U.S. patent application number 14/668036 was filed with the patent office on 2016-02-18 for methods for the treatment of autoimmune disorders using immunosuppressive monoclonal antibodies with reduced toxicity.
The applicant listed for this patent is MacroGenics, Inc.. Invention is credited to Ezio Bonvini, Leslie S. Johnson, Scott Koenig, Ronald Wilder.
Application Number | 20160046714 14/668036 |
Document ID | / |
Family ID | 38832885 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160046714 |
Kind Code |
A1 |
Koenig; Scott ; et
al. |
February 18, 2016 |
Methods for the Treatment of Autoimmune Disorders Using
Immunosuppressive Monoclonal Antibodies with Reduced Toxicity
Abstract
The present invention provides methods of treating, preventing,
slowing the progression of, or ameliorating the symptoms of T cell
mediated immunological diseases, particularly autoimmune diseases
(e.g., autoimmune diabetes (i.e. type 1 diabetes or
insulin-dependent diabetes mellitus (IDDM)) and multiple sclerosis)
through the use of anti-human CD3 antibodies. The antibodies of the
invention of the invention are preferably used in low dose dosing
regimens, chronic dosing regimens or regimens that involve redosing
after a certain period of time. The methods of the invention
provide for administration of antibodies that specifically bind the
epsilon subunit within the human CD3 complex. Such antibodies
modulate the T cell receptor/alloantigen interaction and, thus,
regulate the T cell mediated cytotoxicity associated with
autoimmune disorders. Additionally, the methods of the invention
provide for use of anti-human CD3 antibodies modified such that
they exhibit reduced or eliminated effector function and T cell
activation as compared to non-modified anti-human CD3
antibodies.
Inventors: |
Koenig; Scott; (Rockville,
MD) ; Wilder; Ronald; (Rockville, MD) ;
Bonvini; Ezio; (Rockville, MD) ; Johnson; Leslie
S.; (Darnestown, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MacroGenics, Inc. |
Rockville |
MD |
US |
|
|
Family ID: |
38832885 |
Appl. No.: |
14/668036 |
Filed: |
March 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11763434 |
Jun 14, 2007 |
9056906 |
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14668036 |
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60813903 |
Jun 14, 2006 |
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60871361 |
Dec 21, 2006 |
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Current U.S.
Class: |
424/133.1 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 19/02 20180101; A61K 45/06 20130101; A61P 3/10 20180101; A61P
21/00 20180101; A61P 13/00 20180101; C07K 16/2809 20130101; A61P
35/00 20180101; A61P 1/00 20180101; A61P 37/00 20180101; A61P 1/04
20180101; A61P 43/00 20180101; A61K 2039/505 20130101; A61P 25/04
20180101; A61P 1/10 20180101; A61P 27/16 20180101; A61K 39/3955
20130101; C07K 2317/24 20130101; A61P 27/02 20180101; A61P 37/06
20180101; A61P 25/00 20180101; A61K 2039/545 20130101; A61P 17/00
20180101; A61P 25/28 20180101; A61P 25/24 20180101; C07K 2317/41
20130101; C07K 2317/71 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating, slowing the progression of, or
ameliorating one or more symptoms of an autoimmune disorder in a
patient diagnosed with said disorder, said method comprising
administering to said patient a course of treatment with a
therapeutically effective amount of an anti-human CD3 antibody,
wherein the anti-human CD3 antibody is humanized OKT3.gamma.1
ala-ala; wherein less than 9000 .mu.g/m.sup.2 is administered
parenterally in total during said course of treatment; wherein said
treatment comprises a dosage regimen comprising doses of increasing
amounts of said antibody on at least the initial 4 days of said
course of treatment; wherein said autoimmune disorder is type 1
diabetes; and wherein said patient is in early stages of the
autoimmune disorder before about 80% of .beta.-cells have been
destroyed.
2-11. (canceled)
12. The method of claim 1, wherein the antibody is ChAglyCD3 or
visilizumab.
13-18. (canceled)
19. The method of claim 1, wherein the dose on day 1 is
approximately 51 .mu.g/m.sup.2, the dose on day 2 is approximately
103 .mu.g/m.sup.2, the dose on day 3 is approximately 207
.mu.g/m.sup.2, the dose on day 4 is approximately 413
.mu.g/m.sup.2, and the dose on subsequent days is approximately 826
.mu.g/m.sup.2.
20. (canceled)
21. The method of claim 1, wherein the dose on day 1 is
approximately 17 .mu.g/m.sup.2, the dose on day 2 is approximately
34.3 .mu.g/m.sup.2, the dose on day 3 is approximately 69
.mu.g/m.sup.2, the dose on day 4 is approximately 137
.mu.g/m.sup.2, and the dose on subsequent days is approximately 275
.mu.g/m.sup.2.
22-24. (canceled)
25. The method of claim 1 wherein each dose of said antibody is
administered in one infusion over a period of at least 18
hours.
26. The method of claim 25 in which said administration results in
serum levels of free antihuman CD3 antibody that do not exceed 200
ng/ml.
27-32. (canceled)
33. The method of claim 1 wherein said patient is redosed if the
average daily dose of insulin has increased by 50% or more, if
autoantibodies against one or more islet cell antigens are
detected, if islet cell antigen specific T cells are detected, if
.beta.-cell mass decreases by 50% or more, or if the incidence of
hypoglycemic or ketoacidosis episodes increases by 1 or more
incidents per day in said patient, at least 2 years after initial
administration of said course of treatment.
34-37. (canceled)
38. The method of claim 1, wherein said treatment results in an
increase in the average daily dose of insulin of no more than 0.2
U/kg/day six months after said treatment; in a HA1c of less than
7.5% one year after said treatment; or a C-peptide response to MMTT
twelve months after said treatment that is at least 90% of the
C-peptide response to MTT in said patient before said
treatment.
39-45. (canceled)
46. The method of claim 1, in which said administration does not
result in EBV-induced lymphoproliferative diseases or lymphocyte
counts less than 1000 lymphocytes/.mu.l serum.
47-50. (canceled)
51. The method of claim 55, wherein said patient had been
administered a 6 to 20 day course of treatment with said anti-human
CD3 antibodies prior to said additional round.
52-54. (canceled)
55. The method of claim 1, further comprising redosing said patient
with an additional round of said course of treatment.
56. A method for treating, or slowing the progression of, an
autoimmune disorder in a patient diagnosed with said disorder, or
for preventing or delaying the onset of an autoimmune disorder in a
patient predisposed thereto but not diagnosed with said disorder,
said method comprising: administering to said patient a dosage
regime with a prophylactically or therapeutically effective amount
of an anti-human CD3 antibody, said dosage regimen comprising
administration of a total daily prophylactically or therapeutically
effective amount of 35 .mu.g/kg or less of said antibody; wherein
said antibody is administered at doses that escalate over at least
the initial 4 days of said dosage regimen until the total daily
prophylactically or therapeutically effective amount of said
antibody is achieved; wherein the anti-human CD3 antibody is
humanized OKT3.gamma.1 ala-ala; wherein said autoimmune disorder is
selected from the group consisting of rheumatoid arthritis,
psoriasis, and type 1 diabetes; and wherein said patient is in
early stages of the autoimmune disorder, during which, when the
autoimmune disorder is rheumatoid arthritis, autoreactive cytotoxic
T-lymphocytes are detected in synovial tissues but clinical
symptoms of rheumatoid arthritis have not yet developed and, when
the autoimmune disorder is type 1 diabetes, less than about 80% of
.beta.-cells have been destroyed.
57. The method of claim 56, wherein said antibody is administered
at doses that escalate over the first half of the dosage regimen
until the total daily prophylactically or therapeutically effective
amount of said antibody is achieved.
58. The method of claim 56, wherein said antibody is administered
at doses that escalate by a factor of 2 until the total daily
prophylactically or therapeutically effective amount of said
antibody is achieved.
59. The method of claim 56, wherein the total daily
prophylactically or therapeutically effective amount is 30 .mu.g/kg
or less, 25 .mu.g/kg or less, 20 .mu.g/kg or less, 15 .mu.g/kg or
less, or 10 .mu.g/kg or less, of said antibody.
60. The method of claim 56, wherein said dosage regimen is 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, or 14 days.
61. The method of claim 56, wherein on at least the first day of
said dosage regimen, said antibody is administered in portions at
intervals of 6 hours, 8 hours, or 12 hours.
62. The method of claim 56, wherein said anti-human CD3 antibody is
administered by a route selected from the group consisting of
intravenous, intramuscular, subcutaneous, or oral
administration.
63. The method of claim 62, wherein said anti-human CD3 antibody is
administered by an intravenous route over a period of at least 30
minutes for each administration.
64. The method of claim 56, wherein said anti-human CD3 antibody is
administered in combination with an immunosuppressant.
65. The method of claim 56, wherein said autoimmune disease is Type
1 diabetes and wherein said anti-human CD3 antibody is administered
in combination with administration of insulin, exenatide, or
pramlinitide.
66. The method of claim 56, wherein said autoimmune disease is Type
1 diabetes and, prior to said dosage regime, the HA1c of said
patient is less than 7.5% or the C-peptide response of said patient
to a mixed-meal tolerance test (MMTT) results in a mean area under
the curve of at least 100 pmol/ml.
67. The method of claim 56, wherein said autoimmune disorder is
Type 1 diabetes and said patient is a child.
68. The method of claim 67, wherein said child is between 7 and 20
years of age.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Ser. No. 60/813,903, filed Jun. 14, 2006 and U.S.
Provisional Application Ser. No. 60/871,361, filed Dec. 21, 2006,
the contents of each of which are incorporated herein by reference
for all purposes.
1. INTRODUCTION
[0002] The present invention provides methods of treating,
preventing, slowing the progression of, or ameliorating the
symptoms of T cell mediated immunological diseases, particularly
autoimmune diseases (e.g., autoimmune diabetes (i.e. type 1
diabetes or insulin-dependent diabetes mellitus (IDDM)) and
multiple sclerosis) through the use of anti-human CD3 antibodies.
The antibodies of the invention of the invention are preferably
used in low dose dosing regimens, chronic dosing regimens or
regimens that involve redosing after a certain period of time. The
methods of the invention provide for administration of antibodies
that specifically bind the epsilon subunit within the human CD3
complex. Such antibodies modulate the T cell receptor/alloantigen
interaction and, thus, regulate the T cell mediated cytotoxicity
associated with autoimmune disorders. Additionally, the methods of
the invention provide for use of anti-human CD3 antibodies modified
such that they exhibit reduced or eliminated effector function and
T cell activation as compared to non-modified anti-human CD3
antibodies.
2. BACKGROUND OF THE INVENTION
[0003] 2.1 Autoimmune Diseases
[0004] Autoimmune diseases are caused when the body's immune
system, which normally defends the body against bacteria, viruses
and other infective agents, attacks "self tissue, cells and organs.
The mobilization of the immune system against such self targets is
termed autoimmunity. Although some autoimmunity is present in every
individual, rigid control systems suppress the self-recognizing
cells of the immune system to an extent that the autoimmunity is
normally asymptomatic. Disease states arise when there is some
interruption in the control system, allowing the autoimmune cells
to escape suppression, or when there is some change in a target
tissue such that it is no longer recognized as self. The mechanisms
underlying these changes are not well understood, but have been
theorized to be the result of aberrant immune stimulation in
genetically predisposed individuals.
[0005] Autoimmune diseases can be organ specific or systemic and
are provoked by differing pathogenic mechanisms. Organ specific
autoimmunization is characterized by tolerance and suppression
within the T cell compartment, aberrant expression of
major-histocompatibility complex (MHC) antigens, antigenic mimicry
and allelic variations in MHC genes. Systemic autoimmune diseases
usually involve polyclonal B cell activation and abnormalities of
immunoregulatory T cells, T cell receptors and MHC genes. Examples
of organ specific autoimmune diseases are diabetes, cutaneous
psoriasis, ulcerative colitis, hyperthyroidism, autoimmune adrenal
insufficiency, hemolytic anemia, multiple sclerosis and rheumatic
carditis. Representative systemic autoimmune diseases include
systemic lupus, erythematosus, rheumatoid arthritis, psoriatic
arthritis, Sjogren's syndrome polymyositis, dermatomyositis and
scleroderma.
[0006] Also, while not having an autoimmune disorder, organ
transplant recipients often experience similar symptoms and require
similar therapies to autoimmune patients. Immune system attacks on
the transplanted organ(s) can lead to organ failure or more serious
systemic complications, e.g., graft-vs.-host disease (GVHD) in
bone-marrow transplant recipients.
[0007] There is a clear need for improved strategies to treat
autoimmune disorders and/or to modulate immune response. Currently,
immune system disorders are treated with immunosuppressive agents
such as cortisone, aspirin derivatives, hydroxychloroquine,
methotrexate, azathioprine, cyclophsophamide and various biologics
such as anti TNF antibodies, and/or combinations of the foregoing.
The treatments are varyingly successful, dependent on the
individual patient and disorder. However, a dilemma in the use of
such general immunosuppressive therapies arises in that the greater
the immune-suppression, and thus the increased potential for
successful treatment of the autoimmune disorder, the more at-risk
the patient becomes for developing opportunistic infections.
Further, due to the compromised nature of the patient's immune
system, even a minor infection can rapidly become of serious
concern.
[0008] 2.1.1 Diabetes
[0009] Diabetes is typically classified as one of two types: type 1
or type 2 diabetes. Type 2 diabetes is a non-autoimmune disease
that is typically diagnosed in adults. It is a progressive disease
that develops when the body does not produce sufficient insulin or
fails to efficiently use the insulin it produces (a phenomenon
known as insulin resistance). Patients diagnosed with type 2
diabetes are typically over age 45, overweight (BMI of 25 or
higher) or obese (BMI of 30 or higher), physically inactive, have
hypertension (blood pressure of 140/90 mm Hg or higher in adults),
and have HDL cholesterol of 35 mg/dL or lower and/or triglyceride
level of 250 mg/dL.
[0010] Type 1 diabetes, also known as juvenile diabetes or
insulin-dependent diabetes mellitus, is an autoimmune disease that
is typically diagnosed in children (although Adult-Onset type 1
diabetes may be present in adults). Insulin-dependent diabetes
mellitus (IDDM) affects 15 million people in the United States with
an estimated additional 12 million people who are currently
asymptomatic, and, thus, unaware that they have this disease. Risk
factors for developing type 1 diabetes include presumptive genetic
factors, exposure to childhood viruses or other environmental
factors, and/or the presence of other autoimmune disorders.
Although the genetic factors associated with type 1 diabetes are
not fully understood, risks for the development of the disease have
been linked to both family history and ethnicity. For example, a
child that has a parent or sibling with type 1 diabetes has a
higher risk of developing type 1 diabetes than a child of
non-diabetic parents or with non-diabetic siblings. Further, the
genetic factors associated with the risk for developing type 1
diabetes appear to be linked to a particular HLA type: HLA-DR3 and
DR4 are associated with a higher risk in Caucasians; HLA-DR7 is
associated with a higher risk in people of African decent; and
HLA-DR9 is associated with a higher risk in people of Japanese
descent.
[0011] Unknown factors, including childhood viruses or exposure to
some other environmental factor (e.g., exposure to certain foods or
chemicals), are also theorized to potentiate or activate an
inherited genetic factor and cause the onset of type 1 diabetes.
Viruses that have been associated with type 1 diabetes include
coxsackie B virus, enteroviruses, adenoviruses, rubella,
cytomegalovirus, and Epstein-Barr virus. Last, the presence of
other autoimmune disorders, such as thyroid disease and celiac
disease, raises the risk of developing type 1 diabetes.
[0012] Type 1 Diabetes is caused by an autoimmune response in which
the insulin producing .beta.-cells of the pancreas (also known as
islet cells) are gradually destroyed. The early stage of the
disease, termed insulitis, is characterized by infiltration of
leukocytes into the pancreas and is associated with both pancreatic
inflammation and the release of anti-.beta.-cell cytotoxic
antibodies. As the disease progresses, the injured tissue may also
attract lymphocytes, causing yet further damage to the
.beta.-cells. Also, subsequent general activation of lymphocytes,
for example in response to a viral infection, food allergy,
chemical, or stress, may result in yet more islet cells being
destroyed. Early stages of the disease are often overlooked or
misdiagnosed as clinical symptoms of diabetes typically manifest
only after about 80% of the .beta.-cells have been destroyed. Once
symptoms occur, the type-1 diabetic is normally insulin dependent
for life. The dysregulation of blood-glucose levels associated with
diabetes can lead to blindness, kidney failure, nerve damage and is
a major contributing factor in the etiology of stroke, coronary
heart disease and other blood vessel disorders.
[0013] 2.1.2 Multiple Sclerosis
[0014] Multiple sclerosis (MS) is a chronic, often disabling
inflammatory disease of the central nervous system (CNS). MS is
typified pathologically by multiple inflammatory foci, plaques of
demyelination, gliosis, and axonal pathology within the brain and
spinal cord. Although the causal events that precipitate the
disease are unknown, converging lines of evidence suggest that the
disease is caused by a disturbance in immune function. This
disturbance permits cells of the immune system to attack myelin,
the insulating sheath that surrounds the axons located in the CNS
(i.e., the brain and spinal cord). When observed microscopically,
plaques consist of inflammatory cells, astroglial cells, edema, and
destroyed myelin fragments. When myelin is damaged, electrical
impulses cannot travel quickly along nerve fiber pathways in the
brain and spinal cord. Disruption of electrical conductivity
results in fatigue and disturbances of vision, strength,
coordination, balance, sensations, and bladder and bowel function.
Thus, typical symptoms include one or more of weakness or paralysis
in one or more extremities, tremor in one or more extremities,
muscle spasticity, muscle atropy, dysfunctional movement, numbness
or abnormal sensation in any area, tingling, facial pain, extremity
pain, loss of vision in one or both eyes, double vision, eye
discomfort, uncontrollable rapid eye movements, decreased
coordination, loss of balance, decreased ability to control small
or intricate movements, walking or gait abnormalities, muscle
spasms, dizziness, vertigo, urinary hesitancy, urinary urgency,
increased urinary frequency, incontinence, decreased memory,
decreased spontaneity, decreased judgment, loss of ability to think
abstractly, loss of ability to generalize, depression, decreased
attention span, slurred speech, difficulty speaking or
understanding speech, fatigue, constipation, hearing loss, and/or
positive Babinski's reflex. The symptoms recur periodically, last
days to months, then reduce or disappear. With each recurrence, the
symptoms may vary or be completely different as new areas are
affected.
[0015] Studies of the natural history of MS suggest that there are
different patterns of disease activity. Some patients have rare
attacks, some have frequent attacks, and others gradually but
steadily worsen without experiencing attacks. Patients who have
rare attacks and are minimally disabled ten years after being
diagnosed with MS are said to have benign MS. This group
constitutes only about 10-15% of the total MS patient population,
although there is some evidence suggesting that this course may be
more common than is currently appreciated. Patients who have
attacks with full or partial recovery and are otherwise stable
between attacks are defined as having relapsing-remitting MS.
Approximately 80-90% of patients with MS initially experience a
relapsing-remitting course. Of these, approximately 50% will have
difficulty walking 15 years after onset and 80% will ultimately
(after about 25 years) experience gradual progression of disability
with or without attacks. Patients who first experience
exacerbations and later experience gradual progression of
disability have secondary progressive MS. Approximately 10-15% of
MS patients do not experience an initial attack. Those patients who
gradually worsen after the appearance of the first symptom have
primary progressive MS. A few patients with primary progressive MS
will later experience an exacerbation. These patients have
progressive-relapsing MS.
[0016] There is as yet no cure for MS. Many patients do well with
no therapy at all, especially since many medications have serious
side effects and some carry significant risks. However, three forms
of beta interferon (AVONEX.RTM. (interferon beta-la),
BETASERON.RTM. (interferon beta-1b), and REBIF.RTM. (interferon
beta-la)) have now been approved by the Food and Drug
Administration for treatment of relapsing-remitting MS. Beta
interferon has been shown to reduce the number of exacerbations and
may slow the progression of physical disability. When attacks do
occur, they tend to be shorter and less severe. The FDA also has
approved a synthetic form of myelin basic protein copolymer I,
COPAXONE.RTM. (glatiramer acetate), for the treatment of
relapsing-remitting MS. Copolymer I has few side effects, and
studies indicate that the agent can reduce the relapse rate by
almost one third. An immunosuppressant treatment, NOVATRONE.RTM.
(mitoxantrone), is also approved by the FDA for the treatment of
advanced or chronic MS.
[0017] 2.2 T Cell Functionality in Diabetes and Other Autoimmune
Disorders
[0018] Destruction of .beta.-cells in diabetes, of myelin in
multiple sclerosis, or of the target cells of other autoimmune
disorders is believed largely mediated by cytotoxic T-lymphocytes
(CTLs--also known as CD8+ T cells) that specifically recognize
antigenic, target cell derived peptides. CTLs, as well as other
types of T cells, recognize these antigenic peptides through their
specific T cell receptor (TcR). Unlike antibodies which recognize
soluble whole foreign proteins as antigen, the TcR instead
interacts with small peptidic antigens presented only in complex
with major histocompatibility complex (MHC) proteins.
[0019] Most cells of the body express MHC molecules of various
classes on their surface and, depending on the class of MHC
expressed, will present either soluble antigens, those dispersed
within the lymph and/or circulatory systems, or fragments of their
cytoplasmic proteins. MHC molecules (called human leukocyte
antigens or HLA in humans) and TcRs are extremely polymorphic, each
clonal variation recognizing and binding to a single peptidic
sequence, or set of similar peptidic analogs. Apart from cells
specific to the immune system, i.e. B cells and T cells, cells of
the body express multiple variants of the MHC molecule, each
variant binding to a different peptide sequence. In contrast,
during maturation, B and T cells lose the ability to express
multiple variants of MHC and TcR, respectively. Mature T cells,
therefore, will express only one of the possible variants of the
TcR and will thus recognize/bind a single MHC/antigen complex.
[0020] Binding of a TcR to a MHC/antigen complex elicits an
intracellular signal cascade within the T cell, termed activation,
which results in clonal proliferation of the T cell and
class-specific T cell responses. For example, in CTLs the response
to activation also includes the release of cytotoxic enzymes that
result in apoptosis/destruction of the target cell.
[0021] 2.3 Modulation of T Cell Activation by Monoclonal
Antibodies
[0022] The finding that autoimmune diseases are at least partially
caused by aberrant T cell action has lead to the investigation of
therapies that either eliminate problematic T cell clones (those
expressing TcRs against self antigens) or selectively reduce
undesired T cell activity/activation. T cell activation due to TcR
binding is, however, an unexpectedly complex phenomenon due to the
participation of a variety of cell surface molecules expressed on
the responding T cell population (Billadeau et al., 2002, J. Clin.
Invest. 109:161-168; Weiss, 1990, J. Clin. Invest. 86:1015-1022;
Leo et al., 1987, PNAS 84:1374-1378; Weiss et al., 1984, PNAS
81:4169-4173; Hoffman et al., 1985, J. Immunol. 135:5-8).
[0023] Targeted therapies directed against general T cell
activation were problematic in that the TcR is composed of a
disulfide-linked heterodimer, containing two clonally distributed,
integral membrane glycoprotein chains, .alpha. and .beta., or
.gamma. and .delta.. Most of the research in modulation of T cell
activation was done in connection with improving immune suppression
in organ transplant recipients. One of the first clinically
successful methods of selectively reducing T cell activation was
the use of monoclonal antibodies. U.S. Pat. No. 4,658,019,
describes a novel hybridoma (designated OKT3, ATCC Accession No.
CRL-8001) which produces a murine monoclonal antibody against an
antigen found on essentially all normal human peripheral T cells.
Binding of OKT3 to T cells in vivo produces pronounced, reversible
immunosuppression. OKT3 was found to recognize an epitope on the
8-subunit within the human CD3 complex (Salmeron et al., 1991, J.
Immunol. 147:3047-3052; Transy et al., 1989, Eur. J. Immunol.
19:947-950; see also, U.S. Pat. No. 4,658,019). The CD3 complex
(also known as T3) is comprised of low molecular weight invariant
proteins, which non-covalently associate with the TcR (Samelson et
al., 1985, Cell 43:223-231). The CD3 structures are thought to
represent accessory molecules that may be the transducing elements
of activation signals initiated upon binding of the TcR
.alpha.-.beta. to its ligand.
[0024] OKT3 possesses potent T cell activating and suppressive
properties (Van Seventer, 1987, J. Immunol. 139:2545-2550; Weiss,
1986, Ann. Rev. Immunol. 4:593-619). Fc receptor-mediated
cross-linking of TcR-bound anti-CD3 mAb results in T cell
activation marker expression, and proliferation (Weiss et al.,
1986, Ann. Rev. Immunol. 4:593-619). Similarly, in vivo
administration of OKT3 results in both T cell activation and
suppression of immune responses (Ellenhorn et al., 1990,
Transplantation 50:608-12; Chatenoud, 1990, Transplantation
49:697). Repeated daily administration of OKT3 results in profound
immunosuppression, and provides effective treatment of rejection
following renal transplantation (Thistlethwaite, 1984,
Transplantation 38:695).
[0025] The use of therapeutic mAbs, including, for example, OKT3,
is limited by problems of "first dose" side effects, ranging from
mild flu-like symptoms to severe toxicity. The first dose side
effects are believed to be caused by cytokine production stimulated
by T cell activation. It has been shown that the activating
properties of Anti CD3 monoclonal antibodies result from TcR
cross-linking mediated by the antibodies bound to T cells (via its
variable domain) and to Fc.gamma.R-bearing cells via its Fc domain)
(Palacios et al., 1985, Eur. J. Immunol. 15:645-651; Ceuppens et
al., 1985, J. Immunol. 134:1498-1502; Kan et al., 1986, Cell
Immunol. 98:181-185). For example, the use of OKT3 was found to
trigger activation of mAb-bound T cells and Fc.gamma.R-bearing
cells prior to achieving immune suppression, resulting in a massive
systemic release of cytokines (Abramowicz, 1989, Transplantation
47:P606; Chatenoud, 1989, N. Eng. J. Med. 25:1420-1421). Reported
side effects of OKT3 therapy include flu-like symptoms, respiratory
distress, neurological symptoms, and acute tubular necrosis that
may follow the first and sometimes the second injection of the mAb
(Abramowicz, 1989, Transplantation 47:P606; Chatenoud, 1989, N.
Eng. J. Med. 25:1420-1421; Toussaint, 1989, Transplantation 48:524;
Thistlethwaite, 1988, Am. J. Kid. Dis. 11:112; Goldman, 1990,
Transplantation 50:148).
[0026] Data obtained using experimental models in chimpanzees and
mice have suggested that preventing or neutralizing the cellular
activation induced by anti-CD3 mAbs reduces the toxicity of these
agents (Parleviet, 1990, Transplantation 50:889; Rao, 1991,
Transplantation 52:691; Alegre, 1990, Eur. J. Immunol. 20:707;
Alegre, 1990, Transplant Proc. 22:1920; Alegre, 1991,
Transplantation. 52:674; Alegre, 1991, J. Immun. 146:1184-1191;
Ferran, 1990, Transplantation 50:642). Previous results reported in
mice using F(ab').sub.2 fragments of 145-2C11, a hamster anti-mouse
CD3 that shares many properties with OKT3, have suggested that, in
the absence of Fc.gamma.R binding and cellular activation, anti-CD3
mAbs retain at least some immunosuppressive properties in vivo
(Hirsch, 1991, Transplant Proc. 23:270; Hirsch, 1991, J. Immunol.
147:2088). In addition, administration of anti-CD3 antibodies with
reduced binding to Fc.gamma.R to human patients resulted in
generally only mild side effects and not the severe first class
effects associated with OKT3 administration (Herold et al., 2005,
Diabetes 54:1763).
[0027] 2.4 Immunosuppressive Monoclonal Antibodies Exhibiting
Reduced T Cell Activation
[0028] U.S. Pat. No. 6,491,916, U.S. Pat. Application Pub. No.
2005/0064514 and U.S. Pat. Application Pub. No. 2005/0037000
describe the modification of the Fc regions of immunoglobulins such
that the variant molecules exhibit enhanced or reduced binding to
various Fc receptors when compared to immunoglobulins with wild
type Fc domains. In particular the patents/applications describe
modifications to the Fc regions of IgG antibodies such that the
affinity for the Fc.gamma.R is selectively enhanced or reduced. By
tailoring the affinity for activating or suppressive Fc receptors,
the specific immune response elicited by the therapeutic mAb may be
more selectively controlled. For example, mutations in the CH.sub.2
portion of a humanized OKT3 IgG4 have been identified (P234A and
L235A) that significantly reduced binding of the mAb to human and
murine Fc.gamma.RI and II and lead to a markedly reduced activating
phenotype in vitro (Alegre et al., 1992, 8.sup.th International
Congress of Immunology 23-28; Alegre et al., 1994, Transplantation
57: 1537-1543; Xu et al., 2000, Cell Immunol. 200:16-26).
Importantly, this variant mAb retained the capacity to induce TcR
modulation and immunosuppression (Xu et al., 2000, Cell Immunol.
200:16-26). Other modifications to the Fc domain of anti-CD3
antibodies, such as mutations to make the antibody aglycosylated or
other mutations of the Fc domain residues, to reduce binding to
Fc.gamma.R have been found to reduce toxicity while maintaining
immunosuppressive activity (see, e.g., U.S. Pat. No. 6,491,916;
U.S. Pat. No. 5,834,597, Keymeulen et al., 2005, N. Eng. J. Med.
325:2598, all of which are incorporated by reference herein in
their entireties).
3. SUMMARY OF THE INVENTION
[0029] The present invention provides methods of treating,
preventing, slowing the progression of and ameliorating the
symptoms of T cell mediated immunological diseases, particularly
autoimmune diseases, in subjects diagnosed with such diseases
(and/or in subjects predisposed to developing such diseases or
disorders), by administering to a subject in need thereof a
therapeutically or prophylactically effective amount of an anti-CD3
antibody. In particular, the methods of the invention provide for
administration of antibodies that specifically bind the epsilon
subunit within the human CD3 complex. For example, such antibodies
may be or may be derived from (e.g., humanized or chimerized
versions of) one of the antibodies Leu-4, 500A2, CLB-T3/3, M291,
YTH 12.5 or BMA030, or compete with one of Leu-4, 500A2, CLB-T3/3,
M291, YTH 12.5 or BMA030 for binding (e.g., in an ELISA or
immunoprecipitation assay). In a preferred embodiment, the antibody
has the binding specificity of the murine monoclonal antibody OKT3
(see, e.g., U.S. Pat. Nos. 4,658,019 and 6,113,901, which are
incorporated by reference herein in their entireties), e.g., binds
to the same epitope as OKT3 and/or competes for binding (i.e., in
an ELISA or immunoprecipitation assay) with OKT3, such as a
humanized version of the antibody OKT3, e.g., OKT3-7 (see the
antibodies disclosed in U.S. Pat. No. 6,491,916, which is
incorporated herein by reference in its entirety). In preferred
embodiments, the anti-CD3 antibody of the invention has diminished
(such as, but not limited to, less than 50%, less than 40%, less
than 30%, less than 20%, less than 10%, less than 5% or less than
1% as compared to binding by an antibody having a wild-type,
glycosylated Fc domain) or, more preferably, no detectable binding
to one or more of any Fc.gamma.R (e.g., Fc.gamma.RI, Fc.gamma.RII
or Fc.gamma.RIII) via its Fc domain as determined by assays routine
in the art. In addition or alternatively, the anti-CD3 antibody of
the invention has diminished (such as, but not limited to, less
than 50%, less than 40%, less than 30%, less than 20%, less than
10%, less than 5% or less than 1% as compared to binding by an
antibody having a wild-type, glycosylated Fc domain) or, more
preferably, no detectable binding to any complement receptors, such
as, C1q, as determined in routinely used assays. In particular
embodiments, the antibody is aglycosylated. In other embodiments,
the antibody lacks an Fc domain (e.g., is a Fab fragment,
F(ab').sub.2 or single chain antibody). In other embodiments, the
antibody has an Fc domain having one or more amino acid
modifications that reduce or abolish binding of the Fc domain to
one or more of an Fc.gamma.R. In certain embodiments, the Fc domain
has an amino acid modification (i.e., insertion, substitution,
deletion) at one or more of the residues 234, 235, 236, or 237. In
preferred embodiments, the Fc domain has an alanine at position 234
of the Fc region (CH2) and or an alanine at position 235 of the Fc
region (CH2), in particular having alanine at 234 and 235, such as
OKT3.gamma.1(ala-ala). In other embodiments, the Fc domain has a
glutamate at position 235.
[0030] The invention particularly provides methods of treating,
preventing, slowing the progression of and/or ameliorating the
symptoms of autoimmune diseases such as Type I Diabetes, multiple
sclerosis, psoriasis, rheumatoid arthritis, lupus (particularly,
cutaneous), inflammatory bowel disease (IBD), Crohn's disease,
ulcerative colitis, effects from organ transplantation, graft vs.
host disease (GVHD), etc. Particularly, the methods of the
invention are advantageous in subjects with early stage disease to
slow or reduce the damage from the autoimmunity and maintain a high
level of function and/or reduce the need for other therapy; for
example, in the treatment or prophylaxis of Type I diabetes, the
methods of the invention may reduce the need for exogenous insulin
administration is the subject. In addition, the methods of the
invention advantageously reduce the incidence of or result in no
incidence of cytokine release syndrome previously associated with
administration of therapeutic antibodies, and, in particular,
anti-CD3 antibodies. Cytokine release syndrome is manifested by,
for example, headache, nausea, vomiting, fever, myalgias,
arthralgias and shaking and may be caused by increased serum levels
of, for example, IL-2, IL-6, IL-10, TNF.alpha., and IFN.gamma.. The
methods also reduce the incidence and severity of other adverse
effects, such as, but not limited to, EBV activation,
immunogenicity (production of anti-idiotype antibodies,
particularly IgE anti-idiotype antibodies), lymphopenia,
thrombocytopenia or neutropenia.
[0031] In preferred embodiments, the anti-human CD3 antibodies are
administered are administered at lower dosages or over shorter
periods of time than prior dosing regimens. In particular, the
invention contemplates dosing regimens in which less than 9,000
.mu.g/m.sup.2, and preferably, less than 8,000 .mu.g/m.sup.2, less
than 7,500 .mu.g/m.sup.2, less than 7,000 .mu.g/m.sup.2, or less
than 6,000 .mu.g/m.sup.2 total anti-human CD3 antibody is
administered over the duration of the dosing, particularly of
OKT3.gamma.1 (ala-ala), or of another anti-human CD3 antibody, such
as ChAglyCD3 (TRX4.TM.) or HUM291 (visilizumab; NUVION.TM.). In
certain embodiments, the antibodies of the invention are
administered to the subject in need thereof using a mode other than
intravenous administration that provides the pharmacological
equivalent to the foregoing amounts of OKT3.gamma.1(ala-ala) as
administered intravenously. The invention further contemplates
methods in which the patient is chronically administered low doses
of the anti-human CD3 antibody and methods in which the patient is
administered one or more additional rounds of the anti-human CD3
antibody treatment regimen approximately 6 months, 9 months, 12
months, 18 months, 2 years, 3 years or 5 years after the initial
treatment, optionally depending on clinical parameters, or is
administered another round of treatment with anti-human CD3
antibody every approximately 6 months, 9 months, 12 months, 18
months, 2 years, 3 years or 5 years, optionally depending on
clinical parameters.
[0032] The invention particularly provides methods of treating,
preventing, slowing the progression of or ameliorating the symptoms
of autoimmune diseases and disorders, e.g., type 1 diabetes, by
administration of anti-human CD3 antibodies having reduced
toxicity; e.g. having reduced binding to Fc.gamma.Rs. Particularly,
the methods of the invention are advantageous in subjects with
early stage disease in order to slow or reduce the damage from the
autoimmunity and maintain a high level of function. In certain
embodiments, the methods of the invention may also reduce the need
for additional therapy for the autoimmune disease or disorder; for
example, in the treatment or management of Type I diabetes, the
methods of the invention may reduce or eliminate the need for
administration of exogenous insulin in the subject. In addition,
the methods of the invention advantageously reduce the incidence of
or result in no incidence of cytokine release syndrome previously
associated with administration of anti-human CD3 antibodies such as
OKT3. Cytokine release syndrome is manifested by, for example,
headache, nausea, vomiting, fever, myalgias, arthralgias and
shaking and may be caused by increased serum levels of, for
example, IL-2, IL-6, IL-10, TNF.alpha., and IFN.gamma.. The methods
also reduce the incidence and severity of other adverse effects,
such as, but not limited to, EBV activation, immunogenicity
(production of anti-idiotype antibodies, particularly IgE
anti-idiotype antibodies), lymphopenia, thrombocytopenia or
neutropenia.
[0033] In other embodiments, the invention provides methods of
preventing or delaying the onset of an autoimmune disease or
disorder in a subject predisposed to developing said disease or
disorder, but which subject has yet to experience symptoms of or be
diagnosed with said disease or disorder according to criteria
accepted in the art (e.g., in Type I diabetes, a diagnosis
according to criteria established by the American Diabetes
Association: see, e.g., Mayfield et al., et al., 2006, Am. Fam.
Physician 58:1355-1362, hereby incorporated by reference herein in
its entirety). In other embodiments, administration of an antibody
of the invention prevents onset and/or development of the disorder,
prevents onset of symptoms of the disorder, and/or delays the
positive diagnosis of said disorder by 2 months, 4 months, 6
months, 8, months, 10 months, 12 months, 15 months, 18 months, 21
months, or 24 months relative to a subject with similar clinical
parameters who did not receive treatment.
[0034] According to the invention, the anti-human CD3 antibody is
administered so as to reduce adverse effects, such as the cytokine
release associated with antibody administration, EBV activation (as
evidenced by EBV-induced lymphoproliferative diseases, e.g.,
mononucleosis) or lymphopenia (defined as <1000 lymphocytes/4
serum), associated with administration of anti-human CD3
antibodies, and also reduce the number of doses and duration of the
administration. As used herein, "course of treatment" or "round of
treatment" means administration of anti-human CD3 antibodies every
day, every other day or every 3 or 4 days for a period of time,
e.g. 1 to 30 days. In particular embodiments, the invention
provides a treatment regimen of administration of a dose of the
anti-human CD3 antibody for 2 days, 3 days, 4 days, 5 days, 6 days,
7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14
days. In preferred embodiments, the administration is carried out
on consecutive days but may be carried out on alternate days or on
a schedule that alternates a number of consecutive days in which
the anti-human CD3 antibody is administered with a number of
consecutive days in which there is no antibody administered. In
certain embodiments, the dose administered is the same each day of
the regimen. However, in preferred embodiments the dose
administered escalates over the first few days of the regimen
(e.g., escalates over the first four days of the regimen) to reduce
or eliminate the incidence of cytokine release syndrome.
[0035] In specific embodiments, the dose administered is
approximately 5-50 .mu.g/kg/day, preferably, 20-30 .mu.g/kg/day,
more preferably, approximately 22-28 .mu.g/kg/day or approximately
25-26 .mu.g/kg/day. In other specific embodiments, the dose on day
1 of the regimen is 1-3 .mu.g/kg/day, preferably approximately 1.6
.mu.g/kg/day and escalates to the daily dose by day 3, 4, 5, 6 or
7. For example, on day 1, the subject is administered a dose of
approximately 1.6 .mu.g/kg/day, on day 2 approximately 3.2
.mu.g/kg/day, on day 3 approximately 6.5 .mu.g/kg/day, on day 4
approximately 13 .mu.g/kg/day and on subsequent days of the regimen
26 .mu.g/kg/day. In another example in accordance with this
embodiment, on day 1, the subject may be administered a dose of
approximately 1.42 .mu.g/kg/day, on day 2 approximately 5.7
.mu.g/kg/day, on day 3 approximately 11 .mu.g/kg/day, on day 4
approximately 22.6 .mu.g/kg/day and on subsequent days of the
regimen 45.4 .mu.g/kg/day.
[0036] In specific embodiments, the dose administered is based on
surface area. For example the dose administered is 5-1200
.mu.g/m.sup.2/day, preferably, 51-826 .mu.g/m.sup.2/day. In certain
embodiments, the dose on day 1 of the regimen is 5-100
.mu.g/m.sup.2/day and escalates to the daily dose that is
approximately 2, 4, 5, 8, 10, 12, 15 or 20 times that of the first
day dose, for example a dosage as recited immediately above, by day
3, 4, 5, 6 or 7. For the first 2, 3, 4, or 5 days, the dose may
increase by 1.5-fold, 2-fold, 3-fold, or 4-fold on each subsequent
day. For example, on day 1, the subject is administered a dose of
approximately 51 .mu.g/m.sup.2/day, on day 2 approximately 103
.mu.g/m.sup.2/day, on day 3 approximately 207 .mu.g/m.sup.2/day, on
day 4 approximately 413 .mu.g/m.sup.2/day and on subsequent days of
the regimen (e.g., days 5-14) 826 .mu.g/m.sup.2/day. In another
embodiment, on day 1, the subject is administered a dose of
approximately 227 .mu.g/m.sup.2/day, on day 2 approximately 459
.mu.g/m.sup.2/day, on day 3 and subsequent days, approximately 919
.mu.g/m.sup.2/day. In another embodiment, on day 1, the subject is
administered a dose of approximately 284 .mu.g/m.sup.2/day, on day
2 approximately 574 .mu.g/m.sup.2/day, on day 3 and subsequent
days, approximately 1148 .mu.g/m.sup.2/day.
[0037] In specific embodiments, to reduce the possibility of
cytokine release and other adverse effects, the first 1, 2, 3, or 4
doses or all the doses in the regimen are administered more slowly,
relative to bolus injection, by intravenous administration. For
example, a dose of 51 jag/m.sup.2/day may be administered over
about 5 minutes, about 15 minutes, about 30 minutes, about 45
minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, and about 22 hours.
In certain embodiments, the dose is administered by slow infusion
over a period of, e.g., 20 to 24 hours. In specific embodiments,
the dose is infused by a pump, preferably increasing the
concentration of antibody administered as the infusion
progresses.
[0038] Alternatively, the total daily dose may be divided into two
or more equal portions and administered as bolus infusions over the
day at intervals of 6, 8, 10 or 12 hours. For example, a 13
.mu.g/kg/day dose may be administered in four doses of 3-4 .mu.g/kg
at intervals of 6 hours to reduce the level of cytokine release
caused by administration of the antibody.
[0039] In other embodiments, a set fraction of the doses for the 51
.mu.g/m.sup.2/day to 826 .mu.g/m.sup.2/day regimen described above
is administered in escalating doses. In certain embodiments, the
fraction is 1/10, 1/4, 1/3, 1/2, 2/3 or 3/4 of the daily doses of
the regimen(s) described above. Accordingly, for example, when the
fraction is 1/10, the daily doses will be 5.1 .mu.g/m.sup.2 on day
1, 10.3 .mu.g/m.sup.2 on day 2, 20.7 .mu.g/m.sup.2 on day 3, 41.3
.mu.g/m.sup.2 on day 4 and 82.6 .mu.g/m.sup.2 on days 5 to 14. When
the fraction is 1/4, the doses will be 12.75 .mu.g/m.sup.2 on day
1, 25.5 .mu.g/m.sup.2 on day 2, 51 .mu.g/m.sup.2 on day 3, 103
g/m.sup.2 on day 4, and 207 .mu.g/m.sup.2 on days 5 to 14. When the
fraction is 1/3, the doses will be 17 .mu.g/m.sup.2 on day 1, 34.3
.mu.g/m.sup.2 on day 2, 69 .mu.g/m.sup.2 on day 3, 137.6
.mu.g/m.sup.2 on day 4, and 275.3 .mu.g/m.sup.2 on days 5 to 14.
When the fraction is 1/2, the doses will be 25.5 .mu.g/m.sup.2 on
day 1, 51 .mu.g/m.sup.2 on day 2, 103 .mu.g/m.sup.2 on day 3, 207
.mu.g/m.sup.2 on day 4, and 413 .mu.g/m.sup.2 on days 5 to 14. When
the fraction is 2/3, the doses will be 34 .mu.g/m.sup.2 on day 1,
69 .mu.g/m.sup.2 on day 2, 137.6 .mu.g/m.sup.2 on day 3, 275.3
.mu.g/m.sup.2 on day 4, and 550.1 .mu.g/m.sup.2 on days 5 to 14.
When the fraction is 3/4, the doses will be 38.3 .mu.g/m.sup.2 on
day 1, 77.3 .mu.g/m.sup.2 on day 2, 155.3 .mu.g/m.sup.2 on day 3,
309.8 .mu.g/m.sup.2 on day 4, and 620 .mu.g/m.sup.2 on days 5 to
14. In other embodiments, the regimen is identical to one of those
described above but only over days 1 to 4, days 1 to 5, or days 1
to 6. For example, in a particular embodiment, the doses will be 17
.mu.g/m.sup.2 on day 1, 34.3 .mu.g/m.sup.2 on day 2, 69
.mu.g/m.sup.2 on day 3, 137.6 .mu.g/m.sup.2 on day 4, and 275.3
.mu.g/m.sup.2 on days 5 and 6.
[0040] In other embodiments, doses in the regimen are administered
for a certain number of consecutive days, followed by a certain
number of days without any doses administered, followed again by
doses administered on a certain number of consecutive days and so
on until, for example, 14 (or, e.g., 6, 7, 8, 9, 10, 11, 12, 13,
15, 16, 17, 18, 19 or 20) doses are administered in total. For
example, the day 1, day 2, day 3 and day 4 doses of one of the
regimens described above may be administered over four consecutive
days and then three days without any doses and then the day 5, 6, 7
and 8 doses are administered, followed by another three days
without doses, and then the day 9, 10, 11, 12 day doses, with three
days off, and finally the day 13 and 14 doses.
[0041] In certain embodiments, the antibody administered according
to these regimens is OKT3.gamma.1(ala-ala). In other embodiments
the antibody is not OKT3.gamma.1(ala-ala) and is administered so as
to achieve one or more pharmacokinetic parameters achieved by the
administration of OKT3.gamma.1(ala-ala), preferably, intravenous
administration of OKT3.gamma.1 (ala-ala), such as the serum titer
of the antibody administered at 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2 weeks, 3 weeks or 1 month after the last
day of the dosing regime (i.e., is administered to achieve a
"pharmacologically equivalent" dose).
[0042] In certain embodiments, the anti-human CD3 antibody is
administered so as to achieve a certain level of combined coating
and modulation of T cell receptor complexes on T cells, as
determined by methods well known in the art, see, e.g., Example 11
of U.S. patent application publication US 2003/0108548, which is
hereby incorporated by reference in its entirety. In specific
embodiments, the dosing regimen achieves a combined T cell receptor
coating and modulation of at least 50%, 60%, 70%, 80%, 90%, 95% or
of 100% with, in specific embodiments, little to no detectable free
anti-human CD3 antibody (for example, less than 200 ng/mL the drug
detected in the blood of the patient by standard methods known in
the art).
[0043] In specific embodiments, the anti-human CD3 antibody is not
administered by daily doses over a number of days, but is rather
administered by infusion in an uninterrupted manner over 4 hours, 6
hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours,
24 hours, 30 hours or 36 hours. The infusion may be constant or may
start out at a lower dosage for, for example, the first 1, 2, 3, 5,
6, or 8 hours of the infusion and then increase to a higher dosage
thereafter. Over the course of the infusion, the patient receives a
dose equal to the amount administered, for example, in the 5 to 20
day regimens set forth above. For example, a dose of approximately
150 .mu.g/m.sup.2, 200 .mu.g/m.sup.2, 250 .mu.g/m.sup.2, 500
.mu.g/m.sup.2, 750 .mu.g/m.sup.2, 1000 .mu.g/m.sup.2, 1500
.mu.g/m.sup.2, 2000 .mu.g/m.sup.2, 3000.mu.g/m.sup.2, 4000
.mu.g/m.sup.2, 5000 .mu.g/m.sup.2, 6000 .mu.g/m.sup.2, 7000
.mu.g/m.sup.2, 8000 .mu.g/m.sup.2, or 9000 .mu.g/m.sup.2. In
particular, the speed and duration of the infusion is designed to
minimize the level of free anti-human CD3 antibody in the subject
after administration. In certain embodiments, the level of free
anti-human CD3 antibody should not exceed 200 ng/ml free antibody.
In addition, the infusion is designed to achieve a combined T cell
receptor coating and modulation of at least 50%, 60%, 70%, 80%,
90%, 95% or of 100%.
[0044] In other embodiments, the anti-human CD3 antibody is
administered chronically to treat, manage, maintain, prevent, or
slow the progression of or delay the onset of the autoimmune
disease or disorder. For example, in certain embodiments, a low
dose of the anti-human CD3 antibody is administered once a month,
twice a month, three times per month, once a week or even more
frequently either as an alternative to the 6 to 14 day dosage
regimen discussed above or after administration of such a regimen
to enhance or maintain its therapeutic effect. Such a low dose may
be anywhere from 1 .mu.g/m.sup.2 to 100 .mu.g/m.sup.2, preferably,
approximately 5 .mu.g/m.sup.2, 10 .mu.g/m.sup.2, 15 .mu.g/m.sup.2,
20 .mu.g/m.sup.2, 25 .mu.g/m.sup.2, 30 .mu.g/m.sup.2, 35
.mu.g/m.sup.2, 40 .mu.g/m.sup.2, 45 .mu.g/m.sup.2, or 50
.mu.g/m.sup.2. In certain embodiments, the anti-human CD3 antibody
is administered chronically subsequent to administration of a 1 to
30 day dosing regimen as described above, for example, to maintain
the therapeutic effect of the regimen.
[0045] In other embodiments, the subject may be re-dosed at some
time subsequent to administration of the anti-human CD3 antibody
dosing regimen, preferably, based upon one or more physiological
parameters, but may be done as a matter of course. Such redosing
may be administered and/or the need for such redosing evaluated 6
months, 9 months, 1 year, 15 months, 18 months, 2 years, 30 months
or 3 years after administration of a dosing regimen and may include
administering a course of treatment every 6 months, 9 months, 1
year, 15 months, 18 months, 2 years, 30 months or 3 years.
[0046] In specific embodiments, subjects are administered a
subsequent round of anti-human CD3 antibody treatment based upon
one or a combination of the CD4/CD8 cell ratio, CD8 cell count,
CD4/CD3 inversion, CD4/CD25 cell ratio, CD4/FoxP3 cell ratio,
CD4/CD40 cell ratio, CD4/IL-10 cell ratio, and/or CD4/TGF-.beta.
cell ratio. Other parameters for determining whether to administer
a subsequent round of treatment include an appearance or worsening
of diagnostic indicators for the autoimmune disease or disorder as
described herein and/or known in the art. For example, with respect
to Type I diabetes, an appearance or an increase in anti-islet cell
antibodies, such as GADAs, IA-2 antibodies, ICA antibodies or
anti-insulin antibodies or an appearance or increase in the levels
of T cells specific for islet cell antigens. Further examples with
respect to Type I diabetes include subsequent doses where the
number of .beta.-cells or .beta.-cell activity or function
decreases by 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% as compared
to the .beta.-cell number or activity or function during
administration of the preceding round of treatment. .beta.-cell
function may be determined by any method know in the art, for
example, the C peptide response to MMTT, OGTT, IGTT, or two-phase
glucose clamp, or the First Phase Insulin Release (FPIR) test, as
discussed herein or as is known in the art. Other parameters that
may be used to determine whether to redose during the treatment or
management of Type I diabetes include the HA1 or HA1c levels, the
need for administration of exogenous insulin or increase in the
dosage of exogenous insulin by more than 0.2 U/kg/day, 0.5
U/kg/day, 1 U/kg/day, 2 U/kg/day, 5 U/kg/day, or 10 U/kg/day. In
other embodiments, the further doses may be administered based upon
appearance of or increase in number (such as an increase by, on
average, 1, 2, 3, 4, 5, 8, 10 15, or 20), duration and/or severity
of hypoglycemic episodes or of ketoacidosis episodes on a daily,
weekly or monthly basis.
[0047] In preferred embodiments, the anti-human CD3 antibodies are
administered parenterally, for example, intravenously,
intramuscularly or subcutaneously, or, alternatively, are
administered orally. The anti-human CD3 antibodies may also be
administered as a sustained release formulation.
[0048] Additionally, in certain embodiments, the invention provides
methods and regimens of administering anti-human CD3 antibodies
that reduce the severity and/or incidence of adverse effects such
as, but not limited to, cytokine release, apoptosis, activation of
EBV, immune reaction against the anti-human CD3 antibody,
lymphopenia, anemia, neutropenia, thrombocytopenia or secondary
infection.
[0049] In preferred embodiments of their invention, with respect to
treating, slowing the progression of, delaying the onset of or
preventing type 1 diabetes or disorder, the subject has retained at
least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% .beta.-cell
function prior to initiation of treatment and, in some embodiments,
.beta.-cell function improves over pre-treatment levels by at least
5%, 10%, 20%, 30% or 40%.
[0050] In certain embodiments, the predisposition for development
of Type I diabetes manifests as an impaired fasting glucose level,
i.e., at least one determination of a glucose level of 100-125
mg/dL after fasting (eight hours without food), or is an impaired
glucose tolerance in response to a 75 gram oral glucose tolerance
test (OGTT), i.e., at least one determination of a 2-hour glucose
level of 140-199 mg/dL in response to a 75 OGTT. In other
embodiments, the subjects are positive for one or more
autoantibodies reactive against islet cell antigens, such as, GAD
antibodies, such as GAD 65 and/or GAD 67, IA-2 or anti-insulin
antibodies. In other embodiments, the predisposition for
development of type 1 diabetes is having a first or second degree
relative who is a diagnosed type 1 diabetic. In certain
embodiments, the predisposition is positive diagnosis in the
patient or in a first or second degree relative according to art
accepted criteria of at least one other autoimmune disorder
including, but not limited to, thyroid disease, type 1 diabetes,
rheumatoid arthritis, systemic lupus erythematosus, multiple
endocrine adenopathy, and celiac disease. In some embodiments, the
autoimmune disorder is a MHC DR3- and/or a DR4-related autoimmune
disease.
[0051] With respect to treatment of type 1 diabetes in a diagnosed
patient, and the prevention/delay of symptoms thereof in a
predisposed individual, the anti-human CD3 antibody with reduced
toxicity is administered to achieve, or maintain a level of
glycosylated hemoglobin (HA1 or HA1c) of less than 8%, less than
7.5%, less than 7%, less than 6.5%, less than 6%, less than 5.5% or
5% or less. At the initiation of treatment, patients preferably,
have a HA1 or HA1c level of less than 8%, less than 7.5%, less than
7%, less than 6.5%, less than 6%, or, more preferably, from 4%-6%
(preferably, measured in the absence of other treatment for
diabetes, such as administration of exogenous insulin).
[0052] In certain embodiments, one or more CD3 binding molecules
(e.g., one or more anti-human CD3 antibodies) are administered to
prevent a reduction of .beta.-cell mass associated with autoimmune
diabetes. In some embodiments, after one or more courses of
treatment with an anti-human CD3 antibody according to the
invention, the level of .beta.-cell mass of the patient decreases
by less than 1%, less than 5%, less than 10%, less than 20%, less
than 30%, less than 40%, less than 50%, less than 60%, or less than
70% of the pretreatment levels of at least 3 months, at least 6
months, at least 9 months, at least 1 year, at least 18 months, at
least 2 years, at least 3 years, at least 5 years, at least 7 years
or at least 10 years after initial treatment. In yet another
embodiment of the invention, after one or more courses of treatment
with an anti-CD3 antibody according to the invention, the level of
.beta.-cell mass of the patient is maintained at least 99%, at
least 95%, at least 90%, at least 80%, at least 70%, at least 60%,
at least 50%, at least 40%, or at least 30% of pretreatment levels
for at least 4 months, at least 6 months, at least 9 months, at
least 12 months, at least 18 months, at least 24 months, at least
30 months, at least 3 years, at least 5 years, or at least 10 years
after the first round of treatment.
[0053] In another embodiment of the invention, with respect to the
treatment of type 1 diabetes, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
level of .beta.-cell function of the patient is maintained at least
99%, at least 95%, at least 90%, at least 80%, at least 70%, at
least 60%, or at least 50% of pretreatment levels for at least 4
months, at least 6 months, at least 9 months, at least 12 months,
at least 18 months, at least 24 months, or at least 30 months after
the end of treatment or after the first round of treatment and the
mean lymphocyte count of the patient is not less than 800 cells/ml,
less than 750 cells/ml, less than 700 cells/ml, less than 650
cells/ml, less than 600 cells/ml, less than 550 cells/ml, less than
500 cells/ml, less than 400 cells/ml, less than 300 cells/ml or
less than 200 cells/ml at the same time period. In another
embodiment of the invention, after a course of treatment with an
anti-CD3 antibody according to the invention, the level of
.beta.-cell function of the patient is maintained at at least 99%,
at least 95%, at least 90%, at least 80%, at least 70%, at least
60%, or at least 50% of pretreatment levels for at least 4 months,
at least 6 months, at least 9 months, at least 12 months, at least
18 months, at least 24 months, or at least 30 months after the end
of treatment and the mean platelet count of the patient is not less
than 100,000,000 platelets/ml, less than 75,000,000 platelets/ml,
less than 50,000,000 platelets/ml, less than 25,000,000
platelets/ml, less than 1,000,000 platelets/ml, less than 750,000
platelets/ml, less than 500,000 platelets/ml, less than 250,000
platelets/ml, less than 150,000 platelets/ml or less than 100,000
platelets/ml.
[0054] The administration of the anti-CD3 antibodies prevents
damage to islet cells, thereby delaying onset of the disease or,
once diagnosable disease occurs, disease progression, reducing
and/or delaying the need for insulin administration. In addition,
the invention provides methods of treatment such that a single
round of treatment or round of treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, or every
24 months with an anti-CD3 antibody (preferably, without any
intervening treatment with anti-CD3 antibodies), results in a level
of HA1 or HA1c that is 7% or less, 6.5% or less, 6% or less, 5.5%
or less, or 5% or less 6 months, 9 months, 12 months, 15 months, 18
months, or 24 months after the previous round of treatment or the
first round of treatment. Specifically, in such methods of the
invention a single round of treatment or round of treatment every 6
months, every 9 months, every 12 months, every 15 months, every 18
months, or every 24 months with an anti-CD3 antibody (preferably,
without any intervening treatment with anti-human CD3 antibodies),
decreases the average level of HA1 or HA1c in the patient by about
5%, about 10%, about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about
65% or about 70% as compared to pre-treatment levels at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous round of treatment or first round of treatment. In
addition, after treatment with a CD3 antibody according to the
invention in a single round of treatment or a round of treatment
repeated every 6 months, every 9 months, every 12 months, every 15
months, every 18 months, or every 24 months (preferably, without
any intervening treatment with anti-human CD3 antibodies), the
average level of HA1 or HA1c in the patient only increases by about
0.5%, about 1%, about 2.5%, about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, or
about 50% as compared to pre-treatment levels at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous round of treatment or the first round of treatment. In
other embodiments, after a single round of treatment or rounds of
treatment every 6 months, every 9 months, every 12 months, every 15
months, every 18 months, or every 24 months with an anti-human CD3
antibody according to the methods of the invention (preferably,
without any intervening treatment with anti-human CD3 antibodies),
the average level of HA1 or HA1c in the patient is greater than
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70% or greater than about 100% less than the levels in a
patient that initiated conventional treatment with similar clinical
parameters and was administered conventional treatment after the
same amount of time, which levels were determined at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous round of treatment or the first round of treatment.
[0055] In another embodiment, the anti-human CD3 antibody is
administered to achieve, or maintain the C-peptide response in a
subject who has been diagnosed with autoimmune diabetes, or has a
predisposition thereto, as determined by a mixed-meal tolerance
test (MMTT), oral glucose tolerance test (OGTT), intravenous
tolerance test (IGTT) or two-phase glucose clamp procedure. In
preferred embodiments, the patients have a C-peptide response to
MMTT, OGTT, IGTT, or two-phase glucose clamp procedure (preferably
MMTT) resulting in an area under curve (AUC) of at least 80
pmol/ml/240 min., preferably, at least 90 pmol/ml/240 min., more
preferably at least 100 pmol/ml/240 min., or even at least 110
pmol/ml/240 min. In addition, the invention provides methods of
treatment such that after a single round of treatment or treatment
every 6 months, every 9 months, every 12 months, every 15 months,
every 18 months, or every 24 months with an anti-human CD3 antibody
(preferably, without any intervening treatment with anti-human CD3
antibodies), the level of C-peptide response in the patient is at
least 99%, at least 98%, at least 95%, at least 90%, at least 85%,
at least 80%, at least 75%, at least 70%, at least 65% or at least
60% of the pre-treatment response as determined at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous round of treatment or the first round of treatment.
Specifically, in such methods of the invention, after a single
round of treatment or round of treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, or every
24 months with an anti-human CD3 antibody according to methods of
the invention (preferably, without any intervening treatment with
anti-human CD3 antibodies), the average level of C-peptide response
to a MMTT, OGTT, IGTT, or two-phase glucose clamp procedure in the
patient decreases by less than 1%, less than 5%, less than 10%,
less than 20%, less than 30%, less than 40%, less than 50% of the
pre-treatment levels as determined at 6 months, 9 months, 12
months, 15 months, 18 months, or 24 months after the previous round
of treatment or the first round of treatment. In addition, after a
single round of treatment or round of treatment every 6 months,
every 9 months, every 12 months, every 15 months, every 18 months,
or every 24 months with an anti-human CD3 antibody according to
methods of the invention (preferably, without any intervening
treatment with anti-human CD3 antibodies), the average level of
C-peptide response to a MMTT, OGTT, IGTT or two-phase glucose clamp
procedure in the patient is at least 10%, 20%, 30%, 40%, 50%, 70%
or 100% greater than the levels in a patient who initiated
conventional diabetes therapy with similar clinical parameters and
was administered conventional diabetes therapy over the 6 month, 9
month, 12 month, 15 month or 18 month period or who did not receive
any therapy, said peptide response being determined at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous treatment.
[0056] In specific embodiments, after a single round of treatment
or round of treatment every 6 months, every 9 months, every 12
months, every 15 months, every 18 months, or every 24 months with
an anti-human CD3 antibody according to the methods of the
invention (preferably, without any intervening treatment with
anti-human CD3 antibodies), the patients diagnosed with autoimmune
diabetes, or having a predisposition thereto, have a C-peptide
response to MMTT, OGTT, IGTT or two-phase glucose clamp procedure
(preferably, MMTT) resulting in an AUC of at least 40 pmol/ml/240
min., 50 pmol/ml/240 min, 60 pmol/ml/240 min, 70 pmol/ml/240 min.,
80 pmol/ml/240 min., preferably, at least 90 pmol/ml/240 min., more
preferably at least 100 pmol/ml/240 min., or even at least 110
pmol/ml/240 min, said response determined 6 months, 9 months, 12
months, 15 months, 18 months, or 24 months after the previous round
of treatment or after the previous round of treatment.
[0057] The determination of C-peptide response is a measure of
.beta.-cell function as is known to one skilled in the art. In
other embodiments, .beta.-cell function or residual .beta.-cell
function is determined by First-Phase Insulin Release (FPIR). In
preferred embodiments, the patients prior to treatment with an
anti-human CD3 antibody according to the invention have a FPIR of
at least 300 pmol/1, at least 350 pmol/1,at least 400 pmol/1, at
least 450 pmol/1, at least 500 pmol/l, preferably, at least 550
pmol/1, more preferably at least 600 pmol/1, or even at least 700
pmol/1. In addition, the invention provides methods of treatment
such that after a single round of treatment or a round of treatment
every 6 months, every 9 months, every 12 months, every 15 months,
every 18 months, or every 24 months with an anti-human CD3 antibody
according to the methods of the invention (preferably, without any
intervening treatment with anti-human CD3 antibodies), the FPIR is
at least 99%, at least 98%, at least 95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 65% or at
least 60% of the pre-treatment response, said FPIR determined 6
months, 9 months, 12 months, 15 months, 18 months, or 24 months
after the previous treatment or initial treatment. Specifically, in
such methods of the invention, after a single round of treatment or
round of treatment every 6 months, every 9 months, every 12 months,
every 15 months, every 18 months, or every 24 months with an
anti-human CD3 antibody according to the methods of the invention
(preferably, without any intervening treatment with anti-human CD3
antibodies), the average FPIR in the patient decreases by less than
1%, less than 5%, less than 10%, less than 20%, less than 30%, less
than 40%, less than 50% of the pre-treatment levels, said FPIR
determined 6 months, 9 months, 12 months, 15 months, 18 months, or
24 months after the previous treatment. In addition, after a single
round of treatment or round of treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, or every
24 months with an anti-human CD3 antibody according to the methods
of the invention (preferably, without any intervening treatment
with anti-human CD3 antibodies), the average FPIR in the patient is
at least 10%, 20%, 30%, 40%, 50%, 70% or 100% greater than the
levels in a patient who initiated conventional diabetes therapy
with similar clinical parameters and was administered conventional
diabetes therapy over the 6 month, 9 month, 12 month, 15 month or
18 month period, said FPIR determined 6 months, 9 months, 12
months, 15 months, 18 months, or 24 months after the previous
treatment or initial round of treatment. In specific embodiments,
after a single round of treatment or round of treatment every 6
months, every 9 months, every 12 months, every 15 months, every 18
months, or every 24 months with an anti-human CD3 antibody
according to the methods of the invention (preferably, without any
intervening treatment with anti-human CD3 antibodies), the patients
have a FPIR of at least 300 pmol/1, at least 400 pmol/1,
preferably, at least 500 pmol/1, more preferably at least 600
pmol/1, or even at least 700 pmol/1, said FPIR determined at 6
months, 9 months, 12 months, 15 months, 18 months, or 24 months
after the previous round of treatment or initial round of
treatment.
[0058] In other specific embodiments of the invention with respect
to the treatment of type 1 diabetes, at the initiation of
treatment, the subject does not require administration of insulin
or requires less than 1 U/kg/day, preferably less than 0.5
u/kg/day, even more preferably less than 0.25 U/kg/day, and even
more preferably less than 0.1 U/kg/day. In certain embodiments, a
single treatment or round of treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, or every
24 months with an anti-human CD3 antibody according to the methods
of the invention (preferably, without any intervening treatment
with anti-human CD3 antibodies), prevents the requirement for
administration of insulin or delays the need to administer insulin
by at least 6 months, at least 1 year, at least 18 months, at least
2 years, at least 30 months, at least 3 years, at least 5 years, at
least 7 years or at least 10 years (on average for a population of
type 1 diabetes patients). In other embodiments, a single treatment
or round of treatment every 6 months, every 9 months, every 12
months, every 15 months, every 18 months, or every 24 months with
an anti-human CD3 antibody according to the methods of the
invention (preferably, without any intervening treatment with
anti-human CD3 antibodies), results in either a decrease (for
example, of 10%, 20%, 30%, 40%, or 50%) in the amount of insulin
required on average per day, or no change in the average amount of
insulin required per day, or an increase of less than 1%, less than
5%, less than 10%, less than 20% or less than 30% of insulin
administered, on average, per day as compared to the pre-treatment
average dose of insulin per day. In certain embodiments, a single
round of treatment or round of treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, or every
24 months with an anti-human CD3 antibody according to the methods
of the invention (preferably, without any intervening treatment
with anti-human CD3 antibodies), results in an average daily dose
of insulin that is 10%, 20%, 50%, 75%, 90%, 99% less than the
average daily dose of insulin required for a patient similarly
situated (i.e., similar chemical parameters at the beginning of the
month or year period) that had not received the anti-human CD3
antibody treatment.
[0059] In other embodiments, with respect to the treatment of a
subject diagnosed with autoimmune diabetes, or has a predisposition
thereto, the methods of the invention result in a reduction in
hypoglycemic episodes by 1, 2, 3, 4, 5, 6 or more episodes in a
one-day, two-day, 5-day, 10-day or 15-day period as compared to
similarly situated patients not having been administered the
anti-human CD3 antibody according to the invention.
[0060] In specific embodiments, the subject has received
transplanted islet cells and is administered a prophylactically or
therapeutically effective amount of the anti-CD3 antibody according
to the methods of the invention. In a specific embodiment, the
subject having received transplanted islet cells is an adult. In
other specific embodiments, the subject having received the
transplanted islet cells is younger than 21 years of age or is
younger than 18 years of age.
[0061] With respect to the treatment of multiple sclerosis, the
anti-CD3 antibody is administered to achieve or maintain a
disability score according to the Kurtzke Expanded Disability Scale
(EDSS) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,
6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. In addition, the invention
provides methods such that after a single treatment or treatment
every 6 months, every 9 months, every 12 months, every 15 months,
every 18 months, every 2 years, every 2.5 years or every 3 years
with an anti-CD3 antibody according to the methods of the invention
(preferably, without any intervening treatment with anti-CD3
antibodies), the EDSS score is the same, not more than one-half
step, not more than one step, not more than one and one-half steps,
not more than two steps, not more than two and one-half steps, not
more than three steps, not more than three and one-half steps, not
more than four steps, not more than four an one-half steps, not
more than more than five steps, not more than five and one-half
steps, not more than six steps, not more than six and one-half
steps, not more than seven steps, not more than seven and one-half
steps, not more than eight steps, or not more than eight and
one-half steps greater that the pretreatment EDSS score, said score
determined 6 months, 9 months, 12 months, 15 months, 18 months, or
24 months after the previous treatment. Specifically, in such
methods of the invention, after a single treatment or treatment
every 6 months, every 9 months, every 12 months, every 15 months,
every 18 months, every 2 years, every 2.5 years or every 3 years
with an anti-CD3 antibody according to the methods of the invention
(preferably, without any intervening treatment with anti-CD3
antibodies), the average EDSS score of the patient increases by no
more than one-half step, one step, one and one-half steps, two
steps, two and one-half steps, three steps, three and one-half
steps, four steps, four an one-half steps, five steps, five and
one-half steps, six steps, six and one-half steps, seven steps,
seven and one-half steps, eight steps, or eight and one-half steps
relative to the pre-treatment score, said score determined 6
months, 9 months, 12 months, 15 months, 18 months, or 24 months
after the previous treatment. In addition, after a single treatment
or treatment every 6 months, every 9 months, every 12 months, every
15 months, every 18 months, every 2 years, every 2.5 years or every
3 years with an anti-CD3 antibody according to the methods of the
invention (preferably, without any intervening treatment with
anti-CD3 antibodies), the average EDSS score in the patient is at
least one-half step, one step, one and one-half steps, two steps,
two and one-half steps, three steps, three and one-half steps, four
steps, four an one-half steps, five steps, five and one-half steps,
six steps, six and one-half steps, seven steps, seven and one-half
steps, eight steps, or eight and one-half lower than the score in a
patient who initiated conventional multiple sclerosis therapy with
similar clinical parameters and was administered conventional
multiple sclerosis therapy over the 6 months, 9 months, 12 months,
15 months, 18 months, 2 year, 2.5 year or 3 year period, said score
determined 6 months, 9 months, 12 months, 15 months, 18 months, or
24 months after the previous treatment. In specific embodiments, 6
months, 9 months, 12 months, 15 months, 18 months, 2 years, 2.5
years or 3 years after treatment (preferably, without any
intervening treatment with anti-CD3 antibodies), the patients have
an EDSS score of at least 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.
[0062] In specific embodiments of the invention, patients diagnosed
with multiple sclerosis (e.g., according to McDonald criteria), and
prior to administration of the anti-CD3 antibody of the invention
have an EDSS score of at least 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.5.
[0063] In other embodiments with respect to the treatment of
multiple sclerosis, the anti-CD3 antibody is administered to
achieve reduction in the frequency, duration and/or severity of the
MS attacks relative to the same patient prior to therapy. In
addition, the invention provides methods of treatment such that
after a single treatment or treatment every 6 months, every 9
months, every 12 months, every 15 months, every 18 months, every 2
years, every 2.5 years or every 3 years with an anti-CD3 antibody
according to the methods of the invention (preferably, without any
intervening treatment with anti-CD3 antibodies), the frequency,
duration and/or severity of the MS attacks are reduced by 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to pretreatment
levels, said determinations made 6 months, 9 months, 12 months, 15
months, 18 months, or 24 months after the previous treatment.
Specifically, in such methods of the invention, after a single
treatment or treatment every 6 months, every 9 months, every 12
months, every 15 months, every 18 months, every 2 years, every 2.5
years or every 3 years with an anti-CD3 antibody according to the
methods of the invention (preferably, without any intervening
treatment with anti-CD3 antibodies), the frequency, severity and/or
duration of the MS attacks of the patient increase by no more than
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to
pretreatment conditions, said determinations made at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous treatment. In addition, after a single treatment or
treatment every 6 months, every 9 months, every 12 months, every 15
months, every 18 months, every 2 years, every 2.5 years or every 3
years with an anti-CD3 antibody according to the methods of the
invention (preferably, without any intervening treatment with
anti-CD3 antibodies), the average frequency, severity and/or
duration of the MS attacks is reduced relative to a patient who
initiated conventional multiple sclerosis therapy with similar
clinical parameters and was administered conventional multiple
sclerosis therapy over the 6 months, 9 months, 12 months, 15
months, 18 months, 2 year, 2.5 year or 3 year period, said
determination made at 6 months, 9 months, 12 months, 15 months, 18
months, or 24 months after the previous treatment.
[0064] In other embodiments with respect to the treatment of
psoriasis, the anti-CD3 antibody is administered to achieve a
reduction in the subject's Psoriasis Area and Severity Index (PAST)
score by at least 20%, at least 35%, at least 30%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, or at least 85% relative
to pretreatment conditions, said determinations made at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous treatment. Alternatively, the methods of the invention
improve the global assessment score of a subject by at least 25%,
at least 35%, at least 30%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, or at least
95% relative to pretreatment conditions, said determinations made
at 6 months, 9 months, 12 months, 15 months, 18 months, or 24
months after the previous treatment.
[0065] In other embodiments with respect to the treatment of
rheumatoid arthritis, the anti-CD3 antibody is administered to
achieve an improvement in the subject's condition as assessed by
any arthritis severity scale known in the art (e.g., rheumatoid
arthritis severity scale (RASS)) by at least 25%, at least 35%, at
least 30%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, or at least 100%
relative to pretreatment conditions, said determinations made at 6
months, 9 months, 12 months, 15 months, 18 months, or 24 months
after the previous treatment.
[0066] In certain embodiments, diagnosis of an autoimmune disorder
or manifestation of a predisposition of an autoimmune disorder is
based on the detection of cytotoxic T-lymphocytes ("CTLs") that
recognize donor specific antigens (i.e., autoreactive CTLs) in the
peripheral blood of the subject and/or target tissue of the immune
disorder. In certain embodiments, the anti-CD3 antibody of the
invention is administered to achieve a reduction by at least 10%,
at least 20%, at least 35%, at least 30%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, or at least 85% in absolute
number, or proportion, of the subject's autoreactive CTLs as
determined by immunospot assay (e.g., ELISPOT) relative to the
pretreatment condition, said determinations made at 6 months, 9
months, 12 months, 15 months, 18 months, or 24 months after the
previous treatment.
[0067] In preferred embodiments, the patient is under 21 years of
age, 18 years of age, under 15 years of age, under 12 years of age,
under 9 years of age, or under 5 years of age or from infancy to 3
years of age, from 2 to 5 years of age, from 5 to 9 years of age,
from 9 to 12 years of age, from 12 to 20 years of age. In other
embodiments, the patient is an adult.
[0068] The invention also provides combination therapy methods. The
methods of the invention can be carried out in combination with any
standard treatment for the particular indication, such as standard
immunosuppressant and/or anti-inflammatory treatments administered
for the treatment or amelioration of autoimmune diseases. For
example, with respect to the treatment of Type 1 diabetes, the
anti-human CD3 antibody therapy of the invention may be
administered along with other therapies for diabetes, such as, but
not limited to, administration of insulin, exenatide, pramlintide
or a combination thereof. With respect to the treatment of multiple
sclerosis, the anti-human CD3 antibody therapy of the invention may
be administered with other therapies known in the art for the
treatment of multiple sclerosis, such as, but not limited to,
administration of beta interferon (e.g., AVONEX.RTM.,
BETASERON.RTM., REBIF.RTM.), immunosuppressant (e.g.,
mitoxantrone), myelin basic protein copolymer 1 (e.g.,
COPAXONE.RTM.), or a combination thereof. The CD3 antibodies of the
invention may further be administered with other therapies such as
anti IL-2 antibodies, cytokine antagonists, and steroidal therapies
(for example, but not limited to, glucocorticoids, dexamethasone,
cortisone, hydrocortisone, prednisone, prednisolone, triamcinolone,
azulfidine, etc.), non-steroidal anti-inflammatories (NSAIDS), such
as, but not limited to aspirin, ibuprofen, diclofenac, etodolac,
fenoprofen, indomethacin, ketolorac, oxaprozin, nabumetone,
sulindac, tolmentin, naproxen, or ketoprofen, immunosuppressants,
such as, methotrexate or cyclosporin, and TNF-.alpha. inhibitors
such as, but not limited to, etanercept and infliximab. In certain
embodiments of the invention, subjects which have become refractory
to conventional treatments are treated using methods of the
invention. In certain embodiments, the anti-human CD3 antibody is
administered in combination with one or more islet cell antigens,
such as GAD, IA-2 or other antigens which are bound by autoantigens
found in patients with type 1 diabetes.
[0069] The invention, in other embodiments, provides methods of
producing anti-human CD3 antibodies, particularly OKT3 derived
antibodies, such as, but not limited to, humanized OKT.gamma.1
(ala-ala), in CHO cells. In particular embodiments, the invention
provides methods of producing anti-human CD3 antibodies comprising
(a) culturing CHO cells that have been transfected with the
expression vector pMGX1303, or progeny thereof, in media under
conditions suitable for expression of said anti-human CD3 antibody;
and (b) recovering said anti-human CD3 antibody from said
media.
3.1 Terminology
[0070] As used herein, the term "about" or "approximately," when
used in conjunction with a number, refers to any number within 1, 5
or 10% of the referenced number or within experimental error
typical of methods used for measurement.
[0071] As used herein, the term "analog" in the context of
polypeptides refers to a polypeptide that possesses a similar or
identical function as a second polypeptide but does not necessarily
comprise a similar or identical amino acid sequence of the second
polypeptide, or possess a similar or identical structure of the
second polypeptide. A polypeptide that has a similar amino acid
sequence refers to a second polypeptide that satisfies at least one
of the following: (a) a polypeptide having an amino acid sequence
that is at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99% identical to the amino acid sequence of a
second polypeptide; (b) a polypeptide encoded by a nucleotide
sequence that hybridizes under stringent conditions to a nucleotide
sequence encoding a second polypeptide of at least 5 contiguous
amino acid residues, at least 10 contiguous amino acid residues, at
least 15 contiguous amino acid residues, at least 20 contiguous
amino acid residues, at least 25 contiguous amino acid residues, at
least 40 contiguous amino acid residues, at least 50 contiguous
amino acid residues, at least 60 contiguous amino residues, at
least 70 contiguous amino acid residues, at least 80 contiguous
amino acid residues, at least 90 contiguous amino acid residues, at
least 100 contiguous amino acid residues, at least 125 contiguous
amino acid residues, or at least 150 contiguous amino acid
residues; and (c) a polypeptide encoded by a nucleotide sequence
that is at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95% or at least 99% identical to the nucleotide sequence encoding a
second polypeptide. A polypeptide with similar structure to a
second polypeptide refers to a polypeptide that has a similar
secondary, tertiary or quaternary structure to the second
polypeptide. The structure of a polypeptide can be determined by
methods known to those skilled in the art, including but not
limited to, peptide sequencing, X-ray crystallography, nuclear
magnetic resonance, circular dichroism, and crystallographic
electron microscopy.
[0072] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length.
[0073] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A.
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul et al., 1990, J.
Mol. Biol. 215:403. BLAST nucleotide searches can be performed with
the NBLAST nucleotide program parameters set, e.g., for score=100,
wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid molecules of the present invention. BLAST protein
searches can be performed with the) (BLAST program parameters set,
e.g., to score-50, wordlength=3 to obtain amino acid sequences
homologous to a protein molecule of the present invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., 1997, Nucleic Acids
Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform
an iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used.
[0074] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0075] As used herein, the term "analog" in the context of a
non-proteinaceous analog refers to a second organic or inorganic
molecule which possess a similar or identical function as a first
organic or inorganic molecule and is structurally similar to the
first organic or inorganic molecule.
[0076] As used herein, the terms "antagonist" and "antagonists"
refer to any protein, polypeptide, peptide, antibody, antibody
fragment, large molecule, or small molecule (less than 10 kD) that
blocks, inhibits, reduces or neutralizes the function, activity
and/or expression of another molecule. In various embodiments, an
antagonist reduces the function, activity and/or expression of
another molecule by at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95% or at least 99% relative to a control such as phosphate
buffered saline (PBS).
[0077] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, chimeric antibodies, single-chain
Fvs (scFv), single chain antibodies, Fab fragments, F(ab')
fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. In particular, antibodies include immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site.
Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1 and IgA.sub.2) or subclass.
[0078] As used herein, the term "C-peptide" refers to a 31-amino
acid peptide cleaved from proinsulin as it is converted to insulin.
Proinsulin consists of an A chain, a connecting peptide
(C-peptide), and a B chain. After proinsulin is cleaved, C-peptide
remains in the secretory granules of beta cells in the pancreas
with insulin and is cosecreted with insulin in response to glucose
stimulation. C-peptide is thus released from the pancreas in
equi-molar amounts with insulin and may be used as a marker of
endogenous insulin production.
[0079] As used herein, the term "derivative" in the context of
polypeptides refers to a polypeptide that comprises an amino acid
sequence which has been altered by the introduction of amino acid
residue substitutions, deletions or additions. The term
"derivative" as used herein also refers to a polypeptide that has
been modified, i.e, by the covalent attachment of any type of
molecule to the polypeptide. For example, but not by way of
limitation, an antibody may be modified, e.g., by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. A derivative
polypeptide may be produced by chemical modifications using
techniques known to those of skill in the art, including, but not
limited to specific chemical cleavage, acetylation, formylation,
metabolic synthesis of tunicamycin, etc. Further, a derivative
polypeptide may contain one or more non-classical amino acids. A
polypeptide derivative possesses a similar or identical function as
the polypeptide from which it was derived.
[0080] As used herein, the terms "disorder" and "disease" are used
interchangeably to refer to a condition in a subject. In
particular, the term "autoimmune disease" is used interchangeably
with the term "autoimmune disorder" to refer to a condition in a
subject characterized by cellular, tissue and/or organ injury
caused by an immunologic reaction of the subject to its own cells,
tissues and/or organs.
[0081] As used herein, the term "epitopes" refers to fragments of a
polypeptide or protein having antigenic or immunogenic activity in
an animal, preferably in a mammal, and most preferably in a human.
An epitope having immunogenic activity is a fragment of a
polypeptide or protein that elicits an antibody response in an
animal. An epitope having antigenic activity is a fragment of a
polypeptide or protein to which an antibody immunospecifically
binds as determined by any method well-known to one of skill in the
art, for example by immunoassays. Antigenic epitopes need not
necessarily be immunogenic.
[0082] As used herein, the term "Fc region" is used to define a
C-terminal region of an IgG heavy chain. Although the boundaries
may vary slightly, the human IgG heavy chain Fc region is defined
to stretch from Cys226 to the carboxy terminus. The Fc region of an
IgG comprises two constant domains, CH2 and CH3. The CH2 domain of
a human IgG Fc region usually extends from amino acids 231 to amino
acid 341. The CH3 domain of a human IgG Fc region usually extends
from amino acids 342 to 447. The Fc region of an IgG comprises two
constant domains, CH2 and CH3. The CH2 domain of a human IgG Fc
region (also referred to as "Cy2" domain) usually extends from
amino acid 231-340. The CH2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG.
[0083] Throughout the present specification, the numbering of the
residues in an IgG heavy chain is that of the EU index as in Kabat
et al., Sequences of Proteins of Immunological Interest, 5.sup.th
Ed. Public Health Service, NH1, MD (1991), expressly incorporated
herein by references. The "EU index as in Kabat" refers to the
numbering of the human IgG1 EU antibody.
[0084] The "hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1. Hinge regions of other IgG isotypes
may be aligned with the IgG1 sequence by placing the first and last
cysteine residues forming inter-heavy chain S-S binds in the same
positions.
[0085] As used herein, the term "fragment" refers to a peptide or
polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 40 contiguous amino acid residues, at least 50
contiguous amino acid residues, at least 60 contiguous amino
residues, at least 70 contiguous amino acid residues, at least
contiguous 80 amino acid residues, at least contiguous 90 amino
acid residues, at least contiguous 100 amino acid residues, at
least contiguous 125 amino acid residues, at least 150 contiguous
amino acid residues, at least contiguous 175 amino acid residues,
at least contiguous 200 amino acid residues, or at least contiguous
250 amino acid residues of the amino acid sequence of another
polypeptide. In a specific embodiment, a fragment of a polypeptide
retains at least one function of the polypeptide.
[0086] As used herein, the term "functional fragment" refers to a
peptide or polypeptide comprising an amino acid sequence of at
least 5 contiguous amino acid residues, at least 10 contiguous
amino acid residues, at least 15 contiguous amino acid residues, at
least 20 contiguous amino acid residues, at least 25 contiguous
amino acid residues, at least 40 contiguous amino acid residues, at
least 50 contiguous amino acid residues, at least 60 contiguous
amino residues, at least 70 contiguous amino acid residues, at
least contiguous 80 amino acid residues, at least contiguous 90
amino acid residues, at least contiguous 100 amino acid residues,
at least contiguous 125 amino acid residues, at least 150
contiguous amino acid residues, at least contiguous 175 amino acid
residues, at least contiguous 200 amino acid residues, or at least
contiguous 250 amino acid residues of the amino acid sequence of
second, different polypeptide, wherein said peptide or polypeptide
retains at least one function of the second, different
polypeptide.
[0087] As used herein, the term "fusion protein" refers to a
polypeptide that comprises an amino acid sequence of a first
protein or functional fragment, analog or derivative thereof, and
an amino acid sequence of a heterologous protein (i.e., a second
protein or functional fragment, analog or derivative thereof
different than the first protein or functional fragment, analog or
derivative thereof). In particular embodiments, a fusion protein
comprises a CD3 binding molecule and a heterologous protein,
polypeptide, or peptide.
[0088] As used herein, the term "host cell" refers to the
particular subject cell transfected with a nucleic acid molecule
and the progeny or potential progeny of such a cell. Progeny of
such a cell may not be identical to the parent cell transfected
with the nucleic acid molecule due to mutations or environmental
influences that may occur in succeeding generations or integration
of the nucleic acid molecule into the host cell genome.
[0089] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing
under which nucleotide sequences at least 60% (65%, 70%, preferably
75%, 80% or 85%, and more preferably, 90% or 95%) identical to each
other typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. In one, non-limiting example stringent
hybridization conditions are hybridization at 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.1.times.SSC, 0.2% SDS at about 68.degree.
C. In a preferred, non-limiting example stringent hybridization
conditions are hybridization in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. (i.e., one or more washes at 50.degree. C.,
55.degree. C., 60.degree. C. or 65.degree. C.). It is understood
that the nucleic acids of the invention do not include nucleic acid
molecules that hybridize under these conditions solely to a
nucleotide sequence consisting of only A or T nucleotides.
[0090] As used herein, the term "hypoglycemic episode" refers to a
blood glucose level in a subject of less than 60 mg/dL that results
in typical symptoms of hypoglycemia such as sweatiness, nausea,
blurred vision (e.g., seeing spots), shakiness, numb lips and/or
tongue, irritability, fainting, clammy skin, confusion,
nervousness, weakness, and/or rapid heart beat.
[0091] As used herein, the term "immunomodulatory agent" and
variations thereof refer to an agent that modulates a host's immune
system. In certain embodiments, an immunomodulatory agent is an
immunosuppressant agent. In certain other embodiments, an
immunomodulatory agent is an immunostimulatory agent.
Immunomodulatory agents include, but are not limited to, small
molecules, peptides, polypeptides, fusion proteins, antibodies,
inorganic molecules, mimetic agents, and organic molecules.
[0092] As used herein, the term "immunospecifically binds to an
antigen" and analogous terms refer to peptides, polypeptides,
fusion proteins and antibodies or fragments thereof that
specifically bind to an antigen or a fragment and do not
specifically bind to other antigens. A peptide or polypeptide that
immunospecifically binds to an antigen may bind to other peptides
or polypeptides with lower affinity as determined by, e.g.,
immunoassays, BIAcore, or other assays known in the art. Antibodies
or fragments that immunospecifically bind to an antigen may
cross-reactive with related antigens. Preferably, antibodies or
fragments that immunospecifically bind to an antigen do not
cross-react with other antigens.
[0093] As used herein, the term "immunospecifically binds to a CD3
polypeptide" and analogous terms refer to peptides, polypeptides,
fusion proteins and antibodies or fragments thereof that
specifically bind to a CD3 polypeptide or a fragment thereof and do
not specifically bind to other polypeptides. A peptide or
polypeptide that immunospecifically binds to a CD3 polypeptide may
bind to other peptides or polypeptides with lower affinity as
determined by, e.g., immunoassays, BIAcore, or other assays known
in the art. Antibodies or fragments that immunospecifically bind to
a CD3 polypeptide may be cross-reactive with related antigens.
Preferably, antibodies or fragments that immunospecifically bind to
a CD3 polypeptide or fragment thereof do not cross-react with other
antigens. Antibodies or fragments that immunospecifically bind to a
CD3 polypeptide can be identified, for example, by immunoassays,
BIAcore, or other techniques known to those of skill in the art. An
antibody or fragment thereof binds specifically to a CD3
polypeptide when it binds to a CD3 polypeptide with higher affinity
than to any cross-reactive antigen as determined using experimental
techniques, such as radioimmunoassays (RIA) and enzyme-linked
immunosorbent assays (ELISAs). See, e.g., Paul, ed., 1989,
Fundamental Immunology Second Edition, Raven Press, New York at
pages 332-336 for a discussion regarding antibody specificity.
[0094] As used herein, the term "in combination" refers to the use
of more than one prophylactic and/or therapeutic agent. The use of
the term "in combination" does not restrict the order in which
prophylactic and/or therapeutic agents are administered to a
subject with a disease or disorder. A first prophylactic or
therapeutic agent can be administered prior to (e.g., 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second prophylactic or therapeutic agent
(different from the first prophylactic or therapeutic agen) to a
subject with a disease or disorder.
[0095] As used herein, the term "isolated" in the context of a
peptide, polypeptide, fusion protein or antibody refers to a
peptide, polypeptide, fusion protein or antibody which is
substantially free of cellular material or contaminating proteins
from the cell or tissue source from which it is derived, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of a peptide, polypeptide,
fusion protein or antibody in which the peptide, polypeptide,
fusion protein or antibody is separated from cellular components of
the cells from which it is isolated or recombinantly produced.
Thus, a peptide, polypeptide, fusion protein or antibody that is
substantially free of cellular material includes preparations of a
peptide, polypeptide, fusion protein or antibody having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
peptide, polypeptide, fusion protein or antibody is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the peptide,
polypeptide, fusion protein or antibody is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the peptide, polypeptide, fusion protein or antibody.
Accordingly such preparations of a peptide, polypeptide, fusion
protein or antibody have less than about 30%, 20%, 10%, 5% (by dry
weight) of chemical precursors or compounds other than the peptide,
polypeptide, fusion protein or antibody of interest. In a preferred
embodiment, a CD3 binding molecule is isolated. In another
preferred embodiment, an anti-human CD3 antibody is isolated.
[0096] As used herein, the term "isolated" in the context of
nucleic acid molecules refers to a nucleic acid molecule which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized
and may be free of cDNA or other genomic DNA molecules, e.g., has
been isolated from other clones in a nucleic acid library. In a
preferred embodiment, a nucleic acid molecule encoding a CD3
binding molecule is isolated. In another preferred embodiment, a
nucleic acid molecule encoding an anti-human CD3 antibody is
isolated.
[0097] As used herein, the terms "non-responsive" and refractory"
describe patients treated with a currently available prophylactic
or therapeutic agent for an autoimmune disorder which is not
clinically adequate to relieve one or more symptoms associated with
the autoimmune disorder. Typically, such patients suffer from
severe, persistently active disease and require additional therapy
to ameliorate the symptoms associated with their autoimmune
disorder.
[0098] As used herein, the term "onset" of disease with reference
to Type-1 diabetes refers to a patient meeting the criteria
established for diagnosis of Type-1 diabetes by the American
Diabetes Association (see, Mayfield et al., 2006, Am. Fam.
Physician 58:1355-1362).
[0099] As used herein, the terms "nucleic acids" and "nucleotide
sequences" include DNA molecules (e.g., cDNA or genomic DNA), RNA
molecules (e.g., mRNA), combinations of DNA and RNA molecules or
hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such
analogs can be generated using, for example, nucleotide analogs,
which include, but are not limited to, inosine or tritylated bases.
Such analogs can also comprise DNA or RNA molecules comprising
modified backbones that lend beneficial attributes to the molecules
such as, for example, nuclease resistance or an increased ability
to cross cellular membranes. The nucleic acids or nucleotide
sequences can be single-stranded, double-stranded, may contain both
single-stranded and double-stranded portions, and may contain
triple-stranded portions, but preferably is double-stranded
DNA.
[0100] As used herein, the terms "prophylactic agent" and
"prophylactic agents" refer to CD3 binding molecules which can be
used in the prevention, treatment, management or amelioration of
one or more symptoms of an autoimmune disease. In certain
embodiments, the term "prophylactic agent" refers to anti-human CD3
antibodies (e.g., OKT3 and variants and derivatives thereof).
[0101] As used herein, the term "prophylactically effective amount"
refers to that amount of a CD3 binding molecule sufficient to
prevent the development, recurrence or onset of one or more
symptoms of a disorder. In certain embodiments, the term
"prophylactically effective amount" refers to the amount of an
anti-human CD3 antibody sufficient to prevent the development,
recurrence or onset of one or more symptoms of a disorder.
[0102] As used herein, the terms "prevent", "preventing" and
"prevention" refer to the prevention of the recurrence or onset of
one or more symptoms of an autoimmune or inflammatory disorder in a
subject resulting from the administration of a prophylactic or
therapeutic agent.
[0103] As used herein, a "protocol" includes dosing schedules and
dosing regimens. The protocols herein are methods of use and
include prophylactic and therapeutic protocols. A "dosing regimen"
or "course of treatment" may include administration of several
doses of a therapeutic or prophylactic agent over 1 to 20 days.
[0104] As used herein, the phrase "side effects" encompasses
unwanted and adverse effects of a prophylactic or therapeutic
agent. Adverse effects are always unwanted, but unwanted effects
are not necessarily adverse.
[0105] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, the terms "subject" and "subjects"
refer to an animal, preferably a mammal including a non-primate
(e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate
(e.g., a monkey or a human), and more preferably a human.
[0106] As used herein, the term "synergistic" refers to a
combination of prophylactic or therapeutic agents which is more
effective than the additive effects of the agents in the
combination when administered individually. A synergistic effect of
a combination of prophylactic or therapeutic agents may permit the
use of lower dosages of one or more of the agents and/or less
frequent administration of said agents to a subject with an
autoimmune disorder. The ability to utilize lower dosages of
prophylactic or therapeutic agents and/or to administer said agents
less frequently reduces the toxicity associated with the
administration of said agents to a subjected without reducing the
efficacy of said agents in the prevention or treatment of
autoimmune disorders. In addition, a synergistic effect can result
in improved efficacy of agents in the prevention or treatment of
autoimmune disorders. Finally, synergistic effect of a combination
of prophylactic or therapeutic agents may avoid or reduce adverse
or unwanted side effects associated single agent therapy.
[0107] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to CD3 binding molecules which can be
used in the prevention, treatment, management or amelioration of
one or more symptoms of an autoimmune or inflammatory disease. In
certain embodiments, the term "therapeutic agent" refers to
anti-human CD3 antibodies (e.g., OKT3 and variants or derivatives
thereof).
[0108] As used herein, the term "therapeutically effective amount"
refers to that amount of a therapeutic agent sufficient to result
in amelioration of one or more symptoms of a disorder. With respect
to diabetes, a therapeutically effective amount preferably refers
to the amount of therapeutic agent that reduces a subject's average
daily insulin requirements by at least 20%, by at least 25%, by at
least 30%, by at least 35%, by at least 40%, by at least 45%, by at
least 50%, by at least 55%, by at least 60%, by at least 65%, by at
least 70%, by at least 75%, by at least 80%, by at least 85%, by at
least 90%, by at least 95%.
[0109] As used herein, the terms "treat", "treatment" and
"treating" refer to the amelioration of one or more symptoms
associated with an autoimmune or inflammatory disorder that results
from the administration of one or more CD3 binding molecules. In
particular, such terms refer to the amelioration of one or more
symptoms associated with an autoimmune disorders that results from
the administration of one or more anti-human CD3 antibodies
4. DESCRIPTION OF THE FIGURES
[0110] FIGS. 1A and 1B. Sequences of humanized OKT3 variable
regions. FIG. 1A and FIG. 1B show the alignments of the OKT3 light
chain (FIG. 1A) (SEQ ID NO:1) and the heavy chain (FIG. 1B) (SEQ ID
NO:5) variable domain amino acid sequence (row 1), the variable
domain sequence from the human antibodies chosen as light and heavy
chain acceptor framework (row 2) (SEQ ID NOs:2 and 6,
respectively), and the humanized OKT3 variable domain sequences
(rows 3-5) (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8 and
SEQ ID NO:9). The CDR choices are singly underlined. Rows 3-5 show
only differences from the human acceptor sequence, with the non-CDR
differences shown double underlined. Dashes indicate gaps
introduced in the sequences to maximize the alignment. Numbering is
as in Kabat et al., Sequences of Proteins of Immunological
Interest, 5.sup.th Ed. Public Health Service, NH1, MD (1991), which
is incorporated by reference herein.
[0111] FIGS. 2A-2D. FIGS. 2A and 2B, nucleotide and amino acid
sequences, respectively, of the light chain of humanized
OKT3.gamma.1 (SEQ ID NOs: 10 and 11, respectively). FIGS. 2C and
2D, nucleotide and amino acid sequences, respectively, of the heavy
chain of humanized OKT3.gamma.1 (ala-ala) (SEQ ID NOs: 12 and 13,
respectively).
[0112] FIG. 3. Schematic representation of mammalian expression
vector pMGX1303, containing coding regions for humanized OKT3 and
capable of promoting expression of the humanized antibody in CHO
cells.
5. DETAILED DESCRIPTION OF THE INVENTION
[0113] The present invention provides methods of treating,
preventing, slowing the progression of and ameliorating the
symptoms of autoimmune diseases or disorders using proteins,
particularly, antibodies, directed against the CD3 complex
associated with the human T cell receptor or TcR. In particular
embodiments, the antibody binds to the epsilon subunit of the CD3
complex. The methods of the invention may be used with any
anti-human CD3 antibody presented herein or known in the art, e.g.
OKT3, ChAglyCD3 (TRX4.TM.), HUM291 (visilizumab; Nuvion.TM.),
UCHT1, Leu4, 500A2, CLB-T3/3, BMA030 and YTH 12.5, and variations
or derivatives thereof. In one embodiment of the invention the
antibody is OKT3, preferably humanized versions of OKT3 or an
antibody that competes for binding, for example, as determined by
immunoprecipitation assay or ELISA, with OKT3. In another
embodiment, the antibody is humanized OKT3, which has been modified
at one or more amino acid residues to exhibit reduced T cell
activation and/or FcR binding when compared to a non-modified
humanized OKT3 antibody, such as having an alanine at, e.g.,
residue number 234 of the Fc domain, and an alanine at, e.g.,
residue number 235 of the Fc domain. Preferably, the anti-human CD3
antibodies are administered are administered at lower dosages or
over shorter periods of time than prior dosing regimens. In
particular, the invention contemplates dosing regimens in which
less than 9,000 .mu.g/m.sup.2, preferably, less than 8,000
.mu.g/m.sup.2, less than 7,500 .mu.g/m.sup.2, less than 7,000
.mu.g/m.sup.2, or less than 6,000 .mu.g/m.sup.2 total anti-human
CD3 antibody over the duration of the dosing, particularly of
OKT3.gamma.1 (ala-ala) administered intravenously, or the
pharmacological equivalent amount of another anti-human CD3
antibody of this dose of OKT3.gamma.1 (ala-ala) administered
intravenously and/or any anti-human CD3 antibody administered by a
route of administration other than intravenously, as well as
chronic dosing methods and redosing or repeated dosing methods.
[0114] Anti-CD3 mAbs are potent immunosuppressive agents directed
against an invariant protein complex associated with the human TcR
(Van Wauwe, 1980, J. Immunol. 124:2708). The CD3 complexes are
believed to be accessory structures that transduce the activation
signals initiated upon binding of the TcR to its ligand. Binding of
the anti-CD3 antibody OKT3 to the TcR mediates TcR blockade and
inhibits alloantigen recognition and cell-mediated cytotoxicity
(Landegren et al., 1982, J. Exp. Med. 155:1579; van Seventer et
al., 1987, J. Immunol. 139:2545; Weiss et al., 1986, Ann. Rev.
Immunol. 4:593). However, the administration of some immune-cell
directed antibodies, including OKT3 and other anti-CD3 antibodies,
may induce T cell activation, including the systematic release of
several cytokines, including IL-2, IL-6, TNF-.alpha. and
IFN-.gamma. (Abramowicz, 1989, Transplantation, 47:606-608;
Chatenoud, 1989, New Eng. J. Med. 320:1420-1421). This production
of cytokines has been correlated with the adverse side-effects
frequently observed after the first injection of mAbs (Van Wauwe,
1980, J. Immunol. 124:2708; Chatenoud, 1989, New Eng. J. Med.
320:1420-1421; Thistlethwaite, 1988, Am J Kidney Dis., 11:112-9),
and may augment the production of anti-isotypic and anti-idiotypic
antibodies occurring in some patents after one or two weeks of
treatment. This immune response can neutralize the specific
antibody, as well as other antibodies of the same class (isotype),
and preclude subsequent treatments (Thistlethwaite, 1988, Am J
Kidney Dis. 11:112-9).
[0115] Several pieces of evidence strongly suggest that these
side-effects are a consequence of the cross-linking between T
lymphocytes and Fc receptor (FcR)-bearing cells through the Fc
portion of antibodies, including for example, OKT3, resulting in
activation of both cell types (Debets, 1990, J. Immunol. 144:1304;
Krutman, 1990, J. Immunol. 145:1337): 1) anti-CD3 mAbs did not
stimulate T cell proliferation in vitro, unless the antibody was
immobilized to plastic or bound to FcR+ antigen presenting cells
included in the culture (van Lier, 1989, Immunol. 68:45); 2) the
cross-linking of OKT3 through FcRs I and II enhanced proliferation
in response to IL-2, in vitro (van Lier, 1987, J. Immunol.
139:2873); 3) proliferation of murine T cells induced by 145-2C11,
a hamster monoclonal antibody directed against the murine CD3
complex, could be blocked by the anti-FcR antibody, 2.4G2; 4) the
injection into mice of F(ab').sub.2 fragments of 145-2C11 induced
significant immunosuppression without triggering full T cell
activation (Hirsch, 1990, Transplantation, 49:1117-23) and was less
toxic in mice than the whole antibody (Alegre, 1990, Transplant
Proc. 22:1920-1); and 5) the administration of an OKT3 IgA switch
variant that displayed a reduced FcR-mediated T cell activation as
compared with OKT3 IgG2a, resulted in fewer side effects in
chimpanzees in vivo (Parleviet, 1990, Brief Communications
50:889-892).
[0116] Administration of certain anti-CD3 antibodies has also been
associated with transient retrovirus activation, specifically
activation of dormant Epstein-Barr Virus (EBV) infection. Anti-CD3
antibody treatment has also been found to be lytic to activated T
cells and apoptotic to some T cell populations. The reasons for
these effects are unclear, but they may be dose related and are
probably the result of the modulation of the TcR complex resulting
in suboptimal signaling.
[0117] Thus improvement of anti-CD3 mAb therapy can be obtained by
molecularly modifying the antibody to reduce its affinity for FcRs.
The mutated Ab obtained could lead to lower cellular activation and
reduced toxicity in vivo, but retain the original immunosuppressive
properties of the antibody.
[0118] 5.1 Antibodies that Immunospecifically Bind to CD3
Polypeptides
[0119] It should be recognized that antibodies that
immunospecifically bind to a CD3 polypeptide are known in the art.
Examples of known antibodies that immunospecifically bind to a CD3
polypeptide include, but are not limited to OKT3, HuM291,
ChAglyCD3, UCHT1, Leu4, 500A2, CLB-T3/3, BMA030, YTH 12.5 and rat
CD3 antibody (See Herold et al., 2005, Diabetes 54:1763-1769;
Carpenter et al., 2005, Biol. Blood Marrow Transplant 11:465-471;
Keymeulen et al., 2005, N. Engl. J. Med. 352:26422644; Schwinzer et
al., 1992, J. Immunol. 148:1322-1328; Tsoukas et al., 1985, J.
Immunol. 135:1719-1723; U.S. Pat. No. 6,491,916; Brams et al.,
1989, Immunol., 66:348-353; van Lier et al., 1989, Immunol.
68:45-50; Walker et al., 1987, Eur. J. Immunol. 17:1611-1618;
Routledge et al., 1991, Eur. J. Immunol. 21:2717-2725,
respectively).
[0120] The present invention provides methods of treating,
preventing, slowing the progression of and ameliorating the
symptoms of autoimmune disorders using antibodies that
immunospecifically bind to a CD3 polypeptide expressed by an immune
cell such as a T cell, wherein said antibodies modulate an activity
or function of said T cell. In a specific embodiment, antibodies
that immunospecifically bind to a CD3 polypeptide directly or
indirectly modulate the activity of lymphocytes, preferably
peripheral blood T cells. In particular, the present invention
provides antibodies that immunospecifically bind to a CD3
polypeptide expressed by a T cell, and said antibodies modulate the
activity of peripheral blood T cell.
[0121] In a specific embodiment, antibodies that immunospecifically
bind to a CD3 polypeptide inhibit T cell activation by at least
25%, at least 30%, at least 35%, at least 40%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
98% and inhibit T cell proliferation by at least 25%, at least 30%,
at least 35%, at least 40%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 98% in an in
vivo or in vitro assay described herein or well-known to one of
skill in the art. In another embodiment, antibodies that
immunospecifically bind to a CD3 polypeptide inhibit alloantigen
recognition by T cells by at least 25%, at least 30%, at least 35%,
at least 40%, at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or at least 98% in an in vivo or in vitro
assay described herein or well-known to one of skill in the art. In
another embodiment, antibodies that immunospecifically bind to a
CD3 polypeptide inhibit T cell mediated cytotoxicity by at least
25%, at least 30%, at least 35%, at least 40%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
98% in an in vivo or in vitro assay described herein or well-known
to one of skill in the art.
[0122] In another embodiment, the methods of the invention employ
antibodies that immunospecifically bind to a CD3 polypeptide and do
not induce or have reduced (as compared to unmodified antibodies,
e.g., the murine OKT3 monoclonal antibody) cytokine expression
and/or release in an in vivo or in vitro assay described herein or
well-known to one of skill in the art. In a specific embodiment,
antibodies that immunospecifically bind to a CD3 polypeptide do not
induce an increase in the concentration cytokines such as, e.g.,
IFN-.gamma., IL-2, IL-4, IL-6, IL-9, IL-12, and IL-15 in the serum
of a subject administered such an antibody. In an alternative
embodiment, antibodies that immunospecifically bind to a CD3
polypeptide induce cytokine expression and/or release in an in
vitro or in vivo assay described herein or well-known to one of
skill in the art but at levels less than those induced by
unmodified anti-CD3 antibodies, such as, the murine OKT3 monoclonal
antibody. Serum concentrations of a cytokine can be measured by any
technique well-known to one of skill in the art such as, e.g.,
ELISA.
[0123] In another embodiment, antibodies that immunospecifically
bind to a CD3 polypeptide induce T cell anergy in an in vivo or in
vitro assay described herein or well-known to one of skill in the
art. In an alternative embodiment, antibodies that
immunospecifically bind to a CD3 polypeptide do not induce T cell
anergy in an in vivo or in vitro assay described herein or
well-known to one of skill in the art. In another embodiment,
antibodies that immunospecifically bind to a CD3 polypeptide elicit
a state of antigen-specific unresponsiveness for at least 30
minutes, at least 1 hour, at least 2 hours, at least 6 hours, at
least 12 hours, at least 24 hours, at least 2 days, at least 5
days, at least 7 days, at least 10 days or more in an in vitro
assay described herein or known to one of skill in the art.
[0124] In another embodiment, antibodies that immunospecifically
bind to a CD3 polypeptide inhibit T cell activation by at least
25%, at least 30%, at least 35%, at least 40%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
98% and inhibit T cell proliferation by at least 25%, at least 30%,
at least 35%, at least 40%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 98% in an in
vivo or in vitro assay described herein or well-known to one of
skill in the art.
[0125] In yet another embodiment, antibodies that
immunospecifically bind to a CD3 polypeptide achieve T cell coating
or modulation by at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, or at least 98% and inhibit T cell
proliferation by at least 25%, at least 30%, at least 35%, at least
40%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, and preferably by 100% in
an in vivo or in vitro assay described herein or well-known to one
of skill in the art.
[0126] In another embodiment, the Fc domain of an antibody that
immunospecifically binds to a CD3 polypeptide does not detectably
bind to one or more of the Fc receptors ("FcR") FcRI, FcRII, and/or
FcRIII expressed by an immune cell such as a T cell, monocyte, and
macrophage.
[0127] Antibodies that immunospecifically bind to a CD3 polypeptide
include, but are not limited to, monoclonal antibodies,
multispecific antibodies, human antibodies, humanized antibodies,
chimeric antibodies, single-chain Fvs (scFv), single chain
antibodies, Fab fragments, F(ab') fragments, F(ab').sub.2
fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. In particular, antibodies that immunospecifically
bind to a CD3 polypeptide include immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds to a CD3 polypeptide. The immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1 and IgA.sub.2) or subclass of immunoglobulin
molecule. In a specific embodiment, the antibodies that
immunospecifically bind to a CD3 polypeptide and mediate the
activity of T cells comprise an Fc domain or a fragment thereof
(e.g., the CH2, CH3, and/or hinge regions of an Fc domain). In a
preferred embodiment, the antibodies that immunospecifically bind
to a CD3 polypeptide and mediate the activity of T cells comprise
an Fc domain or fragment thereof that does not detectably bind to
an FcR (e.g., one or more of an FcRI, FcRII or FcRIII) expressed by
an immune cell or has reduced FcR binding as compared to an
antibody with a wild type Fc domain.
[0128] The antibodies that immunospecifically bind to a CD3
polypeptide may be from any animal origin including birds and
mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea
pig, camel, horse, or chicken). Preferably, the antibodies of the
invention are human, humanized or chimeric monoclonal antibodies.
Human antibodies that immunospecifically bind to a CD3 polypeptide
include antibodies having the amino acid sequence of a human
immunoglobulin and antibodies isolated from human immunoglobulin
libraries or from mice that express antibodies from human
genes.
[0129] The antibodies that immunospecifically bind to a CD3
polypeptide may be monospecific, bispecific, trispecific or of
greater multispecificity. Multispecific antibodies may be specific
for different epitopes of a CD3 polypeptide or may be specific for
both a CD3 polypeptide as well as for a heterologous epitope, such
as a heterologous polypeptide or solid support material. See, e.g.,
PCT publications WO 93/17715, WO 92/08802, WO 91/00360, and WO
92/05793; Tutt, et al., J. Immunol. 147:60-69(1991); U.S. Pat. Nos.
4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and
Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0130] The present invention provides for antibodies that have a
high binding affinity for a CD3 polypeptide. In a specific
embodiment, an antibody that immunospecifically binds to a CD3
polypeptide has an association rate constant or k.sub.on rate
(antibody (Ab)+antigen (Ag).sup.k.sup.on.fwdarw.Ab-Ag) of at least
10.sup.5M.sup.-1s.sup.-1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least 5.times.10.sup.6
M.sup.-1s.sup.-1, at least 10.sup.7M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least
10.sup.8M.sup.-1s.sup.-1. In a preferred embodiment, an antibody
that immunospecifically binds to a CD3 polypeptide has a k.sub.on
of at least 2.times.10.sup.5M.sup.-1s.sup.-1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least
5.times.10.sup.6M.sup.-1s.sup.-1, at least 10.sup.7
M.sup.-1s.sup.-1, at least 5.times.10.sup.7M.sup.-1s.sup.-1, or at
least 10.sup.8M.sup.-1s.sup.-1.
[0131] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide has a k.sub.off rate (antibody
(Ab)+antigen (Ag).sup.K.sup.off.sub..fwdarw.Ab-Ag) of less than
10.sup.-1 s.sup.-1, less than 5.times.10.sup.-1 s.sup.-1, less than
10.sup.-2 s.sup.-1, less than 5.times.10.sup.-2 s.sup.-1, less than
10.sup.-3 s.sup.-1, less than 5.times.10.sup.-3 s.sup.-1, less than
10.sup.-4 s.sup.-1, less than 5.times.10.sup.-4 s.sup.-1, less than
10.sup.-5 s.sup.-1, less than 5.times.10.sup.-5 s.sup.-1, less than
10.sup.-6 s.sup.-1, less than 5.times.10.sup.-6 s.sup.-1, less than
10.sup.-7 s.sup.-1, less than 5.times.10.sup.-7 s.sup.-1, less than
10.sup.-8 s.sup.-1, less than 5.times.10.sup.-8 s.sup.-1, less than
10.sup.-9 s.sup.-1, less than 5.times.10.sup.-9 s.sup.-1, or less
than 10.sup.-10 s.sup.-1. In a preferred embodiment, an antibody
that immunospecifically binds to a CD3 polypeptide has a k.sub.on
of less than 5.times.10.sup.-4 s.sup.-1, less than
5.times.10.sup.-5 s.sup.-1, less than 5.times.10.sup.-5 s.sup.-1,
less than 10.sup.-6 s.sup.-1, less than 5.times.10.sup.-6 s.sup.-1,
less than 10.sup.-7 s.sup.-1, less than 5.times.10.sup.-7 s.sup.-1,
less than 10.sup.-8 s.sup.-1, less than 5.times.10.sup.-8 s.sup.1,
less than 10.sup.-9 s.sup.-1, less than 5.times.10.sup.-9 s.sup.-1,
or less than 10.sup.-10 s.sup.-1.
[0132] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide has an affinity constant or K.sub.a
(k.sub.on/k.sub.off) of at least 10.sup.2M.sup.-1, at least
5.times.10.sup.2 M.sup.-1, at least 10.sup.3 M.sup.-1, at least
5.times.10.sup.3M.sup.-1, at least 10.sup.4 M.sup.-1, at least
5.times.10.sup.4 M.sup.-1, at least 10.sup.5 M.sup.-1, at least
5.times.10.sup.5 M.sup.-1, at least 10.sup.6M.sup.-1, at least
5.times.10.sup.6 M.sup.-1, at least 10.sup.7 M.sup.-1, at least
5.times.10.sup.7M.sup.-1, at least 10.sup.8 M.sup.-1, at least
5.times.10.sup.8M.sup.-1, at least 10.sup.9 M.sup.-1, at least
5.times.10.sup.9 M.sup.-1, at least 10.sup.10 M.sup.-1, at least
5.times.10.sup.10 M.sup.-1, at least 10.sup.11 M.sup.-1, at least
5.times.10.sup.11 M.sup.-1, at least 10.sup.12M.sup.-1, at least
5.times.10.sup.12 M.sup.-1, at least 10.sup.13 M.sup.-1, at least
5.times.10.sup.13 M.sup.-1, at least 10.sup.14 M.sup.-1, at least
5.times.10.sup.14M.sup.-1, at least 10.sup.15 M.sup.-1, or at least
5.times.10.sup.15 M.sup.-1. In yet another embodiment, an antibody
that immunospecifically binds to a CD3 polypeptide has a
dissociation constant or K.sub.d (k.sub.off/k.sub.on) of less than
10.sup.-2M, less than 5.times.10.sup.-2M, less than 10.sup.-3 M,
less than 5.times.10.sup.-3 M, less than 10.sup.-4M, less than
5.times.10.sup.-4M, less than 10.sup.-5 M, less than
5.times.10.sup.-5 M, less than 10.sup.-6M, less than
5.times.10.sup.-6 M, less than 10.sup.-7 M, less than
5.times.10.sup.-7 M, less than 10.sup.-8M, less than
5.times.10.sup.-8 M, less than 10.sup.-9M, less than
5.times.10.sup.-9 M, less than 10.sup.-10 M, less than
5.times.10.sup.-10 M, less than 10.sup.-11M, less than
5.times.10.sup.-11 M, less than 10.sup.-12 M, less than
5.times.10.sup.-12M, less than 10.sup.-13 M, less than
5.times.10.sup.-13 M, less than 10.sup.-14 M, less than
5.times.10.sup.-14 M, less than 10.sup.-15 M, or less than
5.times.10.sup.-15 M.
[0133] In a specific embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide is humanized OKT3 or
an antigen-binding fragment thereof e.g., (one or more
complementarity determining regions (CDRs) of humanized OKT3). OKT3
has the amino acid sequence disclosed, e.g., in U.S. Pat. Nos.
4,658,019, 6,113,901 and 6,491,916 (each of which is incorporated
herein by reference in its entirety), or the amino acid sequence of
the monoclonal antibody produced by the cell line deposited with
the American Type Culture Collection (ATCC.RTM.), 10801 University
Boulevard, Manassas, Va. 20110-2209 on Jul. 28, 1993 as Accession
Number CRL-8001 (which is incorporated herein by reference).
Several humanized versions of OKT3 are also reported in U.S. Pat.
No. 6,491,916. In an alternative embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide is not OKT3, a
derivative of OKT3, e.g. humanized OKT3, an antigen-binding
fragment of OKT3, or, more preferably, not a humanized or chimeric
version thereof.
[0134] In a specific embodiment, the present invention also
provides antibodies that immunospecifically bind a CD3 polypeptide,
said antibodies comprising a variable heavy ("VH") domain having an
amino acid sequence of the VH domain of a humanized OKT3 (for
example, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9;
FIG. 1B). In a preferred embodiment, the humanized OKT3 antibody
comprises a heavy chain with the amino acid sequence of
hOKT3.gamma.1(ala-ala) provided in FIG. 2D (SEQ ID NO:13) or
encoded by the nucleotide sequence of hOKT3.gamma.1(ala-ala)
provided in FIG. 2C (SEQ ID NO:12).
[0135] In a specific embodiment, the present invention also
provides antibodies that immunospecifically bind to a CD3
polypeptide, said antibodies comprising a variable light ("VL")
domain having an amino acid sequence of the VL domain for a
humanized OKT3 (for example, SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:4; FIG. 1A). In a preferred embodiment, the humanized OKT3
antibody comprises a light chain with the amino acid sequence of
hOKT3.gamma.1 provided in FIG. 2B (SEQ ID NO:11) or encoded by the
nucleotide sequence of hOKT3.gamma.1 provided in FIG. 2A (SEQ ID
NO:10).
[0136] The present invention also provides antibodies that
immunospecifically bind to a CD3 polypeptide, said antibodies
comprising a VH domain disclosed herein, or a VH domain of an
antibody disclosed herein, combined with a VL domain disclosed
herein, or other VL domain. The present invention further provides
antibodies that immunospecifically bind to a CD3 polypeptide, said
antibodies comprising a VL domain disclosed herein, or a VL domain
of an antibody disclosed herein, combined with a VH domain
disclosed herein, or other VH domain.
[0137] In one embodiment, an isolated nucleic acid molecule encodes
an antibody that immunospecifically binds to a CD3 polypeptide,
said antibody comprising a VH domain having the amino acid sequence
of the VH domain of a humanized OKT3 (for example, SEQ ID NO:5; SEQ
ID NO:7, SEQ ID NO:8; SEQ ID NO:9, FIG. 1B).
[0138] In a preferred embodiment, an isolated nucleic acid molecule
encodes an antibody that immunospecifically binds to a CD3
polypeptide, said antibody comprising a heavy chain having the
amino acid sequence of the heavy chain of hOKT3.gamma.-1 (ala-ala)
disclosed in FIG. 2D (SEQ ID NO:13).
[0139] In one embodiment, an isolated nucleic acid molecule encodes
an antibody that immunospecifically binds to a CD3 polypeptide,
said antibody comprising a VL domain having the amino acid sequence
of the VL domain of a humanized OKT3, for example, SEQ ID NO:1; SEQ
ID NO:3 or 4 (FIG. 1A).
[0140] In a preferred embodiment, an isolated nucleic acid molecule
encodes an antibody that immunospecifically binds to a CD3
polypeptide, said antibody comprising a light chain having the
amino acid sequence of the light chain of hOKT3.gamma.-1 disclosed
in FIG. 2B (SEQ ID NO:11).
[0141] In another embodiment, an isolated nucleic acid molecule
encodes an antibody that immunospecifically binds to a CD3
polypeptide, said antibody comprising a VH domain having the amino
acid sequence of the VII domain of a humanized OKT3, for example,
SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 (FIG. 1B) and a VL domain
having the amino acid sequence of the VL domain of a humanized
OKT3, for example, SEQ ID NO: 3 or SEQ ID NO:4 (FIG. 1A). In
another embodiment, an isolated nucleic acid molecule encodes an
antibody that immunospecifically binds to a CD3 polypeptide, said
antibody comprising a heavy chain having the amino acid sequence of
the heavy chain of a humanized OKT3, for example the amino acid
sequence of hOKT3.gamma.1 heavy chain disclosed in FIG. 2D (SEQ ID
NO:13), and a light chain having the amino acid sequence of the
light chain of a humanized OKT3, for example the amino acid
sequence of hOKT3.gamma.-1 disclosed in FIG. 2B (SEQ ID NO:11).
[0142] In one embodiment, antibodies that immunospecifically bind
to a CD3 polypeptide comprise one or more VH CDRs disclosed in FIG.
1B. In another embodiment, antibodies that immunospecifically bind
to a CD3 polypeptide comprise more than one of the VH CDRs
disclosed in FIG. 1B.
[0143] In one embodiment, antibodies that immunospecifically bind
to a CD3 polypeptide comprise a one or more of the VL CDRs
disclosed in FIG. 1A. In another embodiment, antibodies that
immunospecifically bind to a CD3 polypeptide comprise more than one
of the VL CDRs disclosed in FIG. 1A.
[0144] In another embodiment, antibodies that immunospecifically
bind to a CD3 polypeptide comprise one or more VH CDRs disclosed in
FIG. 1B and one or more VL CDRs disclosed in FIG. 1A. In yet
another embodiment, antibodies that immunospecifically bind to a
CD3 polypeptide comprise more than one of the VH CDRs disclosed in
FIG. 1B and more than one of the VL CDRs disclosed in FIG. 1A.
[0145] The present invention also provides antibodies that
immunospecifically bind to a CD3 polypeptide, said antibodies
comprising derivatives of the VH domains, VH CDRs, VL domains, or
VL CDRs described herein, or available to one of ordinary skill in
the art, that immunospecifically bind to a CD3 polypeptide.
Standard techniques known to those of skill in the art can be used
to introduce mutations in the nucleotide sequence encoding an
antibody of the invention, including, for example, site-directed
mutagenesis and PCR-mediated mutagenesis which results in amino
acid substitutions. Preferably, the derivatives include less than
25 amino acid substitutions, less than 20 amino acid substitutions,
less than 15 amino acid substitutions, less than 10 amino acid
substitutions, less than 5 amino acid substitutions, less than 4
amino acid substitutions, less than 3 amino acid substitutions, or
less than 2 amino acid substitutions relative to the original
molecule. In a preferred embodiment, the derivatives have
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues (i.e., amino acid
residues which are not critical for the antibody to
immunospecifically bind to a CD3 polypeptide). A "conservative
amino acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a side chain with a
similar charge. Families of amino acid residues having side chains
with similar charges have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded antibody can be expressed and the activity of the antibody
can be determined.
[0146] In a specific embodiment, the present invention provides for
antibodies that immunospecifically bind to a CD3 polypeptide, said
antibodies comprising the amino acid sequence of a humanized OKT3
with one or more amino acid residue substitutions in the variable
light (VL) domain and/or variable heavy (VH) domain. The present
invention also provides for antibodies that immunospecifically bind
to a CD3 polypeptide, said antibodies comprising the amino acid
sequence of the heavy and light chains variable domains of murine
(SEQ ID NOs:5 and 1, respectively) with one or more amino acid
residue substitutions in one or more VL CDRs and/or one or more VH
CDRs. The antibody generated by introducing substitutions in the VH
domain, VH CDRs, VL domain and/or VL CDRs of humanized OKT3 can be
tested in vitro and in vivo, for example, for its ability to bind
to a CD3 polypeptide, or for its ability to inhibit T cell
activation, or for its ability to inhibit T cell proliferation, or
for its ability to induce T cell lysis, or for its ability to
prevent, treat or ameliorate one or more symptoms associated with
an autoimmune disorder.
[0147] In a specific embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide comprises a
nucleotide sequence that hybridizes to the nucleotide sequence
encoding the monoclonal antibody produced by the cell line
deposited with the ATCC.RTM. as Accession Number CRL-8001 under
stringent conditions, e.g., hybridization to filter-bound DNA in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C. followed by one or more washes in 0.2.times.SSC/0.1% SDS at
about 50-65.degree. C., under highly stringent conditions, e.g.,
hybridization to filter-bound nucleic acid in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.1.times.SSC/0.2%
SDS at about 68.degree. C., or under other stringent hybridization
conditions which are known to those of skill in the art (see, for
example, Ausubel, F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc. and
John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and
2.10.3).
[0148] In a specific embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide comprises a
nucleotide sequence that hybridizes to the nucleotide sequence
encoding the humanized OKT3 under stringent conditions, e.g.,
hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., under highly stringent conditions, e.g., hybridization to
filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68.degree. C., or under other stringent hybridization conditions
which are known to those of skill in the art (see, for example,
Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley
& Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).
[0149] In a specific embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide comprises an amino
acid sequence of a VH domain or an amino acid sequence a VL domain
encoded by a nucleotide sequence that hybridizes to the nucleotide
sequence encoding the VH or VL domains of humanized OKT3 under
stringent conditions, e.g., hybridization to filter-bound DNA in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C. followed by one or more washes in 0.2.times.SSC/0.1% SDS at
about 50-65.degree. C., under highly stringent conditions, e.g.,
hybridization to filter-bound nucleic acid in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.1.times.SSC/0.2%
SDS at about 68.degree. C., or under other stringent hybridization
conditions which are known to those of skill in the art (see, for
example, Ausubel, F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc. and
John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and
2.10.3).
[0150] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide comprises an amino acid sequence of a VH
CDR or an amino acid sequence of a VL CDR encoded by a nucleotide
sequence that hybridizes to the nucleotide sequence encoding any
one of VH CDRs or VL CDRs of the monoclonal antibody produced by
the cell line deposited with the ATCC.RTM. as Accession Number
CRL-8001 under stringent conditions e.g., hybridization to
filter-bound DNA in 6.times. sodium chloride/sodium citrate (SSC)
at about 45.degree. C. followed by one or more washes in
0.2.times.SSC/0.1% SDS at about 50-65.degree. C., under highly
stringent conditions, e.g., hybridization to filter-bound nucleic
acid in 6.times.SSC at about 45.degree. C. followed by one or more
washes in 0.1.times.SSC/0.2% SDS at about 68.degree. C., or under
other stringent hybridization conditions which are known to those
of skill in the art.
[0151] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide comprises an amino acid sequence of a VH
CDR and an amino acid sequence of a VL CDR encoded by nucleotide
sequences that hybridizes to the nucleotide sequences encoding the
monoclonal antibody produced by the cell line deposited with the
ATCC.RTM. as Accession Number CRL-8001 under stringent conditions,
e.g., hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., under highly stringent conditions, e.g., hybridization to
filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68.degree. C., or under other stringent hybridization conditions
which are known to those of skill in the art.
[0152] In a specific embodiment, an antibody that
immunospecifically binds to a CD3 polypeptide comprises an amino
acid sequence that is at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 99% identical to the amino acid sequence of the
monoclonal antibody produced by the cell line deposited with the
ATCC.RTM. as Accession Number CRL-8001. In another embodiment, an
antibody that immunospecifically binds to a CD3 polypeptide
comprises an amino acid sequence that is at least 35%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least 95%, or at least 99% identical to the amino
acid sequence of humanized OKT3.
[0153] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide comprises an amino acid sequence of a VH
domain that is at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 99% identical to the VH domain of humanized OKT3.
[0154] In another embodiment, an antibody that immunospecifically
binds to a CD3 polypeptide comprises an amino acid sequence of a VL
domain that is at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 99% identical to the VL domain of humanized OKT3.
[0155] The present invention encompasses antibodies that compete
with an antibody described herein for binding to a CD3 polypeptide.
In a specific embodiment, the present invention encompasses
antibodies that compete with anti-CD3 antibodies known in the art,
derivatives thereof or antigen binding fragments thereof. For
example, antibodies provided by the invention compete with OKT3 or
a derivative thereof, e.g. humanized OKT3, or an antigen-binding
fragment thereof for binding to the CD3 polypeptide. In another
specific embodiment, the present invention encompasses antibodies
that compete with ChAglyCD3 or a derivative thereof or an
antigen-binding fragment thereof for binding to the CD3
polypeptide. In another specific embodiment, the present invention
encompasses antibodies that compete with HuM291 or a derivative
thereof or an antigen-binding fragment thereof for binding to the
CD3 polypeptide. In another specific embodiment, the present
invention encompasses antibodies that compete with UCHT1 or a
derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide. In another specific embodiment, the
present invention encompasses antibodies that compete with Leu4 or
a derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide. In another specific embodiment, the
present invention encompasses antibodies that compete with YTH 12.5
or a derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide. In another specific embodiment, the
present invention encompasses antibodies that compete with 500A2 or
a derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide. In another specific embodiment, the
present invention encompasses antibodies that compete with CLB-T3/3
or a derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide. In another specific embodiment, the
present invention encompasses antibodies that compete with BMA030
or a derivative thereof or an antigen-binding fragment thereof for
binding to the CD3 polypeptide.
[0156] The present invention also encompasses VH domains that
compete with the VH domain of the antibodies disclosed herein, or
with the VH domains of other anti-human CD3 antibodies known in the
art, or derivatives or variants thereof for binding to a CD3
polypeptide. In a specific embodiment, the present invention
encompasses VH domains that compete with the VH domain of OKT3 or a
derivative thereof, e.g. humanized OKT3, for binding to a CD3
polypeptide. The present invention also encompasses VL domains that
compete with the VL domain of the antibodies disclosed herein, or
with the VL domains of other anti-human CD3 antibodies known in the
art, or derivatives or variants thereof for binding to a CD3
polypeptide. In a specific embodiment, the present invention
encompasses VL domains that compete with a VL domain of OKT3 or a
derivative thereof, e.g. humanized OKT3, for binding to a CD3
polypeptide.
[0157] The antibodies that immunospecifically bind to a CD3
polypeptide include derivatives that are modified, i.e, by the
covalent attachment of any type of molecule to the antibody such
that covalent attachment. For example, but not by way of
limitation, the antibody derivatives include antibodies that have
been modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative may contain one or more non-classical amino
acids.
[0158] The present invention also provides antibodies that
immunospecifically bind to a CD3 polypeptide, said antibodies
comprising a framework region known to those of skill in the art.
Preferably, the fragment region of an antibody of the invention is
human.
[0159] The present invention also encompasses antibodies, and
methods of use thereof, that immunospecifically bind to a CD3
polypeptide, said antibodies comprising the amino acid sequence of
OKT3 or a derivative thereof, e.g. humanized OKT3, with mutations
(e.g., one or more amino acid substitutions) in the framework
regions. In certain embodiments, antibodies which
immunospecifically bind to a CD3 polypeptide comprise the amino
acid sequence of OKT3 or a derivative thereof, e.g. humanized OKT3,
with one or more amino acid residue substitutions in the framework
regions of the VH and/or VL domains.
[0160] The present invention also encompasses antibodies which
immunospecifically bind to a CD3 polypeptide, said antibodies
comprising the amino acid sequence of OKT3 or a derivative thereof,
e.g. humanized OKT3, with mutations (e.g., one or more amino acid
residue substitutions) in the variable and framework regions.
[0161] The present invention also provides for fusion proteins
comprising an antibody that immunospecifically binds to a CD3
polypeptide and a heterologous polypeptide. Preferably, the
heterologous polypeptide that the antibody is fused to is useful
for targeting the antibody to T cells.
[0162] The antibodies of the invention include derivatives that are
otherwise modified, i.e., by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from binding antigen and/or generating an
anti-idiotypic response. For example, but not by way of limitation,
the antibody derivatives include antibodies that have been
modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative may contain one or more non-classical amino
acids.
[0163] 5.1.1 Polypeptides and Antibodies with Variant Fe
Regions
[0164] The use of therapeutic monoclonal antibodies is limited by
problems of "first dose" side effects. First dose side effects,
range from mild flu-like symptoms to severe toxicity, can be mild
to severe, and include symptoms, such as, high fever,
chills/rigors, headache, tremor, nausea/vomiting, diarrhea,
abdominal pain, malaise, muscle/joint aches and pains, and
generalized weakness. The first dose side effects are believed to
be caused by lymphokine production and cytokine release stimulated
by the Fc region of an antibody binding to and activating an
Fc.gamma.R on an Fc.gamma.R-containing cell.
[0165] The FcR recognizes immunoglobulins of one or more isotypes
through a recognition domain on the .alpha. chain of the Fc
receptor. Fc receptors are defined by their specificity for
immunoglobulin subtypes. For example, Fc receptors for IgG are
referred to as Fc.gamma.R. Different accessory cells bear Fc
receptors for antibodies of different isotype, and the isotype of
the antibody determines which accessory cells will be engaged in a
given response (reviewed by Ravetch J. V. et al. 1991, Annu. Rev.
Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes and
Infection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal of
Clinical Investigation, 2(109): 161-1681; Ravetch J. V. et al.,
2000, Science, 290: 84-89; Ravetch J. V. et al., 2001, Annu. Rev.
Immunol. 19:275-90; Ravetch J. V. 1994, Cell 78: 553-60).
[0166] The invention thus encompasses CD3 binding molecules that
reduce or eliminate at least one symptom associated with first dose
side effects by reducing or eliminating binding of the Fc to one or
more Fc.gamma.R. Such CD3 binding proteins comprise a variant Fc
region having one or more amino acid modifications, relative to a
wild type Fc region. The modification decreases or eliminates
binding of the Fc to one or more Fc.gamma.Rs, relative to a
comparable wild type Fc region. The modification is typically an
amino acid substitution. However, the modification can be an amino
acid insertion and/or deletion. Typically, the modification occurs
in the CH2 and/or hinge region. Alternatively, binding of Fc to one
or more Fc.gamma.Rs can be reduced or eliminated by altering or
eliminating one or more glycosyl groups on the Fc domain. Fc
glycosylation can be altered or eliminated by methods well know in
the art. For example, Fc glycosylation can be altered by producing
the Fc in a cell that is deficient in fucosylation (e.g., fuc6 null
cells), or eliminated by deglycosylation enzymes or an amino acid
modification that alters or eliminates a glycosylation site (e.g.,
the N-X-S/T glycosylation site at positions 297-299 in the CH2
domain). Fc.gamma.R binding can be measured using standard methods
known in the art and exemplified herein. The antibodies of the
invention are thus particularly useful because they have reduced or
no in vivo toxicity caused by lymphokine production or cytokine
release. The affinities and binding properties of the molecules of
the invention for an FcR are initially determined using in vitro
assays (biochemical or immunological based assays) known in the art
for determining Fc-FcR interactions, i.e., specific binding of an
Fc region to an FcR including but not limited to ELISA assay,
surface plasmon resonance assay, immunoprecipitation assays (See
Section 5.4). Preferably, the binding properties of the molecules
of the invention are also characterized by in vitro functional
assays for determining one or more Fc.gamma.R mediator effector
cell functions (See Section 5.4). In most preferred embodiments,
the molecules of the invention have similar binding properties in
in vivo models (such as those described and disclosed herein) as
those in in vitro based assays. However, the present invention does
not exclude molecules of the invention that do not exhibit the
desired phenotype in in vitro based assays but do exhibit the
desired phenotype in vivo.
[0167] 5.1.1.1 Fc.gamma. Receptors
[0168] Each member of this family is an integral membrane
glycoprotein, possessing extracellular domains related to a C2-set
of immunoglobulin-related domains, a single membrane spanning
domain and an intracytoplasmic domain of variable length. There are
three known Fc.gamma.Rs, designated Fc.gamma.RI(CD64),
Fc.gamma.RII(CD32), and Fc.gamma.RIII(CD16), which exhibit
extensive homology but are encoded by distinct genes. Both
activating and inhibitory signals are transduced through the
Fc.gamma.Rs following ligation. These diametrically opposing
functions result from structural differences among the different
receptor isoforms. In general, the binding of a complimentary Fc
domain to Fc.gamma.RI, Fc.gamma.RIIA and Fc.gamma.RIIIA results in
activation of downstream substrates (e.g., PI.sub.3K) and leading
to the release of proinflammatory mediators. In contrast, the
binding of a complimentary Fc domain to Fc.gamma.RIIB results in
phosphorylation of Fc.gamma.RIIB and association with the SH2
domain of the inosital polyphosphate 5'-phosphatase (SHIP). SHIP
hydrolyzes phosphoinositol messengers released as a consequence of
Fc.gamma.RI mediated tyrosine kinase activation, consequently
preventing the influx of intracellular Ca.sup.++. Thus crosslinking
of Fc.gamma.RIIB dampens the activating response to Fc.gamma.R
ligation and inhibits cellular responsiveness.
[0169] Methods of measuring lymphokine production and cytokine
release are known and routine in the art and encompassed herein.
For example, cytokine release may be measured by measuring
secretion of cytokines including but not limited to TNF-.alpha.,
GM-CSF, IFN-.gamma.. See, e.g., U.S. Pat. No. 6,491,916; Isaacs et
al., 2001, Rheumatology, 40: 724-738; each of which is incorporated
herein by reference in its entirety. Lymphokine production may be
measured by measuring secretion of lymphokines including but not
limited to Interleukin-2 (IL-2). Interleukin-4 (IL-4),
Interleukin-6 (IL-6), Interleukin-12 (IL-12), Interleukin-16
(IL-16), PDGF, TGF-.alpha., TGF-.beta., TNF-.alpha., TNF-.beta.,
GCSF, GM-CSF, MCSF, IFN-.alpha., IFN-.beta., TFN-.gamma., IGF-I,
IGF-II. For example, see, Isaacs et al., 2001, Rheumatology, 40:
724-738; Soubrane et al., 1993, Blood, 81(1): 15-19; each of which
is incorporated herein by reference in its entirety.
[0170] As used herein, the term "Fc region" is used to define a
C-terminal region of an IgG heavy chain. Although the boundaries
may vary slightly, the human IgG heavy chain Fc region is defined
to stretch from Cys226 to the carboxy terminus. The Fc region of an
IgG comprises two constant domains, CH2 and CH3. The CH2 domain of
a human IgG Fc region usually extends from amino acids 231 to amino
acid 341. The CH3 domain of a human IgG Fc region usually extends
from amino acids 342 to 447. The CH2 domain of a human IgG Fc
region (also referred to as "Cy2" domain) usually extends from
amino acid 231-340. The CH2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG.
[0171] In preferred embodiments, the invention encompasses
molecules comprising a variant Fc region, wherein said variant Fc
region comprises at least one amino acid modification relative to a
wild-type Fc region, which variant Fc region does not bind any
Fc.gamma.R, as determined by standard assays known in the art and
disclosed herein, relative to a comparable molecule comprising the
wild type Fc region. In a specific embodiment, the one or more
amino acid modifications which abolish binding to all Fc.gamma.Rs
comprise Fc regions which have a phenylalanine at position 233; or
an arginine at position 238; or an alanine at position 265; or a
glutamic acid at position 265; or an alanine at position 270; or an
asparagine at position 270; or an alanine at position 297; or a
glutamine at position 297; or a phenylalanine at position 298; or
an asparagine at position 298; or a any amino acid at position 299
other than serine or threonine; or an alanine at position 265 and
at position 297; or an alanine at position 265 and a glutamine at
position 297; or a glutamic acid at position 265 and an alanine at
position 297; or a glutamic acid at position 265 and a glutamine at
position 297; or an alanine at position 234 and an alanine at
position 235. In another embodiment, the one or more amino acid
modifications which abolish binding to all Fc.gamma.Rs comprise
combinations of the modifications listed herein or combinations of
the modifications listed herein with any that may confer null
binding to Fc.gamma.RIIIA, Fc.gamma.RIIIB, and Fc.gamma.RIIA as
determined by the methods disclosed herein or known to one skilled
in the art.
[0172] The invention encompasses methods for reducing or
eliminating at least one symptom associated with first dose side
effect in a patient comprising administering an effective amount of
one or more antibodies of the invention. The methods of the
invention reduce at least one symptom associated with cytokine
release syndrome including but not limited to high fever,
chills/rigors, headache, tremor, nausea/vomiting, diarrhea,
abdominal pain, malaise, muscle/joint aches and pains, and
generalized weakness.
[0173] The present invention provides for antibodies that
immunospecifically bind to a CD3 polypeptide which have a extended
half-life in vivo. In particular, the present invention provides
antibodies that immunospecifically bind to a CD3 polypeptide which
have a half-life in an animal, preferably a mammal and most
preferably a human, of greater than 3 days, greater than 7 days,
greater than 10 days, preferably greater than 15 days, greater than
25 days, greater than 30 days, greater than 35 days, greater than
40 days, greater than 45 days, greater than 2 months, greater than
3 months, greater than 4 months, or greater than 5 months.
[0174] To prolong the serum circulation of antibodies (e.g.,
monoclonal antibodies, single chain antibodies and Fab fragments)
in vivo, for example, inert polymer molecules such as high
molecular weight polyethyleneglycol (PEG) can be attached to the
antibodies with or without a multifunctional linker either through
site-specific conjugation of the PEG to the N-terminus or
C-terminus of the antibodies or via epsilon-amino groups present on
lysine residues. Linear or branched polymer derivatization that
results in minimal loss of biological activity will be used. The
degree of conjugation can be closely monitored by SDS-PAGE and mass
spectrometry to ensure proper conjugation of PEG molecules to the
antibodies. Unreacted PEG can be separated from antibody-PEG
conjugates by size-exclusion or by ion-exchange chromatography.
PEG-derivatized antibodies can be tested for binding activity as
well as for in vivo efficacy using methods well-known to those of
skill in the art, for example, by immunoassays described
herein.
[0175] Antibodies having an increased half-life in vivo can also be
generated introducing one or more amino acid modifications (i.e.,
substitutions, insertions or deletions) into an IgG constant
domain, or FcRn binding fragment thereof (preferably a Fc or
hinge-Fc domain fragment). See, e.g., International Publication No.
WO 98/23289; International Publication No. WO 97/34631; and U.S.
Pat. No. 6,277,375, each of which is incorporated herein by
reference in its entirety.
[0176] 5.1.2 Antibody Conjugates
[0177] The present invention encompasses antibodies or
antigen-binding fragments thereof that immunospecifically bind to a
CD3 polypeptide recombinantly fused or chemically conjugated
(including both covalently and non-covalently conjugations) to a
heterologous polypeptide (or a fragment thereof, preferably at
least 5, at least 10, at least 20, at least 30, at least 40, at
least 50, at least 60, at least 70, at least 80, at least 90 or at
least 100 contiguous amino acids of the polypeptide) to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. For example, antibodies may
be used to target heterologous polypeptides to particular cell
types (e.g., T cells), either in vitro or in vivo, by fusing or
conjugating the antibodies to antibodies specific for particular
cell surface receptors such as, e.g., CD4 and CD8.
[0178] The present invention also encompasses antibodies or
antigen-binding fragments thereof that immunospecifically bind to a
CD3 polypeptide fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., 1989, Proc. Natl. Acad.
Sci. USA 86:821-824, for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the
hemagglutinin "HA" tag, which corresponds to an epitope derived
from the influenza hemagglutinin protein (Wilson et al., 1984, Cell
37:767) and the "flag" tag.
[0179] The present invention further encompasses antibodies or
antigen-binding fragments thereof that immunospecifically bind to a
CD3 polypeptide conjugated to an agent which has a potential
therapeutic benefit. An antibody or an antigen-binding fragment
thereof that immunospecifically binds to a CD3 polypeptide may be
conjugated to a therapeutic moiety such as a cytotoxin, e.g., a
cytostatic or cytocidal agent, an agent which has a potential
therapeutic benefit, or a radioactive metal ion, e.g.,
alpha-emitters. A cytotoxin or cytotoxic agent includes any agent
that is detrimental to cells. Examples of a cytotoxin or cytotoxic
agent include, but are not limited to, paclitaxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof. Agents which have a potential therapeutic benefit
include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0180] Further, an antibody or an antigen-binding fragment thereof
that immunospecifically binds to a CD3 polypeptide may be
conjugated to a therapeutic agent or drug moiety that modifies a
given biological response. Agents which have a potential
therapeutic benefit or drug moieties are not to be construed as
limited to classical chemical therapeutic agents. For example, the
drug moiety may be a protein or polypeptide possessing a desired
biological activity. Such proteins may include, for example, a
toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria
toxin; a protein such as tumor necrosis factor, interferon-.alpha.
("IFN-.alpha."), interferon-.beta.("IFN-.beta."), nerve growth
factor ("NGF"), platelet derived growth factor ("PDGF"), tissue
plasminogen activator ("TPA"), an apoptotic agent, e.g.,
TNF-.alpha., TNF-.beta., AIM I (see, International Publication No.
WO 97/33899), AIM II (see, International Publication No. WO
97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol.,
6:1567-1574), and VEGF (see, International Publication No. WO
99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,
angiostatin or endostatin; or, a biological response modifier such
as, for example, a lymphokine (e.g., interleukin-1 ("IL-1"), IL-2,
IL-6, IL-10, granulocyte macrophage colony stimulating factor
("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")),
or a growth factor (e.g., growth hormone ("GH")).
[0181] Techniques for conjugating such therapeutic moieties to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985); and Thorpe et al., 1982, Immunol.
Rev. 62:119-58.
[0182] An antibody or an antigen-binding fragment thereof that
immunospecifically binds to a CD3.sup.+ polypeptide can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980, which is
incorporated herein by reference in its entirety.
[0183] Antibodies or antigen-binding fragments thereof that
immunospecifically bind to a CD3 polypeptide may be attached to
solid supports, which are particularly useful for the purification
of CD3.sup.+ immune cells such as T cells. Such solid supports
include, but are not limited to, glass, cellulose, polyacrylamide,
nylon, polystyrene, polyvinyl chloride or polypropylene.
[0184] 5.2 Prophylactic and Therapeutic Methods
[0185] The present invention is directed to therapies which involve
administering CD3 binding molecules, particularly anti-human CD3
antibodies, to a subject, preferably a human subject, for
preventing, treating, delaying the onset of, slowing the
progression of or ameliorating one or more symptoms of an
autoimmune disorder. In particular, the present invention is
directed to therapies which involve administering CD3 binding
molecules, particularly anti-human CD3 antibodies, more
particularly human or humanized forms of anti-human CD3 antibodies,
such as OKT3.gamma.1(ala-ala), ChAglyCD3, and visilizumab that have
Fc domains that do not bind or have significantly reduced binding
to Fc receptors, to a subject, preferably a human subject, for
preventing, treating, delaying the onset of, slowing the
progression of or ameliorating one or more symptoms of an
autoimmune disorder, e.g., type 1 diabetes. The methods disclosed
herein are generally improved methods of administration that permit
administration of lower dosages and/or over shorter periods of time
that still achieve clinical efficacy and avoid toxicity. In
particular, the invention contemplates dosing regimens in which
less than 9,000 .mu.g/m.sup.2, preferably, less than 8,000
.mu.g/m.sup.2, less than 7,500 .mu.g/m.sup.2, less than 7,000
.mu.g/m.sup.2, or less than 6,000 .mu.g/m.sup.2 total anti-human
CD3 antibody over the duration of the dosing, particularly of
OKT3.gamma.1 (ala-ala), or the pharmacological equivalent amount of
another anti-human CD3 antibody, such as ChAglyCD3 (TRX4.TM.) or
HUM291 (visilizumab; NUVION.TM.), or OKT3.gamma.1 (ala-ala)
administered intravenously. The invention further contemplates
methods in which the patient is chronically administered low doses
of the anti-human CD3 antibody and in which the patient is
administered one or more additional rounds of the anti-human CD3
antibody treatment regimen approximately 6 months, 9 months, 12
months, 18 months, 2 years, 3 years or 5 years after the initial
treatment, depending or not on certain clinical parameters, or is
administered another round of treatment with anti-human CD3
antibody every approximately 6 months, 9 months, 12 months, 18
months, 2 years, 3 years or 5 years, depending or not on certain
clinical parameters.
[0186] Examples of autoimmune disorders that may be treated by
administering the molecules of the present invention include, but
are not limited to, alopecia areata, ankylosing spondylitis,
antiphospholipid syndrome, autoimmune Addison's disease, autoimmune
diseases of the adrenal gland, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune oophoritis and orchitis,
autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune
dysfunction syndrome (CFIDS), chronic inflammatory demyelinating
polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid,
CREST syndrome, cold agglutinin disease, Crohn's disease, discoid
lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable
bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen
planus, lupus erthematosus, Meniere's disease, mixed connective
tissue disease, multiple sclerosis, type 1 or immune-mediated
diabetes mellitus, myasthenia gravis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychrondritis,
polyglandular syndromes, polymyalgia rheumatics, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon,
Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma,
SjSgren's syndrome, stiff-man syndrome, systemic lupus
erythematosus, lupus erythematosus, takayasu arteritis, temporal
arteristis/giant cell arteritis, ulcerative colitis, uveitis,
vasculitides such as dermatitis herpetiformis vasculitis, vitiligo,
and Wegener's granulomatosis. Some autoimmune disorders are also
associated with an inflammatory condition. Thus, there is overlap
between what is considered an autoimmune disorder and an
inflammatory disorder. Therefore, some autoimmune disorders may
also be characterized as inflammatory disorders. Examples of
inflammatory disorders which can be prevented, treated or managed
in accordance with the methods of the invention include, but are
not limited to, asthma, encephilitis, inflammatory bowel disease,
chronic obstructive pulmonary disease (COPD), allergic disorders,
pulmonary fibrosis, undifferentitated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections. Examples of the types of psoriasis which can be treated
in accordance with the compositions and methods of the invention
include, but are not limited to, plaque psoriasis, pustular
psoriasis, erythrodermic psoriasis, guttate psoriasis and inverse
psoriasis.
[0187] 5.2.1 Diabetes
[0188] Immune-mediated diabetes mellitus or type 1 diabetes is
caused by an autoimmune response in which the insulin producing
.beta.-cells of the pancreas are gradually destroyed. Destruction
of the .beta.-cells is believed largely mediated by CTLs (CD8+ T
cells). The early stage of the disease, termed insulitis, is
characterized by infiltration of leukocytes into the pancreas and
is associated with both pancreatic inflammation and the release of
anti-.beta.-cell cytotoxic antibodies. Early stages of the disease
are often overlooked or misdiagnosed as clinical symptoms of
diabetes typically manifest only after about 80% of the
.beta.-cells have been destroyed. Even with immunosuppressive
therapy, .beta.-cell populations do not recover to a significant
extent; therefore, once clinical symptoms occur, the type-1
diabetic is normally insulin dependent for life. Insulin is
currently the only standard therapy for treating symptoms of type 1
diabetes. Although immunosuppressive drugs such as methotrexate and
cyclosporin showed early clinical promise in the treatment of type
1 diabetes, e.g., maintenance of .beta.-cell function, as with all
general immunosuppressants, their prolonged use was associated with
a number of severe side effects. Use of the invention in the
context of diabetes therefore encompasses methods to
sustain/protect the levels and functionality of .beta.-cells which
exist at the time of treatment.
[0189] In a specific embodiment, anti-human CD3 antibody therapy is
not used for the treatment of acute diabetes but rather to prevent
progression of the disease in recently diagnosed individuals,
including children diagnosed with juvenile diabetes (see Herold et
al., 2005, Diabetes 54:1763-1769). In a specific embodiment,
anti-human CD3 therapy is used only in patients that have residual
.beta.-cell function as determined by methods described herein or
known to one of ordinary skill in the art. In another embodiment,
anti-human CD3 antibody therapy is used to maintain transplanted
.beta.-cell function in pancreatic transplant recipients.
[0190] In alternate embodiments, the invention encompasses
administration of anti-human CD3 antibodies to individuals
predisposed to develop type 1 diabetes, but do not meet the
diagnosis criteria as established by the American Diabetes
Association or the Immunology of Diabetes Society to prevent or
delay the onset of type 1 diabetes and/or to prevent or delay the
need for administration of insulin to such patients. In certain
embodiments, high-risk factors for identification of predisposed
subjects in accordance with this embodiment are having first or
second degree relatives with diagnosed type-1 diabetes, an impaired
fasting glucose level (i.e., at least one determination of a
glucose level of 100-125 mg/dl after fasting (8 hour with no
food)), an impaired glucose tolerance in response to a 75 g OGTT
(i.e., at least one determination of a 2-hr glucose level of
140-199 mg/dl in response to a 75 g OGTT), an HLA type of DR7 in a
caucasian, an HLA type of DR4 in a person of African descent, an
HLA type of DR9 in a person of Japanese descent, exposure to
childhood viruses (e.g., coxsackie B virus, enteroviruses,
adenoviruses, rubella, cytomegalovirus, Epstein-Barr virus), a
positive diagnosis according to art accepted criteria of at least
one other autoimmune disorder (e.g., thyroid disease, celiac
disease), and/or the detection of autoantibodies, particularly
ICAs, in the serum or other tissues. In certain embodiments, the
subject identified as predisposed to developing type 1 diabetes
according the methods of the invention has at least one of the risk
factors described herein and/or as known in the art. The invention
also encompasses identification of subjects predisposed to
development of type 1 diabetes, wherein said subject presents a
combination of two or more, three or more, four or more, or more
than five of the risk factors disclosed herein or known in the
art.
[0191] Serum autoantibodies associated with type 1 diabetes or with
a predisposition for the development of type 1 diabetes are
islet-cell autoantibodies (e.g., anti-ICA512 autoantibodies),
glutamic acid decarbamylase autoantibodies (e.g., anti-GAD65
autoantibodies), and/or anti-insulin autoantibodies. Accordingly,
in a specific example in accordance with this embodiment, the
invention encompasses the treatment of an individual with
detectable autoantibodies associated with a predisposition to the
development of type 1 diabetes or associated with early stage type
1 diabetes (e.g., anti-IA2, anti-ICA512, anti-GAD or anti-insulin
autoantibodies), wherein said individual has not been diagnosed
with type 1 diabetes and/or is a first or second degree relative of
a type-1 diabetic. In certain embodiments, the presence of the
autoantibodies is detected by ELISA, radioassay (see, e.g., Yu et
al., 1996, J. Clin. Endocrinol. Metab. 81:4264-4267), or by any
other method for immunospecific detection of antibodies described
herein or as known to one of ordinary skill in the art.
[0192] .beta.-cell function prior to, during, and after therapy may
be assessed by methods described herein or by any method known to
one of ordinary skill in the art. For example, the Diabetes Control
and Complications Trial (DCCT) research group has established the
monitoring of percentage glycoslyated hemoglobin (HA1 and HA1c) as
the standard for evaluation of blood glucose control (DCCT, 1993,
N. Engl. J. Med. 329:977-986). Alternatively, characterization of
daily insulin needs, C-peptide levels/response, hypoglycemic
episodes, and/or FPIR may be used as markers of n-cell function or
to establish a therapeutic index (See Keymeulen et al., 2005, N.
Engl. J. Med. 352:2598-2608; Herold et al., 2005, Diabetes
54:1763-1769; U.S. Pat. Appl. Pub. No. 2004/0038867 A1; and
Greenbaum et al., 2001, Diabetes 50:470-476, respectively). For
example, FPIR is calculated as the sum of insulin values at 1 and 3
minutes post IGTT, which are performed according to Islet Cell
Antibody Register User's Study protocols (see, e.g., Bingley et
al., 1996, Diabetes 45:1720-1728 and McCulloch et al., 1993,
Diabetes Care 16:911-915).
[0193] 5.2.2 Multiple Sclerosis
[0194] Diagnosis of MS typically requires multiple neurological
evaluations in order to exclude other more common causes of the
symptoms presented. As of 2001, the International Panel on MS
Diagnosis has recommended revised diagnostic criteria for a
determination of multiple sclerosis that include advances in MRI
technology (e.g., improved imaging of lesions) and other
paraclinical diagnostic methods. The updated criteria have been
called the McDonald criteria after the lead author of the report,
and represent improvements over previous diagnostic criteria, i.e.
the Poser and Schumacher criteria (see, McDonald et al., 2001, Ann.
Neurol. 50:121-127}.
[0195] Studies of the natural history of MS suggest that there are
different patterns of disease activity although four main varieties
are recognized: Relapsing/Remitting MS (RRMS), Secondary
Progressive MS (SPMS), Progressive Relapsing MS (PRMS), and Primary
Progressive MS (PPMS) (see, e.g., Lublin et al., 1996, Neurology
46:907-911. The distinction between the varieties depend on the
frequency and severity of the attacks, and the progression thereof.
RRMS is characterized by full or partial recovery between attacks,
with a general stability of baseline condition are defined as
having relapsing-remitting MS. In some instances, RRMS includes
"benign" MS, characterized by rare attacks and minimum disability
ten years post MS diagnosis. Patients experiencing RRMS constitute
approximately 80-90% of MS sufferers. Of these, approximately 50%
will have difficulty walking 15 years after onset and 80% will
ultimately (after 25 years) experience gradual progression of
disability with or without attacks. Patients who first experience
exacerbations and later experience gradual progression of
disability have SPMS. Approximately 10-15% of MS patients do not
experience an initial attack. Those patients who gradually worsen
after the appearance of the first symptom have PPMS. A few patients
with primary progressive MS will later experience an exacerbation;
these patients have PRMS.
[0196] Clinical tests for diagnosing and/or monitoring MS
progression include, for example, magnetic resonance imaging (MRI)
scan to detect lesions or monitor lesion size, lumbar puncture to
detect evidence of inflammation, an evoked potential test (eye,
ear, or skin) to measure the speed at which messages from the brain
pass along nerves in response to stimuli (visual, auditory, or
pain, respectively), use of the Kurtzke expanded disability status
scale (EDSS) to rate the severity of the symptoms, urinalysis to
measure the level of myelin basic protein-like material present in
a urine sample, measuring atrophy in the brain or spinal cord, and
detecting black holes (areas in the brain that emit very low
signals on an MRI scan). Accordingly, the invention encompasses the
use of an anti-CD3 antibody of the invention for the treatment of
multiple sclerosis in patients exhibiting one or more indications
of having the disease, or wherein a diagnosis according to McDonald
criteria has been established.
[0197] While not being bound by a particular mechanism of action,
administration of anti-CD3 antibody, and in particular, hOKT3
ala-ala, leads to treatment of autoimmune disorders, including, for
example, type-1 diabetes and MS, by induction of immunologic
tolerance. Multiple studies have identified several possible
mechanisms by which hOKT3.gamma.1 ala-ala may induce tolerance
including induction of T cell anergy, a process whereby T cells
become quiescent and fail to react to self antigens; activation of
T.sub.Reg populations, recently identified sub-populations of T
cells which are expanded by hOKT3.gamma.1-ala-ala in vivo and exert
dominant inhibitory effects on autoreactive T cells; and enhanced
production of immunoregulatory Th2-type cytokines such as IL-10 and
TGF(3, which contribute to T.sub.reg-mediated suppression in vivo
(see, e.g., Kohm et al., 2005, Int. Rev. Immunol. 24:361-392,
Filippi et al., 2005, Int. Rev. Immunol. 24:341-360, and Chen et
al., 2004, Cell. Mol. Immunol. 1:328-335).
[0198] 5.2.3 Psoriasis
[0199] Psoriasis is a chronic, inflammatory, hyperproliferative
skin disease that affects approximately 1-2% of the general
population with men and women affected in equal numbers. (Nevitt,
G. J. et al., 1996, British J. of Dermatology 135:533-537).
Approximately 150,000 new cases of psoriasis and approximately 400
deaths from psoriasis are reported each year (Stem, R. S., 1995,
Dermatol. Clin. 13:717-722). The most common type of psoriasis is
chronic plaque syndrome. The condition is chronic for many
sufferers and consists of periods of remission and relapse during
the course of the disease (Ashcroft, D. M., et al., 2000, J. of
Clin. Pharm. And Therap. 25:1-10).
[0200] Psoriasis is characterized by indurated, erythematous
scaling plaques most commonly located on the scalp or the extensor
aspects of the elbows and knees, but may occur at any skin site.
Present treatment options currently available for psoriasis include
topical agents, phototherapy and systemic agents. Topical
treatments are first-line therapy for patients with mild to
moderate plaque psoriasis. Systemic treatment is generally
prescribed for severe cases of psoriasis where topical therapy is
either impractical or ineffective. Phototherapy can be administered
either alone or in combination with either topical or systemic
agents. Unfortunately, each of these treatment options is
associated with severe side effects. Most of the topical agents
available for the treatment of psoriasis are associated with skin
irritation, toxicity and possible carcinogenicity (Ashcroft, D. M.,
et al., 2000, J. of Clin. Pharm, and Therap. 25:1-10).
Phototherapy, either broadband (UVB) or long wave (UVA), is
associated with short term risks such as vesiculation, nausea,
erythema, headache and skin pain as well as long-term risks of
actinic keratoses, premature ageing of the skin, irregular
pigmentation and squamous cell carcinoma which is reported in a
quarter of patients (Stern, R. S., 1994, Cancer 73:2759-2764).
Systemic agents are also associated with adverse side effects, and
most are unavailable to pregnant patients. In particular,
methotrexate, which is considered to be the `gold standard` for
treatment of severe psoriasis, carries a risk of hepatotoxicity
with long-term use. In addition, it is recommended that patients
have a liner biopsy performed at or near the start of each
treatment and after each cumulative dose of 1.0-1.5 mg MTX
(Roenigk, H. H. et al., 1988, J. of the Am. Acad. Of
Dermatology).
[0201] When patients are provided with information regarding the
possible adverse effects of the currently available therapies for
psoriasis, many often choose to live with the condition rather than
undergo treatment (Greaves M. W., 1995, New England J. of Medicine
332:581-588). Thus, there remains a need for better methods of
treating psoriasis than currently available therapies.
[0202] Use of the invention in the context of psoriasis therefore
encompasses methods to treat the acute phases of the disease as
well as to prevent recurrence of symptoms of psoriasis. The
response to anti-CD3 therapy in the context of psoriasis may be
assessed by methods described herein or by any method known to one
of ordinary skill in the art. Common methods used to monitor the
symptoms of psoriasis include, but are not limited to, the
Psoriasis Area and Severity Index (PAST), Physician Global
Assessment (PGA) and NPF Psoriasis Score (NPF-PS) (See Ashcroft et
al., 1999, Br. J. Dermatol. 141:185-191; van der Kerkhof et al.,
1997, Br. J. Dermatol. 137:661-662 and Krueger et al., 1999,
National Psoriasis Foundation Psoriasis Forum 5:1-5.,
respectively).
[0203] 5.2.4 Rheumatoid Arthritis
[0204] Rheumatoid arthritis (RA) is an autoimmune disorder where
the body's immune system improperly identifies the synovial
membranes that secrete the lubricating fluid in the joints as
foreign. Inflammation results, and the cartilage and tissues in and
around the joints are damaged or destroyed. In severe cases, this
inflammation extends to other joint tissues and surrounding
cartilage, where it may erode or destroy bone and cartilage and
lead to joint deformities. The body replaces damaged tissue with
scar tissue, causing the normal spaces within the joints to become
narrow and the bones to fuse together. Rheumatoid arthritis creates
stiffness, swelling, fatigue, anemia, weight loss, fever, and
often, crippling pain. Some common symptoms of rheumatoid arthritis
include joint stiffness upon awakening that lasts an hour or
longer; swelling in a specific finger or wrist joints; swelling in
the soft tissue around the joints; and swelling on both sides of
the joint. Swelling can occur with or without pain, and can worsen
progressively or remain the same for years before progressing.
Besides rheumatoid arthritis, other types of arthritis associated
with autoimmune inflammation include the following: psoriatic
arthritis, Reiter's syndrome and ankylosing spondylitis arthritis.
Rheumatoid arthritis occurs in joints on both sides of the body
(such as both hands, wrists or knees). This symmetry helps
distinguish rheumatoid arthritis from other types of arthritis. In
addition to affecting the joints, rheumatoid arthritis may
occasionally affect the skin, eyes, lungs, heart, blood or
nerves.
[0205] Rheumatoid arthritis affects about 1% of the world's
population and is potentially disabling. There are approximately
2.9 million incidences of rheumatoid arthritis in the United
States. Two to three times more women are affected than men. The
typical age that rheumatoid arthritis occurs is between 25 and 50.
Juvenile rheumatoid arthritis affects 71,000 young Americans (aged
eighteen and under), affecting six times as many girls as boys.
[0206] Currently available therapy for arthritis focuses on
reducing inflammation of the joints with anti-inflammatory or
immunosuppressive medications. The first line of treatment of any
arthritis is usually anti-inflammatories, such as aspirin,
ibuprofen and Cox-2 inhibitors such as celecoxib and rofecoxib.
"Second line drugs" include gold, methotrexate and steroids.
Although these are well-established treatments for arthritis, very
few patients remit on these lines of treatment alone. Recent
advances in the understanding of the pathogenesis of rheumatoid
arthritis have led to the use of methotrexate in combination with
antibodies to cytokines or recombinant soluble receptors. However,
only about 50% of the patients treated with a combination of
methotrexate and anti-TNF-.alpha. agents such as recombinant
soluble receptors for TNF-.alpha. show clinically significant
improvement. Many patients remain refractory despite treatment.
Difficult treatment issues still remain for patients with
rheumatoid arthritis. Many current treatments have a high incidence
of side effects or cannot completely prevent disease
progression.
[0207] Use of the invention in the context of RA therefore
encompasses methods to treat the acute phases of the disease as
well as to prevent recurrence of symptoms of RA. The response to
the therapeutic methods of the invention in the context of RA may
be assessed by methods described herein or by any method known to
one of ordinary skill in the art. For example, many, but not all,
people with rheumatoid arthritis have rheumatoid-factor antibody in
their blood; however, the presence of rheumatoid factor is not in
itself definitive for a positive diagnosis of RA as other
conditions are know which cause the rheumatoid factor to be
produced. Therefore, the diagnosis and assessment of rheumatoid
arthritis is most commonly based on a combination of factors,
including, but not limited to: the specific location and symmetry
of painful joints, the presence of joint stiffness in the morning,
the presence of bumps and nodules under the skin (rheumatoid
nodules), results of X-ray tests that suggest rheumatoid arthritis.
The invention encompasses any method of assessing the severity of
the condition accepted in the art. Common assessment methods are
based on the subjective responses of the subject to pain
questionnaires (e.g., the Health Assessment Questionnaire (HAQ)
with Disability, Pain Severity, and Health State Subscales);
however, such subjective assessments are often confounded by the
psychological functioning of the subject. Accordingly, objective
scales have been developed that allow quantitative assessment of
rheumatoid arthritis severity by the treating physician, e.g., the
Rheumatoid Arthritis Severity Scale ("RASS;" see, e.g., Bardwell et
al., 2002, Rheumatology 41:38-45, hereby incorporated by reference
herein in its entirety.)
[0208] 5.2.5 Tissue Transplantation
[0209] Tissue transplantation between genetically nonidentical
individuals results in immunological rejection of the tissue
through T cell-dependent mechanisms. To prevent allograft
rejection, immunosuppression is achieved with agents that generally
interfere with T cell function by modulating TcR signal
transduction (see, e.g., Borel, J. F., 1989, Pharmacol. Rev.
42:260-372; Moms, P. J., 1991, Curr. Opin. Immunol. 3:748-751;
Sigal et al., 1992, Ann. Rev. Immunol. 10:519-560; and L'Azou et
al., 1999, Arch. Toxicol. 73:337-345). Further, since the effect of
the immunosuppressive agents is short-lasting, transplant
recipients normally require life-long treatment of
immunosuppressive agents to prevent transplant rejection.
Transplant recipients receiving long-term immunosuppressive
treatment have a high risk of developing infections and tumors. For
example, patients receiving immunotherapy are at higher risk of
developing lymphomas, skin tumors and brain tumors (see, e.g.,
Fellstrom et al., 1993, Immunol. Rev. 134:83-98). As an alternative
to the general immunosuppressive agents currently used for the
prevention of allograft rejection, monoclonal antibodies, including
OKT3, have been successfully used to specifically block receptors
involved in T cell stimulation activation.
[0210] Use of the invention in the context of tissue
transplantation therefore encompasses methods to treat the acute
phases of the rejection as well as to prevent recurrence of
symptoms of rejection. The response to the therapeutic methods of
the invention in the context of tissue may be assessed by methods
described herein or by any method known to one of ordinary skill in
the art; however, no general methods, other than detecting an
increasing frequency of CTL that recognize donor antigens
(described infra), presently exist to monitor whether a transplant
is being rejected by a recipient. Although the function of some
transplants may be directly monitored (i.e. kidney or liver), often
the first overt sign of rejection is a complete physiologic failure
of the tissue, at which point the tissue is normally beyond
rescue.
[0211] 5.2.6 Diagnosis. Prediction and Assessment of Autoimmune
Disorders
[0212] Patients with autoimmune disorders generally have an
increasing frequency of CTL that recognize autoantigens. In the
context of tissue transplantation, the patients will exhibit an
increasing frequency of CTL that recognize donor-specific antigens.
Such autoreactive or donor-reactive CTL may be detected in
peripheral blood or target tissues. For example, in the diabetic
patient, autoreactive CTL may be detected in pancreatic islet cell
tissues; in the psoriatic patient, autoreactive CTL may be detected
in epidermal or dermal tissues; in the arthritic patient,
autoreactive CTL may be detected in synovial cell tissues and in
the organ transplant recipient, donor-reactive CTL may be detected
in the transplant graft. Since the generation of autoreactive or
donor-reactive CTL is thought to precede the development of
auto/donor antibodies and other indicia of the clinical symptoms of
immune disorders, detection of specific CTL may in some cases
enable more sensitive and specific diagnosis of the disorder.
[0213] The assays can also be used to quantify both the absolute
number and the proportion of autoreactive CTL present in a sample,
such as a peripheral blood sample, in both pre-clinical subjects
and patients that have received therapy. In some embodiments, both
the severity and course of the autoimmune or allograft disorder may
be predicted and followed using such assays. For example, the human
MHC class I molecule HLA-A 0201 can be used in combination with the
a diabetic autoantigen, for example IA-2, to detect autoreactive
CTL present in a peripheral blood sample of a pre-diabetic subject
or diabetic patient currently undergoing therapy using the methods
of the invention.
[0214] The compounds of the invention can also be used in vivo in
combination with, for example, imaging techniques or other in vivo
detection methods for detecting CTLs labeled by binding with
compounds or formulations of the invention.
[0215] Antigen-specific CTLs can be detected using a wide variety
of assays, including immunospot (e.g., ELISPOT) assays, MHC class I
tetramer assays, or other assays, as described herein or as known
to a person skilled in the art.
[0216] The time period over which any biomarker of disorder
progression or therapeutic effectiveness may be evaluated may be
the time period of a single dose or an extended treatment time
period, e.g. hours, days, weeks or months.
[0217] 5.2.7 Therapeutic and Prophylactic Methods
[0218] The compositions and methods of the invention are
particularly useful for the prevention, treatment or amelioration
of T cell mediated diseases such as autoimmune disorders
characterized by increased T cell infiltration of lymphocytes into
affected tissues, or autoimmune disorders characterized by
increased T cell activation and/or abnormal antigen presentation
and/or recognition. The compositions and methods are also useful
for the prevention, treatment or amelioration of inflammatory
disorders characterized by increased T cell activation and/or
abnormal antigen presentation. In specific embodiments, the
invention provides methods of treating, preventing, managing or
ameliorating the symptoms of an autoimmune disease, particularly,
Type I Diabetes, multiple sclerosis, ulcerative colitis, psoriasis,
rheumatoid arthritis, lupus (particularly, cutaneous), psoriatic
arthritis, inflammatory bowel disease (IBD), effects from organ
transplantation, and graft vs. host disease (GVHD). Preferably, the
treatment regimens described herein are administered to patients in
early stages of the autoimmune disease, exhibiting mild tissue
damage resulting from the immune reaction and requiring minimal
medical intervention, e.g., low doses of standard therapy, to
manage the disease. The treatment regimens described herein
maintain high level functioning and prevent, slow or reduce
additional tissue damage. Thus, the methods of the invention may
reduce the need for additional therapy to treat, manage or
ameliorate the disease or disorder, and/or symptoms thereof.
[0219] In a certain embodiments, pharmaceutical compositions
comprising one or more CD3 binding molecules (e.g., one or more
anti-human CD3 antibodies) are administered one or more times,
preferably in a dosing regimen administered in multiple doses over
a period of 2 to 20 days, to treat, manage or ameliorate the
symptoms of an autoimmune diabetes disorder, to prevent or slow the
decrease in .beta.-cell function associated with autoimmune
diabetes, or to delay or prevent the onset of an autoimmune
diabetes disorder. in a subject with a predisposition for
development of Type-1 diabetes as described herein. In yet another
embodiment, one or more pharmaceutical compositions comprising one
or more CD3 binding molecules (e.g., one or more anti-CD3
antibodies) are administered one or ore times to prevent decrease
in .beta.-cell function associated with diabetes in a subject that
has had an allograft comprising pancreatic islet cell tissue. In
accordance with these embodiments, changes in a subject's
.beta.-cell function may be assessed by characterization of daily
insulin requirements, HA1c levels, C-peptide function/levels,
frequency of hypoglycemic episodes or FPIR as known in the art.
[0220] In certain embodiments, the course of treatment with an
anti-CD3 antibody according to the methods of the invention is
repeated at 2 month, 4 month, 6 month, 8 month, 9 month, 10 month,
12 month, 15 month, 18 month, 24 month, 30 month, or 36 month
intervals. In specific embodiments efficacy of the treatment with
an anti-CD3 antibody of the invention is determined as described
herein or as is known in the art at 2 months, 4 months, 6 months, 9
months, 12 months, 15 months, 18 months, 24 months, 30 months, or
36 months subsequent to the previous treatment.
[0221] In another embodiment, a subject is administered one or more
unit doses of approximately 0.5-50 .mu.g/kg, approximately 0.5-40
.mu.g/kg, approximately 0.5-30 .mu.g/kg, approximately 0.5-20
.mu.g/kg, approximately 0.5-15 .mu.g/kg, approximately 0.5-10
.mu.g/kg, approximately 0.5-5 .mu.g/kg, approximately 1-5 .mu.g/kg,
approximately 1-10 .mu.g/kg, approximately 20-40 .mu.g/kg,
approximately 20-30 .mu.g/kg, approximately 22-28 .mu.g/kg or
approximately 25-26 .mu.g/kg of one or more anti-CD3 antibody to
prevent, treat or ameliorate one or more symptoms of an autoimmune
disorder, particularly diabetes. In another embodiment, a subject
is administered one or more unit doses of 200 .mu.g/kg, 178
.mu.g/kg, 180 .mu.g/kg, 128 .mu.g/kg, 100 .mu.g/kg, 95 .mu.g/kg, 90
.mu.g/kg, 85 .mu.g/kg, 80 .mu.g/kg, 75 .mu.g/kg, 70 .mu.g/kg, 65
.mu.g/kg, 60 .mu.g/kg, 55 .mu.g/kg, 50 .mu.g/kg, 4S .mu.g/kg, 40
.mu.g/kg, 35 .mu.g/kg, 30 .mu.g/kg, 26 .mu.g/kg, 25 .mu.g/kg, 20
.mu.g/kg, 15 .mu.g/kg, 13 .mu.g/kg, 10 .mu.g/kg, 6.5 .mu.g/kg, 5
.mu.g/kg, 3.2 .mu.g/kg, 3 .mu.g/kg, 2.5 .mu.g/kg, 2 .mu.g/kg, 1.6
.mu.g/kg, 1.5 .mu.g/kg, 1 .mu.g/kg, 0.5 .mu.g/kg, 0.25 .mu.g/kg,
0.1 .mu.g/kg, or 0.05 .mu.g/kg of one or more anti-CD3 antibodies
to prevent, treat or ameliorate one or more symptoms of an
autoimmune disorder, particularly diabetes.
[0222] In a one embodiment, a subject is administered one or more
doses of 200 .mu.g/kg or less, 175 .mu.g/kg or less, 150 .mu.g/kg
or less, 128 .mu.g/kg or less, 100 .mu.g/kg or less, 95 .mu.g/kg or
less, 90 .mu.g/kg or less, 85 .mu.g/kg or less, 80 .mu.g/kg or
less, 75 .mu.g/kg or less, 70 .mu.g/kg or less, 65 .mu.g/kg or
less, 60 .mu.g/kg or less, 55 .mu.g/kg or less, 50 .mu.g/kg or
less, 45 .mu.g/kg or less, 40 .mu.g/kg or less, 35 .mu.g/kg or
less, 30 .mu.g/kg or less, 25 .mu.g/kg or less, 20 .mu.g/kg or
less, 15 .mu.g/kg or less, 10 .mu.g/kg or less, 5 .mu.g/kg or less,
2.5 .mu.g/kg or less, 2 .mu.g/kg or less, 1.5 .mu.g/kg or less, 1
.mu.g/kg or less, 0.5 .mu.g/kg or less, 0.25 .mu.g/kg or less, 0.1
.mu.g/kg or less, or 0.05 .mu.g/kg or less of one or more anti-CD3
antibody of the invention to prevent, treat or ameliorate one or
more symptoms of an autoimmune disorder, such as but not limited to
diabetes.
[0223] In particular embodiments, a subject is administered one or
more doses of about 5-1200 .mu.g/m.sup.2, preferably, 51-826
.mu.g/m.sup.2. In another embodiment, a subject is administered one
or more unit doses of 1200 .mu.g/m.sup.2, 1150 .mu.g/m.sup.2, 1100
.mu.g/m.sup.2, 1050 .mu.g/m.sup.2, 1000 .mu.g/m.sup.2, 950
.mu.g/m.sup.2, 900 .mu.g/m.sup.2, 850 .mu.g/m.sup.2, 800
.mu.g/m.sup.2, 750 .mu.g/m.sup.2, 700 .mu.g/m.sup.2, 650
.mu.g/m.sup.2, 600 .mu.g/m.sup.2, 550 .mu.g/m.sup.2, 500
.mu.g/m.sup.2, 450 .mu.g/m.sup.2, 400 .mu.g/m.sup.2, 350
.mu.g/m.sup.2, 300 .mu.g/m.sup.2, 250 .mu.g/m.sup.2, 200
.mu.g/m.sup.2, 150.mu.g/m.sup.2, 100 .mu.g/m.sup.2, 50
.mu.g/m.sup.2, 40 .mu.g/m.sup.2, 30 .mu.g/m.sup.2, 20
.mu.g/m.sup.2, 15 .mu.g/m.sup.2, 10 .mu.g/m.sup.2, or 5
.mu.g/m.sup.2 of one or more anti-human CD3 antibodies to prevent,
treat, slow the progression of, delay the onset of or ameliorate
one or more symptoms of an autoimmune disorder or disease.
[0224] In another embodiment, the subject is administered a
treatment regimen comprising one or more doses of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies, wherein the course of treatment is
administered over 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8
days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In one
embodiment, the treatment regimen comprises administering doses of
the prophylactically or therapeutically effective amount of one or
more anti-human CD3 antibodies every day, every 2.sup.nd day, every
3.sup.rd day or every 4.sup.th day. In certain embodiments, the
treatment regimen comprises administering doses of the
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies on Monday, Tuesday, Wednesday, Thursday
of a given week and not administering doses of the prophylactically
or therapeutically effective amount of one or more anti-human CD3
antibodies on Friday, Saturday, and Sunday of the same week until
14 doses, 13, doses, 13 doses, 12 doses, 11 doses, 10 doses, 9
doses, or 8 doses have been administered. In certain embodiments
the dose administered is the same each day of the regimen. In
certain embodiments, a subject is administered a treatment regimen
comprising one or more doses of a prophylactically or
therapeutically effective amount of one or more anti-human CD3
antibodies, wherein the prophylactically or therapeutically
effective amount is 200 .mu.g/kg/day, 175 .mu.g/kg/day, 150
.mu.g/kg/day, 125 .mu.g/kg/day, 100 .mu.g/kg/day, 95 .mu.g/kg/day,
90 .mu.g/kg/day, 85 .mu.g/kg/day, 80 .mu.g/kg/day, 75 .mu.g/kg/day,
70 .mu.g/kg/day, 65 .mu.g/kg/day, 60 .mu.g/kg/day, 55 .mu.g/kg/day,
50 .mu.s/kg/day, 45 .mu.g/kg/day, 40 .mu.g/kg/day, 35 .mu.g/kg/day,
30 .mu.g/kg/day, 26 .mu.g/kg/day, 25 .mu.g/kg/day, 20 .mu.g/kg/day,
15 .mu.g/kg/day, 13 .mu.g/kg/day, 10 .mu.g/kg/day, 6.5
.mu.g/kg/day, 5 .mu.g/kg/day, 3.2 .mu.g/kg/day, 3 .mu.g/kg/day, 2.5
.mu.g/kg/day, 2 .mu.g/kg/day, 1.6 .mu.g/kg/day, 1.5 .mu.g/kg/day, 1
.mu.g/kg/day, 0.5 .mu.g/kg/day, 0.25 .mu.g/kg/day, 0.1
.mu.g/kg/day, or 0.05 .mu.g/kg/day; and/or wherein the
prophylactically or therapeutically effective amount is 1200
.mu.g/m.sup.2/day, 1150 .mu.g/m.sup.2/day, 1100 .mu.g/m.sup.2/day,
1050 .mu.g/m.sup.2/day, 1000 .mu.g/m.sup.2/day, 950
.mu.g/m.sup.2/day, 900 .mu.g/m.sup.2/day, 850 .mu.g/m.sup.2/day,
800 .mu.g/m.sup.2/day, 750 .mu.g/m.sup.2/day, 700
.mu.g/m.sup.2/day, 650 .mu.g/m.sup.2/day, 600 .mu.g/m.sup.2/day,
550 .mu.g/m.sup.2/day, 500 .mu.g/m.sup.2/day, 450
.mu.g/m.sup.2/day, 400 .mu.g/m.sup.2/day, 350 .mu.g/m.sup.2/day,
300 .mu.g/m.sup.2/day, 250 .mu.g/m.sup.2/day, 200
.mu.g/m.sup.2/day, 150 .mu.g/m.sup.2/day, 100 .mu.g/m.sup.2/day, 50
.mu.g/m.sup.2/day, 40 .mu.g/m.sup.2/day, 30 .mu.g/m.sup.2/day, 20
.mu.g/m.sup.2/day, 15 .mu.g/m.sup.2/day, 10 .mu.g/m.sup.2/day, or 5
.mu.g/m.sup.2/day. In another embodiment, the intravenous dose of
1200 .mu.g/m.sup.2 or less, 1150 .mu.g/m.sup.2 or less, 1100
.mu.g/m.sup.2 or less, 1050 .mu.g/m.sup.2 or less, 1000
.mu.g/m.sup.2 or less, 950 .mu.g/m.sup.2 or less, 900 .mu.g/m.sup.2
or less, 850 .mu.g/m.sup.2 or less, 800 .mu.g/m2 or less, 750
.mu.g/m.sup.2 or less, 700 .mu.g/m.sup.2 or less, 650 .mu.g/m.sup.2
or less, 600 .mu.g/m.sup.2 or less, 550 .mu.g/m.sup.2 or less, 500
.mu.g/m.sup.2 or less, 450 .mu.g/m.sup.2 or less, 400 .mu.g/m.sup.2
or less, 350 .mu.g/m.sup.2 or less, 300 .mu.g/m.sup.2 or less, 250
.mu.g/m2 or less, 200 .mu.g/m.sup.2 or less, 150 .mu.g/m.sup.2 or
less, 100 .mu.g/m.sup.2 or less, 50 .mu.g/m.sup.2 or less, 40
.mu.g/m.sup.2 or less, 30 .mu.g/m.sup.2 or less, 20 .mu.g/m.sup.2
or less, 15 .mu.g/m.sup.2 or less, 10 .mu.g/m.sup.2 or less, or 5
.mu.g/m.sup.2 or less of one or more anti CD3 antibodies is
administered over about 24 hours, about 22 hours, about 20 hours,
about 18 hours, about 16 hours, about 14 hours, about 12 hours,
about 10 hours, about 8 hours, about 6 hours, about 4 hours, about
2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40
minutes, about 30 minutes, about 20 minutes, about 10 minutes,
about 5 minutes, about 2 minutes, about 1 minute, about 30 seconds
or about 10 seconds to prevent, treat or ameliorate one or more
symptoms of type 1 diabetes. The total dosage over the duration of
the regimen is preferably a total of less than 9000 .mu.g/m.sup.2,
8000 .mu.g/m.sup.2, 7000 .mu.g/m.sup.2, 6000 .mu.g/m.sup.2, and may
be less than 5000 .mu.g/m.sup.2, 4000 .mu.g/m.sup.2, 3000
.mu.g/m.sup.2, 2000 .mu.g/m.sup.2, or 1000 .mu.g/m.sup.2. In
specific embodiments, the total dosage administered in the regimen
is 100 .mu.g/m.sup.2 to 200 .mu.g/m.sup.2, 100 .mu.g/m.sup.2 to 500
.mu.g/m.sup.2, 100 .mu.g/m.sup.2 to 1000 .mu.g/m.sup.2, or 500
.mu.g/m.sup.2 to 1000 .mu.g/m.sup.2.
[0225] In preferred embodiments, the dose escalates over the first
fourth, first half or first 2/3 of the doses (e.g., over the first
2, 3, 4, 5, or 6 days of a 10, 12, 14, 16, 18 or 20 day regimen of
one dose per day) of the treatment regimen until the daily
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies is achieved. In certain embodiments, a
subject is administered a treatment regimen comprising one or more
doses of a prophylactically or therapeutically effective amount of
one or more anti-human CD3 antibodies, wherein the prophylactically
or therapeutically effective amount is increased by, e.g., 0.01
.mu.g/kg, 0.02 .mu.g/kg, 0.04 .mu.g/kg, 0.05 .mu.g/kg, 0.06
.mu.g/kg, 0.08 .mu.g/kg, 0.1 .mu.g/kg, 0.2 .mu.g/kg, 0.25 .mu.g/kg,
0.5 .mu.g/kg, 0.75 .mu.g/kg, 1 .mu.g/kg, 1.5 .mu.g/kg, 2 .mu.g/kg,
4 .mu.g/kg, 5 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20 .mu.g/kg, 25
.mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45 .mu.g/kg, 50
.mu.g/kg, 55 .mu.g/kg, 60 .mu.g/kg, 65 .mu.g/kg, 70 .mu.g/kg, 75
.mu.g/kg, 80 .mu.g/kg, 85 .mu.g/kg, 90 .mu.g/kg, 95 .mu.g/kg, 100
.mu.g/kg, or 125 .mu.g/kg each day; or increased by, e.g., 1
.mu.g/m.sup.2, 5 .mu.g/m.sup.2, 10 .mu.g/m.sup.2, 15 .mu.g/m.sup.2,
20 .mu.g/m.sup.2, 30 .mu.g/m.sup.2, 40 .mu.g/m.sup.2, 50
.mu.g/m.sup.2, 60 .mu.g/m.sup.2, 70 .mu.g/m.sup.2, 80
.mu.g/m.sup.2, 90 .mu.g/m.sup.2, 100 .mu.g/m.sup.2, 150
.mu.g/m.sup.2, 200 .mu.g/m.sup.2, 250 .mu.g/m.sup.2, 300
.mu.g/m.sup.2, 350 .mu.g/m.sup.2, 400 .mu.g/m.sup.2, 450
.mu.g/m.sup.2, 500 .mu.g/m.sup.2, 550 .mu.g/m.sup.2, 600
.mu.g/m.sup.2, or 650 .mu.g/m.sup.2, each day as treatment
progresses. In certain embodiments, a subject is administered a
treatment regimen comprising one or more doses of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies, wherein the prophylactically or
therapeutically effective amount is increased by a factor of 1.25,
a factor of 1.5, a factor of 2, a factor of 2.25, a factor of 2.5,
or a factor of 5 until the daily prophylactically or
therapeutically effective amount of one or more anti-human CD3
antibodies is achieved.
[0226] In a specific embodiment, a subject is intramuscularly
administered one or more doses of a 200 .mu.g/kg or less,
preferably 175 .mu.g/kg or less, 150 .mu.g/kg or less, 125 .mu.g/kg
or less, 100 .mu.g/kg or less, 95 .mu.g/kg or less, 90 .mu.g/kg or
less, 85 .mu.g/kg or less, 80 .mu.g/kg or less, 75 .mu.g/kg or
less, 70 .mu.g/kg or less, 65 .mu.g/kg or less, 60 .mu.g/kg or
less, 55 .mu.g/kg or less, 50 .mu.g/kg or less, 45 .mu.g/kg or
less, 40 .mu.g/kg or less, 35 .mu.g/kg or less, 30 .mu.g/kg or
less, 25 .mu.g/kg or less, 20 .mu.g/kg or less, 15 .mu.g/kg or
less, 10 .mu.g/kg or less, 5 .mu.g/kg or less, 2.5 .mu.g/kg or
less, 2 .mu.g/kg or less, 1.5 .mu.g/kg or less, 1 .mu.g/kg or less,
0.5 .mu.g/kg or less, or 0.5 .mu.g/kg or less of one or more
anti-CD3 antibodies to prevent, treat or ameliorate one or more
symptoms of an autoimmune disorder.
[0227] In another embodiment, a subject is subcutaneously
administered one or more doses of a 200 .mu.g/kg or less,
preferably 175 .mu.g/kg or less, 150 .mu.g/kg or less, 125 .mu.g/kg
or less, 100 .mu.g/kg or less, 95 .mu.g/kg or less, 90 .mu.g/kg or
less, 85 .mu.g/kg or less, 80 .mu.g/kg or less, 75 .mu.g/kg or
less, 70 .mu.g/kg or less, 65 .mu.g/kg or less, 60 .mu.g/kg or
less, 55 .mu.g/kg or less, 50 .mu.g/kg or less, 45 tag/kg or less,
40 .mu.g/kg or less, 35 .mu.g/kg or less, 30 .mu.g/kg or less, 25
.mu.g/kg or less, 20 .mu.g/kg or less, 15 .mu.g/kg or less, 10
.mu.g/kg or less, 5 .mu.g/kg or less, 2.5 .mu.g/kg or less, 2
.mu.g/kg or less, 1.5 pig/kg or less, 1 .mu.g/kg or less, 0.5
.mu.g/kg or less, or 0.5 .mu.g/kg or less of one or more anti-CD3
antibodies to prevent, treat or ameliorate one or more symptoms of
an autoimmune disorder.
[0228] In another embodiment, a subject is intravenously
administered one or more doses of a 100 .mu.g/kg or less,
preferably 95 .mu.g/kg or less, 90 .mu.g/kg or less, 85 .mu.g/kg or
less, 80 .mu.g/kg or less, 75 .mu.g/kg or less, 70 .mu.g/kg or
less, 65 .mu.g/kg or less, 60 .mu.g/kg or less, 55 .mu.g/kg or
less, 50 .mu.g/kg or less, 45 .mu.g/kg or less, 40 .mu.g/kg or
less, 35 .mu.g/kg or less, 30 .mu.g/kg or less, 25 .mu.g/kg or
less, 20 .mu.g/kg or less, 15 .mu.g/kg or less, 10 .mu.g/kg or
less, 5 .mu.g/kg or less, 2.5 .mu.g/kg or less, 2 .mu.g/kg or less,
1.5 .mu.g/kg or less, 1 .mu.g/kg or less, 0.5 .mu.g/kg or less, or
0.5 .mu.g/kg or less of one or more anti CD3 antibodies to prevent,
treat or ameliorate one or more symptoms of an autoimmune disorder.
In another embodiment, the intravenous dose of 100 .mu.g/kg or
less, 95 .mu.g/kg or less, 90 .mu.g/kg or less, 85 .mu.g/kg or
less, 80 .mu.g/kg or less, 75 .mu.g/kg or less, 70 .mu.g/kg or
less, 65 .mu.g/kg or less, 60 .mu.g/kg or less, 55 .mu.g/kg or
less, 50 .mu.g/kg or less, 45 .mu.g/kg or less, 40 .mu.g/kg or
less, 35 .mu.g/kg or less, 30 .mu.g/kg or less, 25 .mu.g/kg or
less, 20 .mu.g/kg or less, 15 tug/kg or less, 10 .mu.g/kg or less,
5 .mu.g/kg or less, 2.5 .mu.g/kg or less, 2 .mu.g/kg or less, 1.5
.mu.g/kg or less, 1 .mu.g/kg or less, 0.5 .mu.g/kg or less, or 0.5
.mu.g/kg or less of one or more anti CD3 antibodies is administered
over about 6 hours, about 4 hours, about 2 hours, about 1.5 hours,
about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes,
about 20 minutes, about 10 minutes, about 5 minutes, about 2
minutes, about 1 minute, about 30 seconds or about 10 seconds to
prevent, treat or ameliorate one or more symptoms of an autoimmune
disorder.
[0229] In specific embodiments in which escalating doses are
administered for the first days of the dosing regimen, the dose on
day 1 of the regimen is 5-100 .mu.g/m.sup.2/day, preferably 51
.mu.g/m.sup.2/day and escalates to the daily dose as recited
immediately above by day 3, 4, 5, 6 or 7. For example, on day 1,
the subject is administered a dose of approximately 51
.mu.g/m.sup.2/day, on day 2 approximately 103 .mu.g/m.sup.2/day, on
day 3 approximately 207 .mu.g/m.sup.2/day, on day 4 approximately
413 .mu.g/m.sup.2/day and on subsequent days of the regimen (e.g.,
days 5-14) 826 .mu.g/m.sup.2/day. In another embodiment, on day 1,
the subject is administered a dose of approximately 227
.mu.g/m.sup.2/day, on day 2 approximately 459 .mu.g/m.sup.2/day, on
day 3 and subsequent days, approximately 919 .mu.g/m.sup.2/day. In
another embodiment, on day 1, the subject is administered a dose of
approximately 284 .mu.g/m.sup.2/day, on day 2 approximately 574
.mu.g/m.sup.2/day, on day 3 and subsequent days, approximately 1148
.mu.g/m.sup.2/day.
[0230] In other embodiments, the initial dose is 1/4, to 1/2, to
equal to the daily dose at the end of the regimen but is
administered in portions at intervals of 6, 8, 10 on 12 hours. For
example, a 13 ug/kg/day dose is administered in four doses of 3-4
.mu.g/kg at intervals of 6 hours to reduce the level of cytokine
release caused by administration of the antibody.
[0231] In specific embodiments, to reduce the possibility of
cytokine release and other adverse effects, the first 1, 2, 3, or 4
doses or all the doses in the regimen are administered more slowly
by intravenous administration. For example, a dose of 51
.mu.g/m.sup.2/day may be administered over about 5 minutes, about
15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about
2 hours, about 4 hours, about 6 hours, about 8 hours, about 10
hours, about 12 hours, about 14 hours, about 16 hours, about 18
hours, about 20 hours, and about 22 hours. In certain embodiments,
the dose is administered by slow infusion over a period of, e.g.,
20 to 24 hours. In specific embodiments, the dose is infused in a
pump, preferably increasing the concentration of antibody
administered as the infusion progresses.
[0232] In other embodiments, a set fraction of the doses for the 51
.mu.g/m.sup.2/day to 826 .mu.g/m.sup.2/day regimen described above
is administered in escalating doses. In certain embodiments, the
fraction is 1/10, 1/4, 1/3, 1/2, 2/3 or 3/4 of the daily doses of
the regimens described above. Accordingly, when the fraction is
1/10, the daily doses will be 5.1 .mu.g/m.sup.2 on day 1, 10.3
.mu.g/m.sup.2 on day 2, 20.7 .mu.g/m.sup.2 on day 3, 41.3
.mu.g/m.sup.2 on day 4 and 82.6 .mu.g/m.sup.2 on days 5 to 14. When
the fraction is 1/4, the doses will be 12.75 .mu.g/m.sup.2 on day
1, 25.5 g/m.sup.2 on day 2, 51 .mu.g/m.sup.2 on day 3, 103
.mu.g/m.sup.2 on day 4, and 207 .mu.g/m.sup.2 on days 5 to 14. When
the fraction is 1/3, the doses will be 17 .mu.g/m.sup.2 on day 1,
34.3 .mu.g/m.sup.2 on day 2, 69 .mu.g/m.sup.2 on day 3, 137.6
.mu.g/m.sup.2 on day 4, and 275.3 .mu.g/m.sup.2 on days 5 to 14.
When the fraction is 1/2, the doses will be 25.5 .mu.g/m.sup.2 on
day 1, 51 .mu.g/m.sup.2 fraction is 2/3, the doses will be 34
.mu.g/m.sup.2 on day 1, 69 .mu.g/m.sup.2 on day 2, 137.6
.mu.g/m.sup.2 on day 3, 275.3 .mu.g/m.sup.2 on day 4, and 550.1
.mu.g/m.sup.2 on days 5 to 14. When the fraction is 3/4, the doses
will be 38.3 .mu.g/m.sup.2 on day 1, 77.3 .mu.g/m.sup.2 on day 2,
155.3 .mu.g/m.sup.2 on day 3, 309.8 .mu.g/m.sup.2 on day 4, and 620
.mu.s/m.sup.2 on days 5 to 14. In other embodiments, the regimen is
identical to one of those described above but only over days 1 to
4, days 1 to 5, or days 1 to 6. For example, in a particular
embodiment, the doses will be 17 .mu.g/m.sup.2 on day 1, 34.3
.mu.g/m.sup.2 on day 2, 69 .mu.g/m.sup.2 on day 3, 137.6
.mu.g/m.sup.2 on day 4, and 275.3 .mu.g/m.sup.2 on days 5 and
6.
[0233] In specific embodiments, the anti-human CD3 antibody is not
administered by daily doses over a number of days, but is rather
administered by infusion in an uninterrupted manner over 4 hours, 6
hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours,
24 hours, 30 hours or 36 hours. The infusion may be constant or may
start out at a lower dosage for, for example, the first 1, 2, 3, 5,
6, or 8 hours of the infusion and then increase to a higher dosage
thereafter. Over the course of the infusion, the patient receives a
dose equal to the amount administered in the 5 to 20 day regimens
set forth above. For example, a dose of approximately 150
.mu.g/m.sup.2, 200 .mu.g/m.sup.2, 250 .mu.g/m.sup.2, 500
.mu.g/m.sup.2, 750 .mu.g/m.sup.2, 1000 .mu.g/m.sup.2, 1500
.mu.g/m.sup.2, 2000 .mu.g/m.sup.2, 3000 .mu.g/m.sup.2, 4000
.mu.g/m.sup.2, 5000 .mu.g/m.sup.2, 6000 .mu.g/m.sup.2, 7000
.mu.g/m.sup.2, 8000 .mu.g/m.sup.2, or 9000 .mu.g/m.sup.2. In
particular, the speed and duration of the infusion is designed to
minimize the level of free anti-human CD3 antibody in the subject
after administration. In certain embodiments, the level of free
anti-human CD3 antibody should not exceed 200 ng/ml free antibody.
In addition, the infusion is designed to achieve a combined T cell
receptor coating and modulation of at least 50%, 60%, 70%, 80%,
90%, 95% or of 100%.
[0234] In certain embodiments, the antibody administered according
to these regimens is OKT3.gamma.1(ala-ala). In other embodiments
the antibody is not OKT3.gamma.1(ala-ala) and is administered so as
to achieve one or more pharmacokinetic parameters achieved by the
administration of OKT3.gamma.1(ala-ala), e.g., by intravenous
administration, such as the serum titer of the antibody
administered at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 2 weeks, 3 weeks or 1 month after the last day of the dosing
regime.
[0235] In certain embodiments, the anti-human CD3 antibody is
administered so as to achieve a certain level of combined coating
and modulation of T cell receptor complexes on T cells, as
determined by methods well known in the art, see, e.g., Example 11
of U.S. patent application publication US 2003/0108548, which is
hereby incorporated by reference in its entirety. In specific
embodiments, the dosing regimen achieves a combined T cell receptor
coating and modulation of at least 50%, 60%, 70%, 80%, 90%, 95% or
of 100% with, in specific embodiments, little to no free anti-human
CD3 antibody detected (for example, less than 200 ng/mL of the drug
is detected in the blood of the patient.)
[0236] In other embodiments, the anti-human CD3 antibody is
administered chronically to treat, prevent, or slow or delay the
onset or progression, or ameliorate one or more symptoms of type 1
diabetes. For example, in certain embodiments, a low dose of the
anti-human CD3 antibody is administered once a month, twice a
month, three times per month, once a week or even more frequently
either as an alternative to the 6 to 14 day dosage regimen
discussed above or after administration of such a regimen to
enhance or maintain its therapeutic effect. Such a low dose may be
anywhere from 1 .mu.g/m.sup.2 to 100 .mu.g/m.sup.2, preferably,
approximately 5 .mu.g/m.sup.2, 10 .mu.g/m.sup.2, 15 .mu.g/m.sup.2,
20 .mu.g/m.sup.2, 25 .mu.g/m.sup.2, 30 .mu.g/m.sup.2, 35
.mu.g/m.sup.2, 40 .mu.g/m.sup.2, 45 .mu.g/m.sup.2, or 50
.mu.g/m.sup.2.
[0237] In other embodiments, the subject may be re-dosed at some
time subsequent to administration of the anti-human CD3 antibody
dosing regimen, preferably, based upon one or more physiological
parameters or may be done as a matter of course. Such redosing may
be administered and/or the need for such redosing evaluated 2
months, 4 months, 6 months, 8 months, 9 months, 1 year, 15 months,
18 months, 2 years, 30 months or 3 years after administration of a
dosing regimen and may include administering a course of treatment
every 6 months, 9 months, 1 year, 15 months, 18 months, 2 years, 30
months or 3 years indefinitely.
[0238] In specific embodiments, subjects are administered a
subsequent round of anti-human CD3 antibody treatment based upon
measurements of one or a combination of the following: the CD4/CD8
cell ratio, CD8 cell count, CD4/CD3 inversion, CD4/CD25 cell ratio,
CD4/FoxP3 cell ratio, CD4/CD40 cell ratio, CD4/IL-10 cell ratio,
and/or a CD4/TGF-.beta. cell ratio.
[0239] With respect to the treatment of management of Type 1
diabetes, other parameters for determining whether to administer a
subsequent round of treatment include an appearance or an increase
in anti-islet cell antibodies, such as GADAs, IA-2 antibodies or
anti-insulin antibodies or an appearance or increase in the levels
of T cells specific for islet cell antigens. Subsequent doses may
be administered if the number of .beta.-cells or .beta.-cell
activity or function decreases by 20%, 30%, 40%, 50%, 60%, 70%, 80%
or 90% as compared to the .beta.-cell number or activity or
function during administration of the preceding round of treatment.
.beta.-cell function may be determined by any method know in the
art, for example, the C peptide response to MMTT, OGTT, IGTT, or
two-phase glucose clamp, or the First Phase Insulin Release (FPIR)
test, as discussed above. Other parameters that may be used to
determine whether to redose include the HA1 or HA1c levels, the
need for administration of exogenous insulin or increase in the
dosage of exogenous insulin by more than 0.1 U/kg/day, 0.2
U/kg/day, 0.5 U/kg/day, 0.6 U/kg/day, 1 U/kg/day, or 2 U/kg/day.
For example, a subject may be administered a subsequent round of
treatment when the C-peptide response or FPIR of the patient to
MMTT, OGTT, IGTT or two phase glucose clamp procedure decreases by
more than 1%, more than 5%, more than 10%, more than 20%, more than
30%, more than 40% or more than 50% of pretreatment levels. In
particular embodiments, subjects are redosed if they have a
C-peptide response to MMTT, OGTT, IGTT or two-phase glucose clamp
procedure (preferably, MMTT) resulting in an AUC of less than 40
pmol/ml/240 min., less than 50 pmol/ml/240 min, less than 60
pmol/ml/240 min, less than 70 pmol/ml/240 min., less than 80
pmol/ml/240 min., or less than at least 90 pmol/ml/240 min. In
specific embodiments, subjects may be redosed they have a FPIR of
less than 300 pmol/1, less than 400 pmol/1, less than 500 pmol/1,
less than 600 pmol/1, or less than 700 pmol/1, Also for example, a
subject may be redosed when the subject's HA1. or HA1C levels
increase by at least 5%, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80% or at least 90% compared to pre-treatment levels or the
absolute levels are greater than 8%, greater than 7.5%, or greater
than 7%. In other embodiments, the further doses may be
administered based upon appearance of or increase in number (such
as an increase by, on average, 1, 2, 3, 4, 5, 8, 10 15, or 20),
duration and/or severity of hypoglycemic episodes or of
ketoacidosis episodes on a daily, weekly or monthly basis.
[0240] In a specific embodiment, anti-human CD3 therapy is used in
type 1 diabetes patients that have at least 99%, at least 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 75%, at least 60%, at least 50% residual n-cell function
as compared to an individual with no indicators of diabetes or
predisposition to diabetes in the same population (i.e, age, sex,
race, and general health) and determined by methods described
herein or known to one of ordinary skill in the art. In another
embodiment, after a course of treatment with an anti-human CD3
antibody according to the invention, the level of .beta.-cell
function of the patient decreases by less than 1%, less than 5%,
less than 10%, less than 20%, less than 30%, less than 40% or less
than 50% of the pretreatment levels. In yet another embodiment of
the invention, after a course of treatment with an anti-human CD3
antibody according to the invention, the level of .beta.-cell
function of the patient is maintained at at least 99%, at least
95%, at least 90%, at least 80%, at least 70%, at least 60%, or at
least 50% of pretreatment levels for at least 4 months, at least 6
months, at least 9 months, at least 12 months, at least 18 months,
at least 24 months, or at least 30 months after the end of
treatment. In another embodiment of the invention, after a course
of treatment with an anti-human CD3 antibody according to the
invention, the level of .beta.-cell function of the patient is
maintained at at least 99%, at least 95%, at least 90%, at least
80%, at least 70%, at least 60%, or at least 50% of pretreatment
levels for at least 4 months, at least 6 months, at least 9 months,
at least 12 months, at least 18 months, at least 24 months, or at
least 30 months after the end of treatment and the mean lymphocyte
count of the patient is not less than 800 cells/ml, less than 750
cells/ml, less than 700 cells/ml, less than 650 cells/ml, less than
600 cells/ml, less than 550 cells/ml, less than 500 cells/ml, less
than 400 cells/ml, less than 300 cells/ml or less than 200 cells/ml
at the same time period. In another embodiment of the invention,
after a course of treatment with an anti-human CD3 antibody
according to the invention, the level of .beta.-cell function of
the patient is maintained at at least 99%, at least 95%, at least
90%, at least 80%, at least 70%, at least 60%, or at least 50% of
pretreatment levels for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 18 months, at least 24
months, or at least 30 months after the end of treatment and the
patient's mean platelet count is not less than 100,000,000
platelets/ml, less than 75,000,000 platelets/ml, less than
50,000,000 platelets/ml, less than 25,000,000 platelets/ml, less
than 1,000,000 platelets/ml, less than 750,000 platelets/ml, less
than 500,000 platelets/ml, less than 250,000 platelets/ml, less
than 150,000 platelets/ml or less than 100,000 platelets/ml.
[0241] In certain embodiments, one or more pharmaceutical
compositions comprising one or more CD3 binding molecules (e.g.,
one or more anti-human CD3 antibodies) are administered to a
subject having type 1 diabetes, to prevent or slow the reduction
.beta.-cell mass associated with autoimmune diabetes. In some
embodiments, after a course of treatment with an anti-human CD3
antibody according to the invention, the level of .beta.-cell mass
of the patient decreases by less than 1%, less than 5%, less than
10%, less than 20%, less than 30%, less than 40%, less than 50%,
less than 60%, or less than 70% of the pretreatment levels. In yet
another embodiment of the invention, after a course of treatment
with an anti-human CD3 antibody according to the invention, the
level of .beta.-cell function of the patient is maintained at at
least 99%, at least 95%, at least 90%, at least 80%, at least 70%,
at least 60%, at least 50%, at least 40%, or at least 30% of
pretreatment levels for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 18 months, at least 24
months, or at least 30 months after the end of treatment. In
another embodiment of the invention, after a course of treatment
with an anti-human CD3 antibody according to the invention, the
level of 13 cell function of the patient is maintained at at least
99%, at least 95%, at least 90%, at least 80%, at least 70%, at
least 60%, or at least 50% of pretreatment levels for at least 4
months, at least 6 months, at least 9 months, at least 12 months,
at least 18 months, at least 24 months, or at least 30 months after
the end of treatment and the mean lymphocyte count of the patient
is not less than 800 cells/ml, less than 750 cells/ml, less than
700 cells/ml, less than 650 cells/ml, less than 600 cells/ml, less
than 550 cells/ml, less than 500 cells/ml, less than 400 cells/ml,
less than 300 cells/ml or less than 200 cells/ml over the same time
period. In another embodiment of the invention, after a course of
treatment with an anti-human CD3 antibody according to the
invention the level of .beta.-cell function of the patient is
maintained at least 99%, at least 95%, at least 90%, at least 80%,
at least 70%, at least 60%, or at least 50% of pretreatment levels
for at least 4 months, at least 6 months, at least 9 months, at
least 12 months, at least 18 months, at least 24 months, or at
least 30 months after the end of treatment and the mean platelet
count of the patient is not less than 100,000,000 platelets/ml,
less than 75,000,000 platelets/ml, less than 50,000,000
platelets/ml, less than 25,000,000 platelets/ml, less than
1,000,000 platelets/ml, less than 750,000 platelets/ml, less than
500,000 platelets/ml, less than 250,000 platelets/ml, less than
150,000 platelets/ml or less than 100,000 platelets/ml.
[0242] In the methods of the invention, the anti-human CD3 therapy
is administered in patients that do not require daily insulin, or
that have average insulin requirements of less than 0.05 U/kg/day,
less than 0.1 U/kg/day, less than 0.2 U/kg/day, less than 0.4
U/kg/day, less than 0.6 U/kg/day, less than 0.8 U/kg/day, less than
1 U/kg/day, less than 2 U/kg/day, less than 5 U/kg/day, less than
10 U/kg/day or less than 50 U/kg/day. In another embodiment, a
patient with an autoimmune diabetes disorder is administered a
regimen of doses of a prophylactically or therapeutically effective
amount of one or more anti-human CD3 antibodies to avoid or delay
the need to administer insulin, or increase the dose of insulin
administered for more than 6 months, 1 year, 18 months, 24 months,
30 months, 36 months, 5 years, 7 years or 10. In other embodiments,
in patients who do require exogenous insulin, methods of the
invention achieve a reduction in daily insulin requirement by at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, or at least 90% of pretreatment levels. In yet another
embodiment of the invention in patients who require exogenous
insulin, after a course of treatment with an anti-human CD3
antibody according to the invention, the reduction of a patient's
daily insulin requirements by at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, or at least 90% of
pretreatment levels is maintained for at least 4 months, at least 6
months, at least 9 months, at least 12 months, at least 18 months,
at least 24 months, or at least 30 months after the course of
treatment. In yet another embodiment of the invention, after a
course of treatment with an anti-human CD3 antibody according to
the invention, the reduction of a patient's daily insulin
requirements by at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, or at least 85% of pretreatment levels is maintained
for at least 4 months, at least 6 months, at least 9 months, at
least 12 months, at least 18 months, at least 24 months, or at
least 30 months after the course of treatment and the mean
lymphocyte count of the patient is not less than 800 cells/ml, less
than 750 cells/ml, less than 700 cells/ml, less than 650 cells/ml,
less than 600 cells/ml, less than 550 cells/ml, less than 500
cells/ml, less than 400 cells/ml, less than 300 cells/ml or less
than 200 cells/ml over the same time period.
[0243] In other embodiments, in patients who do require exogenous
insulin, methods of the invention result in an increase in the
daily insulin requirement by no more than 1%, no more than 5%, no
more than 10%, no more than 15%, no more than 20%, no more than
25%, no more than 30%, no more than 40%, no more than 45%, no more
than 50%, no more than 55%, no more than 60%, no more than 65%, no
more than 70%, or no more than 75% as compared to pretreatment
levels. In yet another embodiment of the invention in patients who
require exogenous insulin, after a course of treatment with an
anti-human CD3 antibody according to the invention, the increase in
a patient's daily insulin requirements by no more than 1%, no more
than 5%, no more than 10%, no more than 15%, no more than 20%, no
more than 25%, no more than 30%, no more than 40%, no more than
45%, no more than 50%, no more than 55%, no more than 60%, no more
than 65%, no more than 70%, or no more than 75% of pretreatment
levels is maintained for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 18 months, at least 24
months, or at least 30 months after the course of treatment. In yet
another embodiment of the invention, after a course of treatment
with an anti-human CD3 antibody according to the invention, the
increase in a patient's daily insulin requirements by no more than
1%, no more than 5%, no more than 10%, no more than 15%, no more
than 20%, no more than 25%, no more than 30%, no more than 40%, no
more than 45%, no more than 50%, no more than 55%, no more than
60%, no more than 65%, no more than 70%, or no more than 75% of
pretreatment levels is maintained for at least 4 months, at least 6
months, at least 9 months, at least 12 months, at least 18 months,
at least 24 months, or at least 30 months after the course of
treatment and the mean lymphocyte count of the patient is not less
than 800 cells/ml, less than 750 cells/ml, less than 700 cells/ml,
less than 650 cells/ml, less than 600 cells/ml, less than 550
cells/ml, less than 500 cells/ml, less than 400 cells/ml, less than
300 cells/ml or less than 200 cells/ml over the same time
period.
[0244] In yet another embodiment, a human subject having type 1
diabetes, or a human identified as having a predisposition to
developing type 1 diabetes is administered a course of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies to preserve the subject's C-peptide
response or FPIR to MMTT, OGTT, IGTT or two phase glucose clamp
procedure over about 2 weeks, about 1 month, about 2 months, about
4 months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, about 12
months, about 15 months, about 18 months, about 21 months or about
24 months after treatment. In preferred embodiments, the patients
initially have a C-peptide response to MMTT, OGTT, IGTT, or
two-phase glucose clamp procedure (preferably MMTT) resulting in an
area under curve (AUC) of at least 80 pmol/ml/240 min, preferably,
at least 90 pmol/ml/240 min., more preferably at least 100
pmol/ml/240 min., or even at least 110 pmol/ml/240 min. In
preferred embodiments, the patients prior to treatment with an
anti-human CD3 antibody according to the invention have a FPIR of
at least 300 pmol/1, at least 350 pmol/l, at least 400 pmol/1, at
least 450 pmol/1, at least 500 pmol/1, preferably, at least 550
pmol/l, more preferably at least 600 pmol/1, or even at least 700
pmol/l. In another embodiment of the invention, after a course of
treatment with an anti-human CD3 antibody according to the
invention, the C-peptide response or FPIR of the patient to MMTT,
OGTT, IGTT, or two-phase glucose clamp procedure decreases by less
than 1%, less than 5%, less than 10%, less than 20%, less than 30%,
less than 40% or less than 50% of the pretreatment levels. In yet
another embodiment of the invention, after a course of treatment
with an anti-human CD3 antibody according to the invention, the
C-peptide response or FPIR of the patient to MMTT, OGTT, IGTT or
two phase glucose clamp procedure is maintained at at least 99%, at
least 95%, at least 90%, at least 80%, at least 70%, at least 60%,
or at least 50% of pretreatment levels for at least 4 months, at
least 6 months, at least 9 months, at least 12 months, at least 18
months, at least 24 months, or at least 30 months after the course
of treatment. In another embodiment of the invention, after a
course of treatment with an anti-human CD3 antibody according to
the invention, the C-peptide response or FPIR of the patient to
MMTT, OGTT, IGTT or two phase glucose clamp procedure is maintained
at at least 99%, at least 95%, at least 90%, at least 80%, at least
70%, at least 60%, or at least 50% of pretreatment levels for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 18 months, at least 24 months, or at least 30
months after the end of treatment and the mean lymphocyte count of
the patient is not less than 800 cells/ml, less than 750 cells/ml,
less than 700 cells/ml, less than 650 cells/ml, less than 600
cells/ml, less than 550 cells/ml, less than 500 cells/ml, less than
400 cells/ml, less than 300 cells/ml or less than 200 cells/ml over
the same time period.
[0245] In particular embodiments, the invention provides methods of
treatment such that a single round of treatment or round of
treatment every 6 months, every 9 months, every 12 months, every 15
months, every 18 months, or every 24 months with an anti-human CD3
antibody (preferably, without any intervening treatment with
anti-human CD3 antibodies), results in a level of HA1 or HA1c that
is 7% or less, 6.5% or less, 6% or less, 5.5% or less, or 5% or
less 6 months, 9 months, 12 months, 15 months, 18 months, or 24
months after the previous round of treatment or the first round of
treatment. In specific embodiments, after a single round of
treatment or round of treatment every 6 months, every 9 months,
every 12 months, every 15 months, every 18 months, or every 24
months with an anti-human CD3 antibody according to the methods of
the invention (preferably, without any intervening treatment with
anti-human CD3 antibodies), the patients have a C-peptide response
to MMTT, OGTT, IGTT or two-phase glucose clamp procedure
(preferably, MMTT) resulting in an AUC of at least 40 pmol/ml/240
min., 50 pmol/ml/240 min, 60 pmol/ml/240 min, 70 pmol/ml/240 min.,
80 pmol/ml/240 min., preferably, at least 90 pmol/ml/240 min., more
preferably at least 100 pmol/ml/240 min., or even at least 110
pmol/ml/240 min, said response determined 6 months, 9 months, 12
months, 15 months, 18 months, or 24 months after the previous round
of treatment or after the previous round of treatment. In specific
embodiments, after a single round of treatment or round of
treatment every 6 months, every 9 months, every 12 months, every 15
months, every 18 months, or every 24 months with an anti-human CD3
antibody according to the methods of the invention (preferably,
without any intervening treatment with anti-human CD3 antibodies),
the patients have a FPIR of at least 300 pmol/1, at least 400
pmol/1, preferably, at least 500 pmol/1, more preferably at least
600 pmol/1, or even at least 700 pmol/1, said FPIR determined at 6
months, 9 months, 12 months, 15 months, 18 months, or 24 months
after the previous round of treatment or initial round of
treatment.
[0246] In another embodiment, with respect to the treatment of MS,
pharmaceutical compositions comprising one or more CD3 binding
molecules (e.g., one or more anti-CD3 antibodies) are administered
one or more times to prevent or reduce an increase, or slow or
reduce an increase in EDSS score associated with MS in a subject.
In yet another embodiment, one or more pharmaceutical compositions
comprising one or more CD3 binding molecules (e.g., one or more
anti-CD3 antibodies) are administered one or more times to prevent
an increase in the frequency, severity and/or duration of attacks
associated with MS in a subject. In still other embodiments, one or
more pharmaceutical compositions comprising one or more CD3 binding
molecules (e.g., one or more anti-CD3 antibodies) are administered
one or ore times to prevent an increase in number and/or total
volume of lesions, as detected by, e.g., MRI, associated with MS in
a subject. In accordance with these embodiments, the subject's EDSS
score and/or a determination of the frequency, duration and/or
severity of attacks maybe assessed by a qualified medical
practitioner according to methods commonly accepted and well known
in the art. In certain embodiments, the subject has benign MS. In
other embodiments, the subject has RRMS, SPMS, PRMS, or PPMS. In
certain embodiments, one or more pharmaceutical compositions
comprising one or more CD3 binding molecules (e.g., one or more
anti-CD3 antibodies) are administered one or more times to reduce
the incidence, severity and/or duration of a symptom associated
with MS in a subject, wherein said symptoms are described herein or
are known in the art. In certain embodiments, symptoms associated
with MS include, but are not limited to fatigue, disturbances of
vision, disturbances of strength, disturbances of coordination,
disturbances of balance, disturbances of bladder/bowel function,
weakness or paralysis in one or more extremities, tremor in one or
more extremities, muscle spasticity, muscle atropy, dysfunctional
movement, numbness or abnormal sensation in any area, tingling,
facial pain, extremity pain, loss of vision in one or both eyes,
double vision, eye discomfort, uncontrollable rapid eye movements,
decreased coordination, loss of balance, decreased ability to
control small or intricate movements, walking or gait
abnormalities, muscle spasms, dizziness, vertigo, urinary
hesitancy, urinary urgency, increased urinary frequency,
incontinence, decreased memory, decreased spontaneity, decreased
judgment, loss of ability to think abstractly, loss of ability to
generalize, depression, decreased attention span, slurred speech,
difficulty speaking or understanding speech, fatigue, constipation,
hearing loss, and/or positive Babinski's reflex.
[0247] In a specific embodiment anti-CD3 therapy is used for the
treatment of MS in patients that have a disability score according
to the Kurtzke Expanded Disability Scale (EDSS) of 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0 or 9.5 as determined by methods described herein or known to
one of ordinary skill in the art. In another embodiment, after one
or more courses of treatment with an anti-CD3 antibody according to
the invention the EDSS score of the patient increases by not more
than one-half step, not more than one step, not more than one and
one-half steps, not more than two steps, not more than two and
one-half steps, not more than three steps, not more than three and
one-half steps, not more than four steps, not more than four an
one-half steps, not more than more than five steps, not more than
five and one-half steps, not more than six steps, not more than six
and one-half steps, not more than seven steps, not more than seven
and one-half steps, not more than eight steps, or not more than
eight and one-half steps relative to the pretreatment score.
[0248] In yet another embodiment of the invention, after one or
more courses of treatment with an anti-CD3. antibody according to
the invention the EDSS score of the patient is maintained or
increases by not more than one-half step, not more than one step,
not more than one and one-half steps, not more than two steps, not
more than two and one-half steps, not more than three steps, not
more than three and one-half steps, not more than four steps, not
more than four an one-half steps, not more than more than five
steps, not more than five and one-half steps, not more than six
steps, not more than six and one-half steps, not more than seven
steps, not more than seven and one-half steps, not more than eight
steps, or not more than eight and one-half steps relative to the
pretreatment score for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment.
[0249] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the EDSS score of the patient is maintained or increases
by not more than one-half step, not more than one step, not more
than one and one-half steps, not more than two steps, not more than
two and one-half steps, not more than three steps, not more than
three and one-half steps, not more than four steps, not more than
four an one-half steps, not more than more than five steps, not
more than five and one-half steps, not more than six steps, not
more than six and one-half steps, not more than seven steps, not
more than seven and one-half steps, not more than eight steps, or
not more than eight and one-half steps relative to the pretreatment
score for at least 4 months, at least 6 months, at least 9 months,
at least 12 months, at least 15 months, at least 18 months, at
least 24 months, at least 2.5 years or at least 3 years after the
end of treatment and does not reduce the mean lymphocyte count of
the patient to less than 800 cells/ml, less than 750 cells/ml, less
than 700 cells/ml, less than 650 cells/ml, less than 600 cells/ml,
less than 550 cells/ml, less than 500 cells/ml, less than 400
cells/ml, less than 300 cells/ml or 200 cells/ml or less.
[0250] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the EDSS score of the patient is maintained or increases
by not more than one-half step, not more than one step, not more
than one and one-half steps, not more than two steps, not more than
two and one-half steps, not more than three steps, not more than
three and one-half steps, not more than four steps, not more than
four an one-half steps, not more than more than five steps, not
more than five and one-half steps, not more than six steps, not
more than six and one-half steps, not more than seven steps, not
more than seven and one-half steps, not more than eight steps, or
not more than eight and one-half steps relative to the pretreatment
score for at least 4 months, at least 6 months, at least 9 months,
at least 12 months, at least 15 months, at least 18 months, at
least 24 months, at least 2.5 years or at least 3 years after the
end of treatment and does not reduce the mean platelet count of the
patient to less than 100,000,000 platelets/ml, less than 75,000,000
platelets/ml, less than 50,000,000 platelets/ml, less than
25,000,000 platelets/ml, less than 1,000,000 platelets/ml, less
than 750,000 platelets/ml, less than 500,000 platelets/ml, less
than 250,000 platelets/ml, less than 150,000 platelets/ml or
100,000 platelets/ml or less.
[0251] In other embodiments, after one or more courses of treatment
with an anti-CD3 antibody according to the invention the average
incidence, frequency, severity or duration of symptoms and/or
attacks associated with MS in a patient increases by not more than
2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, b5%,
70% or 75% relative to the pretreatment condition. In yet another
embodiment of the invention, after one or more courses of treatment
with an anti-CD3 antibody according to the invention the average
incidence, frequency, severity or duration of symptoms and/or
attacks associated with MS in a patient increases by not more than
2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70% or 75% relative to the pretreatment condition for at least 4
months, at least 6 months, at least 9 months, at least 12 months,
at least 15 months, at least 18 months, at least 24 months, at
least 2.5 years or at least 3 years after the end of treatment.
[0252] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the average frequency, severity or duration of symptoms
and/or attacks associated with MS in a patient increases by not
more than 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70% or 75% relative to the pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean lymphocyte count of the patient to
less than 800 cells/ml, less than 750 cells/ml, less than 700
cells/ml, less than 650 cells/ml, less than 600 cells/ml, less than
550 cells/ml, less than 500 cells/ml, less than 400 cells/ml, less
than 300 cells/ml or 200 cells/ml or less.
[0253] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the average frequency, severity or duration of symptoms
and/or attacks associated with MS in a patient increases by not
more than 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70% or 75% relative to the pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean platelet count of the patient to less
than 100,000,000 platelets/ml, less than 75,000,000 platelets/ml,
less than 50,000,000 platelets/ml, less than 25,000,000
platelets/ml, less than 1,000,000 platelets/ml, less than 750,000
platelets/ml, less than 500,000 platelets/ml, less than 250,000
platelets/ml, less than 150,000 platelets/ml or 100,000
platelets/ml or less.
[0254] In another specific embodiment, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
number and/or total volume of lesions associated with MS as
determined by MRI in a patient increases by not more than 2%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or
75% of the pretreatment condition. In yet another embodiment of the
invention, after one or more courses of treatment with an anti-CD3
antibody according to the invention the number and/or total volume
of lesions associated with MS as determined by MRI in a patient
increases by not more than 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% relative to the
pretreatment condition for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment.
[0255] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the number and/or total volume of lesions associated with
MS as determined by MRI in a patient increases by not more than 2%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%
or 75% relative to the pretreatment condition for at least 4
months, at least 6 months, at least 9 months, at least 12 months,
at least 15 months, at least 18 months, at least 24 months, at
least 2.5 years or at least 3 years after the end of treatment and
does not reduce the mean lymphocyte count of the patient to less
than 800 cells/ml, less than 750 cells/ml, less than 700 cells/ml,
less than 650 cells/ml, less than 600 cells/ml, less than 550
cells/ml, less than 800 cells/ml, less than 400 cells/ml, less than
300 cells/ml or 200 cells/ml or less.
[0256] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the number and/or total volume of lesions associated with
MS as determined by MRI in a patient increases by not more than 2%,
S %, 10%, 1S %, 20%, 2S %, 30%, 3S %, 40%, 4S %, SO %, SS %, 60%,
6S %, 70% or 7S % relative to the pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean platelet count of the patient to less
than 100,000,000 platelets/ml, less than 75,000,000 platelets/ml,
less than 50,000,000 platelets/ml, less than 25,000,000
platelets/ml, less than 1,000,000 platelets/ml, less than 750,000
platelets/ml, less than 500,000 platelets/ml, less than 250,000
platelets/ml, less than 150,000 platelets/ml or 100,000
platelets/ml or less.
[0257] In another specific embodiment, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
Psoriasis Area and Severity Index (PASI) score of a patient having
psoriasis decreases by at least 20%, at least 35%, at least 30%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, or at least
85% relative to the pretreatment condition. In yet another
embodiment of the invention, after one or more courses of treatment
with an anti-CD3 antibody according to the invention the Psoriasis
Area and Severity Index (PAST) score of a patient having psoriasis
decreases by at least 20%, at least 35%, at least 30%, at least
40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, or at least
85% relative to the pretreatment condition for at least 4 months,
at least 6 months, at least 9 months, at least 12 months, at least
15 months, at least 18 months, at least 24 months, at least 2.5
years or at least 3 years after the end of treatment.
[0258] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the Psoriasis Area and Severity Index (PAST) score of a
patient diagnosed with psoriasis decreases by at least 20%, at
least 35%, at least 30%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, or at least 85% relative to the pretreatment
condition for at least 4 months, at least 6 months, at least 9
months, at least 12 months, at least 15 months, at least 18 months,
at least 24 months, at least 2.5 years or at least 3 years after
the end of treatment and does not reduce the mean lymphocyte count
of the patient to less than 800 cells/ml, less than 750 cells/ml,
less than 700 cells/ml, less than 650 cells/ml, less than 600
cells/ml, less than 550 cells/ml, less than 800 cells/ml, less than
400 cells/ml, less than 300 cells/ml or 200 cells/ml or less.
[0259] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the Psoriasis Area and Severity Index (PASI) score of a
patient diagnosed with psoriasis decreases by at least 20%, at
least 35%, at least 30%, at least 40%, at least 45%, at least 50%,
at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, or at least 85% relative to the pretreatment
condition for at least 4 months, at least 6 months, at least 9
months, at least 12 months, at least 15 months, at least 18 months,
at least 24 months, at least 2.5 years or at least 3 years after
the end of treatment and does not reduce the mean platelet count of
the patient to less than 100,000,000 platelets/ml, less than
75,000,000 platelets/ml, less than 50,000,000 platelets/ml, less
than 25,000,000 platelets/ml, less than 1,000,000 platelets/ml,
less than 750,000 platelets/ml, less than 500,000 platelets/ml,
less than 250,000 platelets/ml, less than 150,000 platelets/ml or
100,000 platelets/ml or less.
[0260] In another specific embodiment, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
global assessment score of a patient diagnosed with psoriasis
improves by at least by at least 25%, at least 35%, at least 30%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95% relative to pretreatment
condition. In yet another embodiment of the invention, after one or
more courses of treatment with an anti-CD3 antibody according to
the invention the global assessment score of a patient diagnosed
with psoriasis improves by at least by at least 25%, at least 35%,
at least 30%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, or at least 95% relative to
pretreatment condition for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment.
[0261] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the global assessment score of a patient diagnosed with
psoriasis improves by at least by at least 25%, at least 35%, at
least 30%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% relative to
pretreatment condition for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment and does not reduce the mean lymphocyte
count of the patient to less than 800 cells/ml, less than 750
cells/ml, less than 700 cells/ml, less than 650 cells/ml, less than
600 cells/ml, less than 550 cells/ml, less than 800 cells/ml, less
than 400 cells/ml, less than 300 cells/ml or 200 cells/ml or
less.
[0262] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the global assessment score of a patient diagnosed with
psoriasis improves by at least by at least 25%, at least 35%, at
least 30%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% relative to
pretreatment condition for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment and does not reduce the mean platelet
count of the patient to less than 100,000,000 platelets/ml, less
than 75,000,000 platelets/ml, less than 50,000,000 platelets/ml,
less than 25,000,000 platelets/ml, less than 1,000,000
platelets/ml, less than 750,000 platelets/ml, less than 500,000
platelets/ml, less than 250,000 platelets/ml, less than 150,000
platelets/ml or 100,000 platelets/ml or less.
[0263] In another specific embodiment, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
subject's condition as assessed by any arthritis severity scale
known in the art (e.g., RASS) improves by at least 25%, at least
35%, at least 30%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
100% relative to pretreatment condition. In yet another embodiment
of the invention, after one or more courses of treatment with an
anti-CD3 antibody according to the invention the subject's
condition as assessed by any arthritis severity scale known in the
art (e.g., RASS) improves by at least 25%, at least 35%, at least
30%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or at least 100% relative
to pretreatment condition for at least 4 months, at least 6 months,
at least 9 months, at least 12 months, at least 15 months, at least
18 months, at least 24 months, at least 2.5 years or at least 3
years after the end of treatment.
[0264] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the subject's condition as assessed by any arthritis
severity scale known in the art (e.g., RASS) improves by at least
25%, at least 35%, at least 30%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 100% relative to pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean lymphocyte count of the patient to
less than 800 cells/ml, less than 750 cells/ml, less than 700
cells/ml, less than 650 cells/ml, less than 600 cells/ml, less than
550 cells/ml, less than 800 cells/ml, less than 400 cells/ml, less
than 300 cells/ml or 200 cells/ml or less.
[0265] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the subject's condition as assessed by any arthritis
severity scale known in the art (e.g., RASS) improves by at least
25%, at least 35%, at least 30%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 100% relative to pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean platelet count of the patient to less
than 100,000,000 platelets/ml, less than 75,000,000 platelets/ml,
less than 50,000,000 platelets/ml, less than 25,000,000
platelets/ml, less than 1,000,000 platelets/ml, less than 750,000
platelets/ml, less than 500,000 platelets/ml, less than 250,000
platelets/ml, less than 150,000 platelets/ml or 100,000
platelets/ml or less.
[0266] In another specific embodiment, after one or more courses of
treatment with an anti-CD3 antibody according to the invention the
absolute number, or proportion, of the subject's autoreactive CTLs
as determined by immunospot assay (e.g., ELISPOT) decreases by at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least
100% relative to pretreatment condition. In yet another embodiment
of the invention, after one or more courses of treatment with an
anti-CD3 antibody according to the invention the absolute number,
or proportion, of the subject's autoreactive CTLs as determined by
immunospot assay (e.g., ELISPOT) decreases by at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 100% relative to
pretreatment condition for at least 4 months, at least 6 months, at
least 9 months, at least 12 months, at least 15 months, at least 18
months, at least 24 months, at least 2.5 years or at least 3 years
after the end of treatment.
[0267] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the absolute number, or proportion, of the subject's
autoreactive CTLs as determined by immunospot assay (e.g., ELISPOT)
decreases by at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 100% relative to pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean lymphocyte count of the patient to
less than 800 cells/ml, less than 750 cells/ml, less than 700
cells/ml, less than 650 cells/ml, less than 600 cells/ml, less than
550 cells/ml, less than 800 cells/ml, less than 400 cells/ml, less
than 300 cells/ml or 200 cells/ml or less.
[0268] In another embodiment of the invention, after one or more
courses of treatment with an anti-CD3 antibody according to the
invention the absolute number, or proportion, of the subject's
autoreactive CTLs as determined by immunospot assay (e.g., ELISPOT)
decreases by at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 100% relative to pretreatment condition for at
least 4 months, at least 6 months, at least 9 months, at least 12
months, at least 15 months, at least 18 months, at least 24 months,
at least 2.5 years or at least 3 years after the end of treatment
and does not reduce the mean platelet count of the patient to less
than 100,000,000 platelets/ml, less than 75,000,000 platelets/ml,
less than 50,000,000 platelets/ml, less than 25,000,000
platelets/ml, less than 1,000,000 platelets/ml, less than 750,000
platelets/ml, less than 500,000 platelets/ml, less than 250,000
platelets/ml, less than 150,000 platelets/ml or 100,000
platelets/ml or less.
[0269] In preferred embodiments, the anti-human CD3 antibodies are
administered parenterally, for example, intravenously,
intramuscularly or subcutaneously, or, alternatively, are
administered orally. The anti-human CD3 antibodies may also be
administered as a sustained release formulation.
[0270] In a specific embodiment, the mean absolute lymphocyte count
in a subject with an autoimmune disorder is assessed before and/or
after the administration of one or more doses of a prophylactically
or therapeutically effective amount of one or more anti-human CD3
antibodies to determine whether one or more subsequent doses of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies should be administered to said subject.
In another embodiment, the mean absolute lymphocyte count in a
subject with an autoimmune disorder is assessed before and/or after
the administration of one or more doses of a prophylactically or
therapeutically effective amount of one or more anti-human CD3
antibodies to determine whether one or more subsequent doses of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies should be administered to said subject.
Preferably, a subsequent dose of a prophylactically or
therapeutically effective amount of one or more anti-human CD3
antibodies is not administered to said subject if the lymphocyte
count is less than 800 cells/mm.sup.3, less than 750
cells/mm.sup.3, less than 700 cells/mm.sup.3, less than 650
cells/mm.sup.3, less than 600 cells/mm.sup.3, less than 500
cells/mm.sup.3, less than 400 cells/mm.sup.3 or less than 300
cells/mm.sup.3
[0271] In another embodiment, the mean absolute lymphocyte count in
a subject with an autoimmune disorder is determined prior to the
administration of a first dose of a prophylactic ally or
therapeutically effective amount of one or more anti-human CD3
antibodies and the mean absolute lymphocyte count is monitored
prior to the administration of one or more subsequent doses of a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies. Preferably, the mean absolute lymphocyte
count in the subject is at least 900 cells/mm.sup.3, preferably at
least 950 cells/mm.sup.3, at least 1000 cells/mm.sup.3, at least
1050 cells/mm.sup.3, at least 1100 cells/mm.sup.3, at least 1200
cells/mm.sup.3, or at least 1250 cells/mm.sup.3 prior to the
administration of a first dose of one or more anti-human CD3
antibodies.
[0272] In another embodiment, a mean absolute lymphocyte count of
approximately 700 cells/ml to approximately 1200 cells/ml,
approximately 700 cells/ml to approximately 1100 cells/ml,
approximately 700 cells/ml to approximately 1000 cells/ml,
approximately 700 to approximately 900 cells/ml, approximately 750
cells/ml to approximately 1200 cells/ml, approximately 750 cells/ml
to approximately 1100 cells/ml, approximately 750 cells/ml to
approximately 1000 cells/ml, approximately 750 cells/ml to
approximately 900 cells/ml, approximately 800 cells/ml to
approximately 1200 cells/ml, approximately 800 cells/ml to
approximately 1100 cells/ml, approximately 800 cells/ml to
approximately 1000 cells/ml, approximately 900 cells/ml to
approximately 1200 cells/ml, approximately 900 cells/ml to
approximately 1100 cells/ml, approximately 900 cells/ml to
approximately 1000 cells/ml, or approximately 1000 cells to
approximately 1200 cells/ml is maintained in a subject having type
1 diabetes disorder by administering one or more doses of a
prophylactic ally or therapeutically effective amount of one or
more anti-human CD3 antibodies. In another embodiment, a mean
absolute lymphocyte count of approximately 700 cells/ml to below
1000 cells/ml is maintained in a subject having an autoimmune
disorder by administering one or more doses of a prophylactic ally
or therapeutically effective amount of one or more anti-human CD3
antibodies.
[0273] In a specific embodiment, the administration of one or more
doses or a dosage regimen of a prophylactically or therapeutically
effective amount of one or more anti-human CD3 antibodies does not
induce or reduces relative to other immunosuppressive agents one or
more of the following unwanted or adverse effects: vital sign
abnormalities (fever, tachycardia, bardycardia, hypertension,
hypotension), hematological events (anemia, lymphopenia,
leukopenia, thrombocytopenia), headache, chills, dizziness, nausea,
asthenia, back pain, chest pain (chest pressure), diarrhea,
myalgia, pain, pruritus, psoriasis, rhinitis, sweating, injection
site reaction, vasodilatation, an increased risk of opportunistic
infection, activation of Epstein Barr Virus, apoptosis of T cells
and an increased risk of developing certain types of cancer. In
another specific embodiment, the administration of one or more
doses of a prophylactically or therapeutically effective amount of
one or more anti-human CD3 antibodies does not induce or reduces
relative to other immunosuppressive agents one or more of the
following unwanted or adverse effects: vital sign abnormalities
(fever, tachycardia, bardycardia, hypertension, hypotension),
hematological events (anemia, lymphopenia, leukopenia,
thrombocytopenia), headache, chills, dizziness, nausea, asthenia,
back pain, chest pain (chest pressure), diarrhea, myalgia, pain,
pruritus, psoriasis, rhinitis, sweating, injection site reaction,
vasodilatation, an increased risk of opportunistic infection,
Epstein Barr Virus activation, apoptosis of T cells, and an
increased risk of developing certain types of cancer.
[0274] In accordance with the invention, the dose or dosage regimen
comprising a prophylactically or therapeutically effective amount
of one or more anti-CD3 antibodies for the treatment of an
autoimmune disorder may be repeated at 1 month, 2 months, 4 months,
6 months, 8 months, 12 months, 15 months, 18 months or 24 months or
longer after the initial or previous dose or dosage regimen
comprising a prophylactically or therapeutically effective amount
of one or more anti-CD3 antibodies. The repeat dose or dosage
regimen may be administered as a matter of course, when symptoms
associated with said autoimmune disorder recur after an improvement
following the initial or previous dose or dosage regimen, or when
symptoms associated with said autoimmune disorder do not improve
after the initial dose or dosage regimen of anti-CD3 antibodies
according to methods of the invention. With respect to diabetes, a
repeat dose or dosage regimen comprising a prophylactically or
therapeutically effective amount of one or more anti-CD3 antibodies
may be administered to a subject when, for example, the subject's
average daily insulin use at 1 month, 2 months, 4 months, 6 months,
8 months, 12 months, 15 months, 18 months or 24 months or longer
after initial or previous treatment with anti-CD3 antibodies does
not decrease by at least 5%, at least 10%, at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80% or at least 90% compared to pre-treatment levels.
Alternatively, with respect to diabetes, a repeat dose or--dosage
regimen comprising a prophylactically or therapeutically effective
amount of one or more anti-CD3 antibodies may be administered to a
subject when, for example, the subject's HA 1 or HA 1 C levels at 1
month, 2 months, 4 months, 6 months, 8 months, 12 months, 15
months, 18 months or 24 months or longer after initial or previous
treatment with anti-CD3 antibodies do not decrease by at least 5%,
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80% or at least 90%
compared to pre-treatment levels. In another embodiment, with
respect to diabetes, a repeat dose or dosage regimen comprising a
prophylactically or therapeutically effective amount of one or more
anti-CD3 antibodies may be administered to a subject when, for
example, the subject's C-peptide response at 1 month, 2 months, 4
months, 6 months, 8 months, 12 months, 15 months, 18 months or 24
months or longer after initial or previous treatment with anti-CD3
antibodies decreases by more than 5%, more than 10%, more than 20%,
more than 30%, more than 40%, more than 50%, more than 60%, more
than 70%, more than 80% or more than 90% compared to pre-treatment
levels.
[0275] 5.2.8 Combinatorial Therapy
[0276] The present invention provides compositions comprising one
or more anti-human CD3 antibody and one or more prophylactic or
therapeutic agents other than anti-human CD3 antibodies, and
methods for preventing, treating, delaying the onset of, slowing
the progression of or ameliorating one or more symptoms associated
with an autoimmune disorder, e.g., an inflammatory autoimmune
disorder, in a subject in need thereof comprising administering to
said subject one or more of said compositions. Therapeutic or
prophylactic agents include, but are not limited to, peptides,
polypeptides, fusion proteins, nucleic acid molecules, small
molecules, mimetic agents, synthetic drugs, inorganic molecules,
and organic molecules. Any agent which is known to be useful, or
which has been used or is currently being used for the prevention,
treatment or amelioration of one or more symptoms associated with
an autoimmune disorder, particularly type 1 diabetes, can be used
in combination with an anti-human CD3 antibody in accordance with
the invention described herein. Examples of such agents include,
but are not limited to antibody fragments, GLP-1 analogs or
derivatives, GLP-1 agonists (e.g. exendin-4; exentatide), amylin
analogs or dericatives, insulin, dermatological agents for rashes
and swellings (e.g., phototherapy (i.e., ultraviolet B radiation),
photochemotherapy (e.g., PUVA) and topical agents such as
emollients, salicylic acid, coal tar, topical steroids, topical
corticosteroids, topical vitamin D3 analogs (e.g., calcipotriene),
tazarotene, and topical retinoids), anti-inflammatory agents (e.g.,
corticosteroids (e.g., prednisone and hydrocortisone},
glucocorticoids, steroids, non-steriodal anti-inflammatory drugs
(e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors),
beta-agonists, anticholinergic agents and methyl xanthines},
immunomodulatory agents (e.g., small organic molecules, a T cell
receptor modulators, cytokine receptor modulators, T cell depleting
agents, cytokine antagonists, monokine antagonists, lymphocyte
inhibitors, or anti-cancer agents), gold injections,
sulphasalazine, penicillamine, anti-angiogenic agents (e.g.,
angiostatin, TNF-.alpha. antagonists (e.g., anti-TNF.alpha.
antibodies), and endostatin), dapsone, psoralens (e.g., methoxalen
and trioxsalen), anti-malarial agents (e.g., hydroxychloroquine),
anti-viral agents, and antibiotics (e.g., erythomycin and
penicillin). Any immunomodulatory agent well-known to one of skill
in the art may also be used in the methods and compositions of the
invention Immunomodulatory agents can affect one or more or all
aspects of the immune response in a subject. Aspects of the immune
response include, but are not limited to, the inflammatory
response, the complement cascade, leukocyte and lymphocyte
differentiation, proliferation, and/or effector function, monocyte
and/or basophil counts, and the cellular communication among cells
of the immune system. In certain embodiments of the invention, an
immunomodulatory agent modulates one aspect of the immune response.
In other embodiments, an immunomodulatory agent modulates more than
one aspect of the immune response. In a preferred embodiment of the
invention, the administration of an immunomodulatory agent to a
subject inhibits or reduces one or more aspects of the subject's
immune response capabilities. In a specific embodiment of the
invention, the immunomodulatory agent inhibits or suppresses the
immune response in a subject. In accordance with the invention, an
immunomodulatory agent is not an anti-human CD3 antibody. In
certain embodiments, an immunomodulatory agent is not an
anti-inflammatory agent. In other embodiments, an immunomodulatory
agent is not a CD3 binding molecule. In yet other embodiments, an
immunomodulatory agent is not OKT3 or a derivative thereof.
[0277] An immunomodulatory agent may be selected to interfere with
the interactions between the T helper subsets (TH1 or TH2) and B
cells to inhibit neutralizing antibody formation. An
immunomodulatory agent may be selected to inhibit the interaction
between TH1 cells and CTLs to reduce the occurrence of CTL-mediated
killing. An immunomodulatory agent may be selected to alter (e.g.,
inhibit or suppress) the proliferation, differentiation, activity
and/or function of the CD4.sup.+ and/or CD8.sup.+ T cells. For
example, antibodies specific for T cells can be used as
immunomodulatory agents to deplete, or alter the proliferation,
differentiation, activity and/or function of CD4.sup.+ and/or
CD8.sup.+ T cells.
[0278] In specific embodiments, the anti-human CD3 binding molecule
is co-administered with a cytokine antagonist. In other
embodiments, the anti-human CD3 binding molecule is co-administered
with an anti-IL-2 antibody, such as, for example, daclizumab,
basiliximab or MT204 (Micromet) or other IL-2 inhibitor, such as
but not limited to rapamycin, cyclosporine, or tacrolimus.
[0279] In other embodiments, the anti-human CD3 binding molecule is
administered in conjunction with an antigen targeted by anti-islet
cell antibodies such as, but not limited to GAD (such as GAD 65),
insulin, IA-2, ICA512 or other antigen against which autoantibodies
are found in type 1 diabetes patients. Such co-administration may
lead to tolerance to the islet cell antigens.
[0280] In a preferred embodiment, proteins, polypeptides or
peptides (including antibodies) that are utilized as prophylactic,
therapeutic or immunomodulatory agents are derived from the same
species as the recipient of the proteins, polypeptides or peptides
so as to reduce the likelihood of an immune response to those
proteins, polypeptides or peptides. In another preferred
embodiment, when the subject is a human, the proteins,
polypeptides, or peptides that are utilized as immunomodulatory
agents are human or humanized.
[0281] In accordance with the invention, one or more prophylactic,
therapeutic or immunomodulatory agents are administered to a
subject with an inflammatory or autoimmune disease prior to,
subsequent to, or concomitantly with the therapeutic and/or
prophylactic agents of the invention. Preferably, one or more
prophylactic, therapeutic or immunomodulatory agents are
administered to a subject with an inflammatory or autoimmune
disease to reduce or inhibit one or more symptoms of the disease or
aspects of the immune response as necessary. Any technique
well-known to one skilled in the art can be used to measure one or
more aspects of the immune response in a particular subject, and
thereby determine when it is necessary to administer an
immunomodulatory agent to said subject. In a preferred embodiment,
an absolute lymphocyte count of approximately 500 cells/mm.sup.3,
preferably 600 cells/mm.sup.3, more 700 cells/mm.sup.3, and most
preferably 800 cells/mm.sup.3 is maintained in a subject. In
another preferred embodiment, a subject with an autoimmune or
inflammatory disorder is not administered an immunomodulatory agent
if their absolute lymphocyte count is 500 cells/mm.sup.3 or less,
S50 cells/mm.sup.3 or less, 600 cells/mm.sup.3 or less, 650
cells/mm.sup.3 or less, 700 cells/mm.sup.3 or less, 750
cells/mm.sup.3 or less, or 800 cells/mm.sup.3 or less.
[0282] In a preferred embodiment, one or more prophylactic,
therapeutic or immunomodulatory agents are administered to a
subject with an inflammatory or autoimmune disease so as to
transiently reduce or inhibit one or more aspects of the disease or
of the immune response. Such a transient inhibition or reduction of
one or more aspects of the disease or of the immune system can last
for hours, days, weeks, or months. Preferably, the transient
inhibition or reduction in one or more aspects of the disease or of
the immune response last for a few hours (e.g., 2 hours, 4 hours, 6
hours, 8 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours,
36 hours, or 48 hours), a few days (e.g., 3 days, 4 days, 5 days, 6
days, 7 days, or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks,
5 weeks or 6 weeks). The transient reduction or inhibition of one
or more aspects of the disease or of the immune response enhances
the prophylactic and/or therapeutic capabilities of an anti-CD3
antibody.
[0283] In accordance with the invention, one or more prophylactic,
therapeutic or immunomodulatory agents are administered to a
subject with type 1 diabetes, or a predisposition thereto, prior
to, subsequent to, or concomitantly with the therapeutic and/or
prophylactic agents of the invention. Such methods may be employed
to treat, prevent, delay the onset of, slow the progression of or
ameliorate one or more symptoms of type 1 diabetes.
[0284] In specific embodiments, the present invention provides a
method for preventing, treating, managing, delaying the onset of,
slowing the progression of, or ameliorating one or more symptoms of
type 1 diabetes, said method comprising administering to said
subject a prophylactically or therapeutically effective amount of
one or more anti-human CD3 antibodies and a prophylactically or
therapeutically effective amount of insulin. In one embodiment, the
present invention provides a method for preventing, treating,
managing, delaying the onset of, slowing the progression of, or
ameliorating one or more symptoms of type 1 diabetes, said method
comprising administering to said subject a prophylactically or
therapeutically effective amount of one or more anti-human CD3
antibodies and a prophylactically or therapeutically effective
amount of GLP1 or GLP1 analog. In one embodiment, the present
invention provides a method for preventing, treating, managing,
delaying the onset of, slowing the progression of, or ameliorating
one or more symptoms of type 1 diabetes, said method comprising
administering to said subject a prophylactically or therapeutically
effective amount of one or more anti-human CD3 antibodies and a
prophylactically or therapeutically effective amount of exendin-4
or analog thereof. In one embodiment, the present invention
provides a method for preventing, treating, managing, delaying the
onset of, slowing the progression of, or ameliorating one or more
symptoms of type 1 diabetes, said method comprising administering
to said subject a prophylactically or therapeutically effective
amount of one or more anti-human CD3 antibodies and a
prophylactically or therapeutically effective amount of amylin or
an analog thereof. In another embodiment, the present invention
provides a method for preventing, treating, managing, delaying the
onset of, slowing the progression of, or ameliorating one or more
symptoms of type 1 diabetes, said method comprising administering
to said subject a prophylactically or therapeutically effective
amount of the humanized anti-human CD3 antibody OKT3 and a
prophylactically or therapeutically effective amount of
insulin.
[0285] Nucleic acid molecules encoding proteins, polypeptides, or
peptides with prophylactic, therapeutic or immunomodulatory
activity or proteins, polypeptides, or peptides with prophylactic,
therapeutic or immunomodulatory activity can be administered to a
subject with an autoimmune disorder in accordance with the methods
of the invention. Further, nucleic acid molecules encoding
derivatives, analogs, fragments or variants of proteins,
polypeptides, or peptides with prophylactic, therapeutic or
immunomodulatory activity, or derivatives, analogs, fragments or
variants of proteins, polypeptides, or peptides with prophylactic,
therapeutic or immunomodulatory activity can be administered to a
subject in accordance with the methods of the invention.
Preferably, such derivatives, analogs, variants and fragments
retain the prophylactic, therapeutic or immunomodulatory activity
of the full-length wild-type protein, polypeptide, or peptide.
[0286] Proteins, polypeptides, or peptides that can be used as
prophylactic, therapeutic or immunomodulatory agents can be
produced by any technique well-known in the art or described
herein. See, e.g., Chapter 16 Ausubel et al. (eds.), 1999, Short
Protocols in Molecular Biology, Fourth Edition, John Wiley &
Sons, NY, which describes methods of producing proteins,
polypeptides, or peptides, and which is incorporated herein by
reference in its entirety. Antibodies which can be used as
prophylactic, therapeutic or immunomodulatory agents can be
produced by, e.g., methods described in U.S. Pat. No. 6,245,527 and
in Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1988, which are
incorporated herein by reference in their entirety. Preferably,
agents that are commercially available and known to function as
prophylactic, therapeutic or immunomodulatory agents are used in
the compositions and methods of the invention. The prophylactic,
therapeutic or immunomodulatory activity of an agent can be
determined in vitro and/or in vivo by any technique well-known to
one skilled in the art, including, e.g., by CTL assays,
proliferation assays, and immunoassays (e.g. ELISAs) for the
expression of particular proteins such as co-stimulatory molecules
and cytokines.
[0287] The combination of one or more anti-human CD3 antibodies and
one or more prophylactic or therapeutic agents other than
anti-human CD3 antibodies produces a better prophylactic or
therapeutic effect in a subject than either treatment alone. In
certain embodiments, the combination of an anti-human CD3 antibody
and a prophylactic or therapeutic agent other than an anti-human
CD3 antibody achieves a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% better prophylactic
or therapeutic effect in a subject with the autoimmune disorder, or
predisposition thereto, than either treatment alone. In particular
embodiments, the combination of one or more anti-CD3 antibodies and
a prophylactic or therapeutic agent other than an anti-CD3 antibody
achieves a 20%, preferably a 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% greater reduction in
the inflammation of a particular organ, tissue or joint in a
subject with an inflammatory disorder or an autoimmune disorder
which is associated with inflammation than either treatment alone.
In other embodiments, the combination of one or more anti-CD3
antibodies and one or more prophylactic or therapeutic agents other
than anti-CD3 antibodies has an a more than additive effect or
synergistic effect in a subject with an autoimmune or inflammatory
disorder.
[0288] The combination therapies of the invention enable lower
dosages of anti-human CD3 antibodies and/or less frequent
administration of anti-human CD3 antibodies to a subject with an
autoimmune disorder to achieve a prophylactic or therapeutic
effect. The combination therapies of the invention enable lower
dosages of the prophylactic or therapeutic agents utilized in
conjunction with anti-human CD3 antibodies and/or less frequent
administration of such prophylactic or therapeutic agents to
achieve a prophylactic or therapeutic effect.
[0289] The prophylactic or therapeutic agents of the combination
therapies of the present invention can be administered
concomitantly, concurrently or sequentially. The prophylactic or
therapeutic agents of the combination therapies of the present
invention can also be cyclically administered. Cycling therapy
involves the administration of a first prophylactic or therapeutic
agent for a period of time, followed by the administration of a
second prophylactic or therapeutic agent for a period of time and
repeating this sequential administration, i.e., the cycle, in order
to reduce the development of resistance to one of the agents, to
avoid or reduce the side effects of one of the agents, and/or to
improve the efficacy of the treatment.
[0290] 5.2.8.1 Methods of Use of Combination Therapy
[0291] In a specific embodiment, the present invention provides a
method for preventing, treating, managing, or ameliorating one or
more symptoms associated with an autoimmune or inflammatory
disorder in a subject, said method comprising administering to said
subject one or more anti-CD3 antibodies and one or more
prophylactic or therapeutic agents other than anti-CD3 antibodies.
In a preferred embodiment, the present invention provides a method
for preventing, treating, managing, or ameliorating one or more
symptoms associated with an autoimmune or inflammatory disorder in
a subject, said method comprising administering to said subject one
or more anti-CD3 antibodies and one or more prophylactic or
therapeutic agents other than anti-CD3 antibodies, wherein at least
one of the anti-CD3 antibodies is a humanized OKT3.
[0292] The present invention provides methods of preventing,
treating, managing or ameliorating one or more symptoms associated
with an autoimmune or inflammatory disorder in a subject, said
methods comprising administering to said subject one or more
anti-CD3 antibodies and one or more prophylactic, therapeutic or
immunomodulatory agents. Preferably, the immunomodulatory agents
are not administered to a subject with an autoimmune or
inflammatory disorder whose absolute lymphocyte count is less than
500 cells/mm.sup.3, less than 550 cells/mm.sup.3, less than 600
cellslmm.sup.3, less than 650 cellslmm.sup.3, less than 700
cells/mm.sup.3, less than 750 cells/mm.sup.3, less than 800
cells/mm.sup.3, less than 850 cells/mm.sup.3 or less than 900
cells/mm.sup.3. Thus, in a preferred embodiment, prior to or
subsequent to the administration of one or more dosages of one or
more immunomodulatory agents to a subject with an autoimmune or
inflammatory disorder, the absolute lymphocyte count of said
subject is determined by techniques well-known to one of skill in
the art, including, e.g., flow cytometry or trypan blue counts.
[0293] In one embodiment, the present invention provides a method
for preventing, treating, managing or ameliorating one or more
symptoms associated with diabetes, said method comprising
administering to said subject a prophylactically or therapeutically
effective amount of one or more anti-CD3 antibodies and a
prophylactically or therapeutically effective amount of insulin. In
one embodiment, the present invention provides a method for
preventing, treating, managing or ameliorating one or more symptoms
associated with diabetes, said method comprising administering to
said subject a prophylactically or therapeutically effective amount
of one or more anti-CD3 antibodies and a prophylactically or
therapeutically effective amount of GLP 1 or GLP 1 analog. In one
embodiment, the present invention provides a method for preventing,
treating, managing or ameliorating one or more symptoms associated
with diabetes, said method comprising administering to said subject
a prophylactically or therapeutically effective amount of one or
more anti-CD3 antibodies and a prophylactically or therapeutically
effective amount of exendin-4 or analog thereof. In one embodiment,
the present invention provides a method for preventing, treating,
managing or ameliorating one or more symptoms associated with
diabetes, said method comprising administering to said subject a
prophylactically or therapeutically effective amount of one or more
anti-CD3 antibodies and a prophylactically or therapeutically
effective amount of amylin or an analog thereof. In another
embodiment, the present invention provides a method for preventing,
treating, managing or ameliorating one or more symptoms associated
with diabetes, said method comprising administering to said subject
a prophylactically or therapeutically effective amount of the
humanized anti-CD3 antibody OKT3 and a prophylactically or
therapeutically effective amount of insulin. In another embodiment,
the present invention provides a method for preventing, treating,
managing or ameliorating one or more symptoms associated with
psoriasis, said method comprising administering to said subject a
prophylactically or therapeutically effective amount of one or more
anti-CD3 antibodies and a prophylactically or therapeutically
effective amount of methotrexate. In another embodiment, the
present invention provides a method for preventing, treating,
managing or ameliorating one or more symptoms associated with
psoriasis in a subject, said method comprising administering to
said subject a prophylactically or therapeutically effective amount
of the humanized anti-CD3 antibody OKT3 and a prophylactically or
therapeutically effective amount of methotrexate.
[0294] 5.3 Pharmaceutical Compositions
[0295] The present invention provides compositions for the
treatment, prophylaxis, and amelioration of one or more symptoms
associated with an autoimmune disorder. In a specific embodiment, a
composition comprises one or more anti-human CD3 antibodies. In
another embodiment, a composition comprises one or more nucleic
acid molecules encoding the heavy and light chains of one or more
anti-human CD3 antibodies.
[0296] In a specific embodiment, a composition comprises an
anti-human CD3 antibody, wherein said anti-human CD3 antibody is a
human or humanized monoclonal antibody, preferably modified to
reduce binding of the Fc domain to Fc receptors and, thereby,
reduce toxicity of the antibody. In yet another preferred
embodiment, a composition comprises humanized OKT3, an analog,
derivative, fragment thereof that immunospecifically binds to CD3
polypeptides, preferably OKT3.gamma.1(ala-ala), but may also
include ChAglyCD3 (TRX4.TM.), or HUM291 (visilizumab;
NUVION.TM.)
[0297] In a preferred embodiment, a composition of the invention is
a pharmaceutical composition. Such compositions comprise a
prophylactically or therapeutically effective amount of one or more
anti-human CD3 antibodies, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant (e.g., Freund's adjuvant (complete and incomplete)),
excipient, or vehicle with which the therapeutic is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like (See, for example, Handbook of Pharmaceutical Excipients,
Arthur H. Kibbe (ed., 2000, which is incorporated by reference
herein in its entirety), Am. Pharmaceutical Association,
Washington, D.C. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders, sustained
release formulations and the like. Oral formulation can include
standard carriers such as pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc. Examples of suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin. Such compositions will contain a prophylactically or
therapeutically effective amount of a prophylactic or therapeutic
agent preferably in purified form, together with a suitable amount
of carrier so as to provide the form for proper administration to
the patient. The formulation should suit the mode of
administration. In a preferred embodiment, the pharmaceutical
compositions are sterile and in suitable form for administration to
a subject, preferably an animal subject, more preferably a
mammalian subject, and most preferably a human subject.
[0298] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion, by injection, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. Preferably, when administering an anti-human
CD3 antibody, care must be taken to use materials to which the
anti-human CD3 antibody does not absorb.
[0299] In another embodiment, the composition can be delivered in a
vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3
17-327; see generally ibid.).
[0300] In yet another embodiment, the composition can be delivered
in a controlled release or sustained release system. In one
embodiment, a pump may be used to achieve controlled or sustained
release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.
Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al.,
1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric
materials can be used to achieve controlled or sustained release of
the antibodies of the invention or fragments thereof (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253. Examples of polymers
used in sustained release formulations include, but are not limited
to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a
preferred embodiment, the polymer used in a sustained release
formulation is inert, free of leachable impurities, stable on
storage, sterile, and biodegradable. In yet another embodiment, a
controlled or sustained release system can be placed in proximity
of the therapeutic target, i.e., the lungs, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984)).
[0301] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more antibodies of the invention or
fragments thereof. See, e.g., U.S. Pat. No. 4,526,938; PCT
publication WO 91/05548; PCT publication WO 96/20698; Ning et al.,
1996, Radiotherapy & Oncology 39:179-189; Song et al., 1995,
PDA Journal of Pharmaceutical Science & Technology 50:372-397;
Cleek et al., 1997, Pro. Intl. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, Proc. Intl. Symp. Control Bioact.
Mater. 24:759-760, each of which is incorporated herein by
reference in its entirety.
[0302] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), intranasal, transdermal (topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal or topical
administration to human beings. In a preferred embodiment, a
pharmaceutical composition is formulated in accordance with routine
procedures for subcutaneous administration to human beings.
Typically, compositions for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lignocamne to ease pain at the site of the
injection.
[0303] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of, e.g.,
an ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 4.sup.th ed., Lea
& Febiger, Philadelphia, Pa. (1985). For non-sprayable topical
dosage forms, viscous to semi-solid or solid forms comprising a
carrier or one or more excipients compatible with topical
application and having a dynamic viscosity preferably greater than
water are typically employed. Suitable formulations include,
without limitation, solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, and the like, which are, if
desired, sterilized or mixed with auxiliary agents (e.g.,
preservatives, stabilizers, wetting agents, buffers, or salts) for
influencing various properties, such as, for example, osmotic
pressure. Other suitable topical dosage forms include sprayable
aerosol preparations wherein the active ingredient, preferably in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon), or in a squeeze bottle. Moisturizers or humectants
can also be added to pharmaceutical compositions and dosage forms
if desired. Examples of such additional ingredients are well-known
in the art.
[0304] If the compositions of the invention are to be administered
intranasally, the compositions can be formulated in an aerosol
form, spray, mist or in the form of drops. In particular,
prophylactic or therapeutic agents for use according to the present
invention can be conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, e.g., gelatin for use
in an inhaler or insufflator may be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or
starch.
[0305] If the compositions of the invention are to be administered
orally, the compositions can be formulated orally in the form of,
e.g., tablets, capsules, cachets, gelcaps, solutions, suspensions
and the like. Tablets or capsules can be prepared by conventional
means with pharmaceutically acceptable excipients such as binding
agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated for slow release, controlled release or
sustained release of a prophylactic or therapeutic agent(s).
[0306] The compositions of the invention may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0307] In specific embodiments, the invention provides dosage forms
that permit administration of the anti-human CD3 antibodies
continuously over a period of hours or days (e.g., associated with
a pump or other device for such delivery), for example, over a
period of 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10
hours, 12 hours. 16 hours, 20 hours, 24 hours, 30 hours, 36 hours,
4 days, 5 days, 7 days, 10 days or 14 days. In other specific
embodiments, the invention provides dosage forms that permit
administration of a continuously increasing dose, for example,
increasing from 51 .mu.g/m.sup.2/day to 826 .mu.g/m.sup.2/day over
a period of 24 hours, 30 hours, 36 hours, 4 days, 5 days, 7 days,
10 days or 14 days.
[0308] The compositions of the invention may also be formulated in
rectal compositions such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0309] In addition to the formulations described previously, the
compositions of the invention may also be formulated as a depot
preparation. Such long acting formulations may be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, the compositions may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0310] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0311] Generally, the ingredients of compositions of the invention
are supplied either separately or mixed together in unit dosage
form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0312] In particular, the invention provides that one or more
anti-human CD3 antibodies, or pharmaceutical compositions of the
invention is packaged in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of the agent. In one
embodiment, one or more of the anti-human CD3 antibodies, or
pharmaceutical compositions of the invention is supplied as a dry
sterilized lyophilized powder or water free concentrate in a
hermetically sealed container and can be reconstituted, e.g., with
water or saline to the appropriate concentration for administration
to a subject. Preferably, one or more of the anti-human CD3
antibodies, or pharmaceutical compositions of the invention is
supplied as a dry sterile lyophilized powder in a hermetically
sealed container at a unit dosage of at least 5 mg, more preferably
at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at
least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.
The lyophilized prophylactic or therapeutic agents, or
pharmaceutical compositions of the invention should be stored at
between 2 and 8.degree. C. in its original container and the
prophylactic or therapeutic agents, or pharmaceutical compositions
of the invention should be administered within 1 week, preferably
within 5 days, within 72 hours, within 48 hours, within 24 hours,
within 12 hours, within 6 hours, within 5 hours, within 3 hours, or
within 1 hour after being reconstituted. In an alternative
embodiment, one or more of the anti-human CD3 antibodies, or
pharmaceutical compositions of the invention is supplied in liquid
form in a hermetically sealed container indicating the quantity and
concentration of the agent. Preferably, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 .mu.g/ml, more preferably at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least 2.5 .mu.g/ml, at least 5
.mu.g/ml, at least 8 .mu.g/ml, at least 10 .mu.g/ml, at least 15
mg/kg, at least 25 .mu.g/ml, at least 50 mg/ml, at least 75
.mu.g/ml or at least 100 .mu.g/ml. The liquid form should be stored
at between 2.degree. C. and 8.degree. C. in its original
container.
[0313] In a preferred embodiment, the invention provides that the
composition of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
anti-human CD3 antibody.
[0314] The compositions may, if desired, be presented in a pack or
dispenser device that may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack.
[0315] Generally, the ingredients of the compositions of the
invention are derived from a subject that is the same species
origin or species reactivity as recipient of such compositions.
Thus, in a preferred embodiment, human or humanized antibodies are
administered to a human patient for therapy or prophylaxis.
[0316] The amount of the composition of the invention which will be
effective in the treatment, prevention or amelioration of one or
more symptoms associated with an autoimmune diabetes disorder can
be determined by standard clinical techniques. The precise dose to
be employed in the formulation will also depend on the route of
administration, and the seriousness of the condition, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems.
[0317] 5.4 Characterization of Anti-CD3 Therapeutic or Prophylactic
Utility
[0318] CD3 binding molecules may be characterized in a variety of
ways. In particular, CD3 binding molecules may be assayed for the
ability to immunospecifically bind to a CD3 polypeptide. Such an
assay may be performed in solution (e.g., Houghten, 1992,
Bio/Techniques 13:412-421), on beads (Lam, 1991, Nature 354:82-84),
on chips (Fodor, 1993, Nature 364:555-556), on bacteria (U.S. Pat.
No. 5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and
5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci.
USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J.
Mol. Biol. 222:301-310) (each of these references is incorporated
herein in its entirety by reference). CD3 binding molecules that
have been identified to immunospecifically bind to a CD3
polypeptide can then be assayed for their specificity and affinity
for a CD3 polypeptide.
[0319] CD3 binding molecules may be assayed for immunospecific
binding to a CD3 polypeptide and cross-reactivity with other
polypeptides by any method known in the art. Immunoassays which can
be used to analyze immunospecific binding and cross-reactivity
include, but are not limited to, competitive and non-competitive
assay systems using techniques such as western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0320] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the CD3 binding molecule of
interest to the cell lysate, incubating for a period of time (e.g.,
1 to 4 hours) at 40.degree. C., adding protein A and/or protein G
sepharose beads to the cell lysate, incubating for about an hour or
more at 40.degree. C., washing the beads in lysis buffer and
resuspending the beads in SDS/sample buffer. The ability of the CD3
binding molecule of interest to immunoprecipitate a particular
antigen can be assessed by, e.g., western blot analysis. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the binding of the CD3 binding molecule
to a CD3 polypeptide and decrease the background (e.g.,
pre-clearing the cell lysate with sepharose beads). For further
discussion regarding immunoprecipitation protocols see, e.g.,
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0321] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with CD3 binding molecule of interest (e.g.,
an antibody of interest) diluted in blocking buffer, washing the
membrane in washing buffer, blocking the membrane with an antibody
(which recognizes the CD3 binding molecule) conjugated to an
enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) or radioactive molecule (e.g., .sup.32P or .sup.125I)
diluted in blocking buffer, washing the membrane in wash buffer,
and detecting the presence of the CD3 polypeptide. One of skill in
the art would be knowledgeable as to the parameters that can be
modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0322] ELISAs comprise preparing CD3 polypeptide, coating the well
of a 96 well microtiter plate with the CD3 polypeptide, adding the
CD3 binding molecule of interest conjugated to a detectable
compound such as an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) to the well and incubating for
a period of time, and detecting the presence of the CD3
polypeptide. In ELISAs the CD3 binding molecule of interest does
not have to be conjugated to a detectable compound; instead, an
antibody (which recognizes the CD3 binding molecule of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the CD3 polypeptide, the
CD3 binding molecule may be coated to the well. In this case, an
antibody conjugated to a detectable compound may be added following
the addition of the CD3 polypeptide to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0323] The binding affinity of a CD3 binding molecule to a CD3
polypeptide and the off-rate of an CD3 binding molecule-CD3
polypeptide interaction can be determined by competitive binding
assays. One example of a competitive binding assay is a
radioimmunoassay comprising the incubation of labeled CD3
polypeptide (e.g., .sup.3H or .sup.125I) with the CD3 binding
molecule of interest in the presence of increasing amounts of
unlabeled CD3 polypeptide, and the detection of the CD3 binding
molecule bound to the labeled CD3 polypeptide. The affinity of a
CD3 binding molecule for a CD3 polypeptide and the binding
off-rates can be determined from the data by scatchard plot
analysis. Competition with a second CD3 binding molecule can also
be determined using radioimmunoassays. In this case, a CD3
polypeptide is incubated with a CD3 binding molecule conjugated to
a labeled compound (e.g., .sup.3H or .sup.125I) in the presence of
increasing amounts of a second unlabeled CD3 binding molecule.
[0324] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of CD3 binding molecules
to a CD3 polypeptide. BIAcore kinetic analysis comprises analyzing
the binding and dissociation of a CD3 polypeptide from chips with
immobilized CD3 binding molecules on their surface.
[0325] The CD3 binding molecules, in particular anti-human CD3
antibodies, and compositions of the invention can also be assayed
for their ability to modulate T cell activation. T cell activation
can be determined by measuring, e.g., changes in the level of
expression of cytokines and/or T cell activation markers.
Techniques known to those of skill in the art, including, but not
limited to, immunoprecipitation followed by western blot analysis,
ELISAs, flow cytometry, Northern blot analysis, and RT-PCR can be
used to measure the expression cytokines and T cell activation
markers. In a preferred embodiment, a CD3 binding molecule or
composition of the invention is tested for its ability to induce
the expression of IFN-.gamma. and/or IL-2.
[0326] The anti-CD3 antibodies, and compositions of the invention
can also be assayed for their ability to induce T cell signaling.
The ability of an anti-CD3 antibody or a composition of the
invention induce T cell signaling can be assayed, e.g., by kinase
assays and electrophoretic shift assays (EMSAs).
[0327] The anti-CD3 antibodies, and compositions of the invention
can be tested in vitro or in vivo for their ability to modulate T
cell proliferation. For example, the ability of an anti-CD3
antibody or a composition of the invention to modulate T cell
proliferation can be assessed by, e.g., .sup.3H-thymidine
incorporation, trypan blue cell counts, and fluorescence activated
cell sorting (FACS).
[0328] The anti-human CD3 antibodies, and compositions of the
invention can be tested in vitro or in vivo for their ability to
induce cytolysis. For example, the ability of an anti-CD3 antibody
or a composition of the invention to induce cytolysis can be
assessed by, e.g., .sup.51Cr-release assays.
[0329] The anti-CD3 antibodies, and compositions of the invention
can be tested in vitro or in vivo for their ability to mediate the
depletion of peripheral blood T cells. For example, the ability of
an anti-CD3 antibody or a composition of the invention to mediate
the depletion of peripheral blood T cells can be assessed by, e.g.,
measuring T cell counts using flow cytometry analysis.
[0330] The anti-CD3 antibodies, and compositions of the invention
can be tested in vivo for their ability to mediate peripheral blood
lymphocyte counts. For example, the ability of an anti-CD3 antibody
or a composition of the invention to mediate peripheral blood
lymphocyte counts can be assessed by, e.g., obtaining a sample of
peripheral blood from a subject, separating the lymphocytes from
other components of peripheral blood such as plasma using, e.g., a
Ficoll gradient, and counting the lymphocytes using trypan
blue.
[0331] 5.4.1 Characterization of Immunoglobulin Molecules with
Variant Fc Regions
[0332] In preferred embodiments, characterization of molecules
comprising variant Fc regions with altered Fc.gamma.R affinities
(e.g., null Fc.gamma.R binding) are done with one or more
biochemical based assays, preferably in a high throughput manner.
The one or more biochemical assays can be any assay known in the
art for identifying Fc-Fc.gamma.R interaction, i.e., specific
binding of an Fc region to an Fc.gamma.R, including, but not
limited to, an ELISA assay, surface plasmon resonance assays,
immunoprecipitation assay, affinity chromatography, and equilibrium
dialysis. The functional based assays can be any assay known in the
art for characterizing one or more Fc.gamma.R mediated effector
cell functions. Comparison of antibodies with altered Fc regions of
the invention to control antibodies provides a measure of the
extent of decrease or elimination of Fc-Fc.gamma.R interaction.
Non-limiting examples of effector cell functions that can be used
in accordance with the methods of the invention, include but are
not limited to, antibody-dependent cell mediated cytotoxicity
(ADCC), antibody-dependent phagocytosis, phagocytosis,
opsonization, opsonophagocytosis, cell binding, rosetting, C1q
binding, and complement dependent cell mediated cytotoxicity. In
preferred embodiments, characterization of molecules comprising
variant Fc regions with altered Fc.gamma.R affinities (e.g., null
FcR binding) are done with one or more biochemical based assays in
combination or in parallel with one or more functional based
assays, preferably in a high throughput manner.
[0333] In some embodiments, characterization of molecules
comprising variant Fc regions with altered Fc.gamma.R affinities
(e.g., null Fc.gamma.R binding) comprise: characterizing the
binding of the molecule comprising the variant Fc region to a
Fc.gamma.R (one or more), using a biochemical assay for determining
Fc-Fc.gamma.R interaction, preferably, an ELISA based assay
followed by comparison of the results to the results of the same
assay obtained with a control, i.e. non-modified, antibody. Once
the molecule comprising a variant Fc region has been characterized
for its interaction with one or more Fc.gamma.Rs and determined to
have null binding to one or more Fc.gamma.Rs, by at least one
biochemical based assay, e.g., an ELISA assay, the molecule maybe
engineered into a complete immunoglobulin, using standard
recombinant DNA technology methods known in the art, and the
immunoglobulin comprising the variant Fc region expressed in
mammalian cells for further biochemical characterization. The
immunoglobulin into which a variant Fc region of the invention is
introduced (e.g., replacing the Fc region of the immunoglobulin)
can be any immunoglobulin including, but not limited to, polyclonal
antibodies, monoclonal antibodies, bispecific antibodies,
multi-specific antibodies, humanized antibodies, and chimeric
antibodies. In preferred embodiments, a variant Fc region is
introduced into an immunoglobulin specific for the CD3 complex
associated with the human TCR.
[0334] The variant Fc regions, preferably in the context of an
immunoglobulin, can be further characterized using one or more
biochemical assays and/or one or more functional assays, preferably
in a high throughput manner. In some alternate embodiments, the
variant Fc regions are not introduced into an immunoglobulin and
are further characterized using one or more biochemical based
assays and/or one or more functional assays, preferably in a high
throughput manner. The one or more biochemical assays can be any
assay known in the art for identifying Fc-Fc.gamma.R interactions,
including, but not limited to, an ELISA assay, and surface plasmon
resonance-based assay for determining the kinetic parameters of
Fc-Fc.gamma.R interaction, e.g., BIAcore assay. The one or more
functional assays can be any assay known in the art for
characterizing one or more Fc.gamma.R mediated effector cell
function as known to one skilled in the art or described herein. In
specific embodiments, the immunoglobulins comprising the variant Fc
regions are assayed in an ELISA assay for binding to one or more
Fc.gamma.Rs, e.g., Fc.gamma.RIIIA, Fc.gamma.RIIA, Fc.gamma.RIIA;
followed by one or more ADCC assays. In some embodiments, the
immunoglobulins comprising the variant Fc regions are assayed
further using a surface plasmon resonance-based assay, e.g.,
BIAcore. For further a detailed discussion of characterization of
immunoglobulins comprising variant Fc regions see U.S. Pat. Appl.
Pub. No. 2005/0064514 A1 and U.S. Pat. Appl. Pub. No. 20050037000
A1.
[0335] The immunoglobulin comprising the variant Fc regions may be
analyzed at any point using a surface plasmon based resonance based
assay, e.g., BIAcore, for defining the kinetic parameters of the
Fc-Fc.gamma.R interaction, using methods known to those of skill in
the art.
[0336] In most preferred embodiments, the immunoglobulin comprising
the variant Fc regions is further characterized in an animal model
for interaction with an Fc.gamma.R. Preferred animal models for use
in the methods of the invention are, for example, transgenic mice
expressing human Fc.gamma.Rs, e.g., any mouse model described in
U.S. Pat. No. 5,877,397, which is incorporated herein by reference
in its entirety. Transgenic mice for use in the methods of the
invention include, but are not limited to, nude knockout
Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIIA; nude knockout
Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIA; nude knockout
Fc.gamma.RIIIA mice carrying human Fc.gamma.RIIB and human
Fc.gamma.RIIIA; nude knockout Fc.gamma.RIIIA mice carrying human
Fc.gamma.RIIB and human Fc.gamma.RIIA.
[0337] 5.4.2 In Vitro and In Vivo Characterization
[0338] Several aspects of the pharmaceutical compositions or the
anti-CD3 antibodies of the invention are preferably tested in
vitro, in a cell culture system, and in an animal model organism,
such as a rodent animal model system, for the desired therapeutic
activity prior to use in humans. For example, assays which can be
used to determine whether administration of a specific
pharmaceutical composition is indicated, include cell culture
assays in which a patient tissue sample is grown in culture, and
exposed to or otherwise contacted with a pharmaceutical
composition, and the effect of such composition upon the tissue
sample is observed. The tissue sample can be obtained by biopsy
from the patient. This test allows the identification of the
therapeutically most effective tumor-targeted bacteria and the
therapeutically most effective therapeutic molecules) for each
individual patient. In various specific embodiments, in vitro
assays can be carried out with representative cells of cell types
involved in an autoimmune or inflammatory disorder (e.g., T cells),
to determine if a pharmaceutical composition of the invention has a
desired effect upon such cell types.
[0339] In accordance with the invention, clinical trials with human
subjects need not be performed in order to demonstrate the
prophylactic and/or therapeutic efficacy of anti-CD3 antibodies. In
vitro and animal model studies using anti-CD3 antibodies can be
extrapolated to humans and are sufficient for demonstrating the
prophylactic and/or therapeutic utility of said anti-CD3
antibodies.
[0340] Anti-CD3 antibodies can be tested in suitable animal model
systems prior to use in humans. Such animal model systems include,
but are not limited to, rats, mice, chicken, cows, monkeys, pigs,
dogs, rabbits, etc. Any animal system well-known in the art may be
used. In a specific embodiment of the invention, CD3 binding
molecules are tested in a mouse model system. Such model systems
are widely used and well-known to the skilled artisan. CD3 binding
molecules can be administered repeatedly. Several aspects of the
procedure may vary. Said aspects include the temporal regime of
administering CD3 binding molecules, and whether such agents are
administered separately or as an admixture.
[0341] The anti-inflammatory activity of anti-CD3 antibodies or
pharmaceutical compositions of invention can be determined by using
various experimental animal models of inflammatory arthritis known
in the art and described in Crofford L. J. and Wilder R. L.,
"Arthritis and Autoimmunity in Animals", in Arthritis and Allied
Conditions: A Textbook of Rheumatology, McCarty et al. (eds.),
Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneous
animal models of inflammatory arthritis and autoimmune rheumatic
diseases can also be used to assess the anti-inflammatory activity
of anti-CD3 antibodies or pharmaceutical compositions of invention.
The following are some assays provided as examples and not by
limitation.
[0342] The principle animal models for arthritis or inflammatory
disease known in the art and widely used include: adjuvant-induced
arthritis rat models, collagen-induced arthritis rat and mouse
models and antigen-induced arthritis rat, rabbit and hamster
models, all described in Crofford L. J. and Wilder R. L.,
"Arthritis and Autoimmunity in Animals", in Arthritis and Allied
Conditions: A Textbook of Rheumatology, McCarty et al. (eds.),
Chapter 30 (Lee and Febiger, 1993), incorporated herein by
reference in its entirety. A collagen-induced arthritis {CIA) is an
animal model for the human autoimmune disease rheumatoid arthritis
(RA) (Trenthorn et al., 1977, J. Exp. Med. 146:857). This disease
can be induced in many "species by the administration of
heterologous type II collagen (Courtenay et al., 1980, Nature
283:665; and Cathcart et at, 1986, Lab. Invest. 54:26). With
respect to animal models of arthritis see, in addition, e.g.,
Holmdahl, R., 1999, Curr. Biol. 15:R528-530.
[0343] Additionally, animal models for inflammatory bowel disease
can also be used to assess the efficacy of the anti-CD3 antibodies
or pharmaceutical compositions of invention (Kim eta 1., 1992,
Scand. J. Gastroentrol. 27:529-537; Strober, 1985, Dig. Dis. Sci.
30(12 Suppl):3S-lOS). Ulcerative cholitis and Crohn's disease are
human inflammatory bowel diseases that can be induced in animals.
Sulfated polysaccharides including, but not limited to amylopectin,
carrageen, amylopectin sulfate, and dextran sulfate or chemical
irritants including but not limited to trinitrobenzenesulphonic
acid (TNBS) and acetic acid can be administered to animals orally
to induce inflammatory bowel diseases.
[0344] Animal models for asthma can also be used to assess the
efficacy of anti-CD3 antibodies or pharmaceutical compositions of
invention. An example of one such model is the marine adoptive
transfer model in which aeroallergen provocation of TH 1 or TH2
recipient mice results in TH effector cell migration to the airways
and is associated with an intense neutrophilic (TH 1) and
eosinophilic (TH2) lung mucosal inflammatory response (Cohn et al.,
1997, J. Exp. Med. 1861737-1747).
[0345] Animal models for autoimmune disorders can also be used to
assess the efficacy of anti-CD3 antibodies or pharmaceutical
compositions of invention. Animal models for autoimmune disorders
such as type 1 diabetes, thyroid autoimmunity, sytemic lupus
eruthematosus, and glomerulonephritis have been developed
(Bluestone et al., 2004, PNAS 101:14622-14626; Flanders et al.,
1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie
81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).
[0346] The efficacy of anti-CD3 antibodies or pharmaceutical
compositions of invention can also be tested in such autoimmune
disorder models as an experimental allergic encephalomyelitis (EAE)
model. EAE is an experimental autoimmune disease of the central
nervous system {CNS) (Zamvil et al, 1990, Ann. Rev, Immunol. 8:579)
and is a disease model for the human autoimmune condition, multiple
sclerosis (MS). EAE is an example of a cell-mediated autoimmune
disorder that is mediated via T cells. EAE is readily induced in
mammalian species by immunizations of myelin basic protein (MBP)
purified from the CNS or an encephalitogenic proteolipid (PLP).
SJL/J mice are a susceptible strain of mice (H-2u) and, upon
induction of EAE, these mice develop an acute paralytic disease and
an acute cellular infiltrate is identifiable within the CNS. EAE
spontaneously develops in MBP1-17 peptide-specific T cell receptor
(TCR) transgenic mice (TgMBP+) of a RAG-1-deficient background
(Lafaille et al., 1994, Cell 78:399).
[0347] Further, any assays known to those skilled in the art can be
used to evaluate anti-CD3 antibodies or the pharmaceutical
compositions disclosed herein for autoimmune and/or inflammatory
diseases.
[0348] The toxicity and/or efficacy of anti-CD3 antibodies or
pharmaceutical compositions of invention can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD.sub.50 (the dose lethal to
50% of the population) and the ED.sub.50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Anti-CD3 antibodies
that exhibit large therapeutic indices are preferred. While
anti-CD3 antibodies that exhibit toxic side effects may be used,
care should be taken to design a delivery system that targets such
agents to the site of affected tissue in order to minimize
potential damage to uninfected cells and, thereby, reduce side
effects.
[0349] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of anti-human
CD3 antibodies for use in humans. The dosage of such agents lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized. For any agent used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0350] Efficacy in preventing or treating an autoimmune disorder
may be demonstrated, e.g., by detecting the ability of a anti-human
CD3 antibodies or composition of the invention to reduce one or
more symptoms of the autoimmune disorder, to reduce mean absolute
lymphocyte counts, to decrease T cell activation, to decrease T
cell proliferation, to reduce cytokine production, or to modulate
one or more particular cytokine profiles. Efficacy in treating
diabetes may be demonstrated, e.g. by detecting the ability of a
anti-human CD3 antibodies or composition of the invention to reduce
one or more symptoms of diabetes, to preserve the C-peptide
response to MMTT, to reduce the level HA1 or HA1c, to reduce the
daily requirement for insulin, or to decrease T cell activation in
pancreatic islet tissue. Efficacy in preventing or treating an
inflammatory disorder may be demonstrated, e.g., by detecting, the
ability of a an anti-CD3 antibody to reduce one or more symptoms of
the inflammatory disorder, to decrease T cell activation, to
decrease T cell proliferation, to modulate one or more cytokine
profiles, to reduce cytokine production, to reduce inflammation of
a joint, organ or tissue or to improve quality of life.
[0351] Changes in inflammatory disease activity may be assessed
through tender and swollen joint counts, patient and physician
global scores for pain and disease activity, and the ESRICRP.
Progression of structural joint damage may be assessed by
quantitative scoring of X-rays of hands, wrists, and feet (Sharp
method). Changes in functional status in humans with inflammatory
disorders may be evaluated using the Health Assessment
Questionnaire (HAQ), and quality of life changes are assessed with
the SF-36.
[0352] 5.5 Methods of Monitoring Lymphocyte Counts and Percent
Binding
[0353] The effect of one or more doses of one or more anti-human
CD3 antibodies or composition on peripheral blood lymphocyte counts
can be monitored/assessed using standard techniques known to one of
skill in the art. Peripheral blood lymphocytes counts in a mammal
can be determined by, e.g., obtaining a sample of peripheral blood
from said mammal, separating the lymphocytes from other components
of peripheral blood such as plasma using, e.g., Ficoll-Hypaque
(Pharmacia) gradient centrifugation, and counting the lymphocytes
using trypan blue. Peripheral blood T cell counts in mammal can be
determined by, e.g., separating the lymphocytes from other
components of peripheral blood such as plasma using, e.g., a use of
Ficoll-Hypaque (Pharmacia) gradient centrifugation, labeling the T
cells with an antibody directed to a T cell antigen such as CD2,
CD3, CD4, and CD8 which is conjugated to FITC or phycoerythrin, and
measuring the number of T cells by FACS. Further, the effect on a
particular subset of T cells (e.g., CD2.sup.+, CD4.sup.+,
CD8.sup.+, CD4.sup.+RO.sup.+, CD8.sup.+RO.sup.+, CD4.sup.+RA.sup.+,
or CD8.sup.+12 A.sup.+) cells can be determined using standard
techniques known to one of skill in the art such as FACS.
[0354] The percentage of CD3 polypeptides expressed by peripheral
blood lymphocytes bound by anti-CD3 antibodies prior or after, or
both prior to and after the administration of one or more doses of
anti-CD3 antibodies can be assessed using standard techniques known
to one of skill in the art. The percentage of CD3 polypeptides
expressed by peripheral blood T cells bound by anti-CD3 antibodies
can be determined by, e.g., obtaining a sample of peripheral blood
from a mammal, separating the lymphocytes from other components of
peripheral blood such as plasma using, e.g., Ficoll-Hypaque
(Pharmacia) gradient centrifugation, and labeling the T cells with
an anti-CD3 binding molecule antibody other than that of the
invention conjugated to FITC and an antibody directed to a T cell
antigen such as CD3, CD4 or CD8 which is conjugated to
phycoerythrin, and determining the number of T cells labeled with
anti-CD3 binding molecule antibody relative to the number of T
cells labeled with an antibody directed to a T cell antigen using
FACS.
[0355] 5.6 Methods of Producing Antibodies
[0356] Antibodies that immunospecifically bind to a CD3 polypeptide
can be produced by any method known in the art for the synthesis of
antibodies, in particular, by chemical synthesis or preferably, by
recombinant expression techniques.
[0357] Polyclonal antibodies that immunospecifically bind to an
antigen can be produced by various procedures well-known in the
art. For example, a human antigen can be administered to various
host animals including, but not limited to, rabbits, mice, rats,
etc. to induce the production of sera containing polyclonal
antibodies specific for the human antigen. Various adjuvants may be
used to increase the immunological response, depending on the host
species, and include but are not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art.
[0358] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T cell Hybridomas 563 681
(Elsevier, N. Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0359] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a CD3 antigen and once an
immune response is detected, e.g., antibodies specific for a CD3
antigen (preferably, CD3 .epsilon. antigen) are detected in the
mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. The hybridoma clones are then assayed by methods
known in the art for cells that secrete antibodies capable of
binding a polypeptide of the invention. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
immunizing mice with positive hybridoma clones.
[0360] Accordingly, the present invention provides methods of
generating antibodies by culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with a CD3 antigen with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind to a CD3 antigen (preferably, CD3
s antigen).
[0361] Antibody fragments which recognize specific CD3 antigens
(preferably, CD3 c antigen) may be generated by any technique known
to those of skill in the art. For example, Fab and F(ab').sub.2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments). F(ab').sub.2 fragments contain the variable region, the
light chain constant region and the CH1 domain of the heavy chain.
Further, the antibodies of the present invention can also be
generated using various phage display methods known in the art.
[0362] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In particular, DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of affected
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector. The vector is electroporated in E. coli and the E. coli is
infected with helper phage. Phage used in these methods are
typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene HI
or gene VIII. Phage expressing an antigen binding domain that binds
to a particular antigen can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Examples of phage display methods that can be used
to make the antibodies of the present invention include those
disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;
Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough
et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997,
Gene 187:9-18; Burton et al., 1994, Advances in Immunology
57:191-280; PCT Application No. PCT/GB91/O1 134; International
Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO
92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844;
and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717,
5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637,
5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0363] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
PCT publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0364] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lamba constant regions. Preferably, the vectors for
expressing the VH or VL domains comprise an EF-1.alpha. promoter, a
secretion signal, a cloning site for the variable domain, constant
domains, and a selection marker such as neomycin. The VH and VL
domains may also be cloned into one vector expressing the necessary
constant regions. The heavy chain conversion vectors and light
chain conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art.
[0365] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use human or
chimeric antibodies. Completely human antibodies are particularly
desirable for therapeutic treatment of human subjects. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See also
U.S. Pat. Nos. 4,444,887 and 4,716,111; and International
Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654,
WO 96/34096, WO 96/33735, and WO 91/10741; each of which is
incorporated herein by reference in its entirety.
[0366] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the J.sub.H
region prevents endogenous antibody production. The modified
embryonic stem cells are expanded and microinjected into
blastocysts to produce chimeric mice. The chimeric mice are then
bred to produce homozygous offspring which express human
antibodies. The transgenic mice are immunized in the normal fashion
with a selected antigen, e.g., all or a portion of a polypeptide of
the invention. Monoclonal antibodies directed against the antigen
can be obtained from the immunized, transgenic mice using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publication Nos. WO
98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos.
5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806,
5,814,318, and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0367] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. Methods for producing chimeric antibodies are known in
the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,
4,816,397, and 6,331,415, which are incorporated herein by
reference in their entirety.
[0368] A humanized antibody is an antibody or its variant or
fragment thereof which is capable of binding to a predetermined
antigen and which comprises a framework region having substantially
the amino acid sequence of a human immunoglobulin and a CDR having
substantially the amino acid sequence of a non-human
immunoglobulin. A humanized antibody comprises substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, Fabc, Fv) in which all or substantially all of the
CDR regions correspond to those of a non human immunoglobulin
(i.e., donor antibody) and all or substantially all of the
framework regions are those of a human immunoglobulin consensus
sequence. Preferably, a humanized antibody also comprises at least
a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin. Ordinarily, the antibody will contain
both the light chain as well as at least the variable domain of a
heavy chain. The antibody also may include the hinge, CH2, CH3, and
CH4 regions of the heavy chain. The humanized antibody can be
selected from any class of immunoglobulins, including IgM, IgG,
IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and
IgG4. Usually the constant domain is a complement fixing constant
domain where it is desired that the humanized antibody exhibit
cytotoxic activity, and the class is typically IgG1. Where such
cytotoxic activity is not desirable, the constant domain may be of
the IgG2 class. Examples of VL and VH constant domains that can be
used in certain embodiments of the invention include, but are not
limited to, C-kappa and C-gamma-1 (nG1m) described in Johnson et
al. (1997) J. Infect. Dis. 176, 1215-1224 and those described in
U.S. Pat. No. 5,824,307. The humanized antibody may comprise
sequences from more than one class or isotype, and selecting
particular constant domains to optimize desired effector functions
is within the ordinary skill in the art. The framework and CDR
regions of a humanized antibody need not correspond precisely to
the parental sequences, e.g., the donor CDR or the consensus
framework may be mutagenized by substitution, insertion or deletion
of at least one residue so that the CDR or framework residue at
that site does not correspond to either the consensus or the import
antibody. Such mutations, however, will not be extensive. Usually,
at least 75% of the humanized antibody residues will correspond to
those of the parental FR and CDR sequences, more often 90%, and
most preferably greater than 95%. Humanized antibody can be
produced using variety of techniques known in the art, including
but not limited to, CDR-grafting (European Patent No. EP 239,400;
International publication No. WO 91/09967; and U.S. Pat. Nos.
5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing
(European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991,
Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994,
Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS
91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and
techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat.
No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119 25
(2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et
al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem.
272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904
(1996), Couto et al., Cancer Res. 55 (23 Supp):5973s-5977s (1995),
Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu J S, Gene
150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol.
235(3):959-73 (1994). See also U.S. Patent Pub. No. US 2005/0042664
A1 (Feb. 24, 2005), which is incorporated by reference herein in
its entirety. Often, framework residues in the framework regions
will be substituted with the corresponding residue from the CDR
donor antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323,
which are incorporated herein by reference in their
entireties.)
[0369] Single domain antibodies, for example, antibodies lacking
the light chains, can be produced by methods well-known in the art.
See Riechmann et al., 1999, J. Immuno. 231:25-38; Nuttall et al.,
2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J.
Biotechnol. 74(4):277302; U.S. Pat. No. 6,005,079; and
International Publication Nos. WO 94/04678, WO 94/25591, and WO
01/44301, each of which is incorporated herein by reference in its
entirety.
[0370] 5.7 Polynucleotides Encoding Antibodies
[0371] The invention provides polynucleotides comprising a
nucleotide sequence encoding an antibody that immunospecifically
binds to a CD3 polypeptide. The invention also encompasses
polynucleotides that hybridize under high stringency, intermediate
or lower stringency hybridization conditions, e.g., as defined
supra, to polynucleotides that encode an antibody of the
invention.
[0372] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. The nucleotide sequence of antibodies immunospecific for a
CD3 polypeptide can be obtained, e.g., from the literature or a
database such as GenBank. Since the amino acid sequences of, e.g.,
humanized OKT3 is known, nucleotide sequences encoding these
antibodies can be determined using methods well known in the art,
i.e., nucleotide codons known to encode particular amino acids are
assembled in such a way to generate a nucleic acid that encodes the
antibody. Such a polynucleotide encoding the antibody may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding the antibody,
annealing and ligating of those oligonucleotides, and then
amplification of the ligated oligonucleotides by PCR.
[0373] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0374] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY, which are both incorporated by reference
herein in their entireties), to generate antibodies having a
different amino acid sequence, for example to create amino acid
substitutions, deletions, and/or insertions.
[0375] In a specific embodiment, one or more of the CDRs is
inserted within framework regions using routine recombinant DNA
techniques. The framework regions may be naturally occurring or
consensus framework regions, and preferably human framework regions
(see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a
listing of human framework regions). Preferably, the polynucleotide
generated by the combination of the framework regions and CDRs
encodes an antibody that specifically binds to a CD3 polypeptide.
Preferably, as discussed supra, one or more amino acid
substitutions may be made within the framework regions, and,
preferably, the amino acid substitutions improve binding of the
antibody to its antigen. Additionally, such methods may be used to
make amino acid substitutions or deletions of one or more variable
region cysteine residues participating in an intrachain disulfide
bond to generate antibody molecules lacking one or more intrachain
disulfide bonds. Other alterations to the polynucleotide are
encompassed by the present invention and within the skill of the
art.
[0376] 5.8 Recombinant Expression of Molecules of the Invention
[0377] Once a nucleic acid sequence encoding molecules of the
invention (i.e., antibodies) has been obtained, the vector for the
production of the molecules may be produced by recombinant DNA
technology using techniques well known in the art. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing the coding sequences for the
molecules of the invention and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. (See, for example, the techniques
described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. and Ausubel et al. eds., 1998, Current Protocols in Molecular
Biology, John Wiley & Sons, NY).
[0378] An expression vector comprising the nucleotide sequence of a
molecule identified by the methods of the invention (i.e., an
antibody) can be transferred to a host cell by conventional
techniques (e.g., electroporation, liposomal transfection, and
calcium phosphate precipitation) and the transfected cells are then
cultured by conventional techniques to produce the molecules of the
invention. In specific embodiments, the expression of the molecules
of the invention is regulated by a constitutive, an inducible or a
tissue, specific promoter. In specific embodiments the expression
vector is pMGX1303 (FIG. 3).
[0379] The host cells used to express the molecules identified by
the methods of the invention may be either bacterial cells such as
Escherichia coli, or, preferably, eukaryotic cells, especially for
the expression of whole recombinant immunoglobulin molecule. In
particular, mammalian cells such as Chinese hamster ovary cells
(CHO), in conjunction with a vector such as the major intermediate
early gene promoter element from human cytomegalovirus is an
effective expression system for immunoglobulins (Foecking et al.,
1998, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
[0380] A variety of host-expression vector systems may be utilized
to express the molecules identified by the methods of the
invention. Such host-expression systems represent vehicles by which
the coding sequences of the molecules of the invention may be
produced and subsequently purified, but also represent cells which
may, when transformed or transfected with the appropriate
nucleotide coding sequences, express the molecules of the invention
in situ. These include, but are not limited to, microorganisms such
as bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing coding sequences for the molecules identified by
the methods of the invention; yeast (e.g., Saccharomyces Pichia)
transformed with recombinant yeast expression vectors containing
sequences encoding the molecules identified by the methods of the
invention; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing the sequences
encoding the molecules identified by the methods of the invention;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic
virus (TMV) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing sequences encoding the
molecules identified by the methods of the invention; or mammalian
cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphotic
cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (human retinal
cells developed by Crucell) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0381] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
molecule being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody, vectors which direct
the expression of high levels of fusion protein products that are
readily purified may be desirable. Such vectors include, but are
not limited, to the E. coli expression vector pUR278 (Ruther et
al., 1983, EMBO J. 2:1791), in which the antibody coding sequence
may be ligated individually into the vector in frame with the lac Z
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to a matrix
glutathione-agarose beads followed by elution in the presence of
free gluta-thione. The pGEX vectors are designed to include
thrombin or factor Xa protease cleavage sites so that the cloned
target gene product can be released from the GST moiety.
[0382] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (e.g., the polyhedrin gene) of the virus and placed under
control of an AcNPV promoter (e.g., the polyhedrin promoter).
[0383] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
immunoglobulin molecule in infected hosts (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific
initiation signals may also be required for efficient translation
of inserted antibody coding sequences. These signals include the
ATG initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:51-544).
[0384] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and
T47D, CRL7030 and Hs578Bst.
[0385] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express an antibody of the invention may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the antibodies of the invention. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that interact directly or indirectly with
the antibodies of the invention.
[0386] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11: 223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48: 202), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et
al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.
Natl. Acad. Sci. USA 78: 2072); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12: 488-505; Wu and Wu,
1991, 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.
32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and
Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH
11(5):155-215). Methods commonly known in the art of recombinant
DNA technology which can be used are described in Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John Wiley
& Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,
Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics,
John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol.
Biol. 150:1; and hygro, which confers resistance to hygromycin
(Santerre et al., 1984, Gene 30:147).
[0387] The expression levels of an antibody of the invention can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3 (Academic Press, New York, 1987). When a marker in the vector
system expressing an antibody is amplifiable, increase in the level
of inhibitor present in culture of host cell will increase the
number of copies of the marker gene. Since the amplified region is
associated with the nucleotide sequence of the antibody, production
of the antibody will also increase (Crouse et al., 1983, Mol. Cell.
Biol. 3:257).
[0388] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0389] Once a molecule of the invention (i.e., antibodies) has been
recombinantly expressed, it may be purified by any method known in
the art for purification of polypeptides or antibodies, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of polypeptides or antibodies.
6. EXAMPLES
6.1 Anti-CD3 Monoclonal Antibody Therapy for Type 1 Patients
[0390] Patients:
[0391] Forty patients with Type 1 diabetes are recruited for
participation according to the following criteria: between 7 and 20
years of age, within 6 weeks of diagnosis according to the American
Diabetes Association criteria, and confirmation of the presence of
anti-GAD65, anti-ICA512, and/or anti-insulin autoantibodies. The
patients remain under the care of their personal physicians during
the course of the study.
[0392] Eligible patients are randomly assigned to a control group
and an anti-human CD3 antibody treatment group. After
randomization, blood samples are drawn to establish baseline HA1c
levels, a pretreatment C-peptide response to a MMTT is established
and a pretreatment FPIR to IGTT is performed. Patients in both
groups are hospitalized to receive either a 6-day course treatment
of the anti-human CD3 monoclonal antibody hOKT3.gamma.1 (ala-ala)
or placebo. The antibody is administered intravenously in the
following dosage: 17 .mu.g/m.sup.2 on day 1, 34.3 .mu.g/m.sup.2 on
day 2, 69 .mu.g/m.sup.2 on day 3, 137.6 .mu.g/m.sup.2 on day 4, and
275.3 .mu.g/m.sup.2 on days 5 and 6. Alternatively, antibody may be
administered intravenously in the following dosage: 1.6
.mu.g/kg/day on day 1; 3.2 .mu.g/kg/day on day 2; 6.5 .mu.g/kg/day
on day 3; 13 .mu.g/kg/day on day 4; and 26 .mu.g/kg/day on days 5
through 14. In dose escalation studies, the treatment may be, e.g.,
1.42 .mu.g/kg/day on day 1; 5.7 .mu.g/kg/day on day 2; 11
.mu.g/kg/day on day 3; 26 .mu.g/kg/day on day 4; and 45.4
.mu.g/kg/day on days 5 through 14. In subsequent studies, the
therapy is altered to increase dosage and/or decrease the time
course of treatment. For example, in subsequent studies patients
may be administered a 4 day treatment: 6.4 .mu.g/kg/day on day 1;
13 .mu.g/kg/day on day 2, and 26 .mu.g/kg/day on days 3 and 4;
during additional dose escalation studies, the treatment may be 8
.mu.g/kg/day on day 1; 16 .mu.g/kg/day on day 2; and 32
.mu.g/kg/day on days 3 and 4.
[0393] During initial studies the antibody dosage on the first
three days of treatment is administered via slow infusion IV over
20 hours to monitor for adverse reactions. Subsequent studies will
decrease the time of administration and/or split the dosage into 2
to 4 equal parts to be administered as bolus injections evenly
distributed over the course of 12 hours. Patients in the control
group undergo metabolic and immunologic tests but do no receive
monoclonal antibodies. Patients are monitored throughout the study
for immunosuppressive effects of the anti-human CD3 monoclonal
antibody hOKT3.gamma.1(ala-ala).
[0394] Patients are monitored for 18 months after the treatment.
.beta.-cell function is determined every 6 months in the case of
impaired glucose tolerance and every 12 months in case of normal
glucose tolerance. Patients are allowed to have a normal diet, and
remain under the care of their personal physician throughout the
duration of the study. Immunological assays are repeated in
intervals of 6 months. Insulin therapy will be given to the
patients as directed by their personal physician.
[0395] .beta.-cell function will be analyzed according to the
changes of the C-peptide levels as measured by radioimmunoassay.
After drawing samples for baseline C-peptide and glucose, the
patients are given a mixed meal. The C-peptide levels are measured
in samples drawn after 15, 30, 60, 90, 120, 150, 180, 210, and 240
min. The C-peptide response to the mixed-meal tolerance test (MMTT)
is expressed as the total area under the response curve (AUC). A
change in the response is considered to have occurred if the
response differs by more than 7.5 percent from the response at
study entry. The patients' C-peptide responses to MMTT are
continuously monitored 6 months, 9 months, 12 months, 15 months and
18 months after the treatment. Alternatively, the .beta.-cell
function is assessed by FPIR to IGTT. Serum insulin levels are
measured by a modification of a double-antibody radioimmunoassay
method using monoiodinated tyrosine A14-labeled insulin (Amersham
Pharmacia). FPIR is calculated as the sum of insulin levels at 1
and 3 minutes after a glucose load (0.5 g/kg). Glycosylated
hemoglobin levels are measured by latex-agglutination inhibition
test.
[0396] Immunological Monitoring:
[0397] The level of autoantibodies against GAD65, IA2/ICA512, and
insulin are measured with radiobinding assays as known in the art
(e.g., Woo et al., 2000, J. Immunol Methods 244:91-103). HLA-DQA
and HLA-DQB genotyping are performed by direct sequencing of exon 2
polymorphisims after PCR amplification. The level of cytokines in
serum after the administration of the monoclonal antibody is
measured by enzyme-linked immunosorbent assay (ELISA). Production
of anti-idotype antibodies is monitored by ELISA assay using a
plate bound hOKT3.gamma.1(ala-ala) or by flow cytometry to measure
blockade of binding of hOKT3.gamma.1(ala-ala)-FITC to CD3.
[0398] Statistical Analysis:
[0399] Data analysis will be conducted on residual beta-cell
function, autoantibody level, cytokine level, and glycosylated
hemoglobin level. A .chi..sup.2 analysis will be performed to test
the effect of drug treatment before and after drug administration.
Comparison between the control group and the treatment group will
be made with the Mann-Whitney U test.
6.2 Anti-CD3 Monoclonal Antibody Therapy in Subjects Predisposed to
Type 1 Diabetes
[0400] Patients:
[0401] Screening for subjects with predisposition for developing
type 1 diabetes is based on first or second degree relationship
with a diagnosed Type-1 diabetic; an impaired fasting glucose
level; an impaired glucose response to OGTT; the presence of serum
autoantibodies against GAD65, against IA2/ICA512, and/or against
insulin; or impaired insulin production after MMTT, OGTT, IGTT or
two phase glucose clamp procedure as determined by C-peptide
response or FPIR. Patients who have been diagnosed with type 1
diabetes according to the criteria established by the American
Diabetes Association by a physician, or who otherwise meet said
criteria, are excluded from this study.
[0402] Patients selected for the study are randomly placed into two
equal-sized groups. Treatment protocols and clinical monitoring are
as described in section 6.1. Additionally, antibody therapy may be
adjusted relative to residual .beta.-cell function, i.e., patients
with more impaired .beta.-cell function as determined by C-peptide
response or FPIR will receive a higher total dose of anti-CD3
monoclonal antibody. For example, given two patients with C-peptide
responses of 40 and 110 pmol/ml/240 min, the patient with impaired
response will be given the higher of the two dosages tested, e.g.,
1.42 .mu.g/kg/day on day 1; 5.7 .mu.g/kg/day on day 2; 11
.mu.g/kg/day on day 3; 26 .mu.g/kg/day on day 4; and 45.4
.mu.g/kg/day on days 5 through 14.
[0403] Patients are monitored for 18 months after the treatment.
.beta.-cell function is determined every 6 months in the case of
impaired glucose tolerance and every 12 months in case of normal
glucose tolerance. Patients are allowed to have a normal diet, and
remain under the care of their personal physician throughout the
duration of the study. Immunological assays are repeated in
intervals of 6 months. Insulin therapy will be given to the
patients as directed by their personal physician.
6.3 Anti-CD3 Monoclonal Antibody Therapy in Multiple Sclerosis
[0404] Patients:
[0405] Patients with relapsing-remitting or secondary progressive
multiple sclerosis, confirmed according to Poser and/or McDonald
criteria are included in this study. Primary selection criteria
also include at least two documented exacerbations in the last two
year, age 18 or above, and baseline EDSS score between 0 and 5.
[0406] The selected patients are randomly assigned into a treatment
group and a control group. Treatment protocols are as outlined in
section 6.1. All patients remain under the care of their personal
physician during the course of the study and receive equivalent
neurological monitoring at equivalent time points.
[0407] Monitoring MS:
[0408] Neurological examinations are scheduled prior to treatment
to establish baseline values, and subsequently every three months
for a total of 36 months. The clinical assessment of patients is
performed by two neurologists to monitor increases in the
frequency, duration and/or severity of attacks, and/or to monitor
for increases in EDSS score. Additionally, Gadolinium-enhanced MRI
scans are performed to obtain baseline measurement of brain or
spinal lesion number and/or volume and are repeated every three
months for a total of 36 months. Patients remain under the care of
personal physicians) during the course of the study. Patients with
relapse are treated with Avonex and reexamined at monthly intervals
for a period of at least 6 months.
[0409] An increase of one point on the EDSS which persists for at
least two scheduled neurological examinations indicates progression
of disability. The efficacy of the treatment is evaluated according
to time to first relapse, relapse rate, and the accumulation of
permanent physical disability. Comparison will be made between the
treatment group and the control group. The extent and number of
active lesions on MRI will also be recorded and compared.
7. EQUIVALENTS
[0410] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0411] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
Sequence CWU 1
1
131107PRTArtificial Sequencehumanized light chain Okt3vl variable
region 1Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro
Gly1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val
Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro
Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val
Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser Gly Met Glu Ala Glu65 70 75 80 Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser
Gly Thr Lys Leu Glu Ile Asn Arg 100 105 2108PRTArtificial
SequenceLight Chain variable domain from REI 2Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ile Lys Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Glu Ala Ser Asn Leu Gln Ala Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu
Gln Pro65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln
Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile
Thr Arg 100 105 3107PRTArtificial SequencegLA -light chain variable
domain of a humanized Okt3vl 3Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln
Thr Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80 Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr
85 90 95 Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg 100 105
4107PRTArtificial SequencegLC -light chain variable domain of a
humanized Okt3vl 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser
Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Thr Pro Gly
Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr
Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80 Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95
Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg 100 105
5119PRTArtificial Sequencehumanized heavy chain Okt3vh variable
region 5Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly
Ala1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr
Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr
Thr Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80 Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr
Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr
Thr Leu Thr Val Ser Ser 115 6126PRTArtificial SequenceLight chain
variable domain from KOL 6Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser
Ser Gly Phe Ile Phe Ser Ser Tyr 20 25 30 Ala Met Tyr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp
Asp Asp Gly Ser Asp Gln His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80
Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys 85
90 95 Ala Arg Asp Gly Gly His Gly Phe Cys Ser Ser Ala Ser Cys Phe
Gly 100 105 110 Pro Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser
Ser 115 120 125 7119PRTArtificial SequencegH -heavy chain variable
domain of a humanized Okt3vh 7Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ser
Ser Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile
Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys
Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75
80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys
85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly
Gln Gly 100 105 110 Thr Pro Val Thr Val Ser Ser 115
8119PRTArtificial SequencegHA -heavy chain variable domain of a
humanized Okt3vh 8Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser
Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe
Thr Ile Ser Thr Asp Lys Ser Lys Ser Thr Ala Phe65 70 75 80 Leu Gln
Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Pro Val Thr Val Ser Ser 115 9119PRTArtificial
SequencegHG -heavy chain variable domain of a humanized Okt3vh 9Gln
Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr
Asn Gln Lys Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Ala Phe65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro
Glu Asp Thr Gly Val Tyr Phe Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp
His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Pro Val Thr
Val Ser Ser 115 10699DNAArtificial SequenceNucleotide sequence of
the Light chain of a humanized OKT3 10atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctacaggtgt ccactccgac 60atccagatga cccagtctcc
ttcttctctg tctgcttctg tcggagacag agtcacaatc 120acatgttctg
cttctagctc tgtctcttac atgaactggt accagcagac acctggaaag
180gctcctaagc ggtggatcta cgacacatct aagctcgctt ctggagtccc
ttctagattc 240tctggttctg gctctggaac agactacaca ttcacaatct
cttctctcca acctgaggac 300atcgctacat actactgcca acagtggtct
agcaatcctt tcacattcgg acagggaaca 360aagctgcaga tcacaagaac
tgtggcggcg ccgtctgtct tcatcttccc gccatctgat 420gagcagttga
aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga
480gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc
ccaggagagt 540gtcacagagc aggacagcaa ggacagcacc tacagcctca
gcagcaccct gacgctgagc 600aaagcagact acgagaaaca caaagtctac
gcctgcgaag tcacccatca gggcctgagc 660tcgcccgtca caaagagctt
caacagggga gagtgttag 69911232PRTArtificial SequenceAmino acid
sequence of the Light chain of a humanized OKT3 11Met Gly Trp Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15 Val His
Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val 35
40 45 Ser Tyr Met Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys
Arg 50 55 60 Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ser Arg Phe65 70 75 80 Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe
Thr Ile Ser Ser Leu 85 90 95 Gln Pro Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn 100 105 110 Pro Phe Thr Phe Gly Gln Gly
Thr Lys Leu Gln Ile Thr Arg Thr Val 115 120 125 Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg145 150 155 160
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165
170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys225
230 121407DNAArtificial SequenceNucleotide sequence of the heavy
chain of a humanized OKT3 12atgggatgga gctgtatcat cctcttcttg
gtagcaacag ctacaggtgt ccactcccag 60gttcagctgg tgcagtctgg aggaggagtc
gtccagcctg gaaggtccct gagactgtct 120tgtaaggctt ctggatacac
cttcactaga tacacaatgc actgggtcag acaggctcct 180ggaaagggac
tcgagtggat tggatacatt aatcctagca gaggttatac taactacaat
240cagaaggtga aggacagatt cacaatttct agagacaatt ctaagaatac
agccttcctg 300cagatggact cactcagacc tgaggatacc ggagtctatt
tttgtgctag atattacgat 360gaccactact gtctggacta ctggggccaa
ggtaccccgg tcaccgtgag ctcagcttcc 420accaagggcc catcggtctt
ccccctggca ccctcctcca agagcacctc tgggggcaca 480gcggccctgg
gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac
540tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc
ctcaggactc 600tactccctca gcagcgtggt gaccgtgccc tccagcagct
tgggcaccca gacctacatc 660tgcaacgtga atcacaagcc cagcaacacc
aaggtggaca agaaagttga gcccaaatct 720tgtgacaaaa ctcacacatg
cccaccgtgc ccagcacctg aggccgcggg aggaccatca 780gtcttcctct
tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc
840acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa
ctggtacgtg 900gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
aggagcagta caacagcacg 960taccgtgtgg tcagcgtcct caccgtcctg
caccaggact ggctgaatgg caaggagtac 1020aagtgcaagg tctccaacaa
agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080aaagggcagc
cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc
1140aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga
catcgccgtg 1200gagtgggaga gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac 1260tccgacggct ccttcttcct ctacagcaag
ctcaccgtgg acaagagcag gtggcagcag 1320gggaacgtct tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1380agcctctccc
tgtctccggg taaatga 140713468PRTArtificial SequenceAmino Acid
sequence of the heavy chain of a humanized OKT3 13Met Gly Trp Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15 Val His
Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln 20 25 30
Pro Gly Arg Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35
40 45 Thr Arg Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
Asn Tyr Asn65 70 75 80 Gln Lys Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn 85 90 95 Thr Ala Phe Leu Gln Met Asp Ser Leu
Arg Pro Glu Asp Thr Gly Val 100 105 110 Tyr Phe Cys Ala Arg Tyr Tyr
Asp Asp His Tyr Cys Leu Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Pro
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165
170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser225 230 235 240 Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 245 250 255 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290
295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val385 390 395 400 Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410
415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys465
* * * * *