U.S. patent application number 15/234575 was filed with the patent office on 2017-07-06 for method for the treatment of multiple sclerosis.
The applicant listed for this patent is The Government of the United States of America as Represented by the Secretary of the Department of. Invention is credited to Bibiana Bielekova, Roland MARTIN, Henry McFarland.
Application Number | 20170190779 15/234575 |
Document ID | / |
Family ID | 30000962 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170190779 |
Kind Code |
A1 |
MARTIN; Roland ; et
al. |
July 6, 2017 |
METHOD FOR THE TREATMENT OF MULTIPLE SCLEROSIS
Abstract
A method for treating a subject with multiple sclerosis is
disclosed herein. In one embodiment, a method is provided for
treating a subject with multiple sclerosis that includes
administering to the subject a therapeutically effective amount of
an IL-21 receptor antagonist, wherein the subject has failed to
respond treatment with beta interferon, thereby treating the
subject.
Inventors: |
MARTIN; Roland; (Bethesda,
MD) ; McFarland; Henry; (Gaithersburg, MD) ;
Bielekova; Bibiana; (Kensington, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America as Represented by
the Secretary of the Department of |
Rockville |
MD |
US |
|
|
Family ID: |
30000962 |
Appl. No.: |
15/234575 |
Filed: |
August 11, 2016 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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14792432 |
Jul 6, 2015 |
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15234575 |
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14487012 |
Sep 15, 2014 |
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14792432 |
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13475688 |
May 18, 2012 |
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14487012 |
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11827876 |
Jul 13, 2007 |
8454965 |
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13475688 |
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10607598 |
Jun 27, 2003 |
7258859 |
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11827876 |
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PCT/US02/38290 |
Nov 27, 2002 |
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10607598 |
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60393021 |
Jun 28, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 5/4848 20130101;
A61K 2039/545 20130101; C07K 2317/34 20130101; C07K 16/2866
20130101; A61K 31/7052 20130101; A61K 2039/505 20130101; A61B 5/055
20130101; A61P 25/28 20180101; A61P 37/02 20180101; C07K 2317/76
20130101; A61P 37/00 20180101; C07K 2317/24 20130101; A61P 43/00
20180101; A61B 5/0042 20130101; A61K 39/39541 20130101; A61K 49/06
20130101; A61K 2039/54 20130101; A61P 37/06 20180101; A61P 25/00
20180101; A61K 38/215 20130101; A61P 21/00 20180101; A61K 39/3955
20130101; A61K 38/215 20130101; A61K 2300/00 20130101; A61K
39/39541 20130101; A61K 2300/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61B 5/055 20060101 A61B005/055; A61B 5/00 20060101
A61B005/00 |
Claims
1. A method for treating a subject with multiple sclerosis,
comprising administering to the subject a therapeutically effective
amount of an IL-2 receptor antagonist in the absence of treatment
with beta interferon, wherein the subject has failed to respond to
previous treatment with beta interferon, thereby ameliorating a
sign or symptom of multiple sclerosis and treating the subject.
2. The method of claim 1, wherein the IL-2 receptor antagonist is
administered intravenously.
3. The method of claim 2, wherein the IL-2 antagonist comprises an
antibody that specifically binds the IL-2 receptor.
4. The method of claim 3, wherein the antibody is a humanized
monoclonal antibody.
5. The method of claim 4, wherein the antibody specifically binds
p55.
6. The method of claim 4, wherein the antibody is administered at a
dose of about 1 to about 3 milligrams per kilogram
intravenously.
7. The method of claim 4, wherein the antibody is administered at a
dose of about 1 per kilogram to about 2 milligrams per kilogram
intravenously.
8. The method of claim 3, wherein the antibody is administered
biweekly.
9. The method of claim 1, wherein treatment of the subject results
in a decreased number of contrast enhancing-lesions as evaluated by
Magnetic Resonance Imaging.
10. The method of claim 1, wherein the treatment with beta
interferon comprises treatment with interferon-beta 1a.
11. The method of claim 1, wherein the treatment with beta
interferon comprises treatment with interferon-beta 1b.
12. The method of claim 1, wherein the subject has
relapsing-remitting multiple sclerosis.
13. The method of claim 1, wherein the subject has progressive
multiple sclerosis.
14. A method for treating a subject with multiple sclerosis,
comprising administering to the subject intravenously a
therapeutically effective amount of a humanized monoclonal antibody
that specifically binds the interleukin-2 receptor, and wherein the
humanized monoclonal antibody is administered at least biweekly for
a period of at least two months, thereby treating the subject.
15. The method of claim 14, wherein the subject is not treated with
interferon-.beta..
16. The method of claim 14, wherein the antibody is administered at
a dose of about 1 to about 3 milligrams per kilogram.
17. The method of claim 14, wherein the antibody is administered at
a dose of about 1 per kilogram to about 2 milligrams per
kilogram.
18. The method of claim 14, wherein the humanized monoclonal
antibody specifically binds p55.
19. The method of claim 15, wherein the subject has
relapsing-remitting multiple sclerosis.
20. A method for identifying a subject responsive to treatment with
an IL-2 receptor antagonist, comprising selecting a subject that
has multiple sclerosis that has not responded to treatment with
interferon-beta, thereby identifying the subject responsive to
treatment with the IL-2 receptor antagonist.
21. The method of claim 20, wherein the subject has
relapsing-remitting multiple sclerosis.
22. The method of claim 20, wherein the IL-2 receptor antagonist
comprises an antibody that specifically binds p55.
23. The method of claim 20, wherein antibody is a monoclonal
antibody.
24. The method of claim 20, wherein the monoclonal antibody is a
humanized monoclonal antibody.
25. The method of claim 20, wherein the interferon-beta comprises
interferon-beta 1a.
26. The method of claim 20, wherein the interferon comprises
interferon-beta 1b.
27. A method for treating a subject with multiple sclerosis,
comprising selecting a subject who has been treated with
interferon-beta and failed to respond to the interferon-beta
treatment; administering to the subject intravenously a
therapeutically effective amount of a humanized monoclonal antibody
that specifically binds the interleukin-2 receptor, wherein the
subject is not treated with interferon-.beta., thereby treating the
subject.
28. The method of claim 27, wherein the humanized monoclonal
antibody is administered at least biweekly for a period of at least
two months.
Description
PRIORITY CLAIM
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/607,598, filed on Jun. 27, 2003, which claims priority
under 35 U.S.C. 365(c) to PCT Application No. PCT/US02/38290, filed
Nov. 27, 2002, which claims the benefit of U.S. Provisional
Application No. 60/393,021, filed Jun. 28, 2002. The prior
applications all are incorporated by reference in their
entirety.
FIELD
[0002] This disclosure relates to the treatment of autoimmune
diseases, specifically to the treatment of multiple sclerosis using
an antagonist of the IL-2 receptor, such as an antibody that binds
the IL-2 receptor (IL-2R).
BACKGROUND
[0003] Multiple sclerosis (MS) is a chronic, neurological,
autoimmune, demyelinating disease. MS can cause blurred vision,
unilateral vision loss (optic neuritis), loss of balance, poor
coordination, slurred speech, tremors, numbness, extreme fatigue,
changes in intellectual function (such as memory and
concentration), muscular weakness, paresthesias, and blindness.
Many subjects develop chronic progressive disabilities, but long
periods of clinical stability may interrupt periods of
deterioration. Neurological deficits may be permanent or
evanescent. In the United States there are about 250,000 to 400,000
persons with MS, and every week about 200 new cases are diagnosed.
Worldwide, MS may affect 2.5 million individuals. Because it is not
contagious, which would require U.S. physicians to report new
cases, and because symptoms can be difficult to detect, the
incidence of disease is only estimated and the actual number of
persons with MS could be much higher.
[0004] The pathology of MS is characterized by an abnormal immune
response directed against the central nervous system. In
particular, T-lymphocytes are activated against the myelin sheath
of the neurons of the central nervous system causing demyelination.
In the demyelination process, myelin is destroyed and replaced by
scars of hardened "sclerotic" tissue which is known as plaque.
These lesions appear in scattered locations throughout the brain,
optic nerve, and spinal cord. Demyelination interferes with
conduction of nerve impulses, which produces the symptoms of
multiple sclerosis. Most subjects recover clinically from
individual bouts of demyelination, producing the classic remitting
and exacerbating course of the most common form of the disease
known as relapsing-remitting multiple sclerosis.
[0005] MS develops in genetically predisposed individuals and is
most likely triggered by environmental agents such as viruses
(Martin et al., Ann. Rev. Immunol. 10:153-187, 1992). According to
current hypotheses, activated autoreactive CD4+ T helper cells (Th1
cells) which preferentially secrete interferon-gamma (IFN-.gamma.)
and tumor necrosis factors alpha/beta (TNF-.alpha./.beta.), induce
inflammation and demyelination in MS (Martin et al., supra).
Available data suggest that the predisposition to mount a Th1-like
response to a number of different antigens is an important aspect
of MS disease pathogenesis. Proinflammatory cytokines (such as
IFN-.gamma., TNF-.alpha./.beta.) and chemokines secreted by Th1
cells contribute to many aspects of lesion development including
opening of the blood-brain-barrier, recruitment of other
inflammatory cells, activation of resident glia (micro- and
astroglia) and the effector phase of myelin damage via nitrogen and
oxygen radicals secreted by activated macrophages (Wekerle et al.,
Trends Neuro Sci. 9:271-277, 1986) (Martin et al., supra).
[0006] The peripheral activation of autoreactive lymphocytes via
molecular mimicry (Wucherpfennig and Strominger, Cell. 80:695-705,
1995; Gran et al., Ann. Neurol. 45:559-567, 1999) is a critical
prerequisite for T cell migration into the CNS compartment
(Calabresi et al., Ann. Neurol. 41:669-674, 1998). Only activated T
cells expressing the necessary adhesion molecules are able to
migrate across the blood-brain-barrier. It has been hypothesized
that T lymphocytes in MS patients as well as in models for MS such
as experimental allergic encephalomyelitis (EAE; in particular in
SJL mice, see Encinas et al. Nature Genet. 21:158-160, 1999) differ
from non-susceptible individuals by being in a different state of
activation (Calabresi ct al., supra), as the cells enter the
cell-cycle more readily, stay longer in growth phase, may exhibit
defects in apoptosis pathways (Zipp et al., Ann. Neurol.
43:116-120, 1998), or are in vivo activated as indicated by higher
mutation rates in the hypoxanthine-phosphoribosyl transferase gene
in myelin-specific T cells (Allegretta et al., Science.
247:718-721, 1990).
[0007] The status of MS patients can be evaluated by longitudinal,
monthly follow-up of magnetic resonance (MRI) activity in the brain
of MS patients. MRI offers a unique set of outcome measures for
phase I/II clinical trials in small cohorts of patients, and is
thus well suited to establish data for proof of principle for novel
therapeutic strategies (e.g., see Harris et al., Ann. Neurol.
29:548-555, 1991; MacFarland et al., Ann. Neurol. 32:758-766, 1992;
Stone et al., Ann. Neurol. 37:611-619, 1995). There are currently
four approved treatments for relapsing-remitting MS, three types of
IFN-.beta. (the Interferon-B multiple sclerosis study group,
Neurology. 43:655-661, 1993; the IFNB Multiple Sclerosis Study
Group and the University of British Columbia MS/MRI Analysis Group,
Neurology. 45:1277-1285, 1995; Jacobs et al., Ann. Neurol.
39:285-294, 1996), and copolymer-1 (Johnson K P, Group. tCMST, J.
Neurol. 242:S38, 1995). Treatment failures have been linked to the
development of neutralizing anti-IFN-.beta. antibodies, although
their role is also not completely understood at present (the IFNB
Multiple Sclerosis Study Group and the University of British
Columbia MS/MRI Analysis Group, Neurology. 47:889-894, 1996).
Failure to respond to IFN-.beta. is not a rare event, and therefore
it is important to identify suitable combinations of standard
IFN-.beta. therapy with other treatment modalities, and new
therapeutic protocols.
SUMMARY
[0008] Methods are disclosed herein for treating a subject, such as
a human subject, with multiple sclerosis.
[0009] In one embodiment, the method includes administering to the
subject a therapeutically effective amount of an IL-2 receptor
(IL-2R) antagonist in the absence of treatment with beta
interferon, thereby ameliorating a symptom or symptoms of multiple
sclerosis and treating the subject. In one example, the subject has
failed to respond to previous treatment with beta interferon. In
another example, the IL-2R antagonist is a monoclonal antibody,
such as a chimeric, humanized or human antibody, that specifically
binds to the .alpha. or p55 (Tac) chain of the IL-2 receptor.
[0010] In another embodiment, a method is provided for treating a
subject with multiple sclerosis, wherein the method includes
administering a therapeutically effective amount of an antibody,
such as a chimeric, humanized, or fully human monoclonal antibody
that specifically binds the interleukin-2 receptor. The monoclonal
antibody is administered at least biweekly for a period of at least
two months. The subject is not treated with interferon-.beta.
during the administration of the monoclonal antibody. In one
example, the monoclonal antibody binds p55. In another specific
non-limiting example, the subject has previously failed to respond
to treatment with interferon-.beta..
[0011] In a further embodiment, a method of treatment is disclosed
in which administration of interferon-beta is combined with
administration of an antagonist of the IL-2R to provide significant
clinical improvement in individuals with MS. In particular
examples, the IL-2R antagonist is an antibody, such as a monoclonal
antibody, for example an anti-p55 antibody, such as daclizumab.
[0012] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a graph of the number of new, total, supertotal
and T2LL lesions in a subject treated with ZENAPAX.RTM. alone over
time. The subject did not respond to previous combination therapy
with ZENAPAX.RTM. and interferon (IFN) beta, as indicated in the
region to the right of the solid vertical line. Initiation of
ZENAPAX.RTM. monotherapy (in the absence of treatment with
interferon-beta) is shown by the arrow. No new lesions were
detected following the initiation of ZENAPAX.RTM. monotherapy.
[0014] FIG. 2 is a graph of the number of new, total, supertotal
and T2LL lesions in a second subject treated with ZENAPAX.RTM.
alone over time. The subject did not respond to previous
combination therapy with ZENAPAX.RTM. and interferon (IFN) beta, as
indicated in the region to the right of the dashed vertical line.
Initiation of ZENAPAX.RTM. monotherapy (in the absence of treatment
with interferon-beta) is shown by the arrow. No new lesions were
detected following the initiation of ZENAPAX.RTM. monotherapy.
[0015] FIG. 3 is a set of graphs showing the changes in new, total
and supertotal contrast enhancing lesions as measured by magnetic
resonance imaging (MRI) scans in subjects treated with a
combination of daclizumab and interferon-beta showing the
difference between a 3-month baseline period of treatment only with
interferon-beta and after combination therapy in eight
subjects.
[0016] FIGS. 4A and 4B are graphs showing changes in neurological
performance as measured by performance on the Expanded Disability
Status Scale (EDSS) (FIG. 4A) and the Scripps Neurologic Rating
Scale (NRS) (FIG. 4B) between the baseline period and after
combination therapy for the same subjects as in FIG. 3.
[0017] FIGS. 5A and 5B are graphs showing changes in neurological
performance as measured by performance on the ambulation index
(FIG. 5A) and the timed 20 m walk (FIG. 5B) between the baseline
period and after combination therapy for the same subjects as in
FIG. 3.
[0018] FIGS. 6A and 6B are graphs showing changes in neurological
performance as measured by the 9-peg hole test times for dominant
(FIG. 6A) and non-dominant (FIG. 6B) hands respectively, between
the baseline period and after combination therapy for the same
subjects as in FIG. 1.
[0019] FIG. 7 is a set of graphs showing changes in the percentage
of CD4+/CD25+ cells and CD8+/CD25+ cells expressing the Tac epitope
between the baseline period and after combination therapy for seven
of the subjects from FIG. 3.
[0020] FIGS. 8A and 8B are graphs showing changes in the number of
CD4 T cell mitoses per one-hundred cells (FIG. 8A) and CD8 T cell
mitoses per one-hundred cells (FIG. 8B) between the baseline period
and after combination therapy for the same subjects as in FIG.
3.
[0021] FIG. 9 is a graph showing changes in the number of CD4 T
cells expressing cytotoxic T lymphocyte-associated antigen 4
(CTLA-4) on their surface as measured by fluorescence-activated
cell sorting of blood samples between the baseline period and after
combination therapy for the same subjects as in FIG. 3.
DETAILED DESCRIPTION
I. Abbreviations
[0022] CDR: complementarity determining region
[0023] CBC: complete blood count
[0024] CNP: Cyclic nucleotide 3'-phosphodiesterase
[0025] EDSS: expanded disability status scale
[0026] FR: framework region
[0027] Gd: gadolinium
[0028] HIV: human immunodeficiency virus
[0029] HV: hypervariable region
[0030] IFN: interferon
[0031] Ig: immunoglobulin
[0032] IL-2: interleukin 2
[0033] IL-2R: interleukin 2 receptor
[0034] kg: kilogram
[0035] KLH: keyhole limpet hemocyanin
[0036] LPS: lippopolysaccharide
[0037] MBP: myelin basic protein
[0038] mg: milligram
[0039] mm: millimeter
[0040] MOG: myelin/oligodendrocyte glycoprotein
[0041] MRI: magnetic resonance imaging
[0042] MS: multiple sclerosis
[0043] NK: natural killer
[0044] NO--: nitric oxide
[0045] PBMC: peripheral blood mononuclear cells
[0046] PLP: myelin proteolipid protein
[0047] SRS: Scripps Neurological Rating Scale
[0048] TGF: transforming growth factor
[0049] TNF: tumor necrosis factor
[0050] VH: variable heavy
[0051] VL: variable light
II. Terms
[0052] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). Definitions and additional information known to one
of skill in the art in immunology can be found, for example, in
Fundamental Immunology, W. E. Paul, ed., fourth edition,
Lippincott-Raven Publishers, 1999.
[0053] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0054] Adverse Effects: Any undesirable signs, including the
clinical manifestations of abnormal laboratory results, or medical
diagnoses noted by medical personnel, or symptoms reported by the
subject that have worsened. Adverse events include, but are not
limited to, life-threatening events, an event that prolongs
hospitalization, or an event that results in medical or surgical
intervention to prevent an undesirable outcome.
[0055] Antagonist of an IL-2 Receptor (IL-2R): An agent that
specifically binds to the IL-2R, or a component thereof, and
inhibits a biological function of the IL-2 receptor or the
component. Exemplary functions that can be inhibited are the
binding of IL-2 to the IL-2R, the intracellular transmission of a
signal from binding of IL-2, and proliferation and/or activation of
lymphocytes such as T cells in response to IL-2. In one embodiment,
IL-2R antagonists of use in the methods disclosed herein inhibit at
least one of these functions. Alternatively, IL-2R antagonist of
use in the methods disclosed herein can inhibit more than one or
all of these functions.
[0056] In one example, an IL-2 receptor antagonist is an antibody
that specifically binds Tac (p55), such as ZENAPAX.RTM. (see
below). Other anti-p55 agents include the chimeric antibody
basiliximab (Simulect.RTM.), BT563 (see Baan et al., Transplant.
Proc. 33:224-2246, 2001), and 7G8. Basiliximab has been reported to
be beneficial in preventing allograft rejection (Kahan et al.,
Transplantation 67:276-84,1999), and treating psoriasis (Owen &
Harrison, Clin. Exp. Dermatol. 25:195-7, 2000). An exemplary human
anti-p55 antibody of use in the methods of the invention is
HuMax-TAC, being developed by Genmab. In another example, an IL-2
receptor antagonist is an antibody that specifically binds the p75
or .beta. subunit of the IL-2R.
[0057] Additional antibodies that specifically bind the IL-2
receptor are known in the art. For example, see U.S. Pat. No.
5,011,684; U.S. Pat. No. 5,152,980; U.S. Pat. No. 5,336,489; U.S.
Pat. No. 5,510,105; U.S. Pat. No. 5,571,507; U.S. Pat. No.
5,587,162; U.S. Pat. No. 5,607,675; U.S. Pat. No. 5,674,494; U.S.
Pat. No. 5,916,559. The mik-.beta.1 antibody is an antagonist that
specifically binds the beta chain of human IL-2R.
[0058] In another example, an IL-2 receptor antagonist is a peptide
antagonist that is not an antibody. Peptide antagonists of the IL-2
receptor, including antagonists of Tac (p55) and p75 (IL-2R.beta.)
are also known. For example, peptide antagonists for p55 and p75
are disclosed in U.S. Pat. No. 5,635,597. These peptides are also
of use in the methods disclosed herein.
[0059] In a further example, an IL-2 receptor antagonist is a
chemical compound or small molecule that specifically binds to the
IL-2 receptor and inhibits a biological function of the
receptor.
[0060] Antibody fragment (fragment with specific antigen binding):
Various fragments of antibodies have been defined, including Fab,
(Fab').sub.2, Fv, and single-chain Fv (scFv). These antibody
fragments are defined as follows: (1) Fab, the fragment that
contains a monovalent antigen-binding fragment of an antibody
molecule produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain or equivalently by genetic engineering; (2) Fab', the
fragment of an antibody molecule obtained by treating whole
antibody with pepsin, followed by reduction, to yield an intact
light chain and a portion of the heavy chain; two Fab' fragments
are obtained per antibody molecule; (3) (Fab').sub.2, the fragment
of the antibody obtained by treating whole antibody with the enzyme
pepsin without subsequent reduction or equivalently by genetic
engineering; (4) F(Ab').sub.2, a dimer of two FAb' fragments held
together by disulfide bonds; (5) Fv, a genetically engineered
fragment containing the variable region of the light chain and the
variable region of the heavy chain expressed as two chains; and (6)
single chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule. Methods of making
these fragments are routine in the art.
[0061] Autoimmune disorder: A disorder in which the immune system
produces an immune response (e.g. a B cell or a T cell response)
against an endogenous antigen, with consequent injury to
tissues.
[0062] Beta interferon: Any beta interferon including
interferon-beta 1a and interferon-beta 1b.
[0063] Interferon-beta 1a is a 166 amino, acid glycoprotein with a
predicted molecular weight of approximately 22,500 daltons. The
interferon-beta 1a known as Avonex.RTM. is produced by recombinant
DNA technology utilizing mammalian cells (Chinese Hamster Ovary
cells) into which the human interferon-beta gene has been
introduced. The amino acid sequence of Avonex.RTM. is identical to
that of natural human interferon-beta. Interferon induced gene
products and markers including 2', 5'-oligoadenylate synthetase,
.beta..sub.2-microglobulin, and neopterin, have been measured in
the serum and cellular fractions of blood collected from patients
treated with Avonex.RTM.. Avonex.RTM. was approved in 1996 and is
marketed by Biogen, Inc. Avonex.RTM. has been demonstrated to
decrease the number of gadolinium (Gd)-enhanced lesions in subjects
who were administered the drug for two years by up to 13% and to
improve approximately 22% of subjects' Expanded Disability Status
Scale (EDSS) scores.
[0064] Another interferon-beta 1a was approved in 2002 and is known
as Rebif.RTM., marketed by Serono, Inc. The interferon-beta 1a
known as Rebif.RTM., has recently been approved for treatment of
relapsing-remitting MS. The primary difference between Avonex.RTM.
and Rebif.RTM. is the approved method of
administration--intramuscular injection for the former and
subcutaneous injection for the latter. According to Samkoff, Hosp.
Phys., p. 21-7 (2002), Rebif.RTM. can reduce relapse rates by 33%
in subjects taking the drug.
[0065] Interferon-beta 1b is a highly purified protein that has 165
amino acids and an approximate molecular weight of 18,500 daltons.
An interferon-beta 1b known as Betaseron.RTM. was approved as a
treatment for MS in 1993 and is marketed by Berlex Laboratories,
Inc. Betaseron.RTM. is manufactured by bacterial fermentation of a
strain of Escherichia coli that bears a genetically engineered
plasmid containing the gene for human interferon-beta. The native
gene was obtained from human fibroblasts and altered to substitute
serine for the cysteine residue found at position 17. According to
the Physicians' Desk Reference (1996), Betaseron.RTM. has been
demonstrated to reduce the exacerbation rate in subjects taking the
drug by about 31%. The mechanisms by which interferon-beta 1b
exerts its actions in multiple sclerosis are not clearly
understood. However, it is known that the biologic
response-modifying properties of interferon-beta 1b are mediated
through its interactions with specific cell receptors. The binding
of interferon-beta 1b to these receptors induces the expression of
a number of interferon induced gene products (e.g.,
2',5'-oligoadenylate synthetase, protein kinase, and indoleamine
2,3-dioxygenase) that are believed to be the mediators of the
biological actions of interferon-beta 1b.
[0066] Complementarity-determining region (CDR): The CDRs are three
hypervariable regions within each of the variable light (VL) and
variable heavy (VH) regions of an antibody molecule that form the
antigen-binding surface that is complementary to the
three-dimensional structure of the bound antigen. Proceeding from
the N-terminus of a heavy or light chain, these
complementarity-determining regions are denoted as "CDR1", "CDR2,"
and "CDR3," respectively. CDRs are involved in antigen-antibody
binding, and the CDR3 comprises a unique region specific for
antigen-antibody binding. An antigen-binding site, therefore, may
include six CDRs, comprising the CDR regions from each of a heavy
and a light chain V region. Alteration of a single amino acid
within a CDR region can destroy the affinity of an antibody for a
specific antigen (see Abbas et al., Cellular and Molecular
Immunology, 4th ed. 143-5, 2000). The locations of the CDRs have
been precisely defined, e.g., by Kabat et al., Sequences of
Proteins of Immunologic Interest, U.S. Department of Health and
Human Services, 1983.
[0067] Epitope: The site on an antigen recognized by an antibody as
determined by the specificity of the amino acid sequence. Two
antibodies are said to bind to the same epitope if each
competitively inhibits (blocks) binding of the other to the antigen
as measured in a competitive binding assay (see, e.g., Junghans et
al., Cancer Res. 50:1495-1502, 1990). Alternatively, two antibodies
have the same epitope if most amino acid mutations in the antigen
that reduce or eliminate binding of one antibody reduce or
eliminate binding of the other. Two antibodies are said to have
overlapping epitopes if each partially inhibits binding of the
other to the antigen, and/or if some amino acid mutations that
reduce or eliminate binding of one antibody reduce or eliminate
binding of the other.
[0068] Framework region (FR): Relatively conserved sequences
flanking the three highly divergent complementarity-determining
regions (CDRs) within the variable regions of the heavy and light
chains of an antibody. Hence, the variable region of an antibody
heavy or light chain consists of a FR and three CDRs. Some FR
residues may contact bound antigen; however, FRs are primarily
responsible for folding the variable region into the
antigen-binding site, particularly the FR residues directly
adjacent to the CDRs. Without being bound by theory, the framework
region of an antibody serves to position and align the CDRs. The
sequences of the framework regions of different light or heavy
chains are relatively conserved within a species. A "human"
framework region is a framework region that is substantially
identical (about 85% or more, usually 90-95% or more) to the
framework region of a naturally occurring human immunoglobulin.
[0069] Immunoglobulin: A protein including one or more polypeptides
substantially encoded by immunoglobulin genes. The recognized
immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma
(IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4), delta (IgD), epsilon
(IgE) and mu (IgM) constant region genes, as well as the myriad
immunoglobulin variable region genes. Full-length immunoglobulin
light chains are generally about 25 Kd or 214 amino acids in
length. Full-length immunoglobulin heavy chains are generally about
50 Kd or 446 amino acid in length. Light chains are encoded by a
variable region gene at the NH2-terminus (about 110 amino acids in
length) and a kappa or lambda constant region gene at the
COOH--terminus. Heavy chains are similarly encoded by a variable
region gene (about 116 amino acids in length) and one of the other
constant region genes.
[0070] The basic structural unit of an antibody is generally a
tetramer that consists of two identical pairs of immunoglobulin
chains, each pair having one light and one heavy chain. In each
pair, the light and heavy chain variable regions bind to an
antigen, and the constant regions mediate effector functions;
Immunoglobulins also exist in a variety of other forms including,
for example, Fv, Fab, and (Fab').sub.2, as well as bifunctional
hybrid antibodies and single chains (e.g., Lanzavecchia et al.,
Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad.
Sci. U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426,
1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984;
Hunkapiller and Hood, Nature 323:15-16, 1986).
[0071] An immunoglobulin light or heavy chain variable region
includes a framework region interrupted by three hypervariable
regions, also called complementarity determining regions (CDR's)
(see, Sequences of Proteins of Immunological Interest, E. Kabat et
al., U.S. Department of Health and Human Services, 1983). As noted
above, the CDRs are primarily responsible for binding to an epitope
of an antigen.
[0072] Chimeric antibodies are antibodies whose light and heavy
chain genes have been constructed, typically by genetic
engineering, from immunoglobulin variable and constant region genes
belonging to different species. For example, the variable segments
of the genes from a mouse monoclonal antibody can be joined to
human constant segments, such as kappa and gamma 1 or gamma 3. In
one example, a therapeutic chimeric antibody is thus a hybrid
protein composed of the variable or antigen-binding domain from a
mouse antibody and the constant or effector domain from a human
antibody (e.g., ATCC Accession No. CRL 9688 secretes an anti-Tac
chimeric antibody), although other mammalian species can be used,
or the variable region can be produced by molecular techniques.
Methods of making chimeric antibodies are well known in the art,
e.g., see U.S. Pat. No. 5,807,715, which is herein incorporated by
reference.
[0073] A "humanized" immunoglobulin is an immunoglobulin including
a human framework region and one or more CDRs from a non-human
(such as a mouse, rat, or synthetic) immunoglobulin. The non-human
immunoglobulin providing the CDRs is termed a "donor" and the human
immunoglobulin providing the framework is termed an "acceptor." In
one embodiment, all the CDRs are from the donor immunoglobulin in a
humanized immunoglobulin. Constant regions need not be present, but
if they are, they must be substantially identical to human
immunoglobulin constant regions, i.e., at least about 85-90%, such
as about 95% or more identical. Hence, all parts of a humanized
immunoglobulin, except possibly the CDRs, are substantially
identical to corresponding parts of natural human immunoglobulin
sequences. A "humanized antibody" is an antibody comprising a
humanized light chain and a humanized heavy chain immunoglobulin. A
humanized antibody binds to the same antigen as the donor antibody
that provides the CDRs. The acceptor framework of a humanized
immunoglobulin or antibody may have a limited number of
substitutions by amino acids taken from the donor framework.
Humanized or other monoclonal antibodies can have additional
conservative amino acid substitutions which have substantially no
effect on antigen binding or other immunoglobulin functions.
Exemplary conservative substitutions are those such as gly, ala;
val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr
(see U.S. Pat. No. 5,585,089, which is incorporated herein by
reference). Humanized immunoglobulins can be constructed by means
of genetic engineering, e.g., see U.S. Pat. No. 5,225,539 and U.S.
Pat. No. 5,585,089, which are herein incorporated by reference.
[0074] A human antibody is an antibody wherein the light and heavy
chain genes are of human origin. Human antibodies can be generated
using methods known in the art. Human antibodies can be produced by
immortalizing a human B cell secreting the antibody of interest.
Immortalization can be accomplished, for example, by EBV infection
or by fusing a human B cell with a myeloma or hybridoma cell to
produce a trioma cell. Human antibodies can also be produced by
phage display methods (see, e.g., Dower et al., PCT Publication No.
WO91/17271; McCafferty et al., PCT Publication No. WO92/001047; and
Winter, PCT Publication No. WO92/20791, which are herein
incorporated by reference), or selected from a human combinatorial
monoclonal antibody library (see the Morphosys website). Human
antibodies can also be prepared by using transgenic animals
carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT
Publication No. WO93/12227; and Kucherlapati, PCT Publication No.
WO91/10741, which are herein incorporated by reference).
[0075] Interleukin 2 (IL-2): A protein of 133 amino acids (15.4
kDa) with a slightly basic pI that does not display sequence
homology to any other factors. Murine and human IL-2 display a
homology of approximately 65%. IL-2 is synthesized as a precursor
protein of 153 amino acids with the first 20 amino terminal amino
acids functioning as a hydrophobic secretory signal sequence. The
protein contains a single disulfide bond (positions Cys58/105)
essential for biological activity. The human IL-2 gene contains
four exons and maps to human chromosome 4q26-28 (murine chromosome
3).
[0076] The biological activities of IL-2 are mediated by a membrane
receptor that is expressed on activated, but not on resting, T
cells and natural killer (NK) cells. Activated B cells and resting
mononuclear leukocytes also rarely express this receptor.
[0077] IL-2 receptor: A cellular receptor that binds IL-2 and
mediates its biological effects. Three different types of IL-2
receptors are distinguished that are expressed differentially and
independently. The high affinity IL-2 receptor (K.sub.d.about.10
pM) constitutes approximately 10% of all IL-2 receptors expressed
by cells. This receptor is a membrane receptor complex consisting
of the two subunits: IL-2R-alpha (also known as T cell activation
(TAC) antigen or p55) and IL-2R-beta (also known as p75 or CD122).
An intermediate affinity IL-2 receptor (K.sub.d=100 pM) consists of
the p75 subunit and a gamma chain, while a low affinity receptor
(K.sub.d=10 nM) is formed by p55 alone.
[0078] p75 is 525 amino acids in length. It has an extracellular
domain of 214 amino acids and a cytoplasmic domain of 286 amino
acids. The p75 gene maps to human chromosome 22q11. 2-q12, contains
10 exons and has a length of approximately 24 kb. p55 is 251 amino
acids in length with an extracellular domain of 219 amino acids and
a very short cytoplasmic domain of 13 amino acids. The gene
encoding p55 maps to human chromosome 10p14-p15.
[0079] p75 is expressed constitutively on resting T-lymphocytes, NK
cells, and a number of other cell types while the expression of p55
is usually observed only after activation. Activated lymphocytes
continuously secrete a 42 kDa fragment of p55 (TAC antigen). This
fragment circulates in the serum and plasma and functions as a
soluble IL2 receptor (see Smith, Ann. Rev. Cell Biol. 5:397-425,
1989; Taniguchi and Minami, Cell 73:5-8, 1993).
[0080] p55 has a length of 251 amino acids with an extracellular
domain of 219 amino acids an a very short cytoplasmic domain of 13
amino acids. The p55 gene maps to human chromosome 10p14-p15. The
expression of p55 is regulated by a nuclear protein called
RPT-1.
[0081] A third 64 kDa subunit of the IL2 receptor, designated
gamma, has been described. This subunit is required for the
generation of high and intermediate affinity IL-2 receptors but
does not bind IL-2 by itself. The gene encoding the gamma subunit
of the IL2 receptor maps to human chromosome Xq13, spans
approximately 4.2 kb and contains eight exons.
[0082] Magnetic Resonance Imaging: Anoninvasive diagnostic
technique that produces computerized images of internal body
tissues and is based on nuclear magnetic resonance of atoms within
the body induced by the application of radio waves.
[0083] Brain MRI is an important tool for understanding the dynamic
pathology of multiple sclerosis. T.sub.2-weighted brain MRI defines
lesions with high sensitivity in multiple sclerosis and is used as
a measure of disease burden. However, such high sensitivity occurs
at the expense of specificity, as T.sub.2 signal changes can
reflect areas of edema, demyelination, gliosis and axonal loss.
Areas of gadolinium (Gd) enhancement demonstrated on
T.sub.1-weighted brain MRI are believed to reflect underlying
blood-brain barrier disruption from active perivascular
inflammation. Such areas of enhancement are transient, typically
lasting <1 month. Gadolinium-enhanced T.sub.1-weighted brain MRI
are therefore used to assess disease activity. Most T2-weighted
(T2) lesions in the central white matter of subjects with multiple
sclerosis begin with a variable period of T1-weighted (T1)
gadolinium (Gd) enhancement and that T1 Gd-enhancing and T2 lesions
represent stages of a single pathological process. The brain MRI
techniques for assessing T1 and T2 Gd-enhancing lesions are
standard (e.g., see Lee et al., Brain 122 (Pt 7):1211-2, 1999).
[0084] Monoclonal antibody: An antibody produced by a single clone
of B-lymphocytes or by a cell into which the light and heavy chain
genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells.
[0085] Multiple sclerosis: An autoimmune disease classically
described as a central nervous system white matter disorder
disseminated in time and space that presents as relapsing-remitting
illness in 80-85% of patients. Diagnosis can be made by brain and
spinal cord magnetic resonance imaging (MRI), analysis of
somatosensory evoked potentials, and analysis of cerebrospinal
fluid to detect increased amounts of immunoglobulin or oligoclonal
bands. MRI is a particularly sensitive diagnostic tool. MRI
abnormalities indicating the presence or progression of MS include
hyperintense white matter signals on T2-weighted and fluid
attenuated inversion recovery images, gadolinium enhancement of
active lesions, hypointensive "black holes" (representing gliosis
and axonal pathology), and brain atrophy on T1-weighted studies.
Serial MRI studies can be used to indicate disease progression.
[0086] Relapsing-remitting multiple sclerosis is a clinical course
of MS that is characterized by clearly defined, acute attacks with
full or partial recovery and no disease progression between
attacks.
[0087] Secondary-progressive multiple sclerosis is a clinical
course of MS that initially is relapsing-remitting, and then
becomes progressive at a variable rate, possibly with an occasional
relapse and minor remission.
[0088] Primary progressive multiple sclerosis presents initially in
the progressive form.
[0089] Polypeptide: A polymer in which the monomers are amino acid
residues that are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used, the L-isomers being preferred.
The terms "polypeptide" or "protein" as used herein is intended to
encompass any amino acid sequence and include modified sequences
such as glycoproteins. The term "polypeptide" is specifically
intended to cover naturally occurring proteins, as well as those
that are recombinantly or synthetically produced.
[0090] The term "fragment" refers to a portion of a polypeptide
that is at least 8, 10, 15, 20 or 25 amino acids in length. The
term "functional fragments of a polypeptide" refers to all
fragments of a polypeptide that retain an activity of the
polypeptide (e.g., the binding of an antigen). Biologically
functional fragments, for example, can vary in size from a
polypeptide fragment as small as an epitope capable of binding an
antibody molecule to a large polypeptide capable of participating
in the characteristic induction or programming of phenotypic
changes within a cell. The term "soluble" refers to a form of a
polypeptide that is not inserted into a cell membrane.
[0091] Pharmaceutical agent or drug: A chemical compound or
composition capable of inducing a desired therapeutic or
prophylactic effect when properly administered to a subject.
[0092] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in the methods disclosed herein are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the IL-2 receptor antagonists herein disclosed.
[0093] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
salts, amino acids, and pH buffering agents and the like, for
example sodium or potassium chloride or phosphate, Tween, sodium
acetate or sorbitan monolaurate.
[0094] Purified: The term purified does not require absolute purity
or isolation; rather, it is intended as a relative term. Thus, for
example, a purified or isolated protein preparation is one in which
protein is more-enriched than the protein is in its generative
environment, for instance within a cell or in a biochemical
reaction chamber. Preferably, a preparation of protein is purified
such that the protein represents at least 50% of the total protein
content of the preparation. For pharmaceuticals, "substantial"
purity of 90%, 95%, 98% or even 99% or higher of the active agent
can be utilized.
[0095] Sequence identity: The similarity between two nucleic acid
sequences, or two amino acid sequences, is expressed in terms of
the similarity between the sequences, otherwise referred to as
sequence identity. Sequence identity is frequently measured in
terms of percentage identity (or similarity or homology); the
higher the percentage, the more similar the two sequences are.
Homologs or orthologs of the IL-2R antibodies or antigen binding
fragments, and the corresponding cDNA sequence, will possess a
relatively high degree of sequence identity when aligned using
standard methods. This homology will be more significant when the
orthologous proteins or cDNAs are derived from species that are
more closely related, compared to species more distantly related
(e.g., human and murine sequences).
[0096] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and
Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and
Sharp, Gene 73:237-244 9, 1988); Higgins and Sharp; CABIOS
5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;
Huang et al., Computer Appls. in the Biosciences 8:155-65, 1992;
and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et
al., J. Mol. Biol. 215:403-410, 1990, presents a detailed
consideration of sequence alignment methods and homology
calculations.
[0097] Specific binding agent: An agent that binds substantially
only to a defined target. Thus an IL-2 receptor-specific binding
agent binds substantially only the IL-2 receptor, or a component
thereof. As used herein, the term "IL-2 receptor-specific binding
agent" includes anti-IL-2 receptor antibodies and other agents that
bind substantially only to an IL-2 receptor or a component thereof
(e.g., p55, p75).
[0098] Anti-IL-2 receptor antibodies may be produced using standard
procedures described in a number of texts, including Harlow and
Lane (Using Antibodies, A Laboratory Manual, CSHL, New York, 1999,
ISBN 0-87969-544-7). In addition, certain techniques may enhance
the production of neutralizing antibodies (U.S. Pat. No. 5,843,454;
U.S. Pat. No. 5,695,927; U.S. Pat. No. 5,643,756; and U.S. Pat. No.
5,013,548). The determination that a particular agent binds
substantially only to an IL-2 receptor component may readily be
made by using or adapting routine procedures. One suitable in vitro
assay makes use of the Western blotting procedure (described in
many standard texts, including Harlow and Lane, 1999). Western
blotting may be used to determine that a given protein binding
agent, such as an anti-IL-2 receptor monoclonal antibody, binds
substantially only to the IL-2 receptor. Antibodies to the IL-2
receptor are well known in the art.
[0099] Shorter fragments of antibodies can also serve as specific
binding agents. For instance, Fabs, Fvs, and single-chain Fvs
(SCFvs) that bind to an IL-2 receptor would be IL-2
receptor-specific binding agents.
[0100] Subject: A human or non-human animal. In one embodiment, the
subject has multiple sclerosis.
[0101] A subject who has multiple sclerosis who has failed a
therapeutic protocol (such as administration of interferon-beta) is
a subject who does not respond or fails to respond adequately to
the therapy, such that their condition has not improved
sufficiently, not changed, or deteriorated in response to treatment
with a therapeutically effective amount of the drug. A subject who
has failed a therapeutic protocol can require escalating doses of
the drug to achieve a desired effect.
[0102] In one example, the failure of a subject with MS to respond
to a therapeutic agent, such as interferon-beta, can be measured as
a recurrence of Gd-contrasting MRI lesions to at least half of the
mean of the baseline monthly contrasting lesions over six months.
In other examples, a subject with MS that fails to respond to a
therapeutic agent, such as interferon-beta treatment, is identified
by the subject experiencing one or more exacerbations in an 18
month period of interferon-beta therapy, exhibiting an increase of
1 point or more on the EDSS over 18 months of treatment, or having
persistence or reoccurrence of contrast enhancing lesions on brain
MRI scans to at least one-half the mean of a baseline of monthly
contrast enhancing lesions established over a 6-month baseline
period measured prior to the beginning of the interferon-beta
therapy.
[0103] Without being bound by theory, a subject can fail to respond
to IFN treatment due to the development of neutralizing antibodies,
although a failure to respond to IFN treatment can also be detected
in the absence of neutralizing antibodies (primary failure). In one
example, a subject who fails treatment with interferon-beta is a
subject who develops neutralizing antibodies that specifically bind
interferon-beta, such that escalating doses are required to see an
effect, or to alter a sign or symptom of MS.
[0104] Symptom and sign: Any subjective evidence of disease or of a
subject's condition, i.e., such evidence as perceived by the
subject; a noticeable change in a subject's condition indicative of
some bodily or mental state. A "sign" is any abnormality indicative
of disease, discoverable on examination or assessment of a subject.
A sign is generally an objective indication of disease. Signs
include, but are not limited to any measurable parameters such as
tests for immunological status or the presence of lesions in a
subject with multiple sclerosis.
[0105] Therapeutically Effective Amount: A dose sufficient to
prevent advancement, or to cause regression of the disease, or
which is capable of reducing symptoms caused by the disease, such
as multiple sclerosis.
[0106] ZENAPAX.RTM. (daclizumab): A particular recombinant,
humanized monoclonal antibody of the human IgG1 isotype that
specifically binds Tac (p55). The recombinant genes encoding
ZENAPAX.RTM. are a composite of human (about 90%) and murine (about
10%) antibody sequences. The donor murine anti-Tac antibody is an
IgG2a monoclonal antibody that specifically binds the IL-2R Tac
protein and inhibits IL-2-mediated biologic responses of lymphoid
cells. The murine anti-Tac antibody was "humanized" by combining
the complementarity-determining regions and other selected residues
of the murine anti-TAC antibody with the framework and constant
regions of the human IgG1 antibody. The humanized anti-Tac antibody
daclizumab is described and its sequence is set forth in U.S. Pat.
No. 5,530,101, see SEQ ID NO: 5 and SEQ ID NO: 7 for the heavy and
light chain variable regions respectively. U.S. Pat. No. 5,530,101
and Queen et al., Proc. Natl. Acad. Sci. 86:1029-1033, 1989 are
both incorporated by reference herein in their entirety. Daclizumab
inhibits IL-2-dependent antigen-induced T cell proliferation and
the mixed lymphocyte response (MLR) (Junghans et al., Cancer
Research 50:1495-1502, 1990), as can other antibodies of use in the
methods disclosed herein.
[0107] ZENAPAX.RTM. has been approved by the U.S. Food and Drug
Administration (FDA) for the prophylaxis of acute organ rejection
in subjects receiving renal transplants, as part of an
immunosuppressive regimen that includes cyclosporine and
coritcosteroids. ZENAPAX.RTM. has been shown to be active in the
treatment of human T cell lymphotrophic virus type 1 associated
myelopathy/topical spastic paraparesis (HAM/TSP, see Lehky et al.,
Ann. Neuro., 44:942-947, 1998). The use of ZENAPAX.RTM. to treat
posterior uveitis has also been described (see Nussenblatt et al.,
Proc. Natl. Acad. Sci., 96:7462-7466, 1999).
[0108] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"oi" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety for all purposes. In case of conflict, the
present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
Methods for Treating Subject with Multiple Sclerosis
[0109] Methods are provided herein for the treatment of subjects
that have multiple sclerosis. In one embodiment the subject has
relapsing-remitting multiple sclerosis. However, the methods
disclosed herein can also be used for the treatment of subjects
with other forms of multiple sclerosis, such as secondary or
primary progressive multiple sclerosis.
[0110] In certain embodiments the method is used to treat subjects
who have failed to respond adequately to interferon-beta treatment
alone. A failure to respond to interferon-beta treatment alone is,
in some examples, demonstrated by the subject experiencing one or
more exacerbations in an 18 month period of interferon-beta
therapy, an increase of 1 point or more on the EDSS over 18 months
of treatment, or persistence or reoccurrence of contrast enhancing
lesions on brain MRI scans to at least one-half the mean of a
baseline of monthly contrast enhancing lesions established over a
6-month baseline period measured prior to the beginning of the
interferon-beta therapy. Other indicators of disease progression or
activity known to those with skill in the art can also be used to
determine whether a subject has failed to respond to
interferon-beta therapy. The interferon-beta therapy can be
treatment with interferon-beta 1b, interferon-beta 1a, or both
types of interferon.
[0111] In a specific embodiment, a therapeutically effective amount
of an IL-2 receptor (IL-2R) antagonist is administered to the
subject without the concurrent administration of interferon-beta. A
single IL-2R antagonist can be utilized, or a combination of IL-2R
antagonists can be utilized in the treatment of multiple sclerosis.
The IL-2R antagonist is any agent that binds to the IL-2R on
activated T-lymphocytes and inhibits the activity of the
receptor.
[0112] In one specific non-limiting example, the IL-2 receptor
antagonist is an antibody, such as a monoclonal antibody, e.g., a
chimeric, humanized or human monoclonal antibody. A specific
example of a humanized monoclonal antibody that specifically binds
p55 is daclizumab, which is described and its sequence is set forth
in U.S. Pat. No. 5,530,101, which is incorporated by reference
herein, and in Queen et al., Proc. Natl. Acad. Sci. 86:1029-1033,
1989. Thus, the antibody can be a humanized immunoglobulin having
complementarity determining regions (CDRs) from a donor
immunoglobulin and heavy and light chain variable region frameworks
from human acceptor immunoglobulin heavy and light chain
frameworks, wherein the humanized immunoglobulin specifically binds
to a human interleukin-2 receptor with an affinity constant of at
least 10.sup.8 M.sup.-1. The sequence of the humanized
immunoglobulin heavy chain variable region framework can be at
least 65% identical to the sequence of the donor immunoglobulin
heavy chain variable region framework. A specific example of the
variable region of the anti-Tac antibody is set forth as SEQ ID NO:
1 and SEQ ID NO: 3 of U.S. Pat. No. 5,520,101 (light and heavy
chain, respectively), and the variable region of the humanized
anti-Tac antibody daclizumab is set forth as SEQ ID NO: 5 and SEQ
ID NO: 7 (heavy and light chain, respectively) of U.S. Pat. No.
5,530,101, which is herein incorporated by reference.
[0113] The antibody can include two light chain/heavy chain dimers,
and specifically binds to either p55 (such as the anti-Tac
antibody) or p75. Il-2R antagonists of use include agents that bind
specifically to p55 (also known as the alpha chain or Tac subunit)
of the human IL-2R. In one example, the agent is a monoclonal
antibody, such as daclizumab, basiliximab, BT563, and 7G8 or their
chimeric or humanized forms. The agent can also be a human
antibody; or a humanized antibody with synthetic CDRs that
specifically binds p55. Antibodies that bind the same (or
overlapping) epitope as daclizumab or basiliximab can also be used
in the methods disclosed herein. In other embodiments, the antibody
will have high sequence identity with daclizumab or basiliximab, at
least 90 or 95%, such as at least 98% or 99% sequence identity,
while retaining the functional properties of the antibody, i.e.,
its antagonist properties to the IL-2R. The antibody may be of any
isotype, but in several embodiment that antibody is an IgG,
including but not limited to, IgG1, IgG2, IgG3 and IgG4.
[0114] In other embodiments the antibody is basilimab, marketed as
Simulect.RTM. by Novartis Pharma AG. Simulect.RTM. is a chimeric
(murine/human) monoclonal antibody (IgG.sub.1.kappa.), produced by
recombinant DNA technology, that functions as an immunostippressive
agent, specifically binding to and blocking the alpha chain of the
IL-2R on the surface of activated T-lymphocytes. Simulect.RTM. is a
glycoprotein obtained from fermentation of an established mouse
myeloma cell line genetically engineered to express plasmids
containing the human heavy and light chain constant region genes
and mouse heavy and light chain variable region genes encoding the
RFT5 antibody that binds selectively to the IL-2R(alpha). Based on
the amino acid sequence, the calculated molecular weight of the
protein is 144 kilodaltons.
[0115] Alternatively, the IL-2R antagonist is a molecule that binds
to other subunits of the IL-2 receptor, such as Mik-.beta.1 or
Mik-.beta.2 or their chimeric or humanized versions, which bind to
the beta chain of human IL-2R, or another antibody that
specifically binds p75 (see U.S. Pat. No. 5,530,101, which is
incorporated herein by reference). The IL-2R antagonist may also be
a fragment of an antibody (e.g., a chimeric, humanized, or human
antibody) such as an Fab, (Fab').sub.2, Fv, or scFv. Further, the
fragment may be pegylated to increase its half-life.
[0116] In some examples, the IL-2R antagonist is a combination of
anti-IL-2R agents. For example, ZENAPAX.RTM. and Simulect.RTM. are
administered together as a cocktail, or the agents are alternated
in the administration schedule.
[0117] The IL-2R antagonist, such as a humanized antibody that
specifically binds the IL-2R, can be used in combination with other
antibodies, particularly human monoclonal antibodies reactive with
other markers on cells responsible for a disease. For example,
suitable T cell markers can include those grouped into the
so-called "Clusters of Differentiation," (CD antigens, see the
First International Leukocyte Differentiation Workshop, Leukocyte
Typing, Bernard, et al., Eds., Springer-Verlag, N.Y., 1984). In
another example, the other antibody binds and inhibits a
lymphokine, such as IFN-gamma, or a lymphokine receptor. In one
example, the other antibody binds .alpha.5.beta.1 integrin (VLA-5),
of which a particularly preferred exemplary antibody is
Antegren.RTM. (Elan Pharmaceuticals and Biogen, Inc.).
[0118] The IL-2R antagonist can be administered parenterally, i.e.,
subcutaneously, intramuscularly or intravenously or by means of a
needle-free injection device. The compositions for parenteral
administration will commonly include a solution of the IL-2R
antagonist (e.g. the antibody) in a pharmaceutically acceptable
carrier as described above. The concentration of antibody in the
formulations can vary widely, i.e., from less than about 0.5%,
usually at or at least about 1% to as much as 15 or 20% by weight
or from 1 mg/mL to 100 mg/mL. The concentration is selected
primarily based on fluid volumes, viscosities, etc., in accordance
with the particular mode of administration selected.
[0119] Methods for preparing pharmaceutical compositions are known
those skilled in the art (see Remington's Pharmaceutical Science,
15th ed., Mack Publishing Company, Easton, Pa., 1980).
[0120] Antibodies of use in the methods disclosed herein can be
frozen or lyophilized for storage and reconstituted in a suitable
carrier prior to use. One of skill in the art can readily design
appropriate lyophilization and reconstitution techniques.
[0121] The IL-2R antagonist can be administered for therapeutic
treatments of a subject with multiple sclerosis. Thus, a
therapeutically effective amount of a composition is administered
to a subject already suffering from MS, in an amount sufficient to
improve a sign or a symptom of the disorder. Generally a suitable
dose of ZENAPAX.RTM. (daclizumab) is about 0.5 milligram per
kilogram (mg/kg) to about 3 mg/kg, such as a dose of about 1 mg/kg,
about 1.5 mg/kg, about 2 mg/kg, or about 2.5 mg/kg administered
intraveneously or subcutaneously. Unit dosage forms are also
possible, for example 50 mg, 100 mg, 150 mg or 200 mg, or up to 400
mg per dose. However, other higher or lower dosages also could be
used, such as from about 0.5 to about 8 mg/kg. It has been
suggested that that serum levels of 5 to 10 .mu.g/mL are necessary
for saturation of the Tac subunit of the IL-2 receptors to block
the responses of activated T lymphocytes. One of skill in the art
will be able to construct an administration regimen to keep serum
levels within that range, although administration resulting in
higher or lower serum levels could be used. Doses of Simulect.RTM.
are likely to be lower, for example 0.25 mg/kg to 1 mg/kg, e.g.,
0.5 mg/kg, or unit doses of 10, 20, 40, 50 or 100 mg. The general
principle of keeping the IL-2R saturated could also be used to
guide the choice of dose levels of other IL-2R antagonists such as
other monoclonal antibodies.
[0122] Single or multiple administrations of the IL-2R antagonist
compositions can be carried out with dose levels and pattern being
selected by the treating physician. Generally, multiple doses are
administered. In several examples, multiple administrations of
ZENAPAX.RTM. (daclizumab) or other IL-2R antibodies are utilized,
such as administration monthly, bimonthly, every 6 weeks, every
other week, weekly or twice per week. An exemplary protocol for
administration of ZENAPAX.RTM. (daclizumab), also applicable to
other IL-2R antibodies, is described in the examples section below.
For an IL-2R antagonist that is not an antibody, more frequent
administration may be necessary, for example, one, two, three of
four or more times per day, or twice per week. Such an IL-2R
antagonist can be administered orally, but subcutaneous or
intravenous administration can also be utilized. Treatment will
typically continue for at least a month, more often for two or
three months, sometimes for six months or a year, and may even
continue indefinitely, i.e., chronically. Repeat courses of
treatment are also possible.
[0123] In one embodiment, the IL-2R antagonist is administered
without concurrent administration of an interferon-beta, such as
interferon-beta-1a or interferon-beta-1b. In one specific,
non-limiting example, ZENAPAX.RTM. (daclizumab) is administered
without concurrent administration of an interferon-beta, such as
interferon-beta-1a or interferon-beta-1b. In another specific,
non-limiting example, ZENAPAX.RTM. (daclizumab) is administered
without concurrent administration of other additional
pharmaceutical agents to treat multiple sclerosis, such as other
immunosuppressive agents.
[0124] In another embodiment, a therapeutically effective amount of
an IL-2 receptor antagonist is administered in combination with an
interferon-beta, such as interferon-beta-1a or
interferon-beta-1b.
[0125] If the interferon-beta is interferon-beta 1b (e.g.,
Betaseron.RTM.), an exemplary dose is 0.25 mg by subcutaneous
injection every other day. However, higher or lower doses can be
used, for example from 0.006 mg to 2 mg daily, biweekly, weekly,
bimonthly or monthly. If the interferon-beta is interferon-beta 1a
and is Avonex.RTM., an exemplary dose is 30 .mu.g injected
intramuscularly once a week. However, higher or lower doses could
be used, for example 15 to 75 .mu.g daily, biweekly, weekly,
bimonthly or monthly. If the interferon-beta 1a is Rebif.RTM., an
exemplary dose is 44 .mu.g three times per week by subcutaneous
injection. However, higher or lower doses can be used, including
treatment daily, biweekly, weekly, bimonthly, or monthly.
Additionally, the dosage may be changed during the course of
therapy. For example, Rebif.RTM. can be administered at an initial
dose of 8.8 .mu.g for the first two weeks, then 22 .mu.g for the
next two weeks, and then at 44 .mu.g for the rest of the therapy
period. In specific embodiments, Avonex.RTM. can be administered at
a dose of 30 .mu.g per week or Betaseron.RTM. can be administered
at a dose of 0.25 mg every other day.
[0126] Administration of interferon-beta also can be performed on
strict or adjustable schedules. For example, interferon-beta is
administered once weekly, every-other-day, or on an adjustable
schedule, for example based on concentration in a subject. One of
skill in that art will realize that the particular administration
schedule will depend on the subject and the dosage being used. The
administration schedule can also be different for individual
subjects or change during the course of the therapy depending on
the subject's reaction. In specific examples, interferon-beta 1a is
administered every other week, or monthly.
[0127] The combined administration of the IL-2R antagonist and
interferon-beta includes administering interferon-beta either
sequentially with the IL-2R antagonist, i.e., the treatment with
one agent first and then the second agent, or administering both
agents at substantially the same time, i.e., an overlap in
performing the administration. With sequential administration a
subject is exposed to the agents at different times so long as some
amount of the first agent remains in the subject (or has a
therapeutic effect) when the other agent is administered. The
treatment with both agents at the same time can be in the same
dose, i.e., physically mixed, or in separate doses administered at
the same time.
[0128] In a particular embodiment interferon-beta 1a (e.g.,
Avonex.RTM.) is administered weekly via intramuscular injection.
The first week of therapy the subject receives an intravenous
infusion of the monoclonal antibody (e.g., ZENAPAX.RTM.) at the
same time as the interferon-beta 1a injection, with a second
humanized anti-Tac monoclonal antibody (e.g., ZENAPAX.RTM.)
infusion being administered two weeks later at the same time as the
interferon-beta 1a (e.g. Avonex.RTM.) injection. Thereafter the
humanized anti-Tac monoclonal antibody (e.g., ZENAPAX.RTM.) is
administered monthly at the same time as the weekly interferon-beta
1a injection. In another embodiment interferon-beta 1b (e.g.
Betaseron.RTM.) is administered every other day via subcutaneous
injection while the humanized anti-Tac monoclonal antibody (e.g.,
ZENAPAX.RTM.) is administered every other week for one month, and
then monthly, with the humanized anti-Tac monoclonal antibody
(e.g., ZENAPAX.RTM.) infusion not necessarily on the same day as
the interferon-beta 1b (e.g., Betaseron.RTM.) injection.
[0129] The IL-2R antagonist may also be used in combination with
one or more other drugs that may be active in treating multiple
sclerosis. These include, but are not limited to, Copaxone.RTM.,
corticosteroids such as prednisone or methylprednisolone;
immunosuppressive agents such as cyclosporine (or other calcineurin
inhibitors, such as Prograf.RTM.), azathioprine, Rapamune.RTM. and
Cellcept.RTM.; anti-metabolites such as methotrexate; and
antineoplastic agents such as mitoxantrone.
[0130] Treatment with the IL-2R antagonist, alone or in combination
with other agents, will on average reduce the number of gadolinium
enhanced MRI lesions by at least 30%. In one embodiment, the
gadolinium enhanced MRI lesions are reduced by at least about 50%
or by at least about 70%, such as a reduction of about 80%, about
90%, or by more than 95%, as compared to baseline measurements for
the same subjects or to measurement in control subjects (e.g.
subjects not receiving the IL-2R antagonist). Similarly, treatment
with the IL-2R antagonist, alone or in combination with other
agents, will reduce the average number of MS exacerbations per
subject in a given period (e.g., 6, 12, 18 or 24 months) by at
least about 25%, such as at least about 40% or at least about 50%.
In one embodiment, the number of MS exacerbations is reduced by at
least about 80%, such as at least about 90%, as compared to control
subjects. The control subjects can be untreated subject or subjects
not receiving the IL-2R antagonist (e.g., subjects receiving other
agents). Treatment with the IL-2R antagonist, alone or in
combination with other agents, can also reduce the average rate of
increase in the subject's disability score over some period (e.g.,
6, 12, 18 or 24 months), e.g., as measured by the EDSS score, by at
least about 10% or about 20%, such as by at least about 30%, 40% or
50%. In one embodiment, the reduction in the average rate of
increase in the ESS score is at least about 60%, at least about
75%, or at least about 90%, or can even lead to actual improvement
in the disability score, compared to control subjects, such as
untreated subjects or subjects not receiving the IL-2R antagonist
but possibly receiving other agents. These benefits can be
demonstrated in one or more randomized, placebo-controlled,
double-blinded, Phase II or III clinical trials and will be
statistically significant (e.g., p<0.05).
[0131] The present disclosure is illustrated by the following
non-limiting Examples.
EXAMPLES
Example 1
Protocol for the Use of a Humanized IL-2R Antibody (ZENAPAX.RTM.)
to Treat Multiple Sclerosis
A. Objectives
[0132] A study was conducted to determine the efficacy of
ZENAPAX.RTM. therapy in subjects with multiple sclerosis who have
failed standard IFN-.beta. therapy by comparing the mean number of
Gd-enhancing lesions during the pre-treatment period to that of the
treatment period. This study was also demonstrated the safety and
tolerability of ZENAPAX.RTM. in subjects with multiple sclerosis
using clinical, MRI, and immunologic measures.
[0133] In order to assess the efficacy of ZENAPAX.RTM. therapy in
subjects with multiple sclerosis who had failed standard IFN-.beta.
therapy the following measures were used: [0134] 1. MRI measures
[0135] T2 lesion load, [0136] Volume of Gd-enhancing fefions,
[0137] Volume of T1 hypointensities (optional); [0138] 2. Clinical
measures, specifically, [0139] Change in EDSS, change in SRS
(Scripps Neurological Rating Scale) Relapse rate; 9-hole peg test
[0140] 3. Immunologic measures, specifically, [0141] Markers of Th1
and Th2 T cell lineages, as well as FACS analysis of various T cell
markers, [0142] Cytokine production by T cells in vitro, [0143]
Proliferation of T cells
[0144] For purposes of the study, failure to respond to standard
IFN-.beta. therapy was defined as a recurrence of Gd-contrasting
MRI lesions to at least half the mean of baseline monthly
Gd-contrasting lesions over 6 months before onset of IFN treatment
or primary non-responsiveness to IFN treatment or the presence of
clinical relapses during the last 12 months. The subjects tested
were primary IFN-.beta. non-responders, i.e., in the absence of
neutralizing antibodies against IFN-.beta., or secondary
non-responders, i.e., in the presence of neutralizing
antibodies.
B. Study Outline
[0145] Subjects were enrolled following completion of all
pre-screening (Week -8) procedures provided that failure to
standard IFN-.beta. therapy was documented. After enrollment,
subjects underwent three Gd-enhanced MRIs at 4-week intervals prior
to the first dose of study drug. Subjects with at least 2
Gd-enhancing lesions or greater in the 3 pre-treatment MRI scans
(an average of at least 0.67 Gd-enhancing lesions per scan) were
eligible to proceed to the treatment phase of the study. During the
treatment phase, subjects received seven IV infusions of 1 mg/kg
body weight anti-interleukin-2 receptor alpha subunit
(IL-2R.alpha.; ZENAPAX.RTM.), day 0, week 2, week 6, week 10, week
14, week 18 and week 22; total of 7 doses) for 5.5 months=22 weeks,
and continued to undergo Gd-enhanced MRIs at 4-week intervals.
Following the last dose of study drug, subjects were monitored for
12 weeks. Some subjects continued to receive standard IFN-.beta.
therapy throughout the trial, while IFN-.beta. therapy was
discontinued in some subjects.
B. 1 Inclusion and Exclusion Criteria for Pre-Treatment
Screening
[0146] Candidates for the study met the following criteria at the
time of enrollment (Table 1):
TABLE-US-00001 TABLE 1 Inclusion Criteria 1) Between the ages of 18
and 65 years, inclusive. 2) Subjects with relapsing-remitting or
secondary progressive MS who had more than one relapse within 18
months preceding study enrollment. Subjects had at least 2
Gd-enhancing lesions or greater in the 3 pre-treatment MRI scans
(an average of at least 0.67 Gd-enhancing lesions per scan). 3)
EDSS score between 1 - 6.5, inclusive. Subjets who have failed
standard IFN-.beta. therapy. IFN-.beta. treatment failures were
specified as follows: Individuals who had received IFN treatment
for at least 6-12 months and had more than one exacerbation during
the past year which required treatment by intravenous steroids.
Subjects currently enrolled in a protocol for the administration of
both ZENAPAX .RTM. and IFN-.beta. were eligible for rollover into
either a dose escalation phase or the ZENAPAX .RTM. single therapy
phase after 5.5 months of therapy. Those subjects that had a 75% or
greater decrease in lesion activity were eligible for the ZENAPAX
.RTM. single dose phase, while those subjects that failed to
achieve at least a 75% reduction in lesion activity were eligible
for the dose escalation phase.
Candidates were excluded from study entry if any of the exclusion
criteria existed at the time of enrollment (Table 2):
TABLE-US-00002 TABLE 2 Exclusion Criteria Medical History 1)
Diagnosis of primary progressive MS, defined as gradual progression
of disability from the onset without relapses. 2) Abnormal
screening/pre-treatment blood tests exceeding any of the limits
defined below: Alanine transaminase (ALT) or aspartate transaminase
(AST) > two times the upper limit of normal (i.e., >2 .times.
ULN) Total white blood cell count <3,000/mm.sup.3 CD4.sup.+
count <320/mm.sup.3 Platelet count <80,000/mm.sup.3
Creatinine >2.0 mg/dL 3) Concurrent, clinically significant (as
determined by the investigator) cardiac, immunologic, pulmonary,
neurologic, renal, and/or other major disease. 4) Any
contraindication to monoclonal antibody therapies. 5) Subjects who
were HIV+. Treatment History 5) If prior treatment was received,
the subject were off treatment for the required period prior to
enrollment (see insert). Restrictions on Treatments Time Required
off Agent Prior Agent to Enrollment Glatiramer acetate (Copaxone
.RTM.), cyclophosphamide 26 weeks (Cytoxan .RTM.) IV Ig,
azathioprine (Imuran .RTM.), methotrexate, plasma 12 weeks
exchange, cyclosporine, oral myelin, cladribine, mitoxantrone
Corticosteroids, ACTH 8 weeks 6) Prior treatment with any other
investigational drug or procedure for MS. 7) History of alcohol or
drug abuse within the 5 years prior to enrollment. 8) Male and
female subjects not practicing adequate contraception. 9) Female
subjects who are not post-menopausal or surgically sterile who are
not using an acceptable method of contraception. Acceptability of
various methods of contraception will be at the discretion of the
investigator. Written documentation that the subject is
post-menopausal or surgically sterile must be available prior to
study start. 10) Unwillingness or inability to comply with the
requirements of this protocol including the presence of any
condition (physical, mental, or social) that is likely to affect
the subject's returning for follow-up visits on schedule. 11)
Previous participation in this study. 12) Breastfeeding
subjects.
[0147] A cohort of subjects entered the protocol described that
failed to show at least a 75% reduction in lesion frequency on
interferon and ZENAPAX.RTM. had the dose of ZENAPAX.RTM. increased
to 2 mg/kg in order to assess whether this dose of ZENAPAX.RTM. is
safe and well tolerated.
B.2 Treatment Agent and Infusion
[0148] Subjects enrolled in the study were given ZENAPAX.RTM. at
designated time points. The anti-Tac formulation contains 5 mg/ml
ZENAPAX.RTM. and 0.2 mg/ml. Polysorbate-80 in 67 mM phosphate
buffer, pH adjusted to 6.9. The formulation was packaged in a 5 ml
volume of appropriate size in flint glass vials. The agent was
stored at 2-8.degree. C. away from light. The appropriate quantity
of antibody solution at 5 mg/ml was diluted with 50 ml of normal
saline in a mini-bag. The diluted antibody was stored for 24 hours
at 2-8.degree. C. before administration. Therapy was administered
intravenously at a dose of 1 mg of ZENAPAX.RTM. per kg, as a 15
minute intravenous infusion. At the end of the infusion, the line
was flushed with 10 ml saline. The time of administrations and vial
signs were recorded on the infusion sheet. Vital-signs were taken
and recorded pre-infusion, immediately post infusion, and 15
minutes after the infusion is completed. The maximum dose of the
study drug was 20 ml, which is the equivalent to 200 mg of
antibody.
[0149] The vials of ZENAPAX.RTM. were vented prior to withdrawing
the contents. A venting needle, or a 20-22G needle attached to a
syringe (without the plunger) was, in some cases, be inserted into
the vials. Air was not injected into the headspace of the vials or
into the solution. After ventilation, the contents were withdrawn
from each of the vials into a syringe (with a 20-22 G needle) large
enough to hold the total calculated dose of ZENAPAX.RTM..
[0150] A syringe and needle was used to remove a volume of saline
equivalent to the calculated dose of ZENAPAX.RTM. (plus any
overfill) from a 150 ml container of sterile water, although
alternatively normal saline (0.9% NaCl USP) can be used. The
contents of the syringe holding the ZENAPAX.RTM. was injected into
the container. The contents were mixed by gently rocking the
container for about 20 seconds, such that the reconstituted product
was ready for infusion. The diluted ZENAPAX.RTM. solution was
stored at room temperature. The diluted solution was completely
infused within 4 hours after dilution.
[0151] Standard clinical practice for ensuring sterility of the
infusion material was followed. ZENAPAX.RTM. was administered by a
dedicated intravenous line at a constant rate over 15 minutes and
was followed by a normal saline flush. To control the rate, an
infusion pump was used. Volume of the saline flush was no less than
the residual volume of the solution retained in the IV tubing. New
tubing was used for each infusion.
[0152] Subjects were required to receive their infusion within 7
days of scheduled appointments. Subjects were examined at each
study visit prior to initiation of the infusion. All subjects used
accepted birth control methods for six months after completion of
treatment, and female subjects were not pregnant.
[0153] ZENAPAX.RTM. was administered as a 15-minute IV infusion of
1 mg/kg (based on ideal body weight) at day 0, week 2, week 6, week
10, week 14, week 18 and week 22; total of 7 doses) for 5.5
months=22 weeks after all other required procedures at each visit
was performed. MRIs occurred within 7 days prior to study drug
dosing. In a few subjects, two additional infusions at 6 weeks
intervals were given at weeks 28 and 34.
C. Treatment Schedule, Including Tests and Evaluations
[0154] The sample size for the initial study disclosed herein, 10
treated subjects, was chosen according to extensive experience
during MS natural history studies, an IFN-.beta.1b MRI study and
statistical evaluation of these data.
[0155] Tests were performed according to the schedule shown in
Table 3.
TABLE-US-00003 TABLE 3 Test and Evaluation Schedule 1. Week -8
(Screening Visit) Unless otherwise specified, the tests and
evaluations were performed within 7 days prior to the subject's
first MRI to determine subject eligibility: A complete medical
history. Vaccination status. A complete physical examination
including measurement of vital signs and body weight. Chest x-ray.
ECG. Blood chemistries. Hematology: CBC with differential and
platelet count. CD4.sup.+ count. Immunologic measures. Urine
pregnancy test for women of child-bearing potential. Testing for
antibodies to ZENAPAX .RTM. (serum stored until analysis).
EDSS/SRS/9-hole peg test. MRI (performed after all other screening
procedures were completed). Skin test with multiple recall
antigens; alternatively performed at week -4 Serum for
determination anti-IL-2R.alpha. serum levels (stored until
analysis) HIV-I status 2. Week -4 Vital signs. Immunologic
measures. Urine pregnancy test for women of child-bearing
potential. EDSS/SRS/9-hole peg test. MRI. Rubeola titer, EBNA titer
(standard). 3. Between Weeks -4 and 0 Optional lumbar puncture.
Lymphacytopheresis. 4. Week 0 Vital signs. Total lymphocyte count
(results were available prior to dosing). Blood chemistries.
Hematology: CBC with differential and platelet count. CD4.sup.+
count. Urine pregnancy test for women of child-bearing potential.
EDSS/SRS/9-hole peg test. MRI. Immunologic measures. Testing for
antibodies to ZENAPAX .RTM. (serum stored until analysis). Serum
for determination anti-IL-2R.alpha. serum levels (stored until
analysis). Subject received first dose of study drug. 5. Week 2
Vital signs. Total lymphocyte count (results were available prior
to dosing). Blood chemistries. Hematology: CBC with differential
and platelet count. CD4.sup.+ count. Immunologic measures. Urine
pregnancy test for women of child-bearing potential.
EDSS/SRS/9-hole peg test. MRI. Infusion of ZENAPAX .RTM. Testing
for antibodies to ZENAPAX .RTM. (serum stored until analysis).
Serum for determination anti-IL-2R.alpha. serum levels (stored
until analysis) 6. Week 4 Vital signs EDSS MRI Testing for
antibodies to ZENAPAX .RTM. (serum stored until analysis). Serum
for determination anti-IL-2R.alpha. serum levels (stored until
analysis) 7. Week 6 Vital signs. Total lymphocyte count (results
were available prior to dosing). Blood chemistries. Hematology: CBC
with differential and platelet count. CD4.sup.+ count. Immunologic
measures. Urine pregnancy test for women of child-bearing
potential. EDSS/SRS/9-hole peg test. MRI. Infusion of ZENAPAX
.RTM.. Testing for antibodies to ZENAPAX .RTM. (serum stored until
analysis). Serum for determination anti-IL-2R.alpha. serum levels
(stored until analysis) 8. Week 10 Vital signs. Total lymphocyte
count (results were available prior to dosing). Blood chemistries.
Hematology: CBC with differential and platelet count. CD4.sup.+
count. Immunologic measures. Urine pregnancy test for women of
child-bearing potential. Testing for antibodies to ZENAPAX .RTM.
EDSS/SRS/9-hole peg test. MRI. Infusion of ZENAPAX .RTM.. Testing
for antibodies to ZENAPAX .RTM. (serum will be stored until
analysis). Serum for determination anti-IL-2R.alpha. serum levels
(stored until analysis) 9. Week 14 Vital signs. Total lymphocyte
count (drawn so that results were available prior to dosing). Blood
chemistries. Hematology: CBC with differential and platelet count.
CD4.sup.+ count. Immunologic measures. Urine pregnancy test for
women of child-bearing potential. EDSS/SRS/9-hole peg test. MRI.
Infusion of ZENAPAX .RTM.. Testing for antibodies to ZENAPAX .RTM.
(serum stored until analysis). Serum for determination
anti-IL-2R.alpha. serum levels (stored until analysis) 10. Week 18
Vital signs. Total lymphocyte count (results were available prior
to dosing). Blood chemistries. Hematology: CBC with differential
and platelet count. CD4.sup.+ count. Immunologic measures. Urine
pregnancy test for women of child-bearing potential.
EDSS/SRS/9-hole peg test. MRI. Infusion of ZENAPAX .RTM.. Testing
for antibodies to ZENAPAX .RTM. (serum stored until analysis).
Serum for determination anti-IL-2R.alpha. serum levels (stored
until analysis) 11. Week 22 Vital signs. Total lymphocyte count
(results were available prior to dosing). Blood chemistries.
Hematology: CBC with differential and platelet count. CD4.sup.+
count. Immunologic measures. Urine pregnancy test for women of
child-bearing potential. EDSS/SRS/9-hole peg test. MRI. Infusion of
ZENAPAX .RTM.. Skin test with multiple antigens (see Appendix I)
Testing for antibodies to ZENAPAX .RTM. (serum stored until
analysis). Serum for determination anti-IL-2R.alpha. serum levels
(stored until analysis) 12. Week 26 Vital signs. Blood chemistries.
Hematology: CBC with differential and platelet count. CD4.sup.+
count. Urine pregnancy test for women of child-bearing potential.
EDSS/SRS/9-hole peg test. MRI. Immunologic measures. Testing for
antibodies to ZENAPAX .RTM. (serum stored until analysis). Serum
for determination anti-IL-2R.alpha. serum levels (stored until
analysis) Optional lumbar puncture. Lymphocytopheresis. 13. Between
Weeks 30 and 34 Immunologic Measures Others (Chest X-ray, EKG)
EDSS/SRS/9-hole peg test. MRI Rubeola titer/EBNA titer (standard).
Testing for antibodies to ZENAPAX .RTM. (serum stored until
analysis). Serum for determination anti-IL-2R.alpha. serum levels
(stored until analysis)
As indicated above, a few subjects received two more ZENAPAX.RTM.
infusions at weeks 28 and 34 and then the same post-treatment
follow-up (see Table 3, #12 and #13).
Example 2
Outcome Measures: Data Analysis
[0156] In addition to the tests and evaluations listed in Table 3,
the following clinical efficacy assessments were performed during
the study: [0157] 1. EDSS/SRS/9-hole peg test--measures of
disability [0158] 2. Number of relapses. Relapses are defined as
new or recurrent neurologic symptoms, not associated with fever or
infection, lasting for at least 48 hours and accompanied by
objective neurological findings upon examination. Clinical safety
was assessed by neurologic status, general physical examination,
measurement of vital signs (temperature, heart rate, and blood
pressure). Adverse events were collected throughout the study.
[0159] The following laboratory efficacy assessments were also
performed during the study: [0160] 1. Brain MRI with and without
gadolinium enhancement; additional MRI parameters [0161] 2.
Immunologic measures. The specific laboratory parameters evaluated
in this study were as follows: [0162] 1. MRI activity as monitored
by the physicians [0163] 2. Blood chemistry: creatinine, total
bilirubin, ALT, AST, alkaline phosphatase, and albumin. Rubella-
and Anti EBV-EBNA antibodies. [0164] 3. Hematology: complete blood
count with differential and platelet count. The safety assessments
were as follows: [0165] 1. Analysis of peripheral CD4.sup.+ subsets
was performed using flow cytometry with well-defined subset markers
for T lymphocytes. [0166] 2. Collection of 4 mL whole blood (to
obtain 2 mL of serum) for determination of antibody formation to
ZENAPAX.RTM.. [0167] 3. Safety in terms of influence of
ZENAPAX.RTM. on CNS inflammatory disease activity was documented
and followed by MRL. An unexpected and potentially alerting
increase in MRI activity was defined as a greater than 3-fold
increase in subjects with mean pre-treatment Gd-lesion loads of
<10 lesions/month. In subjects with mean pre-treatment Gd-lesion
loads <3 lesions/month, a >ten-fold increase raised safety
concerns. If a single new lesion with >5 cm in any diameter
develops, this was considered as a sign of toxicity.
[0168] No concerns as to ZENAPAX.RTM.-related adverse events arose
during the course of these studies.
[0169] The study disclosed herein demonstrated the efficacy of
ZENAPAX.RTM. therapy in subjects with multiple sclerosis by
comparing the mean number of Gd-enhancing lesions during the
pre-treatment period to that of the treatment period. The primary
efficacy endpoint is the number of Gd-enhancing lesions.
[0170] The analyses on the primary endpoint included the following:
[0171] comparison of the mean number of lesions during the
pre-treatment period (Weeks -8, -4, 0) to the mean number of
lesions during the treatment period (Weeks 0 to 22) [0172]
comparison of the mean number of lesions during the pre-treatment
period (Weeks -8, -4, 0) to the mean number of lesions during the
last 12 weeks of the treatment period (Weeks 10-22)
[0173] These comparisons were performed using a paired t-test or
the Wilcoxon signed rank test, depending on the distribution of the
data. The means were based on non-missing evaluations.
[0174] This study also demonstrates the efficacy of ZENAPAX.RTM.
therapy in subjects with multiple sclerosis using the following
measures: [0175] 1. MRI measures [0176] T2 lesion load, [0177]
Volume of Gd-enhancing lesions, [0178] Volume of T1 hypointensities
(optional); [0179] 2. Clinical measures, specifically, [0180]
Change in EDSS/SRS/9-hole peg test [0181] Relapse rate; [0182] 3.
Immunologic measures, specifically, [0183] Markers of Th1 and Th2 T
cell lineages, as well as FACS analysis of various T cell, B cell,
and monocyte subset markers, [0184] Cytokine production by T cells
in vitro
T2 Lesion Load
[0185] The analyses on T2 lesion load included the following:
[0186] comparison of the mean volume of T2 lesions during the
pre-treatment period (Weeks -8, -4, 0) to the mean volume of T2
lesions during the treatment period (Weeks 0-22) [0187] comparison
of the mean volume of T2 lesions during the pre-treatment period
(Weeks -8, -4, 0) to the mean volume of T2 lesions during the last
12 weeks of the treatment period (Weeks 10-22)
[0188] These comparisons were performed using a paired t-test or
the Wilcoxon signed rank test, depending on the distribution of the
data. The means were based on non-missing evaluations.
Volume of Gd-Enhancing Lesions
[0189] The analyses on volume of Gd-enhancing lesions included the
following: [0190] comparison of the mean volume of Gd-enhancing
lesions during the pre-treatment period (Weeks -8, -4, 0) to the
mean volume of Gd-enhancing lesions during the treatment period
(Weeks 0-22) [0191] comparison of the mean volume of Gd-enhancing
lesions during the pre-treatment period (Weeks -8, -4, 0) to the
mean volume of Gd-enhancing lesions during the last 12 weeks of the
treatment period (Weeks 10-22) [0192] These comparisons were
performed using a paired t-test or the Wilcoxon signed rank test,
depending on the distribution of the data. The means were based on
non-missing evaluations.
Volume of T1 Hypointensities
[0193] The analyses on volume of T1 hypointensities included the
following: [0194] comparison of the mean volume of T1
hypointensities during the pre-treatment period (Weeks -8, -4, 0)
to the mean volume of T1 hypointensities during the treatment
period (Weeks 0-22) [0195] comparison of the mean volume of T1
hypointensities during the pre-treatment period (Weeks -8, -4, 0)
to the mean volume of T1 hypointensities during the last 12 weeks
of the treatment period (Weeks 10-22) [0196] These comparisons were
performed using a paired t-test or the Wilcoxon signed rank test,
depending on the distribution of the data. The means were based on
non-missing evaluations.
EDSS
[0197] The change from baseline (Week 0) EDSS to Week 22 and Week
26 were determined. Also, change from baseline to week 22 and 26
for SRS and 9-hole peg test.
Relapses
[0198] The frequency of relapses over the 2 years prior to
receiving study drug were compared to the frequency of relapses on
study drug (Weeks 0 to 22).
Example 3
Outcome Measures: Immunologic Parameters
1. PBMC Cell Surface Expression Analyses
[0199] The analyses for the immunologic parameters were performed
using standard methods. For example, parallel quantitative analysis
of important markers for Th.sub.1/Th.sub.2 T cell development,
effector functions of MS T cells and markers for the biological
activity of the anti-Tac antibody with particular focus on T cell
activation (i.e. determination IL-2 expression, numbers of
CD4.sup.+ and CD3' T cells expressing IL-2R/CD25; in vitro
(proliferation to Tetanus toxoid; Flu-HA peptide 306-318) and in
vivo (skin test) recall responses to standard recall antigens) were
performed in treated subjects.
[0200] Specific Studies Included: [0201] 1. Analysis of changes in
subpopulations of white blood cells (polymorphonuclear cells,
monocytes, NK cells, LAK (lymphocyte-activated killer cells),
lymphocytes--including B-cells, CD4+ and CD8+ subsets of T cells,
NK-T cells, CD4+/CD25+ regulatory T cells) upon in vivo therapy
with daclizumab [0202] 2. Evaluating the changes in surface
expression of multiple activation markers, adhesion molecules,
costimulatory molecules, cytokine- and chemokine receptors etc:
CD95, CTLA-4, CD25 (IL-2R.alpha.-chain), CD122 (IL-2R.beta.-chain),
CD132 (IL-2R.gamma.-chain), CD45RA, CD45RO, CD71, OX-40, CCR5,
CXCR4, CD80, MHC-class II (HLA-DR, DQ, DP), TCR .alpha./.beta., TCR
.gamma./.delta., CD2, CD56, CD161 by flow cytometry. [0203] 3.
Evaluating proliferation of peripheral blood mononuclear cells
(PBMC) to different polyclonal and antigen-specific stimuli
(plate-bound anti-CD3, plate-bound anti-CD3+anti-CD28, IL-2, IL-4,
IL-7, IL-15, myelin basic protein (MBP), tetanus toxoid (TT) by
flow-cytometry based proliferation assay using
5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester
(5(6)-CFDA, SE). Cytokine production (i.e. IL-2, IL-4, IL-6, IL-8,
IL-10, IL-12, IFN-.gamma., Tumor necrosis factor (TNF)-.alpha.,
LT-.alpha., transforming growth factor (TGF)-.beta.) of PBMC
stimulated with these various stimuli by sandwich ELISA [0204] 4.
Longitudinal serum samples were collected from subjects in the
trial to investigate the changes in antibody subtypes,
myelin-specific antibodies, complement and complement-related
markers, markers of oxidative stress and markers indicative of
remyelination and repair. The data obtained demonstrated that in
vivo long-term administration of daclizumab leads to several
immunoregulatory changes. Without being bound by theory, these
changes likely contribute to the positive therapeutic effect of
this drug in MS. The changes noted included: [0205] Mild
(.about.10%) decrease in total lymphocyte count (including CD4+ and
CD8+ T cells and B-cells). [0206] Concomitant increase in
proportion of NK cells and NK-T cells--both of which were shown to
have highly immunoregulatory activity in various animal models of
autoimmunity and in human autoimmune disorders including MS,
insulin dependent diabetes mellitus (IDDM) and systemic lupus
erythematosus (SLE). [0207] Upregulation of CD122 (IL-2R
.beta.-chain) on cell surface of NK cells, NK T cells and
subpopulation of CD8+ lymphocytes that probably underlies increased
proliferative capacity of these cells to IL-2 (via intermediate
affinity IL-2R--i.e. CD122+CD132) and to IL-15 (which shares 2
signaling chains with IL-2R--i.e. CD122 and CD132). [0208] No
significant decrease in proliferation of T cells (both CD4+ and
CD8+ subsets) to strong polyclonal stimuli and to recall antigens
like TT. [0209] Increase of proliferation of NK cells,
.gamma./.delta.-T cells, NK-T cells and subpopulation of CD8+ T
cells to IL-15. 2. cDNA Microarray Expression Analyses
[0210] Daclizumab-induced immunomodulation upon long-term in-vivo
administration in MS subjects was evaluated by cDNA microarrays
performed on cryopreserved PBMC samples from baseline, treatment
and post-treatment phase of clinical trial. The data obtained
indicated that daclizumab therapy leads to upregulation of several
genes of interest, including: suppressor of cytokine signaling 5
(SOCS5), jun-D-proto-oncogene, protein tyrosine
phosphatase--receptor type, CD209-antigen-like, cell division cycle
14 (CDC14), CDC28-protein kinase regulatory subunit 2, and others.
Daclizumab therapy also leads to down-modulation of several genes
closely related to pro-inflammatory immunity, like IFN-.gamma. and
fibroblast growth factor 12 (FGF-12).
3. In Vitro Functional Experiments
[0211] Studies of cryopreserved PBMC samples from subjects in
clinical trial were performed in order to demonstrate in more
detail the changes observed in longitudinal prospective samples and
also add functional components to the observed structural changes:
[0212] a. Proliferation of PBMC was evaluated by flow-cytometry
based proliferation assay using 5-(and-6)-carboxyfluorescein
diacetate, succinimidyl ester (5(6)-CFDA, SE) to additional
stimuli: [0213] b. Plate-bound anti-CD3+anti-CD28-as a potent
polyclonal T-cell activating stimulus [0214] c. Keyhole limpet
hemocyanin (KLH)--as an antigen for CD4+ T cells that humans are
usually not exposed to, i.e. in order to investigate effect of
daclizumab on naive T cell priming [0215] d. Mixture of myelin
antigens myelin basic protein (MBP) (146-170), PLP (139-154), MOG
(35-55) and CNP (343-373)--in order to investigate effect of
daclizumab on autoreactive T cells [0216] e. LPS--as a potent
activator of monocytes and also CD4+/CD25+ regulatory T cells. In
addition, PBMC were seeded with and without exogenous addition of
daclizumab to demonstrate the differences between acute-in-vitro
effects of daclizumab and prolonged--in vivo effects of daclizumab
therapy. PBMC activated with these various stimuli were then
transferred after 72 h into IL-2 or IL-15 or IL-4-enriched media to
observe whether observed upregulation of CD122 and CD132 on cell
surface of these cells resulted in their increased functional
response to cytokines that signal via these signaling molecules.
Proliferation and cell expansion was measured at Day 6 and
functional phenotype of these expanded cells was assessed by
intracellular cytokine staining at Day 10 (measuring production of
IL-2, IL-4, IL-6 and IFN-.gamma.). In addition, supernatants were
collected for evaluation of monocytes-producing cytokines and
markers like IL-1.beta., IL-6, IL-10, TNF-.alpha. and NO. [0217] f.
Immunoregulatory properties of NK cells were assessed in more
detail, e.g., NK T cells and CD4+/CD25+ T regulatory cells upon
daclizumab therapy [0218] g. The gene expression profile from the
cDNA microarray was verified by real-time PCR and by functional
studies
[0219] Results of these experiments indicate that: [0220] The
"acute" effects of in vitro daclizumab administration were
different from prolonged effects of in vivo administration. More
profound inhibition of T cell proliferation to various stimuli was
noted acutely. [0221] Standard doses of daclizumab (i.e. 1 mg/kg/4
weeks IV) were sufficient to block CD25 Tac epitope on T cells, but
were not sufficient to fully block CD25 on activated monocytes.
Without being bound by theory, higher doses of daclizumab have been
needed in many clinical situations (e.g. transplantation) due to
insufficient block of CD25 by this dose. Hence, higher doses would
be useful in very active subjects with autoimmune diseases. [0222]
CD25 epitope was blocked by daclizumab upon in vitro
administration, but the molecule persists on cell surface of cells
in same numbers. However, upon prolonged in vivo administration of
daclizumab, this molecule is downmodulated from the cell surface of
both CD4+ and CD8+ T cells. [0223] Daclizumab administration
influenced T cell priming: CD4+ T cells responding to naive antigen
like KLH produce higher amounts of IL-4 and lower amounts of
IFN-.gamma. following daclizumab treatment. Without being bound by
theory, the effect on T cell priming is believed to control the
pro-inflammatory versus anti-inflammatory balance in MS and other
autoimmune diseases. [0224] Proliferation of T cells and their
functional response to complementary cytokines sharing signaling
chains with IL-2R (i.e. IL-15, IL-4, IL-7 and others) was enhanced
upon daclizumab therapy. [0225] Results also indicate monocyte
activation is modulated upon daclizumab therapy as monocytes
produced lower amounts of cytokines and had a greater response to
IL-4. [0226] Proliferation of CD4+/CD25+ T regulatory cells was
enhanced upon daclizumab therapy (demonstrated with LPS, which
stimulates this T cell subtype via Toll-4 receptor).
Example 4
Subject Assessment
[0227] The mean number of contrast-enhancing lesions between weeks
10-22 (3 months, 4 MRI scans) on combination therapy with weeks
42-62 (5 months, 6 MRI scans) on monotherapy was analyzed. Weeks
10-22 (3 months, 4 MRI scans) on combination therapy was compared
with the entire time (weeks 24-62; 9 months and 10 MRI scans) on
monotherapy.
[0228] The treatment response with single ZENAPAX.RTM. therapy was
considered partial if a reduction of contrast-enhancing lesions
from the baseline treatment, i.e. when subjects were on IFN-.beta.
alone, of >60% was not reached. If a reduction of
contrast-enhancing lesions from baseline of >0, but <60% was
reached, ZENAPAX.RTM. monotherapy was considered partially active.
If disease activity returned to baseline levels, ZENAPAX.RTM.
monotherapy was considered to have failed. However, none of these
outcomes was detected.
[0229] Subjects entering the single therapy phase had lesion
activity assessed monthly. The number of new lesions was evaluated
following each monthly study. If the mean of lesion number over
months 5, 6, 7 and 8 was 50% or less than the 3 months prior to
entering monotherapy, efforts were made to continue the subjects on
ZENAPAX.RTM. therapy until month 10 (week 62) on monotherapy (for
one more year).
[0230] Results from two subjects are shown in FIGS. 1 and 2. The
number of new lesions was assessed by identifying on a single scan
the number of brain lesions that were not previously identified. In
addition the total number of lesions was assessed. These lesions
included contrast enhancing lesions that persist for 1-2 months.
Furthermore, the supertotal number of lesions was assessed. These
included lesions that appeared on more than one scan of the
subject's brain, and provides an indirect measure of the lesion
volume, i.e. via the appearance of one lesion on multiple MRI
slices (supertotal of lesions).
[0231] As indicated in the FIGS. 1 and 2, treatment with
ZENAPAX.RTM. alone (in the absence of IFN-.beta.) resulted in a
dramatic decrease in the number of total lesions. No new lesions
were detected over a period of 5.5 months in any subject treated
with ZENAPAX.RTM. alone (in the absence of IFN-.beta.).
[0232] The data obtained during the last four months of treatment
were compared to four months of baseline treatment. Thus, for each
subject, the results obtained during the period of treatment with
ZENAPAX.RTM. alone (in the absence of IFN-.beta.) were compared to
the results obtained during treatment with ZENAPAX.RTM. and
IFN-.beta.. New Gd lesion number was diminished by 85.95%
(p=0.016). Total number of contrast enhancing lesions was decreased
by 85.75% (p=0.004). The Gd lesion volume was reduced by 87%
(p=0.014). The supertotal number of Gd enhancing lesions were
reduced by 87.4% (p=0.008). The 9-hole peg test was reduced by
5.36% (p=0.004). The annualized relapse rate (number of relapses
per subject per year) was reduced by 88.9% (p=0.047). The SRS was
also reduced by 10.61% (p=0.035). All other measures improved by
did not reach statistical significance. Thus, the primary outcome
was significantly improved when the subjects were treated with
ZENAPAX.RTM. alone.
Example 5
Dose Escalation
[0233] If subjects on the combination of IFN-.beta. and
ZENAPAX.RTM. showed a less than 75% reduction of disease activity
compared to the baseline on IFN-.beta. alone, their ZENAPAX.RTM.
dose was increased to 2 mg/kg (monthly).
[0234] A subject entering the dose escalation was assessed after
three months of therapy on the increased dose. No toxicity was
noted over an 8.5 month trial period. The subject was treated with
2 mg/kg of ZENAPAX.RTM. every other week (4 times the dose
described above). The subject responded to ZENAPAX.RTM. therapy
with a >60% reduction of contrast enhancing lesions.
Example 6
Combined Administration of IFN-.beta. and ZENAPAX.RTM.
[0235] This example illustrates the effects of the combined
administration of interferon-beta and an IL-2R antagonist in
subjects having relapsing-remitting or secondary-progressive
multiple sclerosis. The protocol is generally shown in Example 1,
and is summarized below.
Inclusion Criteria
[0236] Subjects included in the trial were diagnosed with either
relapsing-remitting or secondary-progressive multiple sclerosis;
were between the ages of 16-65; scored between 1 and 6.5 on the
EDSS; failed to respond to interferon-beta treatment alone as
demonstrated by one or more exacerbations in the 18 months prior to
enrollment, an increase of 1 point or more on the EDSS over 18
months of treatment, or persistence or reoccurrence of contrast
enhancing lesions on brain MRI to at least one-half the mean of
baseline monthly contrast enhancing lesions over a 6-month baseline
period measured prior to the beginning of interferon-beta therapy;
and must have had at least 3 gadolinium enhancing lesions in the
first 3 pre-combination therapy MRI scans.
Exclusion Criteria
[0237] Subjects were excluded from the trial if they were diagnosed
with primary-progressive MS; pre-treatment blood tests were
abnormal; diagnosed with a concurrent clinically significant major
disease; contraindications to monoclonal antibody therapies were
observed; determined to be positive for HIV; treated with
glatiramer acetate or cyclophosphamide in the 26 weeks prior to the
trial, or treated with intravenous immunoglobulin (IVIg),
azathioprine (AZA), methotrexate (MTX), cyclosporin,
cyclophosphamide (CTC), cladribine, or mitox in the 12 weeks prior
to the trial, or treated with corticosteroids or
adrenocorticotrophic hormone (ACTH) in the 8 weeks prior to the
trial, or treated with any other investigational drug or procedure
for MS; not practicing adequate contraception; or
breastfeeding.
Course of Treatment
[0238] Ten subjects (one additional one under the abovementioned
single subject exemption with a higher dose) participated in the
trial of the combination therapy. For each subject a baseline
3-month period of treatment with interferon-beta (Avonex.RTM. or
Betaseron.RTM.) was established. Avonex.RTM. was administered as
indicated in the prescribing information supplied by the
manufacturer at a dose of 30 g injected intramuscularly once a
week. Betaseron.RTM. was administered as indicated in the
prescribing information supplied by the manufacturer at a dose of
0.25 mg injected subcutaneously every other day. Four MRI scans
were performed during the baseline period to determine a baseline
number of contrast enhancing lesions, one at the beginning of the
period and then at the end of each month of the baseline period
with the fourth just prior to the beginning of the combination
therapy. Subjects were also evaluated on the EDSS, the Scripps
Neurologic Rating Scale (NRS), and various ambulation and other
motor skill tests.
[0239] Combined therapy began after the 3-month baseline was
established. Interferon-beta treatment was continued and, in
addition, anti-Tac (ZENAPAX.RTM.) was administered for 5.5 months.
During the first month of the combined administration ZENAPAX.RTM.
was administered every other week and thereafter ZENAPAX.RTM. was
administered once a month. ZENAPAX.RTM. was administered
intravenously in the manner described in the manufacturer's
prescribing information at a dose of 1 mg/kg of body weight. One
subject received a dose of 2 mg/kg every other week after showing
no response to the 1 mg/kg dose. MRI scans were performed during
the combined treatment period to determine changes in the number of
contrast enhancing lesions, one every two weeks for the first six
weeks of treatment, and thereafter monthly for a total of 8 MRI
scans. On the same schedule subjects were also evaluated on the
EDSS, the Scripps NRS, and various ambulation and other motor skill
tests.
Results
[0240] The combined administration of interferon-beta and
ZENAPAX.RTM. led to almost complete cessation of disease activity
and clinical improvement in seven of eight subjects. As can be seen
in FIG. 3, seven of eight subjects had either fewer or at least no
increase in both new and total contrast enhancing lesions under the
combination therapy as compared to the baseline period. As shown in
FIG. 4A, four of eight subjects also demonstrated improvement on
the EDSS under the combination therapy as compared to the baseline
period. As shown in FIG. 4B, seven of eight subjects demonstrated
improvement on the Scripps NRS. Referring to FIG. 4A, five of eight
subjects demonstrated improved ambulation on the ambulation index.
As shown in FIG. 5B, five of eight subjects either improved or had
no change in a timed 20 m walk. As shown in FIG. 6A, all subjects
demonstrated improved times with their dominant hand on the peg
hole test. As shown in FIG. 6B, five of eight subjects also
improved with their non-dominant hand on the peg hole test.
Example 7
Effects on T Cells in Combination Therapy
[0241] This example demonstrates the saturation of the Tac epitope
following combination therapy and the parallel decrease in T-cell
proliferation as compared to the baseline period.
[0242] Saturation of the Tac epitope was studied by flow cytometry.
The combined administration of interferon-beta with 1 mg/kg of
ZENAPAX.RTM. caused complete saturation of the Tac epitope on
CD4+/CD25+ and CD8+/CD25+ T-cells (FIG. 7).
[0243] Proliferation of activated T-cells was measured by
carboxyfluorescein succinimidyl ester (CFSE) fluorescence cell
labeling and assessing the number of mitoses in CFSE-labeled cells
by flow cytometry. As shown in FIG. 8A, six of eight subjects
demonstrated decreased proliferation of CD4 T-cells. Referring to
FIG. 8B, all subjects demonstrated a decrease in the proliferation
of CD8 T cells as compared to the baseline period.
Example 8
Upregulation of CTLA-4
[0244] This example demonstrates the unexpected upregulation of
CTLA-4 caused by the combined administration of interferon-beta and
an IL-2R antagonist.
[0245] CLTA-4 surface expression was measured by utilizing
antibodies against CTLA-4 and flow cytometry. For each measurement
of CTLA-4 surface expression, first, a 5 milliliter (ml) tube of
whole blood in ethylene diamine tetra-acetic acid (EDTA) was
obtained from each subject. Then, 42 ml of 1.times. lysing solution
(4.2 ml 10 lysing solution+37.8 ml H.sub.2O) was prepared from
10.times. stock prepared by dissolving in 1 liter of distilled
water: 89.9 g NH.sub.4Cl, 10.0 g KHCO3, 370.0 mg tetrasodium EDTA;
and adjusting the solution to pH 7.3. 3 ml of blood was transferred
by pipette into the 42 ml of 1.times. lysing solution (in 50 ml
Falcon tubes). The mixture was allowed to stand at room temperature
for 3-5 minutes. It was then centrifuged at 300.times. gravity for
5 minutes at room temperature. The supernatant was aspirated and
the pellet was resuspended in 30 ml of cold X-vivo media. The
resuspended mixture was centrifuged at 300.times. gravity for 5
minutes at 2-8.degree. C., the supernatant was aspirated, and the
pellet was resuspended in 2.5 ml of protein-enriched phosphate
buffered saline (PBS) (10 ml of fetal calf serum (FCS) in 500 ml of
1.times.PBS). This cell suspension was divided into 200 .mu.l
aliquots in a 96 well plate, then centrifuged at 300.times. gravity
for 5 minutes. The supernatant was discarded. Staining was
performed by adding 10 microliter (.mu.L)/well of prepared
anti-CTLA-4 antibody mixture. The plate was then incubated for 30
minutes on ice in a dark container. Each well was washed with 200
.mu.l of cold wash-buffer--mixed gently, and spun at 1000 rpm.
Supernatants were removed and each well washed another 2 times with
200 .mu.l of wash-buffer. After the last wash, the pellet was
resuspended in 200 .mu.l of staining buffer and analyzed by
Fluorescence-Activated Cell Sorter (FACS)-Calibur. At least 10000
events gated on lymphocytes and 5000 events gated on monocytes were
acquired.
[0246] As shown in FIG. 9, seven of eight subjects demonstrated
significant upregulation of CTLA-4 during the combined therapy as
compared to the baseline period.
[0247] It will be apparent that the precise details of the methods
or compositions described may be varied or modified without
departing from the spirit of the described invention. We claim all
such modifications and variations that fall within the scope and
spirit of the claims below.
Sequence CWU 1
1
21116PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Arg Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys
Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110 Thr Val Ser Ser 115 2106PRTHomo sapiens 2Asp Ile Gln
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met 20 25
30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
35 40 45 Thr Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg
Ser Thr Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu
Val Lys 100 105
* * * * *