U.S. patent application number 13/645332 was filed with the patent office on 2013-03-14 for tnf-alpha antagonists and methotrexate in the treatment of tnf-mediated disease.
This patent application is currently assigned to The Mathilda and Terence Kennedy Institute of Rheumatology Trust. The applicant listed for this patent is Marc Feldmann, Ravinder Nath Maini. Invention is credited to Marc Feldmann, Ravinder Nath Maini.
Application Number | 20130064816 13/645332 |
Document ID | / |
Family ID | 24773909 |
Filed Date | 2013-03-14 |
United States Patent
Application |
20130064816 |
Kind Code |
A1 |
Feldmann; Marc ; et
al. |
March 14, 2013 |
TNF-alpha ANTAGONISTS AND METHOTREXATE IN THE TREATMENT OF
TNF-MEDIATED DISEASE
Abstract
Methods for treating and/or preventing a TNF-mediated disease in
an individual are disclosed. Also disclosed is a composition
comprising methotrexate and an anti-tumor necrosis factor antibody.
TNF-mediated diseases include rheumatoid arthritis, Crohn's
disease, and acute and chronic immune diseases associated with
transplantation.
Inventors: |
Feldmann; Marc; (London,
GB) ; Maini; Ravinder Nath; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Feldmann; Marc
Maini; Ravinder Nath |
London
London |
|
GB
GB |
|
|
Assignee: |
The Mathilda and Terence Kennedy
Institute of Rheumatology Trust
|
Family ID: |
24773909 |
Appl. No.: |
13/645332 |
Filed: |
October 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12583851 |
Aug 26, 2009 |
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13645332 |
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11225631 |
Sep 12, 2005 |
7846442 |
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12583851 |
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09754004 |
Jan 3, 2001 |
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11225631 |
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08690775 |
Aug 1, 1996 |
6270766 |
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09754004 |
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Current U.S.
Class: |
424/133.1 ;
424/142.1; 424/145.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61P 37/06 20180101; A61K 2039/545 20130101; A61K 38/1793 20130101;
A61K 39/395 20130101; A61K 2039/505 20130101; A61P 37/00 20180101;
A61K 39/39541 20130101; C07K 2319/00 20130101; A61P 19/02 20180101;
Y10S 514/885 20130101; A61K 31/505 20130101; A61K 31/519 20130101;
A61K 45/06 20130101; A61P 1/00 20180101; A61P 29/00 20180101; C07K
16/241 20130101; C07K 2317/24 20130101; A61K 31/505 20130101; A61K
2300/00 20130101; A61K 39/3955 20130101; A61K 2300/00 20130101;
A61K 39/395 20130101; A61K 2300/00 20130101; A61K 38/1793 20130101;
A61K 2300/00 20130101; A61K 39/39541 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/133.1 ;
424/145.1; 424/142.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 29/00 20060101 A61P029/00; A61P 19/02 20060101
A61P019/02; A61P 37/06 20060101 A61P037/06 |
Claims
1-38. (canceled)
39. A method of suppressing an immune response to a recombinant
anti-human tumor necrosis factor-.alpha. monoclonal antibody in an
individual suffering from rheumatoid arthritis to whom the antibody
is administered, which comprises treating the individual according
to a regimen comprising administering to the individual multiple
doses of the antibody at a dosage between 0.01 and 100 mg/kg of the
individual's body weight at intervals of weeks so as to induce
immunological tolerance to the antibody and administering multiple
doses of methotrexate at an interval of a week or weeks so as to
potentiate said immunological tolerance in the individual and
thereby suppress the individual's immune response to the
antibody.
40. The method of claim 39, wherein the antibody is administered
subcutaneously or by intravenous infusion and is a chimeric, human,
or humanized monoclonal antibody.
41. The method of claim 40, wherein the antibody is administered by
intravenous infusion and is a chimeric monoclonal antibody.
42. The method of claim 41, wherein the chimeric monoclonal
antibody is cA2 and is administered as multiple infusions at a
dosage between 1 and 20 mg/kg body weight of the individual.
43. The method of claim 42, wherein the chimeric monoclonal
antibody cA2 is administered as multiple infusions of 3 mg/kg body
weight of the individual.
44. The method of claim 42, wherein the chimeric monoclonal
antibody cA2 is administered as multiple infusions of 10 mg/kg body
weight of the individual.
45. The method of claim 40, wherein the antibody is administered
subcutaneously and is a human or humanized monoclonal antibody.
46. The method of claim 39, wherein the antibody is administered
adjunctively with the administration of methotrexate.
47. The method of claim 40, wherein the antibody is administered
adjunctively with the administration of methotrexate.
48. The method of claim 41, wherein the antibody is administered
adjunctively with the administration of methotrexate.
49. The method of claim 42, wherein the antibody is administered
adjunctively with the administration of methotrexate.
50. The method of claim 43, wherein the antibody is administered
adjunctively with the administration of methotrexate.
51. The method of claim 44, wherein the antibody is administered
adjunctively with the administration of methotrexate.
52. The method of claim 45, wherein the antibody is administered
adjunctively with the administration of methotrexate.
53. The method of claim 39, wherein the antibody is administered
concomitantly with the administration of methotrexate.
54. The method of claim 40, wherein the antibody is administered
concomitantly with the administration of methotrexate.
55. The method of claim 41, wherein the antibody is administered
concomitantly with the administration of methotrexate.
56. The method of claim 42, wherein the antibody is administered
concomitantly with the administration of methotrexate.
57. The method of claim 43, wherein the antibody is administered
concomitantly with the administration of methotrexate.
58. The method of claim 44, wherein the antibody is administered
concomitantly with the administration of methotrexate.
59. The method of claim 45, wherein the antibody is administered
concomitantly with the administration of methotrexate.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 08/690,775, filed Aug. 1, 1996, which is a continuation-in-part
of U.S. application Ser. No. 08/607,419, filed Feb. 28, 1996, now
abandoned, which is a continuation-in-part of International
Application No. PCT/GB94/00462, filed Mar. 10, 1994, which is a
continuation-in-part of U.S. application Ser. No. 08/403,785, now
U.S. Pat. No. 5,741,488, which is the U.S. National Phase of
International Application No. PCT/GB93/02070, filed Oct. 6, 1993,
which is a continuation-in-part of U.S. application Ser. No.
07/958,248, filed Oct. 8, 1992, now abandoned, the teachings of all
of which are entirely incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Monocytes and macrophages secrete cytokines known as tumor
necrosis factor alpha (TNF.alpha.) and tumor necrosis factor beta
(TNF.beta.) in response to endotoxin or other stimuli. TNF.alpha.
is a soluble homotrimer of 17 kD protein subunits (Smith et al., J.
Biol. Client. 262:6951-6954 (1987)). A membrane-bound 26 kD
precursor form of TNF also exists (Kriegler et al., Cell 53:45-53
(1988)). For reviews of TNF, see Beutler et al., Nature 320:584
(1986); Old, Science 230:630 (1986); and Le et al., Lab. Invest.
56:234 (1987).
[0003] Cells other than monocytes or macrophages also produce
TNF.alpha.. For example, human non-monocytic tumor cell lines
produce tumor necrosis factor (TNF) (Rubin et al., J. Exp. Med.
164:1350 (1986); Spriggs et al., Proc. Natl. Acad. Set. USA 84:6563
(1987)). CD4+ and CD8+ peripheral blood T lymphocytes and some
cultured T and B cell lines (Cuturi et al., J. Exp. Med. 165:1581
(1987); Sting et al., J. Exp. Med. 168:1539 (1988); Turner as al.,
Eur. J. Immunol. 17:1807-1814 (1987)) also produce TNF.alpha..
[0004] TNF causes pro-inflammatory actions which result in tissue
injury, such as degradation of cartilage and bone, induction of
adhesion molecules, inducing procoagulant activity on vascular
endothelial cells (Pober et al., J. Immunol. 136:1680 (1986)),
increasing the adherence of neutrophils and lymphocytes (Pober et
al, J. Immunol. 138:3319 (1987)), and stimulating the release of
platelet activating factor from macrophages, neutrophils and
vascular endothelial cells (Camussi et al., J. Exp. Med. 166:1390
(1987)).
[0005] Recent evidence associates TNF with infections (Cerami et
al., Immunol. Today 9:28 (1988)), immune disorders, neoplastic
pathologies (Oliff et al., Cell 50:555 (1987)), autoimmune
pathologies and graft-versus-host pathologies (Piguet et al., J.
Exp. Med. 166:1280 (1987)). The association of TNF with cancer and
infectious pathologies is often related to the host's catabolic
state. Cancer patients suffer from weight loss, usually associated
with anorexia.
[0006] The extensive wasting which is associated with cancer, and
other diseases, is known as "cachexia" (Kern et al., J. Parent.
Enter. Nutr. 12:286-298 (1988)). Cachexia includes progressive
weight loss, anorexia, and persistent erosion of body mass in
response to a malignant growth. The fundamental physiological
derangement can relate to a decline in food intake relative to
energy expenditure. The cachectic state causes most cancer
morbidity and mortality. TNF can mediate cachexia in cancer,
infectious pathology, and other catabolic states.
[0007] TNF also plays a central role in gram-negative sepsis and
endotoxic shock (Michie et al., Br. J. Surg. 76:670-671 (1989);
Debets et al., Second Vienna Shock Forum, p. 463-466 (1989);
Simpson et al., Crit. Care Clin. 5:27-47 (1989)), including fever,
malaise, anorexia, and cachexia. Endotoxin strongly activates
monocyte/macrophage production and secretion of TNF and other
cytokines (Kombluth et al., J. Immunol. 137:2585-2591 (1986)). TNF
and other monocyte-derived cytokines mediate the metabolic and
neurohormonal responses to endotoxin (Michie et al., New Engl. J.
Med. 318:1481-1486 (1988)). Endotoxin administration to human
volunteers produces acute illness with flu-like symptoms including
fever, tachycardia, increased metabolic rate and stress hormone
release (Revhaug et al., Arch. Surg. 123:162-170 (1988)).
Circulating TNF increases in patients suffering from Gram-negative
sepsis (Waage et al., Lancet 1:355-357 (1987); Hammerle et al.,
Second Vienna Shock Forum p. 715-718 (1989); Debets et al., Crit.
Care Med. 17:489-497 (1989); Calandra et al., J. Infect. Dis.
161:982-987 (1990)).
[0008] Thus, TNF.alpha. has been implicated in inflammatory
diseases, autoimmune diseases, viral, bacterial and parasitic
infections, malignancies, and/or neurogenerative diseases and is a
useful target for specific biological therapy in diseases, such as
rheumatoid arthritis and Crohn's disease. Beneficial effects in
open-label trials with a chimeric monoclonal antibody to TNF.alpha.
(cA2) have been reported with suppression of inflammation (Elliott
et al., Arthritis Rheum. 36:1681-1690 (1993); Elliott et al.,
Lancet 344:1125-1127 (1994)). See also, Van Dullemen et al.,
Gastroenterology 109:129-135 (1995). Beneficial results in a
randomized, double-blind, placebo-controlled trial with cA2 have
also been reported with suppression of inflammation (Elliott et
al., Lancet 344:1105-1110 (1994)).
SUMMARY OF THE INVENTION
[0009] The present invention is based on the discovery that
treatment of patients suffering from a TNF-mediated disease with a
tumor necrosis factor antagonist, such as an anti-tumor necrosis
factor antibody, as adjunctive and/or concomitant therapy to
methotrexate therapy produces a rapid and sustained reduction in
the clinical signs and symptoms of the disease. The present
invention is also based on the unexpected and dramatic discovery
that a multiple dose regimen of a tumor necrosis factor antagonist,
such as an anti-tumor necrosis factor antibody, when administered
adjunctively with methotrexate to an individual suffering from a
TNF-mediated disease produces a highly beneficial or synergistic
clinical response for a significantly longer duration compared to
that obtained with a single or multiple dose regimen of the
antagonist administered alone or that obtained with methotrexate
administered alone. As a result of Applicants' invention, a method
is provided herein for treating and/or preventing a TNF-mediated
disease in an individual comprising co-administering an anti-TNF
antibody or a fragment thereof and methotrexate to the individual
in therapeutically effective amounts. In a particular embodiment,
methotrexate is administered in the form of a series of low doses
separated by intervals of days or weeks.
[0010] A method is also provided herein for treating and/or
preventing recurrence of a TNF-mediated disease in an individual
comprising co-administering an anti-TNF antibody or a fragment
thereof and methotrexate to the individual in therapeutically
effective amounts. TNF-mediated diseases include rheumatoid
arthritis, Crohn's disease, and acute and chronic immune diseases
associated with an allogenic transplantation (e.g., renal, cardiac,
bone Marrow, liver, pancreatic, small intestine, skin or lung
transplantation).
[0011] Therefore, in one embodiment, the invention relates to a
method of treating and/or preventing rheumatoid arthritis in an
individual comprising co-administering an anti-TNF antibody or a
fragment thereof and methotrexate to the individual in
therapeutically effective amounts. In a second embodiment, the
invention relates to a method of treating and/or preventing Crohn's
disease in an individual comprising co-administering an anti-TNF
antibody or a fragment thereof and methotrexate to the individual
in therapeutically effective amounts. In a third embodiment, the
invention relates to a method of treating and/or preventing other
autoimmune diseases and/or acute or chronic immune disease
associated with a transplantation in an individual, comprising
co-administering an anti-TNF antibody or a fragment thereof and
methotrexate to the individual in therapeutically effective
amounts.
[0012] A further embodiment of the invention relates to
compositions comprising an anti-TNF antibody or a fragment thereof
and methotrexate.
[0013] In addition to anti-TNF antibodies, TNF antagonists include
anti-TNF antibodies and receptor molecules which bind specifically
to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF
release or its action on target cells, such as thalidomide,
tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and
rolipram), A2b adenosine receptor agonists and A2b adenosine
receptor enhancers; and compounds which prevent and/or inhibit TNF
receptor signalling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-1C are a set of three graphs showing the results
over time for swollen joint count in rheumatoid arthritis (RA)
patients receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10 mg/kg)
with or without methotrexate. Results for the placebo group
(methotrexate alone) are shown with the 1 mg/kg group. The number
of patients with data at each evaluation visit is shown at the
bottom of each graph. White circle=-methotrexate (MTX-); black
circle=+methotrexate (MTX+); square=placebo.
[0015] FIGS. 2A-2C are a set of three graphs showing the results
over time for tender joint count in RA patients receiving cA2
treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or without
methotrexate. Results for the placebo group (methotrexate alone)
are shown with the 1 mg/kg group. The number of patients with data
at each evaluation visit is shown at the bottom of each graph.
White circle=-methotrexate; black circle=+methotrexate;
square=placebo.
[0016] FIGS. 3A-3C are a set of three graphs showing the results
over time for the Physician's Global Disease Assessment in RA
patients receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10 mg/kg)
with or without methotrexate. Results for the placebo group
(methotrexate alone) are shown with the 1 mg/kg group. The number
of patients with data at each evaluation visit is shown at the
bottom of each graph. White circle=-methotrexate; black
circle=+methotrexate; square=placebo.
[0017] FIGS. 4A-4C are a set of three graphs showing the results
over time for the Patient Disease Assessment in RA patients
receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or
without methotrexate. Results for the placebo group (methotrexate
alone) are shown with the 1 mg/kg group. The number of patients
with data at each evaluation visit is shown at the bottom of each
graph. White circle=-methotrexate; black circle=+methotrexate;
square=placebo.
[0018] FIGS. 5A-5C are a set of three graphs allowing the results
over time for C-reactive protein (CRP) concentration in RA patients
receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10 mg/kg) with or
without methotrexate. Results for the placebo group (methotrexate
alone) are shown with the 1 mg/kg group. The number of patients
with data at each evaluation visit is shown at the bottom of each
graph. White circle=-methotrexate; black circle=+methotrexate;
square=placebo.
[0019] FIGS. 6A-6C are a set of three graphs showing the results
over time for the Health Assessment Questionnaire (HAQ) in RA
patients receiving cA2 treatment (1 mg/kg, 3 mg/kg or 10 mg/kg)
with or without methotrexate. Results for the placebo group
(methotrexate alone) are shown with the 1 mg/kg group. The number
of patients with data at each evaluation visit is shown at the
bottom of each graph. White circle=-methotrexate; black
circle=+methotrexate; square=placebo.
[0020] FIGS. 7A-7F are a set of six graphs showing the serum cA2
concentration in each RA patient receiving cA2 treatment (1 mg/kg,
3 mg/kg or 10 mg/kg) with or without methotrexate, plotted over
time. Data plotted are the serum cA2 concentrations obtained just
before the administration of cA2 at weeks 2, 6, 10 and 14 and then
at weeks 18 and 26. The scales for the serum cA2 concentration are
condensed with higher doses of cA2.
[0021] FIGS. 8A and 8B are a set of two graphs showing the median
serum cA2 concentration over time in RA patients receiving 3 mg/kg
cA2 (top panel) or 10 mg/kg cA2 (bottom panel) with or without
methotrexate. Square=+methotrexate; circle or
triangle=-methotrexate.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention relates to the discovery that tumor
necrosis factor antagonists can be administered to patients
suffering from a TNF-mediated disease as adjunctive and/or
concomitant therapy to methotrexate therapy, with good to excellent
alleviation of the signs and symptoms of the disease. The present
invention also relates to the discovery that tumor necrosis factor
antagonists can be administered to patients suffering from a
TNF-mediated disease in multiple doses and as adjunctive and/or
concomitant therapy to methotrexate therapy, with a significant
improvement in duration of clinical response.
[0023] As a result of Applicants' invention, a method is provided
herein for treating and/or preventing a TNF-mediated disease in an
individual, comprising co-administering methotrexate and a tumor
necrosis factor antagonist to the individual in therapeutically
effective, amounts. The TNF antagonist and methotrexate can be
administered simultaneously or sequentially. The TNF antagonist and
methotrexate can each be administered in single or multiple doses.
Multiple TNF antagonists can be co-administered with methotrexate.
Other therapeutic regimens and agents can be used in combination
with the therapeutic co-administration of TNF antagonists and
methotrexate or other drugs that suppress the immune system.
[0024] A method is also provided herein for treating and/or
preventing recurrence of a TNF-mediated disease in an individual
comprising co-administering methotrexate and a TNF antagonist to
the individual in therapeutically effective amounts.
[0025] As used herein, a "TNF-mediated disease" refers to a TNF
related pathology or disease. TNF related pathologies or diseases
include, but are not limited to, the following:
[0026] (A) acute and chronic immune and autoimmune pathologies,
such as, but not limited to, rheumatoid arthritis (RA), juvenile
chronic arthritis (JCA), thyroiditis, graft versus host disease
(GVHD), scleroderma, diabetes mellitus, Graves' disease, allergy,
acute or chronic immune disease associated with an allogenic
transplantation, such as, but not limited to, renal
transplantation, cardiac transplantation, bone marrow
transplantation, liver transplantation, pancreatic transplantation,
small intestine transplantation, lung transplantation and skin
transplantation;
[0027] (B) infections, including, but not limited to, sepsis
syndrome, cachexia, circulatory collapse and shock resulting from
acute or chronic bacterial infection, acute and chronic parasitic
and/or infectious diseases, bacterial, viral or fungal, such as a
human immunodeficiency virus (HIV), acquired immunodeficiency
syndrome (AIDS) (including symptoms of cachexia, autoimmune
disorders, AIDS dementia complex and infections);
[0028] (C) inflammatory diseases, such as chronic inflammatory
pathologies, including chronic inflammatory pathologies such as,
but not limited to sarcoidosis, chronic inflammatory bowel disease,
ulcerative colitis, and Crohn's pathology or disease; vascular
inflammatory pathologies, such as, but not limited to, disseminated
intravascular coagulation, atherosclerosis, Kawasaki's pathology
and vasculitis syndromes, such as, but not limited to,
polyarteritis nodosa, Wegener's granulomatosis, Henoch-Schonlein
purpura, giant cell arthritis and microscopic vasculitis of the
kidneys; chronic active hepatitis; Sjogren's syndrome;
spondyloarthropathies, such as ankylosing spondylitis, psoriatic
arthritis and sporidylitis, enteropathic arthritis and spondylitis,
reactive arthritis and arthritis associated with inflammatory bowel
disease; and uveitis;
[0029] (D) neurodegenerative diseases, including, but not limited
to, demyelinating diseases, such as multiple sclerosis and acute
transverse myelitis; myasthenia gravis; extrapyramidal and
cerebellar disorders, such as lesions of the corticospinal system;
disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders, such as Huntington's chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs which block central nervous system (CNS) dopamine
receptors; hypokinetic movement disorders, such as Parkinson's
disease; progressive supranuclear palsy; cerebellar and
spinocerebellar disorders, such as astructural lesions of the
cerebellum; spinocerebellar degenerations (spinal ataxia,
Friedreich's ataxia, cerebellar cortical degenerations, multiple
systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machadokseph)); and systemic disorders (Refsum's disease,
abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial
multisystem disorder); disorders of the motor unit, such as
neurogenic muscular atrophies (anterior horn cell degeneration,
such as amyotrophic lateral sclerosis, infantile spinal muscular
atrophy and juvenile spinal muscular atrophy); Alzheimer's disease;
Down's syndrome in middle age; diffuse Lewy body disease; senile
dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic
alcoholism; primary biliary cirrhosis; cryptogenic fibrosing
alveolitis and other fibrotic lung diseases; hemolytic anemia;
Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis,
Hallervorden-Spatz disease; and dementia pugilistica, or any subset
thereof;
[0030] (E) malignant pathologies involving TNF-secreting tumors or
other Malignancies involving TNF, such as, but not limited to,
leukemias (acute, chronic myelocytic, chronic lymphocytic and/or
myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's
lymphomas; such as malignant lymphomas (Burkitt's lymphoma or
Mycosis fungoides));
[0031] (F) cachectic syndromes and other pathologies and diseases
involving excess TNF, such as, but not limited to, cachexia of
cancer, parasitic disease and heart failure; and
[0032] (G) alcohol-induced hepatitis and other forms of chronic
hepatitis.
[0033] See, e.g., Berkow at al., Eds., The Merck Manual, 16th
edition, chapter 11, pp. 1380-1529, Merck and Co., Rahway; N.J.,
1992, incorporated herein by reference.
[0034] The terms "recurrence", "flare-up" or "relapse" are defined
to encompass the reappearance of one or more symptoms of the
disease state. For example, in the case of rheumatoid arthritis, a
reoccurrence can include the experience of one or more of swollen
joints, morning stiffness or joint tenderness.
[0035] In one embodiment, the invention relates to a method of
treating and/or preventing rheumatoid arthritis in an individual
comprising co-administering methotrexate and a TNF antagonist to
the individual in therapeutically effective amounts.
[0036] In a second embodiment, the invention relates to a method
far treating and/or preventing Crohn's disease man individual
comprising co-administering a methotrexate and a TNF antagonist to
the individual in therapeutically effective amounts.
[0037] In a third embodiment, the invention relates to a method for
treating and/or preventing an acute or chronic immune disease
associated with an allogenic transplantation in an individual
comprising co-administering methotrexate and a TNF antagonist to
the individual in therapeutically effective amounts. As used
herein, a "transplantation" includes organ, tissue or cell
transplantation, such as renal transplantation, cardiac
transplantation, bone marrow transplantation, liver
transplantation, pancreatic transplantation, small intestine
transplantation, skin transplantation and lung transplantation.
[0038] The benefits of combination therapy with methotrexate and
TNF antagonists include high clinical response rates for
significantly longer durations in comparison with that obtained
with treatment with each therapeutic modality separately. In
addition, methotrexate significantly reduces immunogenicity of
anti-TNF antibodies, thus permitting administration of multiple
dosages of anti-TNF antibodies with enhanced safety. The results
described herein suggest that methotrexate can be used to reduce
immunogenicity of other antibodies or proteins. Based on the
results described herein, methotrexate can be used in other forms
of antibody therapy, such as anti-IL-2 antibody therapy. This
method is particularly pertinent in therapies other than anti-CD4
antibody therapy.
[0039] In a further embodiment, the invention relates to
compositions comprising methotrexate and a TNF antagonist. The
compositions of the present invention are useful for treating a
subject having a pathology or condition associated with abnormal
levels of a substance reactive with a TNF antagonist, in particular
TNF in excess of, or less than, levels present in a normal healthy
subject, where such excess or diminished levels occur in a
systemic, localized or particular tissue type or location in the
body. Such tissue types can include, but are not limited to, blood,
lymph, central nervous system (CNS), liver, kidney, spleen, heart
muscle or blood vessels, brain or spinal cord white matter or grey
matter, cartilage, ligaments, tendons, lung, pancreas, ovary,
testes, prostate. Increased or decreased TNF concentrations
relative to normal levels can also be localized to specific regions
or cells in the body, such as joints, nerve blood vessel junctions,
bones, specific tendons or ligaments, or sites of infection, such
as bacterial or viral infections.
Tumor Necrosis Factor Antagonists
[0040] As used herein, a "tumor necrosis factor antagonist"
decreases, blocks, inhibits, abrogates or interferes with TNF
activity in vivo. For example, a suitable TNF antagonist can bind
TNF and includes anti-TNF antibodies and receptor molecules which
bind specifically to TNF. A suitable TNF antagonist can also
prevent or inhibit TNF synthesis and/or TNF release and includes
compounds such as thalidomide, tenidap, and phosphodiesterase
inhibitors, such as, but not limited to, pentoxifylline and
rolipram. A suitable TNF antagonist that can prevent or inhibit TNF
synthesis and/or TNF release also includes A2b adenosine receptor
enhancers and A2b adenosine receptor agonists (e.g.,
5'-(N-cyclopropyl)-carboxamidoadenosine,
5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine and
R--N.sup.6-phenyl-2-propyladenosine). See, for example, Jacobson
(GB 2 289 218 A), the teachings of which are entirely incorporated
herein by reference. A suitable TNF antagonist can also prevent or
inhibit TNF receptor signalling.
Anti-TNF Antibodies
[0041] As used herein, an "anti-tumor necrosis factor antibody"
decreases, blocks, inhibits, abrogates or interferes with TNF
activity in viva. Anti-TNF antibodies useful in the methods and
compositions of the present invention include monoclonal, chimeric,
humanized, resurfaced and recombinant antibodies and fragments
thereof which are characterized by high affinity binding to TNF and
low toxicity (including human anti-murine antibody (HAMA) and/or
human anti-chimeric antibody (HACA) response). In particular, an
antibody where the individual components, such as the variable
region, constant region and framework, individually and/or
collectively possess low immunogenicity is useful in the present
invention. The antibodies which can be used in the invention are
characterized by their ability to treat patients for extended
periods with good to excellent alleviation of symptoms and low
toxicity. Low immunogenicity and/or high affinity, as well as other
undefined properties, may contribute to the therapeutic results
achieved.
[0042] An example of a high affinity monoclonal antibody useful in
the methods and compositions of the present invention is murine
monoclonal antibody (mAb) A2 and antibodies which will
competitively inhibit in vivo the binding to human TNF.alpha. of
anti-TNF.alpha. murine mAb A2 or an antibody having substantially
the same specific binding characteristics, as well as fragments and
regions thereof. Murine monoclonal antibody A2 and chimeric
derivatives thereof, such as cA2, are described in U.S. application
Ser. No. 08/192,093 (filed Feb. 4, 1994), U.S. application Ser. No.
08/192,102 (filed Feb. 4, 1994; now U.S. Pat. No. 5,656,272), U.S.
application Ser. No. 08/192,861 (filed Feb. 4, 1994; now U.S. Pat.
No. 5,919,452), U.S. application Ser. No. 08/324,799 (filed Oct.
18, 1994; now U.S. Pat. No. 5,698,195), and Le, J. et al.,
International Publication No. WO 92/16553 (published Oct. 1, 1992),
which references are entirely incorporated herein by reference. A
second example of a high affinity monoclonal antibody useful in the
methods and compositions of the present invention is murine mAb 195
and antibodies which will competitively inhibit in vivo the binding
to human TNF.alpha. of anti-TNF.alpha. murine 195 or an antibody
having substantially the same specific binding characteristics, as
well as fragments and regions thereof. Other high affinity
monoclonal antibodies useful in the methods and compositions of the
present invention include murine mAb 114 and murine mAb 199 and
antibodies which will competitively inhibit in vivo the binding to
human TNF.alpha. of anti-TNF.alpha. murine mAb 114 or mAb 199 or an
antibody having substantially the same specific binding
characteristics of mAb 114 or mAb 199, as well as fragments and
regions thereof. Murine monoclonal antibodies 114, 195 and 199 and
the method for producing them are described by Moller, A. et al.
(Cytokine 2(3):162-169 (1990)), the teachings of which are entirely
incorporated herein by reference. Preferred methods for determining
mAb specificity, and affinity by competitive inhibition can be
found in Harlow, et al., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988);
Colligan et al., eds., Current Protocols in Immunology, Greene
Publishing Assoc. and Wiley Interscience, New York (1992, 1993);
Kozbor et al., Immunol. Today 4:72-79 (1983); Ausubel et al. eds.,
Current Protocols in Molecular Biology, Wiley Interscience, New
York (1987, 1992, 1993); and Muller, Meth. Enzymol. 92:589-601
(1983), which references are entirely incorporated herein by
reference.
[0043] Additional examples of monoclonal anti-TNF antibodies that
can be used in the present invention are described in the art (see,
e.g., U.S. application Ser. No. 07/943,852 (filed Sep. 11, 1992);
Rathjen et al., International Publication No. WO 91/02078
(published Feb. 21, 1991); Rubin et al., EPO Patent Publication
0218868 (published Apr. 22, 1987); Yone et al., EPO Patent
Publication No. 0288088 (Oct. 26, 1988); Liang, et al., Biochem.
Biophys. Res. Comm. 137:847-854 (1986); Meager, or al., Hybridoma
6:305-311 (1987); Fendly et al., Hybridoma 6:359-369 (1987);
Bringman, et al., Hybridoma 6:489-507 (1987); Hirai, et al., J.
Immunol. Meth. 96:57-62 (1987); Moller, et al., Cytokine 2:162-169
(1990), which references are entirely incorporated herein by
reference).
[0044] Chimeric antibodies are immunoglobulin molecules
characterized by two or more segments or portions derived from
different animal species. Generally, the variable region of the
chimeric antibody is derived from a non-human mammalian antibody,
such as a murine mAb, and the immunoglobulin constant region is
derived from a human immunoglobulin molecule. Preferably, a
variable region with low immunogenicity is selected and combined
with a human constant region which also has low immunogenicity, the
combination also preferably having low immunogenicity. "Low"
immunogenicity is defined herein as raising significant HACA or
HAMA responses in less than about 75%, or preferably less than
about 50% of the patients treated and/or raising low titres in the
patient treated (less than about 300, preferably less than about
100 measured with a double antigen enzyme immunoassay) (Elliott or
al., Lancet 344:1125-1127 (1994), incorporated herein by
reference).
[0045] As used herein, the term "chimeric antibody" includes
monovalent, divalent or polyvalent immunoglobulins. A monovalent
chimeric antibody is a dimer (HL)) formed by a chimeric H chain
associated through disulfide bridges with a chimeric L chain. A
divalent chimeric antibody is a tetramer (H2L2) formed by two HL
dimers associated through at least one disulfide bridge. A
polyvalent chimeric antibody can also be produced, for example, by
employing a CH region that aggregates (e.g., from an IgM H chain,
or .mu. chain).
[0046] Antibodies comprise individual heavy (H) and/or light (L)
immunoglobulin chains. A chimeric H chain comprises an antigen
binding region derived from the H chain of a non-human antibody
specific for TNF, which is linked to at least a portion of a human
H chain C region (CH), such as CH1 or CH2. A chimeric L chain
comprises an antigen binding region derived from the L chain of a
non-human antibody specific for TNF, linked to at least a portion
of a human L chain C region (CL).
[0047] Chimeric antibodies and methods for their production have
been described in the art (Morrison et al., Proc. Natl. Acad. Sci.
USA 81:6351-6855 (1984); Boulianne as al., Nature 312:643-646
(1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi at
al., European Patent Application No. 171496 (published Feb. 19,
1985); Morrison et al., European Patent Application No. 173494
(published Mar. 5, 1986); Neuberger at al., PCT Application No. WO
86/01533, (published Mar. 13, 1986); Kudo at al., European Patent
Application No. 184187 (published Jun. 11, 1986); Morrison at al.,
European Patent Application No. 173494 (published Mar. 5, 1986);
Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al.,
International Publication No. PCT/US86/02269 (published May 7,
1987); Liu at al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987);
Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better at
al., Science 240:1041-1043 (1988); and Harlow and Lane, Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory, New York,
1988). These references are entirely incorporated herein by
reference.
[0048] The anti-TNF chimeric antibody can comprise, for example,
two light chains and two heavy chains, each of the chains
comprising at least part of a human constant region and at least
part of a variable (V) region of non-human origin having
specificity to human TNF, said antibody binding with high affinity
to an inhibiting and/or neutralizing epitope of human TNF, such as
the antibody cA2. The antibody also includes a fragment or a
derivative of such an antibody, such as one or more portions of the
antibody chain, such as the heavy chain constant or variable
regions, or the light chain constant or variable regions.
[0049] Humanizing and resurfacing the antibody can further reduce
the immunogenicity of the antibody. See, for example, Winter (U.S.
Pat. No. 5,225,539 and EP 239,400 B1), Padlan et al. (EP 519,596
A1) and Pedersen et al. (EP 592,106 A1). These references are
incorporated herein by reference.
[0050] Preferred antibodies useful in the methods and compositions
of the present invention are high affinity human-murine chimeric
anti-TNF antibodies, and fragments or regions thereof, that have
potent inhibiting and/or neutralizing activity in vivo against
human TNF.alpha.. Such antibodies and chimeric antibodies can
include those generated by immunization using purified recombinant
TNF.alpha. or peptide fragments thereof comprising one or more
epitopes.
[0051] An example of such a chimeric antibody is cA2 and antibodies
which will competitively inhibit in vivo the binding to human
TNF.alpha. of anti-TNF.alpha. murine mAb A2, chimeric mAb cA2, or
an antibody having substantially the same specific binding
characteristics, as well as fragments and regions thereof. Chimeric
mAb cA2 has been described, for example, in U.S. application Ser.
No. 08/192,093 (filed Feb. 4, 1994), U.S. application Ser. No.
08/192,102 (filed Feb. 4, 1994; now U.S. Pat. No. 5,656,272), U.S.
application Ser. No. 08/192,861 (filed Feb. 4, 1994; now U.S. Pat.
No. 5,919,452), and U.S. application Ser. No. 08/324,799 (filed
Oct. 18, 1994; now U.S. Pat. No. 5,698,195), and by Le, J. at al.
(International Publication No. WO 92/16553 (published Oct. 1,
1992)); Knight, D. M. et al. (Mol. Immunol. 30:1443-1453 (1993));
and Siegel, S. A. at al. (Cytokine 7(1): 15-25 (1995)). These
references are entirely incorporated herein by reference.
[0052] Chimeric A2 anti-TNF consists of the antigen binding
variable region of the high-affinity neutralizing mouse anti-human
TNF IgG1 antibody, designated A2, and the constant regions of a
human IgG1, kappa immunoglobulin. The human IgG1 Fc region improves
allogeneic antibody effector function, increases the circulating
serum half-life and decreases the immunogenicity of, the antibody.
The avidity and epitope specificity of the chimeric A2 is derived
from the variable region of the murine A2. Chimeric A2 neutralizes
the cytotoxic effect of both natural and recombinant human TNF in a
dose dependent manner. From binding assays of cA2 and recombinant
human TNF, the affinity constant of cA2 was calculated to be
1.8.times.10.sup.9M.sup.-1. Preferred methods for determining mAb
specificity and affinity by competitive inhibition can be found in
Harlow, at al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, 1988; Culligan at
al., eds., Current Protocols in Immunology, Greene Publishing
Assoc. and Wiley Interscience, New York, (1992, 1993); Kozbor at
al., Immunol. Today 4:72-79 (1983); Ausubel et al., eds. Current
Protocols in Molecular Biology, Wiley Interscience, New York (1987,
1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), which
references are entirely incorporated herein by reference.
[0053] As used herein, the term "antigen binding region" refers to
that portion of an antibody molecule which contains the amino acid
residues that interact with an antigen and confer on the antibody
its specificity and affinity for the antigen. The antibody region
includes the "framework" amino acid residues necessary to maintain
the proper conformation of the antigen-binding residues. Generally,
the antigen binding region will be of murine origin. In other
embodiments, the antigen binding region can be derived from other
animal species, such as sheep, rabbit, rat or hamster. Preferred
sources for the DNA encoding such a non-human antibody include cell
lines which produce antibody, preferably hybrid cell lines commonly
known as hybridomas. In one embodiment, a preferred hybridoma is
the A2 hybridoma cell line.
[0054] An "antigen" is a molecule or a portion of a molecule
capable of being bound by an antibody which is additionally capable
of inducing an animal to produce antibody capable of selectively
binding to an epitope of that antigen. An antigen can have one or
more than one epitope.
[0055] The term "epitope" is meant to refer to that portion of the
antigen capable of being recognized by and bound by an antibody at
one or more of the antibody's antigen binding region. Epitopes
usually consist of chemically active surface groupings of molecules
such as amino acids or sugar side chains and have specific three
dimensional structural characteristics as well as specific charge
characteristics. By "inhibiting and/or neutralizing epitope" is
intended an epitope, which, when bound by an antibody, results in
loss of biological activity of the molecule containing the epitope,
in viva or in vitro, more preferably in vivo, including binding of
TNF to a TNF receptor. Epitopes of TNF have been identified within
amino acids 1 to about 20, about 56 to about 77, about 108 to about
127 and about 138 to about 149. Preferably, the antibody binds to
an epitope comprising at least about 5 amino acids of TNF within
TNF residues from about 87 to about 107, about 59 to about 80 or a
combination thereof. Generally, epitopes include at least about 5
amino acids and less than about 22 amino acids embracing or
overlapping one or more of these regions.
[0056] For example, epitopes of TNF which are recognized by, and/or
binds with anti-TNF activity, an antibody, and fragments, and
variable regions thereof, include:
TABLE-US-00001 59-80: (SEQ ID NO: 1)
Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-
Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His- Thr-Ile; and/or 87-108:
(SEQ ID NO: 2) Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-
Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro- Glu-Gly.
[0057] The anti-TNF antibodies, and fragments, and variable regions
thereof, that are recognized by, and/or binds with anti-TNF
activity, these epitopes block the action of TNF.alpha. without
binding to the putative receptor binding locus as presented by Eck
and Sprang (J. Biol. Chem. 264(29): 17595-17605 (1989) (amino acids
11-13, 37-42, 49-57 and 155-157 of hTNF.alpha.). Rathjen et al.,
International Publication No. WO 91/02078 (published Feb. 21,
1991), incorporated herein by reference, discloses TNF ligands
which can bind additional epitopes of TNF.
Antibody Production Using Hybridomas
[0058] The techniques to raise antibodies to small peptide
sequences that recognize and bind to those sequences in the free or
conjugated form or when presented as a native sequence in the
context of a large protein are well known in the art. Such
antibodies can be produced by hybridoma or recombinant techniques
known in the art.
[0059] Murine antibodies which can be used in the preparation of
the antibodies useful in the methods and compositions of the
present invention have also been described in Rubin et al., EP
0218868 (published Apr. 22, 1987); Yone et al., EP 0288088
(published Oct. 26, 1988); Liang, et al., Biochem. Biophys. Res.
Comm. 137:847-854 (1986); Meager, et al., Hybridoma 6:305-311
(1987); Fendly et al., Hybridoma 6:359-369 (1987); Bringman, et
al., Hybridoma 6:489-507 (1987); Hirai, et al., J. Immunol. Meth.
96:57-62 (1987); Willer, et al., Cytokine 2:162-169 (1990).
[0060] The cell fusions are accomplished by standard procedures
well known to those skilled in the field of immunology. Fusion
partner cell lines and methods for fusing and selecting hybridomas
and screening for mAbs are well known in the art. See, e.g, Ausubel
infra, Harlow infra, and Colligan infra, the contents of which
references are incorporated entirely herein by reference.
[0061] The TNF.alpha.-specific murine mAb useful in the methods and
compositions of the present invention can be produced in large
quantities by injecting hybridoma or transfectoma cells secreting
the antibody into the peritoneal cavity of mice and, after
appropriate time, harvesting the ascites fluid which contains a
high titer of the mAb, and isolating the mAb therefrom. For such in
viva production of the mAb with a hybridoma (e.g., rat or human),
hybridoma cells are preferably grown in irradiated or athymic nude
mice. Alternatively, the antibodies can be produced by culturing
hybridoma or transfectoma cells in vitro and isolating secreted mAb
from the cell culture medium or recombinantly, in eukaryotic or
prokaryotic cells.
[0062] In one embodiment, the antibody used in the methods and
compositions of the present invention is a mAb which binds amino
acids of an epitope of TNF recognized by A2, rA2 or cA2, produced
by a hybridoma or by a recombinant host. In another embodiment, the
antibody is a chimeric antibody which recognizes an epitope
recognized by A2. In still another embodiment, the antibody is a
chimeric antibody designated as chimeric A2 (cA2).
[0063] As examples of antibodies useful in the methods and
compositions of the present invention, murine mAb A2 is produced by
a cell line designated c134A.
[0064] "Derivatives" of the antibodies including fragments, regions
or proteins encoded by truncated or modified genes to yield
molecular species functionally resembling the immunoglobulin
fragments are also useful in the methods and compositions of the
present invention. The modifications include, but are not limited
to, addition of genetic sequences coding for cytotoxic proteins
such as plant and bacterial toxins. The fragments and derivatives
can be produced from appropriate cells, as is known in the art.
Alternatively, anti-TNF antibodies, fragments and regions can be
bound to cytotoxic proteins or compounds in vitro, to provide
cytotoxic anti-TNF antibodies which would selectively kill cells
having TNF on their surface.
[0065] "Fragments" of the antibodies include, for example, Fab,
Fab', F(ab').sub.2 and Fv. These fragments lack the Fc fragment of
intact antibody, clear more rapidly from the circulation, and can
have less non-specific tissue binding than an intact antibody (Wahl
et al., J. Nucl. Med. 24:316-325 (1983)). These fragments are
produced from intact antibodies using methods well known in the
art, for example by proteolytic cleavage with enzymes such as
papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments).
Recombinant Expression of Anti-TNF Antibodies
[0066] Recombinant and/or chimeric murine-human or human-human
antibodies that inhibit TNF can be produced using known techniques
based on the teachings provided in U.S. application Ser. No.
08/192,093 (filed Feb. 4, 1994), U.S. application Ser. No.
08/192,102 (filed Feb. 4, 1994; now U.S. Pat. No. 5,656,272), U.S.
application Ser. No. 08/192,861 (filed Feb. 4, 1994; now U.S. Pat.
No. 5,919,452), U.S. application Ser. No. 08/324,799 (filed on Oct.
18, 1994; now U.S. Pat. No. 5,698,195) and Le, J. et al.,
International Publication No. WO 92/16553 (published Oct. 1, 1992),
which references are entirely incorporated herein by reference.
See, e.g., Ausubel at al., eds. Current Protocols in Molecular
Biology, Wiley Interscience, New York (1987, 1992, 1993); and
Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, New York (1989), the contents of which are
entirely incorporated herein by reference. See also, e.g., Knight,
D. M., et al., Mol. Immunol 30:1443-1453 (1993); and Siegel, S. A.,
et al., Cytokine 7(1):15-25 (1995), the contents of which are
entirely incorporated herein by reference.
[0067] The DNA encoding an anti-TNF antibody can be genomic DNA or
cDNA which encodes at least one of the heavy chain constant region
(He), the heavy chain variable region (Hc), the light chain
variable region (Lv) and the light chain constant regions (Lc). A
convenient alternative to the use of chromosomal gene fragments as
the source, of DNA encoding the murine V region antigen-binding
segment is the use of cDNA for the construction of chimeric
immunoglobulin genes, e.g., as reported by Liu et al. (Proc. Natl.
Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987)),
which references are entirely incorporated herein by reference. The
use of cDNA requires that gene expression elements appropriate for
the host cell be combined with the gene in order to achieve
synthesis of the desired protein. The use of cDNA sequences is
advantageous over genomic sequences (which contain introns), in
that cDNA sequences can be expressed in bacteria or other hosts
which lack appropriate RNA splicing systems. An example of such a
preparation is set forth below.
[0068] Because the genetic code is degenerate, more than one codon
can be used to encode a particular amino acid. Using the genetic
code, one or more different oligonucleotides can be identified,
each of which would be capable of encoding the amino acid. The
probability that a particular oligonucleotide will, in fact,
constitute the actual XXX-encoding sequence can be estimated by
considering abnormal base pairing relationships and the frequency
with which a particular codon is actually used (to encode a
particular amino acid) in eukaryotic or prokaryotic cells
expressing an anti-TNF antibody or fragment. Such "codon usage
rules" are disclosed by Lathe, et al., J. Mol. Biol. 183:1-12
(1985). Using the "codon usage rules" of Lathe, a single
oligonucleotide, or a set of oligonucleotides, that contains a
theoretical "most probable" nucleotide sequence capable of encoding
anti-TNF variable or constant region sequences is identified.
[0069] Although occasionally an amino acid sequence can be encoded
by only a single oligonucleotide, frequently the amino acid
sequence can be encoded by any of a set of similar
oligonucleotides. Importantly, whereas all of the members of this
set contain oligonucleotides which are capable of encoding the
peptide fragment and, thus, potentially contain the same
oligonucleotide sequence as the gene which encodes the peptide
fragment, only one member of the set contains the nucleotide
sequence that is identical to the nucleotide sequence of the gene.
Because this member is present within the set, and is capable of
hybridizing to DNA even in the presence of the other members of the
set, it is possible to employ the unfractionated set of
oligonucleotides in the same manner in which one would employ a
single oligonucleotide to clone the gene that encodes the
protein.
[0070] The oligonucleotide, or set of oligonucleotides, containing
the theoretical "most probable" sequence capable of encoding an
anti-TNF antibody or fragment including a variable or constant
region is used to identify the sequence of a complementary
oligonucleotide or set of oligonucleotides which is capable of
hybridizing to the "most probable" sequence, or set of sequences.
An oligonucleotide containing such a complementary sequence can be
employed as a probe to identify and isolate the variable or
constant region anti-TNF gene (Sambrook et al., infra).
[0071] A suitable oligonucleotide, or set of oligonucleotides,
which is capable of encoding a fragment of the variable or constant
anti-TNF region (or which is complementary to such an
oligonucleotide, or set of oligonucleotides) is identified (using
the above-described procedure), synthesized, and hybridized by
means well known in the art, against a DNA or, more preferably, a
cDNA preparation derived from cells which are capable of expressing
anti-TNF antibodies or variable or constant regions thereof. Single
stranded oligonucleotide molecules complementary to the "most
probable" variable or constant anti-TNF region peptide coding
sequences can be synthesized using procedures which are well known
to those of ordinary skill in the art (Belagaje, et al., J. Biol.
Chem. 254:5765-5780 (1979); Maniatis, et al., In: Molecular
Mechanisms in the Control of Gene Expression, Nierlich, et al.,
eds., Acad. Press, New York (1976); Wu, et al., Prog. Nucl. Acid
Res. Molec. Biol. 21:101-141 (1978); Khorana, Science 203:614-625
(1979)). Additionally, DNA synthesis can be achieved through the
use of automated synthesizers. Techniques of nucleic acid
hybridization are disclosed by Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York
(1989); and by Haynes, et al., in: Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, D.C. (1985), which
references are entirely incorporated herein by reference.
Techniques such as, or similar to, those described above have
successfully enabled the cloning of genes for human aldehyde
dehydrogenases (Hsu, at al., Proc. Natl. Acad. Sci. USA
82:3771.3775 (1985)), fibronectin (Suzuki, et al., Bur. Mol. Biol.
Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene
(Waiter, at al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)),
tissue-type plasminogen activator (Penn ice, et al., Nature
301:214-221 (1983)) and human placental alkaline phosphatase
complementary DNA (Keun, at al., Proc. Natl. Acad. Sci. USA
82:8715-8719 (1985)).
[0072] In an alternative way of cloning a polynucleotide encoding
an anti-TNF variable or constant region, a library of expression
vectors is prepared by cloning DNA or, more preferably, cDNA. (from
a cell capable of expressing an anti-TNF antibody or variable or
constant region) into an expression vector. The library is then
screened for members capable of expressing a protein which
competitively inhibits the binding of an anti-TNF antibody, such as
A2 or cA2, and which has a nucleotide sequence that is capable of
encoding polypeptides that have the same amino acid sequence as
anti-TNF antibodies or fragments thereof. In this embodiment, DNA,
or more preferably cDNA, is extracted and purified from a cell
which is capable of expressing an anti-TNF antibody or fragment.
The purified cDNA is fragmentized (by shearing, endonuclease
digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or
cDNA fragments from this pool are then cloned into an expression
vector in order to produce a genomic library of expression vectors
whose members each contain a unique cloned DNA or cDNA fragment
such as in a lambda phage library, expression in prokaryotic cell
(e.g., bacteria) or eukaryotic cells, (e.g., mammalian, yeast,
insect or, fungus). See, e.g., Ausubel, infra. Harlow, infra,
Colligan, infra; Nyyssonen at al. Bio/Technology 11:591-595 (1993);
Marks at al., Bio/Technology 11:1145-1149 (October 1993). Once
nucleic acid encoding such variable or constant anti-TNF regions is
isolated, the nucleic acid can be appropriately expressed in a host
cell, along with other constant or variable heavy or light chain
encoding nucleic acid, in order to provide recombinant monoclonal
antibodies that bind TNF with inhibitory activity. Such antibodies
preferably include a murine or human anti-TNF variable region which
contains a framework residue having complementarity determining
residues which are responsible for antigen binding.
[0073] Human genes which encode the constant (C) regions of the
chimeric antibodies, fragments and regions of the present invention
can be derived from a human fetal liver library, by known methods.
Human C region genes can be derived from any human cell including
those which express and produce human immunoglobulins. The human CH
region can be derived from any of the known classes or isotypes of
human H chains, including gamma, .mu., .alpha., .delta. or
.epsilon., and subtypes thereof, such as G1, G2, G3 and G4. Since
the H chain isotype is responsible for the various effector
functions of an antibody, the choice of CH region will be guided by
the desired effector functions, such as complement fixation, or
activity in antibody-dependent cellular cytotoxicity (ADCC).
Preferably, the CH region is derived from gamma 1 (IgG1), gamma 3
(IgG3), gamma 4 (IgG4), or .mu. (IgM). The human CL region can be
derived from either human L chain isotype, kappa or lambda.
[0074] Genes encoding human immunoglobulin C regions are obtained
from human cells by standard cloning techniques (Sambrook, et al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al.,
eds., Current Protocols in Molecular Biology, Wiley Interscience,
New York (1987-1993)). Human C region genes are readily available
from known clones containing genes representing the two classes of
L chains, the five classes of H chains and subclasses thereof.
Chimeric antibody fragments, such as F(ab').sub.2 and Fab, can be
prepared by designing a chimeric H chain gene which is
appropriately truncated. For example, a chimeric gene encoding an H
chain portion of an F(ab').sub.2 fragment would include DNA
sequences encoding the CH1 domain and hinge region of the H chain,
followed by a translational stop codon to yield the truncated
molecule.
[0075] Generally, the murine, human and chimeric antibodies,
fragments and regions are produced by cloning DNA segments encoding
the H and L chain antigen-binding regions of a TNF-specific
antibody, and joining these DNA segments to DNA segments encoding
CH and CL regions, respectively, to produce murine, human or
chimeric immunoglobulin-encoding genes. Thus, in a preferred
embodiment; a fused chimeric gene is created which comprises a
first DNA segment that encodes at least the antigen-binding region
of non-human origin, such as a functionally rearranged V region
with joining (I) segment, linked to a second DNA segment encoding
at least a part of a human C region.
[0076] Therefore, cDNA encoding the antibody V and C regions and
the method of producing a chimeric antibody can involve several
steps, outlined below: [0077] 1. Isolation of messenger RNA (mRNA)
from the cell line producing an anti-TNF antibody and from optional
additional antibodies supplying heavy and light constant regions;
cloning and cDNA production therefrom; [0078] 2. Preparation of a
full length cDNA library from purified mRNA from which the
appropriate V and/or C region gene segments of the L and H chain
genes can be: (i) identified with appropriate probes, (ii)
sequenced, and (iii) made compatible with a C or V gene segment
from another antibody for a chimeric antibody; [0079] 3.
Construction of complete H or L chain coding sequences by linkage
of the cloned specific V region gene segments to cloned C region
gene, as described above; [0080] 4. Expression and production of L
and H chains in selected hosts, including prokaryotic and
eukaryotic cells to provide murine-murine, human-murine,
human-human or human-murine antibodies.
[0081] One common feature of all immunoglobulin H and L chain genes
and their encoded mRNAs is the J region. H and L chain J regions
have different sequences, but a high of sequence homology exists
(greater than 80%) among each group, especially near the C region.
This homology is exploited in this method and consensus sequences
of H and L chain S regions can be used to design oligonucleotides
for use as primers for introducing useful restriction sites into
the J region for subsequent linkage of V region segments to human C
region segments.
[0082] C region cDNA vectors prepared from human cells can be
modified by site-directed mutagenesis to place a restriction site
at the analogous position in the human sequence. For example, one
can clone the complete human kappa chain C (Ck) region and the
complete human gamma-1 C region (C gamma-1). In this case, the
alternative method based upon genomic C region clones as the source
for C region vectors would not allow these genes to be expressed in
bacterial systems where enzymes needed to remove intervening
sequences are absent. Cloned V region segments are excised and
ligated to L or H chain C region vectors. Alternatively, the human
C gamma-1 region can be modified by introducing a termination codon
thereby generating a gene sequence which encodes the H chain
portion of an Fab molecule. The coding sequences with linked V and
C regions are then transferred into appropriate expression vehicles
for expression in appropriate hosts, prokaryotic or eukaryotic.
[0083] Two coding DNA sequences are said to be "operably linked" if
the linkage results in a continuously translatable sequence without
alteration or interruption of the triplet reading frame. A DNA
coding sequence is operably linked to a gene expression element if
the linkage results in the proper function of that gene expression
element to result in expression of the coding sequence.
[0084] Expression vehicles include plasmids or other vectors.
Preferred among these are vehicles carrying a functionally complete
human CH or CL chain sequence having appropriate restriction sites
engineered so that any VH or VL chain sequence with appropriate
cohesive ends can be easily inserted therein. Human CH or CL chain
sequence-containing vehicles thus serve as intermediates for the
expression of any desired complete H or L chain in any appropriate
host.
[0085] A chimeric antibody, such as a mouse-human or human-human,
will typically be synthesized from genes driven by the chromosomal
gene promoters native to the mouse H and L chain V regions used in
the constructs; splicing usually occurs between the splice donor
site in the mouse J region and the splice acceptor site preceding
the human C region and also at the splice regions that occur within
the human C, region; polyadenylation and transcription termination
occur at native chromosomal sites downstream of the human coding
regions.
[0086] A nucleic acid sequence encoding at least one anti-TNF
antibody fragment may be recombined with vector DNA in accordance
with conventional techniques, including blunt-ended or
staggered-ended termini for ligation, restriction enzyme digestion
to provide appropriate termini, filling in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and ligation with appropriate ligases. Techniques for such
manipulations are disclosed, e.g., by Ausubel, supra, Sambrook,
supra, entirely incorporated herein by reference, and are well
known in the art.
[0087] A nucleic acid molecule, such as DNA, is "capable of
expressing" a polypeptide if it contains nucleotide sequences which
contain transcriptional and translational regulatory information
and such sequences are "operably linked" to nucleotide sequences
which encode the polypeptide. An operable linkage is a linkage in
which the regulatory DNA sequences and the DNA sequence sought to
be expressed are connected in such a way as to permit gene
expression as anti-TNF peptides or antibody fragments in
recoverable amounts. The precise nature of the regulatory regions
needed for gene expression may vary from organism to organism and
is well known in the analogous art. See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York (1989); and Ausubel, eds., Current
Protocols in Molecular Biology, Wiley Interscience, New York (1987,
1993).
[0088] Many vector systems are available for the expression of
cloned anti-TNF peptide H and L chain genes in mammalian cells (see
Glover, ed., DNA Cloning, Vol. II, pp. 143-238, IRL Press,
Washington, D.C., 1985). Different approaches can be followed to
obtain complete H2L2 antibodies. It is possible to co-express H and
L chains in the same cells to achieve intracellular association and
linkage of H and L chains into complete tetrameric H2L2 antibodies.
The co-expression can occur by using either the same or different
plasmids in the same host. Genes for both H and L chains can be
placed into the same plasmid, which's then transfected into cells,
thereby selecting directly for cells that express both chains.
Alternatively, cells can be transfected first with a plasmid
encoding one chain, for example the L chain, followed by
transfection of the resulting cell line with an H chain plasmid
containing a second selectable marker. Cell lines producing H2L2
molecules via either route could be transfected with plasmids
encoding additional copies of peptides, H, L, or H plus L chains in
conjunction with additional selectable markers to generate cell
lines with enhanced properties, such as higher production of
assembled H2L2 antibody molecules or enhanced stability of the
transfected cell lines.
Receptor Molecules
[0089] Receptor molecules (also referred to herein as soluble TNF
receptors) useful in the methods and compositions of the present
invention are those that bind TNF with high affinity (see, e.g.,
Feldmann et al., International Publication No. WO 92/07076.
(published Apr. 30, 1992), incorporated herein by reference) and
possess low immunogenicity. In particular, the 55 kDa (p55 TNF-R)
and the 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in
the present invention. Truncated forms of these receptors,
comprising the extracellular domains (ECD) of the receptors or
functional portions thereof, are also useful in the present
invention. Truncated forms of the TNF receptors, comprising the
ECD, have been detected in urine and serum as 30 kDa and 40 kDa TNF
inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem.
265:1531-1536 (1990)). TNF receptor multimeric molecules and TNF
immunoreceptor fusion molecules, and derivatives and fragments or
portions thereof, are additional examples of receptor molecules
which are useful in the methods and compositions of the present
invention. The receptor molecules which can be used in the
invention are characterized by their ability to treat patients for
extended periods with good to excellent alleviation of symptoms and
low toxicity. Low immunogenicity and/or high affinity, as well as
other undefined properties, may contribute to the therapeutic
results achieved.
[0090] TNF receptor multimeric molecules useful in the present
invention comprise all or a functional portion of the ECD of two or
more TNF receptors linked via one or more polypeptide linkers. The
multimeric molecules can further comprise a signal peptide of a
secreted protein to direct expression of the multimeric molecule.
These multimeric molecules and methods for their production have
been described in U.S. application Ser. No. 08/437,533 (filed May
9, 1995), the content of which is entirely incorporated herein by
reference.
[0091] TNF immunoreceptor fusion molecules useful in the methods
and compositions of the present invention comprise at least one
portion of one or more immunoglobulin molecules and all or a
functional portion of one or more TNF receptors. These
immunoreceptor fusion molecules can be assembled as monomers, or
hetero- or homo-multimers. The immunoreceptor fusion molecules can
also be monovalent or multivalent. An example of such a TNF
immunoreceptor fusion molecule is TNF receptor/IgG fusion
protein.
[0092] TNF immunoreceptor fusion molecules and methods for their
production have been described in the art (Lesslauer et al., Eur.
J. Immunol. 212883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad.
Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med.
1741483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219 (1994); Butler et al., Cytokine 6(6):616-623 (1994);
Baker et al., Eur. J. Immunol, 24:2040-2048 (1994); Beutler et al.,
U.S. Pat. No. 5,447,851; and U.S. application Ser. No. 08/442,133
(filed May 16, 1995)). These references are entirely incorporated
herein by reference. Methods for producing immunoreceptor fusion
molecules can also be found in Capon et al., U.S. Pat. No.
5,116,964; Capon et al., U.S. Pat. No. 5,225,538; and Capon et al.,
Nature 337:525-531 (1989), which references are entirely
incorporated herein by reference.
[0093] Derivatives, fragments, regions and functional portions of
the receptor molecules functionally resemble the receptor molecules
that can be used in the present invention (i.e., they bind TNF with
high affinity and possess low immunogenicity). A functional
equivalent or derivative of the receptor molecule refers to the
portion of the receptor molecule, or the portion of the receptor
molecule sequence which encodes the receptor molecule, that is of
sufficient size and sequences to functionally resemble the receptor
molecules that can be used in the present invention (i.e., bind TNF
with high affinity and possess low immunogenicity). A functional
equivalent of the receptor molecule also includes modified receptor
molecules that functionally resemble the receptor molecules that
can be used in the present invention (i.e., bind TNF with high
affinity and possess low immunogenicity). For example, a functional
equivalent of the receptor molecule can contain a "SILENT" codon or
one or more amino acid substitutions, deletions or additions (e.g.,
substitution of one acidic amino acid for another acidic amino
acid; or substitution of one codon encoding the same or different
hydrophobic amino acid for another codon encoding a hydrophobic
amino acid). See Ausubel at al., Current Protocols in Molecular
Biology, Greene Publishing Assoc. and Wiley-Interscience, New York
(1989).
Methotrexate
[0094] Presently available oral and intravenous formulations of
methotrexate include RHEUMATREX.RTM. methotrexate dose pack
(Lederle Laboratories, Wayne, N.J.); methotrexate tablets (Mylan
Pharmaceuticals Inc., Morgantown, W. Va.; Roxane Laboratories,
Inc., Columbus, Ohio); and methotrexate sodium tablets, for
injection and injection (Immunex Corporation, Seattle, Wash.) and
methotrexate LPF.RTM. sodium (methotrexate sodium injection)
(Immunex Corporation, Seattle, Wash.). Methotrexate is also
available from Pharmacochemie (Netherlands). Methotrexate prodrugs,
homologs and/or analogs (e.g., folate antagonists) can also be used
in the methods and compositions of the present invention.
Alternatively, other immunosuppressive agents (or drugs that
suppress the immune system) can be used in the methods and
compositions of the present invention.
Administration
[0095] TNF antagonists, methotrexate and the compositions of the
present invention can be administered to an individual in a variety
of ways. The routes of administration include intradermal,
transdermal (e.g., in slow release polymers), intramuscular,
intraperitoneal, intravenous, subcutaneous, oral, topical,
epidural, buccal, rectal, vaginal and intranasal routes. Any other
therapeutically efficacious route of administration can be used,
for example, infusion or bolus injection, absorption through
epithelial or mucocutaneous linings, or by gene therapy wherein a
DNA molecule encoding the therapeutic protein or peptide is
administered to the patient, e.g., via a vector, which causes the
protein or peptide to be expressed and secreted at therapeutic
levels in vivo. In addition, the TNF antagonists, methotrexate and
compositions of the present invention can be administered together
with other components of biologically active agents, such as
pharmaceutically acceptable surfactants (e.g., glycerides),
excipients (e.g., lactose), carriers, diluents and vehicles. If
desired, certain sweetening, flavoring and/or coloring agents can
also be added.
[0096] The TNF antagonists and methotrexate can be administered
prophylactically or therapeutically to an individual. TNF
antagonists can be administered prior to, simultaneously with (in
the same or different compositions) or sequentially with the
administration of methotrexate. For example, TNF antagonists can be
administered as adjunctive and/or concomitant therapy to
methotrexate therapy.
[0097] For parenteral (e.g., intravenous, subcutaneous,
intramuscular) administration, TNF antagonists, methotrexate and
the compositions of the present invention can be formulated as a
solution, suspension, emulsion or lyophilized powder in association
with a pharmaceutically acceptable parenteral vehicle. Examples of
such vehicles are water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Liposonies and nonaqueous
vehicles such as fixed oils can also be used. The vehicle or
lyophilized powder can contain additives that maintain isotonicity
(e.g., sodium chloride, mannitol) and chemical stability (e.g.,
buffers and preservatives). The formulation is sterilized by
commonly used techniques.
[0098] Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, A. Osol, a standard reference
text in this field of art.
[0099] For example, a parenteral composition suitable for
administration by injection is prepared by dissolving 1.5% by
weight of active ingredient in 0.9% sodium chloride solution.
[0100] TNF antagonists and methotrexate are administered in
therapeutically effective amounts; the compositions of the present
invention are administered in a therapeutically effective amount.
As used herein, a "therapeutically effective amount" is such that
administration of TNF antagonist and methotrexate, or
administration of a composition of the present invention, results
in inhibition of the biological activity of TNF relative to the
biological activity of TNF when therapeutically effective amounts
of antagonist and methotrexate are not administered, or relative to
the biological activity of TNF when a therapeutically effective
amount of the composition is not administered. A therapeutically
effective amount is preferably an amount of TNF antagonist and
methotrexate necessary to significantly reduce or eliminate signs
and symptoms associated with a particular TNF-mediated disease. As
used herein, a therapeutically effective amount is not necessarily
an amount such that administration of the TNF antagonist alone, or
administration of methotrexate alone, must necessarily result in
inhibition of the biological activity of TNF.
[0101] Once a therapeutically effective amount has been
administered, a maintenance amount of TNF antagonist alone, of
methotrexate alone, or of a combination of TNF antagonist and
methotrexate can be administered to the individual. A maintenance
amount is the amount of TNF antagonist, methotrexate, or
combination of TNF antagonist and methotrexate necessary to
maintain the reduction or elimination of the signs and symptoms
associated with a particular TNF-mediated disease achieved by the
therapeutically effective dose. The maintenance amount can be
administered in the form of a single dose, or a series or doses
separated by intervals of days or weeks.
[0102] The dosage administered to an individual will vary depending
upon a variety of factors, including the pharmacodynamic
characteristics of the particular antagonists, and its mode and
route of administration; size, age, sex, health, body weight and
diet of the recipient; nature and extent of symptoms of the disease
being treated, kind of concurrent treatment, frequency of
treatment, and the effect desired. In vitro and in vivo methods of
determining the inhibition of TNF in an individual are well known
to those of skill in the art. Such in vitro assays can include a
TNF cytotoxicity assay (e.g., the WEHI assay or a radioimmunoassay,
ELISA). In vivo methods can include rodent lethality assays and/or
primate pathology model systems (Mathison et al., J. Clin. Invest.,
81:1925-1937 (1988); Beutler et al., Science 229:869-871 (1985);
Tracey et al., Nature 330:662-664 (1987); Shimamoto et al.,
Immunol. Lett. 17:311-318 (1988); Silva et al., J. Infect. Dis.
162:421-427 (1990); Opal et al., J. Infect. Dis. 161:1148-1152
(1990); Hinshaw et al., Circ. Shock 30:279-292 (1990)).
[0103] TNF antagonist and methotrexate can each be administered in
single or multiple doses depending upon factors such as nature and
extent of symptoms, kind of concurrent treatment and the effect
desired. Thus, other therapeutic regimens or agents (e.g., multiple
drug regimens) can be used in combination with the therapeutic
co-administration of TNF antagonists and methotrexate. In a
particular embodiment, a TNF antagonist is administered in multiple
doses. In another embodiment, methotrexate is administered in the
form of a series of low doses separated by intervals of days or
weeks. Adjustment and manipulation of established dosage ranges are
well within the ability of those skilled in the art.
[0104] Usually a daily dosage of active ingredient can be about
0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 to
40 milligrams per kilogram per day given in divided doses 1 to 6
times a day or in sustained release form is effective to obtain
desired results. Second or subsequent administrations can be
administered at a dosage which is the same, less than or greater
than the initial or previous dose administered to the
individual.
[0105] A second or subsequent administration is preferably during
or immediately prior to relapse or a flare-up of the disease or
symptoms of the disease. For example, second and subsequent
administrations can be given between about one day to 30 weeks from
the previous administration. Two, three, four or more total
administrations can be delivered to the individual, as needed.
[0106] Dosage forms (composition) suitable for internal
administration generally contain from about 0.1 milligram to about
500 milligrams of active ingredient per unit. In these
pharmaceutical compositions the active ingredient will ordinarily
be present in an amount of about 0.5-95% by weight based on the
total weight of the composition.
[0107] The present invention will now be illustrated by the
following example, which is not intended to be limiting in any
way.
EXAMPLES
Example 1
Clinical Treatment of Rheumatoid Arthritis By Multiple Infusions of
an Anti-TNF Antibody with and without Methotrexate
[0108] A randomized, double-blind, placebo controlled study was
conducted to evaluate the safety and efficacy of a chimeric
monoclonal anti-TNF antibody (cA2) following multiple infusions of
1, 3 or 10 mg/kg cA2, alone or in combination with methotrexate,
compared to multiple infusions of placebo in combination with
methotrexate, in the treatment of rheumatoid arthritis (RA) in
patients.
Patients
[0109] One hundred one (101) patients at six European centers who
had been using methotrexate for at least 6 months, had been on a
stable dose of 7.5 mg/wk for at least 4 weeks, and had active
disease (according to the criteria of the American College of
Rheumatology) with erosive changes on X-rays of hands and feet,
were enrolled in the trial. Active disease was defined by the
presence of six or more swollen joints plus at least three of four
secondary criteria (duration of morning stiffness.gtoreq.45
minutes; .gtoreq.6 tender or painful joints; erythrocyte
sedimentation rate (ESR).gtoreq.28 mm/hour; C-reactive protein
(CRP).gtoreq.20 mg/l.
[0110] In patients using corticosteroids (.ltoreq.7.5 mg/day) or
non-steroidal anti-inflammatory drugs (NSAIDs), the doses had been
stable for 4 weeks prior to screening. The dose of corticosteroids
remained stable throughout trial participation. The dose of NSAID
typically also remained stable throughout trial participation.
Study Infusions
[0111] The chimeric monoclonal anti-TNF antibody (cA2) was supplied
as a sterile solution containing 5 mg cA2 per ml of 0.01 M
phosphate-buffered saline in 0.15 M sodium chloride with 0.01%
polysorbate 80, pH 7.2. The placebo vials contained 0.1% human
serum albumin in the same buffer. Before use, the appropriate
amount of cA2 or placebo was diluted to 300 ml in sterile saline by
the pharmacist, and administered intravenously via a 0.2 .mu.m
in-line filter over 2 hours. The characteristics of the placebo and
cA2 infusion bags were identical, and the investigators and
patients did not know which infusion was being administered.
Assessments
[0112] Patients were randomized to one of seven treatment groups.
The number of patients in each dose (or treatment) group is
indicated in Table 1. Each of the 101 patients received multiple
infusions of either 0, 1, 3 or 10 mg/kg cA2. Infusions were to be
administered at weeks 0, 2, 6, 10 and 14. Starting at week 0, the
patients were receiving 7.5 mg/wk of methotrexate (Pharmacochemie,
Netherlands) or 3 placebo tablets/week (Pharmacochemie,
Netherlands). Patients were monitored for adverse events during
infusions and regularly thereafter, by interviews, physical
examination, and laboratory testing.
[0113] The six primary disease-activity assessments were chosen to
allow analysis of the response in individual, patients according to
the Paulus index (Paulus, et al., Arthritis Rheumatism 33:477-484
(1990), the teachings of which are incorporated herein by
reference). The assessments contributing to this index were the
tender joint and swollen joint scores (60 and 58 joints,
respectively, hips not assessed for swelling; graded 0-3), the
duration of morning stiffness (minutes), the patient's and
physician's assessment of disease severity (on a 5-point scale,
ranging from 1 (symptom-free) to 5 (very severe), and erythrocyte
sedimentation rate (ESR). Patients were considered to have
responded if at least four of the six variables improved, defined
as at least 20% improvement in the continuous variables, and at
least two grades of improvement or improvement from grade 2 to 1 in
the two disease-severity assessments (Paulus 20% response).
Improvements of at least 50% in the continuous variables were also
used (Paulus 50% response).
[0114] Other disease-activity assessments included the pain score
(0-10 cm on a visual analogue scale (VAS)), an assessment of
fatigue (0-10 cm VAS), and grip strength (0-300 mm Hg, mean of
three measurements per hand by sphygmomanometer cuff).
[0115] The ESR was measured at each study site with a standard
method (Westergen). C-reactive protein (CRP) was measured by rate
nephelometry (Abbott fluorescent polarizing immunoassay). See also,
Elliott at al., Lancet 344:1105-1110 (1994); Elliott et al., Lancet
344:1125-1127 (1994); and Elliott at al., Arthritis Rheum.
36(12):1681-1690 (1993), which references are entirely incorporated
herein by reference.
[0116] Evaluations were performed at weeks 1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22 and 26.
Results
[0117] The 101 patients were randomized to one of seven treatment
(or dose) groups. The patients enrolled in each dose group were
well matched for baseline demographics. Disease duration and
swollen and tender joint counts at baseline were also well-balanced
across the groups (Table 1). Table 1 also shows the maximum
methotrexate dose administered within 6 months prior to
randomization. Median maximum doses for each group ranged between
10 and 15 mg/week; there were no significant differences amongst
the treatment groups (p=0.404).
TABLE-US-00002 TABLE 1 Baseline Disease Characteristics Joint
Counts Treatment Groups Placebo 1 mg/kg cA2 MTX+ MTX+ MTX- Disease
dur. (yrs) Pts evaluated 14 14 15 Mean .+-. SD 7.6 .+-. 4.0 14.3
.+-. 12.1 7.6 .+-. 6.0 Median 6.9 11.4 5.2 IQ range (4.3, 11.5)
(3.3, 24.7) (3.4, 9.0) Range (1.8, 14.2) (0.7, 37.3) (2.5, 21.3)
Number of Swollen joints, Paulus joint set (0-58) Pts evaluated 14
14 15 Mean .+-. SD 18.1 .+-. 8.6 16.9 .+-. 7.8 21.2 .+-. 11.2
Median 16.5 15.5 20.0 IQ range (12.0, 25.0) (10.0, 25.0) (10.0,
33.0) Range (6.0, 38.0) (6.0, 29.0) (7.0, 40.0) Number of tender
joints, Paulus joint set (0-60) Pts evaluated 14 14 15 Mean .+-. SD
31.5 .+-. 14.2 19.1 .+-. 10.7 29.9 .+-. 17.1 Median 27.0 16.0 30.0
IQ range (22.0, 44.0) (13.0, 30.0) (14.0, 45.0) Range (8.0, 52.0)
(2.0, 39.0) (6.0, 58.0) Max dose MTX prev. 6 mo (mg/kg) Pts
evaluated 14 14 15 Mean .+-. SD 13.8 .+-. 3.9 11.6 .+-. 3.5 12.8
.+-. 5.6 Median 15.0 11.3 12.5 IQ range (10.0, 15.0) (10.0, 12.5)
(10.0, 15.0) Range (7.5, 20.0) (7.5, 20.0) (7.5, 30.0) Treatment
Groups 3 mg/kg cA2 MTX+ MTX- Disease dur. (yrs) Pts evaluated 15 14
Mean .+-. SD 12.1 .+-. 9.0 7.8 .+-. 4.3 Median 11.9 7.7 IQ range
(4.3, 16.4) (4.6, 9.8) Range (0.7, 30.5) (1.4, 17.4) Number of
Swollen joints, Paulus joint set (0-58) Pts evaluated 15 14 Mean
.+-. SD 17.7 .+-. 5.9 19.7 .+-. 9.9 Median 16.0 17.0 IQ range
(13.0, 22.0) (11.0, 32.0) Range (10.0, 29.0) (8.0, 34.0) Number of
tender joints, Paulus joint set (0-60) Pts evaluated 15 14 Mean
.+-. SD 24.5 .+-. 14.4 31.2 .+-. 11.7 Median 21.0 31.0 IQ range
(12.0, 32.0) (23.0, 39.0) Range (10.0, 52.0) (9.0, 52.0) Max dose
MTX prev. 6 mo (mg/kg) Pts evaluated 14 13 Mean .+-. SD 11.6 .+-.
3.3 11.7 .+-. 4.8 Median 10.0 10.0 IQ range (10.0, 15.0) (7.5,
12.5) Range (7.5, 17.5) (7.5, 25.0) Treatment Groups Treatment 10
mg/kg cA2 All effect MTX+ MTX- Patients p-value Disease dur. (yrs)
Pts evaluated 14 15 101 Mean .+-. SD 11.1 .+-. 7.4 9.7 .+-. 7.4
10.0 .+-. 7.8 0.634 Median 10.7 7.6 7.6 IQ Range (4.5, 15.5) (4.9,
14.9) (4.3, 14.4) Range (1.4, 24.1) (1.1, 24.3) (0.7, 37.3) Number
of swollen joints, Paulus joint set (0-58) Pts evaluated 14 15 101
Mean .+-. SD 21.1 .+-. 8.2 17.8 .+-. 8.7 18.9 .+-. 8.7 0.643 Median
19.5 17.0 18.0 IQ Range (15.0, 31.0) (11.0, 21.0) (12.0, 25.0)
Range (10.0, 34.0) (7.0, 41.0) (6.0, 41.0) Number of tender joints,
Paulus joint set (0-60) Pts evaluated 14 15 101 Mean .+-. SD 26.5
.+-. 12.0 26.2 .+-. 11.7 27.0 .+-. 13.5 0.135 Median 25.5 23.0 25.0
IQ Range (21.0, 38.0) (17.0, 35.0) (15.0, 38.0) Range (8.0, 44.0)
(11.0, 48.0) (2.0, 58.0) Max dose MTX prev. 6 mo (mg/kg) Pts
evaluated 14 15 99 Mean .+-. SD 12.7 .+-. 5.0 12.5 .+-. 3.0 12.4
.+-. 4.2 0.404 Median 10.0 12.5 12.5 IQ Range (10.0, 15.0) (10.0,
15.0) (10.0, 15.0) Range (7.5, 25.0) (7.5, 20.0) (7.5, 30.0) MTX =
Methotrexate
[0118] The pre-specified primary analysis in this trial was the
comparison of the total time of clinical response during the
26-week follow-up period. The results for the primary analysis are
shown in Table 2. The duration of response of all cA2-treated
groups, with the exception of the 1 mg/kg group not receiving
methotrexate, was significantly improved (p<0.001) compared to
the placebo group receiving methotrexate alone.
TABLE-US-00003 TABLE 2 Total Time of Response.sup.a Based On Paulus
20% Criteria Treatment Groups Placebo 1 mg/kg cA2 mg/kg cA2 10
mg/kg cA2 Treatment Total time of MTX+ MTX+ MTX- MTX+ MTX- MTX+
MTX- effect response in weeks (n = 14) (n = 14) (n = 15) (n = 15)
(n = 14) (n = 13) (n = 15) p-value Median 0 16.6 2.6 16.5 17.2
>23.1 10.4 <0.001 Minimum 0 0 0 0 0 0 0 25th percentile 0.0
6.2 2.0 7.0 4.0 2.6 6.9 75th percentile 0.0 22.5 8.0 >20.1 20.7
>24.6 >23.1 Maximum >15.1 >26.9 15.1 >24.9 >25.9
>25.6 >26.4 p-value vs. MTX alone <0.001 0.119 <0.001
<0.001 <0.001 <0.001 .sup.aPatients were followed through
26 weeks following the initial infusion of cA2
[0119] The response rates at Paulus 20% are shown in Table 3.
Drop-outs were considered as non-responders subsequent to their
dropping out from the study. With the exception of the 1 mg/kg
group not receiving methotrexate, all of the cA2-treated groups
demonstrated clinical benefit through 14 weeks when the last dose
of cA2 was received. Sustained clinical benefit was observed
through 26 weeks (the last follow-up visit) in patients who
received 3 or 10 mg/kg cA2 with methotrexate. Approximately
one-half of the patients who received 3 mg/kg cA2 with methotrexate
demonstrated continued clinical benefit at 26 weeks.
TABLE-US-00004 TABLE 3 Number of Patients Responding According To
Paulus 20% Criteria At Each Evaluation Visit Treatment Groups
Placebo 1 mg/kg cA2 MTX+ MTX+ MTX- (n = 14) (n = 14) (n = 15) Pts
with any response 21% 93% 80% (3/14) 13/14 12/15 p-value vs MTX
alone <0.001 0.006 Time post-infusion 1 Week 0% 31% 53% (0/14)
(4/13) (8/15) 2 Weeks 7% 64% 57% (1/14) (9/14) (8/14) 4 Weeks.sup.a
0% 79% 33% (0/14) 11/14 (5/15) 6 Weeks 0% 71% 27% (0/14) 10/14
(4/15) 8 Weeks.sup.a 14% 64% 20% (2/14) (9/14) (3/15) 10 Weeks 7%
71% 20% (1/14) 10/14 (3/15) 12 Weeks.sup.a 7% 57% 13% (1/14) (8/14)
(2/15) 14 Weeks 0% 71% 7% (0/14) 10/14 (1/15) 16 Weeks.sup.a 14%
64% 7% (2/14) (9/14) (1/15) 18 Weeks 21% 50% 13% (3/14) (7/14)
(2/15) 20 Weeks 7% 54% 13% (1/14) (7/13) (2/15) 22 Weeks 7% 46% 0%
(1/14) (6/13) (0/15) 26 Weeks.sup.a 7% 21% 7% (1/14) (3/14) (1/15)
Treatment Groups 3 mg/kg cA2 10 mg/kg cA2 Treatment MTX+ MTX- MTX+
MTX- effect (n = 15) (n = 14) (n = 13) (n = 15) p-value Pts with
any response 80% 79% 85% 80% <0.001 12/15 11/14 11/13 12/15
p-value vs MTX alone 0.002 0.002 0.001 0.004 Time post-infusion 1
Week 27% 43% 31% 60% (4/15) (6/14) (4/13) (9/15) 2 Weeks 27% 43%
62% 53% (4/15) (6/14) (8/13) (8/15) 4 Weeks.sup.a 40% 64% 54% 53%
0.002 (6/15) (9/14) (7/13) (8/15) 6 Weeks 47% 50% 54% 47% (7/15)
(7/14) (7/13) (7/15) 8 Weeks.sup.a 60% 71% 69% 40% 0.003 (9/15)
10/14 (9/13) (6/15) 10 Weeks 67% 64% 69% 53% 10/15 (9/14) (9/13)
(8/15) 12 Weeks.sup.a 67% 64% 62% 60% <0.001 10/15 (9/14) (8/13)
(8/13) 14 Weeks 60% 57% 77% 53% (9/15) (8/14) 10/13 (8/15) 16
Weeks.sup.a 67% 64% 54% 67% <0.001 10/15 (9/14) (7/13) 10/15 18
Weeks 71% 69% 62% 57% 10/14 (9/13) (8/13) (8/14) 20 Weeks 53% 43%
54% 53% (8/15) (6/14) (7/13) (8/15) 22 Weeks 47% 36% 54% 33% (7/15)
(5/14) (7/13) (5/15) 26 Weeks.sup.a 47% 21% 54% 33% 0.013 (7/15)
(3/14) (7/13) (5/15) .sup.aEvaluation visits pre-specified for
analysis.
[0120] The response rates at Paulus 50% are shown in Table 4. The
magnitude of the clinical benefit of cA2 treatment was substantial.
The majority of patients were responding to cA2 treatment according
to the 50% Paulus criteria.
TABLE-US-00005 TABLE 4 Number of Patients Responding According To
Paulus 50% Criteria At Each Evaluation Visit Treatment Groups
Placebo 1 mg/kg cA2 MTX+ MTX+ MTX- (n = 14) (n = 14) (n = 15) Pts
with any response 14.3% 85.7% 40.0% (2/14) (12/14) (6/15) p-value
vs MTX alone <0.001 0.079 Time post-infusion 1 Week 0.0% 7.7%
26.7% (0/14) (1/13) (4/15) 2 Weeks 0.0% 21.4% 28.6% (0/14) (3/14)
(4/14) 4 Weeks.sup.a 0.0% 57.1% 13.3% (0/14) (8/14) (2/15) 6 Weeks
0.0% 57.1% 0.0% (0/14) (8/14) (0/15) 8 Weeks.sup.a 7.1% 50.0% 0.0%
(1/14) (7/14) (0/15) 10 Weeks 0.0% 57.1% 0.0% (0/14) (8/14) (0/15)
12 Weeks.sup.a 7.1% 50.0% 6.7% (1/14) (7/14) (1/15) 14 Weeks 0.0%
57.1% 6.7% (0/14) (8/14) (1/15) 16 Weeks.sup.a 0.0% 64.3% 6.7%
(0/14) (9/14) (1/15) 18 Weeks 7.1% 50.0% 6.7% (1/14) (7/14) (1/15)
20 Weeks 7.1% 53.8% 0.0% (1/14) (7/13) (0/15) 22 Weeks 0.0% 38.5%
0.0% (0/14) (5/13) (0/15) 26 Weeks.sup.a 0.0% 21.4% 6.7% (0/14)
(3/14) (1/15) Treatment Groups 3 mg/kg cA2 10 mg/kg cA2 Treatment
MTX+ MTX- MTX+ MTX- effect (n = 15) (n = 14) (n = 13) (n = 15)
p-value Pts with any response 73.3% 64.3% 76.9% 66.7% <0.001
(11/15) (9/14) (10/13) (10/15) p-value vs MTX alone 0.001 0.008
0.002 0.009 Time post-infusion 1 Week 0.0% 35.7% 7.7% 26.7% (0/15)
(5/14) (1/13) (4/15) 2 Weeks 6.7% 28.6% 15.4% 20.0% (1/15) (4/14)
(2/13) (3/15) 4 Weeks.sup.a 13.3% 28.6% 46.2% 40.0% 0.006 (2/15)
(4/14) (6/13) (6/15) 6 Weeks 26.7% 42.9% 38.5% 33.3% (4/15) (6/14)
(5/13) (5/15) 8 Weeks.sup.a 40.0% 50.0% 69.2% 33.3% <0.001
(6/15) (7/14) (9/13) (5/15) 10 Weeks 40.0% 50.0% 69.2% 40.0% (6/15)
(7/14) (9/13) (6/15) 12 Weeks.sup.a 60.0% 35.7% 61.5% 40.0%
<0.001 (9/15) (5/14) (8/13) (6/15) 14 Weeks 40.0% 35.7% 61.5%
40.0% (6/15) (5/14) (8/13) (6/15) 16 Weeks.sup.a 60.0% 50.0% 53.8%
40.0% <0.001 (9/15) (7/14) (7/13) (6/15) 18 Weeks 71.4% 46.2%
61.5% 57.1% (10/14) (6/13) (8/13) (8/14) 20 Weeks 53.3% 35.7% 46.2%
40.0% (8/15) (5/14) (6/13) (6/15) 22 Weeks 46.7% 14.3% 53.8% 26.7%
(7/15) (2/14) (7/13) (4/15) 26 Weeks.sup.a 40.0% 14.3% 46.2% 20.0%
0.008 (6/15) (2/14) (6/13) (3/15) .sup.aEvaluation visits
pre-specified for analysis.
[0121] Commensurate with the clinical response rates shown in
Tables 2-4, most of the patients in the treatment groups
demonstrating effectiveness of cA2 treatment received all 5
infusions of cA2 (Table 5). The principle reason for patients not
receiving the complete dose regimen was because of lack of efficacy
in the placebo group (methotrexate, alone) and in the 1 mg/kg group
not receiving methotrexate. All 15 patients in the 3 mg/kg group
that received methotrexate completed the 5-infusion dose
regimen.
TABLE-US-00006 TABLE 5 Number of Infusions Completed Treatment
Groups Pts with Placebo 1 mg/kg cA2 3 mg/kg cA2 10 mg/kg cA2
complete.sup.a MTX+ MTX+ MTX- MTX+ MTX- MTX+ MTX- Treatment effect
infusions (n = 14) (n = 14) (n = 15) (N = 15) (n = 14) (n = 14) (n
= 15) p-value 5 infusions 6 12 8 15 12 12 12 0.003 (42.86%)
(85.71%) (53.33%) (100.00%) (85.71%) (85.71%) (80.00%) 4 infusions
0 1 0 0 1 1 0 (0.00%) (7.14%) (0.00%) (0.00%) (7.14%) (7.14%)
(0.00%) 3 infusions 2 1 6 0 0 1 1 (14.29%) (7.14%) (40.00%) (0.00%)
(0.00%) (7.14%) (6.67%) 2 infusions 5 0 1 0 1 0 2 (35.71%) (0.00%)
(6.67%) (0.00%) (7.14%) (0.00%) (13.33%) 1 infusion 1 0 0 0 0 0 0
(7.14%) (0.00%) (0.00%) (0.00%) (0.00%) (0.00%) (0.00%)
.sup.aPatients are counted only once for the first group for which
they qualify (5 infusions > 4 infusions etc . . . ). Patients
were only counted if they had completed the entire infusion.
[0122] Results for measures of swollen and tender joint counts and
the physician and patient global assessments are shown in FIGS.
1-4. The median results in FIGS. 1-4 were reported for each
evaluation visit based only on the patients with data collected.
That is, a last observation carried forward approach was not used
for patients who dropped out. Instead, the number of patients with
data that comprise each point on the graph was reported at the
bottom of the figures.
[0123] Despite the number of drop-outs in the placebo group and the
1 mg/kg group not receiving methotrexate, the results in FIGS. 1-4
demonstrate that cA2 treatment in combination with methotrexate
profoundly reduces disease activity for all of the traditional
measurements of disease activity, approaching near remission in
many patients.
[0124] Results for a commonly used serum marker of inflammatory
activity, C-reactive protein (CRP) are shown in FIG. 5. Treatment
with cA2 produced a rapid decrease in CRP concentration which was
sustained through 26 weeks in the patients who received 3 or 10
mg/kg cA2.
[0125] Results for the Health Assessment Questionnaire (HAQ) are
shown in FIG. 6. This measurement of quality of life/disability
demonstrated improvement over time corresponding with the clinical
improvement observed in patients treated with cA2. In the patients
treated with 3 mg/kg cA2 and methotrexate, the HAQ decreased from
2.0 at baseline to 1.1 at 22 weeks.
Pharmacokinetics of cA2
[0126] Serum concentrations of cA2 were obtained in all patients in
this study. The serum concentration in each patient plotted over
time according to the cA2 dose group is shown in FIG. 7. Data
plotted are the serum cA2 concentrations obtained just before the
administration of cA2 at weeks 2, 6, 10 and 14 and then at weeks 18
and 26. These sampling times were selected to best demonstrate the
stability of the cA2 concentration during the multiple dose regimen
and the decline in serum cA2 concentration after the last dose was
administered. For purposes of data presentation, the scales for cA2
concentration for each graph are condensed as the CA2 dose was
increased.
[0127] Substantial differences were observed for the cA2 serum
concentration over time in the 1 mg/kg dose groups according to
whether patients received methotrexate. Most of the patients
receiving 1 mg/kg cA2 with methotrexate demonstrated measurable cA2
concentrations through 18 weeks, although it appeared that there
was a tendency for the concentration to decline over time. In sharp
contrast, the majority of patients who received 1 mg/kg cA2 without
methotrexate were not able to maintain measurable serum
concentrations of cA2 over time. As discussed herein, the inability
to maintain serum cA2 in these patients was associated with a high
rate of neutralizing antibody formation.
[0128] In contrast to the 1 mg/kg groups, patients who received
either 3 mg/kg cA2 or 10 mg/kg cA2 were able to maintain serum cA2
concentrations through the multiple dose regimen. However, even in
those dose groups, there was evidence that concomitant treatment
with methotrexate was associated with high cA2 serum
concentrations. As shown in FIG. 8, the median serum cA2
concentration in both the 3 and 10 mg/kg dose groups receiving
methotrexate was higher than in the corresponding groups not
receiving methotrexate.
Immune Responses to cA2
[0129] Serum samples were collected through 26 weeks from all
patients and analyzed for human anti-chimeric antibodies (HACA) to
cA2. The results for HACA responses for each cA2 treatment group
are shown in Table 6: It should be noted that in several patients
in the 3 mg/kg group and in most patients in the 10 mg/kg group,
cA2 was still present in the 26-week sample and could potentially
interfere with the detection of HACA in the assay. However, it
could also be reasoned that if neutralizing antibodies were present
at 26 weeks, then cA2 should not be present. Therefore, in
presenting the data in Table 6, results for the immune response
rate are shown not including patients with serum cA2 at 26 weeks
and including patients with serum cA2 at 26 weeks, assuming that if
cA2 was present at 26 weeks, the patient did not have a positive
HACA response.
TABLE-US-00007 TABLE 6 HACA Responses 1 mg/kg 3 mg/kg 10 mg/kg MTX+
MTX- MTX+ MTX- MTX+ MTX- HACA 2/13 8/15 0/10 3/12 0/2 1/10
responses not (15.4%) (53.3%) (0%) (25.0%) (0%) ( 10%) including
pts with 26-week serum cA2 HACA 2/13 8/15 0/15 3/14 0/14 1/15
responses (15.4%) (53.3%) (0%) (21.4%) (0%) (6.7%) including pts
with 26-week serum cA2.sup.1 .sup.1Patients with a measurable
26-week serum cA2 concentration were considered negative for a HACA
response for this analysis.
[0130] The results in Table 6 demonstrate that concomitant
methotrexate treatment suppresses the immune response to cA2,
enabling stable pharmacokinetics to be achieved in a multiple dose
regimen of cA2. This effect was also found after combined
anti-CD4/anti-TNF antibody treatment in mice with collagen-induced
arthritis and described in U.S. application Ser. No. 08/607,419,
filed Feb. 28, 1996, the teachings of which are entirely
incorporated herein by reference.
Clinical Safety
[0131] Two out of 86 patients (with most patients receiving 5
treatments) experienced multisystem infusion-related reactions with
retreatment. Multisystem, infusion-related reactions include
headache, fever, facial flushing, pruritus, myalgia, nausea, chest
tightness, dyspnea, vomiting, erythema, abdominal discomfort,
diaphoresis, shivers, hypertension, lightheadedness, hypotension,
palpitations and somnolence.
[0132] Hypersensitivity reactions, as described herein, may occur
whenever protein-containing materials, such as cA2, are
administered. Thus, it is unclear whether these symptoms represent
an immunologic event or physical factors such as infusion rate and
immunoglobulin aggregation. Investigators have reported that
symptoms resolve in some patients by decreasing the rate of the
infusion. Previous literature reports indicate that vasomotor
symptoms have been observed in patients receiving intravenous
immunoglobulin therapy (Berkman et al., Ann. Intern Med.
112:278-292 (1990); Ochs et al., Lancet 2:1158-1159 (1980)).
[0133] One patient developed hypotension during all three infusions
of 10 mg/kg cA2. The patient did not display clinical signs of
hypotension and did not require medical treatment, but, in keeping
with predefined safety criteria, the treatment schedule of this
patient was discontinued.
[0134] One patient treated with 3 infusions of 10 mg/kg of cA2 and
with 7.5 mg/week methotrexate developed symptoms of sepsis as a
result of staphylococcal pneumonia 2 weeks after her last study
visit and 14 weeks after her last infusion with cA2. Six days after
developing symptoms she was admitted to the hospital and treated.
She died one day later. (This patient had not proceeded with the
fourth infusion for reasons unrelated to the sepsis.) Patients with
RA who develop infections have a worse than expected outcome. Wolfe
and coworkers have reported an observed:expected ratio for death
due to pneumonia of 5.307 and an observed:expected ratio for death
due to infections (excluding pneumonia) of 6.213 in RA patients
from the ARAMIS database. (Wolfe et al., Arthritis Rheumatism
4:481-494 (1994)).
[0135] One patient experienced a serious postoperative infection
following cataract surgery 9 weeks after the fifth and last
infusion of 3 mg/kg of cA2 (with 7.5 mg/week methotrexate), leading
to removal of the eye. This patient was receiving prednisolone (7
mg/day). The incidence of endophthalmitis after cataract extraction
has been reported to be between 0.072 and 0.093% (Kattan at al.,
Ophthalmology 98(9):1147-1148 (1991)) and may be heightened in
patients receiving corticosteroid therapy.
[0136] Eight (9%) of 87 patients developed double stranded (ds)-DNA
antibodies following multiple infusions of cA2. Measurements were
performed at baseline, week 8, 16 and 26 (12 weeks following the
last infusion). In these patients with antibodies against ds-DNA,
there was a trend toward a lower level in antibodies at the last
evaluation, with two patients being negative.
[0137] One patient developed dyspnea, pleuritic chest pain and a
rebound of arthritis activity at study week 14 (four weeks after
the fourth infusion of 3 mg/kg of cA2). Symptoms resolved and she
received her fifth dose of cA2. Symptoms recurred 3 weeks later.
Examination of the serial blood samples revealed that the test for
antinuclear antibodies and anti ds-DNA antibodies were negative
prior to treatment, but became positive at week 6 of the study. The
patient's symptoms responded to oral prednisolone 20-30 mg daily.
The working diagnosis was systemic lupus erythematosus (SLE). The
patient currently does not have symptoms of SLE but has active
RA.
[0138] To date, although antibodies to ds-DNA have been detected in
patients treated with cA2, they generally represent transient
increases and only one patient has been symptomatic. In patients
who have had sufficient follow-up, anti-ds-DNA antibodies have
resolved with discontinuation of treatment.
[0139] In summary, treatment with cA2 is well tolerated. The
reductions in disease activity produced by cA2 are significant as
supported by the findings of a low placebo response rate. High
clinical response rates are obtained with a multiple dose regimen
of 3 mg/kg cA2 in combination with 7.5 mg/wk methotrexate and can
be sustained through 26 weeks. This dose regimen is considered
preferable to the 1 mg/kg plus methotrexate regimen because better
pharmacokinetics are obtained, virtually no immune response was
detected and the clinical response is better sustained following
the last treatment with cA2. The clinical benefit obtained by
increasing the dose regimen to 10 mg/kg cA2 plus methotrexate is
similar to that observed with the 3 mg/kg cA2 plus methotrexate
regimen.
[0140] Thus, the results of this study indicate that treatment with
a multiple dose regimen of cA2 as adjunctive and/or concomitant
therapy to methotrexate therapy, in RA patients whose disease is
incompletely controlled by methotrexate, produces a highly
beneficial or synergistic clinical response that can be sustained
through 26 weeks. The benefit produced by cA2 generally exceeds 50%
reductions in the traditional measurements of rheumatoid arthritis
(swollen and tender joints, patient and physician global disease
assessments) and achieves near clinical remission in many patients.
Accordingly, the results of this study indicate that treatment with
multiple infusions of cA2 as adjunctive and/or concomitant therapy
to methotrexate therapy is an important and efficacious therapeutic
approach for treating RA in patients.
Example 2
Clinical Treatment of Rheumatoid Arthritis by Single Infusion of an
Anti-TNF Antibody in Patients Receiving Methotrexate
[0141] A randomized, double-blind, placebo controlled study was
conducted to evaluate the effects of a single infusion of placebo,
5, 10 or 20 mg/kg cA2 in combination with methotrexate,
administered at a dose of 10 mg/week, in the treatment of
rheumatoid arthritis (RA) in patients.
Patients
[0142] Twenty-eight (28) RA patients at three centers in the United
States who, despite receiving three months therapy with
methotrexate administered at a stable dose of 10 mg/wk for at least
4 weeks prior to screening, still had active disease according to
the criteria of the American College of Rheumatology, were enrolled
in the study. Active disease was defined by the presence of six or
more swollen joints plus at least three of four secondary criteria
(duration of morning stiffness.gtoreq.45 minutes; .gtoreq.6 tender
or painful joints; erythrocyte sedimentation rate (ESR) a28
mm/hour; C-reactive protein (CRP).gtoreq.20 mg/l.
[0143] Patients taking NSAIDs and corticosteroids (prednisone) at
screening were allowed to continue at stable doses (7.5
mg/day).
Study Infusions
[0144] The chimeric monoclonal anti-TNF antibody (cA2) was supplied
as a sterile solution containing 5 mg cA2 per ml of 0.01 M
phosphate-buffered saline in 0.15 M sodium chloride with 0.01%
polysorbate 80, pH 7.2. The placebo vials contained 0.1% human
serum albumin in the same buffer. Before use, the appropriate
amount of cA2 or placebo was diluted to 300 ml in sterile saline by
the pharmacist, and administered intravenously via a 0.2 .mu.m
in-line filter over 2 hours. The characteristics of the placebo and
cA2 infusion bags were identical, and the investigators and
patients did not know which infusion was being administered.
Assessments
[0145] Patients were randomized to one of four treatment groups (7
patients per group). Each of the 28 patients received a single dose
of either 0, 5, 10 or 20 mg/kg cA2 and were followed for 12 weeks.
Patients continued treatment with methotrexate (Pharmacochemie,
Netherlands) administered at 10 mg/week throughout the study.
Patients were monitored for adverse events during infusions and
regularly thereafter, by interviews, physical examination, and
laboratory testing.
[0146] The primary measurement of clinical response was defined by
the ACR preliminary definition of response (Felson et al.,
Arthritis Rheumatism 38(6):727-735 (1995)). Patients were
considered to have a response if they had a 20% reduction in
swollen and tender joint count, and had experienced a 20% reduction
in 3 of the 5 following assessments: patient's assessment of pain
(VAS), patient's global assessment of disease activity (VAS),
physician's global assessment of disease activity (VAS), patient's
assessment of physical function (HAQ), and an acute phase reactant
(ESR). The ESR was measured at each study site with a standard
method (Westergen).
[0147] Evaluations were performed at day 3, and at weeks 1, 2, 4,
6, 8, 10, and 12.
Results
[0148] The 28 patients were randomized to one of four treatment (or
dose) groups.
[0149] The clinical response rates over time by ACR 20% criteria in
each of the treatment groups is shown in Table 7.
TABLE-US-00008 TABLE 7 Clinical Response Rates (By ACR 20%
Criteria) In Patients Receiving 10 mg/kg Methotrexate Dose of cA2
cA2 Treated Placebo 5 mg/kg 10 mg/kg 20 mg/kg Patients Pts
evaluated 7 7 7 7 21 Pts with any response 1(14.3%) 6(85.7%)
5(71.4%) 6(85.7%) 17(81.0%) 1 Week 0(0.0%) 4(57.1%) 2(28.6%)
5(71.4%) 11(52.4%) 2 Weeks 0(0.0%) 4(57.1%) 5(71.4%) 5(71.4%)
14(66.7%) 4 Weeks 1(14.3%) 3(42.9%) 5(71.4%) 5(71.4%) 13(61.9%) 6
Weeks 0(0.0%) 3(42.9%) 5(71.4%) 4(57.1%) 12(57.1%) 8 Weeks 1(14.3%)
3(42.9%) 4(57.1%) 4(57.1%) 11(52.4%) 10 Weeks 1(14.3%) 1(14.3%)
4(57.1%) 3(42.9%) 8(38.1%) 12 Weeks 1(14.3%) 2(28.6%) 4(57.1%)
3(42.9%) 9(42.9%)
[0150] Clinical benefit of cA2 treatment was evident at the first
evaluation visit at one week. Although each of the 3 doses of cA2
produced clinical responses in the majority of patients treated,
the duration of clinical response appeared to be better sustained
through 12 weeks in the groups receiving 10 or 20 mg/kg cA2.
Clinical response was achieved much more frequently among patients
receiving cA2 as compared to placebo. That is, 17/21 (81%) patients
in the 3 cA2 groups achieved a response, compared with only 1/7
(14%) placebo treated patients. The magnitude of clinical response
was notable. The mean tender joint count among cA2 treated patients
decreased from 30.1 at baseline to 13.3 at week 12, and mean CRP
decreased from 3.0 at baseline to 1.1 at week 12.
[0151] The duration of clinical response appeared to be dose
dependent. 2/6 (33%) of the responding patients treated with 5
mg/kg cA2 sustained a response through 12 weeks of followup,
compared to 7/11 (64%) of the responding patients who received 10
or 20 mg/kg. Treatment in all groups was generally well
tolerated.
[0152] In summary; the results of this study'indicate that
treatment with cA2 as adjunctive and/or concomitant therapy to
methotrexate therapy is effective in the reduction of the signs and
symptoms of rheumatoid arthritis in patients whose disease is
incompletely controlled by methotrexate. Moreover, the clinical
response achieved by this approach can be sustained for more than
12 weeks after a single treatment. Accordingly, the results of this
study indicate that treatment with cA2 as adjunctive and/or
concomitant therapy to methotrexate therapy is an important and
efficacious therapeutic approach for treating RA in patients.
Example 3
Clinical Treatment of Rheumatoid Arthritis by Repeated Dose
Administration of an Anti-TNF Antibody in Patients Following a
Single Dose, Double-Blind, Placebo-Controlled Trial
[0153] An open label study was conducted to evaluate the effects of
repeated infusions of 10 mg/kg cA2 in combination with
methotrexate, administered at a dose of 10 mg/week, in the
treatment of rheumatoid arthritis in patients.
Patients
[0154] As described in Example 2, a randomized, double-blind,
placebo controlled, 12 week study of cA2 was conducted in RA
patients who had active disease despite receiving three months
therapy with methotrexate administered at a stable dose of 10 mg/wk
for at least 4 weeks prior to screening.
[0155] At week 12, patients who had completed the 12 week
evaluation period and had not experienced adverse events
prohibiting further infusions of cA2, were offered 3 subsequent
open label infusions of cA2, administered at a dose of 10 mg/kg, at
eight week intervals (weeks 12, 20, 28). Twenty-three (23) patients
from the 12 week study were enrolled in this study.
Assessments
[0156] 11/23 patients entering this open label study were evaluated
at 1 of 3 centers in the United States and followed up to 40 weeks
after initial entry. Patients continued treatment with methotrexate
administered at 10 mg/week throughout the study. Repeated
treatments with cA2 were generally well tolerated. Three patients
had transient infusion related symptoms (urticaria,
somnolence).
[0157] The primary measurement of clinical response was defined by
the ACR preliminary definition of response (Felson at al.,
Arthritis Rheumatism 38(6):727-735 (1995)). Patients were
considered to have a response if they had a 20% reduction in
swollen and tender joint count, and had experienced a 20% reduction
in 3 of the 5 following assessments: patient's assessment of pain
(VAS), patient's global assessment of disease activity (VAS),
physician's global assessment of disease activity (VAS), patient's
assessment of physical function (HAQ), and an acute phase reactant
(ESR). The ESR was measured at each study site with a standard
method (Westergen).
Results.
[0158] Of six patients who had all received cA2 during the
double-blinded study described in Example 2 and responded through
the 12 weeks of that study, four patients sustained a response
throughout the 40 week followup. Of the remaining two patients, one
patient is still responding through week 28, and one patient
recently entered this open label trial. For all 4 patients
completing 40 weeks of followup and the patient at week 28, final
tender joint counts were 2 and swollen joint counts 1, compared to
a mean of 23 and 29, respectively, at entry into the double-blinded
study described in Example 2. For 4 of these 5 patients, ESR were
18 mm/hr and CRP 0.7, compared to a mean of 27 and 3.9,
respectively, at entry into the double-blind study described in
Example 2.
[0159] Of two patients who had both received cA2 during the
double-blinded study described in Example 2 and responded only
through week 10 of that study, one patient responded through 36
weeks and one patient is still responding through week 20.
[0160] Of three patients who did not respond during the
double-blinded study described in Example 2 (2 received placebos, 1
received 5 mg/kg cA2), two of these patients experienced a
transient clinical response, and one patient is still responding
through week 20.
[0161] In summary, the preliminary results of this study suggest
that repeated adjunctive and/or concomitant therapy with cA2, in RA
patients whose disease is incompletely controlled by methotrexate,
can result in substantial clinical improvement for a majority of
the patients. Moreover, the clinical response achieved by this
approach can be sustained for up to 40 weeks of followup.
Accordingly, the results of this study indicate that repeated
treatment with cA2 as adjunctive and/or concomitant therapy to
methotrexate therapy is an important and efficacious therapeutic
approach for treating RA in patients.
EQUIVALENTS
[0162] Those skilled in the art will know, or be able to ascertain,
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
2122PRTHomo sapiens 1Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly Cys
Pro Ser Thr His Val1 5 10 15Leu Leu Thr His Thr Ile 20222PRTHomo
sapiens 2Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro
Cys Gln1 5 10 15Arg Glu Thr Pro Glu Gly 20
* * * * *