U.S. patent application number 12/583851 was filed with the patent office on 2011-05-26 for tnfalpha antagonists and methotrexate in the treatment of tnf-mediated disease.
This patent application is currently assigned to The Kennedy Institute of Rheumatology. Invention is credited to Marc Feldmann, Ravinder N. Maini.
Application Number | 20110123543 12/583851 |
Document ID | / |
Family ID | 24773909 |
Filed Date | 2011-05-26 |
United States Patent
Application |
20110123543 |
Kind Code |
A1 |
Feldmann; Marc ; et
al. |
May 26, 2011 |
TNFalpha 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 N.; (London, GB) |
Assignee: |
The Kennedy Institute of
Rheumatology
|
Family ID: |
24773909 |
Appl. No.: |
12/583851 |
Filed: |
August 26, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11225631 |
Sep 12, 2005 |
7846442 |
|
|
12583851 |
|
|
|
|
09754004 |
Jan 3, 2001 |
|
|
|
11225631 |
|
|
|
|
08690775 |
Aug 1, 1996 |
6270766 |
|
|
09754004 |
|
|
|
|
08607419 |
Feb 28, 1996 |
|
|
|
08690775 |
|
|
|
|
PCT/GB94/00462 |
Mar 10, 1994 |
|
|
|
08607419 |
|
|
|
|
08403785 |
May 3, 1995 |
5741488 |
|
|
PCT/GB93/02070 |
Oct 6, 1993 |
|
|
|
PCT/GB94/00462 |
|
|
|
|
07958248 |
Oct 8, 1992 |
|
|
|
08403785 |
|
|
|
|
Current U.S.
Class: |
424/158.1 |
Current CPC
Class: |
A61K 31/505 20130101;
A61K 31/519 20130101; C07K 2317/24 20130101; A61P 19/02 20180101;
C07K 16/241 20130101; C07K 2319/00 20130101; A61K 38/1793 20130101;
A61K 2039/545 20130101; A61K 39/395 20130101; A61K 2039/505
20130101; A61K 39/39541 20130101; A61K 45/06 20130101; A61P 37/00
20180101; A61P 37/06 20180101; Y10S 514/885 20130101; A61P 1/00
20180101; A61P 29/00 20180101; A61K 39/3955 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: |
424/158.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 29/00 20060101 A61P029/00 |
Claims
1-38. (canceled)
39. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective tumor necrosis
factor-.alpha. inhibiting amount of antibody cA2.
40. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective tumor necrosis
factor-.alpha. inhibiting amount of a tumor necrosis factor-.alpha.
antibody, or antigen binding fragment thereof, wherein the antibody
binds to human tumor necrosis factor-.alpha..
41. The method of claim 40, wherein the antibody is cA2.
42. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective TNF.alpha.-inhibiting
amount of an anti-TNF.alpha. antibody or antigen-binding fragment
thereof, said antibody comprising a human constant region, wherein
said antibody or antigen-binding fragment (i) competitively
inhibits binding of A2 (ATCC Accession No. PTA-7045) to human tumor
necrosis factor TNF.alpha., and (ii) binds to human TNF.alpha. with
an affinity of at least 1.times.10.sup.8 liter/mole, measured as an
association constant.
43. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective TNF.alpha.-inhibiting
amount of an anti-TNF.alpha. antibody or antigen-binding fragment
thereof, said antibody comprising a human IgG1 constant region,
wherein said antibody or antigen-binding fragment (i) competitively
inhibits binding of A2 (ATCC Accession No. PTA-7045) to human
TNF.alpha., and (ii) binds to human TNF.alpha. with an affinity of
at least 1.times.10.sup.8 liter/mole, measured as an association
constant (Ka).
44. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective TNF.alpha.-inhibiting
amount of an anti-TNF.alpha. antibody or antigen-binding fragment
thereof, said antibody comprising a human constant region, wherein
said antibody or antigen-binding fragment (i) comprises the
antigen-binding regions of A2 (ATCC Accession No. PTA-7045) and
(ii) binds to human TNF.alpha. with an affinity of at least
1.times.10.sup.8 liter/mole, measured as an association constant
(Ka).
45. A method of treating rheumatoid arthritis in a human comprising
administering to the human an effective TNF.alpha.-inhibiting
amount of an anti-TNF.alpha. antibody or antigen-binding fragment
thereof; said antibody comprising a human IgG1 constant region,
wherein said antibody or antigen-binding fragment (i) comprises the
antigen-binding regions of A2 (ATCC Accession No. PTA-7045), and
(ii) binds to human TNF.alpha. with an affinity of at least
1.times.10.sup.8 liter/mole, measured as an association constant
(Ka).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/225,631, filed Sep. 12, 2005, which is a continuation of U.S.
Ser. No. 09/754,004, filed Jan. 3, 2001, now abandoned, which is a
continuation of U.S. Ser. No. 08/690,775, filed Aug. 1, 1996, now
U.S. Pat. No. 6,270,766 B1, issued Aug. 7, 2001, which (1) is a
continuation-in-part of U.S. Ser. No. 08/607,419, filed Feb. 28,
1996, which is a continuation-in-part of International Application
No. PCT/GB94/00462, filed Mar. 10, 1994, and (2) is a
continuation-in-part of U.S. Ser. No. 08/403,785, filed May 3,
1995, now U.S. Pat. No. 5,741,488, issued Apr. 21, 1998, 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. Ser. No. 07/958,248, filed Oct. 8, 1992, now abandoned, the
entire teachings of all of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Monocytes and macrophages secrete cytokines known as tumor
necrosis factor alpha (TNFO.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. Chem. 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. Sci. 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); Sung et al., J. Exp. Med. 168:1539 (1988); Turner et 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 (Piquet 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
monocytelmacrophage production and secretion of TNF and other
cytokines (Kornbluth 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 showing 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.
[0022] FIG. 1 contains a set of graphs from an experiment which
illustrates the suppression of arthritis as assessed by the
clinical score (FIG. 1a) and pawswelling measurements (FIG. 1b)
after the administration of 50 .mu.g anti-TNF (hamster TN3.19.2)
and 200 .mu.g anti-CD4 to DBA/1 male mice.
[0023] FIG. 2 contains a set of graphs from a second experiment
which illustrates the potentiation of low dose anti-TNF and
anti-CD4 on clinical score (FIG. 2a) and pawswelling measurements
(FIG. 2b) after administration of 50 .mu.g anti-TNF 200 .mu.g
anti-CD4; and the clinical score (FIG. 2c) and pawswelling
measurements (FIG. 2d) after administration of 300 .mu.g anti-TNF
with 200 .mu.g anti-CD4, to DBA/1 male mice.
[0024] FIG. 3 is a graph illustrating the suppression of arthritis
as assessed by pawswelling measurements after the administration of
250 .mu.g cyclosporine A, 50 .mu.g anti-TNF antibody, and a
combination of 250 .mu.g cyclosporine A and 50 .mu.g anti-TNF
antibody to DBA/1 mice. Open squares=control; diamonds=cyclosporine
A; triangles=anti-TNF; closed squares=cyclosporine A/anti-TNF.
[0025] FIG. 6 is a graph showing the effect of administering 300
.mu.g anti-TNF antibody alone, a combination of 250 .mu.g
cyclosporin A and 300 .mu.g anti-TNF antibody to male DBA/1 mice on
the suppression of arthritis as assessed by paw-swelling
measurements. Open square=cyclosporin A plus anti-TNF antibody;
diamond=cyclosporin A plus control antibody; triangle=anti-TNF
antibody.
[0026] FIG. 7 is a graph showing the effect of administering 500
.mu.g cyclosporine A alone, 250 .mu.g anti-TNF antibody alone, and
a combination of 500 .mu.g cyclosporine A and 250 .mu.g anti-TNF
antibody to male DBA/1 mice on the suppression of arthritis as
assessed by clinical score. Open square control; diamond =anti-TNF
antibody; triangle=cyclosporine A; square=cyclosporine A plus
anti-TNF antibody. P<0.05 (vs. PBS treated group)
[0027] FIGS. 9A and 9B are graphs showing results of a
cross-blocking epitope ELISA with murine A2 (mA2) and chimeric
(cA2) antibody competitors.
[0028] FIG. 10A and 10B are graphs of a Scatchard analysis of
.sup.125I-labelled mAb A2 (mA2) and chimeric A2 (cA2) binding to
recombinant human TNF.alpha. immobilized on a microtiter plate.
Each Ka value was calculated from the average of two independent
determinations.
[0029] FIG. 24 is a graphical representation of swollen joint
counts (maximum 28), as recorded by a single observer. Circles
represent individual patients and horizontal bars show median
values at each time point. The screening time point was within 4
weeks of entry to the study (week 0); data from patient 15 were not
included after week 2 (dropout). Significance of the changes,
relative to week 0, by Mann Whitney test, adjusted: week 1,
p>0.05; week 2, p<0.02; weeks 3-4, p<0.002; weeks 6-8,
p>0.001.
[0030] FIG. 25 is a graphical representation of levels of serum
C-reactive protein (CRP)-Serum CRP (normal range 0-10 mg/liter),
measured by nephelometry. Circles represent individual patients and
horizontal bars show median values at each time point. The
screening time point was within 4 weeks of entry to the study (week
0); data from patient 15 were not included after week 2 (dropout).
Significance of the changes, relative to week 0, by Mann-Whitney
test, adjusted: week 1, p<0.001; week 2, p<0.003; week 3,
p<0.002; week 4, p<0.02; week 6,8, p<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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: [0035] (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;
[0036] (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); [0037] (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 spondylitis, enteropathic arthritis and spondylitis,
reactive arthritis and arthritis associated with inflammatory bowel
disease; and uveitis; [0038] (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 MachadoJoseph)); and systemic
disorders (Refsum's disease, abetalipoprotienemia, 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; [0039] (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));
[0040] (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 [0041] (G)
alcohol-induced hepatitis and other forms of chronic hepatitis.
See, e.g., Berkow et al., Eds., The Merck Manual, 16th edition,
chapter 11, pp. 1380-1529, Merck and Co., Rahway, N.J., 1992,
incorporated herein by reference.
[0042] 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.
[0043] 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.
[0044] In a second embodiment, the invention relates to a method
for treating and/or preventing Crohn's disease in an individual
comprising co-administering a methotrexate and a TNF antagonist to
the individual in therapeutically effective amounts.
[0045] 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.
[0046] 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.
[0047] 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
[0048] 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
[0049] As used herein, an "anti-tumor necrosis factor antibody"
decreases, blocks, inhibits, abrogates or interferes with TNF
activity in vivo. 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.
[0050] 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), U.S.
application Ser. No. 08/324,799 (filed Oct. 18, 1994), 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 a of anti-TNF a 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.
[0051] Preferred anti-TNF mAbs are also those 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. Preferred antibodies of the
present invention are those that bind epitopes recognized by A2 and
cA2, which are included in amino acids 59-80 and/or 87-108 of
hTNF.alpha. (as these corresponding amino acids of SEQ ID NO:1),
such that the epitopes consist of at least 5 amino acids which
comprise at least one amino acid from the above portions of human
TNF.alpha..
[0052] The avidity and epitope specificity of the chimeric A2 is
derived from the variable region of the murine A2. In a solid phase
ELISA, cross-competition for TNF was observed between chimeric and
murine A2, indicating an identical epitope specificity of cA2 and
murine A2. The specificity of cA2 for TNF-.alpha. was confirmed by
its inability to neutralize the cytotoxic effects of lymphotoxin
(TNF-.beta.). 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.9
M.sup.1.
[0053] Since circulating concentrations of TNF tend to be extremely
low, in the range of about 10 pg/ml in non-septic individuals, and
reaching about 50 pg/ml in septic patients and above 100 pg/ml in
the sepsis syndrome (Hammerle, A. F. et al., 1989, infra) or can be
only be detectable at sites of TNF-mediated pathology, it is
preferred to use high affinity and/or potent in vivo TNF-inhibiting
and/or neutralizing antibodies, fragments or regions thereof, for
both TNF immunoassays and therapy of TNF-mediated pathology. Such
antibodies, fragments, or regions, will preferably have an affinity
for hTNF.alpha., expressed as Ka, of at least 10.sup.8 M.sup.-1,
more preferably, at least 10.sup.9 M.sup.-1, such as
10.sup.8-10.sup.10 M.sup.-1, 5.times.10.sup.8 M.sup.-1,
8.times.10.sup.8 M.sup.-1, 2.times.10.sup.9 M.sup.-1,
4.times.10.sup.9 M.sup.-1, 6.times.10.sup.9 M.sup.-1,
8.times.10.sup.9 M.sup.-1, or any range or value therein.
[0054] 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, 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); Moller, et al., Cytokine 2:162-169
(1990), which references are entirely incorporated herein by
reference).
[0055] 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 et
al., Lancet 344:1125-1127 (1994), incorporated herein by
reference).
[0056] 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).
[0057] 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).
[0058] Chimeric antibodies and methods for their production have
been described in the art (Morrison et al., Proc. Natl. Acad. Sci.
USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646
(1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et
al., European Patent Application No. 171496 (published Feb. 19,
1985); Morrison et al., European Patent Application No. 173494
(published Mar. 5, 1986); Neuberger et al., PCT Application No. WO
86/01533, (published Mar. 13, 1986); Kudo et al., European Patent
Application No. 184187 (published Jun. 11, 1986); Morrison et 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 et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987);
Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et
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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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), U.S. application Ser. No.
08/192,861 (filed Feb. 4, 1994), and U.S. application Ser. No.
08/324,799 (filed Oct. 18, 1994), and by Le, J. et 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. et al. (Cytokine 7(1):15-25 (1995)). These
references are entirely incorporated herein by reference.
[0063] Chimeric A2 anti-TNF consists of the antigen binding
variable region of the high-affinity neutralizing mouse anti-human
TNF IgGl antibody, designated A2, and the constant regions of a
human IgGl, kappa immunoglobulin. The human IgGl 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.9 M.sup.-1. 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.
[0064] 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.
[0065] 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.
[0066] 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 vivo 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.
[0067] 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.
[0068] 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
[0069] 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.
[0070] 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); Moller, et al., Cytokine 2:162-169 (1990).
[0071] 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.
[0072] 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
vivo 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.
[0073] 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).
[0074] 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.
[0075] "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.
[0076] "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
[0077] 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), U.S. application Ser. No.
08/192,861 (filed Feb. 4, 1994), U.S. application Ser. No.
08/324,799 (filed on Oct. 18, 1994) 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 et 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.
[0078] The DNA encoding an anti-TNF antibody can be genomic DNA or
cDNA which encodes at least one of the heavy chain constant region
(Hc), 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.
[0079] 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.
[0080] 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.
[0081] 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).
[0082] 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, et 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
(Walter, et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)),
tissue-type plasminogen activator (Pennica, et al., Nature
301:214-221 (1983)) and human placental alkaline phosphatase
complementary DNA (Keun, et al., Proc. Natl. Acad. Sci. USA
82:8715-8719 (1985)).
[0083] 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 et al. Bio/Technology 11:591-595 (1993);
Marks et 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.
[0084] 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.
[0085] 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.
[0086] 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 (J) segment, linked to a second DNA segment encoding
at least a part of a human C region.
[0087] Therefore, CDNA encoding the antibody V and C regions and
the method of producing a chimeric antibody can involve several
steps, outlined below: [0088] 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; [0089] 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; [0090] 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; [0091] 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.
[0092] 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 degree 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 J 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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).
[0099] 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 is 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
[0100] 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.
[0101] 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.
[0102] 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.
[0103] TNF immunoreceptor fusion molecules and methods for their
production have been described in the art (Lesslauer et al., Eur.
J. Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl.
Acad. Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med.
174:1483-1489 (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.
[0104] 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 et al., Current Protocols in Molecular
Biology, Greene Publishing Assoc. and Wiley-Interscience, New York
(1989).
Methotrexate
[0105] Presently available oral and intravenous formulations of
methotrexate include Heumatrex.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
[0106] 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.
[0107] 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.
[0108] 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. Liposomes 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.
[0109] Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, A. Osol, a standard reference
text in this field of art.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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., Imunol.
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)).
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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
[0119] 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
[0120] 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/1.
[0121] 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
[0122] 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
[0123] 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.
[0124] 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).
[0125] 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).
[0126] 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 et al., Lancet 344:1105-1110 (1994); Elliott et al., Lancet
344:1125-1127 (1994); and Elliott et al., Arthritis Rheum.
36(12):1681-1690 (1993), which references are entirely incorporated
herein by reference.
[0127] Evaluations were performed at weeks 1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22 and 26.
Results
[0128] 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) MTX = Methotrexate
[0129] 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 3 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
[0130] 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.
[0131] 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 73.3% 64.3% 76.9% 66.7% <0.001 response
(11/15) (9/14) (10/13) (10/15) p-value vs 0.001 0.008 0.002 0.009
MTX alone 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.
[0132] 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 Placebo 1 mg/kg cA2 3 mg/kg cA2 10 mg/kg cA2 MTX+ MTX+ MTX-
MTX+ MTX- MTX+ MTX- (n = 14) (n = 14) (n = 15) (N = 15) (n = 14) (n
= 14) (n = 15) Treatment effect Pts with complete.sup.a infusions
p-value 5 infusions 6 (42.86%) 12 (85.71%) 8 (53.33%) 15 (100.00%)
12 (85.71%) 12 (85.71%) 12 (80.00%) 0.003 4 infusions 0 (0.00%) 1
(7.14%) 0 (0.00%) 0 (0.00%) 1 (7.14%) 1 (7.14%) 0 (0.00%) 3
infusions 2 (14.29%) 1 (7.14%) 6 (40.00%) 0 (0.00%) 0 (0.00%) 1
(7.14%) 1 (6.67%) 2 infusions 5 (35.71%) 0 (0.00%) 1 (6.67%) 0
(0.00%) 1 (7.14%) 0 (0.00%) 2 (13.33%) 1 infusion 1 (7.14%) 0
(0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (0.00%) 0 (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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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
[0137] 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.
[0138] 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.
[0139] 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
[0140] 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 responses not 2/13 (15.4%) 8/15
(53.3%) 0/10 (0%) 3/12 (25.0%) 0/2 (0%) 1/10 (10%) including pts
with 26-week serum cA2 HACA responses 2/13 (15.4%) 8/15 (53.3%)
0/15 (0%) 3/14 (21.4%) 0/14 (0%) 1/15 (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.
[0141] 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
[0142] 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.
[0143] 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)).
[0144] 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.
[0145] 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)).
[0146] 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 et al.,
Ophthalmology 98(9):1147-1148 (1991)) and may be heightened in
patients receiving corticosteroid therapy.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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
[0152] 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
[0153] 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).gtoreq.28
mm/hour; C-reactive protein (CRP).gtoreq.20 mg/l.
[0154] Patients taking NSAIDs and corticosteroids (prednisone) at
screening were allowed to continue at stable doses (7.5
mg/day).
Study Infusions
[0155] 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
[0156] 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.
[0157] 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).
[0158] Evaluations were performed at day 3, and at weeks 1, 2, 4,
6, 8, 10, and 12.
Results
[0159] The 28 patients were randomized to one of four treatment (or
dose) groups.
[0160] 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 7 7 7 7
21 evaluated Pts with 1 (14.3%) 6 (85.7%) 5 (71.4%) 6 (85.7%) 17
(81.0%) any response 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%)
[0161] 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.
[0162] 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.
[0163] 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
[0164] 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
[0165] 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.
[0166] 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
[0167] 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).
[0168] 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).
Results
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
Exemplification: Treatment of Induced Arthritis in a Murine
Model.
[0173] The murine model of collagen type II induced arthritis has
similarities to rheumatoid arthritis (RA) in its marked MHC class
II predisposition, as well as in histology, immunohistology,
erosions of cartilage and bone, and in its response to anti-TNF
therapy. Thus the animal model serves as a good approximation to
human disease. The model of rheumatoid arthritis used herein is
describe by Williams, R. O. et al., (PNAS, in press), i.e., the
collagen type II induced arthritis in the DBA/1 mouse.
[0174] DBA/1 mice, aged 8-12 weeks were immunized intradermally
with 100 .mu.g of bovine type II collagen in complete Freund's
adjuvant, and 21 days later with 100 .mu.g of collagen
intra-peritoneally (i.p.). Immediately after the onset of
clinically evident arthritis (redness and/or swelling in on or more
limbs), which was about 35 days after the initial injection, mice
were injected i.p. with anti-CD4; anti-TNF; anti-CD4 and anti-TNF;
or isotype controls. Arthritis was monitored for clinical score and
paw-swelling for 10 days, after which time the joints were
processed for histology. Antibody was injected on day 1 (onset of
arthritis is day 0), day 4 and day 7.
[0175] Two experiments were completed, assessing clinical score and
pawswelling. In each, 200 .mu.g of anti-CD4 were used pre injection
(rat YTS 191 and YTA 3.1) was used. Clinical score was assessed on
the following scale: 0=normal; 1=slight swelling and/or erythema;
2=pronounced edematoma swelling; and 3=joint rigidity. Each limb
was graded, giving a maximum score of 12 per mouse. Pawswelling was
monitored by measuring the thickness of each affected hind paw with
calipers. The results were expressed as the percentage increment in
paw width relative to the paw width before the onset of
arthritis.
[0176] In the first experiment, a single dose of 50 .mu.g per
injection of anti-TNF (hamster TN3.19.2) was administered to each
of five mice per group. There was no significant effect of anti-CD4
or anti-TNF (TN3.19 given 3 times at 50 .mu.g/mouse). Hence the
benefit of combination therapy, in both clinical score and footpad
swelling, is readily seen (see FIGS. 1a, 1b).
[0177] In the second experiment, either 50 .mu.g and 300 .mu.g of
anti-TNF were administered to each of 7 mice per group. Both
anti-CD4 and anti-TNF at low (50 .mu.g) concentration had some
effect, and benefit of combination therapy of these two
concentrations was noted in pawswelling, not in clinical score.
However, if anti-TNF was injected at 300 .mu.g/mouse, the benefit
of combination therapy with anti-CD4 was seen in both clinical
score and more clearly in paw-swelling (see FIGS. 2a, 2b, 2c,
2d).
[0178] The results of the experiments indicate that there is a
clear benefit to combination therapy with anti-TNF and anti-CD4
antibodies, as measure by clinical score and foot pad swelling.
Study 2
[0179] Male DBA/1 mice were immunized intradermally at 8-12 weeks
of age with 100 .mu.g type II collagen emulsified in Freund's
complete adjuvant. Day one of arthritis was considered to be the
day that erythema and/or swelling was first observed in one or more
limbs. Arthritis became clinically evident around 30 days after
immunization with type II collagen. For each mouse, treatment was
started on the first day that arthritis was observed and continued
over a 10 day period, after which the mice were sacrificed and
joints were processed for histology. Monoclonal antibody treatment
was administered on days 1, 4 and 7. First, a sub-optimal dose of
50 .mu.g of anti-TNF alone (TN3-19.12, hamster IgG1
anti-TNF.alpha./.beta. mAb) was compared with the same does given
together with 200 .mu.g of anti-CD4 (rat IgG2b, a mixture of YTS
191.1.2 and YTA 3.1.2). To verify the results, two separate but
identical experiments were carried out (11-12 mice/group and 7-8
mice/group, respectively). Neither anti-CD4 alone nor sub-optimal
anti-TNF alone were able to significantly reduce paw-swelling (Data
not shown). However, treatment with anti-TNF and anti-CD4 resulted
in a consistently and statistically significant reduction in
paw-swelling relative to the group given control mAb (P<0.001).
Furthermore, in both experiments, combined anti-TNF/anti-CD4
treatment (also referred to herein as anti-CD4/TNF treatment)
produced a significant reduction in paw-swelling relative to
anti-CD4 alone, and anti-TNF alone (P<0.05).
[0180] Next, an optimal dose of anti-TNF (300 .mu.g) alone was
compared in two separate but identical experiments (7-7 mice/group
and 6-7 mice/group, respectively) with the same dose given in
combination with anti-CD4. As before, the combined
anti-TNF/anti-CD4 treatment resulted in a significant reduction in
paw-swelling compared to treatment with the control mAb
(P<0.005; data not shown). In the first experiment, paw-swelling
was also significantly reduced in the combined anti-CD4/anti-TNF
treated group relative to the groups given anti-CD4 alone of
anti-TNF alone (P<0.005). Some reduction in paw-swelling was
observed in mice given either anti-TNF alone or anti-CD4 alone
although the differences were not significant, possibly because of
the small group sizes (6 per group). In the second experiment,
combined anti-CD4/anti-TNF gave significantly reduced paw-swelling
compared to anti-CD4 alone (P<0.05) but not compared to anti-TNF
alone since anti-TNF itself causes a significant reduction in
paw-swelling, as expected from previous work (Williams, R. O. et
al., PNAS 89: 9784-9788 (1992)). In the experiments, the reduction
in paw-swelling attributable to anti-TNF alone was 23% and 33%,
respectively. Thuse, the reduction in paw-swelling attributable to
anti-TNF treatment was broadly comparable with our previously
published findings in which treatment with TN3-119.12 (300
.mu.g/mouse) resulted in a mean reduction in paw-swelling over the
treatment period of around 34% relative to controls (Williams, R.
O. et al., PNAS 89: 9784-9788 (1992)).
[0181] Limb Involvement
[0182] In collagen-induced arthritis, as in RA, it is usually for
additional limbs to become involved after the initial appearance of
clinical disease and new limb involvement is an important indicator
of the progression of the disease. To determine the effect of
anti-CD4/anti-TNF treatment on new limb involvement, the number of
limbs with clinically detectable arthritis at the end of the 10 day
treatment period was compared with the number of arthritis limbs
before treatment. In mice given the control mAb there was an
increase in limb involvement over the 10 day period of
approximately 50%. The results from the two experiments were
pooled, and are shown in table 1.
TABLE-US-00009 TABLE 1 Combined anti-CD4/anti-TNF Inhibits
Progression of Clinical Arthritis Number of Limbs Affected (Mean
.+-. SEM) Increase Treatment Day 1 Day 10 (%) Sub-optimal anti-TNF
(50 .mu.g) anti-CD4 1.30 .+-. 0.10 1.90 .+-. 0.13 46.1 (n = 18)
anti-TNF 1.20 .+-. 0.09 1.65 .+-. 0.17 37.5 (n = 19) anti-CD4/TNF
1.40 .+-. 0.09 1.45 .+-. 0.22 3.4.sup.1 (n = 18) control mAb 1.43
.+-. 0.15 2.24 .+-. 0.18 56.6 (n = 18) Optimal anti-TNF (300 .mu.g)
anti-CD4 1.27 .+-. 0.10 1.80 .+-. 0.14 42.0 (n = 12) anti-TNF 1.50
.+-. 0.17 1.64 .+-. 0.20 9.5.sup.2 (n = 11) anti-CD4/TNF 1.25 .+-.
0.11 1.25 .+-. 0.11 0.sup.3 (n = 13) control mAb 1.53 .+-. 0.19
2.27 .+-. 0.25 47.8 (n = 12) .sup.1P < 0.05 (anti-CD4/TNF vs.
control mAb) .sup.2P < 0.05 (anti-TNF vs. control mAb) .sup.3P
< 0.005 (anti-CD4/TNF vs. control mAb)
[0183] There was some reduction in new limb involvement in the
groups given anti-CD4 alone and sub-optimal anti-TNF alone,
although the differences were not significant. In the group given
optimal anti-TNF the increase in limb involvement was less than 10%
(P<0.05). More striking, however, was the almost complete
absence of new limb involvement in the groups given combined
anti-CD4/anti-TNF. Thus, the increase in new limb involvement was
only 3% in mice given anti-CD4 plus suboptimal anti-TNF (P<0.05)
and 0% in mice given anti-CD4 plus optimal anti-TNF
(P<0.005).
Histology
[0184] After 10 days, the mice were sacrificed; the first limb that
had shown clinical evidence of arthritis was removed from each
mouse, formalin-fixed, decalcified, and wax-embedded before
sectioning and staining with haemotoxylin and eosin. A saggital
section of the proximal interphalangeal (PIP) joint of the middle
digit was studied in a blind fashion for the presence or absence of
erosions in either cartilage or bone (defined as demarcated defects
in cartilage or bone filled with inflammatory tissue). The
comparisons were made only between the same joints, and the
arthritis was of identical duration. Erosions were observed in
almost 100% of the PIP joints from the control groups and in
approximately 70-80% of the joints given either anti-CD4 lone or
sub-optimal anti-THF alone. The results of the two experiments were
pooled, and are shown in Table 2.
TABLE-US-00010 TABLE 2 Proportions of PIP joints showing
Significant Erosion of Cartilage and/or Bone Treatment Joints with
Erosions Sub-optimal anti-TNF (50 .mu.g) Anti-CD4 13/18 (72%)
Anti-TNF 14/19 (74%) Anti-CD4/TNF .sup. 4/18 (22%).sup.1 Control
mAb 17/18 (94%) Optimal anti-TNF (300 .mu.g) Anti-CD4 10/12 (83%)
Anti-TNF .sup. 6/11 (54%).sup.2 Anti-CD4/TNF .sup. 4/13 (31%).sup.3
Control mAb 12/12 (100%) .sup.1P < 0.01 (anti-CD4/TNF vs.
anti-CD4 alone; antiTNF alone and control mAb) .sup.2P < 0.01
(antiTNF alone vs. control mAb) .sup.3P < 0.01 (antiCD4/TNF vs.
antiCD4 alone and control mAb)
[0185] An optimal dose of anti-TNF alone significantly reduced
pathology, as reported previously (Williams, R. O. et al., PNAS 89:
9784-9788 (1992)). Thus, in the mice given optimal anti-TNF alone
the proportion of joints showing erosive changes was reduced to 54%
(P<0.001) whereas in the groups given anti-CD4 plus either
sub-optimal or optimal anti-TNF, only 22% (P<0.01) and 31%
(P>0.01) of the joints, respectively, were eroded. Thus, 300
.mu.g of anti-TNF alone gave a degree of protection against joint
erosion but combined anti-CD4/anti-TNF provided significantly
greater protection.
Example 2
Treatment of Induced Arthritis in a Murine Model using TNF
Receptor/IgG Fusion Protein with Anti-CD4 Antibody
[0186] The murine model of collagen type II induced arthritis,
described above, was used to investigate the efficacy of a human
p55 TNF receptor/IgG fusion protein, in conjunction with anti-CD4
monoclonal antibody (mAb), for its ability to modulate the severity
of joint disease in collagen-induced arthritis. First, a comparison
was made between the efficacy of TNF receptor/IgG fusion protein
treatment, anti-TNF mAb treatment, and high dose corticosteroid
therapy. Subsequently, therapy with TNF receptor/IgG fusion protein
in conjunction with anti-CD4 antibody was investigated.
A. Experimental Procedure
[0187] Male DBA/1 mice were immunized intradermally with 100 .mu.g
of bovine type II collagen emulsified in complete Freund's adjuvant
(Difco Laboratories, East Molsey, UK). The mean day of onset of
arthritis was approximately one month after immunization. After the
onset of clinically evident arthritis (erythema and/or swelling),
mice were injected intraperitoneally with therapeutic agents.
Arthritis was monitored for clinical score and paw swelling
(measured with calipers) for 10 days, after which the mice were
sacrificed and joints were processed for histology. Sera were
collected for analysis on day 10. Therapeutic agents were
administered on day 1 (onset), day 4 and day 7. The therapeutic
agents included TNF receptor/IgG fusion protein (p55-sf2), anti-TNF
antibody, anti-CD4 antibody, and methylprednisolone acetate.
B. Comparison of Treatment with TNF Receptor/IgG Fusion Protein,
Anti-TNF Antibody, or Methylprednisolone Acetate
[0188] Using the Experimental Procedure described above, groups of
mice were subjected to treatment with TNF receptor/IgG protein (2
.mu.g) (18 mice), TNF receptor/IgG protein (20 .mu.g) (18 mice),
TNF receptor/IgG protein (100 .mu.g) (12 mice), anti-TNF monoclonal
antibody (mAb) (300 .mu.g) (17 mice), methylprednisolone acetate (6
mice), an irrelevant human IgG1 monoclonal antibody (mAb) (6 mice),
or saline (control). The TNF receptor/IgG fusion protein, herein
referred to as p55-sf2, (Butler et al., Cytokine (in press):
(1994), was provided by Centocor, Inc., Malvern Pa.; it is dimeric
and consists of the human p55 TNF receptor (extracellular domains)
fused to a partial J sequence followed by the whole of the constant
region of the human IgG1 heavy chain, itself associated with the
constant region of a kappa light chain. The anti-TNP antibody was
TN3-19.12, a neutralizing hamster IgG1 anti-TNF.alpha./.beta.
monoclonal antibody (Sheehan, K. C. et al., J. Immunology
142:3884-3893 (1989)), and was provided by R. Schreiber, Washington
University Medical School (St. Louis, Mo., USA), in conjunction
with Celltech (Slough, UK). Neutralizing titres were defined as the
concentration of TNF.alpha. neutralizing agent required to cause
50% inhibition of killing of WEHI 164 cells by trimeric recombinant
murine TNF.alpha.; the neutralizing titre of p55-sf2 was 0.6 ng/ml,
compared with 62.0 ng/ml for anti-TNF mAb (TN3-19.12), using 60
pg/ml mouse TNF.alpha.. The corticosteroid, mathyl-prednisolone
acetate (Upjohn, Crawley, UK) was administered by intraperitoneal
injection as an aqueous suspension at a dosage level of 2 mg/kg
body weight; using the protocol described above, this dosage is
equivalent to 4.2 mg/kg/week, a dose which is higher than the
typical dose used to treat refractory RA in humans (1-2
mg/kg/week).
Paw-Swelling
[0189] Treatment with p55-sf2 resulted in a dose-dependent
reduction in paw-swelling over the treatment period, with the doses
of 20 .mu.g and 100 .mu.g giving statistically significant
reductions in paw-swelling relative to mice given saline
(P<0.05). The group of mice given an irrelevant human IgG1 mAb
as a control did not show any deviation from the saline-treated
group (data not shown), indicating that the therapeutic effects of
p55-sf2 were attributable to the TNF receptor rather than the human
IgG1 constant region. Similar reductions in paw-swelling were seen
in mice given 300 .mu.g of anti-TNF mAb as in those given 100 .mu.g
of p55-sf2, although anti-TNF mAb was marginally more effective
than p55-sf2 at inhibiting paw-swelling. A reduction in
paw-swelling was observed in the methylprednisolone acetate treated
group that was comparable in magnitude to the reductions given
p55-sf2 at 100 .mu.g or anti-TNF mAb at 300 .mu.g.
Limb Involvement
[0190] The change in the number of arthritic limbs over the 10 day
treatment period was examined. Results are shown in Table 5.
TABLE-US-00011 TABLE 5 Inhibitory Effect of TNF-Targeted Therapy on
Limb Recruitment Limbs Affected Treatment (mean .+-. SEM) (number
of animals) Day 1 Day 10 Increase (%) Saline (n = 12) 1.33 .+-.
0.14 2.25 .+-. 0.18 69% P55-sf2, 1.28 .+-. 0.11 1.94 .+-. 0.17 51%
2 .mu.g (n = 18) P55-sf2, 1.37 .+-. 0.11 1.79 .+-. 0.16 31% 20
.mu.g (n = 18) P55-sf2, 1.17 .+-. 0.17 1.58 .+-. 0.23 35% 100 .mu.g
(n = 12) Control IgG1, 1.00 .+-. 0.00 1.15 .+-. 0.22 50% 100 .mu.g
(n = 6) Anti-TNF mAb, 1.47 .+-. 0.15 .sup. 1.76 .+-. 0.16.sup.1 20%
300 .mu.g (n = 17) Methylpresnisolone 1.00 .+-. 0.00 1.50 .+-. 0.22
33% Acetate (n = 6) P, 0.05 (vs. saline; Mann Whitney Test)
[0191] A strong trend towards reduced limb recruitment was seen in
the groups of mice given p55-sf2, anti-TNF mAb or
methylprednisolone acetate, but only in the anti-TNF mAb treated
group did the reduction reach statistical significance
(P<0.05).
Histology
[0192] After 10 days, the mice were sacrificed; the first limb to
show clinical evidence of arthritis was removed from each mouse,
fixed, decalcified, wax-embedded, and sectioned and stained with
haematoxylon and eosin. Saggital sections of the proximal
interphalangeal (PIP) joint of the middle digit of each mouse were
studied in a blind fashion and classified according to the presence
or absence of erosions, as defined above. Comparisons were thus
made between identical joints, and the arthritis was of equal
duration. Results are shown in Table 6.
TABLE-US-00012 TABLE 6 Histopathology of PIP Joints Treatment PIP
Joints with Erosions Saline 11/12 (92%) P55-sf2, 2 .mu.g 14/18
(78%) P55-sf2, 20 .mu.g 14/18 (78%) P55-sf2, 100 .mu.g 6/12 (50%)
Control IgG1, 100 .mu.g 6/6 (100%) Anti-TNF mAb, 300 .mu.g 7/17
(41%) Methylpresnisolone acetate 4/6 (67%) P < 0.05 (vs.
saline). P < 0.01 (vs. saline). Data were compared by Chi-square
analysis.
[0193] Erosions were present in 92% and 100% of the PIP joints in
the saline treated group and the control human IgG1 treated group,
respectively. However, only 50% (P<0.05) of joints from the mice
treated with p55-sf2 (100 .mu.g) and 41% (P<0.01) of mice given
anti-TNF mAb exhibited erosive changes. Some reductions in the
proportion of eroded joints were observed in mice treated with 2
.mu.g or 20 .mu.g of p55-sf2, but these were not statistically
significant. Similarly, treatment with methylprednisolone acetate
did not significantly reduce joint erosion.
Example 3
Treatment of Induced Arthritis in a Murine Model using Cyclosporin
A and Anti-TNF Antibody
[0194] The murine model of collagen type II induced arthritis,
described above, was used to investigate the efficacy of the CD4+ T
cell inhibiting agent cyclosporin A in conjunction with anti-TNF
monoclonal antibody (mAb), for the ability to modulate the severity
of joint disease in collagen-induced arthritis. A comparison was
made between the efficacy of treatment with cyclosporin A (CsA),
anti-TIF antibody, and combination of CSA and anti-TNF
antibody.
[0195] A. Experimental Procedure
[0196] Male DBA/1 mice were immunized intradermally with 100 .mu.g
of bovine type II collagen emulsified in complete Freund's adjuvant
(Difco Laboratories, East Molsey, UK). The mean day of onset of
arthritis was approximately one month after immunization. After the
onset of clinically evident arthritis (erythema and/or swelling),
groups of mice (11 mice each) were subjected to treatment with one
of the following therapies: 50 .mu.g (2 ag/kg) L2 (the isotype
control for anti-TNF antibody), intraperitoneally once every three
days (days 1, 4 and 7); 250 .mu.g (10 mg/kg) cyclosporin A
intraperitoneally daily; 50 .mu.g (2 mg/kg) anti-TNF mAb TN3-19.12,
intraperitoneally once every three days (days 1, 4 and 7); or 250
.mu.g cyclosporin A intraperitoneally daily in conjunction with 50
.mu.g anti-TNF mAb intraperitoneally once every three days.
Arthritis was monitored for paw swelling (measured with calipers)
for 10 days, after which the mice were sacrificed and joints were
processed for histology.
[0197] Paw-Swelling
[0198] Treatment with cyclosporin A in conjunction with anti-TNF
mAb resulted in a reduction in paw-swelling over the treatment
period, relative to mice treated with control antibody. Results are
shown in FIG. 3.
[0199] Limb Involvement
[0200] As before, the progressive involvement of additional limbs
following the initial appearance of arthritis was studied. Results
are shown in Table 10.
TABLE-US-00013 TABLE 10 Anti-CD4 Antibody and p55-sf2 Prevent New
Limb Recruitment Limbs Affected (mean .+-. SEM) Treatment Day 1 Day
10 Increase (%) Control mAb 1.36 .+-. 0.20 2.45 .+-. 0.28 80.1%
Cyclosporin A 1.36 .+-. 0.15 2.18 .+-. 0.30 60.3% Anti-TNF mAb 1.45
.+-. 0.16 1.9 .+-. 0.21 31.0% CsA/Anti-TNF mAb 1.27 .+-. 0.14 .sup.
1.54 .+-. 0.21.sup.1 21.0% P = 0.03 (vs. control).
[0201] Treatment with cyclosporin A in conjunction with anti-TNF
mAb resulted in statistically significant reductions in limb
involvement in comparison to control monoclonal antibody
(P=0.03).
Example 5
Treatment of Induced Arthritis in a Murine Model using Anti-TNF
Antibody and a Sub-Optimal Dose of Cyclosporin A
[0202] The murine model of collagen Type II induced arthritis,
described above, was used to investigate the ability of
cyclosporine A to prolong the therapeutic effect of a single
injection of anti-TNF antibody to modulate the severity of join t
disease in collagen-induced arthritis. A comparison was made
between the efficacy of treatment with a single injection of 300
.mu.g anti-TNF antibody alone, and a combination of a single
injection of 300 .mu.g anti-TNF antibody and a sub-optimal dose of
CsA.
Experimental Procedure
[0203] Male DBA/1 mice were immunized intradermally with 100 .mu.g
of bovine type II collagen emulsified in complete Freund's adjuvant
(Difco Laboratories, East Molsey, UK). The mean day of onset of
arthritis was approximately one month after immunization. After the
onset of clinically evident arthritis (erythema and/or swelling in
one or more limbs), three groups of mice (10 mice per group) were
subjected to treatment with one of the following therapies: 250
.mu.g (10 mg/kg) cyclosporine A (Sandoz Pharmaceuticals, East
Hanover, N.J.), injected intra-peritoneally in conjunction with 300
.mu.g (12 mg.kg) anti-TNF mAb, injected intra-peritoneally, on day
one; or 300 .mu.g anti-TNF mAb TN3-19.12, injected
intra-peritoneally on day one. Arthritis was monitored for paw
swelling (measured with calipers) for 10 days, after which the mice
were sacrificed and joints were processed for histology.
Paw-Swelling
[0204] Paw swelling was monitored as described in Example 4.
Treatment with a sub-optimal dose of cyclosporine A in conjunction
with a single injection of anti-TNF mAb (300 .mu.g) resulted in a
sustained reduction in paw-swelling over the treatment period,
relative to mice treated with a sub-optimal dose of CsA in
conjunction with the control antibody and mice treated with 300
.mu.g anti-TNF mAb alone. Results are shown in FIG. 6.
Histology
[0205] Saggital sections of the PIP joint of the middle digit of
each mouse (from the first paw with clinical arthritis) were
examined in a blind fashion by microscopy and classified according
to the presence or absence of erosions, using the procedure
described in Example 1. Comparisons were thus made between
identical joints, and the arthritis was of equal duration. Results
are shown in Table 11.
TABLE-US-00014 TABLE 11 PIP Joint Erosions Treatment Incidence of
Erosions L2/CsA 8/10 (80%) TN3 alone 8/9 (89%) CsA/TN3 6/10
(60%)
In mice given a sub-optimal dose of CsA in conjunction with 300
.mu.g of anti-TNF mAb, the proportion of joints showing erosive
changes was reduced to 60% whereas, in the group of mice given a
sub-optimal dose of CsA plus control antibody, 80% of the joints
were eroded, and in the group of mice given 300 .mu.g anti-TNF mAb,
89% of the joints were eroded. Thus, treatment with a sub-optimal
dose of CsA in conjunction with 300 .mu.g anti-TNF mAb provided a
degree of protection against joint erosion.
Example 6
Treatment of Induced Arthritis in a Murine Model using Cyclosporin
A and Anti-TNF Antibody at Effective Doses
[0206] Using the murine model of collagen type II induced
arthritis, described above, a comparison was made between the
efficacy of treatment with CsA alone, anti-TNF antibody, for the
ability to modulate the severity of joint disease in collagen
induced arthritis.
[0207] Experimental Procedure
[0208] Male DBA/1 mice were immunized intradermally with 100 .mu.g
of bovine type II collagen emulsified in complete Freund's adjuvant
(Difco Laboratories, East Molsey, UK). The mean day of onset of
arthritis was approximately one month after immunization. After the
onset of clinically evident arthritis (erythema and/or swelling in
one or more limbs), three groups of mice (11-12 mice per group)
were subjected to treatment with one of the following therapies:
500 .mu.g (20 mg/kg) cyclosporin A (SANDIMMUNE.RTM., Sandoz
Pharmaceuticals, East Hanover, N.J.), injected intra-peritoneally
daily; 250 .mu.g (10 mg/kg) anti-TNF mAb TN3-19.12, injected
intra-peritoneally once every three days (days 1, 4, and 7); or 500
.mu.g cyclosporine A, injected intra-peritoneally daily in
conjunction with 250 .mu.g anti-TNF mAb, injected
intra-peritoneally once every three days. A control group of 24
mice was administered PBS, injected intra-peritoneally daily, after
the onset of clinically evident arthritis. Arthritis was monitored
for paw swelling (measured with calipers) for 10 days, after which
the mice were sacrificed and joints were processed for
histology.
[0209] Clinical Score
[0210] Clinical score was assessed on the following scale:
0=normal; 1=slight swelling and/or erythema; 2=pronounced edematous
swelling; and 3=joint rigidity. Each limb was graded, giving a
maximum score of 12 per mouse.
[0211] The results are presented in FIG. 7 and show that treatment
with 500 .mu.g cyclosporine A plus 250 .mu.g anti-TNF mAb resulted
in a significant reduction in the severity of arthritis over the
treatment period, relative to the control group (PBS treated
group). Treatment with either 250 .mu.g anti-TNF mAb alone of 500
.mu.g cyclosporine A alone also reduced the severity of arthritis.
(P<0.05 and related to differences between the PBS treated group
(Mann-Whitney U Test).
Histology
[0212] For histology, the mice were sacrificed 10 days and the
first limb that had shown clinical evidence of arthritis was
removed from each mouse, formalin-fixed, decalcified, and
waz-embedded before ectioning and staining with haematoxlon and
eosin. A saggital section of the proximal interphalangeal (PIP)
joint of the middle digit was examined by microscopy in a blind
fashion for the presence or absence of erosions in either cartilage
or bone (defined as demarcated defects in cartilage or bone filled
with inflammatory tissue). Comparisons were made between the same
joints, and the arthritis was of identical duration. Erosions were
observed in 9% of the POP joints from the group of mice treated
with a combination of 500 .mu.g (20 mg/kg) CsA and 250 .mu.g (10
mg/kg) anti-TNF mAb compared with in 36% of the PIP joints from the
group of mice treated with 500 .mu.g CsA alone and 42% of the PIP
joints from the group of mice treated with 250 .mu.g anti-TNF
antibody alone. The results of the experiment are shown in Table
13.
TABLE-US-00015 TABLE 13 Therapeutice Effects of Cyclosporin A and
Anti-TNF Monoclonal Antibody in Established Collagen-Induced
Arthritis No. Histology: proportion of Treatment mice per group PIP
joints with erosions PBS 24 23/24 (96%) CsA (20 mg/kg) 12 4/11
(36%) (P < 0.001) Anti-TNF mAb (10 mg/kg) 12 5/12 (42%) (P <
0.001) CsA (20 mg/kg) plus 11 1/11 (9%) Anti-TNF mAb (10 mg/kg) (P
< 0.001) P values refer to comparisions with the PBS-treated
group.
[0213] Treatment with cyclosporine A in conjunction with anti-TNF
antibody provides a greater degree of protection against arthritis
than treatment with either reagent alone. The results show that
there is an additive or synergistic ameliorative effect between
cyclosporine A and anti-TNF antibody.
Example X
Specificity of an Anti-TNF Chimeric Antibody
[0214] Since the antigen binding domain of cA2 was derived from
murine A2, these mAbs would be expected to compete for the same
binding site on TNF. Fixed concentrations of chimeric A2 and murine
mAb A2 were incubated with increasing concentrations of murine and
chimeric A2 competitor, respectively, in a 96-well microtiter plate
coated with rhTNF (Dainippon, Osaka, Japan). Alkaline-phosphatase
conjugated anti-human immunoglobulin and anti-mouse immunoglobulin
second antibodies were used to detect the level of binding of
chimeric and murine A2, respectively. Cross-competition for TNF
antigen was observed in this solid-phase ELISA format (FIG. 9).
This finding is consistent with the expected identical epitope
specificity of cA2 and murine A2.
[0215] The affinity constant for binding of mouse mAb A2 and cA2 to
rhTNF.alpha. was determined by Scatchard analysis (see, for
example, Scatchard, Ann. N.Y. Acad. Sci. 51:660 (1949)). The
results are shown in FIG. 10. This analysis involved measuring the
direct binding of .sup.125I labelled cA2 to immobilized
rhTNF.alpha. in a 96-well plate. The antibodies were each labelled
to a specific activity of about 9.7 .mu.Ci/.mu.g by the iodogen
method. An affinity constant (Ka) of 0.5.times.10.sup.9 liters/mole
was calculated for the mouse mAb A2. Unexpectedly, the chimeric A2
antibody had a higher affinity, with a Ka of 1.8.times.10.sup.9
liters/mole. Thus, the chimeric anti-TNF.alpha. antibody of the
present invention was shown to exhibit a significantly higher
affinity of binding to human TNF.alpha. than did the parental
murine A2 mAb. This finding was surprising, since murine and
chimeric antibodies, fragments and regions would be expected to
have affinities that are equal to or less than that of the parent
mAb.
[0216] Such high affinity anti-TNF antibodies, having affinities of
binding to TNF.alpha. of at least 1.times.10.sup.8 M.sup.-1, more
preferably at least 1.times.10.sup.9 M.sup.-1 (expressed as Ka) are
preferred for immunoassays which detect very low levels of TNF in
biological fluids. In addition, anti-TNF antibodies having such
high affinities are preferred for therapy of TNF-.alpha.-mediated
conditions or pathology states.
[0217] The specificity of cA2 for TNF was confirmed by testing for
cross-neutralization of human lymphotoxin (TNF-.beta.). Lymphotoxin
shares some sequence homology and certain biological activities,
for example, tumor cell cytotoxicity, with TNF (Pennica, et al.,
Nature 312:724-729 (1984)). Cultured human A673 cells were
incubated with increasing concentrations of human lymphotoxin
(GENENTECH, San Francisco, Calif.) with or without 4 .mu.g/ml
chimeric A2 in the presence of 20 .mu.g/ml cycloheximide at
39.degree. C. overnight. Cell death was measured by vital staining
with naphthol blue-black, as above. The results indicated that cA2
was ineffective at inhibiting and/or neutralizing human
lymphotoxin, confirming the TNF.alpha.-specificity of the chimeric
antibody.
[0218] The ability of A2 or cA2 to react with TNF from different
animal species was also evaluated. As mentioned earlier, there are
multiple epitopes on human TNF to which inhibiting and/or
neutralizing mAbs will bind (Moller, et al., infra). Human TNF has
bioactivity in a wide range of host animal species. However,
certain inhibiting and/or neutralizing epitopes on human TNF are
conserved amongst different animal species and others appear to be
restricted to humans and chimpanzees.
[0219] Neutralization experiments utilized endotoxin-activated cell
supernatants from freshly isolated human, chimpanzee, rhesus and
cynomolgus monkey, baboon, pig, dog, rabbit, or rat monocytes as
the TNF source. As discussed above, murine mAb A2 inhibited or
neutralized activity of only human and chimpanzee TNF, and had no
effect on TNF derived from other primates and lower animals. A2
also did not inhibit or neutralize the cytotoxic effect of
recombinant mouse TNF.
[0220] Thus, the epitope recognized by A2 is one shared by human
and chimpanzee TNF.alpha.. Chimeric A2 was also tested in this
manner for cross-reactivity with monocyte-derived TNF from rat,
rabbit, dog and pig, as well as with purified recombinant mouse
TNF.alpha., and natural and recombinant human TNF.alpha.. Chimeric
A2 only inhibited or neutralized natural and recombinant human
TNF.alpha.. Therefore, cA2 appears to share species specificity
with murine A2.
Example XX
Clinical Treatment of Rheumatoid Arthritis by a Anti-TNF Antibody
or Peptide of the Present Invention
[0221] A Phase I open label study was conducted for methods and
compositions of the present invention using a chimeric anti-TNF MAb
for the treatment of patients with severe refractory rheumatoid
arthritis. Nine patients were enrolled in the study. The first five
patients were treated with chimeric anti-TNF antibody (cA2), 10
mg/kg as a single dose infused over a period of two hours. These
patients were subsequently retreated with a second infusion of 10
mg/kg on day 14 of the study. The second group of five patients
received an infusion of 5 mg/kg on the first day of the study. They
were then treated with additional infusions of 5 mg/kg on days 5,
9, and 13. Four of the planned five patients in this second group
have been treated to date.
[0222] Preparation, Administration, and Storage of Test Material
The chimeric monoclonal anti-TNF antibody was supplied in
single-use glass vials containing 20 mL with 100 mg of anti-TNF (5
mg/mL). The anti-TNF antibody was stored at 2-8.degree. C. Prior to
infusion, the antibody was withdrawn from the vials and filtered
through a low-protein-binding 0.22 .mu.m filter. This filtered
antibody was then diluted to a final volume of 300 mL with normal
saline. The 300 mL antibody preparation was then infused via an
in-line filter over a period of not less than two hours.
[0223] Prior to each repeat infusion of study medication a test
dose of 0.1 mL of the infusion was diluted in 10 mL of normal
saline and administered by slow IV push over 5 minutes. The patient
was observed for 15 minutes for signs or symptoms of an immediate
hypersensitivity reaction. If no reaction was observed in this time
period, the full dose was administered as described above.
[0224] Administration Protocol
[0225] Group 1 (patients 1-5): a total of 2 infusions, on day 1 and
day 15 of the trial; dosage 10 mg/kg on each occasion; Group 2
(patients 6-9): a total of 4 infusions, on days 1, 5, and 13 of the
trial; dosage 5 mg/kg on each occasion.
[0226] All infusions were administered iv over 2 hours in a total
volume of cA2+saline of 300 ml. Infusions subsequent to the first
in any patient were preceded by a small test dose administered as
an iv push. All patients had at least three years of disease
activity with rheumatoid arthritis. The patients ranged in age from
23 to 63. All patients had failed therapy with at least three
different DMARD (Disease Modifying Anti-Rheumatic Drug). Six of the
nine patients had serum rheumatoid factors, and all nine patients
had erosions present on X-rays.
Clinical Monitoring
[0227] Patients were monitored during and for 24 hours after
infusions for hemodynamic change, fever or other adverse events.
Clinical and laboratory monitoring for possible adverse events was
undertaken on each follow-up assessment day. Clinical response
parameters were performed at the time-points as specified in the
flow charts present in Tables 9A and 9B. These evaluations were
performed prior to receiving any infusions. [0228] Clinical
response studies will be comprised of the following parameters:
[0229] 1. Number of tender joints and assessment of
pain/tenderness: [0230] The following scoring will be used: [0231]
0=No pain/tenderness [0232] 1=Mild pain. The patient says it is
tender upon questioning. 2=Moderate pain. The patient says it is
tender and winces. 3=Severe pain. The patient says it is tender and
winces and withdraws. [0233] 2. Number of swollen joints [0234]
Both tenderness and swelling will be evaluated for each joint
separately. MCP's, PIP's etc. will not be considered as one joint
for the evaluation. [0235] 3. Duration of morning stiffness (in
minutes) [0236] 4. Grip strength [0237] 5. Visual analog pain scale
(0-10 cm) [0238] 6. Patients and blinded evaluators will be asked
to assess the clinical response to the drug. Clinical response will
be assessed using a subjective scoring system as follows: [0239]
5=Excellent response (best possible anticipated response) [0240]
4=Good response (less than best possible anticipated response)
[0241] 3=Fair response (definite improvement but could be better)
[0242] 2=No response (no effect) [0243] 1=Worsening (disease worse)
[0244] Measurement of index of disease activity is scored according
to the following Table 5.
TABLE-US-00016 [0244] TABLE 5 Clinical Characteristics of Patients
1-5 Previous Di T Pa Age/ Duration Rhe (DMA Number Sex (years) Fac
Nodules only) Therapy 01 48/F 7 -ve/ *Sal, DP, Myo, **Pred 5 , MTX,
, Chl 02 7 -ve + -ve l, M o, DP Para 1-2 g 3 +ve + -ve , C l, My ,
MTX, S l 225 mg 04 56/M 10 +ve -ve My , DP, A , 12.5 mg, Sal 2 g,
Para 1-2 05 28/F 3 -ve M , Sal, DP, P 8 mg, A Para 1-2 , Cod 16
*Sal = ; DP = D- ; Myo = Myo ; Aur = ; MTX = m ; A = ; Ch = . **P =
; P = p ; d = indo ; = ; C = . indicates data missing or illegible
when filed
TABLE-US-00017 TABLE 6 Cli cal Characteristics of P Previous D Tre
C P Age/ Dura Rhe Er (DMARDs Number Sex (y s) Fact N ) Therapy 06 M
3 **Nap 1 g 07 F 7 DP, Myo, Sal, Para 1-2 g A , MTX Oxl 16- mg 08 F
11 Chl, Myo, Sal, Pred 7.5 mg, Dicl MTX, A 100 mg, Para 1-2 g, Dext
100-200 mg 09 F 15 Myo, Chl, DP, Pred 7.5 mg, Dicl MTX 125 mg, Para
1-3 g *Sal = ; Chl = ; A = ; D = D- ; M = My ; A = ; MTX = . **N =
; P = ; Cod = ; P = ; D = dicl ; De = de . indicates data missing
or illegible when filed
TABLE-US-00018 TABLE 7 Pa Number Rit ip CRP M (10-10 A Strength IDA
Index L/R ; normal Number (m ) VAS) (0-28) (0- ) < 10) 1- ) 01
60 3. 19 30 33 5 02 20 2. 25 31 18 14 .0 03 4. 14 16 230/238 48 44
.5 04 30 6. 17 12 204/223 24 35 2.33 05 5. 28 41 52/ 87 107 3.0
indicates data missing or illegible when filed
TABLE-US-00019 TABLE 8 Disease Activity at Entry for Patients 6-9
Pain Grip ESR CRP Morning (0-10 S h (mm (mg IDA P Stiffness cm L/R
(mm/Hg; ranges: F < Number (mins) VAS) (0-2 ) (0- ) ) M < )
1-4) 06 120 5.0 3 4 23 3 07 105 7. 27 31 25 10 2.83 08 270 .3 17 37
73/125 35 31 3.17 09 180 .5 20 26 5 75 15 .5 indicates data missing
or illegible when filed
[0245] All patients have tolerated the infusions of chimeric
anti-CD4 and no serious adverse reactions have been observed.
Specifically, no episodes of hemodynamic instability, fevers, or
allergic reactions were observed in association with the infusions.
Patients have not experienced any infections.
[0246] Although this is a non-blinded study, all patients
experienced improvement in their clinical assessments of disease
status, as well in biochemical parameters of inflammation measured
in their serum.
[0247] Clinical assessments, including the duration of early
morning stiffness; the assessment of pain on a visual analogue
scale; total count of swollen joints; Ritchie articular index (a
scaled score which assesses the total number of tender joints and
the degree of joint tenderness); and Index of Disease Activity (a
scaled score which incorporates several clinical and laboratory
parameters), showed impressive improvements compared to controls.
These improvements were typically in the range of an 80% drop from
the baseline score; a degree of improvement which is well beyond
the amount of improvement that can be attributed to placebo
response. In addition, the duration of these improvements was for
six to eight weeks in most cases, a duration of response far longer
than would be anticipated from a placebo.
[0248] The improvements in clinical assessments were corroborated
by improvements in biochemical inflammatory parameters measured in
serum. The patients showed rapid drops of serum C-reactive protein,
usually in the range of 80% from the baseline. Reductions in the
erythrocyte sedimentation rate, usually in the range of 40%, were
also observed. Circulating soluble TNF receptors were also
decreased following therapy. The durations of the biochemical
responses were similar to the duration of the clinical
responses.
[0249] Preliminary assessment of immune responses to the chimeric
anti-TNF antibody has shown no antibody response in the first four
patients.
[0250] In summary, the preliminary evaluation of the results of
this Phase I trial indicate that treatment of patients with
advanced rheumatoid arthritis with anti-TNF MAb of the present
invention is well tolerated and anti-TNF treatment is associated
with rapid and marked improvement in clinical parameters of disease
activity, including early morning stiffness, pain, and a number of
tender and swollen joints; and is accompanied by improvement of
biochemical parameters of inflammation.
[0251] Although this was an open label study, the magnitude of the
clinical improvements is well beyond the degree of improvement that
would be anticipated from a placebo response, such that the present
invention is shown to have significant clinical efficacy for
treating rheumatoid arthritis.
TABLE-US-00020 TABLE 9A Study TRA Group I day 1 and day 14) Pre-
Screen- Wk 0 Wk 0 Wk 2 Scr ing D 1 D 2 Wk 1 D 14 Wk 3 Wk 4 Wk 6 Wk
8 C X D X Physical X X Ex Pregnancy X Test Weight X X X X Signs X
X* X X X* X X X X X X X X' X X X' X X X X Clinical X X X' X X X X
(Safety) Clinical X X' X X' X X X X (Response) X X7 Response X
Evaluation He X X' X X' X X X X Biochemistry X X' X X' X X X X
Urinalysis X' X X' X X X X X' X X' X X X X X' X X' X X X X PBL X X
X Pharma X.sup.# X.sup.# X.sup.$ HACA Response X' X X' X X X X X =
Vital signs will be obtained , every 30 minutes and every 30
minutes for 2 hours after the infusion; X = Needs to be done prior
to the X = Serum samples will be obtained prior to the infusion and
1, 2, 4, 8, 12, and 24 after the end of the ; X = Serum samples be
obtained to the at 2 hours after the end of the . indicates data
missing or illegible when filed
TABLE-US-00021 TABLE 9B Flowchart for Chimeric Anti-TNF Study
C0168TRA Group II2 ( every day 4 times total) Pre- Screen- Wk 0 Wk
0 Wk 0 Wk 0 Wk 1 Scr ing D 1 D 2 D 5 D 9 D 13 Wk 2 Wk 3 Wk 4 Wk 6
Wk 8 X X Physical X X Examination Pregnancy X Test Weight X X X X X
X Vital Signs X X* X X* X* X* X X X X X Anti-TNF X X X X Labs, X X'
X X' X' X' X X X X X Chart Cl X X' X' X' X X X X X ( ) Clinical X
X' X' X X X X X (Response) X X7 Response X Evaluation + X X' X' X X
X X X ESR Biochemistry X X' X' X X X X X Urinalysis X' X' X X X X X
CRP + RF X' X' X X X X X X' X' X X X X X PBL X X X X
Pharmacokinetic X X X X X.sup.$ Response X' X' X X X X X X = Vital
signs will be obtained prior to infusion, every 30 minutes during
the infusion and every 30 minutes for 2 hours after the infusion; X
= Needs to be prior to the infusion; X = Serum samples will be
obtained prior to the and at 1, 2, 4, 8, 12, and 24 hours after the
end of the infusion; X = Serum samples be obtained to the infusion
and at 2 hours end of the infusion. indicates data missing or
illegible when filed
TABLE-US-00022 TABLE 10 Measurement of the of Disease Activity (DA)
Variables of Di Activity M Grip IDA Stiffness Pain Score (min) ( ,
cm)* (mm ) Index Male Female 1 <10 .sup. 0-2.4 0 >14.1 1.7
0.20 2 10-30 2.5-4.4 50-200 1- 13-14.sup. 10.8-11.6 21-45 3 31-120
4.5-6.4 30-49 8-17 10-12.9 46-80 4 >120 6.5-10 <30 <9.9
<8.3 *Pain was measured on a visual 0-10 cm. indicates data
missing or illegible when filed
[0252] Conclusions (1) [0253] Safety of anti-TNF in RA [0254]
Anti-TNF was safe and very well tolerated: [0255] no hemodynamic,
febrile or allergic episodes; [0256] no infections; [0257] no
clinical adverse events; [0258] a single laboratory adverse event
only, probably unrelated to anti-TNF. [0259] Conclusions (2) [0260]
Efficacy of anti-TNF in RA Anti-TNF therapy resulted in: [0261]
rapid and marked improvements in EMS, pain and articular index in
most patients; [0262] slower but marked improvement in swollen
joint score, maximal by 3-4 weeks; [0263] rapid and impressive
falls in serum CRP, and a slower fall in ESR; [0264] normalization
of CRP and ESR in some patients; [0265] rapid falls in serum C4d (a
complement breakdown product) and IL-6 in patients where these
indices were elevated at entry. [0266] Duration of clinical
improvements variable, with rebound in some patients at 6-8
weeks.
[0267] Accordingly, the present invention has been shown to have
clinical efficacy in human patients for treating TNF involved
pathologies using TNF MAbs of the present invention, such as for
treating rheumatoid arthritis. Additionally, the human clinical use
of TNF antibodies of the present invention in humans is also shown
to correlate with in vitro data and in vivo animal data for the use
of anti-TNF antibodies of the present invention for treating
TNF-related pathologies.
Example XXII
Treatment of Arthritis in Humans using Chimeric Immunoglobulin
Chain of the Present Invention
[0268] Patient Selection
[0269] Twenty patients were recruited, each of whom fulfilled the
revised American Rheumatism Association criteria for the diagnosis
of RA (Arnett et al., Arthritis Rheum. 31:315-324 (1988). The
clinical characteristics of the patients are shown in Table 12. The
study group comprised 15 females and 5 males, with a median age of
51 years (range 23-72), a median disease duration of 10.5 years
(range 3-20) and a history of failed therapy with standard
disease-modifying anti-rheumatic drugs (DMARDs; median number of
failed DMARDs: 4, range 2-7). Seventeen were seropositive at entry
or had been seropositive at some stage of their disease, all had
erosions on X-Rays of hands or feet, and 3 had rheumatoid nodules.
All patients had active disease at trial entry, as defined by an
Index of Disease Activity (IDA; Mallya et al., Rheumatol. Rehab.
20:14-17 (1981) of at least 1.75, together with at least 3 swollen
joints, and were classed as anatomical and functional activity
stage 2 or 3' (Steinbrocker et al., JAMA 140:659-662 (1949). The
pooled data for each of the clinical and laboratory indices of
disease activity at the time of screening for the trial (up to 4
weeks prior to trial entry), and on the day of trial entry itself
(week 0), are shown in Tables 13 and 14.
TABLE-US-00023 TABLE 12 Demographic Features of Falis with Refr Elc
Arthritis Age/ DD P Sex (yr) Previous DMARDs C tant therapy 1 41/F
7 SSZ, DP, GST, AUB_MTX, Pred 5 mg AZA, HCQ 2 63/F 7 SSZ, GST, DP
Pred 1-2 g 3 /M 3 AUR, HCQ, GST, Pred 10 mg; 225 mg MTX, SSZ 4 56/M
10 GST, DP, AZA, SSZ Pred 12.5 mg; 5 g; 1-2 g 5 3 GST, SSZ, DP, AZA
Pred 1 mg; Para 1-2 g; Cod 16 mg 6 /M 3 SSZ, HCQ, AUR 1 g 7 54/F 3
DP, GST, SSZ, Pred 1-2 g; Cod 16-32 mg AZA, MTX 8 /F 11 HCQ, GST,
SSZ, P d 1.5 mg; Diel 1( ) mg; Para MTX, AZA 1-1 g; -20 mg 9 /F 15
GST, HCQ, DP, MTX Pred 1.5 mg, Diel 123 mg; Para 1-3 g 10 41/F 12
SSZ, CYC, MTX 4 g 11 54/F 10 DP, SSZ, MTX Pred 10 mg; Para 1-5 g;
COD 30-93 mg 12 5 /F 12 GST, MTX, TXP, AUR Axp 1-2 g 13 51/F 3 SSZ,
AZA Para 1-4 14 M 11 GST, DP, AZA, MTX Pred 5 mg; Para 1-4 g; C
16-63 mg 15 56/F HCQ, DP, SSZ, MTX A 0.3 g 16 62/F 16 GST, DP, SSZ,
Para 1-4 g; Cod 16-64 mg MTX, AZA 17 46/F 13 SSZ, DP, GST, Pred 1.5
mg; 600 mg; Para MTX, HCQ, AZA 1-2 g; Dex 100-200 mg 18 /F 14 GST,
MTX, DP, SS, Pred 7.5 mg; 100 mg; Para ZAUR, AZA 1-3 g 19 42/F 3
SSZ, MTX Feu 450 mg; B 6 g; Cod 50 mg 20 47/M 20 GST, DP, SSZ, AZA
Pred 10 mg; Para 1-3 g Pat = Patient; DD (yrs) = Disease duration
(years); DMARDs = disease-modifying anti-thematic drugs; SSZ = ; DP
= D-pa ; GST = gird th ; AUR = ; MTX = metiaxtre ; AZA = ; HCQ =
(hy ) ; CYC = cyclophosph ; Pred = pre ( ); Para = pretreatment; I
= ; Ibn = i ; Cod = codeine p ; Nrp = ; Diel = diele ; = ; Bet =
bes ; Axp = rin; etp = eto ; Feu = fre ; indicates data missing or
illegible when filed
TABLE-US-00024 TABLE 13 Changes in Clinical Assessments Following
Treatment of Rheumatoid Arthritis Patients with cA2 Pa Pain Swollen
Strength Strength Morning Score Joints (L) (R) (grades of Stiffness
(0-10) Index (0-28) (0- ) (0-30 ) IDA improved Trial ( ) cm (0-69)
mm Hg mm Hg (1-4) 0-3) Screen 135 7.4 23 16 84 3 NA (0-600) .sup.
(4- ) (4-51) (4- 8) (45- ) (57-300) (2.3-3. ) p value 0 180 7.1 28
1 77 2 2 NA (20-600) (2.7-9. ) (4-52) (3-27) (52-295) (50-293)
.sup. (2-3.5) p value 1 2.6 13 13. 122 133 2 1 (0.6-7.8) (2-28)
(1-25) ( 6-300) 7-300) (1.5-3.3) (1-3) <0.001 <0.001
<0.001; >0.05 >0.05 >0.05 <0.001 NA <0.002 p
value 2 15 3.0 13 11.5 1 9 1 2 1.5 (0-150) (0.2-6.4) (1-28) (1-22)
(75-300) ( ) (1.5-3.2) (1-3) <0.001 <0.001 <0.001
<0.003; <0.03; >0.05 <0.001 NA <0.02 >0.05 p
value 3 5 2.2 8 6 113 2 2 (0-150) (0.2-7.4) (0-22) (1-19) (51-300)
( -300) (1.2- ) (1-2 <0.001 <0.001 <0.001 <0.001;
>0.05 >0.05 <0.001 NA <0.002 p value 4 15 1.90 10 6 124
148 .8 2 (0-90) (0.1-5.6) (0-17) ( ) (79-300) (64-300) (1. -2.7)
(1-2 <0.001 <0.001 <0.001 <0.001; <0.02; <0.03;
<0.001 NA < >0.05 >0.05 p value 5 1.9 6 119 153 1.7 2 (
0) (0.1-6.2) (0-18) (1-14) (68-300) (62-300) (1.3-2.8) (1-2
<0.001 <0.001 <0.001 <0.001 <0.04; <0.05;
<0.001 NA >0.05 >0.05 p value 15 2.1 8 7 117 167 1.8 2
(0-60) (0-2- ) (1-28) (1-18) (69-300) ( -300) (1.5-2.8) <0.001
<0.001 <0.001 <0.001 <0.03; < ; < NA >0.05
>0.0 Datas are expressed as the median ( ) of values from ; data
from 15 were not included after week 2 ( ); P values show
significance by with week 0 values; adjusted for multiple
statistical comparisons. IDA = Index of disease activity; NA = not
applicable. indicates data missing or illegible when filed
TABLE-US-00025 TABLE 14 Changes in Laboratory Measures Following
Treatment of Rh Arthritis pa with cA2 Platelet RF Week of WBC
.times. Count .times. ESR CRP SAA Inverse Trial liter 10/liter
10/liter mm/hour mg/liter mg/ml titer Screen 117 7.9 353 42 ND ND (
) ( .9- 5.2) ( ) P value 0 113 341 55 9.5 245 2.5 ( ) (4. -15.7)
(228-710) (15- 4) (5-107) (18-1900) (160- 0.240) p value 1 114 8.5
351 26 5 58 ND (96-145) ( .6- ) (223- ) (13-100) (0-50) (0-330)
>0.05 >0.05 >0.05 >0.05 <0.001 <0.001; <0.00 p
value 2 112 8.2 296 5.5 9 ND (95-144) (4.3-12.7) ( ) ( ) (0- 0) ( )
>0.05 >0.05 <0.04; <0.02; <0.001; < 2; >0.05
>0.05 <0.003 < 4 p value 3 110 .0 2 9 7 ND ND (89-151) (
-14.4) ( ) ( ) (0- ) >0.05 >0.05 <0.03; <0.04;
<0.01; >0.05 >0.05 <0.002 p value 112 8.2 314 23 10 ND
ND ( -148) (4. ) (186-565) (10-87) (0-91) >0.05 >0.05 > 4;
<0.00 ; >0.0 <0.02 p value 6 116 9.1 339 23 8 ND ND
(91-159) ( ) ( ) (12- ) (0- ) >0.05 >0.05 >0.05 <0.03;
<0.001 >0.05 p value 8 114 7.6 6 ND 480 (94-153) (4.2-13.5)
(210-591) (7-73) (0- ) >0.05 >0.05 >0.05 >0.05
<0.001 >0.05 Data are expressed as the median (range) of
values 20 patients; data from patient 15 not included after week 2
(dropout). For (RF), only 0 the particle agglutination assay were
included (No. = 14). P values significance by Mann-Whitney test
compared with week 0 values; adjusted for multiple statistical ND =
not done. Normal ranges: hem (Hgb) 120-160 g/liter (F), 135-175
(M); white blood cell (WBC) 3-11 .times. 10.sup.9/liter; platelet
150-400 .times. 10.sup.9/liter; erythrocyte sedimentation rate ( )
<15 mm (F), <10 mm/hour (M); C-reactive (CRP) <10 liter;
A(SAA) <10 mg/ml. indicates data missing or illegible when
filed
[0270] All DMARDs were discontinued at least 1 month prior to trial
entry. Patients were allowed to continue on a nonsteroidal
anti-inflammatory drug and/or prednisolone (<12.5 mg/day) during
the trial. The dosage of these agents was kept stable for 1 month
prior to trial entry and during the course of the trial, and no
parenteral corticosteroids were allowed during these periods.
Simple analgesics were allowed ad libitum. Patients with other
serious medical conditions were excluded. Specific exclusions
included serum creatinine>150 umol/liter (normal range 60-120
umol/liter), hemoglobin (Hgb)<90 gm/liter (normal range 120-160
gm/liter [females]; 135-175 gm/liter [males]), white blood cell
count (WBC)<4.times.10 g/liter (normal range
4-11.times.10.sup.9/liter), platelet count<100.times.10 g/liter
(normal range 150-400.times.10.sup.9/liter), and abnormal liver
function tests or active pathology on chest X-Ray.
[0271] All patients gave their informed consent for the trial, and
approval was granted by the local ethics committee.
[0272] Treatment
[0273] The cA2 antibody was stored at 4.degree. C. in 20 ml vials
containing 5 mg of cA2 per milliliter of 0.01 M phosphate buffered
saline in 0.15M sodium chloride at a pH of 7.2 and was filtered
through a 0.2 .mu.m sterile filter before use. The appropriate
amount of cA2 was then diluted to a total volume of 300 ml in
sterile saline and administered intravenously via a 0.2 .mu.m
in-line filter over a 2 hour period.
[0274] Patients were admitted to hospital for 8-24 hours for each
treatment, and were mobile except during infusions. The trial was
of an open, uncontrolled design, with a comparison of two treatment
schedules. Patients 1 to 5 and 11 to 20 received a total of 2
infusions, each of 10 mg/kg cA2, at entry to the study (week 0) and
14 days later (week 2). Patients 6 to 10 received 4 infusions of 5
mg/kg activity included complete blood counts, C-reactive protein
(CRP; by rate nephelometry) and the erythrocyte sedimentation rate
(ESR; Westergren). Follow-up assessments were made at monthly
intervals after the conclusion of the formal trial period, in order
to assess the duration of response.
[0275] Analysis of improvement in individual patients was made
using two separate indices. Firstly, an index of disease activity
(IDA) was calculated for each time point according to the method of
Mallya and Mace (Mallya et al., Rheumatol. Rehab. 20:14-17 (1981),
with input variable of morning stiffness, pain score, Ritchie
Index, grip strength, ESR and Hgb. The second index calculated was
that of Paulus (Paulus et al., Arthritis Rheum. 33:477-484 (1990)
which uses input variables of morning stiffness, ESR, joint
pain/tenderness, joint swelling, patient's and physician's global
assessment of disease severity. In order to calculate the presence
or otherwise of a response according to this index, two
approximations were made to accommodate our data. The 28 swollen
joint count used by us (nongraded; validated in Fuchs et al.,
Arthritis Rheum. 32:531-537 (1989)) was used in place of the more
extensive graded count used by Paulus, and the patient's and
physician's global assessments of response recorded by us were
approximated to the global assessments of disease activity used by
Paulus infra. In addition to determining response according to
these published indices, we selected 6 disease activity assessments
of interest (morning stiffness, pain score, Ritchie index, swollen
joint count, ESR and CRP) and calculated their mean percentage
improvement. We have used FIGS. 24 and 25 to give an indication of
the degree of improvement seen in responding patients.
[0276] Immunological Investigations
[0277] Rheumatoid factors were measured using the rheumatoid
arthritis particle agglutination assay (PAPA, FujiBerio Inc.,
Tokyo, Japan), in which titers of 1/160 or greater were considered
significant. Rheumatoid factor isotypes were measured by ELISA
(Cambridge Life Sciences, Ely, UK). The addition of cA2 at
concentrations of up to 200 .mu.g/ml to these assay cA2, at entry,
and days 4, 8 and 12. The total dose received by the 2 patient
groups was therefore the same at 20 mg/kg.
[0278] Assessment Safety Monitoring
[0279] Vital signs were recorded every 15 to 30 minutes during
infusions, and at intervals for up to 24 hours post infusion.
Patients were questioned concerning possible adverse events before
each infusion and at weeks 1, 2, 3, 4, 6, and 8 of the trial. A
complete physical examination was performed at screening and week
8. In addition, patients were monitored by standard laboratory
tests including complete blood count, C3 and C4 components of
complement, IgG, IgM and IgA, serum electrolytes, creatinine, urea,
alkaline phosphatase, aspartate transaminase and total bilirubin.
Sample times for these tests were between 0800 and 0900 hours
(pre-infusion) and 1200-1400 hours (24 hours post completion of the
infusion). Blood tests subsequent to day 1 were performed in the
morning, usually between 0700 and 1200 hours. Urine analysis and
culture were also performed at each assessment point.
[0280] Response Assessment
[0281] The patients were assessed for response to cA2 at weeks 1,
2, 3, 4, 6 and 8 of the trial. The assessments were all made
between 0700 and 1300 hours by the same observer. The following
clinical assessments were made: duration of morning stiffness
(minutes), pain score (0 to 10 cm on a visual analog scale),
Ritchie Articular Index (maximum 69; Ritchie et al., Quart. J. Med.
147:393-406 (1968)), number of swollen joints (28 joint count;
validated in Fuchs et al., Arthritis Rheum. 32:531-537 (1989), grip
strength (0 to 300 mm Hg, mean of 3 measurements per hand by
sphygmomanometer cuff) and an assessment of function (the Stanford
Health Assessment Questionnaire (HAQ) modified for British
patients; 34). In addition, the patients' global assessments of
response were recorded on a 5-point scale (worse, no response, fair
response, good response, excellent response). Routine laboratory
indicators of disease systems did not alter assay results (data not
shown). Antinuclear antibodies were detected by immunofluorescence
on HEpo 2 cells (Biodiagnostics, Upton, Worcs. UK) and antibodies
to extractable nuclear antigens were measured by counter
immunoelectrophoresis with poly-antigen extract (Biodiagnostics).
Sera positive by immunofluorescence were also screened for
antibodies to DNA by the Farr assay (Kodak Diagnostics, Amersham,
UK). Anti-cardiolipin antibodies were measured by ELISA (Shield
Diagnostics, Dundee, Scotland). Serum amyloid A (SAA) was measured
by sandwich ELISA (Biosource International, Camarillo, Calif.,
USA). Antiglobulin responses to the infused chimeric antibody were
measured by an in-house ELISA, using cA2 as a capture reagent.
[0282] Cytokine Assays
[0283] Bioactive TNF was measured in sera using the WEHI 164 clone
13 cytotoxicity assay (Espevik et al., J. Imm. Methods 95:99-105
(1986). Total IL-6 was measured in sera using a commercial
immunoassay (Medgenix Diagnostics, SA, Belgium) and by a sandwich
ELISA developed "in house" using monoclonal antibodies provided by
Dr. F. di Padova (Basel, Switzerland). Microtiter plates were
coated with monoclonal antibody LNI 314-14 at a concentration of 3
ug/ml for 18 hours at 4.degree. C. and blocked with 3% bovine serum
albumin in 0.1M phosphate buffered saline, pH 7.2. Undiluted sera
or standards (recombinant hIL 6, 0-8.1 ug/ml) were added to the
wells in duplicate and incubated for 18 hours at 4.degree. C. Bound
IL-6 was detected by incubation with monoclonal antibody LNI 110-14
for 90 minutes at 37.degree. C., followed by biotin labeled goat
anti-murine IgG2b for 90 minutes at 37.degree. C. (Southern
Biotechnology, Birmingham, Ala.). The assay was developed using
streptavidin-alkaline phosphatase (Southern Biotechnology) and
p-nitrophenylphosphate as a substrate and the optical density read
at 405 nm.
[0284] Statistics
[0285] Comparisons between week 0 and subsequent time points were
made for each assessment using the Mann-Whitney test. For
comparison of rheumatoid factor (RAPA) titers, the data were
expressed as dilutions before applying the test.
[0286] This was an exploratory study, in which prejudgements about
the optimal times for assessment were not possible. Although it has
not been common practice to adjust for multiple statistical
comparisons in such studies, a conservative statistical approach
would require adjustment of p values to take into account analysis
at several time points. The p values have therefore been presented
in two forms: unadjusted, and after making allowance for analysis
at multiple time points by use of the Bonferroni adjustment. Where
p values remained <0.001 after adjustment, a single value only
is given. A p value of <0.05 is considered significant.
Results
[0287] Safety of cA2
[0288] The administration of cA2 was exceptionally well tolerated,
with no headache, fever, hemodynamic disturbance, allergy or other
acute manifestation. No serious adverse events were recorded during
the 8-week trial. Two minor infective episodes were recorded,
patient 15 presented at week 2 with clinical features of bronchitis
and growth of normal commensals only on sputum culture. She had a
history of smoking and of a similar illness 3 years previously. The
illness responded promptly to treatment with amoxicillin, but her
second cA2 infusion was withheld and the data for this patient are
therefore not analyzed beyond week 2. Patient 18 showed significant
bacteriuria on routine culture at week 6 (>10.sup.5/ml; lactose
fermenting coliform), but was asymptomatic. This condition also
responded promptly to amoxicillin.
[0289] Routine analysis of blood samples showed no consistent
adverse changes in hematological parameters, renal function, liver
function, levels of C3 or C4 or immunoglobulins during the 8 weeks
of the trial. Four minor, isolated and potentially adverse
laboratory disturbances were recorded. Patient 2 experienced a
transient rise in blood urea, from 5.7 mmol/liter to 9.2 mmol/liter
(normal range 2.5 to 7 mmol/liter), with no change in serum
creatinine. This change was associated with the temporary use of a
diuretic, prescribed for a non-rheumatological disorder. The
abnormality normalized within 1 week and was classified as
"probably not" related to cA2. Patient 6 experienced a transient
fall in the peripheral blood lymphocyte count, from 1.6 to
0.8.times.10.sup.9/liter (normal range
1.0-4.8.times.10.sup.9/liter). This abnormality normalized by the
next sample point (2 weeks later), was not associated with any
clinical manifestations and was classified as "possible related" to
cA2. Patients 10 and 18 developed elevated titers of anti-DNA
antibodies at weeks 6 and 8 of the trial, with elevated
anti-cardiolipin antibodies being detected in patient 10 only. Both
patients had a pre-existing positive antinuclear antibody and
patient 10 had a history of borderline lymphocytopenia and high
serum IgM. There were no clinical features of systemic lupus
erythematosus and the laboratory changes were judged `possibly
related` to cA2.
[0290] Efficacy of cA2 Disease Activity
[0291] The pattern of response for each of the clinical assessments
of disease activity and the derived IDA are shown in Table 13. All
clinical assessments showed improvement following treatment with
cA2, with maximal responses from week 3. Morning stiffness fell
from a median of 180 minutes at entry to 5 minutes at week 6
(p<0.001, adjusted), representing an improvement of 73%.
Similarly, the Ritchie Index improved from 28 to 6 at week 6,
(p<0.001, adjusted, 79% improvement) and the swollen joint count
fell from 18 to 5, (p<0.001, adjusted, 72% improvement). The
individual swollen joint counts for all time points are shown in
FIG. 24. Grip strength also improved; the median grip strength rose
from 77 (left) and 92 (right) mm Hg at entry to 119 (left) and 153
(right) mmHg at week 6 (p<0.04, p<0.05, left and right
respectively; p>0.05 after adjustment for multiple comparisons).
The IDA showed a fall from a median of 3 at entry to 1.7 at week 6
(p<0.001, adjusted). Patients were asked to grade their
responses to cA2 on a 5 point scale. No patient recorded a response
of "worse" or "no change" at any point in the trial. "Fair", "good"
and "excellent" responses were classed as improvements of 1, 2 and
3 grades respectively. At week 6, the study group showed a median
of 2 grades of improvement (Table 13).
[0292] We also measured changes in the patients' functional
capacity, using the HAQ modified for British patients (range 0-3).
The median (range) HAQ score improved from 2 (0.9-3) at entry to
1.1 (0-2.6) by week 6, (p<0.001; p<0.002 adjusted).
[0293] The changes in the laboratory tests which reflect disease
activity are shown in Table 14. The most rapid and impressive
changes were seen in serum CRP, which fell from a median of 39.5
mg/liter at entry to 8 mg/liter by week 6 of the trial (p<0.001,
adjusted; normal range <10 mg/liter), representing an
improvement of 80%. Of the 19 patients with elevated CRP at entry,
17 showed falls to the normal range at some point during the trial.
The improvement in CRP was maintained in most patients for the
assessment period (Table 14 and FIG. 25); the exceptions with high
values at 4 and 6 weeks tended to be those with the highest
starting values (data not shown). The ESR also showed improvement,
with a fall from 55 mm/hour at entry to 23 mm/hour at week 6
(p<0.03; p>0.05 adjusted; 58% improvement; normal range
<10 mm/hour, <15 mm/hour, males and females respectively).
SAA levels were elevated in all patients at trial entry, and fell
from a median of 245 mg/ml to 58 mg/ml at week 1 (p<0.003,
adjusted; 76% improvement; normal range <10 mg/ml) and to 80
mg/ml at week 2 (p<0.04, adjusted). No significant changes were
seen in Hgb, WBC or platelet count at week 6, although the latter
did improve at weeks 2 and 3 compared with trial entry (Table
14).
[0294] The response data have also been analyzed for each
individual patient. The majority of patients had their best overall
responses at week 6, at which time 13 assessed their responses as
"good" while 6 assessed their responses as "fair". Eighteen of the
19 patients who completed the treatment schedule achieved an
improvement in the index of Disease Activity (Mallya et al.,
Rheumatol. Rehab. 20:14-17 (1981) of 0.5 or greater at week 6, and
10 achieved an improvement of 1.0 or greater. All patients achieved
a response at week 6 according to the index of Paulus (Paulus et
al., Arthritis Rheum. 33:477-484 (1990). Finally, all patients
showed a mean improvement at week 6 in the 6 selected measures of
disease activity (as presented above) of 30% or greater, with 18 of
the 19 patients showing a mean improvement of 50% or greater.
[0295] Although the study was primarily designed to assess the
short-term effects of cA2 treatment, follow-up clinical and
laboratory data are available for those patients followed for
sufficient time (number=12). The duration of response in these
patients, defined as the duration of a 30% (or greater) mean
improvement in the 6 selected disease activity measures, was
variable, ranging from 8 to 25 (median 14) weeks.
[0296] Comparison of the clinical and laboratory data for patients
treated with 2 infusions of cA2 (each at 10 m/kg) compared with
those treated with 4 infusions (each at 5 mg/kg) showed no
significant differences in the rapidity or extent of response (data
not shown).
[0297] Immunological Investigations and Cytokines
[0298] Measurement of rheumatoid factor by RAPA showed 14 patients
with significant titers (> 1/160) at trial entry. Of these, 6
patients showed a fall of at least 2 titers on treatment with cA2,
while the remaining patients showed a change of 1 titer or less. No
patient showed a significant increase in RF titer during the trial.
The median RF titer in the 11 patients fell from 1/2, 560 at entry
to 1/480 by week 8 (p>0.05; Table 14). Specific RF isotypes were
measured by ELISA, and showed falls in the 6 patients whose RAPA
had declined significantly, as well as in some other patients.
Median values for the three RF isotypes in the 14 patients
seropositive at trial entry were 119, 102 and 62 IU/ml (IgM, IgG
and IgA isotypes respectively) and at week 8 were 81, 64 and 46
IU/ml (p>0.05).
[0299] We tested sera from the first 9 patients for the presence of
bioactive TNF, using the WEHI 164 clone 13 cytotoxicity assay
(Espevik et al., J. Imm. Methods 95:99-105 (1986). In 8 patients,
serum sets spanning the entire trial period were tested, while for
patient 9, one pre-trial, one period were tested, patient, one pre,
one intermediate and the last available sample only were tested.
The levels of bioactive TNF were below the limit of sensitivity of
the assay in the presence of human serum (1 pg/ml). Since
production of CRP and SAA are thought to be regulated in large part
by IL-6, we also measured serum levels of this cytokine, using 2
different assays which measure total IL-6. In the Medgenix assay,
IL-6 was significantly elevated in 17 of the 20 patients at entry.
In this group, levels fell from 60 (18-500) pg/ml to 40 (0-230)
pg/ml at week 1 (p>0.05; normal range <10 pg/ml) and to 32
(0-210) pg/ml at week 2 (p<0.005, p<0.01, adjusted). These
results were supported by measurement of serum IL-6 in the first 16
patients in a separate ELISA developed in-house. IL-6 was
detectable in 11 of the 16, with median (range) levels falling from
210 (25-900) pg/ml at entry to 32 (01, 700) pg/ml at week 1
(p<0.02, p<0.04, adjusted; normal range <10 pg/ml) and to
44 (0-240) pg/ml at week 2 (p<0.02, p<0.03, adjusted).
[0300] We tested sera from the first 10 patients for the presence
of anti-globulin responses to the infused chimeric antibody, but
none were detected. In many patients however, cA2 was still
detectable in serum samples taken at week 8 and this can have
interfered with the ELISA.
[0301] Discussion
[0302] This is the first report describing the use of
anti-TNF.alpha. antibodies in human autoimmune disease. Many
cytokines are produced in rheumatoid synovium, but we chose to
target specifically TNF.alpha. because of mounting evidence that it
was a major molecular regulator in RA. The study results presented
here support that view and allow three important conclusions to be
drawn.
[0303] First, treatment with cA2 was safe and the infusion
procedure was well tolerated. Although fever, headache, chills and
hemodynamic disturbance have all been reported following treatment
with anti CD4 or anti CDw52 in RA, such features were absent in our
patients. Also notable was the absence of any allergic event
despite repeated treatment with the chimeric antibody, although the
interval between initial and repeat infusions can have been too
short to allow maximal expression of any anti-globulin response.
The continuing presence of circulating cA2 at the conclusion of the
trial may have precluded detection of antiglobulin responses, but
also implied that any such responses were likely to be of low titre
and/or affinity. Although we recorded 2 infective episodes amongst
the study group, these were minor and the clinical courses were
unremarkable. TNF.alpha. has been implicated in the control of
listeria and other infections in mice (Havell et al., J. Immunol.
143:2894-2899 (1989), but our limited experience does not suggest
an increased risk of infections after TNF.alpha. blockade in
man.
[0304] The second conclusion concerns the clinical efficacy of cA2.
The patients we treated had long-standing, erosive, and for the
most part seropositive disease, and had each failed therapy with
several standard DMARDs. Despite this, the major clinical
assessments of disease activity and outcome (morning stiffness,
pain score, Ritchie index, swollen joint count and
[0305] HAQ score) showed statistically significant improvement,
even after adjustment for multiple comparisons. All patients graded
their response as at least "fair", with the majority grading it as
"good". In addition, all achieved a response according to the
criteria of Paulus and showed a mean improvement of at least 30% in
6 selected disease activity measures.
[0306] The improvements in clinical assessments following treatment
with cA2 appear to be at least as good as those reported following
treatment of similar patients with antileukocyte antibodies. The
two therapeutic approaches can already be distinguished, however,
by their effects on the acute phase response, since in several
studies of antileukocyte antibodies, no consistent improvements in
CRP or ESR were seen. In contrast, treatment with cA2 resulted in
significant falls in serum CRP and SAA, with normalization of
values in many patients. The changes were rapid and marked, and in
the case of CRP, sustained for the duration of the study (Table
14). The falls in ESR were less marked, achieving statistical
significance only when unadjusted (Table 14).
[0307] These results are consistent with current concepts that
implicate TNF.alpha. in the regulation of hepatic acute phase
protein synthesis, either directly, or by control of other,
secondary, cytokines such as IL-6 (Fong et al., J. Exp. Med.
170:1627-1633 (1989); Guerne et al., J. Clin. Invest. 83:585-592
(1989)). In order to investigate the mechanism of control of the
acute phase response in our patients, we measured serum TNF.alpha.
and IL-6 before and after cA2 treatment. Bioactive TNF.alpha. was
not detectable in baseline or subsequent sera. We used 2 different
assays for IL-6, in view of previous reports of variability between
different immunoassays in the measurement of cytokines in
biological fluids (Roux-Lombard et al., Clin. Exp. Rheum.
10:515-520 (1992), and both demonstrated significant falls in serum
IL-6 by week 2. These findings support the other objective
laboratory changes induced by cA2, and provide in vivo evidence
that TNF.alpha. is a regulatory cytokine for IL-6 in this disease.
Amongst the other laboratory tests performed, rheumatoid factors
fell significantly in 6 patients.
[0308] Neutralization of TNF.alpha. can have a number of beneficial
consequences, including a reduction in the local release of
cytokines such as IL-6 and other inflammatory mediators and
modulation of synovial endothelial/leukocyte interactions. cA2 can
also bind directly to synovial inflammatory cells expressing
membrane TNF.alpha., with subsequent in situ cell lysis.
[0309] The results obtained in this small series have important
implications, both scientifically and clinically. At the scientific
level, the ability of the neutralizing antibody, cA2, to reduce
acute phase protein synthesis, reduce the production of other
cytokines such as IL-6, and significantly improve the clinical
state demonstrates that it is possible to interfere with the
cytokine network in a useful manner without untoward effects. Due
to the many functions and overlapping effects of cytokines such as
IL-1 and TNF.alpha., and the fact that cytokines induce the
production of other cytokines and of themselves, there had been
some pessimism as to whether targeting a single cytokine in vivo
would have any beneficial effect (Kingsley et al., Immunol. Today
12:177-179 (1991), Trentham, Curr. Opin. Rheumatol. 3:369-372
(1991)). This view is clearly refuted. On the clinical side, the
results of short-term treatment with cA2 are significant and
confirm that TNF.alpha. is useful as a new therapeutic target in
RA.
Example XXIV
Treatment of Rheumatoid Arthritis in Humans with cA2 Antibody
Patients
[0310] Patients were recruited from the clinics of four cooperating
trial centers or after referral from outside physicians. Patients
aged 18-75 were included if they met the criteria of the American
College of Rheumatology for the diagnosis of rheumatoid arthritis,
had disease for at least six months, had a history of failed
treatment with at least one disease modifying anti-rheumatic drug
(DMARD) and had evidence of erosive disease on radiography of hands
and feet. In addition, patients had to have active disease, as
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/h; C-reactive protein (CRF)
.gtoreq.20 mg/L). Patients with severe physical incapacity
(Steinbrocker class IV) or with clinically evident joint ankylosis
were excluded. Other exclusion criteria included: severe anemia
(haemoglobin<8.5 g/dL); leucopenia (white
cells<3.5.times.10.sup.9/L, neutrophils<1.5.times.10.sup.9/L)
or thrombocytopenia (100.times.10.sup.9/L); elevation of liver
function tests to over three times the upper limit of normal or of
serum creatine to over 150 .mu.mol/L; or active pathology on chest
film. Patients were also excluded if they had a history of previous
administration of murine monoclonal antibodies, a history of cancer
or HIV infection, or current other serious medical conditions.
Female patients of child-bearing age had to be using an effective
method of birth control and to have a negative pregnancy test
before entry.
[0311] No patient had received other experimental drugs targeted to
TNF (e.g., oxpentifylline) in the previous three months. Patients
taking disease-modifying anti-rheumatic drugs at screening were
withdrawn from their therapy at least four weeks before entry.
Patients taking low-dose oral corticosteroids (prednisolone
.ltoreq.12.5 mg per day) or non-steroidal anti-inflammatory drugs
at screening were allowed to continue on stable doses. Additional
steroids by injection or other routes were not allowed. Simple
analgesics were freely allowed.
[0312] All patients gave their informed consent for the trial,
which was approved by each of the local regional ethics
committees.
[0313] Study Infusions
[0314] The cA2 antibody was supplied as a sterile solution
containing 5 mg cA2 per ml of 0.01 mol/L phosphate-buffered saline
in 0.15 mol/L 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.
[0315] Assessments
[0316] Patients were seen at an initial screening visit and if
eligible, were entered within four weeks. On the day of entry,
patients were admitted to the hospital and randomized (in blocks of
6, stratified for center) to one of three groups (24 per group).
The first group received a single infusion of placebo. The other
two groups received one infusion of cA2, 1 mg/kg ("low dose") and
10 mg/kg ("high dose"). The doses of cA2 were chosen on the basis
of experience in the open-label trial and by extrapolation from the
anti-TNF-treated collagen-arthritis mice.
[0317] Patients were monitored for adverse events during infusions
and regularly thereafter, by interviews, physical examination, and
laboratory testing.
[0318] Before the start of the trial, all clinical observers agreed
on a standard technique to assess joints, and to establish
protocols for the collection of other clinical data. In each
center, patients were assessed by the same clinical observer at
each evaluation visit, usually between 0800 and 1100 hour. Clinical
observers were additionally blinded to the results of laboratory
testing for acute-phase measures (ESR and CRP).
[0319] The six primary disease-activity assessments were chosen to
allow analysis of the response in individual patients according to
the Paulus index. The assessments contributing to this index were
the tender 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
observer's assessment of disease severity (on a point scale,
ranging from 1 (symptom-free) to 5 (very severe) and 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 1 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%).
[0320] 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).
[0321] The ESR was measured at each study site with a standard
method (Westergen). CRP (Abbott fluorescent polarizing immunoassay)
and rheumatoid factor (rheumatoid-arthritis particle-agglutination
assay (RAPA, FujiBerio, Tokyo); titres.gtoreq.160 were taken to be
important) were measured in stored frozen serum samples shipped to
a central laboratory.
[0322] Statistics
[0323] The analysis was on the basis of intention to treat. The
sample size was chosen as having an 80% probability of achieving a
statistically significant (p<0.05) result if the true response
rates were 10% and 40% in the placebo and 10 mg/kg cA2 groups,
respectively. Fisher's exact test was used to compare the groups
for baseline sex ratio and rheumatoid factor status and for Paulus
response rates. Comparisons between groups for other demographic
features and for individual disease activity assessments were by
analysis of variance, or Cochran-Mantel-Haenszel statistics where
appropriate (baseline comparison of disease-modifying
anti-rheumatic drugs usage, patient's and observer's assessments of
disease severity/activity). The Paulus 20% response rate at week 4
was defined as the primary efficacy endpoint, with other time
points and variables considered supportive. Levels of significance
were therefore not adjusted for multiple comparisons.
[0324] Results
[0325] Seventy-two patients were initially randomized. One patient
presented two weeks after treatment with 1 mg/kg cA2 with probable
pneumonia that required admission to the hospital. The patient was
withdrawn and, according to protocol, another patient was
recruited. Thus, the intention-to-treat analysis brought the number
analyzed in the 1 mg/kg group to 25 patients and the total number
to 73.
[0326] The three groups were well-matched at entry, with no
significant differences in age, sex ratio, disease duration, number
of failed disease-modifying anti-rheumatic drugs, or percentage of
patients with significant titre of rheumatoid factor (Table 15).
Demographic data were similar between the four sites. The patients
had active disease at entry, as judged by the presence of multiple
tender and swollen joints, high pain scores, substantial morning
stiffness, raised acute-phase measures (Table 16). Comparison
between groups revealed no significant differences for any of the
clinical and laboratory indices of disease activity at entry.
[0327] The response rates at Paulus 20% and 50% are shown in Table
17. Only 2 of 24 placebo recipients achieved a 20% response at week
4. By contrast, 19 of 24 patients treated with 10 mg/kg cA2
achieved a response by week 4 (p0.0001 compared with placebo). The
response rates in the 1 mg/kg group were intermediate, with 11 of
25 patients responding at week 4 (p=0.0083). Analysis of the Paulus
50% response showed similar differences between the groups, with 14
of 24 high-dose cA2 patients responding (p=0.0005), compared with 2
of 24 patients in the placebo group. Analysis of the response data
for corticosteroid use showed that patients who were taking
steroids behaved no differently in their responses from
non-steroid-treated patients.
[0328] Although secondary to the differences in overall response
rates, analysis of changes in individual disease-activity
assessments was also of interest (Table 16). Significant
improvements were seen in both cA2 groups for each of the clinical
assessments. For many assessments, maximum mean improvements in
cA2-treated groups exceeded 60%. Among the laboratory measures,
significant falls were seen in both cA2 groups for ESR, CRP, and
platelet counts, with the best improvements seen in the high-dose
group. The changes in CRP were particularly rapid in onset and
impressive in extent, with many individual patients achieving
normal concentrations (10 mg/L, data not shown). In addition,
significant improvements relative to placebo were seen for
haemoglobin, especially in the high-dose cA2 group. Trends towards
a fall in white cell count (from increased counts at entry) in both
cA2 groups supported the changes in other laboratory measures, but
did not reach statistical significance (Table 16).
[0329] Detailed time response profiles for six disease-activity
assessments common to the American College of Rheumatology and the
European League Against Rheumatism core-sets showed rapid and
highly significant falls in the cA2-treated groups compared with
placebo, with significant inter-group differences evident as early
as 24 and 72 h (CRP and all other assessments, respectively).
[0330] Seeking possible dose-response relations, we compared
response rates between the cA2 groups. We found no difference in
20% or 50% Paulus responses at week 2, but significantly higher
response rates for the high-dose group at week 4 (likelihood ratio
1.8, 95% CI 1.1, 2.9, p=0.0186; 2.1, 1.1, 4.1, p=0.0450, for Paulus
20% and 50%, respectively). A similar analysis for each of the
individual disease-activity assessments showed no greater benefit
with the higher dose at week 2 of the study, except for haemoglobin
(least squares mean difference 0.5, 95% CI 0.1, 0.9, p=0.021). By
week 4, however, some diminution of the response in the 1 mg/kg
group was evident for several assessments; responses in the 120
mg/kg group were maintained (Table 16). As a result, significantly
better responses were seen at this time in the high-dose group,
including pain score (least-squares mean difference -1.8, 95% CI
-3.4 -0.2, p=0.036), right (28.4, 5.4, 51.3, p=0.018) and left grip
strength (20.6, 3.3, 37.9, p=0.022), observer's assessment of
disease severity (-0.8, -1.3, -0.4, p<0.035), ESR (-15.0, -23.6
to -1.4 p=0.035), CRP (-20.7, -32.1, -9.2, p<0.001), and
haemoglobin (0.5, 0.0, 1.0, p=0.042).
[0331] The infusions of cA2 and placebo were well tolerated, with
no episodes of fever or hemodynamic disturbance. The adverse events
recorded during the 4 weeks after treatment are shown in Table 18.
In all, two-thirds of the adverse events occurred in the cA2
groups. Infections formed the largest group, with 5 infections
recorded in the 1 mg/kg group and 1 each in those receiving 10
mg/kg cA2 and placebo. Of the 72 initially randomized, 2 patients
had severe adverse events. One was the patient with probable
pneumonia. The patient recovered fully with treatment, but was
withdrawn and replaced. This event was judged "possibly" related to
cA2. A second patient presented 1 week after treatment with 10
mg/kg cA2 with a pathological fracture of the clavicle, but
continued in the study. In retrospect, a minor bony abnormality was
evident on an X-ray film taken pretreatment, and the event was
judged "probably not" related to cA2.
TABLE-US-00026 TABLE 15 Demographic Features Group Placebo 1 mg/kg
cA2 10 mg/kg (n = 24) (n = 25) cA2 (n = 24) Age (yr) 48-2 (11-9)
56-2 (12-2) 50-6 (13-1) M/F 7/17 5/25 4/20 Disease 9-0 (7-3) 7-5
(4-8) 7-3 (5-2) Duration Previous 3-7 (1-9) (3-5) 3-1 (1-7) Drugs*
Pneumonia 96% 35% Factor (seropositive) Mean (SC). *Number of
disease-modifying anti-theomatic drugs previously used. indicates
data missing or illegible when filed
TABLE-US-00027 TABLE 16 D Activity Ass Statistical analysis vs.
Placebo Summary 1 10 mg/kg cA2 A Wk acebo 1 mg/kg cA2 10 mg/kg cA2
Le 95% CI p Le 95% CI p Tender joint 0 27.8 (13.5) 29.1 (14.1) 28.1
(12.7) 2 25.7 (16.6) 12.1 (10.2) 11.1 (6.9) -14.8 -21.2, -8.4
<0.001 -14.8 -20.2, -9.5 <0.001 4 26.2 (15.5) 16.9 (12.1)
11.3 (9.8) -10.9 -6.4, -5.3 <0.001 -15.2 -21.2, -9.2 <0.001
Swollen (0-58) 0 23.4 (10.5) 21.4 (10.6) 21.8 (11.5) 2 24.2 (12.1)
11.1 (8.1) 8.2 (5.5) -10.9 -15.6, -6.3 <0.001 -14.4 -19.6, -9.2
<0.001 4 23.0 (11.2) 12.9 (8.8) 8.6 (6.4) -8.2 -12.8, -3.6 0.001
-12.7 -17.8, -7.5 <0.001 Pain Score (0-10 cm) 0 6.8 (2.8) 6.6
(2.6) 6.7 (2.5) 2 6.9 (2.6) 2.5 (2.6) 2.6 (2.1) -1.3 -5.7, -2.9
<0.001 -4.3 -5.8, -2.8 <0.001 4 6.9 (2.5) 4.2 (2.9) 2.5 (1.8)
-2.6 -4.2, -0.9 0.003 -4.3 -5.8, -2.9 <0.001 Morning Stiffness
(min) 0 132.3 (286.7) 142.0 (122.0) 143.1 (105.5) 2 150.6 (284.0)
27.4 (48.7) 10.3 (14.9) -88.9 -147.5, -30.3 0.004 -101.2 -156.4,
-16.1 <0.001 4 172.3 (300.1) 99.6 (286.3) 8.3 (13.6) -33.4
-156.4, 89.6 0.592 -124.8 -188.9, -60.8 <0.001 Fatigue S (0-10
cm) 0 6.3 (2.3) 6.5 (2.6) 5.6 (2.4) 2 5.8 (2.9) 3.2 (2.7) 2.8 (2.3)
-2.6 -4.3, -1.0 -0.003 -2.3 -3.9, -0.7 0.006 4 5.6 (3.0) 3.8 (2.8)
2.3 (1.7) -1.9 -3.6, -0.2 0.028 -2.6 4.3, -1.0 0.003 Grip Strength,
(0-300 mm Hg) 0 120.7 (50.2) 102.4 (48.8) 117.0 (64.1) 2 122.7
(51.5) 161.8 (78.3) 175.3 (79.1) 55.8 32.6, 79.0 <0.001 56.4
35.0, 77.3 <0.001 4 119.1 (50.2) 131.8 (65.0) 175.2 (78.6) 31.3
15.6, 46.9 <0.001 59.9 35.9, 83.9 <0.001 Grip Strength, left
(0-300 mm Hg) 0 120.0 (58.4) 100.8 (46.8) 108.4 (50.5) 2 123.3
(64.9.sup. 152.4 (72.0) 157.2 (65.1) 46.7 23.7, 69.6 <0.001 45.4
28.9, 52.0 <0.001 4 120.9 (58.4) 126.6 (65.8) 155.1 (60.9) 25.0
6.5, 43.5 0.010 45.8 28.9, 62.7 <0.001 Disease Severity, (1-5) 0
3.8 (0.5) 3.7 (0.5) 3.6 (0.6) 2 3.8 (0.8) 3.5 (0.7) 2.6 (1.0) -1.2
-1.6, -0.8 <0.001 -1.1 -1.6 -0.6 <0.001 4 3.3 (0.8) 3.0 (0.8)
2.6 (0.8) -0.7 -1.2, -0.3 0.002 -1.2 -1.7 -0.8 <0.001 Disease
Severity, Observer 0 3.7 (0.7) 3.7 (0.5) 3.6 (0.7) 2 3.5 (0.8) 2.5
(0.8) 2.3 (0.6) -1.0 -1.5, -0.6 <0.001 -1.2 -1.6, -0.8 <0.001
4 3.6 (2.0) 3.0 (1.0) 2.2 (0.6) -0.6 -1.1, -0.1 0.036 -1.4 -1.9,
-1.0 <0.001 SR ( ) 0 63.1 (24.8) 58.1 (25.5) 63.1 (27.6) 2 67.0
(27.4) 41.8 (24.6) 42.4 (25.2) -23.1 -35.9, -10.4 <0.001 -24.9
-39.0, -10.9 <0.001 4 65.1 (29.8) 52.4 (32.3) 42.7 (24.6) -10.7
-26.4, 5.1 0.185 -22.5 -38.7, -6.3 0.009 CRP (mg/L) 0 64 (42) 67
(41) 64 (36) 2 53 (30) 39 (39) 28 (29) -19.1 -34.1, -4.1 0.016
-24.3 -38.8, -9.8 0.002 4 60 (42) 58 (39) 35 (29) -7.7 -20.5, 5.1
0.239 -28.8 -33.7, -12.9 <0.001 H g/dL 0 11.6 (1.6) 11.8 (1.3)
11.0 (1.1) 2 10.9 (1.5) 11.5 (1.2) 11.2 (1.1) 0.4 0.0, 0.7 0.052
0.8 0.5, 1.2 <0.001 4 10.9 (1.7) 11.7 (1.2) 11.4 (1.2) 0.6 0.1,
1.0 0.022 1.1 0.6, 1.5 0.001 WBC (.times.10 /L) 0 10.7 (3.5) 10.1
(3.5) 9.0 (2.1) 2 10.5 (2.9) 9.2 (3.2) 8.4 (2.5) -0. -1.9, 0.4
0.202 -0.4 -1.4, 0.6 0.414 4 10.3 (3.2) 9.3 (4.3) 7.7 (2.0) -0.4
-1.6, 0. 0.500 -0.9 -1.9, 0.2 0.096 P (.times.10 ) 0 447 (126) 421
(132) 400 (127) 2 471 (1 375 (111) 368 (117) -6 -1 , -30 0.001 -56
-89, -22 0.002 4 462 (115) 40 (131) 345 (120) -29 -073, 16 0.208
-69 -103, -36 0.001 Mean (SD) *0.2 1 = ba and after and 4 weeks of
. L . = difference. WBC = white blood cells. Normal values: ESR,
female < , male <10; CRP <10; haemoglobin, female 12-16,
male 13.5-17. ; WBC -11, 150-400. indicates data missing or
illegible when filed
TABLE-US-00028 TABLE 17 Responses According to Paulus 20% and 50%
Criteria at Each Evaluation Point Data Summary Statistical Analysis
vs. Placebo Placebo 1 mg/kg cA2 10 mg/kg cA2 1 mg/kg cA2 10 mg/kg
cA2 n = 24 n = 25 n = 24 LR 95% CI p LR 95% CI p Paulus 20% Day 3 2
( ) 8 (32%) 7 ( %) 1.1, 14.0 0.0738 3. 0.9, 13. 0.13 5 Week 1 2 ( )
13 (52%) 16 (67%) 6. 2.1, 1 .6 0.00 .0, 21.5 0.0001 Week 2 3 (13%)
15 (60%) 18 (75%) 5.0 2.1, 12.2 0.000 6.0 2.7, 1 .5 <0.0001 Week
3 4 (17%) 12 (48%) 21 (88%) 2. 1.2, 7.1 0.0322 5.3 2.7, 10.
<0.0001 Week 4 2 (8%) 11 (44%) 19 (7 %) 5.3 1.7, 16.9 0.00 9.5 ,
23. <0.0001 P s 0% Day 3 1 ( %) 6 (24%) 2 ( %) 5. 1.0, 33.1 0.
2.0 0.2, 1.000 Week 1 1 (4) 11 (44%) 12 (50%) 10.6 2.5, 44.6 0.0019
12.0 3.0, 47.5 0.00 Week 2 0 11 (44%) 12 ( %) NA NA 0.0002 NA NA
<0.0001 Week 3 2 ( %) 7 (28%) 13 (54%) 3. 0.9, 13.0 0.1 6.5 2.2,
19.2 0.0013 Week 4 2 ( %) 7 (28%) 14 (58%) 3. 0.9, 13.0 0.1383 .0
2.5, 0.0005 LR = likelihood ratio, NA = applicable (ratio be
calculated no placebo recipients respon at that time). indicates
data missing or illegible when filed
TABLE-US-00029 TABLE 18 All Adverse Events Recorded During 4 Weeks
After Entry 1 mg/kg 50 mg/kg System Event Placebo cA2 cA2 URTI 1
(0) 2 (0) 1 (1) Probable -- 1 (1) -- Pleu -- 1 (0) -- N 2 (0) -- --
Di 1 (1) -- -- Abdominal Pain -- 2 (0) -- P -- 1 (0) -- Blood loss
per -- 1 (0) -- Cardiovascular Hypertension 1 (0) 1 (1) 1 (1)
peripheral -- 1 (0) 1 (0) Skin H 3 (1) 1 (0) -- Infection -- 2 (2)
-- Injection skin reactivity -- 1 (1) -- Neurological D 3 (1) -- --
Headache -- -- 1 (0) M R -- 1 (0) -- P -- 1 (0) -- Fracture -- -- 1
(0) Other Ma -- 1 (0) -- Ri -- 1 (1) -- -- 1 (0) -- S -- 1 (0) --
Vas 1 (0) -- -- URTI--upper r infection. Those events judged by to
be indicates data missing or illegible when filed
Equivalents
[0332] 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
* * * * *