U.S. patent application number 16/507765 was filed with the patent office on 2020-05-21 for methods for treating progressive multiple sclerosis.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Peter S. Chin, Craig Smith.
Application Number | 20200157235 16/507765 |
Document ID | / |
Family ID | 41571287 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200157235 |
Kind Code |
A1 |
Smith; Craig ; et
al. |
May 21, 2020 |
METHODS FOR TREATING PROGRESSIVE MULTIPLE SCLEROSIS
Abstract
The present invention concerns methods for treating progressive
multiple sclerosis (MS) in a patient, and an article of manufacture
with instructions for such use.
Inventors: |
Smith; Craig; (Seattle,
WA) ; Chin; Peter S.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
41571287 |
Appl. No.: |
16/507765 |
Filed: |
July 10, 2019 |
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Application
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15978064 |
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16507765 |
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15597021 |
May 16, 2017 |
9994642 |
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15978064 |
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15201300 |
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9683047 |
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15597021 |
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14702583 |
May 1, 2015 |
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15201300 |
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14049130 |
Oct 8, 2013 |
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14702583 |
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13414698 |
Mar 7, 2012 |
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14049130 |
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12561131 |
Sep 16, 2009 |
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13414698 |
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61097464 |
Sep 16, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 39/3955 20130101; A61K 39/00 20130101; A61P 25/00 20180101;
A61P 37/02 20180101; C07K 2317/565 20130101; A61K 2039/55 20130101;
A61P 25/28 20180101; G01N 2800/7095 20130101; G06Q 99/00 20130101;
A61P 29/00 20180101; A61K 2039/507 20130101; G01N 2800/52 20130101;
A61K 2039/545 20130101; C07K 2317/76 20130101; A61P 37/00 20180101;
A61K 45/06 20130101; G01N 33/686 20130101; A61K 9/0019 20130101;
C07K 2317/24 20130101; C07K 16/2887 20130101; A61K 2039/505
20130101; A61B 5/055 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 9/00 20060101
A61K009/00; G01N 33/68 20060101 G01N033/68; A61K 39/395 20060101
A61K039/395; A61B 5/055 20060101 A61B005/055; G06Q 99/00 20060101
G06Q099/00; A61K 39/00 20060101 A61K039/00 |
Claims
1. A method of treating progressive multiple sclerosis in a patient
comprising administering to the patient an effective amount of an
anti-CD20 antibody, wherein treatment is based upon the patient
having one or more characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.
2-38. (canceled)
39. A method of treating multiple sclerosis in a patient comprising
administering an effective amount of ocrelizumab to the patient to
provide an initial ocrelizumab exposure of between about 0.3 to
about 0.6 grams followed by a second ocrelizumab exposure of
between about 0.3 to about 0.6 grams, the second exposure not being
provided until from about 16 to 60 weeks from the initial exposure,
and each of the ocrelizumab exposures is provided to the patient as
one or two doses of ocrelizumab.
40-46. (canceled)
47. An article of manufacture comprising: (a) a container
comprising ocrelizumab; and (b) a package insert with instructions
for treating multiple sclerosis in a patient, wherein the
instructions indicate that an amount of ocrelizumab is administered
to the patient that is effective to provide an initial ocrelizumab
exposure of between about 0.3 to about 0.6 grams followed by a
second ocrelizumab exposure of between about 0.3 to about 0.6
grams, the second exposure not being administered until from about
16 to 60 weeks from the initial exposure, and each of the
ocrelizumab exposures is provided to the patient as one or two
doses of ocrelizumab.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit to U.S. Provisional
Application Ser. No. 61/097,464, filed Sep. 16, 2008, the entirety
of which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns methods for treating
progressive multiple sclerosis (MS) in a patient, and an article of
manufacture with instructions for such use.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392006102 SEQLIST.TXT, date recorded: Jul. 13, 2015, size: 38
KB).
BACKGROUND OF THE INVENTION
Multiple Sclerosis
[0004] Multiple Sclerosis (MS) is an inflammatory and demyelinating
degenerative disease of the human central nervous system (CNS). It
is a worldwide disease that affects approximately 300,000 persons
in the United States; it is a disease of young adults, with 70%-80%
having onset between 20 and 40 years old (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) scan assessment, and pathology
analysis of biopsy and autopsy material (Lucchinetti et al. Ann
Neurol 47:707-17 (2000)). The disease manifests itself in a large
number of possible combinations of deficits, including spinal cord,
brainstem, cranial nerve, cerebellar, cerebral, and cognitive
syndromes. Progressive disability is the fate of most patients with
MS, especially when a 25-year perspective is included. Half of MS
patients require a cane to walk within 15 years of disease onset.
MS is a major cause of neurologic disability in young and
middle-aged adults and, until the past decade, has had no known
beneficial treatments. MS is difficult to diagnose because of the
non-specific clinical findings, which led to the development of
highly structured diagnostic criteria that include several
technological advances, consisting of MRI scans, evoked potentials,
and cerebrospinal fluid (CSF) studies. All diagnostic criteria rely
upon the general principles of scattered lesions in the central
white matter occurring at different times and not explained by
other etiologies such as infection, vascular disorder, or
autoimmune disorder (McDonald et al. Ann Neurol 50:121-7 (2001)).
MS has four patterns of disease: relapsing-remitting MS (RRMS;
80%-85% of cases at onset), primary progressive MS (PPMS; 10%-15%
at onset), progressive relapsing MS (PRMS; 5% at 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)). An estimated 50% of patients with RRMS will develop SPMS
in 10 years, and up to 90% of RRMS patients will eventually develop
SPMS (Weinshenker et al. Brain 112(Pt 1):133-46 (1989)).
[0005] Currently, six drugs in four classes are approved in the
United States for the treatment of RRMS, whereas no drugs have been
approved for PPMS. The RRMS treatments include the following:
interferon class, IFN-beta-la (REBIF.RTM. and AVONEX.RTM.) and
IFN-beta-lb (BETASERON.RTM.); glatiramer acetate (COPAXONE.RTM.), a
polypeptide; natalizumab (TYSABRI.RTM.); and mitoxantrone
(NOVANTRONE.RTM.), a cytotoxic agent. Other drugs have been used
with varying degrees of success, including corticosteroids,
methotrexate, cyclophosphamide, azathioprine, and intravenous (IV)
immunoglobulin. The benefits of currently approved treatments are
relatively modest (.about.30%) for relapse rate and prevention of
disability in RRMS as suggested by two meta-analyses (Filippini et
at Lancet 361:545-52 (2003)).
[0006] Other clinical studies evaluated other immunomodulatory
agents in MS, including tumor necrosis factor-.alpha. inhibitors
and altered peptide ligands, which aggravated rather than improved
MS (Lenercept Multiple Sclerosis Study Group and the University of
British Columbia MS/MRI Neurology 53:457-65 (1999); Bielekova et
al. Nat Med 2000; 6:1167-75 (2000), erratum appears in Nat Med
6:1412 (2000)).
[0007] The predominant view of MS pathophysiology has held that
inflammation is principally mediated by CD4.sup.+ Th1 T cells.
Therapeutic approaches based on this theory such as IFN-beta and
glatiramer acetate decrease, but do not fully prevent, occurrence
of exacerbations or accumulation of disability.
[0008] The existence of a humoral component in human MS has been
implicitly recognized for decades, as evidenced by inclusion of CSF
oligoclonal bands and increased intrathecal IgG synthesis in
diagnostic criteria for MS (Siden A. J Neurol 221:39-51(1979);
McDonald et al. Ann Neurol 50:121-7 (2001); Andersson et al Eur J
Neurol 9:243-51 (2002); O'Connor, P. Neurology 59:S1-33 (2002)).
The presence of oligoclonal bands, increased free light chains, and
increased intrathecal IgM synthesis correlates with MS disease
activity and may be a predictor of more severe outcomes (Rudick et
al. Mult Scler 1:150-5 (1995); Zeman et al. Acta Cytol 45:51-9
(2001); Izquierdo et al. Acta Neurol Scand 105:158-63 (2002);
Wolinsky J. J Neurol Sci 206:145-52 (2003); Villar et al. Ann
Neurol 53:222-6 (2003)).
[0009] Anti-myelin antibodies (myelin basic protein (MBP) and
myelin oligodendrocyte glycoprotein (MOG)) have been detected in
the serum of patients with progressive and relapsing forms of MS
(Reindl et al. Brain 122:2047-56 (1999); Egg et al. Mult Scler
7(5):285-9 (2001)). Anti-myelin antibodies have also been detected
in the CSF of MS patients (Reindl et al. Brain 122:2047-56 (1999);
Egg et al. Mult Scler 7(5):285-9 (2001); Andersson et al. Eur J
Neurol 9:243-51 (2002)). Additional types of antibodies such as
anti-ganglioside antibodies or anti-neurofilament antibodies have
been observed in patients with MS (Mata et al. Mult Scler 5:379-88
(1999); Sadatipour et al. Ann Neurol 44:980-3 (1998)). A report
indicated that the presence of serum anti-MOG and anti-MBP
antibodies was a strong predictor of progression from a clinically
isolated demyelinating event to definite RRMS (Berger et al. N Engl
J Med 349:139-45 (2003)). The adjusted hazard ratio for
experiencing an exacerbation was 76.5 for patients who were
seropositive for both antibodies and 31.6 for patients who were
seropositive only for anti-MOG.
[0010] An international pathology consortium found that antibodies
bound to myelin are present in the majority of patients with MS,
with plasma cells and B cells also found in MS lesions, providing
additional evidence for a humoral role in MS (Prineas and Wright,
Lab Invest 38:409-21 (1978); Esiri M. Neuropathol Appl Neurobiol
6:9-21 (1980); Genain et al. Nat Med 5:170-5 (1999); Lucchinetti et
al. Ann Neurol 47:707-17 (2000); Wingerchuk et al. Lab Invest
81:263-81 (2001)). B cells are detectable in the CSF of patients
with MS, and the presence of a relatively high proportion of B
cells may be predictive of more severe disability progression
(Cepok et al. Brain 124(Pt 11):2169-76 (2001)).
[0011] In patients with RRMS or opsoclonus-myoclonus syndrome,
Rituximab reportedly depleted peripheral B-cells in all patients
and decreased the number of CSF B cells in some patients
(Pranzatelli et al. Neurology 60(Suppl1) P05.128:A395 (2003); Cross
et al. "Preliminary Results from a Phase II Trial of Rituximab in
MS" (abstract) Eighth Annual Meeting of the Americas Committees for
Research and Treatment in Multiple Sclerosis ACTRIMS 20-1 (October,
2003); Cross et al. J Neuroimmunol. 180:63-70 (2006)). See also
Cree et al. "Tolerability and Effects of Rituximab "Anti-CD20
Antibody" in Neuromyelitis Optica and Rapidly Worsening Multiple
Sclerosis" Meeting of the Am. Acad. Neurol. (April, 2004); Cree et
al. Neurology 64:1270-2 (2005).
CD20 Antibodies and Therapy Therewith
[0012] Lymphocytes are one of many types of white blood cells
produced in the bone marrow during the process of hematopoiesis.
There are two major populations of lymphocytes: B lymphocytes (B
cells) and T lymphocytes (T cells). The lymphocytes of particular
interest herein are B cells.
[0013] B cells mature within the bone marrow and leave the marrow
expressing an antigen-binding antibody on their cell surface. When
a naive B cell first encounters the antigen for which its
membrane-bound antibody is specific, the cell begins to divide
rapidly and its progeny differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer
life span and continue to express membrane-bound antibody with the
same specificity as the original parent cell. Plasma cells do not
produce membrane-bound antibody but instead produce the antibody in
a form that can be secreted. Secreted antibodies are the major
effector molecule of humoral immunity.
[0014] The CD20 antigen (also called human B-lymphocyte-restricted
differentiation antigen, Bp35) is a hydrophobic transmembrane
protein with a molecular weight of approximately 35 kD located on
pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem.
264(19):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717
(1988)). The antigen is also expressed on greater than 90% of
B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al. Blood
63(6):1424-1433 (1984)), but is not found on hematopoietic stem
cells, pro-B cells, normal plasma cells or other normal tissues
(Tedder et al. J. Immunol. 135(2):973-979 (1985)). CD20 regulates
an early step(s) in the activation process for cell cycle
initiation and differentiation (Tedder et al., supra) and possibly
functions as a calcium ion channel (Tedder et al. J. Cell. Biochem.
14D:195 (1990)).
[0015] Given the expression of CD20 in B-cell lymphomas, this
antigen can serve as a candidate for "targeting" of such lymphomas.
In essence, such targeting can be generalized as follows:
antibodies specific to the CD20 surface antigen of B cells are
administered to a patient. These anti-CD20 antibodies specifically
bind to the CD20 antigen of (ostensibly) both normal and malignant
B cells; the antibody bound to the CD20 surface antigen may lead to
the destruction and depletion of neoplastic B cells. Additionally,
chemical agents or radioactive labels having the potential to
destroy the tumor can be conjugated to the anti-CD20 antibody such
that the agent is specifically "delivered" to the neoplastic B
cells. Irrespective of the approach, a primary goal is to destroy
the tumor; the specific approach can be determined by the
particular anti-CD20 antibody that is utilized and, thus, the
available approaches to targeting the CD20 antigen can vary
considerably.
[0016] The Rituximab (RITUXAN.RTM.) antibody is a genetically
engineered chimeric murine/human monoclonal antibody directed
against the CD20 antigen. Rituximab is the antibody called "C2B8"
in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al.).
RITUXAN.RTM. is indicated for the treatment of patients with
relapsed or refractory low-grade or follicular, CD20-positive,
B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies
have demonstrated that RITUXAN.RTM. binds human complement and
lyses lymphoid B-cell lines through complement-dependent
cytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)).
Additionally, it has significant activity in assays for
antibody-dependent cellular cytotoxicity (ADCC). More recently,
RITUXAN.RTM. has been shown to have anti-proliferative effects in
tritiated thymidine incorporation assays and to induce apoptosis
directly, while other anti-CD19 and CD20 antibodies do not (Maloney
et al. Blood 88(10):637a (1996)). Synergy between RITUXAN.RTM. and
chemotherapies and toxins has also been observed experimentally. In
particular, RITUXAN.RTM. sensitizes drug-resistant human B-cell
lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP,
VP-16, diphtheria toxin and ricin (Demidem et al. Cancer
Chemotherapy & Radiopharmaceuticals 12(3):177-186 (1997)). In
vivo preclinical studies have shown that RITUXAN.RTM. depletes B
cells from the peripheral blood, lymph nodes, and bone marrow of
cynomolgus monkeys, presumably through complement and cell-mediated
processes (Reff et al. Blood 83(2):435-445 (1994)).
[0017] Rituximab was approved in the United States in November 1997
for the treatment of patients with relapsed or refractory low-grade
or follicular CD20.sup.+ B-cell non-Hodgkin's lymphoma (NHL) at a
dose of 375 mg/m.sup.2 weekly for four doses. In April 2001, the
Food and Drug Administration (FDA) approved additional claims for
the treatment of low-grade NHL: retreatment (weekly for four doses)
and an additional dosing regimen (weekly for eight doses). There
have been more than 300,000 patient exposures to Rituximab either
as monotherapy or in combination with immunosuppressant or
chemotherapeutic drugs. Patients have also been treated with
Rituximab as maintenance therapy for up to 2 years (Hainsworth et
al. J Clin Oncol 21:1746-51 (2003); Hainsworth et al. J Clin Oncol
20:4261-7 (2002)).
[0018] Rituximab has also been studied in a variety of
non-malignant autoimmune disorders, in which B cells and
autoantibodies appear to play a role in disease pathophysiology
(Edwards et al. Biochem Soc Trans 30:824-8 (2002)). Rituximab has
been reported to potentially relieve signs and symptoms of
rheumatoid arthritis (RA) (Leandro et al. Ann Rheum Dis. 61:883-8
(2002); Emery et al. Arthritis Rheum 48(9):S439 (2003)), lupus
(Eisenberg R. Arthritis Res Ther 5:157-9 (2003); Leandro et al.
Arthritis Rheum 46:2673-7 (2002)), immune thrombocytopenia (D'Arena
et al. Leuk Lymphoma 44:561-2 (2003)), autoimmune anemia (Zaja et
al. Haematologica 87:189-95 (2002) (erratum appears in
Haematologica 87:336 (2002)), autoimmune neuropathy (Pestronk et
al. J Neurol Neurosurg Psychiatry 74:485-9 (2003)), paraneoplastic
opsoclonus-myoclonus syndrome (Pranzatelli et al. Neurology
60(Suppl1) P05.128:A395 (2003)), and relapsing-remitting multiple
sclerosis (RRMS) (Cross et al. (abstract) Eighth Annual Meeting of
the Americas Committees for Research and Treatment in Multiple
Sclerosis 20-1 (2003)).
[0019] A Phase II study (WA16291) has been conducted in patients
with rheumatoid arthritis (RA), providing 48-week follow-up data on
safety and efficacy of Rituximab (Emery et al. Arthritis Rheum
48(9):5439 (2003); Szczepanski et al. Arthritis Rheum 48(9):5121
(2003)). A total of 161 patients were evenly randomized to four
treatment arms: methotrexate, Rituximab alone, Rituximab plus
methotrexate, Rituximab plus cyclophosphamide (CTX). The treatment
regimen of Rituximab was 1 g administered intravenously on Days 1
and 15. Infusions of Rituximab in most patients with RA were well
tolerated by most patients, with 36% of patients experiencing at
least one adverse event during their first infusion (compared with
30% of patients receiving placebo). Overall, the majority of
adverse events were considered to be mild to moderate in severity
and were well balanced across all treatment groups. There were a
total of 19 serious adverse events across the four arms over the 48
weeks, which were slightly more frequent in the Rituximab/CTX
group. The incidence of infections was well balanced across all
groups. The mean rate of serious infection in this RA patient
population was 4.6 6 per 100 patient-years, which is lower than the
rate of infections requiring hospital admission in RA patients
(9.57 per 100 patient-years) reported in a community-based
epidemiologic study (Doran et al. Arthritis Rheum 46:2287-93
(2002)).
[0020] The reported safety profile of Rituximab in a small number
of patients with neurologic disorders, including autoimmune
neuropathy (Pestronk et al. J Neurol Neurosurg Psychiatry 74:485-9
(2003)), opsoclonus/myoclonus syndrome (Pranzatelli et al.
Neurology 60(Suppl1) P05.128:A395 (2003)), and RRMS (Cross et al.
Preliminary results from a phase II trial of Rituximab in MS
(abstract) Eighth Annual Meeting of the Americas Committees for
Research and Treatment in Multiple Sclerosis 20-1 (2003)), was
reported. In an ongoing investigator-sponsored trial (IST) of
Rituximab in combination with interferon-beta
(IFN-beta.quadrature.) or glatiramer acetate in subjects with RRMS
(Cross et al., supra), 1 of 10 treated subjects was admitted to the
hospital for overnight observation after experiencing moderate
fever and rigors following the first infusion of Rituximab, while
the other 9 subjects completed the four-infusion regimen without
any reported adverse events.
[0021] Patents and patent publications concerning CD20 antibodies,
CD20-binding molecules, and self-antigen vaccines include U.S. Pat.
Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as
well as US 2002/0197255, US 2003/0021781, US 2003/0082172, US
2003/0095963, US 2003/0147885, US 2005/0186205, and WO 1994/11026
(Anderson et al.); U.S. Pat. No. 6,455,043, US 2003/0026804, US
2003/0206903, and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428
(Grillo-Lopez and White); US 2004/0213784 and WO 2000/27433
(Grillo-Lopez and Leonard); WO 2000/44788 (Braslawsky et al.); WO
2001/10462 (Rastetter, W.); WO 2001/10461 (Rastetter and White); WO
2001/10460 (White and Grillo-Lopez); US 2001/0018041, US
2003/0180292, US 2002/0028178, WO 2001/34194, and WO 2002/22212
(Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and
Hariharan); US 2002/0012665, US 2005/0180975, WO 2001/74388, and
U.S. Pat. No. 6,896,885 (Hanna, N.); US 2002/0058029 (Hanna, N.);
US 2003/0103971 (Hariharan and Hanna); US 2005/0123540 (Hanna et
al.); US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO
2001/97858; US 2005/0112060, US 2002/0039557, and U.S. Pat. No.
6,846,476 (White, C.); US 2002/0128448 and WO 2002/34790 (Reff,
M.); WO 2002/060955 (Braslawsky et al.); WO 2002/096948 (Braslawsky
et al.);WO 2002/079255 (Reff and Davies); U.S. Pat. Nos. 6,171,586
and 6,991,790, and WO 1998/56418 (Lam et al.); US 2004/0191256 and
WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642,
U.S. Pat. Nos. 6,194,551, 6,242,195, 6,528,624 and 6,538,124
(Idusogie et al.);U.S. Pat. No. 7,122,637, US 2005/0118174, US
2005/0233382, US 2006/0194291, US 2006/0194290, US 2006/0194957,
and WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO
2001/03734 (Grillo-Lopez et al.); US 2002/0004587, US 2006/0025576,
and WO 2001/77342 (Miller and Presta); US 2002/0197256 and WO
2002/078766 (Grewal, I.); US 2003/0157108 and WO 2003/035835
(Presta, L.); U.S. Pat. Nos. 5,648,267, 5,733,779, 6,017,733, and
6,159,730, and WO 1994/11523 (Reff et al. on expression
technology); U.S. Pat. Nos. 6,565,827, 6,090,365, 6,287,537,
6,015,542, 5,843,398, and 5,595,721 (Kaminski et al.); U.S. Pat.
Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852, and
6,893,625 as well as WO 1988/04936 (Robinson et al.); U.S. Pat. No.
6,410,391 (Zelsacher); U.S. Pat. No. 6,224,866 and WO 2000/20864
(Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO
2000/67795 (Goldenberg); U.S. Pat. No. 7,074,403 (Goldenberg and
Hansen); U.S. Pat. No. 7,151,164 (Hansen et al.); US 2003/0133930;
WO 2000/74718 and US 2005/0191300A1 (Goldenberg and Hansen); US
2003/0219433 and WO 2003/68821 (Hansen et al.); WO 2004/058298
(Goldenberg and Hansen); WO 2000/76542 (Golay et al.);WO 2001/72333
(Wolin and Rosenblatt); U.S. Pat. No. 6,368,596 (Ghetie et al.);
U.S. Pat. No. 6,306,393 and US 2002/0041847 (Goldenberg, D.); US
2003/0026801 (Weiner and Hartmann); WO 2002/102312 (Engleman, E.);
US 2003/0068664 (Albitar et al.); WO 2003/002607 (Leung, S.); WO
2003/049694, US 2002/0009427, and US 2003/0185796 (Wolin et al.);
WO 2003/061694 (Sing and Siegall); US 2003/0219818 (Bohen et al.);
US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818
(Bohen et al.); US 2002/0136719 (Shenoy et al.); WO 2004/032828 and
US 2005/0180972 (Wahl et al.); and WO 2002/56910
(Hayden-Ledbetter). See also U.S. Pat. No. 5,849,898 and EP 330,191
(Seed et al.); EP332,865A2 (Meyer and Weiss); U.S. Pat. No.
4,861,579 (Meyer et al.); US 2001/0056066 (Bugelski et al.); WO
1995/03770 (Bhat et al.); US 2003/0219433 A1 (Hansen et al.); WO
2004/035607 and US 2004/167319 (Teeling et al.); WO 2005/103081
(Teeling et al.); US 2006/0034835, US 2006/0024300, and WO
2004/056312 (Lowman et al.); US 2004/0093621 (Shitara et al.); WO
2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); US
2005/0025764 (Watkins et al.); US 2006/0251652 (Watkins et al.); WO
2005/016969 (Carr et al.); US 2005/0069545 (Carr et al); WO
2005/014618 (Chang et al.); US 2005/0079174 (Barbera-Guillem and
Nelson); US 2005/0106108 (Leung and Hansen); US 2005/0123546 (Umana
et al.); US 2004/0072290 (Umana et al.); US 2003/0175884 (Umana et
al.); and WO 2005/044859 (Umana et al.); WO 2005/070963 (Allan et
al.); US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US
2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/136049
(Ledbetter et al.); US 2003/118592 (Ledbetter et al.); US
2003/133939 (Ledbetter and Hayden-Ledbetter); US 2005/0202012
(Ledbetter and Hayden-Ledbetter); US 2005/0175614 (Ledbetter and
Hayden-Ledbetter); US 2005/0180970 (Ledbetter and
Hayden-Ledbetter); US 2005/0202028 (Hayden-Ledbetter and
Ledbetter); US 2005/0202023 (Hayden-Ledbetter and Ledbetter); WO
2005/017148 (Ledbetter et al.); WO 2005/037989 (Ledbetter et al.);
U.S. Pat. No. 6,183,744 (Goldenberg); U.S. Pat. No. 6,897,044
(Braslawski et al.); WO 2006/005477 (Krause et al.); US
2006/0029543 (Krause et al.); US 2006/0018900 (McCormick et al.);
US 2006/0051349 (Goldenberg and Hansen); WO 2006/042240 (Iyer and
Dunussi-Joannopoulos); US 2006/0121032 (Dahiyat et al.); WO
2006/064121 (Teillaud et al.); US 2006/0153838 (Watkins), CN
1718587 (Chen et al.); WO 2006/084264 (Adams et al.); US
2006/0188495 (Barron et al.); US 2004/0202658 and WO 2004/091657
(Benynes, K.); US 2005/0095243, US 2005/0163775, WO 2005/00351, and
WO 2006/068867 (Chan, A.); US 2006/0135430 and WO 2005/005462 (Chan
et al.); US 2005/0032130 and WO 2005/017529 (Beresini et al.); US
2005/0053602 and WO 2005/023302 (Brunetta, P.); US 2006/0179501 and
WO 2004/060052 (Chan et al.); WO 2004/060053 (Chan et al.); US
2005/0186206 and WO 2005/060999 (Brunetta, P.); US 2005/0191297 and
WO 2005/061542 (Brunetta, P.); US 2006/0002930 and WO 2005/115453
(Brunetta et al.); US 2006/0099662 and WO 2005/108989 (Chuntharapai
et al.); CN 1420129A (Zhongxin Guojian Pharmaceutical); US
2005/0276803 and WO 2005/113003 (Chan et al.); US 2005/0271658 and
WO 2005/117972 (Brunetta et al.); US 2005/0255527 and WO 2005/11428
(Yang, J.); US 2006/0024295 and WO 2005/120437 (Brunetta, P.); US
2006/0051345 and WO 2005/117978 (Frohna, P.); US 2006/0062787 and
WO 2006/012508 (Hitraya, E.); US 2006/0067930 and WO 2006/31370
(Lowman et al.); WO 2006/29224 (Ashkenazi, A.); US 2006/0110387 and
WO 2006/41680 (Brunetta, P.); US 2006/0134111 and WO 2006/066086
(Agarwal, S.); WO 2006/069403 (Ernst and Yansura); US 2006/0188495
and WO 2006/076651 (Dummer, W.); WO 2006/084264 (Lowman, H.); WO
2006/093923 (Quan and Sewell); WO 2006/106959 (Numazaki et al.); WO
2006/126069 (Morawala); WO 2006/130458 (Gazit-Bornstein et al.); US
2006/0275284 (Hanna, G.); US 2007/0014785 (Golay et al.); US
2007/0014720 (Gazit-Bornstein et al.); and US 2007/0020259 (Hansen
et al.); US 2007/0020265 (Goldenberg and Hansen); US 2007/0014797
(Hitraya); US 2007/0224189 (Lazar et al.); WO 2007/014238 (Bruge
and Bruger); and WO 2008/003319 (Parren and Baadsgaard). Certain of
these include, inter alia, treatment of multiple sclerosis.
[0022] Publications concerning therapy with Rituximab include:
Perotta and Abuel "Response of chronic relapsing ITP of 10 years
duration to Rituximab" Abstract #3360 Blood 10(1)(part 1-2): p. 88B
(1998); Stashi et al. "Rituximab chimeric anti-CD20 monoclonal
antibody treatment for adults with chronic idopathic
thrombocytopenic purpura" Blood 98(4):952-957 (2001); Matthews, R.
"Medical Heretics" New Scientist (7 April, 2001); Leandro et al.
"Clinical outcome in 22 patients with rheumatoid arthritis treated
with B lymphocyte depletion" Ann Rheum Dis 61:833-888 (2002);
Leandro et al. "Lymphocyte depletion in rheumatoid arthritis: early
evidence for safety, efficacy and dose response. Arthritis and
Rheumatism 44(9): 5370 (2001); Leandro et al. "An open study of B
lymphocyte depletion in systemic lupus erythematosus", Arthritis
& Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge
"Sustained improvement in rheumatoid arthritis following a protocol
designed to deplete B lymphocytes" Rhematology 40:205-211 (2001);
Edwards et al. "B-lymphocyte depletion therapy in rheumatoid
arthritis and other autoimmune disorders" Biochem. Soc. Trans.
30(4):824-828 (2002); Edwards et al. "Efficacy and safety of
Rituximab, a B-cell targeted chimeric monoclonal antibody: A
randomized, placebo controlled trial in patients with rheumatoid
arthritis. Arthritis and Rheumatism 46(9): S197 (2002); Levine and
Pestronk "IgM antibody-related polyneuropathies: B-cell depletion
chemotherapy using Rituximab" Neurology 52: 1701-1704 (1999);
DeVita et al. "Efficacy of selective B cell blockade in the
treatment of rheumatoid arthritis" Arthritis & Rheum
46:2029-2033 (2002); Hidashida et al. "Treatment of
DMARD-Refractory rheumatoid arthritis with Rituximab." Presented at
the Annual Scientific Meeting of the American College of
Rheumatology; October 24-29; New Orleans, L A 2002; Tuscano, J.
"Successful treatment of Infliximab-refractory rheumatoid arthritis
with Rituximab" Presented at the Annual Scientific Meeting of the
American College of Rheumatology; October 24-29; New Orleans, L A
2002; Specks et al. "Response of Wegener's granulomatosis to
anti-CD20 chimeric monoclonal antibody therapy" Arthritis &
Rheumatism 44(12):2836-2840 (2001); Anolik et al., "B lympocyte
Depletion in the Treatment of Systemic Lupus (SLE): Phase I/II
Trial of Rituximab (RITUXAN.RTM.) in SLE" Arthritis And Rheumatism,
46(9), S289-S289 Abstract 717 (October, 2002), and Albert et al.,
"A Phase I Trial of Rituximab (Anti-CD20) for Treatment of Systemic
Lupus Erythematosus" Arthritis And Rheumatism, 48(12): 3659-3659,
Abstract LB9 (December, 2003); Martin and Chan "Pathogenic Roles of
B cells in Human Autoimmunity: Insights from the Clinic" Immunity
20:517-527 (2004); Cree et al. "An open label study of the effects
of rituximab in neuromyelitis optica." Neurology 64(7):1270-2
(2005); Cross et al. "Rituximab reduces B cells and T cells in
cerebrospinal fluid of multiple sclerosis patients." J
Neuroimmunol, 180(1-2):63-70 (2006); Bar-Or A. et al., "Safety,
pharmacodynamics, and activity of Rituximab in patients with
relapsing-remitting multiple sclerosis: a phase I, multicentre,
open-label clinical trial." Ann Neurol 63(3):395-400 (2008); Hauser
S. et al., "B-cell depletion with Rituximab in relapsing-remitting
multiple sclerosis." NEJM, 358(7):676-88, (2008); Hawker K et al.,
"Efficacy and Safety of rituximab in patients with primary
progressive multiple sclerosis: results of a randomized,
double-blind, placebo-controlled, multicenter trial." Multiple
Sclerosis 14(1):5299 (2008), Abstract; Hawker K et al., "Efficacy
and Safety of rituximab in patients with primary progressive
multiple sclerosis: results of a randomized, double-blind,
placebo-controlled, multicenter trial." Neurology 72(S3):A254
(2009), Abstract.
BRIEF SUMMARY OF THE INVENTION
[0023] The present invention provides methods of treating
progressive multiple sclerosis in a patient comprising
administering to the patient an effective amount of an anti-CD20
antibody, wherein treatment is based upon the patient having one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0024] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0025] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0026] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0027] In some embodiments, the age of the patient is less than
about 51.
[0028] In some embodiments, the treatment reduces the time to
confirmed disease progression. In some embodiments, the confirmed
disease progression is an increase in EDSS that is sustained for
twelve weeks. In some embodiments, the confirmed disease
progression is an increase in EDSS that is sustained for
twenty-four weeks.
[0029] In some embodiments, the effective amount of the anti-CD20
antibody is administered to the patient to provide an initial
anti-CD20 antibody exposure of between about 0.3 to about 4.0 grams
followed by a second anti-CD20 antibody exposure of between about
0.3 to about 4.0 grams. In some embodiments, the initial anti-CD20
antibody exposure and/or the second anti-CD20 antibody exposure is
between about 0.3 to about 1.5 grams. In some embodiments, the
second exposure not being provided until from about 16 to 60 weeks
from the initial exposure. In some embodiments, each of the
anti-CD20 antibody exposures is provided to the patient as one or
two doses of anti-CD20 antibody.
[0030] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0031] The present invention provides methods of treating
progressive multiple sclerosis in a patient provided that the
patient has been found to have one or more characteristics selected
from the group consisting of (a) an age less than about 55 years,
(b) one or more gadolinium staining lesions, (c) at least about a
one point increase in Expanded Disability Status Scale (EDSS) over
two years prior to starting treatment, and (d) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points, the treatment
comprising administering to the patient an effective amount of an
anti-CD20 antibody.
[0032] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0033] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0034] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0035] In some embodiments, the age of the patient is less than
about 51.
[0036] In some embodiments, the treatment reduces the time to
confirmed disease progression. In some embodiments, the confirmed
disease progression is an increase in EDSS that is sustained for
twelve weeks. In some embodiments, the confirmed disease
progression is an increase in EDSS that is sustained for
twenty-four weeks.
[0037] In some embodiments, the effective amount of the anti-CD20
antibody is administered to the patient to provide an initial
anti-CD20 antibody exposure of between about 0.3 to about 4.0 grams
followed by a second anti-CD20 antibody exposure of between about
0.3 to about 4.0 grams. In some embodiments, the initial anti-CD20
antibody exposure and/or the second anti-CD20 antibody exposure is
between about 0.3 to about 1.5 grams. In some embodiments, the
second exposure not being provided until from about 16 to 60 weeks
from the initial exposure. In some embodiments, each of the
anti-CD20 antibody exposures is provided to the patient as one or
two doses of anti-CD20 antibody.
[0038] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0039] The present invention provides methods of treating
progressive multiple sclerosis, comprising: (a) selecting a patient
having progressive multiple sclerosis, wherein said patient has one
or more characteristics selected from the group consisting of (i)
an age less than about 55 years, (ii) one or more gadolinium
staining lesions, (iii) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment, and (iv) a Multiple Sclerosis Severity Score
(MSSS) greater than about 5 points; and (b) administering to the
patient thus selected an effective amount of an anti-CD20
antibody.
[0040] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0041] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0042] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0043] In some embodiments, the age of the patient is less than
about 51.
[0044] In some embodiments, the treatment reduces the time to
confirmed disease progression. In some embodiments, the confirmed
disease progression is an increase in EDSS that is sustained for
twelve weeks. In some embodiments, the confirmed disease
progression is an increase in EDSS that is sustained for
twenty-four weeks.
[0045] In some embodiments, the effective amount of the anti-CD20
antibody is administered to the patient to provide an initial
anti-CD20 antibody exposure of between about 0.3 to about 4.0 grams
followed by a second anti-CD20 antibody exposure of between about
0.3 to about 4.0 grams. In some embodiments, the initial anti-CD20
antibody exposure and/or the second anti-CD20 antibody exposure is
between about 0.3 to about 1.5 grams. In some embodiments, the
second exposure not being provided until from about 16 to 60 weeks
from the initial exposure. In some embodiments, each of the
anti-CD20 antibody exposures is provided to the patient as one or
two doses of anti-CD20 antibody.
[0046] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0047] The present invention also provides methods of assessing
whether a patient with progressive multiple sclerosis will respond
to treatment with an anti-CD20 antibody comprising assessing one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points, wherein one or more of the
characteristics in the patient indicates the patient will be
responsive to the treatment.
[0048] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0049] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0050] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0051] In some embodiments, the age of the patient is less than
about 51.
[0052] In some embodiments, the method further comprises advising a
patient.
[0053] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0054] The present invention also provides methods of identifying a
patient with progressive multiple sclerosis likely to respond to
anti-CD20 antibody treatment comprising: (a) assessing one or more
characteristics selected from the group consisting of (i) an age
less than about 55 years, (ii) one or more gadolinium staining
lesions, (iii) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (iv) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points; and (b) identifying the patient having
one or more characteristics selected from the group consisting of
(i) an age less than about 55 years, (i) one or more gadolinium
staining lesions, (iii) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting the anti-CD20 treatment, and (iv) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points.
[0055] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0056] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0057] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0058] In some embodiments, the age of the patient is less than
about 51.
[0059] In some embodiments, the method further comprises advising a
patient.
[0060] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0061] The present invention further provides methods for marketing
an anti-CD20 antibody or a pharmaceutically acceptable composition
thereof for use in a progressive multiple sclerosis patient
subpopulation, the methods comprising informing a target audience
about the use of the anti-CD20 antibody for treating the patient
subpopulation characterized by the patients of such subpopulation
having one or more characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.
[0062] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient subpopulation is not diagnosed with relapsing remitting
multiple sclerosis when starting treatment.
[0063] In some embodiments, the patient subpopulation further has
evidence of inflammation in a sample. In some embodiments, the
sample is a cerebrospinal fluid sample. In some embodiments, the
evidence of inflammation is indicated by an elevated IgG index. In
some embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0064] In some embodiments, the patient subpopulation had an EDSS
of greater than about 5.0 for less than about 15 years. In some
embodiments, the patient subpopulation had an EDSS less than or
equal to about 5.0 for less than about 10 years. In some
embodiments, the increase in EDSS over two years prior to starting
treatment is not attributable to relapse. In some embodiments, the
increase in EDSS is at least about a 1.5 point increase in EDSS
over two years prior to starting treatment. In some embodiments,
the at least about a 1.5 point increase in EDSS over two years
prior to starting treatment is not attributable to relapse. In some
embodiments, the patient subpopulation further had two or more
relapses within two years prior to starting treatment. In some
embodiments, the EDSS when starting treatment is between about 3.0
and about 6.5.
[0065] In some embodiments, the age of the patient subpopulation is
less than about 51.
[0066] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0067] The present invention provides articles of manufacture
comprising, packaged together, a pharmaceutical composition
comprising an anti-CD20 antibody and a pharmaceutically acceptable
carrier and a label denoting (i.e., indicating) that the anti-CD20
antibody or pharmaceutical composition is indicated for treating
patients with multiple sclerosis having one or more characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, (c) at least
about a one point increase in Expanded Disability Status Scale
(EDSS) over two years prior to starting the treatment, and (d) a
Multiple Sclerosis Severity Score (MSSS) greater than about 5
points.
[0068] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the patient is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0069] In some embodiments, the patient further has evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0070] In some embodiments, the patient has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the patient has had an EDSS less than or equal to about 5.0 for
less than about 10 years. In some embodiments, the increase in EDSS
over two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the patient further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0071] In some embodiments, the age of the patient is less than
about 51.
[0072] In some embodiments, the pharmaceutical composition
comprising the anti-CD20 antibody and the pharmaceutically
acceptable carrier is in a container. In some embodiments, the
container comprises between about 0.3 to about 4.0 grams of the
anti-CD20 antibody. In some embodiments, the container comprises
between about 0.3 to about 1.5 grams of the anti-CD20 antibody.
[0073] In some embodiments, the label provides instructions,
wherein the instructions indicate that an effective amount of the
anti-CD20 antibody is administered to the patient to provide an
initial anti-CD20 antibody exposure of between about 0.3 to about
4.0 grams followed by a second anti-CD20 antibody exposure of
between about 0.3 to about 4.0 grams. In some embodiments, the
initial anti-CD20 antibody exposure and/or the second anti-CD20
antibody exposure is between about 0.3 to about 1.5 grams. In some
embodiments, the second exposure not being provided until from
about 16 to 60 weeks from the initial exposure. In some
embodiments, each of the anti-CD20 antibody exposures is provided
to the patient as one or two doses of anti-CD20 antibody.
[0074] In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0075] The present invention also provides methods for predicting
whether a subject with progressive multiple sclerosis will respond
to a treatment with a drug used to treat multiple sclerosis, the
methods comprising assessing one or more characteristics selected
from the group consisting of (a) an age less than about 55 years,
(b) one or more gadolinium staining lesions, (c) at least about a
one point increase in Expanded Disability Status Scale (EDSS) over
two years prior to starting treatment, and (d) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points, whereby the age,
the gadolinium staining lesions, the increase in EDDS over two
years prior to starting the treatment, the MSSS, or a combination
thereof indicates that the subject will respond to the
treatment.
[0076] In some embodiments, the progressive multiple sclerosis is
primary progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is secondary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is progressive relapsing multiple sclerosis. In some embodiments,
the subject is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0077] In some embodiments, the subject further has had evidence of
inflammation in a sample. In some embodiments, the sample is a
cerebrospinal fluid sample. In some embodiments, the evidence of
inflammation is indicated by an elevated IgG index. In some
embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing.
[0078] In some embodiments, the subject has had an EDSS of greater
than about 5.0 for less than about 15 years. In some embodiments,
the subject had an EDSS less than or equal to about 5.0 for less
than about 10 years. In some embodiments, the increase in EDSS over
two years prior to starting treatment is not attributable to
relapse. In some embodiments, the increase in EDSS is at least
about a 1.5 point increase in EDSS over two years prior to starting
treatment. In some embodiments, the at least about a 1.5 point
increase in EDSS over two years prior to starting treatment is not
attributable to relapse. In some embodiments, the subject further
had two or more relapses within two years prior to starting
treatment. In some embodiments, the EDSS when starting treatment is
between about 3.0 and about 6.5.
[0079] In some embodiments, the age of the subject is less than
about 51.
[0080] In some embodiments of any of the methods or articles of
manufacture described herein, the patients have one or more
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, and (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment.
[0081] The invention further provides methods of treating multiple
sclerosis in a patient comprising administering an effective amount
of ocrelizumab to the patient to provide an initial ocrelizumab
exposure of between about 0.3 to about 0.6 grams followed by a
second ocrelizumab exposure of between about 0.3 to about 0.6
grams, the second exposure not being provided until from about 16
to 60 weeks from the initial exposure, and each of the ocrelizumab
exposures is provided to the patient as one or two doses of
ocrelizumab.
[0082] In some embodiments, the initial ocrelizumab exposure is
about 0.6 grams. In some embodiments, the second ocrelizumab
exposure is about 0.6 grams. In some embodiments, the second
exposure is administered from about 24 weeks from the initial
exposure. In some embodiments, one or more of the ocrelizumab
exposures are provided to the patient as one dose of ocrelizumab.
In some embodiments, one or more of the ocrelizumab exposures are
provided to the patient as two doses of ocrelizumab. In some
embodiments, the initial ocrelizumab exposure comprises a first
dose and a second dose of ocrelizumab, wherein the first dose and
second dose of ocrelizumab is about 0.3 grams. In some embodiments,
the second ocrelizumab exposure comprises a single dose of
ocrelizumab, wherein the single dose of ocrelizumab is 0.6 grams.
In some embodiments, the methods further comprising providing a
third ocrelizumab exposure. In some embodiments, the methods
further comprising providing a fourth ocrelizumab exposure. In some
embodiments, the methods further comprising providing a fifth
ocrelizumab exposure. In some embodiments of any of the methods,
the methods further comprising providing between about one to about
three subsequent ocrelizumab exposures.
[0083] The invention also provides articles of manufacture
comprising: (a) a container comprising ocrelizumab; and (b) a
package insert with instructions for treating multiple sclerosis in
a patient, wherein the instructions denote that an amount of
ocrelizumab is administered to the patient that is effective to
provide an initial ocrelizumab exposure of between about 0.3 to
about 0.6 grams followed by a second ocrelizumab exposure of
between about 0.3 to about 0.6 grams, the second exposure not being
administered until from about 16 to 60 weeks from the initial
exposure, and each of the ocrelizumab exposures is provided to the
patient as one or two doses of ocrelizumab.
[0084] In some embodiments, the initial ocrelizumab exposure is
about 0.6 grams. In some embodiments, the second ocrelizumab
exposure is about 0.6 grams. In some embodiments, the second
exposure is administered from about 24 weeks from the initial
exposure. In some embodiments, one or more of the ocrelizumab
exposures are provided to the patient as one dose of ocrelizumab.
In some embodiments, one or more of the ocrelizumab exposures are
provided to the patient as two doses of ocrelizumab. In some
embodiments, the initial ocrelizumab exposure comprises a first
dose and a second dose of ocrelizumab, wherein the first dose and
second dose of ocrelizumab is about 0.3 grams. In some embodiments,
the instructions further comprising providing a third ocrelizumab
exposure. In some embodiments, the instructions further comprising
providing a fourth ocrelizumab exposure. In some embodiments, the
instructions further comprising providing a fifth ocrelizumab
exposure. In some embodiments of any of the methods, the
instructions further comprising providing between about one to
about three subsequent ocrelizumab exposures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1A is a sequence alignment comparing the amino acid
sequences of the light chain variable domain (V.sub.L) of each of
murine 2H7 (SEQ ID NO:1), humanized 2H7.v16 variant (SEQ ID NO:2),
and the human kappa light chain subgroup I (SEQ ID NO:3). The CDRs
of V.sub.L of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO:4),
CDR2 (SEQ ID NO:5), and CDR3 (SEQ ID NO:6).
[0086] FIG. 1B is a sequence alignment comparing the amino acid
sequences of the heavy chain variable domain (V.sub.H) of each of
murine 2H7 (SEQ ID NO:7), humanized 2H7.v16 variant (SEQ ID NO:8),
and the human consensus sequence of the heavy chain subgroup III
(SEQ ID NO:9). The CDRs of V.sub.H of 2H7 and hu2H7.v16 are as
follows: CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11), and CDR3 (SEQ ID
NO:12).
[0087] In FIG. 1A and FIG. 1B, the CDR1, CDR2 and CDR3 in each
chain are enclosed within brackets, flanked by the framework
regions, FR1-FR4, as indicated. 2H7 refers to the murine 2H7
antibody. The asterisks in between two rows of sequences indicate
the positions that are different between the two sequences. Residue
numbering is according to Kabat et al. Sequences of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), with insertions shown as a, b, c, d,
and e.
[0088] FIG. 2 shows the amino acid sequence of the mature 2H7.v16
light chain (SEQ ID NO:13)
[0089] FIG. 3 shows the amino acid sequence of the mature 2H7.v16
heavy chain (SEQ ID NO:14).
[0090] FIG. 4 shows the amino acid sequence of the mature 2H7.v31
heavy chain (SEQ ID NO:15). The L chain of 2H7.v31 is the same as
for 2H7.v16.
[0091] FIG. 5 shows an alignment of the mature 2H7.v16 and 2H7.v511
light chains (SEQ ID NOS. 13 and 16, respectively), with Kabat
variable domain residue numbering and Eu constant domain residue
numbering.
[0092] FIG. 6 shows an alignment of the mature 2H7.v16 and 2H7.v511
heavy chains (SEQ ID NOS. 14 and 17, respectively), with Kabat
variable domain residue numbering and Eu constant domain residue
numbering.
[0093] FIG. 7 shows an overview of the study design for treating
relapsing-remitting multiple sclerosis using ocrelizumab.
[0094] FIG. 8 shows Kaplan Meier plots of the time to confirmed
disease progression for subjects in the placebo and rituximab
groups.
[0095] FIG. 9 shows the median change in T2 lesion volume from
baseline to week 96. The Y-axis shows the T2 Lesion Volume
mm.sup.3.
[0096] FIG. 10 shows a summary of baseline characteristics and
hazard ratio of subjects in the placebo and rituximab groups.
[0097] FIGS. 11A-11D show multivariate analysis of additive
predictive effects of age and gadolinium (Gd) lesion at baseline
for the treatment effect in the placebo and rituximab groups. FIG.
11A shows multivariate analysis of age <51 and Gd lesions at
baseline=0. FIG. 11B shows multivariate analysis of age .gtoreq.51
and Gd lesions at baseline=0. FIG. 11C shows multivariate analysis
of age <51 and Gd lesions at baseline .gtoreq.1. FIG. 11D shows
multivariate analysis of age .gtoreq.51 and Gd lesions at baseline
.gtoreq.1.
[0098] FIGS. 12A-12D show multivariate analysis of additive
predictive effects of age and Multiple Sclerosis Severity Score
(MSSS) for the treatment effect in the placebo and rituximab
groups. FIG. 12A shows multivariate analysis of age .ltoreq.55 and
MSSS<5. FIG. 12B shows multivariate analysis of age >55 and
MSSS<5. FIG. 12C shows multivariate analysis of age <55 and
MSSS.gtoreq.5. FIG. 12D shows multivariate analysis of age >55
and MSSS.gtoreq.5.
[0099] FIG. 13 shows Kaplan Meier plots for the time to confirmed
disease progression of subjects in the placebo and rituximab groups
with the following characteristics: age .ltoreq.55;
3.ltoreq.baseline EDSS.ltoreq.6.5; excluding patients with disease
duration >10 years if their baseline EDSS<5 or disease
duration >15 if their baseline EDSS.gtoreq.5.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0100] A "B-cell" is a lymphocyte that matures within the bone
marrow, and includes a naive B cell, memory B cell, or effector B
cell (plasma cells). The B-cell herein may be a normal or
non-malignant B cell.
[0101] A "B-cell surface marker" or "B-cell surface antigen" herein
is an antigen expressed on the surface of a B cell that can be
targeted with an antibody that binds thereto. Exemplary B-cell
surface markers include the CD10, CD19, CD20, CD21, CD22, CD23,
CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,
CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86
leukocyte surface markers (for descriptions, see The Leukocyte
Antigen Facts Book, 2.sup.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, CXCRS, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2,
ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The B-cell surface
marker of particular interest herein is preferentially expressed on
B cells compared to other non-B-cell tissues of a mammal and may be
expressed on both precursor B cells and mature B cells. The
preferred B-cell surface marker herein is CD20.
[0102] The "CD20" antigen, or "CD20," is an about 35-kDa,
non-glycosylated phosphoprotein found on the surface of greater
than 90% of B cells from peripheral blood or lymphoid organs. CD20
is present on both normal B cells as well as malignant B cells, but
is not expressed on stem cells. Other names for CD20 in the
literature include "B-lymphocyte-restricted antigen" and "Bp35".
The CD20 antigen is described in Clark et al. Proc. Natl. Acad.
Sci. (USA) 82:1766 (1985), for example.
[0103] An "antibody antagonist" herein is a antibody that, upon
binding 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, e.g. by reducing or preventing a humoral response
elicited by the B cell. The antibody antagonist preferably is able
to deplete B cells (i.e. reduce circulating B-cell levels) in a
mammal treated therewith. Such depletion may be achieved via
various mechanisms such antibody-dependent cell-mediated
cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC),
inhibition of B-cell proliferation and/or induction of B-cell death
(e.g. via apoptosis).
[0104] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991). To assess ADCC activity of a molecule of interest,
an in vitro ADCC assay, such as that described in U.S. Pat. No.
5,500,362 or 5,821,337 may be performed. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0105] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. In some embodiments, the
cells express at least Fc.gamma.RIII and carry out 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 PBMCs and
NK cells being preferred.
[0106] 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 one that binds an IgG antibody (a gamma receptor)
and includes receptors of the Fc.gamma.RI, Fc.gamma.RII, and
Fc.gamma.RIII subclasses, including allelic variants and
alternatively spliced forms of these receptors. Fc.gamma.RII
receptors include Fc.gamma.RIIA (an "activating receptor") and
Fc.gamma.RIIB (an "inhibiting receptor"), which have similar amino
acid sequences that differ primarily in the cytoplasmic domains
thereof. Activating receptor Fc.gamma.RIIA contains an
immunoreceptor tyrosine-based activation motif (ITAM) in its
cytoplasmic domain. Inhibiting receptor Fc.gamma.RIIB contains an
immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic domain. (see Daeron, Annu. Rev. Immunol. 15:203-234
(1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol
9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de
Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,
including those to be identified in the future, are encompassed by
the term "FcR" herein. The term also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al, J. Immunol. 24:249 (1994)).
[0107] "Complement dependent cytotoxicity" or "CDC" refer to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (Clq) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0108] "Growth inhibitory" antibodies are those that prevent or
reduce proliferation of a cell expressing an antigen to which the
antibody binds. For example, the antibody may prevent or reduce
proliferation of B cells in vitro and/or in vivo.
[0109] Antibodies that "induce apoptosis" are those that induce
programmed cell death, e.g. of a B cell, as determined by standard
apoptosis assays, such as binding of annexin V, fragmentation of
DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
[0110] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0111] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0112] For the purposes herein, an "intact antibody" is one
comprising heavy and light variable domains as well as an Fc
region.
[0113] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0114] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0115] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-binding sites
and is still capable of cross-linking antigen.
[0116] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. 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.sub.H-V.sub.L dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0117] 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 carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments that have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0118] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.kappa.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains.
[0119] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0120] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. In some embodiments, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0121] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0122] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0123] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant region sequences (U.S. Pat.
No. 5,693,780). "Humanized" forms of non-human (e.g., murine)
antibodies are chimeric antibodies that contain minimal sequence
derived from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a hypervariable region of the recipient are replaced
by residues from a hypervariable region of a non-human species
(donor antibody) such as mouse, rat, rabbit or nonhuman primate
having the desired specificity, affinity, and capacity. In some
instances, framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence, except for FR
substitution(s) as noted above. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin. For further
details, see Jones et at, Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0124] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "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),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0125] A "naked antibody" is an antibody (as herein defined) that
is not conjugated to a heterologous molecule, such as a cytotoxic
moiety or radiolabel.
[0126] Examples of Examples of anti-CD20 antibodies include:
"C2B8," which is now called "rituximab"
("RITUXAN.RTM./MABTHERA.RTM.") (U.S. Pat. No. 5,736,137); the
yttrium-[90]-labelled 2B8 murine antibody designated "Y2B8" or
"Ibritumomab Tiuxetan" (ZEVALIN.RTM.) commercially available from
Biogen Idec, Inc. (e.g., U.S. Pat. No. 5,736,137; 2B8 deposited
with ATCC under accession no. HB11388 on Jun. 22, 1993); murine
IgG2a "B1," also called "Tositumomab," optionally labelled with
.sup.131I to generate the "131I-B1" or "iodine I131 tositumomab"
antibody (BEXXAR.TM.) commercially available from Corixa (see,
also, e.g., U.S. Pat. No. 5,595,721); murine monoclonal antibody
"1F5" (e.g., Press et al. Blood 69(2):584-591 (1987) and variants
thereof including "framework patched" or humanized 1F5 (e.g., WO
2003/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and
chimeric 2H7 antibody (e.g., U.S. Pat. No. 5,677,180); a 2H7
antibody (e.g., WO 2004/056312 (Lowman et al.) and as set forth
below); HUMAX-CD20.sup.Im (ofatumumab) fully human, high-affinity
antibody targeted at the CD20 molecule in the cell membrane of
B-cells (Genmab, Denmark; see, for example, Glennie and van de
Winkel, Drug Discovery Today 8: 503-510 (2003) and Cragg et al.,
Blood 101: 1045-1052 (2003)); the human monoclonal antibodies set
forth in WO 2004/035607 and WO 2005/103081 (Teeling et al.,
GenMab/Medarex); the antibodies having complex N-glycoside-linked
sugar chains bound to the Fc region described in US 2004/0093621
(Shitara et al.); a chimerized or humanized monoclonal antibody
having a high binding affinity to an extracellular epitope of a
CD20 antigen described in WO 2006/106959 (Numazaki et al.,
Biomedics Inc.); monoclonal antibodies and antigen-binding
fragments binding to CD20 (e.g., WO 2005/000901, Tedder et al.)
such as HB20-3, HB20-4, HB20-25, and MB20-11; single-chain proteins
binding to CD20 including, but not limited to, TRU-015 (e.g., US
2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534
(Hayden-Ledbetter and Ledbetter); US 2005/0202028 (Hayden-Ledbetter
and Ledbetter); US 2005/136049 (Ledbetter et al.); US 2005/0202023
(Hayden-Ledbetter and Ledbetter)--Trubion Pharm Inc.); CD20-binding
molecules such as the AME series of antibodies, e.g., AME-133
antibodies as set forth, for example, in WO 2004/103404; US
2005/0025764; and US 2006/0251652 (Watkins et al., Applied
Molecular Evolution, Inc.) and the anti-CD20 antibodies with Fc
mutations as set forth, for example, in WO 2005/070963 (Allan et
al., Applied Molecular Evolution, Inc.); CD20-binding molecules
such as those described in WO 2005/016969 and US 2005/0069545 (Carr
et al.); bispecific antibodies as set forth, for example, in WO
2005/014618 (Chang et al.); humanized LL2 monoclonal antibodies and
other anti-CD20 antibodies as described, for example, in U.S. Pat.
No. 7,151,164 (Hansen et al., Immunomedics; US 2005/0106108 (Leung
and Hansen; Immunomedics); fully human antibodies against CD20 as
described, e.g., in WO 2006/130458; Gazit et al.,
Amgen/AstraZeneca); antibodies against CD20 as described, for
example, in WO 2006/126069 (Morawala, Avestha Gengraine
Technologies Pvt Ltd.); chimeric or humanized B-Ly1 antibodies to
CD20 (e.g., GA-101) as described, for example, in WO 2005/044859;
US 2005/0123546; US 2004/0072290; and US 2003/0175884 (Umana et
al.; GlycArt Biotechnology AG); A20 antibody or variants thereof
such as chimeric or humanized A20 antibody (cA20, hA20,
respectively) and IMMUN-106 (e.g., US 2003/0219433, Immunomedics);
and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2
available from the International Leukocyte Typing Workshop (e.g.,
Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440,
Oxford University Press (1987)). In some embodiments,the anti-CD20
antibodies herein are chimeric, humanized, or human anti-CD20
antibodies, more preferably rituximab, a 2H7 antibody, chimeric or
humanized A20 antibody (Immunomedics), and HUMAX-CD20.TM. human
anti-CD20 antibody (Genmab).
[0127] The terms "Rituximab" or "RITUXAN.RTM." herein refer to the
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S.
Pat. No. 5,736,137, including fragments thereof that retain the
ability to bind CD20. Rituximab is commercially available from
Genentech.
[0128] Purely for the purposes herein and unless indicated
otherwise, "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, the
antibody comprising in the H chain variable region (V.sub.H)
thereof at least a CDR H3 sequence of SEQ ID NO:12 (FIG. 1B) from
an anti-human CD20 antibody and substantially the human consensus
framework (FR) residues of the human heavy-chain subgroup III
(V.sub.HIII). In some embodiments, this antibody further comprises
the H chain CDR H1 sequence of SEQ ID NO:10 and CDR H2 sequence of
SEQ ID NO:11, and, in some embodiments, further comprises the L
chain CDR L1 sequence of SEQ ID NO:4, CDR L2 sequence of SEQ ID
NO:5, CDR L3 sequence of SEQ ID NO:6 and substantially the human
consensus framework (FR) residues of the human light chain
.quadrature. subgroup I (V.quadrature.I), wherein the V.sub.H
region may be joined to a human IgG chain constant region, wherein
the region may be, for example, IgG1 or IgG3. In some embodiments,
such antibody comprises the V.sub.H sequence of SEQ ID NO:8 (v16,
as shown in FIG. 1B), optionally also comprising the V.sub.L
sequence of SEQ ID NO:2 (v16, as shown in FIG. 1A), which may have
the amino acid substitutions of D56A and N100A in the H chain and
S92A in the L chain (v96). In some embodiments, the antibody is an
intact antibody comprising the light and heavy chain amino acid
sequences of SEQ ID NOS:13 and 14, respectively, as shown in FIGS.
2 and 3. In some embodiments, the antibody is 2H7.v31 comprising
the light and heavy chain amino acid sequences of SEQ ID NOS:13 and
15, respectively, as shown in FIGS. 2 and 4. The antibody herein
may further comprise at least one amino acid substitution in the Fc
region that improves ADCC and/or CDC activity, such as one wherein
the amino acid substitutions are S298A/E333A/K334A, and in some
embodiments, the 2H7.v31 having the heavy chain amino acid sequence
of SEQ ID NO:15 (as shown in FIG. 4). Any of these antibodies may
further comprise at least one amino acid substitution in the Fc
region that decreases CDC activity, for example, comprising at
least the substitution K322A. See U.S. Pat. No. 6,528,624B1
(Idusogie et al.).
[0129] The term "Ocrelizumab" herein refers to the genetically
engineered humanized monoclonal antibody directed against the CD20
antigen and comprising (a) a light chain comprising the amino acid
sequence of SEQ ID NO: 13 and (b) a heavy chain comprising the
amino acid sequence of SEQ ID NO:14, including fragments thereof
that retain the ability to bind CD20. Ocrelizumab is available from
Genentech.
[0130] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. In some embodiments,
the antibody will be purified (1) to greater than 95% by weight of
antibody as determined by the Lowry method, and in some
embodiments, more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, in some embodiments, silver stain. Isolated
antibody includes the antibody in situ within recombinant cells
since at least one component of the antibody's natural environment
will not be present. Ordinarily, however, isolated antibody will be
prepared by at least one purification step.
[0131] A "subject" or "patient" herein is a human subject or
patient. Generally, the subject or patient is eligible for
treatment for multiple sclerosis. For the purposes herein, such
eligible subject or patient is one who is experiencing, has
experienced, or is likely to experience, one or more signs,
symptoms or other indicators of multiple sclerosis; has been
diagnosed with multiple sclerosis, whether, for example, newly
diagnosed (with "new onset" MS), previously diagnosed with a new
relapse or exacerbation, previously diagnosed and in remission,
etc; and/or is at risk for developing multiple sclerosis. One
suffering from or at risk for suffering from multiple sclerosis may
optionally be identified as one who has been screened for elevated
levels of CD20-positive B cells in serum, cerebrospinal fluid (CSF)
and/or MS lesion(s) and/or is screened for using an assay to detect
autoantibodies, assessed qualitatively, and preferably
quantitatively. Exemplary such autoantibodies associated with
multiple sclerosis include anti-myelin basic protein (MBP),
anti-myelin oligodendrocytic glycoprotein (MOG), anti-ganglioside
and/or anti-neurofilament antibodies. Such autoantibodies may be
detected in the subject's serum, cerebrospinal fluid (CSF) and/or
MS lesion. By "elevated" autoantibody or B cell level(s) herein is
meant level(s) of such autoantibodies or B cells which
significantly exceed the level(s) in an individual without MS.
[0132] As used herein, "treatment" or "treating" is an approach for
obtaining beneficial or desired results including clinical results.
For purposes of this invention, beneficial or desired clinical
results include, but are not limited to, one or more of the
following: decreasing one or more symptoms resulting from the
disease, diminishing the extent of the disease, stabilizing the
disease (e.g., preventing or delaying the worsening of the
disease), delay or slowing the progression of the disease,
ameliorating the disease state, decreasing the dose of one or more
other medications required to treat the disease, and/or increasing
the quality of life.
[0133] As used herein, "delaying" the progression of multiple
sclerosis means to defer, hinder, slow, retard, stabilize, and/or
postpone development of the disease. This delay can be of varying
lengths of time, depending on the history of the disease and/or
individual being treated.
[0134] As used herein, "at the time of starting treatment" refers
to the time period at or prior to the first exposure to a multiple
sclerosis drug, such as an anti-CD20 antibody. In some embodiments,
"at the time of starting treatment" is about any of one year, nine
months, six months, three months, second months, or one month prior
to a multiple sclerosis drug, such as an anti-CD20 antibody. In
some embodiments, "at the time of starting treatment" is
immediately prior to coincidental with the first exposure to a
multiple sclerosis drug, such as an anti-CD20 antibody.
[0135] As used herein, "based upon" includes (1) assessing,
determining, or measuring the patient characteristics as described
herein (and preferably selecting a patient suitable for receiving
treatment; and (2) administering the treatment(s) as described
herein.
[0136] A "symptom" of MS is any morbid phenomenon or departure from
the normal in structure, function, or sensation, experienced by the
subject and indicative of MS.
[0137] "Multiple sclerosis" refers to the chronic and often
disabling disease of the central nervous system characterized by
the progressive destruction of the 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).
[0138] "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 documented, irreversible
loss of neurological function persisting for .gtoreq.6 months that
cannot be attributed to clinical relapse.
[0139] "Primary progressive multiple sclerosis" or "PPMS" is
characterized by a gradual progression of the disease from its
onset with no superimposed relapses and remissions at all. There
may be periods of a leveling off of disease activity and there may
be good and bad days or weeks. PPMS differs from RRMS and SPMS in
that onset is typically in the late thirties or early forties, men
are as likely women to develop it, and initial disease activity is
often in the spinal cord and not in the brain. PPMS often migrates
into the brain, but is less likely to damage brain areas than RRMS
or SPMS. For example, people with PPMS are less likely to develop
cognitive problems than those with RRMS or SPMS. PPMS is the
sub-type of MS that is least likely to show inflammatory
(gadolinium enhancing) lesions on MRI scans. The Primary
Progressive form of the disease affects between 10 and 15% of all
people with multiple sclerosis. PPMS may be defined according to
the criteria in McDonald et al. Ann Neurol 50:121-7 (2001). The
subject with PPMS treated herein is usually one with probable or
definitive diagnosis of PPMS.
[0140] "Relapsing-remitting multiple sclerosis" or "RRMS" is
characterized by relapses (also known as exacerbations) during
which time new symptoms can appear and old ones resurface or
worsen. The relapses are followed by periods of remission, during
which time the person fully or partially recovers from the deficits
acquired during the relapse. Relapses can last for days, weeks or
months and recovery can be slow and gradual or almost
instantaneous. The vast majority of people presenting with MS are
first diagnosed with RRMS. This is typically when they are in their
twenties or thirties, though diagnoses much earlier or later are
known. Twice as many women as men present with this sub-type of MS.
During relapses, myelin, a protective insulating sheath around the
nerve fibers (neurons) in the white matter regions of the central
nervous system (CNS), may be damaged in an inflammatory response by
the body's own immune system. This causes a wide variety of
neurological symptoms that vary considerably depending on which
areas of the CNS are damaged. Immediately after a relapse, the
inflammatory response dies down and a special type of glial cell in
the CNS (called an oligodendrocyte) sponsors remyelination--a
process whereby the myelin sheath around the axon may be repaired.
It is this remyelination that may be responsible for the remission.
Approximately 50% of patients with RRMS convert to SPMS within 10
years of disease onset. After 30 years, this figure rises to 90%.
At any one time, the relapsing-remitting form of the disease
accounts around 55% of all people with MS.
[0141] "Secondary progressive multiple sclerosis" or "SPMS" is
characterized by a steady progression of clinical neurological
damage with or without superimposed relapses and minor remissions
and plateaux. People who develop SPMS will have previously
experienced a period of RRMS which may have lasted anything from
two to forty years or more. Any superimposed relapses and
remissions there are, tend to tail off over time. From the onset of
the secondary progressive phase of the disease, disability starts
advancing much quicker than it did during RRMS though the progress
can still be quite slow in some individuals. After 10 years, 50% of
people with RRMS will have developed SPMS. By 25 to 30 years, that
figure will have risen to 90%. SPMS tends to be associated with
lower levels of inflammatory lesion formation than in RRMS but the
total burden of disease continues to progress. At any one time,
SPMS accounts around 30% of all people with multiple sclerosis.
[0142] "Progressive relapsing multiple sclerosis" refers to "PRMS"
is characterized by a steady progression of clinical neurological
damage with superimposed relapses and remissions. There is
significant recovery immediately following a relapse but between
relapses there is a gradual worsening of symptoms. PRMS affects
around 5% of all people with multiple sclerosis. Some neurologists
believe PRMS is a variant of PPMS.
[0143] The expression "effective amount" refers to an amount of the
antibody (or other drug) that is effective for ameliorating or
treating the multiple sclerosis. Such an effective amount will
generally result in an improvement in the signs, symptoms or other
indicators of MS, such as reducing relapse rate, preventing
disability, reducing number and/or volume of brain MRI lesions,
improving timed 25-foot walk, slow or delay the progression of the
disease such as extending the time to disease progression (e.g.
using Expanded Disability Status Scale, EDSS), etc.
[0144] "Antibody exposure" refers to contact with or exposure to
the antibody herein in one or more doses administered over a period
of time of about 1-20 days. The doses may be given at one time or
at fixed or irregular time intervals over this period of exposure.
Initial and later (e.g. second or third) antibody exposures are
separated in time from each other as described in detail
herein.
[0145] The term "immunosuppressive agent" as used herein for
adjunct therapy refers to substances that act to suppress or mask
the immune system of the mammal being treated herein. This would
include substances that suppress cytokine production, down-regulate
or suppress self-antigen expression, or mask the MHC antigens.
Examples of such agents include 2-amino-6-aryl-5-substituted
pyrimidines (see U.S. Pat. No. 4,665,077); nonsteroidal
anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,
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; bromocryptine; danazol; dapsone;
glutaraldehyde (which masks the MHC antigens, as described in U.S.
Pat. No. 4,120,649); anti-idiotypic antibodies for MEC antigens and
MEC fragments; cyclosporin A; steroids such as corticosteroids or
glucocorticosteroids or glucocorticoid analogs, e.g., prednisone,
methylprednisolone, and dexamethasone; dihydrofolate reductase
inhibitors such as methotrexate (oral or subcutaneous);
hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine
receptor antagonists including anti-interferon-alpha, -beta, or
-gamma antibodies, anti-tumor necrosis factor-alpha antibodies
(infliximab or adalimumab), anti-TNF-alpha immunoahesin
(etanercept), anti-tumor necrosis factor-beta antibodies,
anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-LFA-1 antibodies, including anti-CD11a and anti-CD18
antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte
globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a
antibodies; soluble peptide containing a LFA-3 binding domain (WO
90/08187 published Jul. 26, 1990); streptokinase; TGF-beta;
streptodornase; RNA or DNA from the host; FK506; RS-61443;
deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S.
Pat. No. 5,114,721); T-cell receptor fragments (Offner et al,
Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341:
482 (1989); and WO 91/01133); and T cell receptor antibodies (EP
340,109) such as T10B9.
[0146] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, or fragments
thereof.
[0147] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegall (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0148] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; vaccines such as gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1
inhibitor; ABARELIX.RTM. rmRH; and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0149] The term "cytokine" is a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines;
interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines, including synthetically produced small-molecule
entities and pharmaceutically acceptable derivatives and salts
thereof.
[0150] The term "hormone" refers to polypeptide hormones, which are
generally secreted by glandular organs with ducts. Included among
the hormones are, for example, growth hormone such as human growth
hormone, N-methionyl human growth hormone, and bovine growth
hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); prolactin, placental lactogen, mouse
gonadotropin-associated peptide, inhibin; activin;
mullerian-inhibiting substance; and thrombopoietin. As used herein,
the term hormone includes proteins from natural sources or from
recombinant cell culture and biologically active equivalents of the
native sequence hormone, including synthetically produced
small-molecule entities and pharmaceutically acceptable derivatives
and salts thereof.
[0151] 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-.beta.; platelet-derived growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; and colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF).
As used herein, the term growth factor includes proteins from
natural sources or from recombinant cell culture and biologically
active equivalents of the native sequence growth factor, including
synthetically produced small-molecule entities and pharmaceutically
acceptable derivatives and salts thereof.
[0152] The term "integrin" refers to a receptor protein that allows
cells both to bind to and to respond to the extracellular matrix
and is involved in a variety of cellular functions such as wound
healing, cell differentiation, homing of tumor cells 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 consist of two transmembrane glycoprotein
subunits, called alpha and beta that are non-covalently bound. The
alpha subunits all share some homology to each other, as do the
beta subunits. The receptors always contain one alpha chain and one
beta chain. Examples include Alpha6beta1, Alpha3beta1, Alpha7beta1,
LFA-1, alpha 4 integrin etc. As used herein, the term integrin
includes proteins from natural sources or from recombinant cell
culture and biologically active equivalents of the native sequence
integrin, including synthetically produced small-molecule entities
and pharmaceutically acceptable derivatives and salts thereof.
[0153] Examples of "integrin antagonists or antibodies" herein
include an LFA-1 antibody such as Efalizumab (RAPTIVA.RTM.)
commercially available from Genentech; an alpha 4 integrin antibody
such as natalizumab (TYSABRI.RTM.) 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);
propanoic acid derivatives (WO 2000/37444); alkanoic acid
derivatives (WO 2000/32575); substituted phenyl derivatives (U.S.
Pat. Nos. 6,677,339 and 6,348,463); aromatic amine derivatives
(U.S. Pat. No. 6,369,229); and ADAM disintegrin domain polypeptide
(US2002/0042368), antibodies to alphavbeta3 integrin (EP 633945);
aza-bridged bicyclic amino acid derivatives (WO 2002/02556)
etc.
[0154] For the purposes herein, "tumor necrosis factor alpha
(TNF-alpha)" refers to a human TNF-alpha molecule comprising the
amino acid sequence as described in Pennica et al., Nature, 312:721
(1984) or Aggarwal et al., JBC, 260:2345 (1985).
[0155] A "TNF-alpha inhibitor" herein is an agent that inhibits, to
some extent, a biological function of TNF-alpha, generally through
binding to TNF-alpha and neutralizing its activity. Examples of TNF
inhibitors specifically contemplated herein are Etanercept
(ENBREL.RTM.), Infliximab (REMICADE.RTM.) and Adalimumab
(HUMMIRA.TM.).
[0156] Examples of "disease-modifying anti-rheumatic drugs" or
"DMARDs" include hydroxycloroquine, sulfasalazine, methotrexate,
leflunomide, etanercept, infliximab (plus oral and subcutaneous
methrotrexate), azathioprine, D-penicillamine, Gold (oral), Gold
(intramuscular), minocycline, cyclosporine, Staphylococcal protein
A immunoadsorption, including salts and derivatives thereof,
etc.
[0157] Examples of "nonsteroidal anti-inflammatory drugs" or
"NSAIDs" are acetylsalicylic acid, ibuprofen, naproxen,
indomethacin, sulindac, tolmetin, including salts and derivatives
thereof, etc.
[0158] "Corticosteroid" refers to any one of several synthetic or
naturally occurring substances with the general chemical structure
of steroids that mimic or augment the effects of the naturally
occurring corticosteroids. Examples of synthetic corticosteroids
include prednisone, prednisolone (including methylprednisolone),
dexamethasone, glucocorticoid and betamethasone.
[0159] A "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications, other therapeutic
products to be combined with the packaged product, and/or warnings
concerning the use of such therapeutic products, etc.
[0160] A "label" is used herein to refer to information customarily
included with commercial packages of pharmaceutical formulations
including containers such as vials and package inserts, as well as
other types of packaging.
[0161] Reference to "about" a value or parameter herein includes
(and describes) variations that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X".'
[0162] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise. It is understood that aspects
and variations of the invention described herein include
"consisting" and/or "consisting essentially of" aspects and
variations.
II. Methods of Treatment
[0163] The present invention provides methods of treating
progressive multiple sclerosis in a patient comprising
administering to the patient an effective amount of an anti-CD20
antibody.
[0164] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis in a patient comprising
administering to the patient an effective amount of an anti-CD20
antibody, wherein treatment is based upon the patient having one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0165] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis in a patient provided that
the patient has been found to have one or more characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, (c) at least
about a one point increase in Expanded Disability Status Scale
(EDSS) over two years prior to starting the treatment, and (d) a
Multiple Sclerosis Severity Score (MSSS) greater than about 5
points, the treatment comprising administering to the patient an
effective amount of an anti-CD20 antibody.
[0166] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis in a patient comprising
administering to the patient an effective amount of an anti-CD20
antibody, wherein the patient has at the time of starting treatment
one or more characteristics selected from the group consisting of
(a) an age less than about 55 years, (b) one or more gadolinium
staining lesions, (c) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment, and (d) a Multiple Sclerosis Severity Score
(MSSS) greater than about 5 points, whereby evidence of the age,
the gadolinium staining lesions, the increase in EDDS over two
years prior to starting the treatment, MSSS, or a combination
thereof indicates that the patient will respond to treatment with
the anti-CD20 antibody.
[0167] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis, comprising: (a) selecting
a patient has one or more characteristics selected from the group
consisting of (i) an age less than about 55 years, (ii) one or more
gadolinium staining lesions, (iii) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (iv) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points; and (b) administering to
the patient thus selected an effective amount of an anti-CD20
antibody.
[0168] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis in a patient, comprising
administering to the patient an effective amount of an anti-CD20
antibody, and wherein one or more characteristics selected from the
group consisting of (a) an age less than about 55 years, (b) one or
more gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.is used as a basis for
selecting the patient to receive the treatment, and wherein said
treatment comprises administering an effective amount of the
anti-CD20 antibody to the patient.
[0169] The invention further provides methods of treating multiple
sclerosis in a patient comprising administering an effective amount
of ocrelizumab to the patient to provide an initial ocrelizumab
exposure of between about 0.3 to about 0.6 grams followed by a
second ocrelizumab exposure of between about 0.3 to about 0.6
grams, the second exposure not being provided until from about 16
to 60 weeks from the initial exposure, and each of the ocrelizumab
exposures is provided to the patient as one or two doses of
ocrelizumab. In some embodiments, the initial ocrelizumab exposure
is about 0.6 grams. In some embodiments, the second ocrelizumab
exposure is about 0.6 grams. In some embodiments, the second
exposure is administered from about 24 weeks from the initial
exposure. In some embodiments, one or more of the ocrelizumab
exposures are provided to the patient as one dose of ocrelizumab.
In some embodiments, one or more of the ocrelizumab exposures are
provided to the patient as two doses of ocrelizumab. In some
embodiments, the two doses of ocrelizumab comprise about 0.3 grams
of ocrelizumab.
[0170] The present invention also provides methods of assessing
and/or predicting responsiveness of a patient with progressive
multiple sclerosis to an anti-CD20 antibody treatment.
[0171] In some embodiments, the invention provides methods of
assessing whether a patient with progressive multiple sclerosis
will respond to treatment with an anti-CD20 antibody comprising
assessing one or more characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points, wherein one or more of
the characteristics in the patient indicates the patient will be
responsive to the treatment.
[0172] In some embodiments, the invention provides methods of
assessing whether a patient with progressive multiple sclerosis
will respond to treatment with an anti-CD20 antibody comprising:
(a) assessing one or more characteristics selected from the group
consisting of (i) an age less than about 55 years, (ii) one or more
gadolinium staining lesions, (iii) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (iv) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points; (b) implementing an
algorithm to determine that the patient is responsive to said
treatment; and (c) recording a result specific to the patient being
tested.
[0173] In some embodiments, the invention provides methods of
treating progressive multiple sclerosis in a patient comprising:
(a) assessing one or more characteristics selected from the group
consisting of (i) an age less than about 55 years, (i) one or more
gadolinium staining lesions, (iii) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (iv) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points, wherein the patient is
selected for treatment based on the patient having one or more
characteristics selected from the group consisting of (i) an age
less than about 55 years, (ii) one or more gadolinium staining
lesions, (iii) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (iv) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points; and (b) treating said selected patient
by administering to said selected patient an effective amount of an
anti-CD20 antibody.
[0174] In some embodiments, the invention provides methods for
selecting a therapy for a patient and/or a patient population with
progressive multiple sclerosis comprising: (a) assessing one or
more characteristics selected from the group consisting of (i) an
age less than about 55 years, (ii) one or more gadolinium staining
lesions, (iii) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (iv) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points; and (b) selecting an anti-CD20
antibody for treatment if the patient or patient population has one
or more characteristics selected from the group consisting of (i)
an age less than about 55 years, (ii) one or more gadolinium
staining lesions, (iii) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting the anti-CD20 treatment, and (iv) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points.
[0175] In some embodiments, the invention provides methods for
predicting whether a patient with progressive multiple sclerosis
will respond to an anti-CD20 antibody, the methods comprising
assessing one or more characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points, whereby the age, the
gadolinium staining lesions, the increase in EDDS over two years
prior to starting treatment, MSSS, or a combination thereof
indicates that the patient will respond to the anti-CD20
antibody.
[0176] In some embodiments, the invention provides methods of
identifying a patient with progressive multiple sclerosis likely to
respond to anti-CD20 antibody treatment comprising: (a) assessing
one or more characteristics selected from the group consisting of
(i) an age less than about 55 years, (ii) one or more gadolinium
staining lesions, (iii) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment, and (iv) a Multiple Sclerosis Severity Score
(MSSS) greater than about 5 points; and (b) identifying the patient
having one or more characteristics selected from the group
consisting of (i) an age less than about 55 years, (ii) one or more
gadolinium staining lesions, (iii) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (iv) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.
[0177] In some embodiments of any of the methods of treating,
assessing, and/or predicting responsiveness described herein, the
patients have one or more characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, and (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment.
[0178] In some embodiments of any of the methods of assessing
and/or predicting responsiveness described herein, the methods
further comprise advising a patient.
[0179] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient has more than one characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points. In some embodiments of
any of the methods of treating and assessing and/or predicting
responsiveness described herein, the patient has two
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points. In some embodiments, the patient has
three characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, and (c) at least about a one point increase in EDSS over
two years prior to starting treatment. In some embodiments, the
patient has (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.
[0180] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
progressive multiple sclerosis is primary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is secondary progressive multiple sclerosis. In some embodiments,
the progressive multiple sclerosis is progressive relapsing
multiple sclerosis. In some embodiments, the patient and/or patient
population is not diagnosed with relapsing remitting multiple
sclerosis when starting treatment.
[0181] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient and/or patient population further has evidence of
inflammation in a sample. Evidence of inflammation is indicated by
assessing one or more indicia of inflammation. The sample can be
any suitable sample to assess inflammation. In some embodiments,
the sample is tissue or fluid. In some embodiments, the fluid
sample is a cerebrospinal fluid sample. In some embodiments, the
evidence of inflammation is indicated by an elevated IgG index. In
some embodiments, the evidence of inflammation is indicated by IgG
oligoclonal bands detected by isoelectric focusing. In some
embodiments, the evidence of inflammation is detected by MRI. In
some embodiments, the evidence of inflammation is detected by the
presence of Gd-enhancing lesions or T2 lesions. Other methods of
assessing evidence of inflammation are known in the art.
[0182] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient and/or patient population is characterized by a change in
EDSS over a period of time. In some embodiments, the patient and/or
patient population is characterized by at least about any of 1
point, 1.25 point, 1.5 point, 1.75 point, 2 point, 2.25 point, 2.5
point, 2.75 point, or 3 point increase in EDSS over two years prior
to starting the anti-CD20 antibody treatment. In some embodiments,
the increase in EDSS is at least about a 1.5 point increase in EDSS
over two years prior to starting treatment. In some embodiments,
the increase in EDSS over two years prior to starting treatment is
not attributable to relapse. In some embodiments, the patient
and/or patient population is characterized by having had an EDSS of
greater than about 5.0 for less than about any of 5 years, 6 years,
7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years,
14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20
years. In some embodiments, the patient and/or patient population
is characterized by having had an EDSS of greater than about 5.0
for less than about 15 years. In some embodiments, the patient
and/or patient population is characterized by having had an EDSS
less than or equal to about 5.0 for less than about any of 5 years,
6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years,
13 years, 14 years, or 15 years. In some embodiments, the patient
and/or patient population is characterized by having had an EDSS
less than or equal to about 5.0 for less than about 10 years.
[0183] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient and/or patient population is characterized by having an
EDSS of between about any of 1.5 points to 7 points, 1.5 points to
6.5 points, 2 points to 6.5 points, or 3 points to 6.5 points. In
some embodiments, the patient and/or patient population is
characterized by having an EDSS when starting treatment is between
about 3.0 and about 6.5.
[0184] In some embodiments of any of the methods of treating,
assessing and/or predicting responsiveness described herein, the
patient and/or patient population is characterized by having an
MSSS of greater than about any of 6, 7, 8, or 9. In some
embodiments of any of the methods of treating and assessing and/or
predicting responsiveness described herein, the patient and/or
patient population is characterized by having an MSSS of greater
than about 9.
[0185] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient and/or patient population is further characterized by
having two or more relapses within two years prior to starting
treatment. In some embodiments, the patient and/or patient
population is further characterized by having any of 2 relapses, 3
relapses, 4 relapses, or 5 relapses within two years prior to
starting treatment.
[0186] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
patient and/or patient population is characterized by an age. In
some embodiments, the patient and/or patient population is
characterized by having an age less than about any of 55 years, 54
years, 53 years, 52 years, 51 years, or 50 years. In some
embodiments, the patient and/or patient population is characterized
by having an age of less than about 51 years.
[0187] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, the
treatment reduces time to confirmed disease progression. In some
embodiments, the confirmed disease progression is an increase in
EDSS that is sustained for about any of 4 weeks, 8 weeks, 12 weeks,
16 weeks, 20 weeks, 24 weeks, 28 weeks, or 32 weeks. In some
embodiments, the confirmed disease progression is an increase in
EDSS that is sustained for twelve weeks. In some embodiments, the
confirmed disease progression is an increase in EDSS that is
sustained for twenty-four weeks.
[0188] In some embodiments of any of the methods of treating and
assessing and/or predicting responsiveness described herein, 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 of any of the methods
described herein, the anti-CD20 antibody is ocrelizumab. In some
embodiments of any of the methods described herein, the anti-CD20
antibody is rituximab. In some embodiments of any of the methods
described herein, the anti-CD20 antibody is ofatumumab. In some
embodiments of any of the methods described herein, the anti-CD20
antibody is TRU-015 or SBI-087. In some embodiments of any of the
methods described herein, the anti-CD20 antibody is GA101. In some
embodiments of any of the methods described herein, the anti-CD20
antibody is hA20.
[0189] The methods described herein may encompass any combination
of the embodiments described herein. For example, the methods
include methods of treatment and assessing and/or predicting
wherein the patient has (a) an age less than about 55 years and (b)
one or more gadolinium staining lesions.
III. Dosages
[0190] According to some embodiments of any of the methods or
articles of manufacture described herein, the method or
instructions comprises administering an effective amount of an
anti-CD20 antibody to the multiple sclerosis patient 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 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), the second antibody exposure not being
provided until from about 16 to about 60 weeks from the initial
antibody exposure. For purposes of this invention, the second
antibody exposure is the next time the patient is treated with the
anti-CD20 antibody after the initial antibody exposure, there being
no intervening anti-CD20 antibody treatment or exposure between the
initial and second exposures. In some embodiments, the initial
antibody exposure and/or the second antibody exposure is about any
of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8
grams, 0.9 grams, or 1.0 grams.
[0191] The interval between the initial and second or subsequent
antibody exposures can be measured from either the first or second
dose of the initial antibody exposure, but in some embodiments,
from the first dose of the initial antibody exposure.
[0192] In some embodiments, the antibody exposures are
approximately 24 weeks or 6 months apart; or approximately 48 weeks
or 12 months apart.
[0193] In one embodiment, the second antibody exposure is not
provided until about 20 to about 30 weeks from the initial
exposure, optionally followed by a third antibody exposure of about
0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams), the
third exposure not being administered until from about 46 to 60
weeks (preferably from about 46 to 54 weeks) from the initial
exposure, and then, in some embodiments, no further antibody
exposure is provided until at least about 70-75 weeks from the
initial exposure. In some embodiments, the third antibody exposure
is about any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7
grams, 0.8 grams, 0.9 grams, or 1.0 grams.
[0194] In an alternative embodiment, the second antibody exposure
is not provided until about 46 to 60 weeks from the initial
exposure, and subsequent antibody exposures, if any, are not
provided until about 46 to 60 weeks from the previous antibody
exposure.
[0195] Any one or more of the antibody exposures herein may be
provided to the patient as a single dose of antibody, or as two
separate doses of the antibody (i.e., constituting a first and
second dose). The particular number of doses (whether one or two)
employed for each antibody exposure is dependent, for example, on
the type of MS treated, the type of antibody employed, whether and
what type of second medicament is employed, and the method and
frequency of administration. Where two separate doses are
administered, the second dose is preferably administered from about
3 to 17 days, more preferably from about 6 to 16 days, and most
preferably from about 13 to 16 days from the time the first dose
was administered. In some embodiments, where two separate doses are
administered, the second dose is about 14 days. Where two separate
doses are administered, the first and second dose of the antibody
is preferably about 0.3 to 1.5 grams, more preferably about 0.3 to
about 1.0 grams. In some embodiments, where two separate doses are
administered, the first and second dose of the antibody is about
any of 0.3 grams, 0.4 grams, 0.5 grams, or 0.6 grams. In some
embodiments, the initial ocrelizumab exposure comprises a first
dose and a second dose of ocrelizumab, wherein the first dose and
second dose of ocrelizumab is about 0.3 grams. In some embodiments,
the second ocrelizumab exposure comprises a single dose of
ocrelizumab, wherein the single dose of ocrelizumab is 0.6
grams.
[0196] In one embodiment, the patient is provided at least about
three, at least about four, or at least about five exposures of the
antibody, for example, from about 3 to 60 exposures, and more
particularly about 3 to 40 exposures, most particularly, about 3 to
20 exposures. In some embodiments of any of the methods, the
methods further comprising providing between about one to about
three subsequent ocrelizumab exposures. In some embodiments, such
exposures are administered at intervals each of approximately 24
weeks or 6 months, or 48 weeks or 12 months. In one embodiment,
each antibody exposure is provided as a single dose of the
antibody. In an alternative embodiment, each antibody exposure is
provided as two separate doses of the antibody. However, not every
antibody exposure need be provided as a single dose or as two
separate doses.
[0197] The antibody may be a naked antibody or may be conjugated
with another molecule such as a cytotoxic agent such as a
radioactive compound. In some embodiments, the antibody is
Rituximab, humanized 2H7 (e.g. comprising the variable domain
sequences in SEQ ID NOS. 2 and 8) or humanized 2H7 comprising the
variable domain sequences in SEQ ID NOS. 23 and 24, or huMax-CD20
(Genmab). In some embodiments, the antibody is ocrelizumab (e.g.,
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).
[0198] In one embodiment, the patient has never been previously
treated with drug(s), such as immunosuppressive agent(s), to treat
the multiple sclerosis and/or has never been previously treated
with an antibody to a B-cell surface marker (e.g. never previously
treated with a CD20 antibody).
[0199] The antibody is administered by any suitable means,
including parenteral, 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, e.g., US
Patent Appln No. 2002/0009444, Grillo-Lopez, A concerning
intrathecal delivery of a CD20 antibody). In addition, the antibody
may suitably be administered by pulse infusion, e.g., with
declining doses of the antibody. In some embodiments, the dosing is
given intravenously, subcutaneously or intrathecally. In some
embodiments, the dosing is given by intravenous infusion(s).
[0200] While the CD20 antibody may be the only drug administered to
the patient to treat the multiple sclerosis, one may optionally
administer a second medicament, such as a cytotoxic agent,
chemotherapeutic agent, immunosuppressive agent, cytokine, cytokine
antagonist or antibody, growth factor, hormone, integrin, integrin
antagonist or antibody (e.g. an LFA-1 antibody such as efalizumab
(RAPTIVA.RTM.) commercially available from Genentech, or an alpha 4
integrin antibody such as natalizumab (TYSABRI.RTM.) available from
Biogen Idec/Elan Pharmaceuticals, Inc) etc, with the antibody that
binds a B cell surface marker (e.g. with the CD20 antibody).
[0201] In some embodiments of combination therapy, the antibody is
combined with an interferon class drug such as IFN-beta-la
(REBIF.RTM. and AVONEX.RTM.) or IFN-beta-lb (BETASERON.RTM.); an
oligopeptide such a glatiramer acetate (COPAXONE.RTM.); a cytotoxic
agent such as mitoxantrone (NOVANTRONE.RTM.), methotrexate,
cyclophosphamide, chlorambucil, azathioprine; intravenous
immunoglobulin (gamma globulin); lymphocyte-depleting therapy
(e.g., mitoxantrone, cyclophosphamide, Campath, anti-CD4,
cladribine, total body irradiation, bone marrow transplantation);
corticosteroid (e.g. methylprednisolone, prednisone, dexamethasone,
or glucorticoid), including systemic corticosteroid therapy;
non-lymphocyte-depleting immunosuppressive therapy (e.g.,
mycophenolate mofetil (MMF) or cyclosporine); cholesterol-lowering
drug of the "statin" class, which includes cerivastatin
(BAYCOL.RTM.), fluvastatin (LESCOL.RTM.), atorvastatin
(LIPITOR.RTM.), lovastatin (MEVACOR.RTM.), pravastatin
(PRAVACHOL.RTM.), Simvastatin (ZOCOR.RTM.); estradiol; testosterone
(optionally at elevated dosages; Stuve et al. Neurology 8:290-301
(2002)); hormone replacement therapy; treatment for symptoms
secondary or related to MS (e.g., spasticity, incontinence, pain,
fatigue); a TNF inhibitor; disease-modifying anti-rheumatic drug
(DMARD); non-steroidal anti-inflammatory drug (NSAID);
plasmapheresis; levothyroxine; cyclosporin A; somatastatin
analogue; cytokine or cytokine receptor antagonist;
anti-metabolite; immunosuppressive agent; rehabilitative surgery;
radioiodine; thyroidectomy; another B-cell surface
antagonist/antibody; etc.
[0202] The second medicament is administered with the initial
exposure and/or later exposures of the CD20 antibody, such combined
administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0203] Aside from administration of antibodies to the patient, the
present application contemplates administration of antibodies by
gene therapy. Such administration of nucleic acid encoding the
antibody is encompassed by the expression administering an
"effective amount" of an antibody. See, for example, WO96/07321
published Mar. 14, 1996 concerning the use of gene therapy to
generate intracellular antibodies.
[0204] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes that
are implanted into the patient (see, e.g. U.S. Pat. Nos. 4,892,538
and 5,283,187). There are a variety of techniques available for
introducing nucleic acids into viable cells. The techniques vary
depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or in vivo in the cells of the intended
host. Techniques suitable for the transfer of nucleic acid into
mammalian cells in vitro include the use of liposomes,
electroporation, microinjection, cell fusion, DEAE-dextran, the
calcium phosphate precipitation method, etc. A commonly used vector
for ex vivo delivery of the gene is a retrovirus.
[0205] In some embodiments, the in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). In some
situations it is desirable to provide the nucleic acid source with
an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a
ligand for a receptor on the target cell, etc. Where liposomes are
employed, proteins that bind to a cell surface membrane protein
associated with endocytosis may be used for targeting and/or to
facilitate uptake, e.g. capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins that undergo
internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of
receptor-mediated endocytosis 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 review of the
currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
IV. Antibodies and their Production
[0206] The methods and articles of manufacture of the present
invention use, or incorporate, an antibody that binds to a B-cell
surface marker, especially one that binds to CD20. Accordingly,
methods for generating such antibodies will be described here.
[0207] The B cell surface marker to be used for production of, or
screening for, antibodies may be, e.g., a soluble form of the
marker or a portion thereof, containing the desired epitope.
Alternatively, or additionally, cells expressing the marker at
their cell surface can be used to generate, or screen for,
antibodies. Other forms of the B cell surface marker useful for
generating antibodies will be apparent to those skilled in the
art.
[0208] A description follows as to exemplary techniques for the
production of the antibodies used in accordance with the present
invention.
(i) Polyclonal Antibodies
[0209] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R'N.dbd.C.dbd.NR, where R and
R.sup.1 are different alkyl groups.
[0210] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. In some embodiments, the animal
is boosted with the conjugate of the same antigen, but conjugated
to a different protein and/or through a different cross-linking
reagent. Conjugates also can be made in recombinant cell culture as
protein fusions. Also, aggregating agents such as alum are suitably
used to enhance the immune response.
(ii) Monoclonal antibodies
[0211] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical and/or bind the
same epitope except for possible variants that arise during
production of the monoclonal antibody, such variants generally
being present in minor amounts. Thus, the modifier "monoclonal"
indicates the character of the antibody as not being a mixture of
discrete or polyclonal antibodies.
[0212] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0213] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as herein described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0214] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0215] In some embodiments, the myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. Among these, in some embodiments, the
myeloma cell lines are murine myeloma lines, such as those derived
from MOPC-21 and MPC-11 mouse tumors available from the Salk
Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2
or X63-Ag8-653 cells available from the American Type Culture
Collection, Rockville, Md. USA. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0216] Culture medium in which hybridoma cells are growing is
assayed for 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 by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0217] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al,
Anal. Biochem., 107:220 (1980).
[0218] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0219] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0220] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies). In
some embodiments, the hybridoma cells serve as a source of such
DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transfected into host cells such as E. coli cells,
simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma
cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Review articles on recombinant expression in bacteria
of DNA encoding the antibody include Skerra et al., Curr. Opinion
in Immunol, 5:256-262 (1993) and Pluckthun, Immunol. Revs.,
130:151-188 (1992).
[0221] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al,
J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine
and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by 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 techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0222] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0223] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
(iii) Humanized Antibodies
[0224] Methods for humanizing non-human antibodies have been
described in the art. In some embodiments, a humanized antibody has
one or more amino acid residues introduced into it from a source
that is non-human. These non-human amino acid residues are often
referred to as "import" residues, which are typically taken from an
"import" variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-327
(1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0225] The choice of human variable domains, both light and heavy,
to be used in making the 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 closest to that of the rodent
is then accepted as the human framework region (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 particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chain variable regions. The same framework may 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)).
[0226] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, in some embodiments of
the methods, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available that illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0227] In some embodiments, the humanized anti-CD20 antibody is a
humanized 2H7 antibody. In some embodiments, the humanized 2H7
antibody preferably comprises one, two, three, four, five or six of
the following CDR sequences: [0228] CDR L1 sequence RASSSVSYXH
wherein X is M or L (SEQ ID NO. 18), for example SEQ ID NO:4 (FIG.
1A), [0229] CDR L2 sequence of SEQ ID NO:5 (FIG. 1A), [0230] CDR L3
sequence QQWXFNPPT wherein X is S or A (SEQ ID NO. 19), for example
SEQ ID NO:6 (FIG. 1A), [0231] CDR H1 sequence of SEQ ID NO:10 (FIG.
1B), [0232] CDR H2 sequence of AIYPGNGXTSYNQKFKG wherein X is D or
A (SEQ ID NO. 20), for example SEQ ID NO:11 (FIG. 1B), and [0233]
CDR H3 sequence of VVYYSXXYWYFDV wherein the X at position 6 is N,
A, Y, W or D, and the X as position 7 is S or R (SEQ ID NO. 21),
for example SEQ ID NO:12 (FIG. 1B).
[0234] The CDR sequences above are generally present within human
variable light and variable heavy framework sequences, such as
substantially the human consensus FR residues of human light chain
kappa subgroup I (V.sub.L6I), and substantially the human consensus
FR residues of human heavy chain subgroup III (V.sub.HIII). See
also WO 2004/056312 (Lowman et al.).
[0235] In some embodiments, the variable heavy region may be joined
to a human IgG chain constant region, wherein the region may be,
for example, IgG1 or IgG3, including native sequence and variant
constant regions.
[0236] In some embodiments, such antibody comprises the variable
heavy domain sequence of SEQ ID NO:8 (v16, as shown in FIG. 1B),
optionally also comprising the variable light domain sequence of
SEQ ID NO:2 (v16, as shown in FIG. 1A), which optionally comprises
one or more amino acid substitution(s) at positions 56, 100, and/or
100a, e.g. D56A, N100A or N100Y, and/or S100aR in the variable
heavy domain and one or more amino acid substitution(s) at
positions 32 and/or 92, e.g. M32L and/or S92A, in the variable
light domain. In some embodiments, the antibody is an intact
antibody comprising the light chain amino acid sequences of SEQ ID
NOs. 13 or 16, and 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).
[0237] In the embodiments, the humanized 2H7 is an intact antibody
or antibody fragment comprising the variable light chain
sequence:
TABLE-US-00001 (SEQ ID NO: 2)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKP
LIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQW SFNPPTFGQGTKVEIKR;
and the variable heavy chain sequence: (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMWVRQAPGKGLE
WVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDT
AVYYCARVVYYSNSYWYFDVWGQGTLVTVSS.
[0238] In some embodiments, the humanized 2H7 antibody is an intact
antibody, in some embodiments, it comprises the light chain amino
acid sequence:
TABLE-US-00002 (SEQ ID NO: 13)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPK
PLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
QWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; and the heavy chain amino
acid sequence: (SEQ ID NO: 14)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPGK
GLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLR
AEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK or the
heavy chain amino acid sequence: (SEQ ID NO: 15)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPGK
GLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLR
AEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK.
[0239] In some embodiments, the humanized 2H7 antibody comprises
2H7.v511 variable light domain sequence:
TABLE-US-00003 (SEQ ID NO: 23)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPK
PLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QWAFNPPTFGQGTKVEIKR
and 2H7.v511 variable heavy domain sequence: (SEQ ID NO. 24)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEWVRQAPGKG
LEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRA
EDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS.
[0240] In some embodiments, the humanized 2H7.v511 antibody is an
intact antibody, it may comprise the light chain amino acid
sequence:
TABLE-US-00004 (SEQ ID NO: 16)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAP
KPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
CQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC and the heavy chain amino
acid sequence of SEQ ID NO. 17 or: (SEQ ID NO. 25)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPG
KGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNS
LRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVSC
SVMHEALHNHYTQKSLSLSPG.
[0241] In some embodiments, the antibody herein may further
comprise at least one amino acid substitution in the Fc region that
improves ADCC activity, such as one wherein the amino acid
substitutions are at positions 298, 333, and 334, preferably S298A,
E333A, and K334A, using Eu numbering of heavy chain residues. See
also U.S. Pat. No. 6,737,056B1, Presta. Any of these antibodies may
comprise at least one substitution in the Fc region that improves
FcRn binding or serum half-life, for example a substitution at
heavy chain position 434, such as N434W. See also U.S. Pat. No.
6,737,056B1, Presta. Any of these antibodies may further comprise
at least one amino acid substitution in the Fc region that
increases CDC activity, for example, comprising at least a
substitution at position 326, preferably K326A or K326W. See also
U.S. Pat. No. 6,528,624B1 (Idusogie et al.).
[0242] In some embodiments, the humanized 2H7 variants are those
comprising the variable light domain of SEQ ID NO:2 and the
variable heavy domain of SEQ ID NO:8, including those with or
without substitutions in an Fc region (if present), and those
comprising a variable heavy domain with alteration N100A; or D56A
and N100A; or D56A, N100Y, and S100aR; in SEQ ID NO:8 and a
variable light domain with alteration M32L; or S92A; or M32L and
S92A; in SEQ ID NO:2. M34 in the variable heavy domain of 2H7.v16
has been identified as a potential source of antibody stability and
is another potential candidate for substitution.
[0243] In some embodiments of the invention, the variable region of
variants based on 2H7.v16 comprise the amino acid sequences of v16
except at the positions of amino acid substitutions that are
indicated in the table below. Unless otherwise indicated, the 2H7
variants will have the same light chain as that of v16.
TABLE-US-00005 TABLE 1 Exemplary Humanized 2H7 Antibody Variants
2H7 Heavy chain Light chain Version (V.sub.H) changes (V.sub.L)
changes Fc changes 16 for 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, M32L, S92A S298A, E333A, K334A, S100aR K326A
(iv) Human Antibodies
[0244] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al,
Nature, 362:255-258 (1993); Bruggermann et al, Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
[0245] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al,
EMBO J. 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0246] Human antibodies may also be generated by in vitro activated
B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
(v) Antibody Fragments
[0247] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
The antibody fragment may also be a "linear antibody", e.g., as
described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
(vi) Bispecific Antibodies
[0248] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the B
cell surface marker. Other such antibodies may bind the B cell
surface marker and further bind a second different B-cell surface
marker. Alternatively, an anti-B cell surface marker binding arm
may be combined with an arm that binds to a triggering molecule on
a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3),
or Fc receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so as to focus
cellular defense mechanisms to the B cell. Bispecific antibodies
may also be used to localize cytotoxic agents to the B cell. These
antibodies possess a B cell surface marker-binding arm and an arm
that binds the cytotoxic agent (e.g. saporin,
anti-interferon-.alpha., vinca alkaloid, ricin A chain,
methotrexate or radioactive isotope hapten). Bispecific antibodies
can be prepared as full length antibodies or antibody fragments
(e.g. F(ab').sub.2 bispecific antibodies).
[0249] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO 1,10:3655-3659 (1991).
[0250] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. In
some embodiments, the fusion is with an immunoglobulin heavy chain
constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. In some embodiments, the first heavy chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0251] In some embodiments of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al,
Methods in Enzymology, 121:210 (1986).
[0252] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers that are
recovered from recombinant cell culture. In some embodiments, the
interface comprises at least a part of the C.sub.H3 domain of an
antibody constant domain. In this method, one or more small amino
acid side chains from the interface of the first antibody molecule
are replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g. alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers.
[0253] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0254] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0255] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy chain variable domain (V.sub.H) connected to a light chain
variable domain (V.sub.L) by a linker that is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al, J. Immunol.,
152:5368 (1994).
[0256] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
V. Conjugates and Other Modifications of the Antibody
[0257] The antibody used in the methods or included in the articles
of manufacture herein is optionally conjugated to a cytotoxic
agent. For instance, the antibody may be conjugated to a drug as
described in WO2004/032828.
[0258] Chemotherapeutic agents useful in the generation of such
antibody-cytotoxic agent conjugates have been described above.
[0259] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, a maytansine (U.S. Pat. No.
5,208,020), a trichothene, and CC1065 are also contemplated herein.
In one embodiment of the invention, the antibody is conjugated to
one or more maytansine molecules (e.g. about 1 to about 10
maytansine molecules per antibody molecule). Maytansine may, for
example, be converted to May-SS-Me, which may be reduced to May-SH3
and reacted with modified antibody (Chari et al. Cancer Research
52: 127-131 (1992)) to generate a maytansinoid-antibody
conjugate.
[0260] Alternatively, the antibody is conjugated to one or more
calicheamicin molecules. The calicheamicin family of antibiotics is
capable of producing double-stranded DNA breaks at sub-picomolar
concentrations. Structural analogues of calicheamicin that may be
used include, but are not limited to, .gamma..sub.1.sup.I,
.alpha..sub.2.sup.I, .alpha..sub.3.sup.I,
N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sub.1.sup.I (Hinman
et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer
Research 58: 2925-2928 (1998)).
[0261] Enzymatically active toxins and fragments thereof that can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,
curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0262] The present invention further contemplates antibody
conjugated with a compound with nucleolytic activity (e.g. a
ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0263] A variety of radioactive isotopes are available for the
production of radioconjugated antibodies. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of
Lu.
[0264] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling 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 dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, dimethyl linker or disulfide-containing linker (Chari et
al. Cancer Research 52: 127-131 (1992)) may be used.
[0265] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g. by recombinant techniques or
peptide synthesis.
[0266] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) that is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
[0267] The antibodies of the present invention may also be
conjugated with a prodrug-activating enzyme that converts a prodrug
(e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an
active anti-cancer drug. See, for example, WO 88/07378 and U.S.
Pat. No. 4,975,278.
[0268] The enzyme component of such conjugates includes any enzyme
capable of acting on a prodrug in such a way so as to covert it
into its more active, cytotoxic form.
[0269] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0270] The enzymes of this invention can be covalently bound to the
antibody by techniques well known in the art such as the use of the
heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the 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, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
[0271] Other modifications of the antibody are contemplated herein.
For example, the antibody may be linked to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol. In some embodiments,
the antibody fragments, such as Fab', are linked to one or more PEG
molecules.
[0272] The antibodies disclosed herein may also be formulated as
liposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0273] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of an antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
[0274] Amino acid sequence modification(s) of the antibody are
contemplated. For example, it may be desirable to improve the
binding affinity and/or other biological properties of the
antibody. Amino acid sequence variants of the antibody are prepared
by introducing appropriate nucleotide changes into the antibody
nucleic acid, or by peptide synthesis. Such modifications include,
for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
[0275] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0276] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody of an
enzyme, or a polypeptide that increases the serum half-life of the
antibody.
[0277] 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 different residue. The sites of
greatest interest for substitutional mutagenesis of antibody
antibodies include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 2 under the heading of "preferred substitutions". If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table
2, may be introduced and the products screened.
TABLE-US-00006 TABLE 2 Original Exemplary Preferred Residue
Substitutions 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; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe 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; Leu Ala; Norleucine
[0278] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0279] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0280] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (Q)
[0281] (3) acidic: Asp (D), Glu (E)
[0282] (4) basic: Lys (K), Arg (R), His(H)
[0283] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0284] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0285] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0286] (3) acidic: Asp, Glu;
[0287] (4) basic: His, Lys, Arg;
[0288] (5) residues that influence chain orientation: Gly, Pro;
[0289] (6) aromatic: Trp, Tyr, Phe.
[0290] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0291] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[0292] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody. Generally, the resulting variant(s) selected for
further development will have improved biological properties
relative to the parent antibody from which they are generated. A
convenient way for generating such substitutional variants is
affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g. 6-7 sites) are mutated to generate
all possible amino substitutions at each site. The antibody
variants thus generated are displayed in a monovalent fashion from
filamentous phage particles as fusions to the gene III product of
M13 packaged within each particle. The phage-displayed variants are
then screened for their biological activity (e.g. binding affinity)
as herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in
additionally, it may be beneficial to analyze a crystal structure
of the antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is patiented to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0293] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. Such altering
includes deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0294] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0295] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0296] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody are described in US Pat Appl No US
2003/0157108 A1 (Presta, L.); see also US 2004/0093621 A1 (Kyowa
Hakko Kogyo Co., Ltd) concerning a CD20 antibody composition.
Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the
carbohydrate attached to an Fc region of the antibody are
referenced in WO03/011878, Jean-Mairet et al. and U.S. Pat. No.
6,602,684, Umana et al. Antibodies with at least one galactose
residue in the oligosaccharide attached to an Fc region of the
antibody are reported in WO97/30087 (Patel et al.); see also
WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) concerning
antibodies with altered carbohydrate attached to the Fc region
thereof.
[0297] In some embodiments, the glycosylation variant herein
comprises an Fc region, wherein a carbohydrate structure attached
to the Fc region lacks fucose. Such variants have improved ADCC
function. Optionally, the Fc region further comprises one or more
amino acid substitutions therein which further improve ADCC, for
example, substitutions at positions 298, 333, and/or 334 of the Fc
region (Eu numbering of residues). Examples of publications related
to "defucosylated" or "fucose-deficient" antibodies include: US
Pat. Appl. No. US 2003/0157108 A1, Presta, L; WO 00/61739A1;
WO01/29246A1; US2003/0115614A1; US2002/0164328A1; US2004/0093621A1;
US2004/0132140A1; US2004/0110704A1; US2004/0110282A1;
US2004/0109865A1; WO03/085119A1; WO03/084570A1; WO2005/035778;
WO2005/035586 (describing RNA inhibition (RNAi) of fucosylation);
Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et
al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines
producing defucosylated antibodies include Lec13 CHO cells
deficient in protein fucosylation (Ripka et al. Arch. Biochem.
Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1,
Presta, L; and WO 2004/056312 A1, Adams et al., especially at
Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells
(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).
[0298] Nucleic acid molecules encoding amino acid sequence variants
of the 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 preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0299] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of an antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability 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 may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
that has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug
Design 3:219-230 (1989).
[0300] WO00/42072 (Presta, L.) describes antibodies with improved
ADCC function in the presence of human effector cells, where the
antibodies comprise amino acid substitutions in the Fc region
thereof. In some embodiments, the antibody with improved ADCC
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 comprising or consisting of substitutions at one, two or
three of these positions.
[0301] Antibodies with altered Clq binding and/or complement
dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.). The
antibodies comprise an amino acid substitution at one or more of
amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334
of the Fc region thereof.
[0302] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule. Antibodies with substitutions in an Fc region thereof
and increased serum half-lives are also described in WO00/42072
(Presta, L.).
[0303] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appln
No. US2002/0004587 A1, Miller et al.).
VI. Pharmaceutical Formulations
[0304] Therapeutic formulations of the antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of 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 nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0305] Exemplary anti-CD20 antibody formulations are described in
WO98/56418. This publication describes a liquid multidose
formulation comprising 40 mg/mL Rituximab, 25 mM acetate, 150 mM
trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that
has a minimum shelf life of two years storage at 2-BBC. Another
anti-CD20 formulation of interest comprises 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.
[0306] Lyophilized formulations adapted for subcutaneous
administration are described in U.S. Pat. No. 6,267,958 (Andya et
al.). Such lyophilized formulations may be reconstituted with a
suitable diluent to a high protein concentration and the
reconstituted formulation may be administered subcutaneously to the
mammal to be treated herein.
[0307] Crystalized forms of the antibody or antibody are also
contemplated. See, for example, US 2002/0136719A1 (Shenoy et
al).
[0308] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
in some embodiments, those with complementary activities that do
not adversely affect each other. For example, it may be desirable
to further provide a cytotoxic agent; chemotherapeutic agent;
immunosuppressive agent; cytokine; cytokine antagonist or antibody;
growth factor; hormone; integrin; integrin antagonist or antibody
(e.g. an LFA-1 antibody such as efalizumab/RAPTIVA commercially
available from Genentech, or an alpha 4 integrin antibody such as
natalizumab/TYSABRI.RTM.) available from Biogen Idec/Elan
Pharmaceuticals, Inc.); interferon class drug such as IFN-beta-1a
(REBIF.RTM. and AVONEX.RTM.) or IFN-beta-1b (BETASERON.RTM.); an
oligopeptide such a glatiramer acetate (COPAXONE.RTM.); a cytotoxic
agent such as mitoxantrone (NOVANTRONE.RTM.), methotrexate,
cyclophosphamide, chlorambucil, or azathioprine; intravenous
immunoglobulin (gamma globulin); lymphocyte-depleting drug (e.g.,
mitoxantrone, cyclophosphamide, Campath, anti-CD4, or cladribine);
non-lymphocyte-depleting immunosuppressive drug (e.g.,
mycophenolate mofetil (MMF) or cyclosporine); cholesterol-lowering
drug of the "statin" class; estradiol; testosterone; hormone
replacement therapy; drug that treats symptoms secondary or related
to MS (e.g., spasticity, incontinence, pain, fatigue); a TNF
inhibitor; disease-modifying anti-rheumatic drug (DMARD);
non-steroidal anti-inflammatory drug (NSAID); corticosteroid (e.g.
methylprednisolone, prednisone, dexamethasone, or glucorticoid);
levothyroxine; cyclosporin A; somatastatin analogue; cytokine
antagonist; anti-metabolite; immunosuppressive agent; integrin
antagonist or antibody (e.g. an 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 formulation. 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 clinical parameters
of the patients. These are generally used in the same dosages and
with administration routes as used hereinbefore or about from 1 to
99% of the heretofore employed dosages.
[0309] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0310] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0311] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0312] In some embodiments, the formulation comprises one or more
of the group consisting of a 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 the administration of the anti-CD20 antibody are found in Andya
et al., US2006/0088523, which is incorporated by reference in its
entirety with respect to formulations.
[0313] Exemplary anti-CD20 antibody formulations are described in
Andya et al., US2006/0088523 and WO98/56418, which are incorporated
by reference in its entirety. In some embodiments, formulation is a
liquid multidose formulation comprising the anti-CD20 antibody at
40 mg/mL, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol,
0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of two
years storage at 2-8.degree. C. In some embodiments, anti-CD20
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 in an aqueous pharmaceutical
formulation comprising 10-30 mM sodium acetate from about pH 4.8 to
about pH 5.5, preferably at pH5.5, polysorbate as a surfactant in a
an amount of about 0.01-0.1% v/v, trehalose at an amount of about
2-10% w/v, and benzyl alcohol as a preservative (U.S. Pat. No.
6,171,586, which is incorporated by reference in its entirety).
Lyophilized formulations adapted for subcutaneous administration
are described in WO97/04801, which is incorporated by reference in
its entirety. Such lyophilized formulations may be reconstituted
with a suitable diluent to a high protein concentration and the
reconstituted formulation may be administered subcutaneously to the
mammal to be treated herein.
[0314] In some embodiments, the humanized 2H7 variants formulation
is antibody at 12-14 mg/mL in 10 mM histidine, 6% sucrose, 0.02%
polysorbate 20, pH 5.8. In a specific embodiment, 2H7 variants and
in particular 2H7.v16 is formulated at 20 mg/mL antibody in 10 mM
histidine sulfate, 60 mg/ml sucrose., 0.2 mg/ml polysorbate 20, and
Sterile Water for Injection, at pH5.8. 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.TM.), pH 5.3. In some embodiments, the humanized 2H7.v511
variant formulation 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
pH5.8. In yet another embodiment, the formulation for 2H7 variants
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, 2H7.v 114
formulation is antibody at 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.
VII. Articles of Manufacture and Methods of Manufacture
[0315] The invention provides articles of manufacture comprising:
(a) a container comprising ocrelizumab; and (b) a package insert
with instructions for treating multiple sclerosis in a patient,
wherein the instructions denote (i.e., indicate) that an amount of
ocrelizumab is administered to the patient that is effective to
provide an initial ocrelizumab exposure of between about 0.3 to
about 0.6 grams followed by a second ocrelizumab exposure of
between about 0.3 to about 0.6 grams, the second exposure not being
administered until from about 16 to 60 weeks from the initial
exposure, and each of the ocrelizumab exposures is provided to the
patient as one or two doses of ocrelizumab. In some embodiments,
the initial ocrelizumab exposure is about 0.6 grams. In some
embodiments, the second ocrelizumab exposure is about 0.6 grams. In
some embodiments, the second exposure is administered from about 24
weeks from the initial exposure. In some embodiments, one or more
of the ocrelizumab exposures are provided to the patient as one
dose of ocrelizumab. In some embodiments, one or more of the
ocrelizumab exposures are provided to the patient as two doses of
ocrelizumab. In some embodiments, the two doses of ocrelizumab
comprise about 0.3 grams of ocrelizumab.
[0316] The invention further provides articles of manufacture
containing materials useful for the treatment of progressive
multiple sclerosis described herein. In some embodiments, the
article of manufacture comprising, packaged together, a
pharmaceutical composition comprising an anti-CD20 antibody and a
pharmaceutically acceptable carrier and a label denoting that the
anti-CD20 antibody or pharmaceutical composition is indicated for
treating patients with multiple sclerosis having one or more
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting the
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0317] In some embodiments, the article of manufacture comprising,
packaged together, a pharmaceutical composition comprising an
anti-CD20 antibody and a pharmaceutically acceptable carrier and a
label denoting that administration of the anti-CD20 antibody or
pharmaceutical composition is based upon the patient having one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0318] In some embodiments, the article of manufacture comprising,
packaged together, a pharmaceutical composition comprising an
anti-CD20 antibody and a pharmaceutically acceptable carrier and a
label denoting that the pharmaceutical composition is administered
to a selected patient, wherein the selected patient has one or more
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0319] In some embodiments of any of the articles of manufacture
described herein, the patients have one or more characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, and (c) at
least about a one point increase in Expanded Disability Status
Scale (EDSS) over two years prior to starting treatment.
[0320] In some embodiments of any of the articles of manufacture
described herein, the patient has more than one characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, (c) at least
about a one point increase in Expanded Disability Status Scale
(EDSS) over two years prior to starting treatment, and (d) a
Multiple Sclerosis Severity Score (MSSS) greater than about 5
points. In some embodiments of any of the articles of manufacture
described herein, the patient has two characteristics selected from
the group consisting of (a) an age less than about 55 years, (b)
one or more gadolinium staining lesions, and (c) at least about a
one point increase in EDSS over two years prior to starting
treatment. In some embodiments, the patient has three
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points. In some embodiments, the patient has
(a) an age less than about 55 years, (b) one or more gadolinium
staining lesions, (c) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment, and (d) a Multiple Sclerosis Severity Score
(MSSS) greater than about 5 points.
[0321] In some embodiments of any of the articles of manufacture,
the progressive multiple sclerosis is primary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is secondary progressive multiple sclerosis. In some embodiments,
the progressive multiple sclerosis is progressive relapsing
multiple sclerosis. In some embodiments, the patient is not
diagnosed with relapsing remitting multiple sclerosis when starting
treatment.
[0322] In some embodiments of any of the articles of manufacture,
the patient further has evidence of inflammation in a sample. In
some embodiments, the sample is a cerebrospinal fluid sample. In
some embodiments, the evidence of inflammation is indicated by an
elevated IgG index. In some embodiments, the evidence of
inflammation is indicated by IgG oligoclonal bands detected by
isoelectric focusing.
[0323] In some embodiments of any of the articles of manufacture,
the increase in EDSS over two years prior to starting treatment is
not attributable to relapse. In some embodiments, the patient has
had an EDSS of greater than about 5.0 for less than about 15 years.
In some embodiments, the patient has had an EDSS less than or equal
to about 5.0 for less than about 10 years. In some embodiments, the
EDSS when starting treatment is between about 3.0 and about 6.5. In
some embodiments, the increase in EDSS is at least about a 1.5
point increase in EDSS over two years prior to starting treatment.
In some embodiments, the 1.5 point increase in EDSS over two years
prior to starting treatment is not attributable to relapse. In some
embodiments, the patient further had two or more relapses within
two years prior to starting treatment.
[0324] In some embodiments of any of the articles of manufacture
described herein, the patient and/or patient population is
characterized by having an MSSS of greater than about any of 6, 7,
8, or 9. In some embodiments of any of the articles of manufacture,
the patient and/or patient population is characterized by having an
MSSS of greater than about 9.
[0325] In some embodiments of any of the articles of manufacture,
the age of the patient is less than about 51.
[0326] In some embodiments of any of the articles of manufacture,
the treatment reduces the time to confirmed disease progression. In
some embodiments, the confirmed disease progression is an increase
in EDSS that is sustained for twelve weeks. In some embodiments,
the confirmed disease progression is an increase in EDSS that is
sustained for twenty-four weeks.
[0327] 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. In some embodiments, the anti-CD20 antibody is
rituximab. In some embodiments, the anti-CD20 antibody is
ofatumumab. In some embodiments, the anti-CD20 antibody is TRU-015
or SBI-087. In some embodiments, the anti-CD20 antibody is GA101.
In some embodiments, the anti-CD20 antibody is hA20.
[0328] The article of manufacture comprises a container and a label
or package insert on or associated with the container. Suitable
containers include, for example, bottles, vials, syringes, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds or contains a composition that is
effective for treating the multiple sclerosis and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is the antibody. In some embodiments, the container
comprises between about 0.3 to about 4.0 grams of the anti-CD20
antibody. In some embodiments, the container comprises between
about 0.3 to about 1.5 grams of the anti-CD20 antibody.
[0329] The label or package insert indicates that the composition
is used for treating multiple sclerosis in a patient suffering
therefrom with specific guidance regarding dosing amounts and
intervals of antibody and any other drug being provided. The
article of manufacture may further comprise a second container
comprising a pharmaceutically acceptable diluent buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. The article of
manufacture may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0330] Optionally, the article of manufacture herein further
comprises a container comprising an agent other than the antibody
for treatment and further comprising instructions on treating the
patient with such agent, such agent preferably being a
chemotherapeutic agent or immunosuppressive agent, interferon class
drug such as IFN-beta-1a (REBIF.RTM. and AVONEX.RTM.) or
IFN-beta-1b (BETASERON.RTM.); an oligopeptide such a glatiramer
acetate (COPAXONE.RTM.); a cytotoxic agent such as mitoxantrone
(NOVANTRONE.RTM.), methotrexate, cyclophosphamide, chlorambucil, or
azathioprine; intravenous immunoglobulin (gamma globulin);
lymphocyte-depleting drug (e.g., mitoxantrone, cyclophosphamide,
Campath, anti-CD4, or cladribine); non-lymphocyte-depleting
immunosuppressive drug (e.g., mycophenolate mofetil (MMF) or
cyclosporine); cholesterol-lowering drug of the "statin" class;
estradiol; hormone replacement therapy; drug that treats symptoms
secondary or related to MS (e.g., spasticity, incontinence, pain,
fatigue); a TNF inhibitor; disease-modifying anti-rheumatic drug
(DMARD); non-steroidal anti-inflammatory drug (NSAID);
corticosteroid (e.g. methylprednisolone, prednisone, dexamethasone,
or glucorticoid); levothyroxine; cyclosporin A; somatastatin
analogue; cytokine or cytokine receptor antagonist;
anti-metabolite; immunosuppressive agent; integrin antagonist or
antibody (e.g. an LFA-1 antibody, such as efalizumab or an alpha 4
integrin antibody such as natalizumab); and another B-cell surface
marker antibody; etc.
[0331] In some embodiments, the label may further denote any of the
embodiments described herein. For example, the label may denote
that the patient has (a) an age less than about 55 years and (b)
one or more gadolinium staining lesions.
[0332] In another embodiment of the invention, a method for
manufacturing containing materials useful for the treatment of
progressive multiple sclerosis described herein is provided. In
some embodiments, the method for manufacturing an anti-CD20
antibody or a pharmaceutical composition thereof comprising
combining in a package the anti-CD20 antibody or pharmaceutical
composition and a label denoting that the anti-CD20 antibody or
pharmaceutical composition is indicated for treating patients with
progressive multiple sclerosis, wherein the patients have one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0333] In some embodiments of any of the methods of manufacturing
described herein, the patients have one or more characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, and (c) at
least about a one point increase in Expanded Disability Status
Scale (EDSS) over two years prior to starting treatment.
VIII. Methods of Advertising and Marketing
[0334] The present invention also provides methods for advertising
an anti-CD20 antibody or a pharmaceutically acceptable composition
thereof comprising promoting, to a target audience, the use of the
anti-CD20 antibody or pharmaceutical composition thereof for
treating a patient or patient population with progressive multiple
sclerosis, wherein the patient or patient population has one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0335] Provided herein are also methods for marketing an anti-CD20
antibody or a pharmaceutically acceptable composition thereof for
use in a progressive multiple sclerosis patient subpopulation, the
method comprising informing a target audience about the use of the
anti-CD20 antibody for treating the patient subpopulation
characterized by the patients of such subpopulation having one or
more characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0336] In addition, the invention provides methods of specifying an
anti-CD20 antibody for use in a progressive multiple sclerosis
patient subpopulation, the method comprising providing instruction
to administer the anti-CD20 antibody or a pharmaceutically
acceptable composition thereof to the patient subpopulation
characterized by the subpopulation has one or more characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, (c) at least
about a one point increase in Expanded Disability Status Scale
(EDSS) over two years prior to starting treatment, and (d) a
Multiple Sclerosis Severity Score (MSSS) greater than about 5
points.
[0337] The invention further provides methods of providing a
treatment option for patients with progressive multiple sclerosis
comprising packaging an anti-CD20 antibody in a vial with a package
insert containing instructions to treat patients with progressive
multiple sclerosis, wherein the patients have one or more
characteristics selected from the group consisting of (a) an age
less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points.
[0338] In some embodiments of any of the methods, the patients have
one or more characteristics selected from the group consisting of
(a) an age less than about 55 years, (b) one or more gadolinium
staining lesions, and (c) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment.
[0339] In some embodiments of any of the methods, the patient has
two characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points. In some embodiments, the patient has
three characteristics selected from the group consisting of (a) an
age less than about 55 years, (b) one or more gadolinium staining
lesions, (c) at least about a one point increase in Expanded
Disability Status Scale (EDSS) over two years prior to starting
treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)
greater than about 5 points. In some embodiments, the patient has
(a) an age less than about 55 years, (b) one or more gadolinium
staining lesions, (c) at least about a one point increase in
Expanded Disability Status Scale (EDSS) over two years prior to
starting treatment, and (d) a Multiple Sclerosis Severity Score
(MSSS) greater than about 5 points.
[0340] In some embodiments of any of the methods, the progressive
multiple sclerosis is primary progressive multiple sclerosis. In
some embodiments, the progressive multiple sclerosis is secondary
progressive multiple sclerosis. In some embodiments, the
progressive multiple sclerosis is progressive relapsing multiple
sclerosis. In some embodiments, the patient is not diagnosed with
relapsing remitting multiple sclerosis when starting treatment.
[0341] In some embodiments of any of the methods, the patient
further has evidence of inflammation in a sample. In some
embodiments, the sample is a cerebrospinal fluid sample. In some
embodiments, the evidence of inflammation is indicated by an
elevated IgG index. In some embodiments, the evidence of
inflammation is indicated by IgG oligoclonal bands detected by
isoelectric focusing.
[0342] In some embodiments of any of the methods, the increase in
EDSS over two years prior to starting treatment is not attributable
to relapse. In some embodiments, the patient has had an EDSS of
greater than about 5.0 for less than about 15 years. In some
embodiments, the patient has had an EDSS less than or equal to
about 5.0 for less than about 10 years. In some embodiments, the
EDSS when starting treatment is between about 3.0 and about 6.5. In
some embodiments, the increase in EDSS is at least about a 1.5
point increase in EDSS over two years prior to starting treatment.
In some embodiments, the 1.5 point increase in EDSS over two years
prior to starting treatment is not attributable to relapse. In some
embodiments, the patient further had two or more relapses within
two years prior to starting treatment.
[0343] In some embodiments of any of the methods, the age of the
patient is less than about 51.
[0344] In some embodiments of any of the methods, the treatment
reduces the time to confirmed disease progression. In some
embodiments, the confirmed disease progression is an increase in
EDSS that is sustained for twelve weeks. In some embodiments, the
confirmed disease progression is an increase in EDSS that is
sustained for twenty-four weeks.
[0345] In some embodiments of any of the methods, the anti-CD20
antibody is ocrelizumab. In some embodiments of any of the methods,
the anti-CD20 antibody is rituximab. In some embodiments of any of
the methods, the anti-CD20 antibody is ofatumumab. In some
embodiments of any of the methods, the anti-CD20 antibody is
TRU-015 or SBI-087. In some embodiments of any of the methods, the
anti-CD20 antibody is GA101. In some embodiments of any of the
methods, the anti-CD20 antibody is hA20.
[0346] The methods described herein may encompass any combination
of the embodiments described herein. For example, the methods
include methods, wherein the patient (a) an age less than about 55
years and (b) one or more gadolinium staining lesions.
IX. Systems and Methods for Predicting Responsiveness to Multiple
Sclerosis Treatment
[0347] The invention also provides systems and methods for
analyzing whether a subject and/or patient with progressive
multiple sclerosis will respond to a treatment with a drug used to
treat multiple sclerosis. The invention provides systems for
analyzing responsiveness of a patient with progressive multiple
sclerosis to treatment with a drug used to treat multiple sclerosis
comprising: (a) assessing one or more characteristics selected from
the group consisting of (i) an age less than about 55 years, (ii)
one or more gadolinium staining lesions, (iii) at least about a one
point increase in Expanded Disability Status Scale (EDSS) over two
years prior to starting treatment, and (iv) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points; (b) hardware to
perform the assessment of (a); and (c) computational means to
perform an algorithm to determine if the patient is susceptible or
responsive to said treatment.
[0348] The invention further provides methods for predicting
whether a subject with progressive multiple sclerosis will respond
to a treatment with a drug used to treat multiple sclerosis, the
methods comprising assessing one or more characteristics selected
from the group consisting of (a) an age less than about 55 years,
(b) one or more gadolinium staining lesions, (c) at least about a
one point increase in Expanded Disability Status Scale (EDSS) over
two years prior to starting treatment, and (d) a Multiple Sclerosis
Severity Score (MSSS) greater than about 5 points, whereby the age,
the gadolinium staining lesions, the increase in EDDS over two
years prior to starting the treatment, MSSS, or a combination
thereof indicates that the subject will respond to the
treatment.
[0349] In some embodiments of any of the systems and/or methods,
the patient has more than one characteristics selected from the
group consisting of (a) an age less than about 55 years, (b) one or
more gadolinium staining lesions, and (c) at least about a one
point increase in Expanded Disability Status Scale (EDSS) over two
years prior to starting treatment.
[0350] In some embodiments of any of the systems and/or methods
described herein, the patient has more than one characteristics
selected from the group consisting of (a) an age less than about 55
years, (b) one or more gadolinium staining lesions, (c) at least
about a one point increase in Expanded Disability Status Scale
(EDSS) over two years prior to starting treatment, and (d) a
Multiple Sclerosis Severity Score (MSSS) greater than about 5
points. In some embodiments of any of the systems and/or methods,
the patient has two characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points. In some embodiments, the
patient has three characteristics selected from the group
consisting of (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points. In some embodiments, the
patient has (a) an age less than about 55 years, (b) one or more
gadolinium staining lesions, (c) at least about a one point
increase in Expanded Disability Status Scale (EDSS) over two years
prior to starting treatment, and (d) a Multiple Sclerosis Severity
Score (MSSS) greater than about 5 points.
[0351] In some embodiments of any of the systems and/or methods,
the progressive multiple sclerosis is primary progressive multiple
sclerosis. In some embodiments, the progressive multiple sclerosis
is secondary progressive multiple sclerosis. In some embodiments,
the progressive multiple sclerosis is progressive relapsing
multiple sclerosis. In some embodiments, the patient is not
diagnosed with relapsing remitting multiple sclerosis when starting
treatment.
[0352] In some embodiments of any of the systems or methods, the
patient further has evidence of inflammation in a sample. In some
embodiments, the sample is a cerebrospinal fluid sample. In some
embodiments, the evidence of inflammation is indicated by an
elevated IgG index. In some embodiments, the evidence of
inflammation is indicated by IgG oligoclonal bands detected by
isoelectric focusing.
[0353] In some embodiments of any of the systems and/or methods,
the increase in EDSS over two years prior to starting treatment is
not attributable to relapse. In some embodiments, the patient has
had an EDSS of greater than about 5.0 for less than about 15 years.
In some embodiments, the patient has had an EDSS less than or equal
to about 5.0 for less than about 10 years. In some embodiments, the
EDSS when starting treatment is between about 3.0 and about 6.5. In
some embodiments, the increase in EDSS is at least about a 1.5
point increase in EDSS over two years prior to starting treatment.
In some embodiments, the 1.5 point increase in EDSS over two years
prior to starting treatment is not attributable to relapse. In some
embodiments, the patient further had two or more relapses within
two years prior to starting treatment.
[0354] In some embodiments of any of the systems and/or methods,
the age of the patient is less than about 51.
[0355] In some embodiments of any of the systems and/or methods,
the systems or methods further comprises advising the patient.
[0356] In some embodiments of any of the systems and/or methods,
the treatment reduces the time to confirmed disease progression. In
some embodiments, the confirmed disease progression is an increase
in EDSS that is sustained for twelve weeks. In some embodiments,
the confirmed disease progression is an increase in EDSS that is
sustained for twenty-four weeks.
[0357] In some embodiments of any of the systems and/or methods,
the drug is a Interferon beta-1b (e.g., Betaseron.RTM.), Interferon
beta-la (e.g., Avonex.RTM. or Rebif.RTM.), Glatiramer (e.g.,
Copaxone.RTM.), Mitoxantrone (e.g., Novantrone), corticosteroids
(e.g., ethylprednisolone, prednisone, dexamethasone), 3-4
diaminopyridine, ABT-874, Alemtuzumab, Albuterol (Proventil.RTM.),
ATL1102, Atorvastatin (Lipitor.RTM.), Azathioprine, BG00012
(dimethyl fumarate), BHT-3009, Botulinum toxin A (Botox.RTM.),
C-105, cannador, dronabinol, tetrahydrocannabinol, cannabidiol,
CDP323, Cladribine, CNTO 1275, Cyclophosphamide, Daclizumab,
Dextromethorphan/quinidine (AVP-923, Zenvia.TM.), Donepezil
(Aricept.RTM.), Doxycycline, Estradiol, Estriol, Estroprogestins,
Fampridine-SR (4-aminopyridine, sustained release), Fingolimod
(FTY720), Interferon tau, Lamotrigine (Lamictal.RTM.), Laquinimod,
Lidocaine+prilocaine (EMLA), MBP8298 (synthetic myelin basic
protein peptide), Memantine (Namenda.RTM.), Methylprednisolone,
MN-166, Modafinil (Provigil.RTM.), Mycophenolate mofetil
(Cellcept.RTM.), naltrexone, Natalizumab (Tysabri.RTM.), Paroxetine
(Paxil.RTM.), PI-2301 (copolymer), Pioglitazone (Actos.RTM.),
Pixantrone (BBR 2778), Pravastatin (Pravachol.RTM.), Pregabalin
(Lyrica.RTM.), Progesterone, RG2077, Riluzole (Rilutek.RTM.),
Rolipram (phosphodiesterase-4 inhibitor), RTL1000, SB-683699,
Simvastatin (Zocor.RTM.), T cell receptor peptide vaccine
(NeuroVax.TM.), Teriflunomide, Testosterone gel (Androgel.RTM.), or
Trimethoprim.
[0358] In some embodiments of any of the systems and/or methods,
the drug used to treat multiple sclerosis is an anti-CD20 antibody.
In some embodiments, 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. In some embodiments, the
anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20
antibody is ofatumumab. In some embodiments, the anti-CD20 antibody
is TRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody
is GA101. In some embodiments, the anti-CD20 antibody is hA20.
[0359] The systems and/or methods described herein may encompass
any combination of the embodiments described herein. For example,
the methods include methods, wherein the patient (a) an age less
than about 55 years and (b) one or more gadolinium staining
lesions.
[0360] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
EXAMPLES
[0361] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and
embodiments of the invention discussed above. The foregoing
examples and detailed description are offered by way of
illustration and not by way of limitation.
Example 1: A Phase II Study of Ocrelizumab in Relapsing-Remitting
Multiple Sclerosis (RRMS)
[0362] A phase II, multicenter, randomized, parallel-group,
partially blinded, placebo and Avonex controlled dose finding study
to evaluate the efficacy as measured by brain Magnetic Resonance
Imaging (MRI) lesions, and safety of two dose regimens of
ocrelizumab in patients with Relapsing-Remitting Multiple Sclerosis
(RRMS) is performed.
[0363] The two ocrelizumab dose regimens under investigation are as
follows: 1) ocrelizumab 1000 mg dose regimen: consisting of a dual
infusion of 1000 mg for the first treatment cycle followed by
single infusions of 1000 mg for the subsequent treatment cycles and
2) ocrelizumab 600 mg dose regimen: consisting of a dual infusion
of 300 mg for the first treatment cycle followed by single
infusions of 600 mg for the subsequent treatment cycles.
[0364] Eligible patients are randomized (1:1:1:1) into one of four
treatment groups A, B, C or D as described in FIG. 7. The overview
of the study design is illustrated in FIG. 7.
[0365] Group A (ocrelizumab 1000 mg): Two intravenous (i.v.)
infusions of ocrelizumab each of 1000 mg separated by 14 days for
the 1st treatment cycle. Patients then receive the maintenance dose
regimen, i.e., a single infusion of 1000 mg for every subsequent
24-week treatment cycle. Subsequently, in order to maintain the
study double-blind during the 2nd treatment cycle, patients receive
two infusions separated by 14 days, the first infusion is
ocrelizumab 1000 mg and the second infusion placebo. In the 3rd and
4th treatment cycles, patients are treated with a single 1000 mg
infusion, without a second placebo infusion, in a double-blind
manner until a preferred dose is chosen on the basis of the primary
analysis.
[0366] Group B (ocrelizumab 600 mg): Two i.v. infusions of
ocrelizumab each of 300 mg separated by 14 days for the 1st
treatment cycle. Patients then receive the maintenance dose
regimen, i.e., a single infusion of 600 mg for every subsequent
24-week treatment cycle. Subsequently, in order to maintain the
study double-blind during the 2nd treatment cycle, patients receive
two infusions separated by 14 days, the first infusion is
ocrelizumab 600 mg and the second infusion placebo. In the 3rd and
4th treatment cycles, patients are treated with a single 600 mg
infusion, without a second placebo infusion in a double-blind
manner until a preferred dose is chosen on the basis of the primary
analysis.
[0367] Group C (placebo): Two i.v. infusions of placebo separated
by 14 days for the 1st treatment cycle. Thereafter, patients are
placed on the 600 mg dose regimen of ocrelizumab starting with two
double-blind i.v. infusions of ocrelizumab 300 mg separated by 14
days at the start of the 2nd treatment cycle. Patients then receive
the maintenance dose regimen, i.e., a single infusion of 600 mg
administered in a double-blind manner for the 3rd and 4th treatment
cycles, until a preferred dose is chosen on the basis of the
primary analysis.
[0368] Group D (Avonex): Avonex 30 .mu.g intra-muscular (i.m.)
weekly for the 1st treatment cycle. Thereafter, patients are
offered, on a voluntary and open label basis, the 600 mg dose
regimen of ocrelizumab starting with two i.v. infusions of
ocrelizumab 300 mg separated by 14 days at the start of the 2nd
treatment cycle. Patients in the 3rd and 4th treatment cycles are
treated with a single 600 mg infusion until a preferred dose is
chosen on the basis of the primary analysis.
[0369] For all groups, after investigators and ethics committees
are informed of the preferred dose, patients receive the preferred
dose (600 mg or 1000 mg) as a single infusion at their next
successive treatment cycle(s).
[0370] The first administration of study medication whether an i.v.
infusion (ocrelizumab or placebo) or the first i.m. injection of
Avonex will define the start of the Treatment Period (Day 1). All
patients also receive an i.v. infusion of methylprednisolone 100 mg
on study day 1 and with each subsequent ocrelizumab or placebo
infusion or, for patients receiving Avonex (Group D) according to
the time points required for the ocrelizumab infusions.
[0371] There are four treatment cycles, i.e. cycle 1=Baseline to
Week 24; cycle 2=Week 24 to Week 48; cycle 3=Week 48 to Week 72;
cycle 4=Week 72 to Week 96. After the 1st cycle infusion visits
(Visits 2 and 3, Day 1 and Week 2, respectively), visits occur at
Week 4 and every 4 weeks thereafter for the first 24 weeks. After
the 2nd cycle infusion visits (Visits 9 and 10, Week 24 and 26,
respectively), visits occur at Week 36 and every 12 weeks
thereafter through the end of the Treatment and Follow-up Periods.
All effort should be made to schedule the visits within the
provided windows. Additional unscheduled visits for the assessment
of potential relapses, new neurological symptoms or safety events
may occur at any time.
Study Population and Selection Criteria
[0372] Men and women from 18 to 55 years of age inclusive, who are
diagnosed with relapsing-remitting multiple sclerosis (RRMS) in
accordance with the revised McDonald criteria (2005) and who meet
the inclusion/exclusion criteria provided below are eligible for
enrollment into the study.
[0373] Inclusion Criteria:
[0374] Patients must meet the following criteria to be eligible for
study entry: [0375] 1. RRMS in accordance with the revised McDonald
criteria (2005); [0376] 2. Ages 18-55 years inclusive; [0377] 3. At
least two documented relapses within the last 3 years prior to
screening, at least one of which occurred within the last year
prior to screening; [0378] 4. Expanded Disability Status Scale
(EDSS) at baseline from 1.0 to 6.0 points; [0379] 5. Evidence of
Multiple Sclerosis (MS) disease burden as defined below: [0380] a.
At least six T2 lesions on an MRI scan done in the year prior to
screening, based on local reading. Should an MRI scan be
unavailable within the last year or showing less than six T2
lesions, a screening MRI scan with at least six T2 lesions is
required for the patient to be eligible, OR [0381] b. Patient had 2
documented relapses within the year prior to screening.
[0382] Exclusion Criteria
[0383] Patients who meet the following criteria will be excluded
from study entry:
[0384] 1. Secondary or primary progressive multiple sclerosis at
screening (Visit 1);
[0385] 2. Disease duration of more than 15 years in patients with
an EDSS.ltoreq.2.0.
Efficacy Analysis
[0386] The primary objective in this study is to investigate the
effect of ocrelizumab given as two dose regimens of 600 or 1000 mg
intravenously (see FIG. 7) on the total number of
gadolinium-enhancing T1 lesions observed on MRI scans of the brain
at weeks 12, 16, 20 and 24 as compared with placebo.
[0387] The secondary objectives of this study are to evaluate the
efficacy and safety of ocrelizumab compared with placebo, as
reflected by the following: the annualized protocol defined relapse
rate by Week 24; proportion of patients who remain relapse-free by
Week 24 (protocol defined relapses); the total number of
gadolinium-enhancing T1 lesions observed on MRI scans of the brain
at Weeks 4, 8, 12, 16, 20 and 24; the total number of new and/or
persisting gadolinium-enhancing T1 lesions on MRI scans of the
brain at Weeks 4, 8, 12, 16, 20 and 24; change in total volume of
T2 lesions on MRI scans of the brain from baseline to Week 24, to
evaluate the safety and tolerability of two dose regimens of
ocrelizumab in patients with RRMS as compared with placebo and
Avonex at Week 24 and the overall safety of ocrelizumab
administered for up to 96 weeks, and to investigate the
pharmacokinetics and other pharmacodynamic study endpoints of
ocrelizumab.
[0388] In this Example, a relapse is defined as the occurrence of
new or worsening neurological symptoms attributable to MS and
immediately preceded by a relatively stable or improving
neurological state of least 30 days. Symptoms must persist for
>24 hours and should not be attributable to confounding clinical
factors (e.g., fever, infection, injury, adverse reactions to
concomitant medications). The new or worsening neurological
symptoms must be accompanied by objective neurological worsening
consistent with an increase of at least half a step on the EDSS, or
2 points on one of the appropriate Functional System Scores (FSS),
or 1 point on two or more of the appropriate FSS. The change must
affect the selected FSS (i.e., pyramidal, gait, cerebellar,
brainstem, sensory, or visual). Sensory changes, episodic spasms,
fatigue, mood change or bladder or bowel urgency or incontinence do
not suffice to establish a relapse. The examining investigator
confirms those relapses that adhere to the above criteria.
[0389] The exploratory objectives in this study will include, but
may not be limited to: change in brain volume on MRI scans of the
brain from the baseline scan to Week 12; change in brain volume on
MRI scans of the brain from Week 12 to Week 96 in a subgroup of
patients receiving ocrelizumab; the total number of new and/or
enlarging T2 lesions observed on MRI scans of the brain at Weeks 4,
8, 12, 16, 20 and 24; the proportion of patients who remain free of
new gadolinium-enhancing T1 lesions by Week 24; time to first new
gadolinium-enhancing T1 lesions developing over 24 weeks; to
evaluate the treatment withdrawal effect by means of the total
number of gadolinium-enhancing T1 lesions 48 weeks after receiving
up to 4 treatment cycles of ocrelizumab in a subgroup of patients;
proportion of patients who remain free from relapses (clinical and
protocol-defined relapses) during each treatment cycle and at Weeks
48 and 96; proportion of patients requiring systemic
methylprednisolone treatment for an MS relapse during each
treatment cycle and at Weeks 48 and 96; annualized clinical and
protocol-defined relapse rate during each treatment cycle and at
Weeks 48 and 96; time to first protocol-defined relapse by Week 24;
time to first protocol-defined relapse by Week 96; time to onset of
sustained disability progression as defined by the sustained
worsening in EDSS of 1.0 point or more for 12 weeks through Week
96; time to onset of sustained disability progression as defined by
the sustained worsening in EDSS of 1.0 point or more for 24 weeks
through Week 96; to explore the effects of ocrelizumab on the
primary and secondary study endpoints vs. Avonex; to explore the
correlation of polymorphic variants in genes associated with RRMS
susceptibility and ocrelizumab activity and therapeutic response to
ocrelizumab in RRMS patients; to explore the relationship between
circulating biomarkers associated with RRMS susceptibility and
ocrelizumab activity and therapeutic response to ocrelizumab
treatment in RRMS patients; change in the Modified Fatigue Impact
Scale (MFIS) from baseline to Weeks 24 and 48; change in the
Fatigue Scale for Motor and Cognitive Functions (FSMC) from
baseline to Weeks 24 and 48; change in the proportion of patients
who moved from "severe" to "moderate" and from "moderate" to "mild"
fatigue on the FSMC, comparing baseline to Weeks 24 and 48; change
in the Center for Epidemiological Studies-Depression Scale (CES-D)
from baseline to Weeks 24 and 48; and change in the proportion of
patients who moved from a state of greater depressive
symptomatology to a state of less depressive symptomatology on the
CES-D, comparing baseline to Weeks 24 and 48.
[0390] Brain MRI
[0391] MRI is a useful tool for monitoring central nervous system
(CNS) lesions in MS. Brain MRI scans are only obtained at screening
in some patients (see Secondary Endpoints) and in all patients at
baseline and at four-week intervals between baseline and week 24.
In addition, in a subgroup of patients (Groups A and B), a brain
MRI scan is performed at weeks 96 (Visit 16) and 48 weeks later,
i.e., week 144.
[0392] The MRI includes the acquisition of the following scans at
each time point: T2-weighted MRI scan, T1-weighted MRI scan
(without gadolinium-enhancement), and T1-weighted MRI scan (with
gadolinium-enhancement).
[0393] Assessment of Disability
[0394] Disability progression as measured by EDSS is assessed in
all patients by the independent examining investigator at screening
and every 12 weeks throughout the study until the Observation
Period at which time disability progression is assessed after 24
weeks.
[0395] Disability progression is defined as an increase of
.gtoreq.1.0 point from the baseline EDSS score that is not
attributable to another etiology (e.g. fever, concurrent illness,
or concomitant medication) when the baseline score is 5.0 or less,
and .gtoreq.0.5 when the baseline score is 5.5 or more. Disease
progression is considered sustained when the increase in the EDSS
is confirmed at a regularly scheduled visit at least 12 weeks after
the initial documentation of the progression. An alternative
definition of sustained disability progression requires that the
increase in EDSS be confirmed at least 24 weeks after the initial
documentation of the progression.
[0396] The EDSS is based on a standard neurological examination;
the seven categories of the EDSS representing functional systems
(pyramidal, cerebellar, brainstem, sensory, bowel and bladder,
visual, and cerebral and/or mental, plus "other") are rated and
scored (collectively, functional system scores or FSS). Each score
of the FSS is an ordinal clinical rating scale ranging from 0 to 5
or 6. These ratings are then used in conjunction with observations
and information concerning ambulation and use of assistive devices
to determine the EDSS score. The EDSS is a disability scale that
ranges in 0.5-point steps from 0 (normal) to 10 (death).
Example 2: A Phase II/III Study of Rituximab in Primary Progressive
Multiple Sclerosis (PPMS)
[0397] A randomized, double-blind, parallel group, placebo
controlled, multicenter Phase II/III study (U2786g) to evaluate the
safety and efficacy of rituximab in adults with Primary Progressive
Multiple Sclerosis (PPMS) as defined by McDonald et al (Ann Neurol
50:121-7(2001)) was performed.
[0398] Subjects were randomized in a 2:1 ratio to receive either
rituximab or placebo. Rituximab, commercially available from
Genentech, was formulated for i.v. administration as a sterile
product in 9.0 mg/ml sodium chloride, 0.7 mg/ml polysorbate 80,
7.35 mg/ml sodium citrate dehydrate, and sterile water for
injection (pH 6.5). Each course of study drug consisted of two i.v.
infusions (separated by 14 days) of 1000 mg rituximab or placebo.
Subjects received the first course of treatment at Days 1 and 15
and received additional courses at Weeks 24, 48, and 72. Subjects
received acetaminophen (1 g) and diphenhydramine HCl (50 mg), or
equivalent, by mouth 30-60 min prior to start of each infusion.
Glucocorticoids were not administered prior to infusion. In 96
weeks of trial duration, subjects were seen at regularly scheduled
visits for physical examinations, neurologic and MRI assessments,
to collect adverse events and vital signs, and to complete routine
hematology, serum chemistries, and urinalysis lab tests.
[0399] Baseline demographics of Intent-To-Treat (ITT) subjects are
presented in Table 3.
TABLE-US-00007 TABLE 3 Demographic and Baseline Characteristics:
Intent-to-Treat Subjects. Placebo Rituximab All Subjects
Characteristic (n = 147) (n = 292) (n = 439) Age (yr) n 147 292 439
Mean (SD) 49.6 (8.69) 50.1 (9.02) 49.9 (8.90) Median 51.0 51.0 51.0
Minimum to 20-66 18-66 18-66 maximum 18-<140 20 (13.6%) 40
(13.7%) 60 (13.7%) 40-<55 80 (54.4%) 145 (49.7%) 225 (51.3%)
.gtoreq.55 47 (32.0%) 107 (36.6%) 154 (35.1%)
[0400] MS disease duration was similar in both treatment groups.
Baseline MRI results are summarized in Table 4. The baseline MRI
characteristics were similar in placebo and rituximab groups.
TABLE-US-00008 TABLE 4 Baseline MRI Results: Intent-to-Treat
Subjects. MRI Placebo Rituximab All Subjects Endpoint (N = 147) (N
= 292) (N = 439) Total 2 gadolinium- enhancing lesion count N 147
290 437 Mean (SD) 0.5 (1.26) 0.7 (2.96) 0.7 (2.52) Median 0.0 0.0
0.0 Minimum to 0-8 0-32 0-32 maximum 0 110 (74.8%) 220 (75.9%) 330
(75.5%) 1 23 (15.6%) 44 (15.2%) 67 (15.3%) 2 5 (3.4%) 10 (3.4%) 15
(3.4%) 3 5 (3.4%) 5 (1.7%) 10 (2.3%) .gtoreq.4 4 (2.7%) 11 (3.8%)
15 (3.4%) Total gadolinium- enhancing lesion volume (mm.sup.3) N
147 290 437 Mean (SD) 27.56 (81.86) 49.50 (220.32) 42.12 (185.82)
Median 0.00 0.00 0.00 Minimum to 0.00-556.40 0.00-2660.00
0.00-2660.00 maximum T2 lesion volume (mm.sup.3) N 147 290 437 Mean
(SD) 8850.86 9336.66 9173.25 (11808.95) (13744.94) (13113.98)
Median 5199.50 5240.50 5220.70 Minimum to 73.83-74534.0
174.00-155303.0 73.83-155303.0 maximum Brain volume (cc) N 130 237
367 Mean (SD) 1210.91 (128.89) 1202.92 (120.23) 1205.75 (123.25)
Median 1209.5 1204.0 1207.0 Minimum to 642.0-1522.0 712.60-1508.0
642.0-1522.0 maximum MRI = magnetic resonance imaging.
[0401] Randomization was stratified according to study site;
baseline disease severity defined by EDSS (.ltoreq.4.0, >4.0).
Baseline EDSS is summarized in Table 5. As a result of the dynamic
randomization, the percentage of subjects in each treatment group
was similar at all levels of the stratification factors.
TABLE-US-00009 TABLE 5 Baseline Stratification Factors, Disease
Severity Intent-to-Treat Subjects. Stratification Placebo Rituximab
All Subjects Factor (n = 147) (n = 292) (n = 439) EDSS N 147 292
439 2 6 (4.1%) 8 (2.7%) 14 (3.2%) 2.5 5 (3.4%) 11 (3.8%) 16 (3.6%)
3 11 (7.5%) 22 (7.5%) 33 (7.5%) 3.5 21 (14.3%) 36 (12.3%) 57
(13.0%) 4 25 (17.0%) 47 (16.1%) 72 (16.4%) 4.5 8 (5.4%) 19 (6.5%)
27 (6.2%) 5 6 (4.1%) 10 (3.4%) 16 (3.6%) 5.5 10 (6.8%) 10 (3.4%) 20
(4.6%) 6 28 (19.0%) 81 (27.7%) 109 (24.8%) 6.5 27 (18.4%) 48
(16.4%) 75 (17.1%) Mean (SD) 4.73 (1.395) 4.84 (1.369) 4.80 (1.377)
Median 4.50 5.00 5.00 Minimum to 2.0-6.5 2.0-6.5 2.0-6.5 maximum
EDSS = Expanded Disability Status Scale.
[0402] Additionally, the two treatment groups were similar in other
baseline disease severity measures: EDSS, Kurtzke Functional System
Scores, the Multiple Sclerosis Functional Composite Scale (MSFCS)
score and the MSFCS components (Timed 25-Foot Walk, 9-Hole Peg
Test, and PASAT-3).
Efficacy Results
[0403] The primary efficacy analysis for this trial compared the
time to confirmed disease progression, during the 96 week treatment
period, between rituximab and placebo. Disease progression is
defined as an increase of .gtoreq.1.0 point from baseline EDSS
(Kurtzke I Neurology 33(11):1444-52 (1983)), if the baseline EDSS
is between 2.0 and 5.5 points (inclusive), or an increase of
.gtoreq.0.5 point if the baseline EDSS is >5.5 points, for which
change is not attributable to another etiology (e.g., fever,
concurrent illness, MS relapse or exacerbation, or concomitant
medication).
[0404] Stratified analysis showed that rituximab did not
significantly delay the confirmed disease progression compared with
placebo (p=0.1442, stratified log-rank). The percentage of patients
progressing by 96 weeks was estimated to be 38.5% and 30.2% for the
placebo and rituximab groups, respectively (Table 6). Kaplan-Meier
plots for the time to confirmed disease progression are shown in
the FIG. 8.
TABLE-US-00010 TABLE 6 The Time to Confirmed Disease Progression
during Treatment Period Intent-to-Treat Subjects. Placebo Rituximab
(n = 147) (n = 292) No. of subjects who had CDP (%) 53 (36.1%) 83
(28.4%) No. of subjects who censored (%) 94 (63.9%) 209 (71.6%)
Stratified p-value Log-rank test 0.1442 Stratified hazard ratio
0.773 (rel to placebo) 95% CI for (0.546-1.093) Proportion of
subjects with CDP at week 24 6.9% 9.1% at week 48 19.3% 20.2% at
week 72 30.3% 28.0% at week 96 38.5% 30.2%
[0405] Secondary efficacy endpoints included change from baseline
to Week 96 in the total volume of T2 lesions and change from
baseline to Week 96 in the brain volume. A Hochberg-Bonferroni
procedure was used to control the type I error rate in testing
these two secondary endpoints. The change from baseline to Week 96
in the brain volume was not significantly different in the two
treatment groups (p=0.6237). See Table 7.
TABLE-US-00011 TABLE 7 Change in Brain Volume from Baseline to Week
96. Placebo Rituximab (N = 130) (N = 237) P-value Volume at
baseline (cm3) mean (SD) 1211 (129) 1203 (120) median 1209.5 1204.0
Volume change from baseline to week 96 (LOCF) mean (SD) -9.9 (37.0)
-10.8 (40.3) 0.62 median -14.0 -13.1
[0406] A significant difference was observed between the two
treatments for the change in T2 lesion volume from baseline to week
96 (p=0.0008). The median increase in volume of the T2 lesion was
809.50 mm.sup.3 and 301.95 mm.sup.3 in the placebo and rituximab
groups, respectively. (See Table 8 and FIG. 9).
TABLE-US-00012 Change from Baseline to Week 96 in the Total Volume
of T2 Lesions on Brain MRI Scans Intent-to-Treat Subjects Placebo
Rituximab (n = 147) (n = 292) p-Value Total volume of T2 lesions on
brain MRI scan (mm3) Baseline N 147 290 Mean (SD) 8850.86
(11808.95) 9336.66 (13744.94) Median 5199.50 5240.50 Range
73.83-74534.00 174.00-155303.0 SE 973.99 807.13 95% CI
(6925.93-10775.79) (7748.06-10925.26) Week 96 N 147 290 Mean (SD)
11055.55 (14536.29) 10843.80 (15827.44) Median 5526.60 5569.35
Range 94.92-86232.00 179.30-170464.0 SE 1198.93 929.42 95% CI
(8686.04-13425.05) (9014.51-12673.09) Change from baseline to week
96 N 147 290 Mean (SD) 2204.69 (4306.24) 1507.14 (3739.45) Median
809.50 301.95 Range -8557.00-26367.00 -4031.00-24076.00 SE 355.17
219.59 95% CI (1502.74-2906.63) (1074.94-1939.33) Treatment -718.24
difference in LS means (vs. placebo) 95% CI of the (-1504.48,
68.00) difference in LS means ANOVA t-test 0.0733 (stratified)
Friedman ranked 0.0008 ANOVA test ANOVA t-test 0.0006 (stratified)
on percent change from baseline to week 96 Friedman ranked 0.0005
ANOVA test on percent change from baseline to week 96
[0407] Analyses of all exploratory endpoints except the change in
T2 lesion volume, enlarging T2 lesion and new T2 lesion showed
statistically non-significant differences between placebo and
rituximab arms. Compared to placebo, the rituximab group
experienced significantly less increase in T2 lesion volume at Week
48 and 122 (p=0.0051 and 0.0222, respectively); had less new T2
lesion at week 48 and 96 (p<0.001); had less enlarging T2 lesion
count at week 48 and 96 (p=0.008 and 0.072, respectively).
Subgroup Analysis
[0408] Subgroup analysis for the primary endpoints included time to
confirmed disease progression according to the following
demographic and baseline disease characteristics: sites, age,
gender, race, prior MS therapies, baseline EDSS, duration since MS
symptom onset and baseline gadolinium (Gd) lesion, and baseline
Multiple Sclerosis Severity Score (MSSS) (an index of how fast the
patient progressed; see Roxburgh et al. Neurology 64; 1144-1151
(2005)).
[0409] The subgroup analysis results suggest a potential treatment
effect in patients who are younger, progressed more rapidly (higher
MSSS) or with Gd lesions at baseline (FIG. 10). Moreover, additive
predictive effects of age, Gd lesion at baseline and MSSS for the
treatment effect have been verified using multivariate analysis
method (FIG. 11 and FIG. 12). See also Table 9. Based on these
findings with MSSS, a subgroup of the study population excluding
older patients with longstanding disease and slow progression was
selected using modified inclusion/exclusion criteria (age
.ltoreq.55, 3.ltoreq.baseline EDSS.ltoreq.6.5, excluding patients
with disease duration >10 if their baseline EDSS<5 or disease
duration >15 if their baseline EDSS.gtoreq.5). A significant
treatment effect was also shown for this subgroup (stratified
log-rank test P-value=0.01; FIG. 13).
TABLE-US-00013 TABLE 9 Time to Confirmed Disease Progression
Subgroup Result Summary. Total CDP@wk 96 CDP@wk 96 P-value
Subgroups N Placebo Rituximab HR HR 95% CI (Log-rank) All patients
439 38.5% 30.2% n/a n/a 0.1442 (Primary analysis) Age <51 215
44.9% 27.5% 0.52 (0.32, 0.86) 0.0101 Gd+ 107 52.8% 27.4% 0.41
(0.21, 0.80) 0.0069 Gd+ and age <51 72 51.6% 24.6% 0.33 (0.14,
0.79) 0.0088 Gd+ and age <55 93 49.5% 29.1% 0.40 (0.19, 0.84)
0.0126 MSSS >= 5 and 251 46.8% 29.6% 0.59 (0.38, 0.91) .0163 age
<55
[0410] Subgroup analyses suggest that PPMS patients with evidence
of active disease show significant clinical signs of treatment
related benefit as measured by time to confirmed disease
progression, as well as change from baseline in EDSS, MSFC, and T2
lesions on brain MRI (data not shown). Independent factors that
appeared prognostic of disease progression in the placebo group and
potentially predictive of treatment response in the rituximab group
included the following: younger age, particularly age less than 51;
presence of contrast enhancing lesions at baseline on brain MRI;
and higher MS severity score. These observations serve a hypothesis
generating role supporting a potential therapeutic benefit of
B-cell depletion on confirmed disease progression in appropriately
selected progressive onset MS patients.
[0411] While this study failed to demonstrate primary efficacy in
the overall PPMS population, subgroup efficacy analyses indicated
that patients with contrast enhancing brain MRI lesions at baseline
potentially responded to treatment with rituximab, with a 57%
relative reduction in the hazard (1-HR) of confirmed disease
progression in the treated group versus placebo, which is largely
but not entirely driven by a very high placebo Confirmed Disease
Progression (CDP) rate of 52.8% at 96 weeks (FIG. 10). PPMS
patients aged less than 51 may also have benefited, with a 43%
relative reduction in the hazard of confirmed disease progression
and a placebo progression rate of 44.9%. While the presence of
contrast enhancing lesions and age <51 were correlated, a
post-hoc analysis of the 72 patients exhibiting both
characteristics revealed a more pronounced apparent effect, with a
77% relative reduction in the hazard of confirmed disease
progression (Table 9). In this subgroup the placebo progression
rate of 51.6% was not higher than the rate for all patients with
enhancing MRI lesions at baseline, but a lower rate of progression
in the rituximab group (24.6%) accounts for the potentially greater
risk reduction with treatment. These OLYMPUS placebo data
corroborate natural history observations on the clinical and MRI
heterogeneity of PPMS patients (Sastre-Garriga et al. Neurology
65(4):633-5 (2005), Ingle et al. Brain 126(Pt 11):2528-36 (2003),
Tremlett et al. Mult Scler. 14(3):314-24 (2008), Tremlett et al.
Neurology 65(12):1919-23 (2005), Kremenchutzky et al. Brain 129(Pt
3):584-94. (2006)). Furthermore, the MAGNIMS clinical and MRI
cohort study described a subset of PPMS patients with more
inflammatory MRI activity early in the disease course and a worse
prognosis for disability progression (Ingle et al. J. Neurol
Neurosurg Psychiatry 76(9):1255-8 (2005)); the OLYMPUS placebo data
appear to confirm these observations for the first time.
Example 3: A Phase III Study of Ocrelizumab in Progressive Multiple
Sclerosis
[0412] A Phase III, randomized, double-blind, parallel-group,
multicenter study to evaluate the safety and efficacy of 600 mg of
ocrelizumab as compared with placebo in adults with progressive MS
is performed.
[0413] A total of 630 progressive MS patients (315 with progressive
onset and 315 with relapsing onset MS) are enrolled and assigned
(2:1 randomization) to either ocrelizumab arm or placebo arm
stratified by site and type of multiple sclerosis. This study
consists of the following three periods that apply to all patients:
a screening period, a treatment period and a treatment free follow
up period. In the first course of study drug treatment (300 mg
ocrelizumab or placebo infusion.times.2) are administered on Days 1
and 15. In subsequent treatment courses, patients are dosed (600 mg
ocrelizumab single infusion) every 24 weeks until the last patient
enrolled receives his/her last course of treatment to be
administered at Week 96.
[0414] Prior to each study drug infusion, patients receive
treatment with an analgesic/antipyretic such as
acetaminophen/paracetamol (1 grams) and an i.v. or oral
antihistamine (such as diphenhydramine 50 mg), and 100 mg
methylprednisolone intravenously, or equivalents, to reduce the
incidence of potential infusion reactions. In patients with Common
Terminology for Adverse Events (CTCAE) Grade 3 or higher (severe)
infusion reactions with associated respiratory symptoms (stridor,
wheeze or bronchospasm), additional treatment with bronchodilators
may be indicated.
[0415] Routine laboratory studies are obtained throughout the
study, with additional tests following courses of study drug
treatment. Immune panel, serum human anti-human antibody (HAHA),
and thyroid tests are also conducted. Serum samples of all patients
are collected for pharmacokinetic analysis and blood samples are
collected for B-cell count determination. B-cell counts are
followed as a pharmacodynamic marker of ocrelizumab.
Patient Population and Selection Criteria
[0416] The target population for this study includes patients with
progressive MS with or without a history of superimposed relapses.
Patients with progressive MS eligible for this study are
characterized by a diagnosis in accordance with the revised
McDonald criteria (2005) and a period of 6 months or greater of
documented irreversible loss of neurological function in the
absence of relapses. Patients are selected with evidence of active
disease and higher risk for more rapid disability progression,
using criteria identified as potential risk factors in previous
clinical trials with progressive MS patients. These factors include
younger age, evidence of inflammation in the cerebrospinal fluid
(CSF) (oligoclonal bands or elevated IgG index), contrast enhancing
lesions on brain MRI, high relapse activity superimposed on
non-relapse related progression, and more rapid historical
accumulation of disability.
[0417] All patients volunteering and eligible for participation in
the study are screened for conformance with the following inclusion
and exclusion criteria:
[0418] Inclusion Criteria include:
1. Diagnosis of Multiple Sclerosis in accordance with the revised
McDonald criteria (2005). 2. Progressive MS, characterized by
documented, irreversible loss of neurological function persisting
for .gtoreq.6 months that cannot be attributed to clinical relapse.
3. Ages 18-55 years inclusive. 4. EDSS at screening from 3.0 to 6.5
points. 5. Score of .gtoreq.2.0 on the Functional Systems (FS)
scale for the pyramidal system or gait that is due to lower
extremity findings. 6. Presence of at least one of the following
laboratory findings in a CSF specimen obtained during the screening
period or documented within the previous 6 months as indicated by,
for example, elevated IgG index and/or IgG oligoclonal bands
detected by isoelectric focusing. 7. Presence of at least one of
the following criteria: [0419] Age <50 [0420] Gd+ lesions on
brain MRI at screening or within 6 months of screening [0421] At
least 1.5 point increase in EDSS over past 2 yrs not attributable
to relapse [0422] Two relapses in past two years
[0423] Exclusion criteria include:
1. Relapsing remitting multiple sclerosis at screening (Visit 1) 2.
Disease duration from the onset of MS symptoms: more than 15 years
in patients with an EDSS at screening >5.0 or more than 10 years
in patients with an EDSS at screening <5.0.
Efficacy Analysis
[0424] The primary efficacy endpoint is the time to confirmed
disease progression. Disease progression is defined as an increase
of .gtoreq.1.0 point from baseline EDSS, if the baseline EDSS is
between 2.0 and 5.5 points (inclusive), or an increase of
.gtoreq.0.5 points, if the baseline EDSS is >5.5 points, for
which change is not attributable to another etiology (e.g., fever,
concurrent illness, MS relapse or exacerbation, or concomitant
medication).
[0425] The EDSS is based on a standard neurological examination;
the seven categories of the EDSS representing functional systems
(pyramidal, cerebellar, brainstem, sensory, bowel and bladder,
visual, and cerebral and/or mental, plus "other") are rated and
scored (collectively, functional system scores or FSS). Each score
of the FSS is an ordinal clinical rating scale ranging from 0 to 5
or 6. These ratings are then used in conjunction with observations
and information concerning ambulation and use of assistive devices
to determine the EDSS score. The EDSS is a disability scale that
ranges in 0.5-point steps from 0 (normal) to 10 (death).
[0426] The secondary efficacy endpoints in support of the primary
efficacy endpoint include: change from baseline to Week 120 in the
total volume of T2 lesions on brain MRI scan, change from baseline
to Week 120 in the 25-foot timed walk, time to confirmed disease
progression, with confirmation occurring at least 24 weeks
(.gtoreq.168 days) after initial disease progression.
[0427] Assessment of Relapse
[0428] Patients are evaluated for relapses by the treating
investigator at each visit throughout the study and, if necessary,
at unscheduled visits to confirm relapses occurring between the
visits. To meet the criteria for a protocol-defined relapse, the
relapse is defined as the occurrence of new or worsening
neurological symptoms attributable to MS and immediately preceded
by a relatively stable or improving neurological state of least 30
days. Symptoms must persist for >24 hours and should not be
attributable to confounding clinical factors (e.g., fever,
infection, injury, adverse reactions to concomitant medications).
The new or worsening neurological symptoms must be accompanied by
objective neurological worsening consistent with an increase of at
least half a step on the EDSS, or 2 points on one of the
appropriate FSS, or 1 point on two or more of the appropriate FSS.
The change must affect the selected FSS (i.e., pyramidal,
ambulation, cerebellar, brainstem, sensory, or visual). Episodic
spasms, sexual dysfunction, fatigue, mood change or bladder or
bowel urgency or incontinence do not suffice to establish a
relapse.
[0429] Brain MRI Imaging
[0430] Magnetic resonance imaging of the brain and cervical spinal
cord are obtained at multiple time points during this study,
including at baseline. The brain MRI includes the acquisition of
the following scans at each time point: T2-weighted MRI scan and
T1-weighted MRI scan (without gadolinium-enhancement).
[0431] Change from baseline to Week 120 in the total volume of T2
lesions and timed 25 foot walk are compared between ocrelizumab and
placebo using ranked analysis of variance. The model includes the
two stratification factors noted in the primary analysis.
Example 4: A Phase III Study of Ocrelizumab in Primary Progressive
Multiple Sclerosis
[0432] A Phase III, randomized, double-blind, parallel-group,
multicenter study to evaluate the safety and efficacy of one of two
dose regimens of ocrelizumab as compared with placebo in adults
with primary progressive MS is performed.
The two ocrelizumab dose regimens under investigation are as
follows: 1) ocrelizumab 1000 mg dose regimen: consisting of a dual
infusion of 1000 mg for the first treatment cycle followed by
single infusions of 1000 mg for the subsequent treatment cycles and
2) ocrelizumab 600 mg dose regimen: consisting of a dual infusion
of 300 mg for the first treatment cycle followed by single
infusions of 600 mg for the subsequent treatment cycles.
[0433] A total of 630 primary progressive MS patients are enrolled
and assigned (2:1 randomization) to either ocrelizumab arm or
placebo arm stratified by site and type of multiple sclerosis. This
study consists of the following three periods that apply to all
patients: a screening period, a treatment period and a safety
follow up period. In the first course of study drug treatment
(ocrelizumab or placebo infusion x 2) are administered on Days 1
and 15. In subsequent treatment courses, patients are dosed
(ocrelizumab or placebo single infusion) every 24 weeks until the
last patient enrolled receives his/her last course of treatment to
be administered at Week 96.
[0434] Prior to each study drug infusion, patients receive
treatment with an analgesic/antipyretic such as
acetaminophen/paracetamol (1 grams) and an i.v. or oral
antihistamine (such as diphenhydramine 50 mg), and 100 mg
methylprednisolone intravenously, or equivalents, to reduce the
incidence of potential infusion reactions. In patients with Common
Terminology for Adverse Events (CTCAE) Grade 3 or higher (severe)
infusion reactions with associated respiratory symptoms (stridor,
wheeze or bronchospasm), additional treatment with bronchodilators
may be indicated.
[0435] Routine laboratory studies are obtained throughout the
study, with additional tests following courses of study drug
treatment. Immune panel, serum human anti-human antibody (HAHA),
and thyroid tests are also conducted. Serum samples of all patients
are collected for pharmacokinetic analysis and blood samples are
collected for B-cell count determination. B-cell counts are
followed as a pharmacodynamic marker of ocrelizumab.
Patient Population and Selection Criteria
[0436] The target population for this study includes patients with
primary progressive MS. Patients with primary progressive MS
eligible for this study are characterized by a diagnosis in
accordance with the revised McDonald criteria (2005). Patients are
selected with evidence of active disease and higher risk for more
rapid disability progression, using criteria identified as
potential risk factors in previous clinical trials with progressive
MS patients. These factors include younger age, evidence of
inflammation in the cerebrospinal fluid (CSF) (oligoclonal bands or
elevated IgG index), and more rapid historical accumulation of
disability.
[0437] All patients volunteering and eligible for participation in
the study are screened for conformance with the following inclusion
and exclusion criteria:
[0438] Inclusion Criteria include:
1. Diagnosis of Primary Progressive Multiple Sclerosis in
accordance with the revised McDonald criteria (2005). 2. Ages 18-55
years inclusive. 3. EDSS at screening from 3.0 to 6.5 points. 4.
Score of .gtoreq.2.0 on the Functional Systems (FS) scale for the
pyramidal system that is due to lower extremity findings. 5.
Documented history or presence at screening of at least one of the
following laboratory findings in a CSF specimen as indicated by,
elevated IgG index and/or IgG oligoclonal bands detected by
isoelectric focusing. 6. Disease duration from the onset of MS
symptoms: less than 15 years in patients with an EDSS at screening
>5.0 or less than 10 years in patients with an EDSS at screening
.ltoreq.5.0.
[0439] Exclusion criteria include:
1. History of relapsing remitting, secondary progressive, or
progressive relapsing multiple sclerosis at screening (Visit
1).
Efficacy Analysis
[0440] The primary efficacy endpoint is the time to confirmed
disease progression. Disease progression is defined as an increase
of .gtoreq.1.0 point from baseline EDSS, if the baseline EDSS is
between 2.0 and 5.5 points (inclusive), or an increase of
.gtoreq.0.5 points, if the baseline EDSS is >5.5 points, for
which change is not attributable to another etiology (e.g., fever,
concurrent illness, MS relapse or exacerbation, or concomitant
medication).
[0441] The EDSS is based on a standard neurological examination;
the seven categories of the EDSS representing functional systems
(pyramidal, cerebellar, brainstem, sensory, bowel and bladder,
visual, and cerebral and/or mental, plus "other") are rated and
scored (collectively, functional system scores or FSS). Each score
of the FSS is an ordinal clinical rating scale ranging from 0 to 5
or 6. These ratings are then used in conjunction with observations
and information concerning ambulation and use of assistive devices
to determine the EDSS score. The EDSS is a disability scale that
ranges in 0.5-point steps from 0 (normal) to 10 (death).
[0442] The secondary efficacy endpoints in support of the primary
efficacy endpoint include: change from baseline to Week 120 in the
total volume of T2 lesions on brain MRI scan, change from baseline
to Week 120 in the 25-foot timed walk, time to confirmed disease
progression, with confirmation occurring at least 24 weeks
(.gtoreq.168 days) after initial disease progression.
[0443] Assessment of Relapse
[0444] Patients are evaluated for relapses by the treating
investigator at each visit throughout the study and, if necessary,
at unscheduled visits to confirm relapses occurring between the
visits. To meet the criteria for a protocol-defined relapse, the
relapse is defined as the occurrence of new or worsening
neurological symptoms attributable to MS and immediately preceded
by a relatively stable or improving neurological state of least 30
days. Symptoms must persist for >24 hours and should not be
attributable to confounding clinical factors (e.g., fever,
infection, injury, adverse reactions to concomitant medications).
The new or worsening neurological symptoms must be accompanied by
objective neurological worsening consistent with an increase of at
least half a step on the EDSS, or 2 points on one of the
appropriate FSS, or 1 point on two or more of the appropriate FSS.
The change must affect the selected FSS (i.e., pyramidal,
ambulation, cerebellar, brainstem, sensory, or visual). Episodic
spasms, sexual dysfunction, fatigue, mood change or bladder or
bowel urgency or incontinence do not suffice to establish a
relapse.
[0445] Brain MRI Imaging
[0446] Magnetic resonance imaging of the brain and cervical spinal
cord are obtained at multiple time points during this study,
including at baseline. The brain MRI includes the acquisition of
the following scans at each time point: T2-weighted MRI scan and
T1-weighted MRI scan (without gadolinium-enhancement).
[0447] Change from baseline to Week 120 in the total volume of T2
lesions and timed 25 foot walk are compared between ocrelizumab and
placebo using ranked analysis of variance. The model includes the
two stratification factors noted in the primary analysis.
Sequence CWU 1
1
251107PRTMus musculus 1Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu
Ser Ala Ser Pro1 5 10 15Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
Ser Ser Val Ser 20 25 30Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser
Ser Pro Lys Pro 35 40 45Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly
Val Pro Ala Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser65 70 75Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp 80 85 90Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu 95 100 105Lys Arg2107PRTArtificial
sequenceSynthetic construct 2Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Ser Ser Val Ser 20 25 30Tyr Met His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Pro 35 40 45Leu Ile Tyr Ala Pro Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Trp 80 85 90Ser Phe Asn Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 95 100 105Lys Arg3108PRTArtificial
SequenceSynthetic construct 3Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser 20 25 30Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ala Ala Ser Ser Leu
Glu Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile65 70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Asn Ser Leu Pro Trp Thr Phe Gly
Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg410PRTMus musculus
4Arg Ala Ser Ser Ser Val Ser Tyr Met His1 5 1057PRTMus musculus
5Ala Pro Ser Asn Leu Ala Ser1 569PRTMus musculus 6Gln Gln Trp Ser
Phe Asn Pro Pro Thr1 57122PRTMus musculus 7Gln Ala Tyr Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly1 5 10 15Ala Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser Tyr Asn Met His Trp
Val Lys Gln Thr Pro Arg Gln Gly Leu 35 40 45Glu Trp Ile Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr 50 55 60Asn Gln Lys Phe Lys Gly
Lys Ala Thr Leu Thr Val Asp Lys Ser65 70 75Ser Ser Thr Ala Tyr Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp 80 85 90Ser Ala Val Tyr Phe Cys
Ala Arg Val Val Tyr Tyr Ser Asn Ser 95 100 105Tyr Trp Tyr Phe Asp
Val Trp Gly Thr Gly Thr Thr Val Thr Val 110 115 120Ser
Ser8122PRTArtificial sequenceSynthetic construct 8Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser Tyr Asn Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr 50 55 60Asn Gln Lys Phe
Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser 95 100 105Tyr Trp Tyr
Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120Ser
Ser9119PRTArtificial SequenceSynthetic construct 9Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Ala
Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr 50 55 60Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser65 70 75Lys Asn Thr Leu
Thr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu 95 100 105Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 1151010PRTMus
musculus 10Gly Tyr Thr Phe Thr Ser Tyr Asn Met His1 5 101117PRTMus
musculus 11Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys
Phe1 5 10 15Lys Gly1213PRTMus musculus 12Val Val Tyr Tyr Ser Asn
Ser Tyr Trp Tyr Phe Asp Val1 5 1013213PRTArtificial
sequenceSynthetic construct 13Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Ser Ser Val Ser 20 25 30Tyr Met His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Pro 35 40 45Leu Ile Tyr Ala Pro Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Trp 80 85 90Ser Phe Asn Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 95 100 105Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser 110 115 120Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu 125 130 135Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp140 145 150Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 155 160 165Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 170 175 180Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 185 190 195Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 200 205 210Gly
Glu Cys14452PRTArtificial sequenceSynthetic construct 14Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser Tyr Asn
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr 50 55 60Asn Gln Lys
Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser 95 100 105Tyr Trp
Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 125 130
135Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu140
145 150Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
155 160 165Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln 170 175 180Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 185 190 195Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 200 205 210Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys215 220 225Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln290 295 300Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 320 325 330Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 335 340 345Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 350 355 360Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys365 370
375Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 380
385 390Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
395 400 405Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 410 415 420Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 425 430 435Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro440 445 450Gly Lys15452PRTArtificial sequenceSynthetic
construct 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe
Thr 20 25 30Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 35 40 45Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser
Tyr 50 55 60Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys
Ser65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn
Ser 95 100 105Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val
Thr Val 110 115 120Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 125 130 135Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu140 145 150Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser 155 160 165Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln 170 175 180Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 185 190 195Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys 200 205 210Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys215 220 225Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 230 235 240Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln290 295
300Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305
310 315Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
320 325 330Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala
Lys 335 340 345Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg 350 355 360Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys365 370 375Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly 380 385 390Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 395 400 405Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser 410 415 420Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 425 430 435Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro440 445 450Gly Lys16213PRTArtificial
sequenceSynthetic construct 16Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Ser Ser Val Ser 20 25 30Tyr Leu His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Pro 35 40 45Leu Ile Tyr Ala Pro Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Trp 80 85 90Ala Phe Asn Pro Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 95 100 105Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser 110 115 120Asp Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu 125 130 135Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp140 145 150Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 155 160 165Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 170 175 180Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 185 190 195Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 200 205 210Gly
Glu Cys17452PRTArtificial sequenceSynthetic construct 17Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser Tyr Asn
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val
Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr 50 55 60Asn Gln Lys
Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg 95 100 105Tyr Trp
Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 125 130
135Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu140
145 150Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
155 160 165Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln 170 175 180Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 185 190 195Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 200 205 210Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys215 220 225Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln290 295 300Tyr Asn Ala Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 320 325 330Ala Ala Leu
Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys 335
340 345Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
350 355 360Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys365 370 375Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 380 385 390Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 395 400 405Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 410 415 420Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 425 430 435Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro440 445 450Gly Lys1810PRTArtificial
sequenceSynthetic constructVariant9Xaa is M or L 18Arg Ala Ser Ser
Ser Val Ser Tyr Xaa His1 5 10199PRTArtificial sequenceSynthetic
constructVariant4Xaa is S or A 19Gln Gln Trp Xaa Phe Asn Pro Pro
Thr1 52017PRTArtificial sequenceSynthetic constructVariant8Xaa is D
or A 20Ala Ile Tyr Pro Gly Asn Gly Xaa Thr Ser Tyr Asn Gln Lys Phe1
5 10 15Lys Gly2113PRTArtificial sequenceSynthetic
constructVariant6Xaa is N, A, Y, W or DVariant7Xaa is S or R 21Val
Val Tyr Tyr Ser Xaa Xaa Tyr Trp Tyr Phe Asp Val1 5
1022451PRTArtificial sequenceSynthetic construct 22Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser Tyr Asn Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr 50 55 60Asn Gln Lys Phe
Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser 95 100 105Tyr Trp Tyr
Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110 115 120Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 125 130 135Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu140 145
150Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 155
160 165Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
170 175 180Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 185 190 195Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 200 205 210Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys215 220 225Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln290 295 300Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His 305 310 315Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 320 325 330Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 335 340 345Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 350 355 360Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys365 370 375Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 380 385
390Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 395
400 405Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
410 415 420Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 425 430 435Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro440 445 450Gly23107PRTArtificial sequenceSynthetic construct
23Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10
15Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser 20 25
30Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro 35 40
45Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg 50 55
60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70
75Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp 80 85
90Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 95
100 105Lys Arg24122PRTArtificial sequenceSynthetic construct 24Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser
Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu
Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr 50 55 60Asn
Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr
Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg 95 100
105Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110
115 120Ser Ser25451PRTArtificial sequenceSynthetic construct 25Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Ser
Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu
Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr 50 55 60Asn
Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser65 70 75Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr
Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg 95 100
105Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val 110
115 120Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
125 130 135Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu140 145 150Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser 155 160 165Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln 170 175 180Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 185 190 195Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 200 205 210Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys215 220 225Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln290 295 300Tyr Asn Ala
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 320 325 330Ala
Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys 335 340
345Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 350
355 360Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys365 370 375Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 380 385 390Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 395 400 405Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 410 415 420Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 425 430 435Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro440 445 450Gly
* * * * *