RU2757961C2 - Method for treating multiple sclerosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a method for improving functional capacity in a patient having multiple sclerosis, comprising administering to the patient an effective amount of anti-CD20 antibody, wherein the patient has improved functional capacity after administration and where the functional capacity improvement is maintained for at least 12 weeks; where anti-CD20 antibody contains: a) a heavy chain variable region comprising CDR1 having the amino acid sequence SEQ ID NO: 10, CDR2, having amino acid sequence SEQ ID NO: 11, and CDR3 having the amino acid sequence SEQ ID NO: 12, and b) a light chain variable region containing CDR1 having the amino acid sequence SEQ ID NO: 4, CDR2 having the amino acid sequence SEQ ID NO: 5, and CDR3, having amino acid sequence SEQ ID NO: 6. Group of inventions also relates to a method of reducing the average annual recurrence rate in a human patient having multiple sclerosis; kit for treating multiple sclerosis in a patient in accordance with said method.

EFFECT: group of inventions provides improved functional capacity in the patient suffering multiple sclerosis.

58 cl, 34 dwg, 14 tbl, 2 ex

Description

FIELD OF THE INVENTION

[0001] The present invention relates to methods for treating multiple sclerosis (MS) in a patient and to a device with instructions for such use.

BACKGROUND OF THE INVENTION

[0002] Multiple sclerosis (MS) is an inflammatory and demyelinating degenerative disease of the human central nervous system (CNS). It is a common disease worldwide, affecting approximately 300,000 people in the United States; it is a disease of young adults, with 70% to 80% of onset 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 disorder based on clinical course, magnetic resonance imaging (MRI) assessment and pathology analysis of biopsy and sectional material (Lucchinetti et al. Ann Neurol 47: 707-17 (2000)). The disease manifests itself in a large number of possible combinations of disorders, including syndromes of the spinal cord, brainstem, cranial nerves, cerebellum, cerebral hemispheres, and cognitive syndromes. Progressive disability is the fate of most MS patients, especially over the 25-year horizon. Half of MS patients require a walking stick within 15 years of onset. MS is a leading cause of neurological disability in young adults and middle-aged people and had no known beneficial treatments until the last decade. MS is difficult to diagnose due to nonspecific clinical findings, which has led to the development of highly structured diagnostic criteria that include several technological advances consisting of MRI scans, evoked potential studies, and cerebrospinal fluid (CSF) studies. All diagnostic criteria are based on the general principles of disseminated plaques in the central white matter, appearing at different times and unexplained by other etiological factors such as infection, vascular disorder or autoimmune disorder (McDonald et al. Ann Neurol 50: 121-7 (2001)) ... MS has four forms of the disease: relapsing-remitting MS (RRMS; 80% -85% of cases upon onset), primary progressive MS (PPMS; 10% -15% upon onset), progressive-relapsing MS (PRMS; 5% upon onset ); 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)). It is estimated that 50% of RRMS patients develop SPMS within 10 years and up to 90% of RRMS patients eventually develop SPMS (Weinshenker et al. Brain 112 (Pt 1): 133-46 (1989)).

[0003] In the United States, several disease-modifying drugs belonging to five classes are approved for the treatment of RRMS, while there are no approved drugs for PPMS. Methods RRMS treatments include the following: interferon class, ^{IFN-beta-1a (REBIF ®, Extavia,} AVONEX ® and PLEGRIDY ™), and IFN-beta-1b (BETASERON ^®); glatiramer acetate (COPAXONE ^®), polypeptide; natalizumab (TYSABRI ^®), alemtuzumab (LEMTRADA ^®, both are monoclonal antibodies); dimethylfumarate (TECFIDERA ^®) and fingolimod (GILENYA ^®), both of which are small molecule agents, and mitoxantrone (NOVANTRON ^®), a cytotoxic agent; teriflunomide (AUBAGIO ^®). Other drugs for disease modification have been used with varying degrees of success, including methotrexate, cyclophosphamide, azathioprine, and intravenous (IV) immunoglobulin. Current therapies are not effective enough to treat 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 therapies with an acceptable benefit-risk profile that can be delivered early in the course of the disease to reduce the long-term effects of increased disability and improve the quality of life of patients.

[0004] All references cited herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

[0005] Within the scope of the invention, there are provided methods for treating multiple sclerosis in a human patient comprising administering an effective amount of an anti-CD20 antibody. Any of the anti-CD20 antibodies described herein can be administered. In some embodiments, an 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.

[0006] In one aspect, the present invention provides a method of improving functional ability in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient has an improvement in functional ability 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, the patient has a confirmed reduction in disability within 12 weeks. In some embodiments, the patient has a confirmed reduction in disability at 24 weeks after treatment. In some embodiments, the implementation of the improvement in functional ability in the patient is maintained for at least 12 weeks. In some embodiments, the implementation of the improvement in functional ability in the patient is maintained for at least 24 weeks. In some embodiments, improvement in functional ability is measured by a 25 foot time walk test (T-25FW) or EDSS score.

[0007] In some embodiments, the patient has gadolinium stained T1 plaques at baseline. In some embodiments, the patient does not have gadolinium staining T1 plaques at baseline.

[0008] In another aspect, the present invention provides a method of suppressing the progression of disability 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 decrease in confirmed cases of progression of disability, and 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 decrease in the confirmed 12-week progression of disability. In some embodiments, administration results in a decrease in the confirmed 24 week progression of disability. In some embodiments, confirmed progression of disability is determined by an Extended Disability Assessment Scale (EDSS) score.

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

[0010] In another aspect, the present invention provides a method of slowing the onset of confirmed progression of disability or reducing the risk of confirmed progression of disability 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) variable a heavy chain region containing 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 containing CDR1, having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. disability progression. In some embodiments, administration results in a delay in the onset or reduction in the risk of a confirmed 24 week progression of disability.

[0011] In some embodiments, the patient has gadolinium stained T1 plaques at baseline. In some embodiments, the patient does not have gadolinium staining T1 plaques at baseline.

[0012] Within the scope of the present invention, there is provided a method for reducing the volume of T2 plaques 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 containing CDR1 having an amino acid a 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, 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 gadolinium stained T1 plaques at baseline. In certain embodiments, the patient does not have gadolinium staining T1 plaques at baseline.

[0013] In another aspect, the present invention provides a method for slowing or preventing a decrease in brain volume in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the decrease in brain volume is slowed down or prevented in the patient; and 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, further volume reduction is prevented brain in a patient who has a decrease in brain volume.

[0014] Also within the scope of the present invention, there is provided a method of reducing brain atrophy in a human patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the brain atrophy is slowed down or prevented, wherein the anti-CD20 antibody comprises: a) a severe variable region a chain containing 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 containing CDR1 having the amino acid 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 is prevented in a patient who has brain atrophy.

[0015] Within the scope of the present invention, there is provided 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 evidence of disease activity (NEDA) for at least 12 weeks, 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, treatment results in no evidence of disease activity (NEDA) for at least 24 weeks.

[0016] Also within the scope of the present invention is 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 the patient achieving a plaque-free status after treatment for 96 weeks, 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, treatment results in the patient achieving a plaque-free status after 48 weeks of treatment. In certain embodiments, the treatment results in the patient achieving a plaque-free status after 24 weeks of treatment. In certain embodiments, the treatment results in the patient being free of gadolinium staining plaques. In certain embodiments, the treatment results in the patient being free of T2 plaques.

[0017] Within the scope of the present invention, there is provided a method of treating a human patient with recurrent multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in one or more of:

a) the patient is free of relapse after 96 weeks;

b) the patient has no confirmed progression of disability after 96 weeks;

c) the patient has no gadolinium-enhanced T1 plaques at 96 weeks;

d) the patient has no new and / or increasing T2 plaques 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.

[0018] In another aspect, the present invention provides 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 containing CDR1 having the amino acid sequence 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, 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 highly active multiple sclerosis patient has an inadequate response to another multiple sclerosis therapy. In some embodiments, the highly active multiple sclerosis patient has not previously been treated with another multiple sclerosis therapy. In some embodiments, another method of treating multiple sclerosis is therapy with interferon or glatiramer acetate. In some embodiments, administration of an anti-CD20 antibody is effective against one or more of the following: (1) reducing the amount of plaque in the patient's brain; (2) a decrease in the average annual relapse rate; (3) reducing the progression of disability; and (4) increased functional ability. In some embodiments, the method further comprises performing an MRI scan and determining whether the patient has highly active multiple sclerosis prior to administering the anti-CD20 antibody to the patient.

[0019] In another aspect, the present invention provides 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 containing CDR1 having an amino acid sequence 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 containing 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 method further comprises diagnosing a patient having early stage multiple sclerosis prior to administering an anti-CD20 antibody to the patient. In some embodiments, the patient is diagnosed with multiple sclerosis but has not been treated for at least two years prior to administration of the anti-CD20 antibody.

[0020] In another aspect, the present invention provides 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 evidence of disease activity (NEDA) in the patient, and 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 protocol detectable relapses, no events CDP, no new or increasing T2 plaques, and no whiskers of injected Gd T1 plaques.

[0021] In certain embodiments, treatment or administration of an anti-CD20 antibody to a patient with recurrent multiple sclerosis results in one or more of the following:

a) approximately 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 average annual relapse rate (eg, over a period of at least about 1 year, 1.5 years, or two years) compared to the patient (s) receiving a treatment with interferon beta-1a (such as REBIF ^®), including any range between these values;

b) approximately any of 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 progression of disability for at least 12 weeks compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®) , including any range between these values;

c) approximately any of 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 progression of disability for at least 24 weeks compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®) , including any range between these values;

d) approximately any of a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction in the total number of gadolinium ⁺ T1 plaques compared to the patient (s), being treated with interferon ^{beta-1a (such as REBIF ®)} , including any range between these values;

e) approximately any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% reduction in mean gadolinium ⁺ T1 plaque counts at 24 weeks, 48 weeks, and / or 96 weeks compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®), including any range between these values;

f) approximately any of 70%, 71%, 72% 73% 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85% reducing the number of new and / or enlarging T2-hyperintense plaques compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®), including any range between these values;

g) approximately any of 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%, 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% decrease in the average number of new and / or increasing hyperintense T2 plaques through 24 weeks, 48 weeks, and / or 96 weeks compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®), including any range between these values;

h) approximately any of 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% reduction in the rate of decrease in the volume of the whole brain compared with the patient ( s) that is not being treated, including any range between these values;

i) approximately any of 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%, or 65% reduction in proven reduction in disability persisting for at least 12 weeks compared to patient (s) treated with interferon beta-1a ( such as REBIF ^®), including any range between these values;

j) approximately any of 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 hypointense T1 plaques compared to the patient (s) being treated interferon beta-1a (such as REBIF ^®), including any range between these values; and

k) approximately any of 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 signs of disease activity),

wherein NEDA defined as the absence of defined relapse protocol, no CDP events, no new or enlarging T2-plaque and absence of amplified Gd T1-plaques compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®), including any range between these values.

[0022] In certain embodiments, treatment additionally or alternatively may result in about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21% , 22%, 23%, 24% or 25% reduction in brain atrophy as compared with the patient (s) being treated with interferon beta-1a (such as REBIF ^®), including any range between these values.

[0023] In certain embodiments, treatment or administration of an anti-CD20 antibody to a patient with primary progressive multiple sclerosis results in one or more of the following:

a) approximately any of 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% reduction in risk of confirmed progression of disability for at least 12 weeks compared to untreated patient (s), including any range between these values;

b) approximately any of 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% reduction in risk of confirmed progression of disability for at least 24 weeks compared to untreated patient (s), including any range between these values;

c) approximately any of 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% reduction in the rate of progression of deterioration in walking time when measured using 25 feet of walking time compared to untreated patient (s), including any range between these values;

d) approximately any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% reduction T2 plaque volume compared to untreated patient (s), including any range between these values;

e) approximately any of 1%, 1.5%, 2%, 2.5%, 3%, 3.4%, 3.5%, 4%, 4.5%, or 5% reduction in hyperintense T2 plaque volume from baseline at approximately 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 44 weeks, 48 weeks, 52 weeks, 56 weeks, 60 weeks, 64 weeks, 68 weeks, 72 weeks, 76 weeks, Week 80, Week 84, Week 88, Week 92, Week 96, Week 100, Week 104, Week 108, Week 112, Week 116, and Week 120; and

f) approximately any of 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 in the rate of decrease in total brain volume compared to untreated patient (s), including any range between these values.

[0024] In certain embodiments of any of the methods, the patient retains the ability to induce a humoral response to an antigen during treatment. In certain embodiments, the antigen is mumps antigen, rubella antigen, chickenpox antigen, S. pneumonia antigen, tetanus toxoid antigen, pneumococcal antigen, and influenza antigen.

[0025] In some embodiments of the methods described above and herein, upon initial exposure or subsequent exposures, a second drug is administered, wherein the anti-CD20 antibody is the first drug. In some embodiments, the second drug is selected from the group consisting of interferon, glatiramer acetate, cytotoxic agent, chemotherapeutic agent, mitoxantrone, methotrexate, cyclophosphamide, chlorambucil, azathioprine, gamma globulin, campata, anti-CD4 antibody, cladribine, corticolate mofetila (MMF), cyclosporin, a cholesterol-lowering drug of the statin class, estradiol, testosterone, hormone replacement drug, TNF inhibitor, disease-modifying antirheumatic drug (DMARD), non-steroidal anti-inflammatory drug (NSAID), levothyroxine, cyclosporin A analogue , antagonist of a cytokine or cytokine receptor, antimetabolite, immunosuppressive agent, antagonist of integrins or antibodies against them, antibodies against LFA-1, efalizumab, antibodies against alpha-4-integrin, natalizumab and other antibodies against a surface marker spines of B cells.

[0026] In some embodiments of the methods described above and herein, multiple sclerosis is a recurrent form of multiple sclerosis. In some embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the recurrent form of multiple sclerosis is recurrent secondary progressive multiple sclerosis (rSPMS). In some embodiments, the multiple sclerosis is progressive multiple sclerosis. In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis (PPMS).

[0027] In some embodiments of the methods described above and herein, an anti-CD20 antibody is administered to a patient to provide an initial exposure to an anti-CD20 antibody followed by one or more exposures of an anti-CD20 antibody, where each exposure is about 600 mg of the antibody and each exposure provide the patient as one or two doses of anti-CD20 antibody, and wherein the interval between exposures is about 24 weeks or about 6 months. In some embodiments, the initial exposure comprises a first dose and a second dose of anti-CD20 antibody, where each dose is approximately 300 mg and the interval between the first dose and the second dose is approximately 14 days. In some embodiments, the additional exposure comprises a single dose of about 600 mg. In some embodiments, the initial exposure and one or more side effects comprise a first dose and a second dose of anti-CD20 antibody, where each dose is approximately 300 mg and the interval between the first dose and the second dose is approximately 14 days.

[0028] 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 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 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.

[0029] 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 at each exposure to the antibody. In some embodiments of the methods described herein above, the antibody is administered subcutaneously. In some embodiments, the anti-CD20 antibody is administered subcutaneously at each exposure to the antibody.

[0030] In some embodiments, the implementation of the present invention provides a composition comprising an antibody against CD20, which 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 SEQ ID NO: 6, for use in the treatment of multiple sclerosis in a patient according to any of the methods described above.

[0031] In some embodiments, the present invention provides an anti-CD20 antibody that 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, for use in the manufacture of a medicament for the treatment of multiple sclerosis in a patient according to any of the methods described above.

[0032] In certain embodiments of any of the methods described above and / or in the present description, the decrease, or decrease, or improvement after administration of the anti-CD20 antibody can be compared with the baseline level, with the level in the patient (s) without treatment and / or with the level in a patient (s), e.g., such as average, weighted average or median level in the group of patients undergoing different treatments (such as interferon beta-1a or REBIF ^®).

[0033] In another aspect, the present invention provides an article of manufacture comprising: (a) a container containing an anti-CD20 antibody (eg, ocrelizumab); and (b) a package insert with instructions for treating multiple sclerosis in a patient according to any of the methods described above and herein.

[0034] It should be understood that one, some or all of the properties of the various embodiments described herein can be combined to provide other embodiments of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1A shows a sequence alignment comparing the amino acid sequences of the light chain variable domain (V _L ) of each of mouse 2H7 (SEQ ID NO: 1), humanized 2H7.v16 variant (SEQ ID NO: 2) and kappa light chain human subgroup I (SEQ ID NO: 3). CDR V _L 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 6).

[0036] Figure 1B shows a sequence alignment comparing the amino acid sequences of the heavy chain variable domain (V _H ) of each of the mouse 2H7 (SEQ ID NO: 7), the humanized 2H7.v16 variant (SEQ ID NO: 8), and the consensus sequence of the heavy human subgroup III chains (SEQ ID NO: 9). CDR V _H 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12).

[0037] In Figures 1A and 1B, CDR1, CDR2, and CDR3 in each strand are bracketed and flanked by framework regions FR1-FR4 as indicated. 2H7 refers to the mouse 2H7 antibody. Asterisks between two rows of sequences indicate a position that differs between the two sequences. The residues are numbered according to Kabat et al. Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions shown as a, b, c, d and e.

[0038] Figure 2 shows the amino acid sequence of the mature light chain 2H7.v16 (SEQ ID NO: 13).

[0039] Figure 3 shows the amino acid sequence of the mature heavy chain 2H7.v16 (SEQ ID NO: 14).

[0040] Figure 4 shows the amino acid sequence of the mature heavy chain 2H7.v31 (SEQ ID NO: 15). The L-chain of 2H7.v31 is the same as that of 2H7.v16.

[0041] Figure 5 shows the alignment of mature light chains 2H7.v16 and 2H7.v511 (SEQ ID NOs. 13 and 16, respectively) with variable domain residue numbering according to Kabat and constant domain residue numbering according to Eu.

[0042] Figure 6 shows the alignment of mature heavy chains 2H7.v16 and 2H7.v511 (SEQ ID NO. 14 and 17, respectively) with variable domain numbering according to Kabat and constant domain numbering according to Eu.

[0043] Figure 7 provides an overview of the test circuit for testing two identical phase III (i.e., Test I and Test II) for ocrelizumab as compared to interferon beta-1a (REBIF ^®) in patients with relapsing multiple sclerosis.

[0044] Figure 8 shows a hierarchical statistical analysis plan for Test I and Test II.

[0045] Figure 9 shows the distribution of patients in Trial I and Trial II.

[0046] Figure 10A shows the mean annual relapse rate (ARR) at 96 weeks in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial I.

[0047] Figure 10B shows the mean annual relapse rate (ARR) at 96 weeks for patients treated with ocrelizumab versus patients treated with interferon beta-1a in trial II.

[0048] Figure 11 shows the time to onset of confirmed disability progression (CDP) for at least 12 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a (pooled data tests I and tests II).

[0049] Figure 12A shows the time to confirmed progression of disability for at least 12 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a in trial I.

[0050] FIG. 12B depicts the time to confirmed progression of disability for at least 12 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a in trial II.

[0051] FIG. 13 shows the time to onset of confirmed progression of disability for at least 24 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a (data pooled from Trial I and tests II).

[0052] Figure 14A shows the time to confirmed progression of disability for at least 24 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a in trial I.

[0053] FIG. 14B depicts the time to confirmed progression of disability for at least 24 weeks over a 96 week period in patients treated with ocrelizumab versus patients treated with interferon beta-1a in trial II.

[0054] Figure 15 depicts the proportion of patients (baseline EDSS of 2.0 or less) with a confirmed reduction in disability (ie, CDI) for at least 12 weeks in ocrelizumab-treated patients compared to patients injected with interferon beta-1a (data pooled from trial I and trial II).

[0055] Figure 16A shows the proportion of patients (baseline EDSS of 2.0 or less) with a confirmed reduction in disability (ie, CDI) for at least 12 weeks in ocrelizumab-treated patients compared to patients injected with interferon beta-1a in trial I.

[0056] Figure 16B depicts the proportion of patients (baseline EDSS of 2.0 or less) with a confirmed reduction in disability (ie, CDI) for at least 12 weeks in ocrelizumab-treated patients compared to patients injected with interferon beta-1a in trial II.

[0057] Figure 16C depicts the change in the multiple sclerosis composite functional score from baseline to week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial I.

[0058] Figure 16D depicts the change in multiple sclerosis complex functional assessment from baseline to week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial II.

[0059] Figure 17A shows the total number of gadolinium-enhanced T1 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial I.

[0060] Figure 17B shows the total number of gadolinium-enhanced T1 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial II.

[0061] Figure 18A shows the average total number of gadolinium-enhanced T1 plaques in ocrelizumab-treated patients at week 24, week 48, and at week 96 compared to IFNβ-1a-treated patients in trial I.

[0062] Figure 18B shows the average total number of gadolinium-enhanced T1 plaques in ocrelizumab-treated patients at week 24, week 48 and at week 96 compared to IFNβ-1a-treated patients in trial II.

[0063] Figure 19A shows the total number of new and / or increasing hyperintense T2 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients. test I and test II.

[0064] Figure 19B depicts the total number of new and / or increasing hyperintense T2 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients. test II.

[0065] Figure 19C shows the mean number of new and / or increasing hyperintense T2 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients. test I.

[0066] Figure 19D shows the mean number of new and / or increasing hyperintense T2 plaques found at week 24, week 48, and week 96 in ocrelizumab-treated patients versus interferon beta-1a-treated patients in test II.

[0067] Figure 20A shows the rate of decrease in brain volume from week 24 to week 96 in ocrelizumab-treated patients compared to interferon beta-1a-treated patients in trial I.

[0068] Figure 20B shows the rate of decrease in brain volume from week 24 to week 96 in ocrelizumab-treated patients compared to interferon beta-1a patients in trial II.

[0069] Figure 21A shows the rate of decrease in brain volume from baseline to week 96 in ocrelizumab-treated patients compared to interferon beta-1a-treated patients in trial I.

[0070] Figure 21B shows the rate of decrease in brain volume from baseline to week 96 in ocrelizumab-treated patients compared to interferon beta-1a-treated patients in trial II.

[0071] Figure 22A shows the total number of new hypointense T1 plaques on MRI at 24 weeks, 48 weeks and 96 weeks in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial I ...

[0072] Figure 22B shows the total number of new hypointense T1 plaques on MRI at 24 weeks, 48 weeks and 96 weeks in ocrelizumab-treated patients versus interferon beta-1a-treated patients in trial II ...

[0073] Figure 23A shows infusion-related responses in IFNβ-1a-treated patients in Test I and Test II (pooled).

[0074] Figure 23B depicts infusion-mediated responses in ocrelizumab-treated patients in trial I and trial II (pooled).

[0075] Figure 24 is a test design for a phase III trial for ocrelizumab in patients with primary progressive multiple sclerosis (PPMS).

[0076] Figure 25 shows the statistical hierarchy of a Phase III trial for ocrelizumab in PPMS patients.

[0077] Figure 26 shows the distribution of patients at the clinical discontinuation date of the Phase III PPMS trial.

[0078] Figure 27 shows the time to onset of confirmed progression of disability for at least 12 weeks in ocrelizumab-treated patients versus placebo-treated patients for a Phase III PPMS trial.

[0079] Figure 28 depicts the time to onset of confirmed progression of disability for at least 24 weeks in ocrelizumab-treated patients versus placebo-treated patients for a Phase III PPMS trial.

[0080] FIG. 29 depicts the rate of decline in walking speed as measured in a 25 feet timed gait test in patients treated with 600 mg ocrelizumab versus placebo patients, from baseline to week 120 for the PPMS trial. phase III.

[0081] Figure 30 depicts the rate of decrease in walking speed at 120 weeks from baseline in patients treated with 600 mg ocrelizumab versus placebo in a Phase III PPMS trial.

[0082] Figure 31 depicts the percent change in total brain volume from week 24 to week 96 in patients treated with 600 mg ocrelizumab versus placebo patients for a Phase III PPMS trial.

[0083] Figure 32 depicts the change in T2 plaque volume in patients treated with 600 mg ocrelizumab versus placebo patients for a Phase III PPMS trial.

[0084] Figure 33 depicts infusion-mediated responses (IRRs) in patients treated with 600 mg ocrelizumab versus placebo-treated patients for exposure and severity to date of clinical discontinuation in a Phase III PPMS trial.

[0085] Figure 34A shows the proportion of patients with no signs of disease activity (NEDA) treated with IFNβ-1a compared to the proportion of patients with NEDA treated with ocrelizumab in trial I.

[0086] FIG. 34B shows the proportion of patients without signs of disease activity (NEDA) treated with IFNβ-1a compared to the proportion of patients with NEDA treated with ocrelizumab in trial II.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

[0087] A "B cell" is a lymphocyte that matures in the bone marrow and includes a naive B cell, a memory B cell, or an effector B cell (plasma cells). A B cell in the context of the present invention can be a normal or non-cancerous B cell.

[0088] A "B-cell surface marker" or "B-cell surface antigen" in the context of the present invention is an antigen expressed on the surface of a B-cell, which can be targeted by an antibody that binds thereto. Exemplary B cell surface markers include leukocyte surface markers 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 (see for description. The Leukocyte Antigen Facts Book, 2 ^nd 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, BCMA and FcRH6 239287. Within the scope of the present invention, a B cell surface marker of particular interest is preferably expressed on B cells over other mammalian B cell tissues, and can be expressed on both B progenitor cells and mature B cells. The preferred B-cell surface marker in the context of the present invention is CD20.

[0089] The antigen "CD20" 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 B cells and malignant B cells, but it is not expressed on stem cells. Other names for CD20 in the literature include "B-lymphacyte-limited antigen" and "Bp35". The CD20 antigen is described, for example, in Clark et al. Proc. Natl. Acad. Sci. (USA) 82: 1766 (1985).

[0090] An "antagonist antibody" in the context of the present invention is an antibody that, when bound to a B cell surface marker on B cells, destroys or depletes B cells in a mammal and / or interferes with one or more B cell functions, for example , by reducing or preventing the humoral response induced by the B cell. The antagonist antibody is preferably capable of depleting B cells (ie, reducing circulating B cell levels) in a mammal that has been treated with them. Such depletion can be achieved through various mechanisms such as 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., through apoptosis).

[0091] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on the target cell, and then lysis of the target cell is performed. The main cells for mediating ADCC - NK cells - express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464 Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay such as described in US Pat. No. 5,500,362 or 5,821,337 can be performed. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al, Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998).

[0092] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; with PBMC and NK cells being preferred.

[0093] 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. Moreover, a preferred FcR is an FcR that binds an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-binding activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB in its cytoplasmic domain contains an immunoreceptor tyrosine-binding inhibitory motif (ITIM), (see Daëron, 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). The term "FcR" as used herein refers to other FcRs, including FcRs, that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J. Immunol. 117: 587 (1976) and Kim et al, J. Immunol. 24: 249 (1994)).

[0094] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation cascade is initiated by the binding of the first component of the complement system (C1q) to a molecule (eg, a polypeptide (eg, an antibody)) in complex with an antigen recognized by it. 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).

[0095] "Growth inhibitory" antibodies are antibodies that prevent or reduce the proliferation of a cell expressing the antigen to which the antibody binds. For example, an antibody can prevent or reduce B cell proliferation in vitro and / or in vivo.

[0096] Antibodies that "induce apoptosis" are antibodies that induce programmed cell death, eg, B cells, as determined by standard apoptosis assays such as annexin V binding, DNA fragmentation, cell shrinkage, endoplasmic reticulum dilation, cell fragmentation, and / or the formation of membrane vesicles (called apoptotic bodies).

[0097] The term "antibody" in the framework of the present invention is used in its broadest sense and, in particular, it includes monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, and antibody fragments provided that they exhibit the desired biological activity.

[0098] "Antibody fragments" comprise a portion of an intact antibody, preferably containing an antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') _2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and polyspecific antibodies formed by antibody fragments.

[0099] For the purposes of the present description, "intact antibody" is an antibody containing the variable domains of the heavy and light chains, as well as an Fc region.

[0100] "Native antibodies" are usually heterotetrameric glycoproteins weighing approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced interchain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V _L ) and a constant domain at the other end; the constant domain of the light chain is parallel to the first constant domain of the heavy chain, and the variable domain of the light chain is parallel to the variable domain of the heavy chain. It is believed that particular amino acid residues form a contact surface between the variable domains of the light chain and the heavy chain.

[0101] The term "variable" refers to the fact that the sequences of certain segments of the variable domains differ significantly among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniform throughout the region of the variable domains of antibodies. It concentrates in three segments, called hypervariable regions, in the variable domains of both the light chain and the heavy chain. The more highly conserved portions of the variable domains are called framework regions (FRs). Each of the variable domains of native heavy and light chains contains four FRs, mainly conforming to the β-sheet configuration, connected by three hypervariable regions, which form loops that unite the β-sheet structure, and, in some cases, form part of it. The hypervariable regions in each chain are located together in close proximity to the FR and, together with the hypervariable regions of the other chain, participate in the formation of the antigen-binding center of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in antibody-antigen binding, but they exhibit various effector functions such as the involvement of antibodies in antibody-dependent cellular cytotoxicity (ADCC).

[0102] The cleavage of antibodies with papain provides two identical antigen-binding fragments, called "Fab" fragments, each of which has one antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to rapidly crystallize. Pepsin treatment provides an F (ab ') ₂ fragment that has two antigen binding sites and is still capable of binding antigen.

[0103] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer consisting of one heavy chain variable domain and one light chain variable domain linked by a strong non-covalent bond. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Collectively, the six hypervariable regions confer antigen-binding capacity to the antibody. However, even one 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 lower affinity than the entire binding site.

[0104] the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the C-terminus _{of the heavy chain C H} 1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH is used herein for Fab 'in which the cysteine residue (s) of the constant domains have a free thiol group. F (ab ') ₂ antibody fragments were originally prepared as pairs of Fab' fragments that have hinge cysteine residues between them. Other methods of chemically linking antibody fragments are also known.

[0105] "Light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two different types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0106] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further subdivided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of various classes of immunoglobulins are well known.

[0107] "Single chain Fv" or antibody fragments "scFv" contain the V _H and V _L domains of an antibody, where these domains are present as a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains that allows the formation of the scFv structure required for antigen binding. For an overview of scFv, see Pliickthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0108] The term "diabodies" refers to small antibody fragments with two antigen binding regions that contain a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) in the same polypeptide chain (V _H -V _L ). By using a linker that is too short to allow the pairing of two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger et ah, Proc. Natl. Acad. Set USA 90: 6444-6448 (1993).

[0109] The term "monoclonal antibody", as used herein, refers to an antibody prepared from a collection of substantially homogeneous antibodies, i. E. the individual antibodies constituting the population are identical and / or bind the same epitope, with the exception of possible variants that may arise during the preparation of the monoclonal antibody, which are mainly present in small quantities. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they generally do not contain impurities of other immunoglobulins. The term "monoclonal" indicates that an antibody is derived from a substantially homogeneous population of antibodies, and does not imply that the antibody is to be obtained by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by a variety of methods, including, for example, the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or they can be produced by recombinant DNA methods (see ., for example, US patent No. 4816567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using methods described, for example, in Clackson et al, Nature 352: 624-628 (1991) and Marks et al, J. Mol. Biol. 222: 581-597 (1991).

[0110] Monoclonal antibodies within the scope of the present invention particularly include "chimeric" antibodies (immunoglobulins) in which part of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) is identical or homologous to the corresponding sequences in antibodies obtained from a different species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the required biological activity (US Patent No. 4,816,567 ; and Morrison et al., Proc Natl Acad Sci USA 81: 6851-6855 (1984)). Chimeric antibodies of interest within the scope of the present invention include "primatized" antibodies comprising antigen-binding variable domain sequences from a non-human primate (e.g., an old-fashioned monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant domain sequences (US Patent No. 5,693,780 ).

[0111] "Humanized" forms of non-human antibodies (eg, mice) are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. Basically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primates that possess the required specificity, affinity and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Moreover, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody contains substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin and all or substantially all of the FR The β-regions are FR regions from the human immunoglobulin sequence, with the exception of the FR substitution (s) as indicated above. Also, a humanized antibody optionally contains at least a portion of an immunoglobulin constant region, typically a human immunoglobulin constant domain. For more 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).

[0112] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may contain amino acid residues from the "complementarity determining region" or "CDR" (e.g., about the region of residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in V _L , and about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in V _H (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 loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain, and 26-32 (H1 ), 52A-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). "Framework" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein.

[0113] A "simple antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule such as a cytotoxic moiety or radioactive label.

[0114] For the purposes of this disclosure solely and unless otherwise indicated, "humanized 2H7" refers to a humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody is effective to deplete primate B cells in vivo, wherein the antibody comprises in its H chain variable region (V _H ) at least the CDR H3 sequence of SEQ ID NO: 12 (FIG. 1B) from an anti-human CD20 antibody and substantially consensus human framework (FR) residues from the heavy chain of subgroup III (V _H III) of a person. In some embodiments, the antibody further comprises the H chain CDR H1 sequence SEQ ID NO: 10 and the H2 CDR sequence SEQ ID NO: 11, and, in some embodiments, further comprises the L chain CDR L1 sequence SEQ ID NO: 4, the CDR sequence L2 SEQ ID NO: 5, CDR sequence L3 SEQ ID NO: 6 and substantially consensus human framework (FR) residues from human kappa light chain subgroup I (V _κ I), wherein the V _H region may be linked to a constant region of the chain Human IgG, where this region can be, for example, IgG1 or IgG3. In some embodiments, such an antibody comprises the V _H sequence of SEQ ID NO: 8 (v16, as shown in FIG. 1B), optionally also comprising the V _L sequence of SEQ ID NO: 2 (v16, as shown in FIG. 1A), which can have amino acid substitutions 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, respectively, as shown in FIGS. 2 and 3. In some embodiments, the antibody is 2H7.v31 comprising the amino acid sequences light and heavy chains SEQ ID NO: 13 and 15, respectively, as shown in Figs. 2 and 4. An antibody in the framework of the present invention may further contain at least one amino acid substitution in the Fc region that increases the activity of ADCC and / or CDC, such as an antibody in which the amino acid substitutions are S298A / E333A / K334A, and in some embodiments, 2H7.v31 having the heavy chain amino acid sequence SEQ ID NO: 15 (as shown in FIG. 4). Any of these antibodies, in addition, may contain at least one amino acid substitution in the Fc region, which reduces the activity of CDC, for example, contains at least one substitution K322A. See US patent No. 6528624B1 (Idusogie et al.).

[0115] The term "ocrelizumab" (CAS accession number 637334-45-3) as used herein refers to a genetically engineered humanized monoclonal antibody directed against the CD20 antigen and comprising (a) a light chain containing the amino acid sequence SEQ ID NO: 13, and (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 14, including fragments thereof that retain the ability to bind CD20. Ocrelizumab is available from Genentech.

[0116] An "isolated" antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of the natural environment are materials that interfere with diagnostic or therapeutic uses of 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 of the antibody, as determined by the Lowry method, and in some embodiments, to greater than 99% by weight, (2) to an extent sufficient to provide at least 15 residues N-terminal or internal amino acid sequence using a rotating beaker sequencer, or (3) homogeneous on SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Isolated antibody includes the antibody in situ in recombinant cells since at least one component of the antibody's natural environment is missing. Typically, however, isolated antibody is obtained through at least one purification step.

[0117] An "individual" or "patient" in the context of the present invention is a human individual or patient. Typically, the individual or patient is suitable for the treatment of multiple sclerosis. For the purposes of this description, such a suitable individual or patient is an individual or patient who experiences, has experienced, or is likely to experience one or more signs, symptoms or other signs of multiple sclerosis; who have been diagnosed with multiple sclerosis, for example, newly diagnosed (in the case of "newly diagnosed" MS), previously diagnosed with a new relapse or exacerbation, previously diagnosed and in remission, etc .; and / or there is a risk of developing multiple sclerosis. An individual at risk of having multiple sclerosis may optionally be identified as an individual who has been screened for increased levels of CD20 positive B cells in serum, cerebrospinal fluid (CSF) and / or MS plaque (s), and / or subjected to screening using an assay to detect autoantibodies assessed qualitatively and preferably quantitatively. Illustrative such autoantibodies associated with multiple sclerosis include antibodies against myelin basic protein (MBP), antibodies against myelin oligodendrocyte glycoprotein (MOG), antibodies against ganglioside, and / or antibodies against neurofilaments. Such autoantibodies can be found in an individual's serum, cerebrospinal fluid (CSF) and / or MS plaque. By "elevated" level (s) of autoantibodies or B cells, as used herein, is meant the level (s) of such autoantibodies or B cells that are significantly higher than the level (s) in an individual without MS.

[0118] As used in the framework of the invention, "treatment" or "treatment" is an approach for obtaining beneficial or desirable results, including clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: amelioration of one or more symptoms of a disease, a decrease in the severity of a disease, stabilization of a disease (e.g., preventing or slowing down the worsening of a disease), delaying or slowing the progression of a disease , alleviating the disease state, reducing the dose of one or more other drugs required to treat the disease, and / or improving the quality of life.

[0119] As used in the framework of the invention, "delaying" or "slowing down" the progression of multiple sclerosis means delaying, arresting, slowing down, inhibiting, stabilizing and / or delaying the development of the disease. This delay can be of varying duration, depending on the medical history and / or the individual being treated.

[0120] As used in the framework of the invention, "at the time of initiation of treatment" refers to the period of time at or before the first exposure to a drug against multiple sclerosis, such as an anti-CD20 antibody. In some embodiments, “at the time of initiation of treatment” is any of one year, nine months, six months, three months, two months, or one month prior to administration of an anti-multiple sclerosis agent, such as an anti-CD20 antibody. In some embodiments, “at the time of initiation of treatment” means from immediately prior to the time coinciding with the first exposure to an anti-multiple sclerosis drug, such as an anti-CD20 antibody.

[0121] As used in the framework of the invention, "based on" includes (1) evaluating, determining or measuring characteristics of a patient as described herein (and preferably selecting a patient suitable for treatment); and (2) administering treatment (s) as described herein.

[0122] A "symptom" of MS is any pathological phenomenon or abnormality in structure, function, or sensation experienced by an individual and indicative of MS.

[0123] "Multiple sclerosis" refers to a chronic and often disabling disease of the central nervous system, characterized by progressive destruction of myelin. There are four internationally recognized forms of MS, namely, primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS).

[0124] "Progressive multiple sclerosis" as used herein 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 ≥ 6 months that cannot be attributed to clinical relapse.

[0125] "Primary progressive multiple sclerosis" or "PPMS" is characterized by the gradual progression of the disease from its onset with rare concomitant relapses and remissions. There may be periods of stabilization of disease activity and there may be good and bad days or weeks. PPMS differs from RRMS and SPMS in that onset tends to occur in the late thirties or early forties, males are as likely to develop it as females, and the initial activity of the disease is often located in the spinal cord but not in the brain. brain. PPMS disease activity can also be observed (or detected) in the brain. PPMS is the subtype of MS that is least likely to show inflammatory (gadolinium-enhanced) plaques on MRI scans. The primary progressive form of the disease affects 10 to 15% of all people with multiple sclerosis. PPMS can be determined according to the criteria of et al. Ann Neurol 69: 292-392 (2010). An individual with PPMS being treated in the framework of the invention is usually an individual with a probable or definitive diagnosis of PPMS.

[0126] "Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by relapses (also known as flare-ups), during which new symptoms may appear and old ones may reappear or worsen. Relapses are followed by periods of remission, during which the individual fully or partially recovers from the disorders acquired during the relapse. Relapses can last for days, weeks, or months, and recovery can be slow or gradual, or almost instantaneous. Most people with MS are first diagnosed with RRMS. This usually occurs at the age of twenties or thirties, although much earlier or later diagnoses are known. This subtype of MS is present in women twice as often as in men. During relapses, myelin, the protective insulating sheath around nerve fibers (neurons) in the white matter areas of the central nervous system (CNS), can be damaged by an inflammatory response by the body's own immune system. This causes a wide variety of neurological symptoms that can vary greatly depending on which areas of the CNS are damaged. Immediately after relapse, the inflammatory response fades and a particular type of glial cell in the CNS (called oligodendrocytes) promotes remyelination, a process by which the myelin sheath around an axon can be repaired. It is remyelination that may be responsible for remission. Approximately 50% of RRMS patients become SPMS patients within 10 years of the onset of the disease. After 30 years, this figure reaches 90%. At any given time, people with relapsing-remitting disease represent approximately 55% of all people with MS.

[0127] "Secondary progressive multiple sclerosis" or "SPMS" is characterized by sustained progression of clinical neurological damage with or without concomitant relapses and with little remission and stabilization. People who develop SPMS have previously had an RRMS period that can last anywhere from two to forty years or more. Any concomitant relapses and remissions tend to subside over time. From the onset of the secondary progressive phase of the disease, disability progresses significantly faster than with RRMS, although progression may still be quite slow in some individuals. After 10 years, 50% of people with RRMS develop SPMS. By the age of 25 to 30, this figure reaches 90%. SPMS tends to be associated with lower levels of inflammatory plaque formation than RRMS, but the overall severity of the disease continues to increase. At any given time, people with SPMS account for approximately 30% of all people with multiple sclerosis.

[0128] "Progressive-relapsing multiple sclerosis", referred to as "PRMS", is characterized by sustained progression of clinical neurological damage with concomitant relapses and remissions. Significant recovery occurs immediately after relapse, but symptoms gradually worsen between relapses. PRMS affects approximately 5% of all people with multiple sclerosis. Some neurologists believe that PRMS is a variant of PPMS. The expression "effective amount" refers to the amount of an antibody (or other drug) that is effective to alleviate or treat multiple sclerosis. Such an effective amount generally results in an improvement in signs, symptoms, or other indicators of MS, such as a decrease in relapse rate, prevention of disability, a decrease in the number and / or volume of brain plaques on MRI, an improvement in walking 25 feet at a time, slowing down, or delaying disease progression, such as increasing the time to disease progression (for example, using the extended disability assessment scale EDSS), etc.

[0129] "Antibody exposure" refers to exposure to an antibody or exposure to an antibody as described herein in one or more doses administered over a period of about 1-20 days. Doses can be administered at the same time or at fixed or irregular time intervals during this exposure period. Initial and subsequent (eg, second or third) exposure to antibodies are timed from each other, as detailed herein.

[0130] As used in the framework of the invention, "interval" between antibody exposures refers to the time period between earlier antibody exposure and later antibody exposure. The exposure to the antibody of the present invention may include one or two doses. In cases where antibody exposure comprises a single dose, the interval between two antibody exposures refers to the amount of time elapsed between one antibody exposure dose (eg, 1 day) and the next antibody exposure dose. If one antibody exposure comprises two doses and the next antibody exposure comprises one dose, the interval between the two antibody exposures refers to the amount of time elapsed between the first of two doses of the first antibody exposure (e.g., day 1) and the next antibody exposure. In cases where each of the two antibody exposures contains two doses, the interval between antibody exposures refers to the amount of time elapsed between the first of the two doses of the first antibody exposure (eg, Day 1) and the first dose of the two doses of the second antibody exposure. For example, if the method of the present invention comprises a first exposure to a two-dose antibody and a second exposure to a two-dose antibody and the second exposure to an antibody is not performed until approximately 24 weeks or 6 months after the first exposure to the antibody, then the interval between the first dose of the first exposure to the antibody and the first dose of the second exposure to the antibody is approximately 24 weeks or 6 months.

[0131] The term "immunosuppressive agent" as used in the context of the invention for adjuvant therapy refers to substances that act to suppress or mask the immune system of a mammal being treated within the framework of the present invention. It may include substances that suppress cytokine production, decrease or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see US Pat. No. 4,665,077); non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide; bromocriptine; danazol; dapzon; glutaraldehyde (which masks MHC antigens as described in US Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs such as prednisone, methylprednisolone and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); hydroxychloroquine; sulfasalazine; leflunomide; antagonists of cytokines or cytokine receptors, including antibodies against interferon-alpha, -beta or -gamma, antibodies against tumor necrosis factor-alpha (infliximab or adalimumab), immunoadhesin against TNF-alpha (etanercept), antibodies against tumor necrosis factor-beta, antibodies anti-interleukin-2 and anti-IL-2 receptor antibodies; antibodies against LFA-1, including antibodies against CD11a and anti-CD18; antibodies against L3T4; heterologous anti-lymphocyte globulin; general antibodies against T cells, preferably antibodies against CD3 or against CD4 / CD4a; a soluble peptide containing the binding domain of LFA-3 (WO 90/08187 published 7/26/90); streptokinase; TGF beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualine; rapamycin; T cell receptor (Cohen et al., US patent No. 5114721); fragments of the T cell receptor (Offner et al., Science, 251: 430-432 (1991); WO 90/11294; Janeway, Nature, 341: 482 (1989); and WO 91/01133); and anti-T cell receptor antibodies (EP 340109) such as T10B9.

[0132] The term "cytotoxic agent" as used herein refers to a substance that inhibits or interferes with the functioning of cells and / or causes destruction of cells. The term is meant to include 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 Lu), chemotherapeutic agents, and toxins such as low molecular weight toxins or enzymatically active toxins from bacteria, fungi, plants or animals, or fragments thereof.

[0133] A "chemotherapeutic agent" is a chemical compound useful for treating cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide CYTOXAN ^®; alkyl sulfonates such as bisulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetoginins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs based on adoselesin, carzelesin and bisellesin); cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmicin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodictyin; spongystatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterol, prednemustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as endyne antibiotics (for example, calicheamicin, especially calicheamicin gamma1I and calicheamicin omega1I (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; bisphosphonates such as clodronate; esperamycin; as well as neocarcinostatin-based chromophores and similar endyin-based chromoprotein chromophores), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, chromophore, carcinomycin, dycinomycin, dycin, dycin -diazo-5-oxo-L-norleucine, doxorubicin ADRIAMYCIN ^® (including morfolinodoksorubitsin, tsianomorfolinodoksorubitsin, 2-pyrrolino and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marsellomitsin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin , potfiromycin, puromycin, quelamycin, rhodorubicin, 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 analogs such as antsitabine, azacytidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostan, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defopamine; demecolcine; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguason; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyirubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex ^{PSK ® (JHS Natural Products, Eugene} , OR); razoxane; rhizoxin; sizophyran; spirogermanium; tenuazonic acid; triaziquon; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and angvidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside"("Ara);cyclophosphamide;thiotepa; taxoids, e.g., TAXOL ^® paclitaxel (Bristol-Myers Squibb Oncology, Princeton , NJ), free of Cremophor modified albumin composition nanoparticle paclitaxel ^{ABRAXANE TM (American Pharmaceutical Partners, Schaumberg} , Illinois) , and doxetaxel TAXOTERE ^® (Rhône - Poulenc Rorer, Antony, France); hloranbutsil; gemcitabine GEMZAR ^®; 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; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine and pharmaceutically acceptable salts, acids or derivatives of any of those described above.

[0134] Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as antiestrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen (including tamoxifen NOLVADEX ^®), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4 (5) -imidazoles, aminoglutethimide, magestrola acetate MEGASE ^®, exemestane AROMASIN ^®, formestane, fadrozole, vorozole RIVISOR ^®, lektrozol FEMARA ^®, and anastrozole ARIMIDEX ^®; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (nucleoside analogue of cytosine 1,3-dioxolane); antisense oligonucleotides, in particular oligonucleotides that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as PKC-alpha, Ralf and H-Ras; vaccines such as vaccines for gene therapy, for example, ALLOVECTIN ^® vaccine, LEUVECTIN ^® vaccine, and VAXID ^® vaccine; PROLEUKIN ^® rIL-2; topoisomerase 1 inhibitor LURTOTECAN ^®; ABARELIX ^® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0135] The term "cytokine" is a general term for proteins released by one population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, interleukins (IL) 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, IL-15; tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used in the framework of the invention, the term "cytokine" includes proteins from natural sources or from recombinant cell cultures and biologically active equivalents of native sequence cytokines, including synthetically obtained low molecular weight compounds and their pharmaceutically acceptable derivatives and salts.

[0136] The term "hormone" refers to polypeptide hormones that are typically secreted by ductal glandular organs. Hormones include, for example, growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; estradiol; hormone replacement therapy; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostan, or testolactone; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); prolactin, placental lactogen, mouse gonadotropin-associated peptide, gonadotropin-releasing factor; inhibin; activin; Müllerian inhibiting substance and thrombopoietin. As used in the framework of the present invention, the term "hormone" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the hormone with a native sequence, including synthetically produced low molecular weight compounds and their pharmaceutically acceptable derivatives and salts.

[0137] The term "growth factor" refers to proteins that promote growth and include, for example, hepatic growth factor; fibroblast growth factor; vascular endothelial growth factor; nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -γ; and colony stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte macrophage CSF (GM-CSF) and granulocytic CSF (G-CSF). As used in the framework of the present invention, the term "growth factor" includes proteins from natural sources or from culture of recombinant cells and biologically active equivalents of the native sequence growth factor, including synthetically produced low molecular weight compounds and their pharmaceutically acceptable derivatives and salts.

[0138] The term "integrin" refers to a receptor protein that allows cells to both bind to and respond to the extracellular matrix and is 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 that are involved in cell-extracellular matrix and cell-cell interactions. Functional integrins are composed of two transmembrane glycoprotein subunits called alpha and beta, which are non-covalently linked. Alpha subunits share homology with each other, as well as beta subunits. Receptors always contain one alpha chain and one beta chain. Examples include alpha-6-beta-1, alpha-3-beta-1, alpha-7-beta1, LFA-1, etc. As used in the framework of the invention, the term "integrin" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence integrin, including synthetically produced low molecular weight compounds and their pharmaceutically acceptable derivatives and salts.

[0139] Examples of "integrin antagonists or anti-integrin antibodies" in the context of the present invention include anti-LFA-1 antibody; anti-alpha-4 integrin, 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); derivatives of propionic acid (WO 2000/37444); alkanoic acid derivatives (WO 2000/32575); substituted phenyl derivatives (US Pat. Nos. 6677339 and 6348463); derivatives of aromatic amines (US patent No. 6369229) and polypeptide disintegrin domain ADAM (US2002 / 0042368), antibodies to integrin alpha-v-beta-3 (EP 633945); aza-linked bicyclic amino acid derivatives (WO 2002/02556), etc.

[0140] For the purposes of this description, "tumor necrosis factor alpha (TNF-alpha)" refers to a human TNF-alpha molecule containing the amino acid sequence as described in Pennica et al., Nature, 312: 721 (1984) or Aggarwal et al., JBC 260: 2345 (1985).

[0141] A "TNF-alpha inhibitor" in the context of the present invention is an agent that inhibits to some extent the biological function of TNF-alpha, typically by binding to TNF-alpha and neutralizing its activity. Examples of TNF inhibitors specifically contemplated in the present invention are etanercept (ENBREL ^®), infliximab (REMICADE ^®), and adalimumab (HUMIRA ™).

[0142] Examples of "disease-modifying antirheumatic drugs" or "DMARDs" include hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate), azathioprine, D-penicillamine, gold) (intramuscular) , minocycline, cyclosporine, immunosorption with staphylococcal protein A, including its salts and derivatives, etc.

[0143] Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" are acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, tolmetin, including salts and derivatives thereof, and the like.

[0144] "Corticosteroid" refers to any of several synthetic or naturally occurring substances with the general chemical structure of steroids that mimic or enhance the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone), dexamethasone, glucocorticoid, and betamethasone.

[0145] "Package insert" refers to instructions commonly included in commercial packaging of therapeutic products that include information on indications, use, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and / or warnings regarding the use of such therapeutic products, etc.

[0146] "Label" is used within the scope of the present invention to refer to information commonly included in commercial packaging of pharmaceutical formulations, including containers such as vials and package inserts, as well as other types of packaging.

[0147] Reference to "about" a value or parameter within the scope of the present invention includes (and describes) variation related to that value or parameter itself. For example, a description indicating "about X" includes the description "X".

[0148] As used within the scope of the invention and in the appended claims, the singular includes the plural of the referenced subject unless the context clearly dictates otherwise. It is understood that aspects and variations of the invention described herein include "consisting" and / or "essentially consisting" of aspects and variations.

[0149] It should be understood that one, some or all of the properties of the various embodiments described herein may be combined to provide embodiments of the present invention. These and other aspects of the invention will become apparent to those skilled in the art.

[0150] All references cited in this specification, including patent applications and publications, are incorporated by reference in their entirety.

II. Treatment methods

[0151] In certain embodiments, there is provided a method of providing an improvement in functional ability in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient has an improvement in functional ability 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.

[0152] In certain embodiments, the patient after treatment has a confirmed reduction in disability for at least about 12 weeks. In certain embodiments, the patient after treatment has a confirmed reduction in disability for at least 24 weeks. In certain embodiments, a confirmed reduction in disability is determined by an Extended Disability Scale (EDSS) score. In certain embodiments, the patient's EDSS decreases by at least about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0 , 7, approximately 0.8, approximately 0.9, approximately 1.0, or more than approximately 1.0 (such as approximately 1.1, approximately 1.2, approximately 1.3 , approximately 1.4 or approximately 1.5 points).

[0153] In certain embodiments, the improvement in functional ability 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 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 total, at least approximately 23 weeks including any range between these values. In certain embodiments, the improvement in functional ability 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 range between these values.

[0154] In certain embodiments, the increase in functional ability is measured using a 25 foot timed walking test (T25-FW). In certain embodiments, the 25 feet walking time after starting 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, approximately 7.5 minutes, approximately 8 minutes, approximately 8.5 minutes, approximately 9 minutes, approximately 9.5 minutes, or approximately 10 minutes relative to the 25-foot walk time immediately before starting treatment.

[0155] In certain embodiments, the increase in functional activity is demonstrated by the absence of signs of disease activity (NEDA). In certain embodiments, NEDA is demonstrated by the absence of new or increasing T2 plaques or gadolinium-enhanced T1 plaques on magnetic resonance imaging. In certain embodiments, the implementation of NEDA is demonstrated by the absence of relapse. In certain embodiments, the implementation of NEDA is demonstrated by the absence of progression. In certain embodiments, the implementation of NEDA is demonstrated by the absence of deterioration in EDSS. In certain embodiments, NEDA is defined as: no protocol-defined relapses, no CDP events, no new or increasing T2 plaques, and no gadolinium-enhanced T1 plaques. In certain embodiments, 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 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 approximately 21 weeks, at least approximately 22 weeks, at least approximately Leaving 23 weeks, including any range between these values. In certain embodiments, the increase in functional ability 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 range between these values.

[0156] In certain embodiments, the patient has gadolinium staining T1 plaques at baseline (ie, prior to treatment). In certain embodiments, the patient does not have gadolinium staining T1 plaques at baseline (ie, prior to treatment).

[0157] In certain embodiments, the present invention provides a method of suppressing the progression of disability in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the administration results in a decrease in confirmed cases of progression of disability, and 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, administration results in a decrease in the 12-week confirmed progression work capacity. In certain embodiments, administration results in a decrease in 24-week confirmed progression of disability.

[0158] In certain embodiments, the patient has gadolinium staining T1 plaques at baseline (ie, prior to treatment). In certain embodiments, the patient does not have gadolinium staining T1 plaques at baseline (ie, prior to treatment).

[0159] In certain embodiments, the present invention provides a method of slowing the onset of a confirmed progression of disability 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 CDR1 having the amino acid sequence of SEQ ID NO: 10, 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 containing 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.

[0160] In certain embodiments, confirmed progression of disability is determined by an Extended Disability Scale (EDSS) score. In certain embodiments, the patient's EDSS increases by at least about 1.0 points from a baseline EDSS of about 5.5 or less. In certain embodiments, the patient's EDSS increases by about 0.5 points from a baseline EDSS greater than 5.5. In certain embodiments, an increase in EDSS is confirmed at least 12 weeks after the initial neurological deterioration.

[0161] In certain embodiments, the patient has gadolinium staining T1 plaques at baseline (ie, prior to treatment). In certain embodiments, the patient does not have gadolinium staining T1 plaques at baseline (ie, prior to treatment).

[0162] In certain embodiments, the present invention provides a method for reducing T2 plaque volume 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 CDR1 having the amino acid sequence of SEQ ID NO: 10, 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 containing 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 gadolinium stained T1 plaques at baseline. In certain embodiments, the patient does not have gadolinium staining T1 plaques at baseline.

[0163] In certain embodiments, there is provided a method of slowing or preventing a decrease in brain volume in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the decrease in brain volume is slowed down or prevented in the patient; and 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, a decrease in brain volume slows down. In certain embodiments, the decrease in brain volume is prevented in a patient who has not had a decrease in brain volume. In certain embodiments, a further decrease in brain volume is prevented in a patient who has a decrease in brain volume.

[0164] In certain embodiments, there is provided a method of slowing or preventing cerebral atrophy in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient slows or prevents brain atrophy; and 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, brain atrophy is slowed down ... In certain embodiments, brain atrophy is prevented in a patient who does not have brain atrophy. In certain embodiments, further brain atrophy is prevented in a patient who has brain atrophy.

[0165] In certain embodiments, there is provided 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 evidence of disease activity (NEDA) for at least 12 weeks, 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, treatment results in no evidence of disease activity (NEDA) for at least 24 weeks.

[0166] A method of treating a human patient with multiple sclerosis is provided, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in the patient achieving a plaque-free status after 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 containing 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, treatment results in the patient achieving a plaque-free status after 48 weeks of treatment. In certain embodiments, treatment results in a plaque-free status after 24 weeks of treatment. In certain embodiments, the treatment results in the patient being free of gadolinium staining plaques. In certain embodiments, the treatment results in the patient being free of T2 plaques.

[0167] In certain embodiments, a method of treating a human patient with recurrent multiple sclerosis is provided, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein treatment results in one or more of the following: a) the patient is relapse-free after 96 weeks; b) the patient has no confirmed events of progression of disability after 96 weeks; c) the patient has no gadolinium-enhanced T1 plaques at 96 weeks; d) the patient has no new and / or increasing T2 plaques 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.

[0168] In certain embodiments, the patient has not previously been treated with another therapy for multiple sclerosis (ie, "naive patient"). In certain embodiments, the naive patient experiences at least 2 relapses in the 2 years prior to starting treatment. In certain embodiments, the naive patient experiences at least 1 relapse in the last year prior to starting treatment.

[0169] In certain embodiments, the patient has a poor response to another therapy for multiple sclerosis. In certain embodiments, the patient who has a deficient response has been previously treated with interferon beta-1a or glatiramer acetate for at least 1 year. In certain embodiments, a patient who has an inadequate response, has at least one relapse, or has at least 1 initial gadolinium-enhanced plaque while being treated with another therapy for multiple sclerosis.

[0170] In certain embodiments, a method of treating a human patient with highly active multiple sclerosis is provided, 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 containing 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 SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0171] In certain embodiments, the highly active multiple sclerosis patient has not previously been treated with another multiple sclerosis therapy (ie, "naive patient"). In certain embodiments, naive patients with highly active multiple sclerosis experience at least 2 relapses in the last year prior to randomization, and have either (a) at least 1 baseline gadolinium-enhanced plaque, or (b) an increase in the number of T2 plaques at the initial visit ( change in category from 0-5 to 6-9 plaques or from 6-9 plaques to> 9 plaques), compared to the previous MRI.

[0172] In certain embodiments, a patient with highly active multiple sclerosis has an inadequate response to another therapy for multiple sclerosis. In certain embodiments, the highly active multiple sclerosis patient who has an inadequate response has been previously treated with interferon beta-1a or glatiramer acetate for at least 1 year. In certain embodiments, a highly active multiple sclerosis patient who has an inadequate response, has experienced at least one relapse, and either (a) had at least nine T2 plaques, or (b) had at least one gadolinium-enhanced plaque at baseline in while he was being treated with another therapy for multiple sclerosis.

[0173] In certain embodiments, administration of an anti-CD20 antibody to a patient with highly active multiple sclerosis is effective for one or more of the following: (1) reducing the amount of plaque in the patient's brain; (2) a decrease in the average annual relapse rate; (3) reduced progression of disability; and (4) improving functional ability. In certain embodiments, a method of treating a patient with highly active multiple sclerosis further comprises performing an MRI scan and determining whether the patient has highly active multiple sclerosis prior to administering an anti-CD20 antibody to the patient.

[0174] In certain embodiments, there is provided a method of treating a human patient with an early stage of 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 containing 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 a sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the method further comprises diagnosing a patient having early stage multiple sclerosis prior to administering an anti-CD20 antibody to the patient.

[0175] In certain embodiments, there is provided 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 evidence of disease activity in the patient, and wherein the anti-CD20 antibody comprises: a) a severe variable region a chain containing 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 containing CDR1 having the amino acid a 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.

[0176] In certain embodiments, the patient has a recurrent form of multiple sclerosis. In some embodiments, recurrent MS (RMS) refers to a sample of patients, typically consisting of both RRMS patients and SPMS patients with concomitant relapses (also commonly referred to as "recurrent SPMS"). In certain embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). In certain embodiments, the recurrent form of multiple sclerosis is secondary progressive multiple sclerosis with associated relapses (rSPMS). In certain embodiments, the patient has progressive multiple sclerosis. In certain embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In certain embodiments, the patient is less than 18 years of age. In certain embodiments, the patient is 18 to 55 years of age. In certain embodiments, the patient is over 55 years of age.

[0177] In some embodiments, "confirmed progression of disability" or "CDP" refers to an increase of at least 1.0 point from baseline EDSS in patients with a baseline of 5.5 or less, or an increase of 0.5 points in patients with a baseline of more than 5.5 at 96 weeks, where an increase in EDSS is confirmed at regular scheduled visits at least 12 weeks after the initial neurological deterioration.

[0178] In some embodiments, "confirmed reduction in disability" or "CDI" refers to a decrease in EDSS score of at least 1.0 points from baseline in patients with baseline EDSS of 5.5 or less, or a reduction of 0 , 5 points in patients with baseline EDSS above 5.5.

[0179] In some embodiments, the brain atrophy refers to one or more of the following: loss of axons in the brain, loss of tissue in gray matter or white matter plaques, plaque-associated Waller pathway degeneration, or plaque loading. In certain embodiments, cerebral atrophy refers to a decrease in the volume of the entire brain. In certain embodiments, brain atrophy refers to a decrease in the volume of one or more brain structures (including, but not limited to, cerebral hemispheres, cerebellum, thalamus, frontotemporal neocortex, brainstem, hippocampus, parietal lobe, and / or hypothalamus ). In certain embodiments, cerebral atrophy refers to a thinning of the cortex in the precentral gyrus, superior frontal gyrus, thalamus, and / or shell. In certain embodiments, cerebral atrophy refers to a loss of at least about 0.4%, at least about 0.5%, at least about 0.6%, or at least 0.7% of brain volume per year ... The following details regarding cerebral atrophy are detailed, for example, in Riley et al. (2012) Expert Rev Neurother 12 (3), 323-333.

[0180] In some embodiments, the patient or individual has highly active multiple sclerosis. In some embodiments, "highly active multiple sclerosis" in the treatment of naive patients refers to patients who have not previously been treated with another multiple sclerosis therapy and who have experienced at least 2 relapses in the last year prior to randomization, and either (a) have at least 1 baseline gadolinium-enhanced plaque, or (b) have an increase in T2 plaque count at baseline visit (change from 0-5 plaque to 6-9 plaque category or from 6-9 plaque to> 9 plaque category) compared to the previous MRI study. In some embodiments, "highly active multiple sclerosis" refers to patients who have previously been treated with another therapy for multiple sclerosis and who have had at least one relapse in the past year and either (a) have at least nine T2 plaques, or (b) have at least one gadolinium-enhanced plaque at baseline.

[0181] In certain embodiments, the patient baseline refers to the level before administration or treatment with the anti-CD20 antibody to the patient, for example, 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, about 1 day before administration or treatment patient with anti-CD20 antibody.

[0182] In certain embodiments, the patient retains the ability to induce a humoral immune response to an antigen during treatment. In certain embodiments, the antigen is a mumps antigen, rubella antigen, chickenpox antigen, S. pneumonia antigen, tetanus toxoid antigen, pneumococcal antigen, or influenza antigen.

[0183] The methods described herein may encompass any combination of the embodiments described herein.

III. Dosage

[0184] In some embodiments of any of the methods and articles described herein, the method or instructions comprise administering an effective amount of an anti-CD20 antibody to a patient with multiple sclerosis to provide an initial antibody exposure of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, such as about 0.6 grams or about 1.0 grams) followed by a second exposure to antibody of about 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams, such as about 0.6 grams or about 1.0 grams), with no exposure to the second antibody occurring until about 16 to about 60 weeks after the initial exposure to the antibody. For the purposes of the present invention, the second exposure to the antibody is the next time the patient is treated with the anti-CD20 antibody, after the initial exposure to the antibody, with no intermediate treatment or exposure to the anti-CD20 antibody between the initial and second exposures. In some embodiments, the initial antibody exposure and / or the second antibody exposure is any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams, 0.9 gram or 1.0 gram.

[0185] The interval between initial and second or subsequent exposures to the antibody can be measured from the first dose of the initial exposure to the antibody.

[0186] In some embodiments, exposure to antibodies is performed at intervals of about 24 weeks or 6 months; or approximately 48 weeks or 12 months.

[0187] In one embodiment, the second exposure to the antibody is not performed until about 20 to about 30 weeks after the initial exposure, optionally followed by a third exposure to about 0.3 to about 4 grams of antibody (preferably from about 0.3 to about about 1.5 grams), with the third exposure not administered until about 46 to 60 weeks (preferably from about 46 to 54 weeks) after the initial exposure, and then, in some embodiments, no further exposure to the antibody until at least about 70 75 weeks after initial exposure. In some embodiments, the third exposure to the antibody is in any amount 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.

[0188] In an alternative embodiment, the second exposure to the antibody is not performed until about 46 to 60 weeks after the initial exposure, and subsequent exposure to the antibody is not performed until about 46 to 60 weeks after the initial exposure to the antibody.

[0189] Any one or more of the effects of antibodies within the framework of the present invention can be provided to a patient as a single dose of antibody or as two separate doses of antibody (ie, consisting of a first and a second dose). The specific number of doses (one or two) used for each antibody exposure may depend on, for example, the type of MS being treated, the type of antibody used, the use and type of second drug used, and the mode and frequency of administration. When two separate doses are administered, the second dose is preferably administered over about 3 to 17 days, more preferably about 6 to 16 days, and most preferably about 13 to 16 days after the first dose. In some embodiments, when two separate doses are administered, the second dose is administered approximately 14 days later. When two separate doses are administered, the first and second doses of antibody are preferably from about 0.3 to 1.5 grams, more preferably from about 0.3 to about 1.0 grams. In some embodiments, where two separate doses are administered, the first and second doses of the antibody are any of 0.3 grams, 0.4 grams, 0.5 grams, or 0.6 grams. In some embodiments, the initial exposure to ocrelizumab comprises a first dose and a second dose of ocrelizumab, wherein the first dose and second dose of ocrelizumab are approximately 0.3 grams. In some embodiments, the second exposure to ocrelizumab comprises a single dose of ocrelizumab, wherein the single dose of ocrelizumab is 0.6 grams.

In one embodiment, at least about three, at least about four, or at least about five exposures of the antibody to a patient are provided, for example, from about 3 to 60 exposures, and more specifically from about 3 to 40 exposures, most often approximately 3 to 20 exposures. In some embodiments of any of the methods, the methods further comprise providing about one to about three subsequent exposures to ocrelizumab. In some embodiments, the implementation of such exposure is at intervals of about 24 weeks or 6 months, or 48 weeks or 12 months. 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 certain embodiments, more than one 1 interval is shortened by about 4 weeks, by about 3.5 weeks, by about 3 weeks, by about 2.5 weeks, by about 2 weeks, by about 1.5 weeks, by 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, the interval is 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. In certain embodiments, more than one interval is lengthened by about 4 weeks, by about 3.5 weeks, by about 3 weeks, by about 2.5 weeks, by about 2 weeks, by about 1.5 weeks, by about 1 week, by about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In certain embodiments, more than 1 interval is shortened by about 4 weeks, by about 3.5 weeks, by about 3 weeks, by about 2.5 weeks, by about 2 weeks, by about 1.5 weeks, by about 1 week, by about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day, or 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 ...

[0190] In one embodiment, each exposure to the antibody is performed as a single dose of the antibody. In an alternative embodiment, each exposure to the antibody is administered as two separate doses of the antibody. In some embodiments, some exposure is administered as a single dose or as two separate doses.

[0191] The antibody can be a simple 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 (eg, containing the variable domain sequences of SEQ ID NO. 2 and 8) or humanized 2H7 containing the variable domain sequences of SEQ ID NO. 23 and 24, or huMax-CD20 (Genmab). In some embodiments, the antibody is ocrelizumab (eg, 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).

[0192] In one embodiment, the patient has never previously been treated with a drug (s), such as an immunosuppressive agent (s), for the treatment of multiple sclerosis and / or has never previously been treated with an antibody to a B-cell surface marker (e.g., never previously treated anti-CD20 antibody).

[0193] The antibody is administered by any suitable means, including perennial, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and / or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Intrathecal administration is also contemplated (see, for example, US Patent Application No. 2002/0009444, Grillo-Lopez, A concerning intrathecal delivery of a CD20 antibody). In addition, the antibody can be administered by pulse infusion, for example, with decreasing doses of the antibody. In some embodiments, dosing is administered intravenously, subcutaneously, or intrathecally. In some embodiments, dosing is by intravenous infusion (s).

[0194] Although an anti-CD20 antibody may be the only drug administered to a patient to treat multiple sclerosis, a second drug such as a cytotoxic agent, chemotherapeutic agent, immunosuppressive agent, cytokine, cytokine antagonist or antibody, growth factor, may optionally be administered. hormone, integrin, integrin antagonist or antibody thereto (e.g., anti-LFA-1 or an antibody against alpha 4 integrin, such as natalizumab (TYSABRI ^®), available from Biogen Idec / Elan Pharmaceuticals, Inc), etc., together with an antibody that binds a B-cell surface marker (eg, an anti-CD20 antibody).

[0195] In certain embodiments of combination therapy, the antibody may be combined with interferon class drug such as IFN-beta-1a (REBIF ^® and AVONEX ^®) or IFN-beta-1b (BETASERON ^®); an oligopeptide such as glatiramer acetate (COPAXONE ^®); a cytotoxic agent such as mitoxantrone (NOVANTRONE ^®), methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenous immunoglobulin (gamma globulin); lymphocyte depleting therapy (e.g., mitoxantrone, cyclophosphamide, alemtuzumab (Campath ^®, LEMTRADA ^TM), anti-CD4, cladribine, total body irradiation only, bone marrow transplantation); a corticosteroid (eg, methylprednisolone, prednisone, dexamethasone, or glucocorticoid), including systemic corticosteroid therapy; non-lymphocyte-depleting immunosuppressive therapy (eg, mycophenolate mofetil (MMF) or cyclosporine); cholesterol-lowering drugs class "statins" which include cerivastatin (BAYCOL ^®), fluvastatin (LESCOL ^®), atorvastatin (LIPITOR ^®), lovastatin (MEVACOR ^®), pravastatin (PRAVACHOL ^®), simvastatin (ZOCOR ^®); estradiol; testosterone (optionally at higher doses; Stuve et al. Neurology 8: 290-301 (2002)); hormone replacement therapy; treating symptoms secondary to or associated with MS (eg, spasticity, incontinence, pain, fatigue); a TNF inhibitor; a disease modifying antirheumatic agent (DMARD); a non-steroidal anti-inflammatory drug (NSAID); plasmapheresis; levothyroxine; cyclosporin A; a somatostatin analog; a cytokine or cytokine receptor antagonist; antimetabolite; an immunosuppressive agent; rehabilitating surgery; radioactive iodine; thyroidectomy; another B-cell surface antagonist / antibody to it; etc.

[0196] The second drug is administered upon initial exposure and / or subsequent exposures of the anti-CD20 antibody, such combined administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in any order, where preferably there is a period of time when both (or all) active substances simultaneously exhibit their types of biological activity.

[0197] In addition to administering antibodies to a patient, the present invention provides for administration by gene therapy. Such introduction of a nucleic acid encoding an antibody is encompassed by the expression introduction of an "effective amount" of an antibody. See, for example, WO96 / 07321, published March 14, 1996, regarding the use of gene therapy for the production of intracellular antibodies.

[0198] There are two main approaches to introducing nucleic acid (optionally contained in a vector) into patient cells: in vivo and ex vivo. For in vivo delivery, the nucleic acid is injected directly into the patient, usually in the region where the antibody is required. For ex vivo treatment, the patient's cells are removed, nucleic acid is introduced into the isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated in porous membranes that are implanted into the patient (see, for example, US Pat. Nos. 4,892,538 and 5,283,187). There are many methods available for introducing nucleic acids into living cells. The methods vary depending on whether the nucleic acid is transferred into cultured cells in vitro or into cells of the intended host in vivo. Methods suitable for transferring nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, use of DEAE-dextran, calcium phosphate precipitation method, etc. A commonly used vector for ex vivo gene delivery is a retrovirus.

[0199] In some embodiments, in vivo nucleic acid transfer methods include transfection with viral vectors (such as adenovirus, herpes simplex virus I, or adeno-associated virus) and lipid-based systems (suitable lipids for lipid-mediated gene transfer are, for example, DOTMA, DOPE and DC-Chol). In some situations, it is desirable to provide a nucleic acid source with an agent that targets target cells, such as an antibody specific for a cell surface membrane protein or target cell, a ligand for a receptor on a target cell, etc. When liposomes are used, proteins that bind to the cell surface membrane associated with endocytosis can be used to target and / or facilitate uptake, for example, capsid proteins or fragments thereof tropic to a particular cell type, antibodies for proteins that undergo internalization upon cycling, and proteins that target intracellular localization and increase intracellular half-life. A receptor-mediated endocytosis method is described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990). For an overview of currently known gene labeling and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992). Also see WO 93/25673 and the references cited therein.

IV. Antibodies and their production

[0200] The methods and articles of the present invention use or comprise an antibody that binds to a B-cell surface marker, in particular an antibody that binds to CD20. Thus, methods for making such antibodies are described herein.

[0201] In some embodiments, the anti-CD20 antibody used in the methods described herein is prepared by a method comprising expressing a nucleic acid encoding a humanized antibody comprising the heavy and light chain amino acid sequences of SEQ ID NO: 14 or 13, respectively, in the host cell and the isolation of the humanized antibody or antigen-binding fragment thereof expressed in the host cell. In some embodiments, the host cell is a mammalian cell (eg, a CHO cell), an insect cell, or a plant cell. In some embodiments, the host cell is a bacterial cell. Methods for making antibodies against CD20 are further described, for example, in US Pat. No. 7,799,900.

[0202] A B-cell surface marker for use in producing or screening an antibody can be, for example, a soluble form of the marker or a portion thereof containing the desired epitope. Alternatively or additionally, cells expressing a marker on their cell surface can be used to generate or screen antibodies. Other forms of the B-cell surface marker suitable for the production of antibodies will be apparent to those skilled in the art.

[0203] The description provides illustrative methods for producing antibodies used in accordance with the present invention.

(i) Polyclonal antibodies

[0204] Polyclonal antibodies are preferably induced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the appropriate antigen and adjuvant. It may be suitable to conjugate the appropriate antigen to a protein that is immunogenic in the species to be immunized (for example, snail lymph hemocyaninoma (KLH), serum albumin, bovine thyroglobulin, or a soybean trypsin inhibitor, using a bifunctional or derivatizing agent such as male conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N = C = NR, where R and R ¹ are different alkyl groups.

[0205] Animals are immunized with antigen, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally into multiple sites. One month later, animals receive a booster injection of 1/5 to 1/10 of the initial amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection into multiple areas. After seven to 14 days, blood is taken from the animals and the serum is analyzed in relation to the antibody titer. Auxiliary injections in animals are carried out until the titer reaches a plateau. In some embodiments, the animal is immunized with a conjugate of the same antigen but with a different protein and / or through a different linker reagent. Conjugates can also be produced in recombinant cell culture as fusion proteins. Aggregating agents such as alum can also be used to enhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are prepared from a collection of substantially homogeneous antibodies, i. E. the individual antibodies that make up the population are identical and / or bind the same epitope, except for possible variants that arise during the production of the monoclonal antibody, and such variants are usually present in small quantities. Thus, the term "monoclonal" indicates that an antibody is not a mixture of different or polyclonal antibodies.

[0206] For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be produced by recombinant DNA methods (US Pat. No. 4,816,567).

[0207] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable cell fusion agent such as polyethylene glycol to produce hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0208] The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused parental myeloma cells. For example, if parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), selective culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0209] In some embodiments, the myeloma cells are cells that fuse efficiently, maintain stable antibody production at a high level by selected antibody-producing cells, and are sensitive to a medium such as HAT. Among these, in some embodiments, the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 murine tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP cells -2 or X63-Ag8-653, available from American Type Culture Collection, Rockville, Mariland, USA. Also for the production of human monoclonal antibodies, myeloma cell lines and mouse-human heteromyeloma lines have been described (Kozbor, J. Immunol, 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0210] The culture medium in which the hybridoma cells are grown is analyzed for the production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro binding assay, such as a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0211] The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard assay described in Munson et al., Anal. Biochem. 107: 220 (1980).

[0212] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution methods and grown by standard methods (Goding, Monoclonal antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 media. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors.

[0213] Monoclonal antibodies secreted by subclones are conveniently isolated from culture medium, ascites fluid or serum by conventional antibody purification methods such as protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0214] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional techniques (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells are the source of such DNA. To facilitate the synthesis of monoclonal antibodies in recombinant host cells, once isolated, the DNA can be inserted into expression vectors, which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. which otherwise do not produce an immunoglobulin protein. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol 5: 256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992).

[0215] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries created using the methods 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. More recent publications describe the production of high affinity (nM order) human antibodies through chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)). Thus, these methods represent acceptable alternatives to conventional hybridoma monoclonal antibody methods for isolating monoclonal antibodies.

[0216] DNA can also be modified, for example, by replacing human heavy and light chain constant domain coding sequences with homologous mouse sequences (US Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci USA, 81: 6851 (1984 )), or covalently linking to an immunoglobulin coding sequence of all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0217] Typically, such non-immunoglobulin polypeptide sequences are substituted for the constant domains of an antibody, or they replace the variable domains of one antigen-binding region of an antibody to produce a chimeric bivalent antibody containing one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for another antigen.

(iii) Humanized antibodies

[0218] Methods for humanizing non-human antibodies are described in the art. In some embodiments, a humanized antibody has one or more amino acid residues inserted therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from an "import" variable domain. Humanization can mainly be carried out in accordance with the method of Winter and colleagues (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 replacing the hypervariable region sequences with the corresponding human antibody sequences. Thus, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567), where substantially less than an entire human variable domain has been replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues have been replaced by residues from analogous regions of rodent antibodies.

[0219] The choice of human variable domains, both light and heavy chains, for use in the production of humanized antibodies is very important to reduce antigenicity. According to 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 most similar to the rodent sequence is then adopted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol, 151: 2296 (1993); Chothia et al., J. Mol. Biol 196: 901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies from a specific subset of light or heavy chain variable regions. The same framework region 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) ).

[0220] In addition, it is important that antibodies are humanized while maintaining high affinity for the antigen and other positive biological properties. To achieve this goal, in some embodiments, the implementation of humanized antibodies are obtained using a process of analysis of the parental sequences and various putative humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are widely available and known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of this displayed data allows analysis of the possible role of residues in the functioning of putative immunoglobulin sequences, i. E. analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody properties, such as increased affinity for the target antigen (s), are achieved. As a rule, the residues of the hypervariable region have the direct and most significant effect on antigen binding.

[0221] In some embodiments, the anti-CD20 antibody is a humanized anti-2H7 antibody. In some embodiments, a humanized anti-2H7 antibody preferably comprises one, two, three, four, five, or six CDR sequences as shown in FIGS. 1A and 1B. In some embodiments, the implementation of the humanized 2H7 antibody preferably contains 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) (FIG.1A),

CDR sequence L2 APSNLAS (SEQ ID NO: 5) (Figure 1A),

CDR sequence L3 QQWXFNPPT, where X is S or A (SEQ ID NO: 19), e.g. QQWSFNPPT (SEQ ID NO: 6) (FIG. 1A),

CDR sequence H1 GYTFTSYNMH (SEQ ID NO: 10) (Figure 1B),

CDR sequence H2 AIYPGNGXTSYNQKFKG, where X is D or A (SEQ ID NO: 20), for example AIYPGNGDTSYNQKFKG (SEQ ID NO: 11) (Figure 1B), and

CDR sequence H3 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) ( 1B).

[0222] The CDR sequences described above are typically present in light chain variable region and heavy chain variable region framework sequences, such as essentially human consensus FR residues of the kappa light chain subgroup I (V _L 6I) of a human, and essentially human _{human subgroup III (V H} III) heavy chain FR consensus residues. Also see WO 2004/056312 (Lowman et al.).

[0223] In some embodiments, the heavy chain variable region may be linked to a human IgG constant region, where the region may be, for example, an IgG1 or IgG3 region, including native sequence and constant region variants.

[0224] In some embodiments, such an antibody comprises the heavy chain variable domain sequence SEQ ID NO: 8 (v16, as shown in FIG. 1B), and optionally also contains the light chain variable domain sequence SEQ ID NO: 2 (v16, as shown 1A), which optionally contains one or more amino acid substitution (s) at positions 56, 100 and / or 100a, for example, D56A, N100A or N100Y, and / or S100aR in the variable domain of the heavy chain and one or more amino acid substitutions (substitutions) at positions 32 and / or 92, for example M32L and / or S92A, the variable domain of the light chain. In some embodiments, the antibody is an intact antibody comprising the light chain amino acid sequences of SEQ ID NO: 13 or 16 and the heavy chain amino acid sequences of SEQ ID NO: 14, 15, 17, 22, or 25. In some embodiments, the humanized 2H7 antibody is ocrelizumab (Genentech).

[0225] In embodiments, the humanized 2H7 is an intact antibody or antibody fragment comprising a light chain variable region sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 2);

and the sequence of the variable region of the heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTV: 8

[0226] In some embodiments, the humanized 2H7 antibody is an intact antibody, in some embodiments it comprises the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNKLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13);

and the heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14)

or the heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 26)

or the heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15)

or the heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 27).

[0227] In some embodiments, the amino acid K at the C-terminus of the heavy chain is removed.

[0228] In some embodiments, the humanized 2H7 antibody comprises a light chain variable domain sequence 2H7.v511:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 23)

and the sequence of the variable domain of the heavy chain 2H7.v511:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS (SEE 24).

[0229] In some embodiments, the implementation of the humanized antibody 2H7.v511 is an intact antibody, it may contain the amino acid sequence of the light chain:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNKLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16)

and the amino acid sequence of the heavy chain SEQ ID NO: 17 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 25).

[0230] In some embodiments, an antibody within the scope of the present invention further comprises at least one amino acid substitution in the Fc region that enhances ADCC activity, such as amino acid substitutions at positions 298, 333, and 334, preferably S298A, E333A and K334A, with using Eu numbering for heavy chain residues. See also U.S. Patent No. 6,737,056B1, Presta. Any of these antibodies may contain at least one Fc region substitution that increases FcRn binding or serum half-life, for example, a substitution at position 434 of the heavy chain such as N434W. See also U.S. Patent No. 6,737,056B1, Presta. Any of these antibodies, in addition, may contain at least one amino acid substitution in the Fc region that increases the activity of CDC, for example, may contain at least a substitution at position 326, preferably K326A or K326W. See also US Patent No. 6528624B1 (Idusogie et al.).

[0231] in some embodiments, the humanized 2H7 variants are variants comprising the light chain variable domain of SEQ ID NO: 2 and the heavy chain variable domain of SEQ ID NO: 8, including variants with or without Fc region substitutions (if present), and variants containing the variable domain of the heavy chain with an N100A alteration; or D56A and N100A; or D56A, N100Y, and S100aR; in SEQ ID NO: 8 and the variable domain of the light chain with a change in M32L; or S92A; or M32L and S92A; in SEQ ID NO: 2. M34 in the variable domain of the heavy chain 2H7.v16 has been identified as a potential source of antibody stability and is another potential candidate for replacement.

[0232] In some embodiments, the variable region of the 2H7.v16-based variants contains the v16 amino acid sequences except for the amino acid substitution positions that are indicated in Table 1 below. Unless otherwise indicated, the 2H7 variants have the same light chain as v16.

Table 1
Exemplary Variants of Humanized 2H7 Antibody Version 2H7 Heavy chain changes Light chain changes Fc changes (V _H ) (V _L ) 16 as reference - 31 - - S298A, E333A, K334A 73 N100A M32L 75 N100A M32L S298A, E333A, K334A 96 D56A, N100A S92A 114 D56A, N100A M32L, S92A S298A, E333A, K334A 115 D56A, N100A M32L, S92A S298A, E333A, K334A, E356D, M358L 116 D56A, N100A M32L, S92A S298A, K334A, K322A 138 D56A, N100A M32L, S92A S298A, E333A, K334A, K326A 477 D56A, N100A M32L, S92A S298A, E333A, K334A, K326A, N434W 375 - - K334L 588 - - S298A, E333A, K334A, K326A 511 D56A, N100Y, S100aR M32L, S92A S298A, E333A, K334A, K326A

(iv) Human antibodies

[0233] As an alternative to humanization, you can get human antibodies. For example, it is currently possible to obtain transgenic animals (eg, mice) that are capable, upon immunization, of producing a complete set of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain junction (J _H ) gene in chimeric mice and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene system in such germline mutant mice results in the production of human antibodies upon antigenic stimulation. See, for example, 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 US Pat. Nos. 5,591,669; 5589369 and 5545807.

[0234] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) from immunoglobulin variable (V) domain gene sets from non-immunized donors can be used to generate human antibodies and antibody fragments in vitro. In accordance with this method, antibody V domain genes are cloned in frame of either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and are displayed as functional antibody fragments on the surface of the phage particle. Due to the fact that the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody will also lead to selection of the gene encoding the antibody exhibiting such properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in various forms; for a review, see, for example, 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 variety of anti-oxazolone antibodies from a small random combinatorial V-gene library obtained from the spleens of immunized mice. A set of V genes can be constructed from non-immunized human donors and antibodies against a wide range of antigens (including self antigens) can be isolated, principally according to the methods described by Marks et al., J. Mol Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Nos. 5,565,332 and 5,573,905.

[0235] Human antibodies can also be obtained through activated B cells in vitro (see US patents 5567610 and 5229275).

(v) Antibody fragments

[0236] Various methods have been developed to obtain antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of full-length antibodies (see, for example, 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 obtained directly using recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be isolated directly from E. coli and chemically coupled to generate F (ab ') ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') ₂ fragments can be isolated directly from a recombinant host cell culture. Other methods of obtaining fragments of antibodies will be obvious to the skilled artisan. In other embodiments, the preferred antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US patent No. 5571894; and US Pat. No. 5,587,458. The antibody fragment can also be a "linear antibody", for example, as described in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

(vi) Bispecific antibodies

[0237] Bispecific antibodies are antibodies that have binding specificity for at least two different epitopes. Illustrative bispecific antibodies can bind to two different epitopes of the B-cell surface marker. Other such antibodies can bind a B-cell surface marker and, in addition, bind a second distinct B-cell surface marker. Alternatively, an arm that binds to a cell surface marker B can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or IgG Fc receptors (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so that the cellular defense mechanisms are focused on the B cell. Bispecific antibodies can also be used to localize cytotoxic agents to a B cell. These antibodies have an arm that binds a B-cell surface marker and an arm that binds a cytotoxic agent (eg, saporin, anti-interferon-α antibody, vinca alkaloid, ricin A chain, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be produced as full length antibodies or antibody fragments (eg, bispecific antibodies F (ab ') ₂ ).

[0238] Methods for producing bispecific antibodies are known in the art. A common method for producing full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Due to the random assembly of heavy and light chains of immunoglobulins, such hybridomas (quadromas) potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecules, which is usually carried out by means of affinity chromatography steps, is rather laborious and the product yield is low. Similar methods are described in WO 93/08829, and Traunecker et al., EMBO J., 10: 3655-3659 (1991).

[0239] In accordance with another approach, the antibody variable domains with the desired binding specificity (antibody-antigen binding sites) are fused with immunoglobulin constant domain sequences. In some embodiments, the implementation is fusion with a constant domain of the heavy chain of an immunoglobulin containing at least a portion of the hinge region, CH2 and CH3. In some embodiments, at least one of the fusion components has a first heavy chain constant domain (CH1) containing a site required for light chain binding. DNA encoding the components of the immunoglobulin heavy chains and, if desired, the immunoglobulin light chain to be fusion, are inserted into separate expression vectors and cotransfected into a suitable host organism. This provides significant flexibility in adjusting the ratios of the three polypeptide fragments to each other in embodiments where unequal ratios of the three polypeptide chains in the construct provide optimal yield. However, it is possible to insert two or all three polypeptide chains into one expression vector if the expression of at least two polypeptide chains in equal proportions results in a high yield of the product or if the proportions are not particularly important.

[0240] In some embodiments of this approach, the bispecific antibodies consist of a hybrid immunoglobulin heavy chain with one binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (having a different binding specificity) on the other arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound and unwanted immunoglobulin chain combinations, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides an easy way of separation. This approach is described in WO 94/04690. For a more detailed description of the preparation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0241] In accordance with another approach described in US Pat. No. 5,731,168, to maximize the percentage of heterodimers that are isolated from recombinant cell cultures, an interface between a pair of antibody molecules can be engineered. In some embodiments, the interface comprises at least a portion of the C _H 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the contact area of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensating "cavities" of identical or similar size to the large side chain (s) are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller chains (eg, alanine or threonine). This provides a mechanism to increase the yield of heterodimer over other unwanted end products such as homodimers.

[0242] Bispecific antibodies include cross-linked antibodies or "heteroconjugates" of antibodies. For example, one of the antibodies in the heteroconjugate may bind to avidin and the other to biotin. Such antibodies, for example, have been proposed for targeting cells of the immune system against unwanted cells (US patent No. 4676980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugated antibodies can be prepared using any suitable cross-linking method. Suitable crosslinking agents are well known in the art and are described in US Pat. No. 4,676,980, in conjunction with a number of crosslinking techniques.

[0243] Methods for producing bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be produced using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a method wherein full-length antibodies are proteolytically cleaved to generate F (ab ') ₂ fragments. These fragments are reduced in the presence of a sodium arsenite compound that forms complexes with dithiols to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide. The resulting Fab 'fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then re-converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to obtain a bispecific antibody. The obtained bispecific antibodies can be used as agents for selective immobilization of enzymes.

[0244] Various methods have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kositelny et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab 'regions of two different antibodies through gene fusion. Antibody homodimers were reduced in the hinge region to give monomers and then reoxidized to give antibody heterodimers. This method can also be used to obtain homodimers of antibodies. The diabody method described by Hollinger et al., Proc. Natl. Acad. Sci USA 90: 6444-6448 (1993) provides an alternative mechanism for producing bispecific antibody fragments. Fragments contain a heavy chain variable domain (V _H ) linked to a light chain variable domain (V _L ) via a linker that is too short to allow pairing between two domains on the same chain. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of another fragment, thus forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single chain Fv (sFv) dimers has also been described. See Gruber et al., J. Immunol, 152: 5368 (1994).

[0245] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be produced. Tutt et al. J. Immunol. 147: 60 (1991).

V. Conjugates and Other Antibody Modifications

[0246] An antibody used in the methods and included in the articles of manufacture described herein is optionally conjugated to a cytotoxic agent. For example, an antibody can be conjugated to a drug as described in WO2004 / 032828.

[0247] Chemotherapeutic agents suitable for preparing such antibody-cytotoxic agent conjugates are described above.

[0248] Also provided within the scope of the present invention are conjugates of an antibody and one or more low molecular weight toxins such as calicheamicin, maytansine (US Pat. No. 5,208,020), trichotene and CC1065. In one embodiment, the antibody is conjugated to one or more maytansine molecules (eg, about 1 to about 10 maytansine molecules per antibody molecule). Maytansine, for example, can be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with a modified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) to form a maytansinoid-antibody conjugate.

[0249] Alternatively, the antibody is conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family are capable of forming double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG, and θ ^I ₁ (Hinman et al., Cancer Res. 53 : 3336-3342 (1993) and Lode et al., Cancer Res. 58: 2925-2928 (1998)).

[0250] Enzymatically active toxins and fragments thereof that can be used include A-chain of diphtheria toxin, non-binding active fragments of diphtheria toxin, A-chain of exotoxin (from Pseudomonas aeruginosa), A-chain of ricin, A-chain of abrin, A-chain modecin, alpha-sarcin, Aleurites fordii proteins, diantan proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restriction triceccinome, phenycinome ... See, for example, WO 93/21232 published October 28, 1993.

[0251] In addition, the present invention provides an antibody conjugated to a compound with nucleolytic activity (eg, ribonuclease or a DNA endonuclease such as deoxyribonuclease; DNase).

[0252] A variety of radioactive isotopes 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 Lu radioactive isotopes.

[0253] Conjugates of an antibody and a cytotoxic agent can be prepared using various bifunctional protein binding agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1- carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidylsubrate), aldehydes (such as glutaraldehyde), bis-azidobenzanediamino derivatives, bis-diazonium (such as bis- (p-diazonium benzoyl) ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and secondary active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin-based immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatebenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an illustrative chelating agent for conjugating a radionucleotide to an antibody. See WO94 / 11026. The linker can be a "cleavage linker" that facilitates the release of a cytotoxic drug into the cell. For example, an acid labile linker, a peptidase sensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al. Cancer Research 52: 127-131 (1992)).

[0254] Alternatively, a fusion protein containing an antibody and a cytotoxic agent can be produced, for example, by recombinant methods or peptide synthesis.

[0255] In another embodiment, an antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-targeting, where the antibody-receptor conjugate is administered to a patient and then the unbound conjugate is removed from the circulation using a clearance agent, and then a "ligand" (eg, avidin) that is conjugated to a cytotoxic agent (eg, a radionuclide) is administered.

[0256] The antibodies of the present invention can also be conjugated to a prodrug activating enzyme that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO81 / 01145) into an active anti-cancer drug. See, for example, WO 88/07378 and US patent No. 4975278.

[0257] The enzyme component of such conjugates includes any enzyme capable of acting on a prodrug so that it is converted to its more active cytotoxic form.

[0258] Enzymes that are useful in the method of the present invention include, but are not limited to, alkaline phosphatase, useful for converting phosphate-containing prodrugs to free drugs; arylsulfatase, which is useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminase, which is useful for converting non-toxic 5-fluorocytosine into an anti-cancer drug, 5-fluorouracil; proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs; D-alanyl carboxypeptidases, which are useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-degrading enzymes such as β-galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs to free drugs; β-lactamase, which is suitable for converting β-lactam derived drugs into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are useful for converting drugs derivatized at their amino nitrogen atoms 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 prodrugs to free active drugs (see, eg, Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein to deliver the abzyme to a population of tumor cells.

[0259] The enzymes of the present invention can be covalently linked to an antibody by methods well known in the art, such as the use of the heterobifunctional cross-linking reagents discussed above. Alternatively, fusion proteins containing at least an antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, for example, Neuberger et al., Nature 312: 604-608 (1984)).

[0260] Other modifications of the antibody are provided within the scope of the present invention. For example, the antibody can be linked to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, antibody fragments, such as Fab ', are linked to one or more PEG molecules.

[0261] The antibodies described herein can also be formulated as liposomes. Liposomes containing the antibody are 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); US Pat. Nos. 4,485,045 and 4,544,545; and WO97 / 38731 published October 23, 1997. Liposomes with extended circulation times are described in US Pat. No. 5,013,556.

[0262] Particularly useful liposomes can be prepared by a reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a defined pore size to obtain liposomes of the desired diameter. Fab 'fragments of an antibody of the present invention can be conjugated to liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide exchange reaction. The chemotherapeutic agent is not necessarily contained in the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

[0263] Modification (s) of the amino acid sequence of the antibody is contemplated. For example, it may be desirable to increase the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide substitutions into an antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution is performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid substitutions can also alter the post-translational processes of antibodies, such as changing the number or position of glycosylation sites.

[0264] A suitable method for identifying specific residues or regions of an antibody that are preferred regions for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). In this method, a target residue or group of residues (for example, charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to disrupt the interaction of amino acids with the antigen ... Those amino acid positions that demonstrate functional susceptibility to substitution are then confirmed by introducing additional or different variants at, or instead of, the sites of substitution. Thus, since the site for making a change in the amino acid sequence is predetermined, there is no need for an immediate predetermination of the nature of the mutation. For example, to analyze the effect of a mutation in a given region, ala scanning or random mutagenesis is performed at a target codon or site, and the expressed antibody variants are screened for the desired activity.

[0265] Amino acid sequence insertions include N- and / or C-terminal fusion constructs ranging in length from one residue to polypeptides containing one hundred or more residues, intrasequence insertions of one or more amino acid residues. Examples of terminal inserts include an N-terminal methionyl residue antibody or an antibody fused to a cytotoxic polypeptide. Other variants of the antibody molecule with inserts include fusion constructs of the N- or C-terminus of an antibody with an enzyme or polypeptide that increases the half-life of the antibody.

[0266] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by another residue. Sites of greatest interest for mutagenesis with substitutions include hypervariable regions, however, FR changes are also contemplated. Conservative substitutions are presented in Table 2 under the heading "Preferred Substitutions". If such substitutions result in a change in biological activity, then more substantial changes, referred to as “exemplary substitutions” in Table 2, can be made and the products screened.

table 2 Original residue Illustrative replacements Preferred substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0267] Substantial modifications to the biological properties of an antibody are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, such as, for example, a layer or helical conformation, (b) the charge or hydrophobicity of the molecule in site of the target, or (c) the volume of the side chain. Amino acids can be classified into groups based on the similarity of their side chains: (A. L. Lehninger, 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 polar: 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)

[0268] Alternatively naturally occurring residues can be subdivided into groups based on the general properties of the side chains:

(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.

[0269] Non-conservative substitutions involve replacing a member of one of these classes with a member from another class.

[0270] Any cysteine residue not involved in maintaining the proper conformation of the antibody can also be replaced, mainly by serine, to increase the oxidative stability of the molecule and to prevent aberrant crosslinking. In contrast, cysteine bond (s) can be added to the polypeptide to increase its stability (in particular, where the antibody is an antibody fragment, such as an Fv fragment).

[0271] A particularly preferred type of substitution variant comprises the substitution of one or more hypervariable region residues of the parent antibody. Typically, the resulting variant (s) selected for further development will have improved biological properties relative to the parent antibody from which it is derived. A convenient way to obtain such substitutional variants involves affinity maturation using phage display. Briefly, several regions of the hypervariable region (eg, 6-7 regions) are mutated to obtain all possible amino acid substitutions in each region. The antibody variants thus obtained are displayed monovalently on filamentous phage particles as molecules fused to the M13 gene III product packaged in each particle. Phage-exposed variants are then screened for their biological activity (eg, binding affinity) as described herein. Alanine scanning mutagenesis can be performed to identify candidate hypervariable region regions for modification, identifying hypervariable region residues that significantly contribute to antigen binding. Alternatively or additionally, it may be useful to analyze the crystal structure of the antigen-antibody complex in order to identify points of contact between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to methods known in the art, including those described herein. Once such variants are obtained, a panel of variants is screened as described herein and the antibodies with the best properties in one or more of the appropriate assays can be selected for further development.

[0272] Another type of amino acid variant of the polypeptide alters the original glycosylation pattern of the antibody. Such a change includes the deletion of one or more carbohydrate moieties found on the antibody and / or the addition of one or more glycosylation sites that are not present on the antibody.

[0273] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a 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 recognized sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any 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, usually serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0274] The addition of glycosylation sites to a polypeptide is conveniently performed by changing the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can also be carried out by adding or replacing one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

[0275] When the antibody contains an Fc region, you can change the carbohydrate associated with it. For example, antibodies with a mature carbohydrate structure that lacks fucose associated with the Fc region of the antibody are described in US Patent Application No. US 2003/0157108 A1 (Presta, L.); also see US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd) regarding CD20 antibody composition. Antibodies with bisecting N-acetylglucosamine (GlcNAc) in carbohydrate linked to the Fc region of the antibody are described in WO03 / 011878, Jean-Mairet et al. and US Pat. No. 6,602,684 to Umana et al. Antibodies with at least one galactose residue in the oligosaccharide linked to the Fc region of the antibody are described in WO97 / 30087 (Patel et al.); also see WO98 / 58964 (Raju, S.) and WO99 / 22764 (Raju, S.) concerning antibodies with altered carbohydrate linked to the Fc region.

[0276] In some embodiments, the glycosylation variant described herein comprises an Fc region, where the carbohydrate structure associated with the Fc region is fucose free. These variants have an improved ADCC function. Optionally, the Fc region further contains one or more amino acid substitutions that further enhance ADCC, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (residue numbering according to Eu). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US Patent Application No. US 2003/0157108 A1, Presta, L; WO 00 / 61739A1; WO 01 / 29246A1; US 2003/0115614A1; US 2002/0164328A1; US 2004 / 0093621A1; US 2004/0132140A1; US 2004 / 0110704A1; US 2004/0110282A1; US 2004 / 0109865A1; WO 03 / 085119A1; WO 03 / 084570A1; WO 2005/035778; WO 2005/035586 (describing RNA inhibition of (RNA-i) fucosylation); Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L ; and WO 2004/056312 A1, Adams et al., especially Example 11), and knockout cell lines such as CHO cells with a knockout of the alpha-1,6-fucosyltransferase FUT8 gene (Yamane-Ohnuki et al. Biotech. Bioeng . 87: 614 (2004)).

[0277] Nucleic acid molecules encoding amino acid sequence variants of an antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or obtaining by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of a previously obtained variant or not a variant of the antibody version.

[0278] It may be desirable to modify an antibody of the invention with respect to effector function, for example, to enhance 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 substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residue (s) can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. A homodimeric antibody thus obtained may have increased internalization capacity and / or increased complement mediated cell killing and antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional crosslinking agents as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has two Fc regions and thus can have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

[0279] WO 00/42072 (Presta, L.) describes antibodies with enhanced ADCC function in the presence of human effector cells, where the antibodies contain amino acid substitutions in their Fc region. In some embodiments, the ADCC-enhanced antibody comprises substitutions at positions 298, 333, and / or 334 of the Fc region. In some embodiments, the altered Fc region is a human IgG1 Fc region containing or consisting of substitutions at one, two, or three of these positions.

[0280] Antibodies with altered C1q binding and / or complement dependent cytotoxicity (CDC) are described in WO99 / 51642, US patent No. 6194551B1, US patent No. 6242195B1, US patent No. 6528624B1 and US patent No. 6538124 (Idusogie et al.). Antibodies contain an amino acid substitution at one or more of the amino acid positions 270, 322, 326, 327, 329, 313, 333 and / or 334 of their Fc region.

[0281] To increase the serum half-life of an antibody, a salvage receptor binding epitope may be included in an antibody (especially an antibody fragment), as described, for example, in US Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (eg, IgG ₁ , IgG ₂ , IgG _3, or IgG ₄ ) that is responsible for increasing the serum half-life of an IgG molecule in vivo. Antibodies with Fc region substitutions and extended half-lives are also described in WO 0/42072 (Presta, L.).

[0282] Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Application No. US2002 / 0004587 A1, Miller et al.).

Vi. Pharmaceutical formulations

[0283] Therapeutic formulations of antibodies used in accordance with the present invention are prepared for storage by mixing antibodies of the required purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980 )), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl- or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

[0284] Exemplary anti-CD20 antibody formulations are described in WO 98/56418. This publication describes a liquid multi-dose formulation containing 40 mg / ml rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0, which has a minimum shelf life of two years at 2-8 ° C. Another anti-CD20 antibody formulation of interest contains 10 mg / ml rituximab in 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.

[0285] Lyophilized formulations adapted for subcutaneous administration are described in US Pat. No. 6,267,958 (Andya et al.). Such lyophilized formulations can be diluted with a suitable diluent to a high protein concentration, and the diluted formulation can be administered subcutaneously to a mammal to be treated in accordance with the present invention.

[0286] Crystallized forms of the antibody are also contemplated. See, for example, US 2002/0136719 A1 (Shenoy et al.).

[0287] A formulation within the scope of the present invention may also contain more than one active compound, if necessary for the particular indication being treated, in some embodiments, compounds with complementary activity that do not adversely affect each other. For example, it may be desirable to additionally provide a cytotoxic agent; a chemotherapeutic agent; an immunosuppressive agent; cytokine; a cytokine antagonist or antibodies to it; growth factor; hormone; integrin; integrin antagonist or antibody thereto (e.g., anti-LFA-1 or anti-alpha-4 integrin (such as natalizumab / TYSABRI ^®), available from Biogen Idec / Elan Pharmaceuticals, Inc.) ; interferon class drug such as IFN-beta-1a (REBIF ^® and AVONEX ^®) or IFN-beta-1b (BETASERON ^®); an oligopeptide such as glatiramer acetate (COPAXONE ^®); a cytotoxic agent, such as (NOVANTRONE ^®), methotrexate, cyclophosphamide, chlorambucil, or azathioprine; intravenous immunoglobulin (gamma globulin); a lymphocyte-depleting drug (eg, mitoxantrone, cyclophosphamide, campat, anti-CD4 antibody, or cladribine); a non-lymphocyte-depleting immunosuppressive drug (eg, mycophenolate mofetil (MMF) or cyclosporine); a cholesterol-lowering drug of the "statin"class;estradiol;testosterone; hormone replacement therapy; a drug that treats symptoms secondary to or associated with MS (eg, spasticity, incontinence, pain, fatigue); a TNF inhibitor; a disease modifying antirheumatic drug (DMARD); non-steroidal anti-inflammatory drug (NSAID); a corticosteroid (eg, methylprednisolone, prednisone, dexamethasone, or glucocorticoid); levothyroxine; cyclosporin A; a somatostatin analog; a cytokine antagonist; antimetabolite; an immunosuppressive agent; an integrin antagonist or antibodies thereto (for example, an anti-LFA-1 antibody such as efalizumab or an alpha-4 integrin antibody such as natalizumab); or another B-cell surface antagonist / antibody; etc. in the composition. The type and effective amounts of such other agents depend, for example, on the amount of antibody present in the formulation, the type of multiple sclerosis being treated, and the clinical parameters of the patients. They are usually used in the same dosages and with the same routes of administration as used previously, or from about 1 to 99% of the previously used dosages.

[0288] The active ingredients can also be enclosed in microcapsules obtained, for example, by coacervation or inter-surface polymerization methods, such as microcapsules based on hydroxymethyl cellulose or gelatin and microcapsules based on polymethyl methacrylate, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0289] You can get drugs with sustained release. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyethers, hydrogels (e.g., poly- (2-hydroxyethyl methacrylate) or polyvinyl alcohol, polylactides (U.S. Patent No. lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ (injection microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid.

[0290] Formulations for use for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

[0291] In some embodiments, the formulation comprises 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 administering an anti-CD20 antibody are provided in Andya et al., US 2006/0088523, which is incorporated herein by reference in its entirety with respect to formulations.

[0292] Exemplary anti-CD20 antibody formulations are described in Andya et al., US 2006/0088523 and WO 98/56418, which are incorporated by reference in their entirety. In some embodiments, the formulation is a liquid multi-dose formulation containing anti-CD20 antibody at a dose of 40 mg / ml, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0, which has a minimum shelf life of two years at 2-8 ° C. In some embodiments, the anti-CD20 antibody formulation of interest comprises 10 mg / ml antibody in 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 an aqueous pharmaceutical formulation containing 10-30 mM sodium acetate at about pH 4.8 to about pH 5.5, preferably at pH 5.5, polysorbate 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. 6171586, which is incorporated herein by reference in its entirety). Lyophilized formulations adapted for subcutaneous administration are described in WO 97/04801, which is incorporated by reference in its entirety. Such lyophilized formulations can be diluted with a suitable diluent to a high protein concentration, and the diluted formulation can be administered subcutaneously to a mammal to be treated in accordance with the present invention.

[0293] In some embodiments, the formulation of the humanized 2H7 variants is antibodies at a dose of 12-14 mg / ml in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH 5.8. In a specific embodiment, variants 2H7 and in particular 2H7.v16 are formulated at a dose of 20 mg / ml of antibody in 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20 and sterile water for injection at pH 5 ,eight. In a specific embodiment, one IV formulation of humanized 2H7.v16 is: 30 mg / ml antibody in 20 mM sodium acetate, 4% trehalose dihydrate, 0.02% polysorbate 20 (Tween 20 ™), pH 5.3. In some embodiments, the formulation of the humanized 2H7.v511 variant is 15-30 mg / ml antibody, preferably 20 mg / ml antibody, in 10 mM histidine sulfate, 60 mg / ml sucrose (6%), 0.2 mg / ml polysorbate 20 (0.02%) and sterile water for injection at pH 5.8. In another embodiment, the formulation for variants 2H7 and in particular 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 an antibody at a concentration of 15-25 mg / ml, preferably 20 mg / ml, in 20 mM sodium acetate, 240 mM (8%) trehalose dihydrate, 0.02% polysorbate 20, pH 5 , 3. In some embodiments, the anti-CD20 antibody (eg, 2H7.v16) is formulated containing 30 mg / ml antibody, 20 mM sodium acetate, 106 mM trehalose, 0.02% polysorbate 20, and pH 5.3. The liquid formulation containing the antibody can be in an amount of 300 mg / vial and can be stored at 2-8 ° C, protected from light. In some embodiments, the antibody formulation is diluted with normal saline (0.9% sodium chloride) in an IV bag by infusion prior to administration.

Vii. Products and manufacturing methods

[0294] In addition, the invention relates to articles containing materials suitable for the treatment of progressive multiple sclerosis described herein. In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of patients with multiple sclerosis and provides an improvement in functional ability in patients, having multiple sclerosis.

[0295] In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of patients with multiple sclerosis and inhibits the progression of disability in patients with multiple sclerosis.

[0296] In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of patients with multiple sclerosis and slows the onset of confirmed progression disability in patients with multiple sclerosis. In some embodiments, the confirmed disease progression is an increase in EDSS that persists for twelve weeks. In some embodiments, the confirmed disease progression is an increase in EDSS that persists for twenty-four weeks.

[0297] In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of patients with multiple sclerosis and slows or prevents a decrease in the volume of the brain in patients with multiple sclerosis.

[0298] In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of 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 highly active multiple sclerosis patients who have not previously been treated with another multiple sclerosis therapy. In some embodiments, the label further indicates that the anti-CD20 antibody or pharmaceutical composition is indicated for treating highly active multiple sclerosis patients who have previously been treated with another multiple sclerosis therapy. 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 an inadequate response to another multiple sclerosis therapy.

[0299] In some embodiments, the article of manufacture contains, packaged together, a pharmaceutical composition comprising an anti-CD20 antibody and a pharmaceutically acceptable carrier and a label indicating that the anti-CD20 antibody or pharmaceutical composition is indicated for the treatment of patients with multiple sclerosis and that the anti-CD20 antibody or the pharmaceutical composition is effective for one or more of the following: (1) reducing the amount of plaque in the patient's brain; (2) a decrease in the average annual relapse rate; (3) reducing the progression of disability; and (4) increased functional ability.

[0300] In some embodiments of any of the articles, the multiple sclerosis is progressive multiple sclerosis. In certain embodiments, progressive multiple sclerosis is primary progressive multiple sclerosis. In some embodiments, multiple sclerosis is a recurrent form of multiple sclerosis. In certain embodiments, the relapsing form of multiple sclerosis is relapsing-remitting multiple sclerosis. In certain embodiments, the recurrent form of multiple sclerosis is secondary progressive multiple sclerosis with associated relapses (rSPMS).

[0301] In some embodiments of any of the articles of manufacture, 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.

[0302] In certain embodiments, the package insert has instructions that indicate (i.e. indicate) that an amount of ocrelizumab is administered to the patient that is effective to provide an initial exposure to ocrelizumab of about 0.3 to about 0.6 grams per second followed by a second exposure to ocrelizumab from about 0.3 to about 0.6 grams, with the second exposure not applied until about 16 to 60 weeks after the initial exposure, and each exposure to ocrelizumab is provided to the patient as one or two doses of ocrelizumab. In some embodiments, the initial exposure to ocrelizumab is approximately 0.6 grams. In some embodiments, the second exposure to ocrelizumab is approximately 0.6 grams. In some embodiments, the second exposure occurs approximately 24 weeks after the initial exposure. In some embodiments, one or more treatments of ocrelizumab are provided to the patient as a single dose of ocrelizumab. In some embodiments, one or more treatments of ocrelizumab are provided to the patient as two doses of ocrelizumab. In some embodiments, the two doses of ocrelizumab contain about 0.3 grams of ocrelizumab.

[0303] The product contains a container and a container label or package insert attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be made from a variety of materials such as glass or plastic. The container contains or contains a composition that is effective for treating multiple sclerosis and can have a sterile access opening (for example, the container can be an intravenous solution bag or a vial containing a permeable hypodermic needle plug). At least one active substance 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.

[0304] The label or package insert indicates that the composition is used to treat multiple sclerosis in a patient suffering from it, with specific guidance regarding dosage amounts and dosage intervals of the antibody and any other medication provided. In addition, the article may contain a second container containing a pharmaceutically acceptable diluent or buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, the article may include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0305] Optionally, an article of manufacture within the scope of the present invention further comprises a container containing an agent other than an antibody for treatment and further contains instructions for treating a patient with such an agent, which agent is preferably a chemotherapeutic agent or immunosuppressive agent, a drug of the class interferons, such as IFN-beta-1a (REBIF ^® and AVONEX ^®) or IFN-beta-1b (BETASERON ^®); an oligopeptide such as glatiramer acetate (COPAXONE ^®); a cytotoxic agent such as mitoxantrone (NOVANTRONE ^®), methotrexate, cyclophosphamide, chlorambucil, or azathioprine; intravenous immunoglobulin (gamma globulin); a lymphocyte-depleting drug (eg, mitoxantrone, cyclophosphamide, campat, anti-CD4 antibody, or cladribine); a non-lymphocyte-depleting immunosuppressive drug (eg, mycophenolate mofetil (MMF) or cyclosporine); a cholesterol-lowering drug of the "statin"class;estradiol; hormone replacement therapy; a drug that treats symptoms secondary to or associated with MS (eg, spasticity, incontinence, pain, fatigue); a TNF inhibitor; disease modifying antirheumatic drug (DMARD); non-steroidal anti-inflammatory drug (NSAID); a corticosteroid (eg, methylprednisolone, prednisone, dexamethasone, or glucocorticoid); levothyroxine; cyclosporin A; a somastatin analog; a cytokine or cytokine receptor antagonist; antimetabolite; immunosuppressive agent; an integrin antagonist or antibody thereto (for example, an anti-LFA-1 antibody such as efalizumab or an anti-alpha 4 integrin antibody such as natalizumab); and another antibody against a B cell surface marker; etc.

EXAMPLES

Example 1: Phase III Trials of Ocrelizumab Compared to Interferon Beta-1a (Rebif) in Patients with Recurrent Multiple Sclerosis

[0306] Multiple sclerosis is a heterogeneous disease with an unpredictable 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 for recurrent forms of multiple sclerosis that have become available in recent years, neurological disability continues to increase in many patients, thus 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 to date has hindered their early use (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).

[0307] B cells are a key contributor to the pathogenesis of multiple sclerosis (Monson (2005) J Neuroimmunol. 158, 170-81; Hauser SL. (2015) Mult Scler. 21, 8-21). B cells are rarely seen in the cerebrospinal fluid of healthy controls, but they are often found in 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), their increased levels in cerebrospinal fluid correlate with more rapid disease progression in relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis (Cepok (2005) Brain. 128, 1667-76). B cells influence the underlying pathogenesis of multiple sclerosis through a number of 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). Interest in B cells has increased following proof-of-concept and experimental studies, and interest in their applicability in multiple sclerosis has increased 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).

[0308] 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 and plasmacytes (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, a chimeric anti-CD20 monoclonal antibody, significantly reduced inflammatory plaques in the brain and clinical relapses compared to placebo in patients with relapsing-remitting multiple sclerosis; thus providing evidence that selective CD20 ⁺ B cell depletion is a potentially effective treatment approach for multiple sclerosis (Kappos et al. (2011) Lancet. 378, 1779-87).

[0309] 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 maintaining the ability to repair B cells 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 to the large extracellular loop CD20 with high affinity, selectively depleting 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. 5 22-33).

[0310] Conducted two identical multicenter, randomized, double-blind, placebo-controlled phase 3 trials with parallel groups (trial I and trial II) to investigate the efficacy and safety of ocrelizumab versus interferon (IFN) β-1a in patients with recurrent forms of multiple sclerosis. The results of these two studies are described below.

The ways

Eligibility criteria and exclusions

[0311] Key eligibility criteria included: age 18 to 55; 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); an Extended Disability Assessment Scale (EDSS, see world-wide-web.neurostatus.org) score of 0 to 5.5 at screening; at least two documented clinical attacks in the previous 2 years or one clinical attack in the year before screening (but not within 30 days before screening); documented MRI examination of the brain with abnormalities consistent with multiple sclerosis; neurological stability for at least 30 days prior to both screening and baseline visit.

[0312] Key exclusion criteria included: diagnosis of primary progressive multiple sclerosis; patients with a disease duration of more than 10 years in combination with an EDSS of less than or equal to 2.0 at screening; the known presence of other neurological disorders that can mimic multiple sclerosis; pregnancy or lactation; prior treatment with any B-cell targeted therapies or other contraindicated drugs (i.e. need for long-term treatment with systemic corticosteroids or immunosuppressants during the study, prior or secondary immunodeficiency in history or currently active, active infection or recurrent or chronic a history of infection or its known presence (eg, hepatitis B or C, HIV, syphilis, tuberculosis), a history of progressive multifocal leukoencephalopathy, contraindications or intolerance to oral or intravenous corticosteroids, or contraindications to Rebif Rebif or incompatibility with the use of Rebif Rebif).

Study design

[0313] Patients were randomly assigned (1: 1) to either ocrelizumab, 600 mg, by intravenous infusion every 24 weeks (administered as two infusions of 300 mg on days 1 and 15 for the first dose, and as a single infusion of 600 mg on day 1 for each 24-week course of treatment thereafter), or subcutaneous IFNβ-1a three times a week at a dose of 44 μg over a 96-week treatment period. See Fig. 7. Patients in the ocrelizumab group and in the IFNβ-1a group were also injected subcutaneously and intravenously with placebo, respectively. The randomization was carried out by a centrally independent organizer. Patients were stratified by region (US / rest of the world) and baseline EDSS (less than 4 / greater or equal to 4).

[0314] To maintain the concealment of study group allocation, each study center had separate treating investigators (neurologists experienced in the treatment of multiple sclerosis) and evaluating researchers (neurologists or other health care practitioners), all of whom the entire trial was was blind. Treating investigators had access to safety data and latent efficacy data and made treatment decisions based on the patient's clinical response data and laboratory data. Evaluating investigators performed neurological assessments, including EDSS scores (Kurtze (1983) "Rating neurologic impairment in multiple sclerosis: an Expanded Disability Status Scale (EDSS)." Neurology. 33, 1444-52), functional systemic indicators (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), complex functional scale for multiple sclerosis (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), low contrast visual acuity testing is associated with cognitive performance in multiple sclerosis: a pilot study. BMC Neurology. 13, 167), vol. eats on character-number matching (SDMT) (Smith A. (1982). 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 assessment was carried out independently by a central MRI investigator who was blinded for treatment allocation.

[0315] Patients who completed the 96-week treatment period were eligible for inclusion in the open, extended phase of the trial. Patients who discontinued participation prematurely or who did not want to participate in the open renewal were included in a 48-week follow-up phase for a safety assessment that included B-cell monitoring.

Test methods

[0316] EDSS scores were determined at screening, at baseline and every 12 weeks; MSFC, LCVA and SDMT scores were measured at baseline and every 12 weeks; and the Karnofsky General Condition Scale was assessed at baseline and every 24 weeks. MRIs were performed at baseline and at 24, 48 and 96 weeks. Adverse events were monitored throughout the study.

[0317] The primary outcome was the average annual rate of protocol-defined relapses at 96 weeks, where relapses were defined as new or worsening neurological symptoms persisting for more than 24 hours that are only explainable by multiple sclerosis. New or worsening neurological symptoms should be accompanied by objective neurological deterioration consistent with an increase in EDSS of at least half a grade, an increase of 2 points in the functional systemic EDSS score, or 1 point in two or more functional systemic EDSS scores. Relapses were classified as protocol-defined relapses using an automated algorithm following the rules described above. The algorithm was written before closing the database and disclosing the data.

[0318] Key secondary outcomes included: time to onset of confirmed 12-week disability progression (ie, CDP), defined in the trial as an increase of at least 1.0 points from baseline EDSS in patients with a baseline of 5.5 or less, or an increase of 0.5 points in patients with a baseline above 5.5, at 96 weeks, where the increase in EDSS was confirmed at regular scheduled visits at least 12 weeks after the initial neurological deterioration; the total number of gadolinium-enhanced T1 plaques at 24, 48, and 96 weeks; the total number of new and / or increasing hyperintense T2 plaques at 24, 48 and 96 weeks; proportion of patients who had a confirmed 12-week reduction in disability (ie, CDI) over 96 weeks (analyzed only for a subgroup of patients with a baseline EDSS of at least 2.0); time to onset of 24-week CDP, confirmed 24 weeks after initial neurological deterioration, over 96 weeks; the pharmacokinetics, immunogenicity and pharmacodynamics of ocrelizumab; and the safety and tolerability of ocrelizumab. CDI in a trial is defined as a decrease in EDSS of at least 1.0 points from baseline in patients with a baseline EDSS of 5.5 or less, or a decrease of 0.5 in patients with a baseline EDSS greater than 5.5 ...

Statistical analysis

[0319] The statistical hierarchy is shown in FIG. 8. To achieve sufficient statistical power to investigate the effects of ocrelizumab on CDP and CDI, it was planned in advance that these results would be pooled for both phase III trials. All other results were analyzed separately for each trial. Primary and secondary efficacy analyzes were performed on a sample of all patients with intent to be treated.

[0320] All efficacy analyzes were performed on a sample of all intent-to-treat patients. The mean annual relapse rate (ARR), which is the primary outcome of efficacy, was analyzed using a negative binomial model, which for each patient included the occurrence between the date of randomization and the date of early discontinuation / 96 weeks in statistical analysis, considering the duration of exposure, treatment group, region (USA / rest) and baseline EDSS (less than 4.0 / greater than or equal to 4.0) as covariates. A significant result at a two-tailed alpha value of 0.05 demonstrates the superior effect of ocrelizumab in reducing ARR.

[0321] The sample size for each trial was based on an estimated mean annual 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 concluded that a sample of 400 patients per group would provide 84% statistical power to maintain a Type I error rate of 0.05 and detect a 50% relative reduction for ocrelizumab versus IFNβ-1a (assuming a dropout rate of 20 %). Sensitivity analysis per protocol assessed the effect of significant protocol violations on the primary outcome.

[0322] Ten secondary efficacy outcomes were investigated in a hierarchical order of decreasing clinical importance of two-tailed alpha 0.05 (see FIG. 8; ARR = annual mean relapse rate; CDI = confirmed decline in disability; CDP = confirmed progression of disability; Gd = gadolinium; MSFC = complex functional scale for multiple sclerosis; NEDA = no evidence of disease activity; SF-36 PCS = short form 36, physical health indicator). NEDA is defined as: no protocol-defined relapses, no CDP events, no new or increasing T2 plaques, and no Gd amplified T1 plaques. The analysis of secondary efficacy outcomes at the individual study level is as follows:

[0323] - For the first secondary efficacy result (time to onset of confirmed progression of disability for at least 12 weeks), the p-value at the trial level is interpreted as unconfirming due to insufficient statistical power at the trial level to detect corresponding treatment differences.

[0324] - A second secondary efficacy outcome (total Gd T1 plaques amplified at 24, 48, and 96 weeks) is examined as confirmation if and only if, in the analysis of both trials together, the first secondary efficacy outcome reaches a significance level of 0.05 (i.e. e. p of pooled analysis ≤ 0.05). If, in the analysis of pooled trials, the p-value of the pooled analysis of the first secondary performance outcome is p> 0.05, then the p-values of the second and subsequent secondary performance outcomes in the hierarchy are interpreted as unconfirming.

[0325] - The third secondary efficacy outcome (total number of new and / or increasing hyperintense T2 plaques at 24, 48 and 96 weeks) is investigated as confirmation if and only if the second secondary efficacy outcome (total number of Gd T1 amplified plaques per 24, 48 and 96 weeks) reaches a significance level of 0.05 (i.e., p≤0.05). If the p-value of the second secondary performance outcome p> 0.05, then the p-values of the third and subsequent secondary performance outcomes in the hierarchy are interpreted as unconfirming.

[0326] - For a fourth (proportion of patients who have a confirmed decline in disability for at least 12 weeks) and a fifth (time to a confirmed progression of disability for at least 24 weeks) secondary efficacy results, the p-value at the trial level is interpreted as unconfirming, due to insufficient statistical power at the trial level to detect corresponding treatment differences.

[0327] - The sixth secondary efficacy outcome (total number of new hypointense T1 plaques (chronic black holes) at 24, 48 and 96 weeks) is investigated as confirmation if and only if the fifth secondary efficacy outcome in the pooled trial analysis (time before the onset of confirmed progression of disability for at least 12 weeks) reaches the significance level of 0.05 (i.e., the p-value of the pooled analysis p≤0.05). If, in the analysis of both trials together, the p-value of the pooled analysis of the fifth secondary efficacy result is p> 0.05, then the p-values of the sixth and subsequent efficacy results in the hierarchy are interpreted as unconfirming.

[0328] - A seventh (and subsequent) secondary efficacy outcome is validated if and only if the sixth (or immediately preceding) secondary efficacy outcome reaches a significance level of 0.05 (ie, p≤0.05). If the p-value of the sixth (or immediately preceding) secondary performance result p> 0.05, then the p-values of the seventh (or current) and all subsequent secondary performance results in the hierarchy are interpreted as unconfirming.

[0329] Moreover, for analyzes of secondary efficacy outcomes where data from both trials are pooled (so that there is sufficient statistical power to compare all primary and secondary efficacy outcomes):

[0330] - The first secondary efficacy outcome (time to onset of confirmed progression of disability for at least 12 weeks) is investigated as confirmation if and only if the primary efficacy outcome (mean annual protocol-defined relapse rate over 2 years) reaches a significance level of 0 , 05 (i.e. p-value in the pooled analysis p≤0.05). If the p-value of the pooled analysis of the primary efficacy result p> 0.05, then all p-values of the pooled analysis of the secondary efficacy results are interpreted as nonconfirming.

[0331] - The second (and subsequent) secondary performance result is investigated as confirmation if and only if the first (or immediately preceding) secondary performance result reaches a significance level of 0.05 (i.e., the p-value of the pooled analysis p≤0.05 ). If the p-value of the pooled analysis of the first (or immediately preceding) secondary performance result p> 0.05, then the p-values of the pooled analysis of the second (or current) and all subsequent secondary performance results in the hierarchy are interpreted as unconfirming.

[0332] The safety sample was used for all analyzes of safety data, and it included all patients who received any study treatment.

results

Patients

[0333] A total of 1656 patients were included in the first (N = 821) and second (N = 835) Phase III trials (sample of all patients with intent to be treated). Baseline demographic and disease characteristics were similar among populations in each trial and between the two trials (see Table 3).

Table 3.
Baseline demographic and disease characteristics CHARACTERISTIC TEST I TEST II IFNβ-1a, 44 μg (n = 411) Ocrelizumab 600 mg (n = 410) IFNβ-1a, 44 μg (n = 418) Ocrelizumab 600 mg (n = 417) Age, years, mean (SD) 36.9 (9.3) 37.1 (9.3) 37.4 (9.0) 37.2 (9.1) Female gender, n (%) 272 (66.2) 270 (65.9) 280 (67.0) 271 (65.0) Time since occurrence, years, average (SD) 6.3 (6.0) 6.7 (6.4) 6.7 (6.1) 6.7 (6.1) Time since diagnosis, years, mean (SD) 3.7 (3.6) 3.8 (4.8) 4.1 (5.1) 4.2 (5.0) Relapses in the last 12 months, mean (SD) 1.3 (0.6) 1.3 (0.7) 1.3 (0.7) 1.3 (0.7) Previously untreated, no. (%) ^† 292 (71.4) 301 (73.8) 314 (75.3) 304 (72.9) Average EDSS 2.8 (1.3) 2.9 (1.2) 2.8 (1.4) 2.8 (1.3) Gadolinium-reinforced T1 plaques, amount (%) 0 252 (61.9) 233 (57.5) 243 (58.6) 252 (61.0) 1 52 (12.8) 64 (15.8) 62 (14.9) 58 (14.0) 2 30 (7.4) 30 (7.4) 38 (9.2) 33 (8.0) 3 16 (3.9) 16 (3.9) 14 (3.4) 15 (3.6) > 4 57 (14.0) 57 (14.0) 58 (14.0) 55 (13.3) T2 plaques - quantity 51.06 ± 39.91 51.04 ± 39.00 51.01 ± 35.69 49.26 ± 38.59 The volume of T2 plaques - cm ³ 9.74 ± 11.28 10.84 ± 13.90 10.61 ± 12.30 10.73 ± 14.28 Normalized brain volume - cm ³ 1499.18 ± 87.68 1500.93 ± 84.10 1501 ± 90.98 1503 ± 92.63 Patients without Gd ⁺ plaques, n (%) 252 (61.9) 233 (57.5) 243 (58.6) 252 (61.0) Gd ^{+ quantity}
T1 plaque mean (SD) 1.9 (5.2) 1.7 (4.2) 2.0 (4.9) 1.8 (5.0) T2 plaque count, mean (SD) 51.1 (39.9) 51.0 (39.0) 51.0 (35.7) 49.3 (38.6) † Patients not treated with disease-modifying therapy within 2 days prior to enrollment

[0334] A total of 366 (89%) and 340 (83%) patients in the ocrelizumab and IFNβ-1a groups, respectively, completed the first trial (see FIG. 9), and 360 (86%) and 320 (77%) in the the ocrelizumab and IFNβ-1a groups completed the second trial (see FIG. 9). More than 85% of patients in the ocrelizumab groups completed Test I and Test II. All randomized patients were included in the ITT sample. Patients who were prematurely excluded from the trial and patients who were not assessed for any reason were nevertheless included in the ITT (intent to treat) analysis.

Efficiency

[0335] Clinical and MRI outcomes for the first and second trials are summarized in Table 4. The MS relapse rate decreased with ocrelizumab in both trials, with a mean annual relapse rate (ARR) after adjustment at 96 weeks (primary outcome) 0.156 for ocrelizumab (versus 0.292 for IFNβ-1a) in Test I and 0.155 for ocrelizumab (versus 0.290 for IFNβ-1a) in Test II (see Table 4 and FIGS. 10A and 10B). The median annual relapse rates (based on protocol-defined relapses) in ocrelizumab-treated patients were reduced by 46% and 47% versus IFNβ-1 in trial I and trial II, respectively (p <0.0001 for both comparisons). The adjusted ARR was calculated using binomial regression and corrected for baseline EDSS (<4.0 vs. ≥4.0) and geographic location (US vs. rest of the world). The average annual 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 IFNβ-1 in trial I and trial II, respectively.

Table 4.
Clinical and MRI results from 96-week trial (ITT sample) * Result Combined Trials I and II Challenge I Challenge II IFNβ-1a, 44 μg (n = 829) Ocrelizumab 600 mg (n = 827) IFNβ-1a, 44 μg (n = 411) Ocrelizumab 600 mg (n = 410) IFNβ-1a, 44 μg (n = 418) Ocrelizumab 600 mg (n = 417) Clinical results Average annual relapse rate at 96 weeks Frequency (95% CI) 0.292
(0.235-0.361) 0.156
(0.122-0.200) 0.290
(0.234-0.361) 0.155
(0.121-0.198) Risk reduction against IFNβ-1a -% (95% CI; p-value) 46 p <0.0001) 47; p <0.0001) Confirmed progression of disability for at least 12 weeks ^† Patients with event -% 13.6 9.1 Hazard Ratio vs IFNβ-1a (95% CI) 0.60
(0.45-0.81) 0.57
(0.37-0.90) 0.63
(0.42-0.92) Risk reduction against IFNβ-1a -% (p-value) 0 (p = 0.0006) 43 (p = 0.0139) 37 (p <0.0169) Confirmed progression of disability for at least 24 weeks ^† Patients with event -% 10.5 6.9 Odds ratio against IFNβ-1a (95% CI) 0.60
(0.43-0.84) 0.57
(0.34-0.95) 0.63
(0.40-0.98) Risk reduction against IFNβ-1a -% (p-value) 40 (p = 0.0025) 43 (p = 0.0278) 37 (p = 0.0370) Confirmed reduction in disability for at least 12 weeks ^{†, ‡} Number of patients evaluated 614 628 Patients with improvement lasting 12 weeks -% 15.6 20,7 Relative improvement against IFNβ-1a -% (p-value) 33 (p = 0.0194) 61 (p = 0.0106) 14 (p = 0.4019) Multiple Sclerosis Complex Functional Scale Mean change in MSFC from baseline to week 96 0.174 ± 0.031 0.213 ± 0.031) 0.169 ± 0.029 0.276 ± 0.028 Difference in corrected mean (95% CI; p-value) 0.039 ± 0.039
(-0.039-0.166; p = 0.3261) 0.107 ± 0.037
(0.034-0.180; p = 0040) NEDA Week 96 ^{‡, §} Number of patients evaluated 291 289 270 289 Patients with NEDA -% 27.1 47.4 24.1 43.9 Relative improvement against IFNβ-1a -% (p-value) 74 (p <0.0001) 81 (p <0.0001) NEDA Components at 96 ‡, § Week -% Relapse-free patients 66.7 80.4 64.3 78.9 Patients free from confirmed progression of disability 87.8 92.4 84.9 89.4 Patients free of gadolinium-enhanced T1 plaques 69.8 91,7 63.9 90.2 Patients free of new and / or increasing T2 plaques 38.7 61.7 38.0 60.9 MRI Results Gadolinium-enhanced T1 plaques at 24, 48 and 96 weeks Patients with any gadolinium-enhanced T1 plaques 30.2 8.3 36.1 9.8 Average number of plaques (95% CI) 0.286
(0.200-0.409) 0.016
(0.0009-0.030) 0.416
(0.309-0.561) 0.021
(0.012-0.036) Risk reduction vs. IFNβ-1a -% (p value) 94 (p <0.0001) 95 (p <0.0001) New and / or increasing hyperintense T2 plaques at 24, 48, and 96 weeks ^¶ Patients with new and / or increasing hyperintense T2 plaques -% 61.3 38.3 62.0 39.1 Average number of plaques (95% CI) 1.413
(1,123-1,777) 0.323
(0.256-0.407) 1.904
(1,536-2,359) 0.325
(0.259-0.409) Frequency ratio (95% CI) 0.229
(0.174-0.300) 0.171
(0.130-0.225) Risk reduction against IFNβ-1a -% (p-value) 77 (P <0.0001) 83 (P <0.0001) Hypointense T1 plaques at 24, 48 and 96 weeks ^¶ Average number of plaques (95% CI) 0.982
(0.780-1.237) 0.420
(0.337-0.524) 1,255
(1.003-1.571) 0.449
(0.357-0.560) Frequency ratio (95% CI) 0.428
(0.328-0.557) 0.357
(0.272-0.470) Risk reduction against IFNβ-1a -% (p-value) 57 (P <0.0001) 64 (P <0.0001) Brain volume at 24-96 weeks Average change -0.741 ± 0.046 -0.572 ± 0.044 -0.750 ± 0.051 0.638 ± 0.049 p value P = 0.0042 P = 0.0900 Mean difference of corrected means (95% CI) 0.168
(0.053-0.283) 0.112
(-0.018-0.241) Brain volume from baseline to 96 put on ** Average percentage change -1.233 ± 0.055 -0.943 ± 0.054 -1.354 ± 0.064 -1.032 ± 0.062 p value p <0.0001 p <0.0001 Mean difference of corrected means (95% CI) 0.290
(0.148-0.432) 0.322
(0.156-0.488) Patient-reported outcomes Short form of the 36 components of physical health from baseline to 96 weeks Mean 45.399 ± 0.529 45.065 ± 0.507 44.552 ± 0.544 44.307 ± 0.541 p value P = 0.2193 P = 0.0404 Difference in averaged values 0.693
(-0.414-1.800) 1.159
(0.051-2.269) * Plus or minus values represent means ± standard deviation
† Planned combined results are shown in bold
‡ In patients with a baseline EDSS of at least 2.0
§NEDA is defined as: no protocol-defined relapses, no CDP events, no new or increasing T2 plaques, and no gadolinium-enhanced plaques
¶After corrected for baseline plaque count, EDSS (<4.0 / ≥4.0) and geographic region (US / ROW)

Disability

[0336] Compared to IFNβ-1a, ocrelizumab reduced the risk of confirmed disability progression (i.e., CDP) that persisted for 12 weeks by 40% over the 96-week trial period (trial I and trial II, pooled data ; p = 0.0006; FIG. 11, CI = Confidence Interval; HR = Hazard Ratio). Non-pooled data for Trial I and Trial II demonstrating that ocrelizumab reduced the risk of confirmed disability progression (i.e., CDP) that persisted for 12 weeks, compared to IFNβ-1a, are presented in FIG. 12A. (trial I) and Fig. 12B (trial II). Ocrelizumab also reduced the risk of CDP, which persisted for 24 weeks, by 40% against IFNβ-1a over a 96 week trial period (trial I and trial II, pooled data; p = 0.0025; FIG. 13). Non-pooled data for Trial I and Trial II demonstrating that ocrelizumab reduced the risk of confirmed disability progression (i.e., CDP) that persisted for 24 weeks, compared to IFNβ-1a, are shown in FIG. 14A and Fig. 14B. The proportion of patients (with a baseline EDSS of 2.0 or less) with a confirmed reduction in disability (i.e., CDI) for at least 12 weeks was 20.7% in patients treated with ocrelizumab (n = 628) versus 15 , 6% in patients treated with IFNβ-1a (n = 614), corresponding to a 33% risk reduction for ocrelizumab (trial I and trial II, pooled data; p = 0.0194). See Fig. 15. Non-pooled data for the proportion of patients (baseline EDSS of 2.0 or less) with a confirmed reduction in disability for at least 12 weeks in trial I and trial II are shown in FIG. 16A and FIG. 16B. Non-pooled data for the multiple sclerosis functional scale results are presented in FIGS. 16C and 16D. 16C and 16D include patients with a baseline score and at least one value after baseline. Estimates derived from a mixed effects model for repeated measurements (MMRM) analysis using an unstructured covariance matrix: change = baseline MSFCS + geographic region (US vs. rest) + baseline EDSS score (<4.0 vs.> = 4, 0) + week + treatment + treatment * week (repeating values throughout the week) + baseline MSFCS * week. 15.6% of patients treated with ocrelizumab achieved CDI at week 24 (versus 11.6% for patients treated with IFNβ-1a), corresponding to a relative improvement of 36% (relative risk 1.36 [p = 0.0343] ).

[0337] Compared with IFNβ-1a, higher proportions of patients treated with ocrelizumab had improved EDSS scores (20.2% [n = 146] versus 15.0% [n = 98]; adjusted odds ratio [aOR ] 1.288; p = 0.0866]). Significantly fewer patients in the ocrelizumab group had deterioration in EDSS scores compared with IFNβ-1a (10.1% [n = 73] versus 16.6% [n = 109]; aOR 0.575, p = 0.0009).

MRI-related results

[0338] Compared to IFNβ-1a, ocrelizumab reduced the total number of gadolinium-enhanced T1 plaques at 24, 48, and 96 weeks by 94% in trial I and 95% in trial II (p <0.0001 for both trials; see Table 2). table 4 and Figures 17A and 17B). Ocrelizumab reduced the mean total number of gadolinium-enhanced T1 plaques by 91% at 24 weeks, 98% at 48 weeks, and 95% at 96 weeks compared with IFNβ-1a from Trial I (p <0.0001 at all time points , see Fig. 18A). Ocrelizumab reduced the mean total number of gadolinium-enhanced T1 plaques by 92% at week 24, 96% at week 48, and 97% at week 96 compared to IFNβ-1a in trial II (p <0.0001 at all time points. see Fig. 18B). The results in Figures 17A-B and 18A-B were corrected for mean values calculated by negative binomial regression and corrected for baseline Gd-enhanced T1 plaques (present or not), baseline EDSS score (<4.0 vs. ≥4.0) and geographic location (US v. Rest of the world).

[0339] Ocrelizumab also reduced the total number of new / increasing hyperintense T2 plaques at 24, 48 and 96 weeks by 77% compared to IFNβ-1a in trial I and by 83% compared to IFNβ-1a in trial II (p < 0.0001 for both comparisons; see Table 5 and Figures 19A and 19B). As shown in FIG. 19C, ocrelizumab reduced the mean number of new / increasing hyperintense T2 plaques at week 24 by 41%; at 48 weeks by 94% and at 96 weeks by 98% compared to IFNβ-1a in trial I. As shown in Fig.19D, ocrelizumab reduced the mean number of new / increasing hyperintense T2 plaques at 24 weeks by 61%; at 48 weeks by 96%; and at 97 weeks by 97% compared to IFNβ-1a in trial II. (After adjusting for negative binomial regression means and after adjusting for baseline T2 plaque counts, baseline EDSS (<4.0 vs. ≥4.0), and geographic area (US vs. ROW).) EDSS, an expanded scale for assessing the state of disability; IFN, interferon; MRI, magnetic resonance imaging; ROW, other countries. Ocrelizumab reduced the appearance of more chronic and growing areas of MS-related brain damage (hyperintense T2 plaques) at 24, 48 and 96 weeks by approximately 80% compared to IFNβ-1a (trial I and trial II combined). The results in FIGS. 19A-D were corrected for mean values calculated by negative binomial regression and corrected for baseline T2 plaque count, baseline EDSS score (<4.0 vs. ≥ 4.0), and geographic location (US vs. other countries).

[0340] Ocrelizumab slowed down the decrease in brain volume from week 24 to week 96 compared to IFNβ-1a (Test I, p = 0.0042; Test II, p = 0.0900; FIGS. 20A and 20B). Ocrelizumab also slowed the decline in brain volume from baseline to week 96 compared to IFNβ-1a (Test I, p = <0.0001; Test II, p = 0.0001; Figures 21A and 21B). The results presented in Figures 20A-B and 21A-B were compared using the Cochran-Mantel-Hänsel test stratified by geographic area (US versus rest) and baseline EDSS (<4.0 versus ≥4.0). The results in Figures 20A-B and 21A-B were compared using the Cochran-Mantel-Hänsel test stratified by geographic area (US versus rest) and baseline EDSS (<4.0 versus> 4.0). Ocrelizumab reduced the rate of decrease in brain volume from week 24 to week 96 by 22.7% in trial I (p = 0.0042) and by 14.9% in trial II (p = 0.0900) compared with IFNβ-1a ... See Fig. 20. In trial I, the decrease in brain volume (from baseline to week 96) in patients treated with ocrelizumab was reduced by 23.5% relative to patients treated with IFNβ-1a (P <0.0001). In Trial II, the decrease in brain volume (from baseline to week 96) in patients treated with ocrelizumab was reduced by 23.8% relative to patients treated with IFNβ-1a (P = 0.0001). See Fig. 21.

[0341] Patients treated with ocrelizumab showed an 18.8% reduction in brain atrophy compared to patients treated with IFNβ-1a.

[0342] Ocrelizumab reduced the total number of new hypointense T1 plaques by 57% compared to IFNβ-1a in trial I and by 64% compared to IFNβ-1a in trial II. See Figures 22A and 22B.

[0343] The efficacy results for Test I and Test II are summarized in Table 5.

Table 5.
Efficacy Summary for Test I and Test II TEST I TEST II Joint trials Result Risk reduction, p-value Risk reduction, p-value Risk reduction, p-value ARR 46%,
<0.0001 47%,
<0.0001 Time to CDP
12 weeks 43%,
0.0139 37%,
0.0169 40%,
0.0006 The total amount of amplified Gd
T1 plaques
(24, 48, 96 weeks) 94%, <0.0001 95%, <0.0001 Total new / increasing T2 plaques
(24, 48, 96 weeks) 77%, <0.0001 83%, <0.0001 CDI, 12 weeks improvement 61%, 0.0106 improvement 14%, 0.4019 improvement 33%, 0.0194 Time to CDP
24 weeks 43%, 0.0278 37%, 0.0370 40%, 0.0025 The total number of hypointense
T1 plaques
(24, 48, 96 weeks) 57%, <0.0001 64%, <0.0001 Mean MSFC Difference (Baseline to Week 96) p = 0.3261 p = 0.0040 Difference in average brain volumes (from 24 weeks to 96 weeks) p = 0.0042 p = 0.090 SF-36 PCS Change (Baseline to Week 96) p = 0.2193 p = 0.0404 NEDA at week 96 improvement 74%, <0.0001 improvement 81%, <0.0001

[0344] In trial I, 80.4% of patients treated with ocrelizumab had no relapse within 96 weeks versus 66.7% of patients treated with IFNβ-1a. In Trial II, 78.9% of patients treated with ocrelizumab had no relapse at 96 weeks versus 64.5% of patients treated with IFNβ-1a.

[0345] In trial I, 92.4% of patients treated with ocrelizumab had no CDP at 96 weeks versus 87.8% of patients treated with IFNβ. In Trial II, 89.4% of patients treated with ocrelizumab had no CDP at 96 weeks versus 84.9% of patients treated with IFNβ.

[0346] In trial I, 92.3% of patients treated with ocrelizumab had a reduction / stabilization of disability versus 86.1% of patients treated with IFNβ-1a. In Trial II, 87.5% of patients treated with ocrelizumab had a reduction / stabilization of disability versus 80.4% of patients treated with IFNβ-1a.

[0347] In trial I, 7.7% of patients treated with OCR had an increase in disability versus 13.9% of patients treated with IFNβ-1a (odds ratio adjusted 0.559; p = 0.0242). In trial II, 12.5% of patients treated with OCR had an increase in disability versus 19.6% of patients treated with IFNβ-1a (odds ratio adjusted 0.577; p = 0.0121).

[0348] In trial I, 91.7% of patients treated with ocrelizumab had no gadolinium-enhanced T1 plaques at 96 weeks versus 69.8% of patients treated with IFNβ. In trial II, 90.2% of patients treated with ocrelizumab had no gadolinium-enhanced T1 plaques at 96 weeks versus 63.9% of patients treated with IFNβ.

[0349] In trial I, 61.7% of patients treated with ocrelizumab had no new / increasing T2 plaques at 96 weeks versus 38.7% of patients treated with IFNβ. In Trial II, 60.9% of patients treated with ocrelizumab had no new / increasing T2 plaques at 96 weeks versus 38.0% of patients treated with IFNβ.

[0350] At week 24, ≥96.0% of all patients treated with OCR had no new / increasing T2 plaques compared with 60.8-70.9% of patients treated with IFNβ1-a.

Security

Adverse events

[0351] The overall incidence of adverse events was 83.3% and 83.3% for patients treated with IFNβ-1a and ocrelizumab, respectively, in trial I and trial II (pooled). (See Table 6A).

Table 6A.
Adverse events (safety sample). n (%) COMBINED TEST I AND TEST II IFNβ-1a, 44 μg (n = 826) Ocrelizumab 600 mg (n = 825) Total number of patients with ≥ 1 adverse event (AE) 688 (83.3) 687 (83.3) Total number of patients with ≥ 1 AE occurring at a relative frequency of ≥ 5% 539 (65.3) 544 (65.9) Damage, poisoning and complications caused by procedures 155 (18.8) 333 (40.4) Infusion-related reaction 80 (9.7) 283 (34.3) General disorders and conditions of the area of introduction 396 (47.9) 173 (21.0) Flu-like disorders 177 (21.4) 38 (4.6) Erythema of the injection site 127 (15.4) 1 (0.1) Fatigue 64 (7.7) 64 (7.8) Injection site reaction 45 (5.4) 2 (0.2) Infections and infestations 433 (52.4) 482 (58.4) Upper respiratory tract infection 87 (10.5) 125 (15.2) Nasopharyngitis 84 (10.2) 122 (14.8) Urinary tract infection 100 (12.1) 96 (11.6) Sinusitis 45 (5.4) 46 (5.6) Bronchitis 29 (3.5) 42 (5.1) Nervous system disorders 252 (30.5) 224 (27.2) Headache 124 (15.0) 93 (11.3) Psychiatric disorders 144 (17.4) 149 (18.1) Depression 54 (6.5) 64 (7.8) Insomnia 38 (4.6) 46 (5.6) Musculoskeletal and connective tissue disorders 207 (25.1) 204 (24.7) Backache 37 (4.5) 53 (6.4) Arthralgia 51 (6.2) 46 (5.6)

Table 6A includes only pooled AEs occurring in ≥5% of patients in at least one treatment group and corresponding organ system class.

Table 6B.
All severe adverse events n (%) IFNβ-1a,
44 μg (n = 826) Ocrelizumab,
600 mg (n = 825) Total number of patients with ≥ SAE 72 (8.7) 57 (6.9) Infections and infestations 24 (2.9) 11 (1.3) Nervous system disorders 11 (1.3) 8 (1.0) Damage, poisoning and complications caused by procedures 10 (1.2) 6 (0.7)

[0352] Serious adverse events were reported for 8.7% of patients treated with IFNβ-1a and 6.9% of patients treated with ocrelizumab in trial I and trial II (pooled). (See Table 6B).

Infections

[0353] The incidence of infections was 52.8% for IFNβ-1a and 56.6% for ocrelizumab in trial I and 52.0% for IFNβ-1a and 60.2% for ocrelizumab in trial II. The most common infections (reported in at least 10% of patients in at least one treatment group) were urinary tract infections, upper respiratory tract infections, and nasopharyngitis. The incidence of serious infections in both trials was low (1.2 to 2.9% for all treatment groups in both trials). No serious opportunistic infections were reported in either group during the 96 weeks trial. Overall, herpes virus infections have been reported for 28 patients treated with both IFNβ-1a and 50 patients treated with ocrelizumab. All cases were mild to moderate, with the exception of one case of herpes simplex in a patient treated with ocrelizumab in trial I, which was classified as severe.

Infusion-related reactions

[0354] The number of patients with at least one infusion-mediated response (IRR) was higher in patients treated with ocrelizumab versus IFNβ-1a (30.9% for ocrelizumab versus 7.3% for IFNβ-1a in trial I; 37.6% for ocrelizumab versus 12.0% for IFNβ-1a in Test II; 34.3% for ocrelizumab versus 9.7% for IFNβ-1 in both Test I and Test II (pooled)). One patient treated with OCR had severe IRR on first infusion with life-threatening brochospasm; despite the resolution of this case, subsequent treatment was canceled according to the protocol. Withdrawal of treatment due to IRR on the first infusion occurred in 11 patients (1.3%) in the ocrelizumab group alone. The incidence of IRR in patients treated with ocrelizumab was highest at the first infusion (27.5%) and significantly decreased with subsequent dosing (13.7% at 2 doses). Most of the infusion-related reactions were grade 1 or 2 reactions and were reported for the first infusion of the first dose (see FIGS. 23A and 23B, pooled data for Test I and Test II). The numbers in the bars represent the proportion of patients experiencing some degree of IRR. Common Criteria for Terminology (CTCAE) classifications: grade 1, mild: asymptomatic or mild symptoms; grade 2, moderate: minimal, local, or non-invasive intervention indicated; grade 3, severe or clinically significant, but not directly life threatening; Grade 4, life-threatening consequences, urgent intervention indicated; grade 5, death attributable to AE. Note: All received 100 mg of methylprednisolone IV. At 96 weeks, 9.7% of patients treated with IFNβ-1a (n = 80/826) and 34.3% of patients treated with ocrelizumab (n = 283/825) had at least 1 IRR; most had mild to moderate severity (99% [n = 79] and 93% [n = 262], respectively). Most IRR symptoms in patients treated with ocrelizumab included itching, rash, throat irritation, and flushing. IRRs most frequently occurred during infusion (IFNβ-1a, 46.3%; ocrelizumab, 80.6%) and were controlled by adjusting the infusion and symptomatic treatment (42.5% of patients who had IRRs in the IFNβ-1a group and 65 , 4% in the ocrelizumab group received treatment).

Malignant tumors

[0355] A total of two malignant tumors were reported in the case of IFNβ-1a (one mantle cell carcinoma and one squamous cell carcinoma) and four malignant tumors were reported in patients treated with ocrelizumab (two cases of invasive breast duct carcinoma, one renal cell carcinoma and one malignant melanoma).

Laboratory evaluation

[0356] On laboratory assessment at 2 weeks, mean CD19 levels decreased to negligible in patients treated with ocrelizumab. There was no effect on CD3-, CD4-, CD8-, CD16- and CD56-positive cells, which confirms that ocrelizumab does not affect innate immunity and total T-cell counts. There was no effect of ocrelizumab on existing antibody titers against mumps, rubella, chickenpox and pneumococcus.

[0357] Although only direct comparisons of ocrelizumab to IFNβ-1a were performed in Trial I and Trial II, these efficacy and safety results indicate that the benefit / risk profile of ocrelizumab over a 2-year period is the best for all disease-modifying therapies ( DMT) available to treat RMS patients.

Effects of ocrelizumab on humoral immunity

[0358] Before enrollment in the trial, physicians were advised to review the patient's immunization status and follow local vaccination recommendations; immunizations must have been completed ≥6 weeks prior to treatment. Measurements of antibody titers (Ab) against mumps, rubella, chickenpox and Streptococcus pneumoniae were performed at baseline and at 12, 24, 48, 72 and 96 weeks. The proportion of patients with Ab levels that could be considered protective was assessed for each treatment group over time.

[0359] Piggy: 94.1% of patients treated with IFNβ and 93.6% of patients treated with ocrelizumab had positive anti-mumps Ab levels at baseline. (This proportion varied (minimum-maximum) 92.7-94.8% (IFNβ-1a) and 91.8-93.5% (ocrelizumab) over the six measurements taken during the trial's 96-week treatment period. )

[0360] Rubella: 87.9% of patients treated with IFNβ-1a and 89.0% of patients treated with ocrelizumab had positive anti-rubella Ab levels, and they were in the (minimum-maximum) range of 89.8-90.8% (IFNβ -1a) and 88.7-89.4% (ocrelizumab) over six measurements taken during the treatment period.

[0361] Chickenpox: 95.5% of both treatment groups had positive anti-chickenpox Ab levels at baseline, and they were in the range (minimum-maximum) 96.2-97.5% (IFNβ-1a) and 94.8- 95.6% (ocrelizumab) over six measurements taken during the treatment period.

[0362] S. pneumonia: Among the patients evaluated, the mean (standard deviation) of Ab against S. pneumonia at baseline was 53.67 (54.13) mg / ml for patients treated with IFNβ-1a, and 55.35 (67.00) mg / ml for patients treated with ocrelizumab. At 96 weeks, the mean change from baseline was -1.13 (40.25) mg / ml for patients treated with IFNβ-1a and -1.99 (59.60) mg / ml for patients treated with ocrelizumab.

[0363] In an open-label phase III trial to evaluate the effect of ocrelizumab on immune responses in patients with relapsing multiple sclerosis, approximately 100 patients will be randomly assigned (2: 1) to administer ocrelizumab 600 mg as two 300 mg intravenous infusions on day 1 and 15 days. In group A will administer ocrelizumab prior to immunization. Group B will be left naive about treatment / will continue to be treated with interferon during immunization.

[0364] Patients in Group A will be immunized with tetanus toxoid (TT) adsorbed vaccine, 23-valent pneumococcal polysaccharide (23-PPV) vaccine, and KLH at 85 days / 12 weeks (12 weeks after ocrelizumab treatment). Group A patients further will be subdivided into receiving any of:

- booster 13-valent pneumococcal conjugate vaccine (13-PCV; group A1); or

- influenza virus vaccine (group A2).

[0365] Patients in Group B will immunize with TT, 23-PPV, influenza vaccine and re-injections of snail lymph hemocyanin (KLH) at 1 day / 1 week.

[0366] All groups will be evaluated after immunization until the end of the test period (group A : 169 days / 24 weeks; group B : 84 days / 12 weeks). Key eligibility criteria include at least 1 prior immunization against TT, tetanus, or diphtheria; or tetanus, diphtheria, and acellular pertussis vaccine. The primary outcome measure will compare the proportion of patients in groups A1 and A2 with a positive response to TT (IgG) 8 weeks after immunization with patients in group B.

[0367] Key secondary outcomes include: proportions of patients in groups A1 and A2 against group B with a positive response of the individual serotype antibodies against pneumococcus 4 weeks after 23-PPV; mean levels of anti-KLH antibodies (IgG) in group A against group B 4 weeks after the last administration of KLH; the proportion of patients in group A1 against group B with positive responses of the individual serotype of antibodies against pneumococcus 4 weeks after booster administration of 13-PCV; the proportion of patients in group A2 versus group B who achieved seroprotection 4 weeks after administration of the influenza vaccine; indices of B- and T-cell subgroups and natural killer cells; and overall security.

Generalization

[0368] Trial I and Trial II showed that in patients with recurrent forms of MS, ocrelizumab significantly reduced the average annual relapse rate and the risk of confirmed progression of disability at 12 and 24 weeks compared to IFNβ-1a. Ocrelizumab is the first treatment for multiple sclerosis that significantly reduces proven progression of disability at both 12 and 24 weeks, in two separate phase III trials and against an active comparator, demonstrating a consistent effect. The proportion of patients who achieved a reduction in disability for at least 12 weeks was also significantly increased among ocrelizumab-treated patients compared with IFNβ-1a. These clinical findings are supported by MRI findings that ocrelizumab significantly reduced the number of gadolinium-enhanced T1 plaques and new / increasing T2 plaques in the brain compared to IFNβ-1a. The effect on gadolinium-enhanced T1 plaques and new / increasing T2 plaques is of a magnitude that indicates that focal inflammation and disease activity are largely stopped / arrested.

[0369] In exploratory analyzes, ocrelizumab reduced the decrease in brain volume (see Figs. 21A-D) and increased the proportion of patients without evidence of disease activity (NEDA) in the ITT sample with an improvement in EDSS ≥ 2.0 (see Tables 4 and 5 and Fig. 34) compared to IFNβ-1a. (In FIG. 34, the proportion of patients with NEDA was compared using the Cochran-Mantel-Hänsel β2 test stratified by geographic region (US versus rest) and baseline EDSS (<4.0 versus ≥4.0)).

[0370] These efficacy results, which demonstrate a reduction in the impairment of disability and, in some patients, improved functioning, support the hypothesis that ocrelizumab has effects on markers of inflammation and neuronal damage. The efficacy demonstrated by ocrelizumab on both inflammatory and degenerative outcomes indicates that ocrelizumab treatment has potent anti-inflammatory effects and may also have neuroprotective effects.

[0371] The benefit of ocrelizumab in trial I and trial II was observed early and persisted over the 96-week treatment period; based on earlier experience, after stopping treatment, the risk of recurrence is not expected (Kappos et al. (2012) "Long term safety and efficacy of ocrelizumab in patients with relapsing-remitting multiple sclerosis: Week 144 results of a phase II, randomized, multicenter trial. "28th Congress of the European Committee for Treatment and Research in Multiple Sclerosis).

[0372] Adverse events observed in patients treated with ocrelizumab were consistent with adverse events reported for the phase II ocrelizumab trial (Kappos et al. (2011) Lancet. 378, 1779-87). There were no cases of severe opportunistic infections reported in the 96 weeks trial. The results to date indicate that ocrelizumab has no significant effect on immune surveillance. Overall, ocrelizumab had a similar safety profile to IFNβ-1a at 96 weeks in both Test I and Test II.

[0373] As a class of drugs, CD20 ⁺ targeting drugs have a low risk of progressive multifocal leukoencephalopathy (<1 in 25,000) (Clifford et al. (2011) Arch Neurol. 68, 1156-64). In trials to date, there have been no reports of cases of progressive multifocal leukoencephalopathy.

[0374] As expected, infusion-related reactions were more frequently observed with ocrelizumab. In a Phase II trial with the 600 mg ocrelizumab dose, these reactions were mostly observed with the first dose and decreased with subsequent doses (Hauser et al. (2008) N Engl J Med. 358, 676-88). This confirms the rationale for dividing the first 600 mg dose into two separate 300 mg infusions approximately two weeks apart. Infusion-related reactions were controlled by premedication, infusion adjustments, and symptomatic treatment.

[0375] Clinical and MRI data collectively indicate that ocrelizumab is at least as effective as any other agent tested for the treatment of multiple sclerosis, including agents that are considered highly effective therapies. Although Trial I and Trial II only compared head-to-head with IFNβ-1a, these efficacy and safety results indicate that the benefit / risk profile of ocrelizumab over a 2-year period is the best of all disease-modifying therapies available to treat patients. with RMS. High efficacy is not associated with an increased safety risk compared to IFNβ-1a. Other benefits include early onset of treatment benefits and no recurrence of symptoms after stopping ocrelizumab treatment.

[0376] The data obtained for Test I and Test II demonstrate that targeting CD20 ⁺ B cells can be effective for the treatment of recurrent MS.

Subgroup analysis

[0377] Four additional subgroups were identified to assess the relative benefit / risk with varying degrees of disease activity and prior treatment response prior to enrollment:

[0378] Active naive about treatment; defined as having no prior treatment, having had at least 2 relapses in the last 2 years, and at least 1 relapse in the last year before randomization.

[0379] Active insufficiently responding; defined as patients previously treated with IFNβ-1a or glatiramer acetate for at least 1 year, and either (a) experienced at least one relapse in the past year, or (b) had at least 1 gadolinium-enhanced plaque at baseline ...

[0380] Highly active treatment-naive patients; defined as treatment-naive patients who experienced at least 2 relapses in the last year prior to randomization, and either (a) have at least 1 baseline gadolinium-enhanced plaque, or (b) have an increase in T2 plaques at baseline level (change in category from 0-5 to 6-9 plaques or from 6-9 plaques to> 9 plaques), compared with the previous MRI examination.

[0381] Highly active insufficiently responding; are defined as patients who have been previously treated with interferon or glatiramer acetate for at least 1 year, who have had at least one relapse in the past year, and either (a) have at least nine T2 plaques, or b) have at least at least one Gd-enhanced plaque at the initial visit.

[0382] Test I and Test II were combined to increase power to detect differences among 4 subgroups. There were at least 100 patients in each subgroup to ensure that differences between treatments were sufficiently identified.

[0383] A study of the average annual relapse rate per unit / group was conducted to determine if the trends for insufficient response were determined by the absolute outcomes for ocrelizumab and IFNβ-1a. Overall, ocrelizumab showed a significant effect of reducing the mean annual relapse rate in all subgroups compared to IFNβ-1a (see Table 7). The underresponders in the interferon group had a higher ARR value than the ITT group, while the ocrelizumab-treated patients in the same subgroup had a lower ARR value than the ITT group. The group of active naive patients with regard to treatment had a higher ARR value in both the interferon and ocrelizumab groups.

[0384] For all 4 subgroups, ocrelizumab had a higher treatment effect than IFNβ-1a on ARR, CDP for at least 12 weeks, CDP for at least 24 weeks, CDI and MRI (see tables 7-11 above). For ARR and MRI, the difference was statistically significant among all 4 subgroups. For CDP for at least 12 weeks, there were statistically significant effects for both active and highly active underresponsive patients, demonstrating that ocrelizumab has stable treatment effects compared to IFNβ-1a. For CDP for at least 24 weeks, these groups showed a trend towards significance (p = 0.075 and p = 0.082, respectively). Compared to objective measures such as Gd-enhanced T1 plaques (indicating acute inflammatory effects consistent with the mechanism of action for OCR), the persistent and stable effects of ocrelizumab versus IFNβ-1a are evident across all subgroups.

[0385] Similarly, safety outcomes were analyzed for 4 subgroups and compared with the overall safety sample. Due to the relatively small size of the groups, comparisons between groups were limited so as not to draw inappropriate conclusions from the small number of events. The safety results in the 4 groups were consistent with each other and were similar to the overall sample ("safety sample" defined as any patient with any OCR exposure) with a generally low proportion of patients who experienced any AE, SAE, and AE leading to exception. Consistent with observations in the safety sample, the ocrelizumab group had more infections than the interferon group, while the proportion of patients with severe infections in the ocrelizumab group was similar or lower than interferon in all subgroups.

[0386] The overall efficacy and safety outcomes for ocrelizumab were comparable to the ITT / safety sample in each subgroup. Thus, ocrelizumab showed a favorable benefit / risk profile across all subgroups. The consistency of data across subgroups further confirms the observed effects in the overall sample.

Example 2: Phase III trial for ocrelizumab in patients with primary progressive multiple sclerosis

[0387] A randomized, double-blind, placebo-controlled, parallel-group trial was conducted to evaluate the efficacy and safety of ocrelizumab versus placebo in patients with primary progressive multiple sclerosis.

[0388] Clinical trials of progressive forms of MS typically last> 2 years due to heterogeneity in disease progression rates, which are sometimes historically lower than expected, resulting in failure to demonstrate a treatment effect. This trial was an event-driven model, which means that if the target number of confirmed disability progression events were not achieved by week 120 due to slow disease progression, the treatment period was extended until there were enough progression events to maintain statistical power to detect differences. ways of treatment. As a result, the blind treatment period was at least 120 weeks from the time of the last enrolled patient; the average treatment period was 3.5 years. Most patients will continue to be followed up for a period of 3-4 years in the blind phase of treatment.

The ways

Eligibility criteria and exclusions

[0389] Key eligibility criteria included: age 18 to 55; 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); an Extended Disability Assessment Scale (EDSS) score of 3 to 6.5 on screening; a documented history of, or the presence on screening, of an elevated IgG index in a cerebrospinal fluid (CSF) sample and / or one or more oligoclonal IgG bands detected by isoelectric focusing in a cerebrospinal fluid (CSF) sample; no history of relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), or progressive-relapsing multiple sclerosis (PRMS); duration of illness from onset of MS symptoms (a) less than 15 years in patients with EDSS at screening> 5.0 or (b) less than 10 years in patients with EDSS at screening ≤ 5.0; and no treatment with other disease-modifying agents for multiple sclerosis at screening.

[0390] Key exclusion criteria included: relapsing-remitting multiple sclerosis, history of secondary-progressive or progressive-relapsing multiple sclerosis at screening; contraindications to magnetic resonance imaging (MRI); the known presence of other neurological disorders; known active infection or a history or presence of a current recurrent or chronic infection; a history of malignant tumor, including solid tumors and hematologic malignancies (with the exception of basal cell in situ squamous cell carcinoma of the skin and carcinoma in situ of the cervix, which have been excised and removed); previous treatment with B-cell targeted therapies (eg, rituximab, ocrelizumab, atacicept, belimumab, or ofatumumab); any previous treatment blockers lymphocytes transport (e.g., natalizumab, FTY720), any previous treatment with alemtuzumab (CAMPATH ^®, LEMTRADA ™), anti-CD4, cladribine, cyclophosphamide, mitoxantrone, azathioprine, mycophenolate mofetil, cyclosporine, methotrexate, total body irradiation or transplantation bone marrow; treatment with interferons β, glatiramer acetate, iv immunoglobulin, plasmapheresis, or other immunomodulatory therapies within 12 weeks prior to randomization; systemic corticosteroid therapy within 4 weeks before screening; and any concomitant disease that may require long-term treatment with systemic corticosteroids or immunosuppressants during the trial. The screening period could be lengthened (but cannot exceed 8 weeks) for patients who used systemic anti-MS corticosteroids before screening. To enroll patients, systemic corticosteroids should not be administered between screening and initial visit.

Test scheme

[0391] Patients were randomly assigned 2: 1 to be treated with either ocrelizumab (300 mg intravenously, 2 infusions 14 days apart in each course of treatment (ie exposure)) or placebo. Patients were stratified by age (≤45 versus> 45) and region (US versus rest of the countries).

[0392] The test consisted of the following periods (summarized in FIG. 24):

Screening up to 4 weeks.

Blind Treatment Period: All patients underwent study treatment for at least 120 weeks, corresponding to five courses of treatment, each course lasting 24 weeks. As patients were recruited over a period of 12-18 months, this blind treatment period extended up to 3.5-4 years for the first group of patients included in the trial. As shown in FIG. 24, ocrelizumab doses were administered as double intravenous infusions of 300 mg x 2 at 14 day intervals. Thirty minutes before the start of each infusion of ocrelizumab or placebo, patients received 100 mg of methylprednisolone intravenously. Pre-infusion treatment with an oral pain reliever / antipyretic agent (eg, acetaminophen) and an oral antihistamine (eg, diphenhydramine) has also been recommended.

Open treatment period: After initial unmasking, patients who benefited from the treatment were openly treated with ocrelizumab. Patients randomized to the ocrelizumab group continued to be openly treated with a double infusion of ocrelizumab (300 mg IV infusions given 14 days apart) every 24 weeks. Patients randomized to placebo received a double infusion of ocrelizumab during the first open course of treatment, i.e. two intravenous infusions, given at an interval of 14 days, starting from the next regular scheduled visit after the intermediate blocking of the database and the initial analysis.

Test methods

[0393] The primary efficacy outcome was the time to confirmed progression of disability during the treatment period, defined as an increase in EDSS that persisted for at least 12 weeks based on regularly scheduled visits.

[0394] Secondary outcomes included: time to confirmed progression of disability during the treatment period, defined as an increase in EDSS that persisted for at least 24 weeks based on regular scheduled visits; change in the test results of walking 25 feet for time (T25-FW) from baseline to 120 weeks; percent change in total T2 plaque volume at MRI from baseline to week 120; percent change in total brain volume at MRI from week 24 to week 120; and changing the short form 36 (SF-36) of the physical component score from baseline to week 120.

Statistical analysis

[0395] The statistical hierarchy is shown in FIG. 25.

results

Patients

[0396] As shown in Table 12, the medical history of patients with multiple sclerosis and disease characteristics were well balanced.

Table 12.
Patient demographics and baseline disease characteristics Placebo, n = 244 Ocrelizumab 600 mg, N = 488 Age, years, mean (SD) 44.4 (8.3) 44.7 (7.9) Women, n (%) 124 (50.8) 237 (48.6) Time from symptom onset, years, mean (SD) 6.1 (3.6) 6.7 (4.0) Time since diagnosis, years, mean (SD) 2.8 (3.3) 2.9 (3.2) Naive about treatment with a disease-modifying agent against MS, n (%) 214 (87.7) 433 (88.7) EDSS mean (SD) 4.7 (1.2) 4.7 (1.2) MRI data Patients Gd ⁺ plaque, n (%) 60 (24.7%) 133 (27.5) Number of Gd ⁺ T1 plaques, mean (SD) 0.6 (1.6) 1.2 (5.1) T2-plaque volume, cm ³ , mean value (SD) 10.9 (13.0) 12.7 (15.1) Normalized brain volume, cm ³ , mean (SD) 1469.9 (88.7) 1462.9 (83.9)

[0397] As shown in Fig. 26, the characteristics of the sample of all patients with intent to be treated were well balanced.

Disability

[0398] The time to confirmed progression of disability that persisted for at least 12 weeks in patients treated with 600 mg ocrelizumab versus placebo treated patients is shown in FIG. 27 (HR odds ratio). The time to confirmed progression of disability, which persisted for at least 24 weeks, in patients treated with 600 mg of ocrelizumab compared to patients treated with placebo is shown in FIG. 28. ITT patients with an initial progression of disability who discontinued treatment early without a confirmatory EDSS score were considered to have confirmed progression of disability, a p-value based on a log-rank test stratified by geographic region and age. Ocrelizumab reduced the risk of CDP persisting for ≥ 12 weeks by 24% versus placebo and reduced the risk of CDP persisting for ≥ 24 weeks by 25% versus placebo. The analyzes in FIGS. 27 and 28 were based on the ITT sample; the p-value was based on a log-rank test, stratified by geographic region and age. Patients with an initial progression of disability who discontinued treatment early and lacked a confirmatory EDSS score were considered to have a confirmed progression of disability.

[0399] The risk of CDP within 12 weeks was reduced by 35% in patients who received ocrelizumab with baseline Gd ⁺ T1 plaques (odds ratio 0.65; 95% CI, 0.40-1.06; p = 0.0826 ) versus 16% in ocrelizumab-treated patients without baseline Gd ⁺ T1 plaques (odds ratio 0.84; 95% CI, 0.62-1.13; p = 0.2441).

[0400] The risk of CDP within 24 weeks was reduced by 33% in ocrelizumab-treated patients with baseline Gd ⁺ T1 plaques (odds ratio 0.67; 95% CI, 0.40-1.14; p = 0, 1417) versus 19% in ocrelizumab-treated patients without baseline Gd ⁺ T1 plaques (odds ratio 0.81; 95% CI, 0.59-1.10; p = 0.1783).

[0401] FIG. 29 depicts a rate of decrease in walking speed (ie, decrease in the rate of progression of walking time) as measured by the 25 feet timed gait test in patients treated with 600 mg ocrelizumab compared to patients treated with placebo, from baseline to 120 weeks. Ocrelizumab reduced the rate of decrease in walking speed by 29% versus placebo. The analysis would be based on an ITT sample; p-value was based on rank ANCOVA at week 120 visit corrected for baseline 25-foot gait test result for time, geographic region and age, with missing data entered by last observation carryover (ie, LOCF). The point estimate and 95% CI were based on log-transformed data. The analysis in FIG. 19 is based on ITT (sample of all patients with intent to be treated); p-value was based on rank ANCOVA at week 120, adjusted for baseline 25-foot walking test result for time, geographic region, and age, with missing data entered by LOCF (Last Observation Carry-over). The point score and 95% CI (confidence interval) were based on MMRM analysis and log-transformed data, which were corrected for the baseline 25-foot gait test result, geographic region, and age. Figure 30 shows the rate of decrease in walking speed at 120 weeks from baseline in patients treated with 600 mg ocrelizumab compared to patients treated with placebo. Ocrelizumab reduced the rate of decrease in walking speed by 10% versus placebo (p = 0.404).

MRI Results

[0402] Figure 31 shows the percentage change in total brain volume from week 24 to week 96 in patients treated with 600 mg ocrelizumab compared to patients treated with placebo. Ocrelizumab reduced the rate of total brain volume loss from week 24 to week 96 by 17.5% compared with placebo (p = 0.0206). The analysis was based on an ITT sample with a score at 24 weeks and at least one score after 24 weeks; p-value was based on MMRM at week 120 visit, adjusted for brain volume at week 24, geographic region, and age.

[0403] Patients who received 600 mg of ocrelizumab showed a significant decrease in T2 plaque volume from baseline to 120 weeks compared to patients who received placebo. As shown in FIG. 32, T2 plaque volume increased by 7.4% in patients in the placebo group, while T2 plaque volume in patients treated with ocrelizumab decreased by 3.4% (p <0.001). The analysis was based on a sample of ITT; p-value was based on ranked ANCOVA at week 120, adjusted for baseline T2 plaque volume, geographic region, and age, with missing data entered by LOCF (last observation carryover). The point estimate and 95% CI (confidence intervals) were based on MMRM analysis and log-transformed data corrected for baseline T2-plaque volume, geographic region, and age.

[0404] In patients with ^{baseline Gd +} T1 plaques, total T2 plaque volume decreased by 3.8% in the ocrelizumab group (95% CI, -7.0 to -0.5) ... In contrast, total T2 plaque volume increased by 12.0% in placebo-treated patients (95% CI, 7.2-17.1; p <0.001). In patients without Gd ⁺ T1 plaques at baseline, the total T2 plaque volume decreased by 3.1% in patients treated with ocrelizumab (95% CI, -5.0 to -1.1). In contrast, total T2 plaque volume increased by 6.1% in placebo-treated patients (95% CI, 3.3-9.0; p <0.001).

Efficiency

[0405] The efficacy results for this test are summarized in Table 13.

Table 13
Generalization of efficiency Result Risk reduction P value Time to CDP in 12 weeks 24% 0.321 Time to CDP in 24 weeks 25% 0.0365 Change in 25ft Walk Test Results over Time (Baseline to 120 Weeks) 29% 0.0404 Percentage change in total T2 plaque volume on MRI (from baseline to 120 weeks) placebo: an increase of 7.4%, ocrelizumab: a decrease of 3.4% <0.0001 Percentage Change in Total Brain Volume by MRI (Week 24 to Week 120) 17.5% 0.0206 SF-36 PCS Change (Baseline to Week 120)

Security

Infusion-related reactions

[0406] 725 patients were included in the safety analysis (ie, 239 in the placebo group and 486 in the ocrelizumab group). Over a median treatment duration of approximately 3 years, the proportion of patients with at least one infusion-mediated response (IRR) was 25.5% (n = 61) for placebo-treated patients and 39.9% (n = 194 ) for patients treated with ocrelizumab; most of them had mild to moderate severity (93.4% [n = 57] and 96.9% [n = 188], respectively). The most common symptoms of IRR included skin and subcutaneous tissue disorders (45.9% [n = 89] for ocrelizumab-treated patients and 13.1% [n = 8] for placebo-treated patients). There were no life-threatening or fatal IRRs. The incidence of IRR in patients treated with ocrelizumab was highest with the first infusion (27.4%) and significantly decreased with subsequent dosing (11.6% with the first infusion of dose 2). Overall, most IRRs occurred during infusion in patients treated with ocrelizumab (61.3% versus 37.7% in patients treated with placebo). One patient (0.2%) refused ocrelizumab treatment due to IRR on the first infusion.

Adverse events

[0407] Adverse events reported for ≥10% of patients in each treatment group before the last clinical date are presented in Table 14A.

Table 14A. n (%) Placebo (n = 239) Ocrelizumab 600 mg (n = 486) All patients with ≥1 AE 215 (90.0) 462 (95.1) Infections and Infestations * 162 (67.8) 339 (69.8) Nasopharyngitis 65 (27.2) 110 (22.6) Urinary tract infections 54 (22.6) 96 (19.8) Flu 21 (8.8) 56 (11.5) Upper respiratory tract infection 14 (5.9) 53 (10.9) Bronchitis 12 (5.0) 30 (6.2) Gastroenteritis 12 (5.0) 20 (4.1) Damage, poisoning and complications caused by procedures 104 (43.5) 263 (54.1) Musculoskeletal and connective tissue disorders 98 (41.0) 181 (37.2) Nervous system disorders 79 (33.1) 174 (35.8) General violations and violations of the area of introduction 60 (25.1) 130 (26.7) Gastrointestinal disorders 60 (25.1) 126 (25.9) Psychiatric disorders 59 (24.7) 89 (18.3) Disorders of the skin and subcutaneous tissue 44 (18.4) 99 (20.4) Respiratory, thoracic and mediastinal disorders 35 (14.6) 87 (17.9) Metabolic and nutritional disorders 28 (11.7) 56 (11.5) Kidney and urinary tract disorders 30 (12.6) 51 (10.5) Vascular disorders 26 (10.9) 54 (11.1) The study 20 (8.4) 58 (11.9) ^* For SOC infections and infestations only: events reported for at least 5% of patients in one treatment group are presented.

[0408] The percentage of all patients who experienced more than 1 severe adverse event was 22.2% in the placebo group versus 20.4% in the administration group. See Table 14B. In general, the number of severe adverse events was low and they were similar in both groups.

[0409] The most common adverse event associated with ocrelizumab was infusion-related reactions (39.9% versus 25.5% for placebo). One patient (0.2%) refused ocrelizumab treatment due to IRR on the first infusion. As shown in FIG. 33, the severity of infusion-related reactions (IRR) decreased with each successive course and with each dose in the same course. Ocrelizumab has a safety profile similar to placebo.

Table 14B.
Common severe adverse events n (%) Placebo, n = 239 Ocrelizumab, n = 486 All patients with ≥1 SAE 53 (22.2) 99 (20.4) Infections and infestations 14 (5.9) 30 (6.2) Damage, poisoning and complications caused by procedures 11 (4.6) 19 (3.9) Nervous system disorders 9 (3.8) 18 (3.7) Neoplasms: benign, malignant, and unspecified (including cysts and polyps) 7 (2.9) 8 (1.6) Gastrointestinal disorders 3 (1.3) 10 (2.1) Musculoskeletal and connective tissue disorders 6 (2.5) 6 (1.2) Musculoskeletal and connective tissue disorders 3 (1.3) 6 (1.2) Kidney and urinary tract disorders 3 (1.3) 5 (1.0)

[0410] The data obtained in this trial demonstrate that B cells may play a role in the pathophysiology of PPMS. Ocrelizumab is the first investigational drug to show a consistent statistically significant clinical effect on PPMS in a large phase III trial compared with placebo. Ocrelizumab significantly reduced clinical progression of disability persisting for 12 weeks (primary outcome), decreased clinical progression of disability persisting for 24 weeks, altered walking test result 25 feet for a time, altered T2 plaque volume, and decreased brain volume shrinkage ... Patients treated with ocrelizumab consistently met key secondary outcomes. Ocrelizumab has shown a favorable safety profile over an average treatment duration of approximately 3 years. Overall, the incidence of adverse events associated with ocrelizumab was similar to placebo. The most common adverse events were mild to moderate infusion-related reactions. The incidence of severe adverse events associated with ocrelizumabol, including severe infections, was similar to placebo.

[0411] Examples, which are intended only to illustrate the invention, and thus should not be construed as limiting the invention in any way, also describe and detail the aspects and embodiments of the invention discussed above. The above examples and detailed description are presented by way of illustration and not for limitation.

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SEQUENCE LIST

<110> Hideki GARREN

Jean-Paul PFEFEN

Anne-Marie LI-KWAI-CHEUNG

Michele LIBONATI

<120> METHOD FOR MULTIPLE SCLEROSIS TREATMENT

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His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 14

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 15

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 16

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ala Phe Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 17

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro

325 330 335

Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 18

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 9

<223> Xaa = Met or Leu

<400> 18

Arg Ala Ser Ser Ser Val Ser Tyr Xaa His

1 5 10

<210> 19

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 4

<223> Xaa = Ser or Ala

<400> 19

Gln Gln Trp Xaa Phe Asn Pro Pro Thr

15

<210> 20

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 8

<223> Xaa = Asp or Ala

<400> 20

Ala Ile Tyr Pro Gly Asn Gly Xaa Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 21

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OPTION

<222> 6

<223> Xaa = Asn, Ala, Tyr, Trp, or Asp

<220>

<221> OPTION

<222> 7

<223> Xaa = Ser or Arg

<400> 21

Val Val Tyr Tyr Ser Xaa Xaa Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 22

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Trp Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Trp Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 23

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Leu

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr

35 40 45

Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ala Phe Asn Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 24

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro

325 330 335

Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 26

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<210> 27

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly

450

<---

Claims (83)

1. A method of improving functional ability in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, where the patient has an improvement in functional ability after administration and where the improvement in functional ability persists for at least 12 weeks; 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. 2. The method of claim 1, wherein the patient after treatment has a confirmed 12 week reduction in disability. 3. The method of claim 1, wherein the patient has a confirmed 24 week reduction in disability after treatment. 4. The method of claim 1, wherein the improvement in functional ability in the patient is maintained for at least 24 weeks. 5. A method according to any one of claims 1 to 4, wherein the improvement in functional ability is measured by a 25 foot time walk test (T-25FW) or an EDSS score. 6. A method of reducing the average annual relapse rate in a human patient with multiple sclerosis by at least 30%, comprising administering to the patient an effective amount of an anti-CD20 antibody, where the administration leads to a decrease in the average annual relapse rate by at least 30%, where the anti-CD20 antibody CD20 contains: 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 variable region a light chain containing 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. 7. A method of reducing the risk of confirmed progression of disability in a human patient with multiple sclerosis, comprising administering to the patient an effective amount of anti-CD20 antibody, wherein the risk of confirmed progression of disability for at least 12 weeks is reduced: (i) by at least 30% in a patient with relapsing multiple sclerosis compared with a patient with a relapsing form who is being treated with interferon beta-1a, or (ii) at least 15% in a patient with primary progressive multiple sclerosis compared with a patient with primary progressive multiple sclerosis who do not carry out treatment; and 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. 8. The method of claim 7, wherein the risk of confirmed progression of disability over at least 24 weeks is reduced: (i) by at least 30% in a patient with relapsing multiple sclerosis compared to a patient with recurrent multiple sclerosis undergoing treatment with interferon beta-1a, or (ii) at least 15% in a patient with primary progressive multiple sclerosis compared to a patient with primary progressive multiple sclerosis who is not being treated. 9. The method according to any one of claims 7, 8, wherein the patient has gadolinium stained T1 plaques at baseline. 10. The method according to any one of claims 7, 8, wherein the patient does not have gadolinium staining T1 plaques at baseline. 11. A method for reducing the volume of T2 plaques in a human patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the patient's T2 plaque volume after administration is reduced by at least 1%, and 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. 12. The method of claim 11, wherein the patient has gadolinium stained T1 plaques at baseline. 13. The method of claim 11, wherein the patient has no baseline gadolinium stained T1 plaques. 14. A method of reducing the rate of brain volume loss in a patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, where the rate of brain volume loss in the patient is reduced by at least 10%; and 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. 15. The method of claim 14, wherein a further decrease in brain volume is prevented in a patient who has a decrease in brain volume. 16. A method of reducing brain atrophy in a human patient having multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the brain atrophy is reduced by at least 10% after administration, wherein the anti-CD20 antibody comprises: a) a severe variable region a chain containing 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 containing CDR1 having the amino acid a 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. 17. The method of claim 16, wherein further cerebral atrophy is prevented in a patient who has cerebral atrophy. 18. 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 evidence of disease activity (NEDA) for at least 12 weeks, wherein the anti-CD20 antibody comprises: a) a severe variable region a chain containing 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 containing CDR1 having the amino acid a 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. 19. The method of claim 18, wherein the treatment results in no evidence of disease activity (NEDA) for at least 24 weeks. 20. 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 the patient achieving a plaque-free status after treatment for 96 weeks, 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 containing 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. 21. The method of claim 20, wherein the treatment results in the patient achieving a plaque-free status after treatment for 48 weeks. 22. The method of claim 20, wherein the treatment results in the patient achieving a plaque-free status after treatment for 24 weeks. 23. A method according to any one of claims 20 to 22, wherein the treatment results in the patient being free of gadolinium stained plaques. 24. A method according to any one of claims 20-22, wherein the treatment results in the patient being free of T2 plaques. 25. A method of treating a human patient with recurrent multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in one or more of: a) the patient is free of relapse after 96 weeks; b) the patient has no confirmed events of progression of disability after 96 weeks; c) the patient has no gadolinium-enhanced T1 plaques at 96 weeks; d) the patient has no new and / or increasing T2 plaques 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. 26. 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 containing CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having an amino acid a 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 CDR3 having the amino acid sequence of SEQ ID NO: 6. 27. The method of claim 26, wherein the highly active multiple sclerosis patient is not responding well to another multiple sclerosis therapy. 28. The method of claim 26, wherein the highly active multiple sclerosis patient has not previously been treated with another multiple sclerosis therapy. 29. The method of claim 27, wherein the other therapy for multiple sclerosis is interferon or glatiramer acetate. 30. A method according to any one of claims 26-29, wherein the administration of an anti-CD20 antibody is effective for one or more of the following: (1) reducing the amount of plaque in the patient's brain; (2) a decrease in the average annual relapse rate; (3) reduced progression of disability; and (4) improved functional ability. 31. The method of any one of claims 26-30, further comprising performing an MRI scan and determining if the patient has highly active multiple sclerosis prior to administering the anti-CD20 antibody to the patient. 32. 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 containing CDR1 having the amino acid sequence of SEQ ID NO: 10, 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. 33. The method of claim 32, further comprising diagnosing the patient having early stage multiple sclerosis prior to administering the anti-CD20 antibody to the patient. 34. 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 evidence of disease activity (NEDA) in the patient, and wherein the anti-CD20 antibody comprises: a) a heavy chain variable region containing CDR1, having the amino acid sequence of SEQ ID NO: 10, 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 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. 35. A method of treating a human patient with recurrent multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in one or more of: a) at least a 30% reduction in the average annual relapse rate; b) at least 30% reduction in the risk of confirmed progression of disability for at least 12 weeks; c) at least 30% reduction in the risk of confirmed progression of disability for at least 24 weeks; d) at least 90% reduction in the number of gadolinium ⁺ T1 plaques; e) at least 90% reduction in the mean number of gadolinium ⁺ T1 plaques at 24 weeks, 48 weeks and / or 96 weeks; f) at least 70% reduction in the number of new and / or increasing hyperintense T2 plaques; g) at least 40% reduction in the average number of new and / or increasing T2 plaques at 24 weeks, 48 weeks and / or 96 weeks; h) at least 10% reduction in the rate of decrease in the volume of the whole brain; i) at least 10% reduction in proven disability persisting for at least 12 weeks; j) at least 50% reduction in the number of new hypointense T1 plaques; and k) at least 55% improvement in NEDA (no evidence of disease activity); wherein the reduction or improvement in one or more of a) to k) is compared to a patient (s) with relapsing multiple sclerosis treated with interferon beta-1a, and wherein the anti-CD20 antibody comprises: 1) a heavy chain variable region containing CDR1 having the amino acid sequence of SEQ ID NO: 10, 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 containing 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. 36. A method of treating a human patient with recurrent multiple sclerosis, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the treatment results in at least 15% reduction in cerebral atrophy, wherein the reduction is compared to a patient (s) with recurrent multiple sclerosis, which is treated with interferon beta-1a, and 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 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. 37. 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 results in one or more of: a) at least 15% reduction in the risk of confirmed progression of disability for at least 12 weeks compared to untreated patient (s); b) at least 15% reduction in the risk of confirmed progression of disability for at least 24 weeks compared to the patient (s) who is not being treated; c) at least 15% reduction in progression in walking time as measured in the 25 foot timed walking test compared to untreated patient (s); d) at least 10% reduction in T2 plaque volume compared to untreated patient (s); e) at least 3% reduction in T2 plaque volume from baseline to 24 weeks; and f) at least 12% reduction in the rate of decrease in the volume of the whole brain; where the decrease in one or more of a) -f) is compared to the patient (s) with a recurrent form of multiple sclerosis, which is not being treated; 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. 38. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the patient retains the ability to induce a humoral immune response to an antigen during treatment. 39. The method of claim 38, wherein the antigen is a mumps antigen, rubella antigen, chickenpox antigen, S. pneumonia antigen, tetanus toxoid antigen, pneumococcal antigen, or influenza virus antigen. 40. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein during the initial exposure or subsequent exposures, a second drug is administered, wherein the anti-CD20 antibody represents the first drug. 41. The method according to any one of claims 1, 6, 11, 14, 16, 18, 20, 26, 32 and 34, wherein the multiple sclerosis is a recurrent form of multiple sclerosis. 42. The method of claim 41, wherein the recurrent form of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS). 43. The method of claim 42, wherein the recurrent form of multiple sclerosis is secondary progressive multiple sclerosis with concomitant relapses (rSPMS). 44. The method of any one of claims 1, 6, 11, 14, 16, 18, 20, 26, 32 and 34, wherein the multiple sclerosis is progressive multiple sclerosis. 45. The method of claim 44, wherein the multiple sclerosis is primary progressive multiple sclerosis (PPMS). 46. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the anti-CD20 antibody is administered to the patient to provide an initial exposure to the anti-CD20 antibody, followed by followed by one or more additional exposures of anti-CD20 antibody, where each exposure is 600 mg of antibody, and each exposure is performed on the patient as one or two doses of anti-CD20 antibody, and where the interval between exposures is 24 weeks or 6 months. 47. The method of claim 46, wherein the initial exposure comprises a first dose and a second dose of anti-CD20 antibody, wherein each dose is 300 mg and the first dose and second dose are separated by 14 days. 48. The method of claim 47, wherein the additional treatments comprise a single dose of 600 mg. 49. The method of claim 48, wherein the initial exposure and one or more additional exposures comprise a first dose and a second dose of anti-CD20 antibody, wherein each dose is 300 mg and the first dose and second dose are separated by 14 days. 50. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO : 8, and the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 2. 51. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein 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 containing the amino acid sequence of SEQ ID NO: 13. 52. The method of any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the anti-CD20 antibody is ocrelizumab. 53. The method of any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the anti-CD20 antibody is a pharmaceutically acceptable composition. 54. The method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the anti-CD20 antibody is administered intravenously. 55. The method of claim 54, wherein the anti-CD20 antibody is administered intravenously at each exposure to the antibody. 56. The method of any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, wherein the antibody is administered subcutaneously. 57. The method of claim 56, wherein the anti-CD20 antibody is administered subcutaneously at each exposure to the antibody. 58. A kit for the treatment of multiple sclerosis in a patient in accordance with the method according to any one of claims 1, 6, 7, 11, 14, 16, 18, 20, 25, 26, 32 and 34-37, containing: (a) a container containing ocrelizumab; and (b) a package insert with instructions for treating a patient with multiple sclerosis.

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