposure to one, two, three, and / or four anti-CD20 antibodies.

89. 89. The method of any one of claims 76 to 88, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

90. A method of treating a human patient with early stage multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

91. 91. The method of claim 90, further comprising diagnosing the patient as having early stage multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

92. 92. The method of any one of claims 90-91, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20-24 weeks or about 5-6 months.

93. 93. The method of claim 92, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

94. 94. The method of claim 93, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

95. 95. The method of any one of claims 92-94, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and wherein the first and second doses are separated by about 2 weeks or about 14 days.

96. 96. The method of claim 95, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

97. 95. The method of any one of claims 92 to 94, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 14 days.

98. 98. The method of any one of claims 90 to 97, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

99. A method of treating a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in no observed disease activity (NEDA) in the patient, and wherein the anti-CD20 antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

100. 100. The method of claim 99, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

101. 101. The method of claim 100, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

102. 102. The method of claim 101, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

103. 102. The method of any one of claims 100-101, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

104. 104. The method of claim 103, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

105. 102. The method of any one of claims 100 to 101, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

106. 106. The method of any one of claims 100 to 105, wherein the treatment results in NEDA after one, two, three, and / or four exposures to anti-CD20 antibodies.

107. 107. The method of any one of claims 99 to 106, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

108. 1. A method of treating a human patient with a relapsing form of multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment comprises: a) a reduction in annualized relapse rate of at least about 30%; b) at least about a 30% reduction in the risk of progression of confirmed disability for at least 12 weeks; c) at least about a 30% reduction in the risk of confirmed disability progression for at least 24 weeks; d) T1 gadolinium ＋ a reduction in the number of lesions by at least about 90%; e) T1 gadolinium at 24, 48, and / or 96 weeks ＋ a reduction of at least about 90% in the average number of lesions; f) at least about a 70% reduction in the number of new and / or enlarging T2 hyperintense lesions; g) at least about a 40% reduction in the mean number of new and / or enlarging T2 hyperintense lesions at Weeks 24, 48, and / or 96; h) a reduction of at least about 10% in the rate of total brain volume loss; i) at least about a 10% improvement in confirmed disability improvement sustained for at least 12 weeks; j) at least about a 50% reduction in the number of new T1 hypointense lesions; and k) at least about a 55% improvement in NEDA (No Disease Activity); wherein the reduction or improvement in a) through k) is compared to patient(s) receiving treatment with interferon beta-1a, and the anti-CD20 antibody comprises 1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and 2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

109. 109. The method of claim 108, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

110. 110. The method of claim 109, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

111. 111. The method of claim 110, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

112. 112. The method of any one of claims 109-111, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

113. 113. The method of claim 112, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

114. 112. The method of any one of claims 109-111, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

115. 115. The method of any one of claims 109 to 114, wherein the treatment results in one or more of a) through k) after one, two, three, and / or four exposures of anti-CD20 antibodies.

116. 116. The method of any one of claims 108 to 115, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

117. A method of treating a human patient with primary progressive multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment a) at least about a 15% reduction in the risk of confirmed disability progression for at least 12 weeks compared to a patient(s) not receiving treatment; b) at least about a 15% reduction in the risk of confirmed disability progression for at least 24 weeks compared to untreated patient(s); c) at least about a 15% decrease in the rate of increase in walking time as measured by the Time 25-Foot Walk compared to untreated patient(s); d) at least about a 10% reduction in T2 lesion volume compared to untreated patient(s); e) a reduction in T2 lesion volume of at least about 3% from baseline to week 24; and f) at least about a 12% decrease in the rate of total brain volume loss compared to a patient(s) not receiving treatment; wherein the anti-CD20 antibody comprises 1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and 2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

118. 118. The method of claim 117, wherein the anti-CD20 antibody is administered to the patient to provide a first anti-CD20 antibody exposure followed by a second anti-CD20 antibody exposure, wherein the first and second exposures are each about 600 mg of antibody, and the interval between the first and second exposures is about 20 to 24 weeks or about 5 to 6 months.

119. 119. The method of claim 118, wherein an anti-CD20 antibody is administered to the patient to provide a third anti-CD20 antibody exposure, wherein the third exposure is about 600 mg of antibody, and the interval between the second and third exposures is about 20 to 24 weeks or about 5 to 6 months.

120. 120. The method of claim 119, wherein the anti-CD20 antibody is administered to the patient to provide a fourth anti-CD20 antibody exposure, wherein the fourth exposure is about 600 mg of antibody, and the interval between the third and fourth exposures is about 20 to 24 weeks or about 5 to 6 months.

121. 121. The method of any one of claims 118-120, wherein the first exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

122. 122. The method of claim 121, wherein the second, third, and / or fourth exposure comprises a single dose of about 600 mg.

123. 121. The method of any one of claims 118-120, wherein the initial exposure and the second, third, and / or fourth additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

124. 124. The method of any one of claims 118 to 123, wherein the treatment results in one or more of a) through f) after one, two, three, and / or four exposures of anti-CD20 antibodies.

125. 125. The method of any one of claims 117 to 124, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 26, and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

126. 126. The method of any one of claims 1 to 125, wherein the patient maintains the ability to mount a humoral immune response to the antigen during treatment.

127. 127. The method of claim 126, wherein the antigen is a mumps antigen, rubella antigen, varicella antigen, Streptococcus pneumoniae antigen, tetanus toxoid antigen, pneumococcal antigen, or influenza antigen.

128. 128. The method of any one of claims 1 to 127, wherein the anti-CD20 antibody is the first medicament and the second medicament is administered with the initial exposure or a subsequent exposure.

129. 129. The method of any one of claims 1 to 116 and 126 to 128, wherein the multiple sclerosis is a relapsing form of multiple sclerosis.

130. 130. The method of claim 129, wherein the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).

131. 130. The method of claim 129, wherein the relapsing form of multiple sclerosis is secondary progressive multiple sclerosis with overlapping relapses (rSPMS).

132. 129. The method of any one of claims 1 to 65 and 76 to 128, wherein the multiple sclerosis is progressive multiple sclerosis.

133. 133. The method of claim 132, wherein the multiple sclerosis is primary progressive multiple sclerosis (PPMS).

134. 120. The method of any one of claims 10, 22, 34, 47, 60, 70, 84, 94, 102, 111, and 120, wherein the anti-CD20 antibody is administered to the patient to provide one or more additional anti-CD20 antibody exposures after the fourth exposure, wherein the one or more additional exposures after the fourth exposure are about 600 mg of antibody, and the interval between each exposure is about 20 to 24 weeks or about 5 to 6 months.

135. 135. The method of claim 134, wherein the one or more additional anti-CD20 antibody exposures after the fourth exposure comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is about 300 mg, and the first and second doses are separated by about 2 weeks or about 14 days.

136. 135. The method of claim 134, wherein the one or more additional anti-CD20 antibody exposures after the fourth exposure comprise a single dose of about 600 mg.

137. 137. The method of any one of claims 1 to 136, wherein the anti-CD20 antibody is ocrelizumab.

138. 138. The method of any one of claims 1 to 137, wherein the anti-CD20 antibody is an antigen-binding fragment thereof.

139. 139. The method of any one of claims 1 to 138, wherein the anti-CD20 antibody is present in a pharmaceutically acceptable composition.

140. 140. The method of any one of claims 1 to 139, wherein the anti-CD20 antibody is administered intravenously.

141. 141. The method of claim 140, wherein the anti-CD20 antibody is administered intravenously for each antibody exposure.

142. 140. The method of any one of claims 1 to 139, wherein the antibody is administered subcutaneously.

143. 143. The method of claim 142, wherein the anti-CD20 antibody is administered subcutaneously for each antibody exposure.

144. 144. An anti-CD20 antibody for use in accordance with the method of any one of claims 1 to 143, wherein the anti-CD20 antibody comprises 1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8, and 2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

2.

145. 145. An article of manufacture comprising: (a) a container containing ocrelizumab; and (b) a package insert containing instructions for treating multiple sclerosis in a patient described in any one of claims 1 to 144.

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