U.S. patent application number 10/762096 was filed with the patent office on 2004-11-18 for treatment of tumor necrosis factor-mediated diseases.
This patent application is currently assigned to The Kennedy Institute of Rheumatology. Invention is credited to Feldmann, Marc, Maini, Ravinder N., Williams, Richard O..
Application Number | 20040228863 10/762096 |
Document ID | / |
Family ID | 34812139 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040228863 |
Kind Code |
A1 |
Feldmann, Marc ; et
al. |
November 18, 2004 |
Treatment of tumor necrosis factor-mediated diseases
Abstract
A method of treating and/or preventing a TNF-mediated disease in
an individual is disclosed. Also disclosed is a composition
comprising a tumor necrosis factor antagonist and a CD4+ T cell
inhibiting agent. 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) ;
Williams, Richard O.; (London, GB) |
Correspondence
Address: |
John P. White, Esq.
Cooper & Dunham, LLP
23rd Floor
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
The Kennedy Institute of
Rheumatology
|
Family ID: |
34812139 |
Appl. No.: |
10/762096 |
Filed: |
January 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10762096 |
Jan 20, 2004 |
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09093450 |
Jun 8, 1998 |
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6770279 |
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09093450 |
Jun 8, 1998 |
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08607419 |
Feb 28, 1996 |
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08607419 |
Feb 28, 1996 |
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PCT/GB94/00462 |
Mar 10, 1994 |
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08607419 |
Feb 28, 1996 |
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08403785 |
May 3, 1995 |
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5741488 |
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08403785 |
May 3, 1995 |
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PCT/GB93/02070 |
Oct 6, 1993 |
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08403785 |
May 3, 1995 |
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07958248 |
Oct 8, 1992 |
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Current U.S.
Class: |
424/145.1 ;
514/16.6; 514/20.5 |
Current CPC
Class: |
A61K 39/395 20130101;
A61K 39/395 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/145.1 ;
514/011 |
International
Class: |
A61K 039/395; A61K
038/13 |
Claims
1.-22. (Canceled)
23. A method for treating or preventing Crohn's disease in an
individual in need thereof comprising administering to the
individual cyclosporin or analog thereof in combination with a
tumor necrosis factor antagonist, in therapeutically effective
amounts.
24. A method of claim 23 wherein the tumor necrosis factor
antagonist is an anti-tumor necrosis factor antibody or fragment
thereof.
25. A method of claim 24 wherein the antibody is a chimeric
antibody.
26. A method of claim 23 wherein the tumor necrosis factor
antagonist is a receptor molecule which binds to tumor necrosis
factor.
27. A method of claim 26 wherein the receptor molecule is a tumor
necrosis factor receptor/immunoglobulin G fusion protein.
28. A method of claim 23 wherein the tumor necrosis factor
antagonist prevents or inhibits tumor necrosis factor synthesis,
tumor necrosis factor release or its action on target cells.
29. A method of claim 28 wherein the tumor necrosis factor
antagonist is a phosphodiesterase inhibitor.
30. A method of claim 29 wherein the phosphodiesterase inhibitor is
pentoxifylline.
31.-45. (Canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application 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, which is a
continuation-in-part of U.S. application Ser. No. 08/403,785, which
is the U.S. National Phase of International; Application No.
PCT/GB93/02070, filed Oct. 6, 1993, which is a continuation-in-part
of U.S. application Ser. No. 07/958,248, filed Oct. 8, 1992, now
abandoned, the entire teachings of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Monocytes and macrophages secrete cytokines known as tumor
necrosis factor alpha (TNF.alpha.) and tumor necrosis factor beta
(TNF.beta.) in response to endotoxin or other stimuli. TNF.alpha.
is a soluble homotrimer of 17 kD protein subunits (Smith et al., J.
Biol. 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 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 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 (Oliffet al., Cell 50:555 (1987)), autoimmune
pathologies and graft-versus-host pathologies (Piguet et al., J.
Exp. Med 166:1280 (1987)). The association of TNF with cancer and
infectious pathologies is often related to the host's catabolic
state. Cancer patients suffer from weight loss, usually associated
with anorexia.
[0006] The extensive wasting which is associated with cancer, and
other diseases, is known as "cachexia" (Kem et al, J. Parent.
Enter. Nutr. 12:286-298 (1988)). Cachexia includes progressive
weight loss, anorexia, and persistent erosion of lean 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, pp.463-466 (1989);
Simpson et al., Crit. Care Clin. 5:27-47 (1989)), including fever,
malaise, anorexia, and cachexia. Endotoxin strongly activates
monocyte/macrophage roduction 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., N. 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 pp. 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 an
open-label trial with a chimeric antibody to TNF.alpha. (cA2) have
been reported with suppression of inflammation (Elliott et al.,
Arthritis Rheum. 36:1681-1690 (1993)).
SUMMARY OF THE INVENTION
[0009] The present invention pertains to the discovery that
co-administration of a CD4+ T cell inhibiting agent and a tumor
necrosis factor (TNF) antagonist to an individual suffering from a
TNF-mediated disease produces a significantly improved response
compared to that obtained with administration of the inhibiting
agent alone or that obtained with administration of the antagonist
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 a CD4+ T cell inhibiting
agent and a TNF antagonist to the individual in therapeutically
effective amounts. The present invention further relates to a
method for treating and/or preventing recurrence of a TNF-mediated
disease in an individual comprising co-administering a CD4+ T cell
inhibiting agent and a TNF antagonist 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).
[0010] Therefore, in one embodiment, the invention relates to a
method of treating and/or preventing rheumatoid arthritis in an
individual comprising co-administering a CD4+ T cell inhibiting
agent and a TNF antagonist 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 a CD4+ T cell inhibiting
agent and a TNF antagonist to the individual in therapeutically
effective amounts. In a third embodiment, the invention relates to
a method of treating and/or preventing acute or chronic immune
disease associated with a transplantation in an individual
comprising co-administering a CD4+ T cell inhibiting agent and a
TNF antagonist to the individual in therapeutically effective
amounts.
[0011] A further embodiment of the invention relates to
compositions comprising a CD4+ T cell inhibiting agent and a TNF
antagonist. CD4+ T cell inhibiting agents useful in the methods and
compositions of the present invention include antibodies to T cells
or to their receptors, antibodies to antigen presenting cells (APC)
or to their receptors, peptides and small molecules blocking the T
cell/APC interaction, including those which block the BLA class II
groove, or block signal transduction in T-cell activation, such as
cyclosporin and cyclosporin analogs, and antibodies to B cells.
[0012] TNF antagonists useful in the methods and compositions of
the present invention include anti-TNF antibodies and receptor
molecules which bind specifically to TNF; compounds which prevent
and/or inhibit TNF synthesis, T release or its action on target
cells, such as thalidomide, tenidap, phosphodiesterase inhibitors
(e.g, pentoxifyrlline and rolipram), A2b adenosine receptor
agonists and A2b adenosine receptor enhancers; and compounds which
prevent and/or inhibit TNF receptor signalling.
[0013] In a particular embodiment of the invention, an inflammatory
mediator other than a TNF antagonist can be used instead of or in
addition to the TNF antagonist.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a graph showing the effect of administering a
sub-optimal dose of anti-TNF antibody (50 .mu.g hamster TN3.19.2)
alone and in combination with anti-CD4 antibody (200 .mu.g) to male
DBA/1 mice on the suppression of arthritis as assessed by
paw-swelling measurements. Triangle=control antibody;
diamond=anti-CD4 antibody; open square=anti-TNF antibody; closed
square=anti-CD4 antibody plus anti-TNF antibody. An asterisk
indicates a significant reduction compared to the group of mice
administered control antibody (P<0.05; two-sample t test).
Arrows indicate times of injection.
[0015] FIG. 2 is a graph showing the effect of administering an
optimal dose (300 .mu.g) of anti-TNF antibody alone and in
combination with anti-CD4 antibody (200 .mu.g) to male DBA/1 mice
on the suppression of arthritis as assessed by paw-swelling
measurements. Triangle=control antibody; diamond=anti-CD4 antibody;
open square=anti-TNF antibody; closed square=anti-CD4 antibody plus
anti-TNF antibody. An asterisk indicates a significant reduction
compared to the group of mice administered control antibody
(P<0.05; two-sample t test). Arrows indicate times of
injection.
[0016] FIG. 3 is a graph showing the effect of administering 100
.mu.g TNF receptor/IgG fusion protein alone, and a combination of
100 .mu.g TNF receptor/IgG fusion protein plus either 6 .mu.g, 25
.mu.g, 100 .mu.g or 400 .mu.g anti-CD4 antibody to male DBA/1 mice
on the suppression of arthritis as assessed by clinical score.
[0017] FIG. 4 is a graph showing the effect of administering 250
.mu.g cyclosporin A, 50 .mu.g anti-TNF antibody, and a combination
of 250 .mu.g cyclosporin A and 50 .mu.g anti-TNF antibody to male
DBA/1 mice on the suppression of arthritis as assessed by
paw-swelling measurements. Open square=control; diamond=cyclosporin
A; triangle=anti-TNF antibody; closed square=cyclosporin A plus
anti-TNF antibody.
[0018] FIG. 5 is a graph showing the effect of administering 250
.mu.g cyclosporin A, alone, 50 .mu.g anti-TNF antibody alone, and a
combination of 250 .mu.g cyclosporin A and 50 .mu.g anti-TNF
antibody to male DBA/1 mice on the suppression of arthritis as
assessed by clinical score. Open square=control;
triangle=cyclosporin A; diamond=anti-TNF antibody;
square=cyclosporin A plus anti-TNF antibody. P<0.05 (vs. PBS
treated group).
[0019] 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 control antibody L2, and 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.
[0020] FIG. 7 is a graph showing the effect of administering 500
.mu.g cyclosporin A alone, 250 .mu.g anti-TNF antibody alone, and a
combination of 500 .mu.g cyclosporin 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=cyclosporin A; square=cyclosporin A plus
anti-TNF antibody. P<0.05 (vs. PBS treated group).
[0021] FIG. 8 is a graph comparing the effects of administering
Cremophor EL.RTM. (negative control) alone, either 3 mg/kg, 5 mg/kg
or 10 mg/kg of rolipram alone, and 300 .mu.g anti-TNF antibody
alone to male DBA/1 mice on the suppression of arthritis as
assessed by clinical score. Square with black dot=negative control;
diamond=3 mg/kg rolipram; x=rolipram (5 mg/kg); square=rolipram (10
mg/kg); circle=anti-TNF antibody.
[0022] FIG. 9 is a graph showing the effect of administering
Cremophor EL.RTM. (negative control) alone, either 0.5 mg/kg, 3
mg/kg or 5 mg/kg of rolipram alone, 300 .mu.g anti-TNF antibody
alone, and a combination of 5 mg/kg rolipram and 300 .mu.g anti-TNF
antibody to male DBA/1 mice on the suppression of arthritis as
assessed by clinical score. Square with black dot=negative control;
diamond=anti-TNF antibody; square with white dot=rolipram plus
anti-TNF antibody; diamond with white dot=rolipram (0.5 mg/kg);
square=rolipram (3 mg/kg); open square=rolipram (5 mg/kg).
[0023] FIGS. 10A and 10B are graphs showing the effect of
administering Cremophor EL.RTM. (negative control) alone, 5 mg/kg
of rolipram alone, 50 .mu.g anti-CD4 antibody alone, and a
combination of 5 mg/kg rolipram and 50 .mu.g anti-CD4 antibody to
male DBA/1 mice on the suppression of arthritis as assessed by
clinical score FIG. 10A) and paw-swelling (FIG. 11B). Square with
black dot=negative control; diamond=rolipram; square with white
dot=rolipram plus anti-CD4 antibody; diamond with white
dot=anti-CD4 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The nature of autoantigens responsible for autoimmune
disorders is not known, nor is the action which triggers the
autoimmune response. One theory involves the similarity of a viral
protein to a self antigen, which results in autoreactive T cells or
B cells recognizing a self antigen. Whereas B lymphocytes produce
antibodies, thymus-derived or "T cells" are associated with
cell-mediated immune functions. T cells recognize antigens
presented on the surface of cells and carry out their functions
with these antigen-presenting cells.
[0025] Various markers have been used to define human T cell
populations. CD4 is a non-polymorphic surface glycoprotein receptor
with partial sequence identity to immunoglobulins. CD4 receptors
define distinct subsets of mature peripheral T cells. In general,
CD4 T cells expressing helper or regulatory functions interact with
B cells in immune responses, while T cells expressing the CD8
surface antigen function as cytotoxic T cells and have regulatory
effects on immune responses. Since T cell receptors are the pathway
through which stimuli augment or modulate T cell responses, they
present a potential target for immunological intervention.
[0026] Of the cellular interactions, that of CD4+ T cells with
antigen presenting cells (APC) lies at the root of the immune
response. Many aspects of the autoimmune response are essentially
similar to that of normal immune responses. Thus, CD4+ autoantigen
reactive T cells are restimulated by APC expressing class II with
autoantigen peptides in the binding groove. In certain human
diseases the evidence that this occurs has been provided: in
Graves' disease of the thyroid, in vivo activated T cells are
present in the glands that are removed for refractory disease, and
many of these cells after cloning can be shown to recognize
autologous thyrocytes,(as APC) not extrinsically supplied with any
antigen, or APC supplied with the thyroid specific antigens thyroid
peroxidase or thyroglobulin (Londei, M. et al., Science 228:85-89
(1985); Dayan, C. M. et al., Proc. Natl. Acad. Sci. USA
88:7415-7419 (1991)). Similarly, in rheumatoid arthritis (RA), in
vivo activated T cells recognizing collagen type II have been
isolated from joints of an RA patient in three consecutive
operations during the course of three years (Londei, M. et al,
Proc. Natl. Acad. Sci. USA 86:636-646 (1989)). In other human
diseases displaying autoimmune characteristics, CD4+ T cells from
the blood have been cloned, including CD4+ T cells recognizing the
acetylcholine receptor in myasthenia gravis (Hohlfeld, R. et al.,
Nature 310:224-246 (1984)); myelin basic protein in multiple
sclerosis (Hafler, D. A. et al., J. Immunol. 139:68-72 (1987)); or
islet cell membranes in insulin dependent diabetes mellitus (De
Berardinis, P. et al., Lancet II:823-824 (1988); Kontiainen, S. et
al., Autoimmunity 8:193-197 (1991)).
[0027] The present invention is directed to a method for treating
and/or preventing a TNF-mediated disease in an individual,
comprising co-administering a tumor necrosis factor antagonist and
a CD4+ T cell inhibiting agent to the individual in therapeutically
effective amounts. The TNF antagonist and CD4+ T cell inhibiting
agent can be administered simultaneously or sequentially. Multiple
CD4+ T cell inhibiting agents and multiple TNF antagonists can be
co-administered. Other therapeutic regimens can be used in
combination with the therapeutic co-administration of TNF
antagonists and CD4+ T cell inhibiting agents.
[0028] Inflammatory mediators other than TNF antagonists can be
used instead of or in addition to TNF antagonists. As used herein,
the term "inflammatory mediator" refers to an agent which
decreases, blocks, inhibits, abrogates or interferes with
pro-inflammatory mediator activity. Blocking TNF activity in
rheumatoid joint cell cultures results in down-regulation of
interleukin-1 (IL-1) production (Brennan et al., Lancet 11:244-247
(1989)) and down-regulation of the pro-inflammatory cytokine
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Haworth
et al., Eur. J. Immunol. 21:2575-2579 (1991); Butler et al., Eur.
Cytokine Network 6:225-230 (1995)). Blocking TNF activity also
blocks IL-6 and IL-8 production. These cytokine "networks" or
"hierarchies" also operate in vivo; rheumatoid arthritis patients
treated with anti-TNF antibody reduced their serum IL-6 levels, as
well as levels of IL-6 dependent acute phase proteins such as C
reactive protein, in the weeks following treatment (Elliott, M. J.
et al., Arthritis & Rheumatism 36:1681-1690 (1993)). Since the
pro-inflammatory mediators TNF, IL-1, GM-CSF, IL-6 and IL-8 are
part of the same network or hierarchy, blocking any of these can
have comparable effects. Thus, agents which block TNF, IL-1,
GM-CSF, IL-6 and/or IL-8 are useful as the inflammatory mediators
of the present invention.
[0029] Representative inflammatory mediators that can be used in
the present invention include agents which decrease, block,
inhibit, abrogate or interfere with IL-1 activity, synthesis, or
receptor signalling, such as anti-IL-1 antibody, soluble IL-IR,
IL-1 receptor antagonist, or other appropriate peptides and small
molecules; agents which decrease, block, inhibit, abrogate or
interfere with IL-6 activity, synthesis, or receptor signalling,
such as anti-IL-6 antibody, anti-gp130, or other appropriate
peptides and small molecules; modalities which decrease, block,
inhibit, abrogate or interfere with the activity, synthesis, or
receptor signalling of other pro-inflammatory mediators, such as
GM-CSF and members of the chemokine (IL-8) family; and cytokines
with anti-inflammatory properties, such as IL-4, IL-10, IL-13, and
TGF.beta.. In addition, other anti-inflammatory agents, such as the
anti-rheumatic agent methotrexate, can be administered in
conjunction with the CD4+ T cell inhibiting agent and/or the TNF
antagonist.
[0030] The present invention is further directed to a method for
treating and/or preventing recurrence of a TNF-mediated disease in
an individual comprising co-administering a CD4+ T cell inhibiting
agent and a TNF antagonist to the individual in therapeutically
effective amounts.
[0031] 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:
[0032] (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;
[0033] (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 (HMV), acquired immunodeficiency
syndrome (AIDS) (including symptoms of cachexia, autoimmune
disorders, AIDS dementia complex and infections);
[0034] (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; 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;
[0035] (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) dopamiine
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,
abetalipoproteinemia, ataxia, telangiectasia, and mitochondrial
multisystem disorder); disorders of the motor unit, such as
neurogenic muscular atrophies (anterior horn cell degeneration,
such as amyotrophic lateral sclerosis, infantile spinal muscular
atrophy and juvenile spinal muscular atrophy); Alzheimer's disease;
Down's syndrome in middle age; diffuse Lewy body disease; senile
dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic
alcoholism; primary biliary cirrhosis; cryptogenic fibrosing
alveolitis and other fibrotic lung diseases; hemolytic anemia;
Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis,
Hallervorden-Spatz disease; and dementia pugilistica, or any subset
thereof;
[0036] (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));
[0037] (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
[0038] (G) alcohol-induced hepatitis and other forms of chronic
hepatitis.
[0039] 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.
[0040] 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
recurrence can include the experience of one or more of swollen
joints, morning stiffness or joint tenderness.
[0041] In one embodiment, the invention relates to a method of
treating and/or preventing rheumatoid arthritis in an individual
comprising co-administering a CD4+ T cell inhibiting agent and a
TNF antagonist to the individual in therapeutically effective
amounts.
[0042] 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 CD4+ T cell inhibiting agent and a
TNF antagonist to the individual in therapeutically effective
amounts.
[0043] 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 a CD4+ T cell inhibiting agent and a
TNF antagonist to the individual in therapeutically effective
amounts. As used herein, a "transplantation" includes renal
transplantation, cardiac transplantation, bone marrow
transplantation, liver transplantation, pancreatic transplantation,
small intestine transplantation, skin transplantation and lung
transplantation.
[0044] The benefits of combination therapy with TNF antagonists and
CD4+ T cell inhibiting agents include significantly improved
response in comparison with the effects of treatment with each
therapeutic modality separately. In addition, lower dosages can be
used to provide the same reduction of the immune and inflammatory
response, thus increasing the therapeutic window between a
therapeutic and a toxic effect. Lower doses also results in lower
financial costs to the patient, and potentially fewer side effects.
For example, immune and/or allergic responses to TNF antagonists
can be reduced, thus enhancing safety and therapeutic efficacy.
[0045] In a further embodiment, the invention relates to
compositions comprising a TNF antagonist and a CD4+ T cell
inhibiting agent. 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.
[0046] Tumor Necrosis Factor Antagonists
[0047] 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)-carboxaridoadenosine,
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.
[0048] Anti-TNF Antibodies
[0049] 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. 08/192,102 (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. 08/192,093 (filed Feb. 4, 1994),
U.S. application Ser. No. 08/324,799 (filed Oct. 18, 1994; now U.S.
Pat. No. 5,698,195) and Le, J. et al., International Publication
No. WO 92/16553 (published Oct. 1, 1992), which references are
entirely incorporated herein by reference. A second example of a
high affinity monoclonal antibody useful in the methods and
compositions of the present invention is murine mAb 195 and
antibodies which will competitively inhibit in vivo the binding to
human TNF.alpha. of anti-TNF.alpha. murine 195 or an antibody
having substantially the same specific binding characteristics, as
well as fragments and regions thereof. Other high affinity
monoclonal antibodies useful in the methods and compositions of the
present invention include murine mAb 114 and murine mAb 199 and
antibodies which will competitively inhibit in vivo the binding to
human TNF.alpha. of anti-TNF.alpha. murine mAb 114 or mAb 199 or an
antibody having substantially the same specific binding
characteristics of mAb 114 or mAb 199, as well as fragments and
regions thereof. Murine monoclonal antibodies 114, 195 and 199 and
the method for producing them are described by Moller, A. et al.
(Cytokine 2(3):162-169 (1990)), the teachings of which are entirely
incorporated herein by reference. Preferred methods for determining
mAb specificity and affinity by competitive inhibition can be found
in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988); Colligan
et al., Eds., Current Protocols in Immunology, Greene Publishing
Assoc. and Wiley Interscience, New York (1992, 1993); Kozbor et
al., Immunol. Today 4:72-79 (1983); Ausubel et al., eds. Current
Protocols in Molecular Biology; Wiley Interscience, New York (1987,
1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), which
references are entirely incorporated herein by reference.
[0051] 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 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).
[0052] 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).
[0053] 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).
[0054] 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).
[0055] 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 171496 (published Feb. 19, 1985);
Morrison et al., European Patent Application 173494 (published Mar.
5, 1986); Neuberger et al., PCT Application WO 86/01533, (published
Mar. 13, 1986); Kudo et al., European Patent Application 184187
(published Jun. 11, 1986); Morrison et al., European Patent
Application 173494 (published Mar. 5, 1986); Sahagan et al., J.
Immunol. 137:1066-1074 (1986); Robinson et al., International
Publication No. PCT/US86/02269 (published 7 May 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.
[0056] 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.
[0057] 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) incorporated herein by
reference.
[0058] 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.
[0059] 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. 08/192,102 (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. 08/192,093 (filed Feb. 4, 1994) and U.S.
application Ser. No. 08/324,799 (filed on Oct. 18, 1994; now U.S.
Pat. No. 5,698,195) 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)), which references are entirely
incorporated herein by reference.
[0060] Chimeric A2 anti-TNF consists of the antigen binding
variable region of the high-affinity neutralizing mouse antihuman
TNF IgG1 antibody, designated A2, and the constant regions of a
human IgG1, kappa immunoglobulin. The human IgG1 Fc region improves
allogeneic antibody effector function, increases the circulating
serum half-life and decreases the immunogenicity of the antibody.
The avidity and epitope specificity of the chimeric A2 is derived
from the variable region of the murine A2. Chimeric A2 neutralizes
the cytotoxic effect of both natural and recombinant human TNF in a
dose dependent manner. From binding assays of cA2 and recombinant
human TNF, the affinity constant of cA2 was calculated to be
1.8.times.10.sup.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; Coiligan 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] For example, epitopes of TNF which are recognized by, and/or
binds with anti-TNF activity, an antibody, fragments, and variable
regions thereof, include:
1 59-80: Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-
(SEQ ID NO:1) His-Val-Leu-Leu-Thr-His-Thr-Ile; and/or 87-108:
Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser- -Ala-Ile-Lys-Ser-Pro-Cys- (SEQ
ID NO:2) Gln-Arg-Glu-Thr-Pro-Glu-Gly.
[0065] The anti-TNF antibodies, fragments, and variable regions
thereof, that are recognized by, and/or bind 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 WO 91/02078 (published Feb. 21, 1991),
incorporated herein by reference, discloses TNF ligands which can
bind additional epitopes of TNF.
[0066] Antibody Production Using Hybridomas
[0067] 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.
[0068] 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.,
EP0218868 (published Apr. 22, 1987); Yone et al., EP0288088
(published Oct. 26, 1988); Liang et al., Biochem. Biophys. Res.
Comm. 137:847-854 (1986); Meager, et al., Hybridoma 6:305-.sub.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).
[0069] 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.
[0070] 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
nice. 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.
[0071] 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).
[0072] 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. Chimeric antibody cA2 is produced by
a cell line designated c168A.
[0073] "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.
[0074] "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).
[0075] Recombinant Expression of Anti-TNF Antibodies
[0076] Recombinant and/or chimeric murine-human or human-human
antibodies that inhibit TNF can be provided using known techniques
based on the teachings provided in 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. 08/192,093 (filed Feb. 4, 1994), U.S.
application Ser. No. 08/324,799 (filed on Oct. 18, 1994; now U.S.
Pat. No. 5,698,195) and Le, J. et al., International Publication
No. WO 92/16553 (published Oct. 1, 1992), which references are
entirely incorporated herein by reference. See, e.g., Ausubel 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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).
[0081] 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. Mol. 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)).
[0082] 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.
[0083] 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 A (IgM). The human CL region can be
derived from either human L chain isotype, kappa or lambda.
[0084] Genes encoding human immunoglobulin C regions are obtained
from human cells by standard cloning techniques (see, e.g.,
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.
[0085] 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.
[0086] Therefore, cDNA encoding the antibody V and C regions and
the method of producing a chimeric antibody can involve several
steps, outlined below:
[0087] 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;
[0088] 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;
[0089] 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;
[0090] 4. Expression and production of L and H chains in selected
hosts, including prokaryotic and eukaryotic cells to provide
murine-murine, human-murine or human-human antibodies.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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 (1989); and Ausubel et al., Eds. Current Protocols
in Molecular Biology, Wiley Interscience, New York (1987,
1993).
[0098] 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.
[0099] 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); Beutier 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).
[0105] CD4+ T Cell Inhibiting Agents
[0106] As used herein, a "CD4+ T cell inhibiting agent" decreases,
blocks, inhibits, abrogates or interferes with the activation of
CD4+ T cells or the interaction of CD4+ T cells with antigen
presenting cells (APC). CD4+ T cell inhibiting agents include
antibodies to T cells or to their receptors, such as anti-CD4,
anti-CD28, anti-CD52 (e.g., CAMPATH-1H) and anti-IL-2R antibodies;
antibodies to APC or to their receptors, such as anti-class II,
anti-ICAM-1, anti-LFA-3, and anti-LFA-1 antibodies; peptides and
small molecules blocking the T cell/APC interaction, including
those which block the HLA class II groove, CD4 or block signal
transduction in T-cell activation, such as cyclosporin and
cyclosporin analogs, particularly cyclosporin A, or FK-506; and
antibodies to B cells including CD5+ B cells, such as CD19, CD20,
CD21, CD23 and BB/7 or B1 antibodies, ligands for CD28, and
inhibitors of B7/CD28, such as CTLA4-Ig. B cells, including CD5+ B
cells, are considered to be an important type of APC in disease
processes (Plater-Zyberk et al., Ann. N.Y Acad. Sci. 651:540-555
(1992)), and thus, anti-B cell antibodies can be particularly
useful in the methods and compositions of the present
invention.
[0107] Anti-CD4 Antibodies
[0108] Anti-CD4 antibodies useful in the present invention include
polyclonal, monoclonal, chimeric, humanized, resurfaced and
recombinant antibodies and fragments thereof which are
characterized by high affinity binding to CD4 and low toxicity
(including HAMA and/or 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.
[0109] Techniques described herein for producing anti-TNF
antibodies can be employed in producing anti-CD4 antibodies that
can be used in the present invention.
[0110] Monoclonal antibodies reactive with CD4 can be produced
using somatic cell hybridization-techniques (Kohler and Milstein,
Nature 256: 495-497 (1975)) or other techniques. In a typical
hybridization procedure, a crude or purified protein or peptide
comprising at least a portion of CD4 can be used as the immunogen.
An animal is vaccinated with the immunogen to obtain anti-CD4
antibody-producing spleen cells. The species of animal immunized
will vary depending on the species of monoclonal antibody desired.
The antibody producing cell is fused with an immortalizing cell
(e.g., myeloma cell) to create a hybridoma capable of secreting
anti-CD4 antibodies. The unfused residual antibody-producing cells
and immortalizing cells are eliminated. Hybridomas producing
desired antibodies are selected using conventional techniques and
the selected hybridomas are cloned and cultured.
[0111] Polyclonal antibodies can be prepared by immunizing an
animal with a crude or purified protein or peptide comprising at
least a portion of CD4. The animal is maintained under conditions
whereby antibodies reactive with either CD4 are produced. Blood is
collected from the animal upon reaching a desired titre of
antibodies. The serum containing the polyclonal antibodies
(antisera) is separated from the other blood components. The
polyclonal antibody-containing serum can optionally be further
separated into fractions of particular types of antibodies (e.g.,
IgG, IgM).
[0112] Examples of anti-CD4 antibodies that can be used in the
present invention are described in the art (see, e.g., U.S.
application Ser. No. 07/867,100 (filed Jun. 25, 1992); Grayheb et
al., J. Autoimmunity 2:627-642 (1989); Ranges et al, J. Exp. Med
162: 1105-1110 (1985); Hom et al., Eur. J. Immunol. 18: 881-888
(1988); Wooley et al., J. Immunol. 134: 2366-2374 (1985); Cooper et
al., J. Immunol. 141: 1958-1962 (1988); Van den Broek et al., Eur.
J. Immunol. 22: 57-61 (1992); Wofsy et al., J. Immunol. 134:
852-857 (1985); Wofsy et al., J. Immunol. 136: 4554-4560 (1986);
Ermak et al., Laboratory Investigation 61: 447-456 (1989); Reiter
et al., 34:525-532 (1991); Herzog et al., J. Autoimmun. 2:627
(1989); Ouyang et al., Dig. Dis. Sci. 33:1528-1536 (1988); Herzog
et al, Lancet ii: 1461 (Dec. 19, 1987); Emmrich et al., Lancet
338:570-571 (1991), which references are entirely incorporated
herein by reference).
[0113] Administration
[0114] TNF antagonists, CD4+ T cell inhibiting agents, 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, including infusion
and/or bolus injection, subcutaneous, oral, topical, epidural,
buccal, rectal, vaginal and intranasal routes. Other suitable
routes of administration can also be used, for example, to achieve
absorption through epithelial or mucocutaneous linings. TNF
antagonists, CD4+ T cell inhibiting agents, and the compositions of
the present invention can also be administered 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, CD4+ T cell
inhibiting agents 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.
[0115] The TNF antagonists and CD4+ T cell inhibiting agents 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 a CD4+ T cell inhibiting agent.
[0116] For parenteral (e.g., intravenous, subcutaneous,
intramuscular) administration, TNF antagonists, CD4+ T cell
inhibiting agents 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.
[0117] Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, A. Osol, a standard reference
in this field of art.
[0118] 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.
[0119] In a particular embodiment, cyclosporin and cyclosporin
analogs can be administered to an individual orally or
intravenously. However, other therapeutically efficacious routes of
administration can also be used, such as those described above.
Cyclosporin, or a cyclosporin analog, can be administered alone,
but is generally administered with a pharmaceutical carrier
selected on the basis of the chosen route of administration and
standard pharmaceutical practice. Presently available oral and
intravenous formulations of cyclosporin include SANDIMMUNE.RTM.
soft gelatin capsules, oral solution and injection (Sandoz
Pharmaceuticals/Consumer Division, East Hanover, N.J.).
[0120] TNF antagonists and CD4+ T cell inhibiting agents are
co-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 co-administration
of TNF antagonist and CD4+ T cell inhibiting agent, 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 TNF antagonist and CD4+ T cell inhibiting agent are not
co-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 that amount of
TNF antagonist and CD4+ T cell inhibiting agent necessary to
significantly reduce or eliminate symptoms associated with a
particular TNF-mediated disease. As used herein, a therapeutically
effective amount is not an amount such that administration of the
TNF antagonist alone, or administration of the CD4+ T cell
inhibiting agent alone, must necessarily result in inhibition of
the biological activity of TNF or in immunosuppressive
activity.
[0121] Once a therapeutically effective amount has been
administered, a maintenance amount of TNF antagonist alone, of CD4+
T cell inhibiting agent alone, or of a combination of TNF
antagonist and CD4+ T cell inhibiting agent can be administered to
the individual. A maintenance amount is the amount of TNF
antagonist, CD4+ T cell inhibiting agent, or combination of TNF
antagonist and CD4+ T cell inhibiting agent necessary to maintain
the reduction or elimination of symptoms 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.
[0122] 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, health, sex, body weight and
diet of the recipient; nature and extent of symptoms of the disease
being treated, kind of concurrent treatment, frequency of
treatment, and the effect desired. In vitro and in vivo methods of
determining the inhibition of TNF in an individual are well known
to those of skill in the art. Such in vitro assays can include a
TNF cytotoxicity assay (e.g., the WEHI assay or a radioimmunoassay,
ELISA). In vivo methods can include rodent lethality assays and/or
primate pathology model systems (Mathison et al., J. Clin. Invest.
81:.1925-1937 (1988); Beutler et al., Science 229: 869-871 (1985);
Tracey et al., Nature 330: 662-664 (1987); Shimamoto et al.,
Immunol. Lett. 17: 311-318 (1988); Silva et al., J. Infect. Dis.
162: 421-427 (1990); Opal et al, J. Infect. Dis. 161: 1148-1152
(1990); Hinshaw et al., Circ. Shock 30: 279-292 (1990)).
[0123] TNF antagonists and CD4+ T cell inhibiting agents can be
co-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 CD4+ T cell
inhibiting agents. Adjustment and manipulation of established
dosage ranges are well within the ability of those skilled in the
art.
[0124] In a particular embodiment, TNF antagonist and cyclosporin
(or cyclosporin analog) can be co-administered in single or
multiple doses depending upon factors such as nature and extent of
symptoms, kind of concurrent treatment and the effect desired.
Other therapeutic regimens or agents (e.g., multiple drug regimens)
can be used in combination with the therapeutic co-administration
of TNF antagonists and cyclosporin (or cyclosporin analog).
Adjustment and manipulation of established dosage ranges are well
within the ability of those skilled in the art.
[0125] 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.
[0126] 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, the 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.
[0127] 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.
[0128] The present invention will now be illustrated by the
following examples, which are not intended to be limiting in any
way.
EXAMPLES
Example 1
Treatment of Induced Arthritis in a Murine Model Using Anti-CD4
Antibody and Anti-TNF Antibody
[0129] 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, and
erosions of cartilage and bone. Furthermore, there is a good
correlation of therapeutic response with human rheumatoid
arthritis. For example, in both diseases anti-TNF antibody has
beneficial effects (Williams, R. O. et al., Proc. Natl. Acad Sci.
USA 89:9784-9788 (1992); Elliott, M. J. et al., Arthritis &
Rheumatism 36:1681-1690 (1993)), and anti-CD4 antibody has minimal
effect in mouse arthritis as well as in human arthritis (Williams,
R. O. et al., Proc. Natl. Acad Sci. USA 91:2762-2766 (1994);
Horneff, G. et al., Arthritis & Rheumatism 34:129-140 (1991)).
Thus, the animal model serves as a good approximation to human
disease.
[0130] The model of rheumatoid arthritis used herein, i.e., the
collagen type II induced arthritis in the DBA/1 mouse, is described
by Williams, R. O. et al. (Proc. Natl. Acad. Sci. USA 89:9784-9788
(1992)). Type II collagen was purified from bovine articular
cartilage by limited pepsin solubilization and salt fractionation
as described by Miller (Biochemistry 11:4903-4909 (1972)).
[0131] Experimental Procedure
[0132] 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 (Difco Laboratories, East Molsey, UK). 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 (mAb) treatment was administered on
days 1, 4, and 7. For anti-TNF antibody, TN3-19.12, a neutralizing
hamster IgG anti-TNF.alpha./.beta. monoclonal antibody (mAb), was
used (Sheehan, K. C. et al., J. Immunology 142:3884-3893 (1989)).
The isotype control was L2. The anti-TNF antibody and the isotype
control were provided by R. Schreiber, Washington University
Medical School (St. Louis, Mo., USA), in conjunction with Celltech
(Slough, UK). The cell-depleting anti-CD4 monoclonal antibody (rat
IgG2b) consisted of a 1:1 mixture of YTS 191.1.2 and YTA 3.1.2,
provided by H. Waldmann (University of Cambridge, UK) (Galfre, G.
et al., Nature 277: 131-133 (1979); Cobbold, S. P. et al., Nature
312: 548-551 (1984); Qin, S. et al, European J. Immunology
17:1159-1165 (1987)).
[0133] Paw-Swelling
[0134] First, a sub-optimal dose of 50 .mu.g of anti-TNF antibody
alone was compared with the same dose given together with 200 .mu.g
of anti-CD4 antibody. To verify the results, two separate but
identical experiments were carried out (18-19 mice/group).
Paw-swelling was monitored for 10 days by measuring the thickness
of each affected hind paw with calipers. Neither anti-CD4 antibody
alone nor sub-optimal anti-TNF antibody alone were able to
significantly reduce paw-swelling (FIG. 1). However, treatment with
anti-TNF antibody and anti-CD4 antibody resulted in a consistently
and statistically significant reduction in paw-swelling relative to
the group given control rb (P<0.01). Furthermore, in both
experiments, combined anti-TNF/anti-CD4 antibody treatment (also
referred to herein as anti-CD4/TNF antibody treatment) produced a
significant reduction in paw-swelling relative to anti-CD4 antibody
alone (P<0.05), and anti-TNF antibody alone (P<0.05).
[0135] Next, an optimal dose of anti-TNF antibody (300 .mu.g) alone
was compared in two separate but identical experiments (11-13
mice/group) with the same dose given in combination with anti-CD4
antibody. As before, the combined anti-TNF/anti-CD4 antibody
treatment resulted in a significant reduction in paw-swelling
compared to treatment-with the control mAb (P<0.01; FIG. 2). In
addition, paw swelling was significantly reduced in the combined
anti-CD4/anti-TNF antibody treated group relative to the groups
administered anti-CD4 antibody alone (P<0.01) or anti-TNF
antibody alone (P<0.01). A reduction in paw swelling was also
observed in the mice administered anti-CD4 antibody alone and in
the mice administered anti-TNF antibody alone. The reduction in paw
swelling attributable to anti-TNF antibody treatment was broadly
comparable with previously published findings in which treatment
with TN3-19.12 (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., Proc. Natl. Acad. Sci. USA
89:9784-9788 (1992)).
[0136] Limb Involvement
[0137] In collagen-induced arthritis, as in RA, it is usual 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 antibody 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
arthritic limbs before treatment. In mice given the control mAb
there was an increase in limb involvement over the 10 day period of
approximately 50%. Results are shown in Table 1.
2TABLE 1 Combined anti-CD4/anti-TNF Antibody 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 (n = 18) 1.30 .+-. 0.10 1.90 .+-. 0.12 46.1 anti-TNF (n =
19) 1.20 .+-. 0.09 1.65 .+-. 0.17 37.5 anti-CD4/TNF 1.40 .+-. 0.17
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 (n =
12) 1.27 .+-. 0.10 1.80 .+-. 0.14 42.0 anti-TNF (n = 11) 1.50 .+-.
0.17 1.64 .+-. 0.20 9.5.sup.2 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 antibodies vs.
control mAb) .sup.2P < 0.05 (anti-TNF antibodies vs. control
mAb) .sup.3P < 0.005 (anti-CD4/TNF antibodies vs. control
mAb)
[0138] There was some reduction in new limb involvement in the
groups given anti-CD4 antibody alone and sub-optimal anti-TNF
antibody alone, although the differences were not significant. In
the group given optimal anti-TNF antibody, 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 antibodies. Thus, the
increase in new limb involvement was only 3% in mice given anti-CD4
antibody plus suboptimal anti-TNF antibody (P<0.05) and 0% in
mice given anti-CD4 antibody plus optimal anti-TNF antibody
(P<0.005).
[0139] Histology
[0140] 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 haematoxylon and eosin. A sagittal
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 antibody
alone or sub-optimal anti-TNF antibody alone. Results are shown in
Table 2.
3TABLE 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 4/18 (22%).sup.1 control mAb 17/18 (94%) Optimal
anti-TNF (300 .mu.g) anti-CD4 10/12 (83%) anti-TNF 6/11 (54%).sup.2
anti-CD4/TNF 4/13 (31%).sup.3 control mAb 12/12 (100%) .sup.1P <
0.01 (anti-CD4/TNF antibodies vs. anti-CD4 antibody alone; anti-TNF
antibody alone and control mAb) .sup.2P < 0.01 (anti-TNF
antibody alone vs. control mAb) .sup.3P < 0.01 (anti-CD4/TNF
antibodies vs. anti-CD4 antibody alone and control mAb)
[0141] An optimal dose of anti-TNF antibody alone significantly
reduced pathology, as reported previously (Williams, R. O. et al.,
Proc. Natl. Acad. Sci. USA 89: 9784-9788 (1992)). Thus, in the mice
given optimal anti-TNF antibody alone the proportion of joints
showing erosive changes was reduced to 54% (P<0.001) whereas in
the groups given anti-CD4 antibody plus either sub-optimal or
optimal anti-TNF antibody, only 22% (P<0.01) and 31% (P<0.01)
of thejoints, respectively, were eroded. Thus, 300 .mu.g of
anti-TNF antibody alone gave a degree of protection against joint
erosion but combined anti-CD4/anti-TNF antibodies provided
significantly greater protection.
[0142] Depletion of CD4+ T Cells
[0143] The extent to which anti-CD4 antibody treatment depleted
peripheral CD4+ T cells was determined by flow cytometry. To
enumerate the proportion of CD4+ lymphocytes in disassociated
spleen populations or peripheral blood, cells were incubated with
phycoerythrin-conjugated anti-CD4 (Becton Dickinson, Oxford, UK),
then analyzed by flow cytometry using a flow cytometer sold under
the trademark "FACScan" (Becton Dickinson) with scatter gates set
on the lymphocyte fraction. Anti-CD4 antibody treatment resulted in
98% (.+-.1%) depletion of CD4+ T cells in the spleen and 96%.
(.+-.3%) depletion of CD4+ T cells in the blood.
[0144] Immunohistochemistry
[0145] The possible persistence of CD4+ T cells in the joint
despite virtual elimination of peripheral CD4+ T cells was next
investigated by immunohistochemical analysis of sections from
treated arthritic mice. Wax-embedded sections were de-waxed,
trypsin digested, then incubated with anti-CD4 mAb (YTS 191.1.2/YTA
3.1.2). To confirm the T cell identity of the CD4+ T cells,
sequential sections were stained with anti-Thy-1 mAb (YTS 154.7)
(Cobbold, S. P. et al., Nature 312:548-551 (1984)). Control
sections were incubated with HRPN11/12a (an isotype control mAb; a
gift from Stephen Hobbs, Institute of Cancer Research, London).
Detection of bound antibody was by alkaline phosphatase/rat
anti-alkaline phosphatase complex (APAAP; Dako, High Wycombe, UK)
and fast red substrate as described (Deleuran, B. W. et al.,
Arthritis & Rheumatism 34:1125-1132(1991)). Small numbers of
CD4+ T cells were detected in the joints, not only of mice given
control mAb, but also of those treated with anti-CD4 antibody.
[0146] Furthermore, within the small number of mice that were
studied (four per treatment group), it was not possible to detect
significantly reduced numbers of CD4+ T cells in the groups given
anti-CD4 antibody alone or anti-CD4 antibody plus anti-TNF
antibody. Anti-CD4 antibody treatment did not, therefore, eliminate
CD4+ T cells from the joint.
[0147] Anti-collagen IgG Levels
[0148] Serum anti-collagen IgG levels were measured by
enzyme-linked immunosorbent assay (ELISA). Microtitre plates were
coated with bovine type II collagen (2 .mu.g/ml), blocked, then
incubated with test sera in serial dilution steps. Detection of
bound IgG was by incubation with alkaline phosphatase-conjugated
goat anti-mouse IgG, followed by substrate (dinitrophenol
phosphate). Optical densities were read at 405 nm. A reference
sample, consisting of affinity-purified mouse anti-type II collagen
antibody, was included on each plate. Results are shown in Table
3.
4TABLE 3 Serum Levels of Anti-type II collagen IgG Anti-collagen
IgG (Mean .+-. SEM) Treatment (.mu.g/ml) Sub-optimal anti-TNF (50
.mu.g) anti-CD4 (n = 18) 285 .+-. 37 anti-TNF (n = 19) 208 .+-. 29
anti-CD4/TNF (n = 18) 208 .+-. 34 control mAb (n = 18) 238 .+-. 36
Optimal anti-TNF (300 .mu.g) anti-CD4 (n = 12) 288 .+-. 39 anti-TNF
(n = 11) 315 .+-. 49 anti-CD4/TNF (n = 13) 203 .+-. 33 control mAb
(n = 12) 262 .+-. 47
[0149] Serum levels of anti-type II collagen IgG were not
significantly altered within the 10 day treatment period by
anti-CD4 antibody alone, anti-TNF antibody alone, or anti-CD4
antibody plus anti-TNF antibody.
[0150] Anti-globulin Response
[0151] To find out whether anti-CD4 antibody treatment prevented a
neutralizing anti-globulin response against the anti-TNF mAb, IgM
anti-TN3-19.12 levels on day 10, as measured by ELISA, were
compared. At this time, an IgG anti-TN3-19.12 response was not
detected. Microtitre plates were coated with TN3-19.12 (5 .mu.g/
ml), blocked, then incubated with serially diluted test sera. Bound
IgM was detected by goat anti-mouse IgM-alkaline phosphatase
conjugate, followed by substrate. The results demonstrated that
anti-CD4 antibody was highly effective in preventing the
development of an anti-TN3-19.12 antibody response (Table 4). Next,
to determine whether anti-CD4 antibody treatment led to increased
levels of circulating TNF.alpha. (by reducing the antibody response
to the hamster anti-TNF antibody), an ELISA was carried out in
which recombinant murine TNF.alpha. was used to detect free
TN3-19.12 in the sera of mice on day 10 of the experiment.
Microtitre plates were coated with recombinant murine TNF.alpha.
(Genentech Inc., South San Francisco, Calif.), blocked, then
incubated with test sera. Goat anti-hamster IgG-alkaline
phosphatase conjugate (adsorbed against murine IgG) was then
applied, followed by substrate. Quantitation was by reference to a
sample of known concentration of TN3-19.12. Results are shown in
Table 4.
5TABLE 4 IgM anti-TN3 Titres and Levels of Unbound TN3 Unbound TN3
Reciprocal of Anti- (Mean .+-. SEM) Treatment TN3 Titre (Mean)
(.mu.g/ml) Sub-optimal anti-TNF (50 .mu.g) anti-TNF (n = 12) 242
8.6 .+-. 2.0 anti-CD4/TNF (n = 12) 84.sup.1 12.1 .+-. 1.9 Optimal
anti-TNF (300 .mu.g) anti-TNF (n = 12) 528 90.7 .+-. 11.9
anti-CD4/TNF (n = 12) 91.sup.1 102.7 .+-. 12.5 .sup.1Significantly
reduced anti-TN3 titre (P < 0.005; Mann-Whitney test)
[0152] Levels of TN3-19.12 were slightly elevated in the groups
given anti-CD4 antibody plus anti-TNF antibody compared to anti-TNF
antibody alone, although the differences were not significantly
different.
Example 2
Treatment of Induced Arthritis in a Murine Model Using TNF
Receptor/IgG Fusion Protein With Anti-CD4 Antibody
[0153] 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 T 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.
[0154] A. Experimental Procedure
[0155] 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.
[0156] B. Comparison of Treatment With TNF Receptor/IgG Fusion
Protein, Anti-TNF Antibody, or Methylprednisolone Acetate
[0157] 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 was provided by Centocor, Inc., Malvern Pa.
(Butler et al., Cytokine 6:616-623 (1994); Scallon et al., Cytokine
7:759-770 (1995)); 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-TNF 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 trirneric 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 .mu.g/ml mouse TNF.alpha.. The
corticosteroid, methyl-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).
[0158] Paw-Swelling
[0159] 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, 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 i
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 observed in the mice
administered p55-sf2 at 100 .mu.g or anti-TNF mAb at 300 .mu.g.
[0160] Limb Involvement
[0161] The change in the number of arthritic limbs over the 10 day
treatment period was examined. Results are shown in Table 5.
6TABLE 5 Inhibitory Effect of TNF-Targeted Therapy on Limb
Recruitment Limbs Affected Treatment (number of (mean .+-. SEM)
Increase animals) Day 1 Day 10 (%) 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 0.15 .+-. 0.22 50% 100 .mu.g (n =
6) Anti-TNF mAb, 1.47 .+-. 0.15 1.76 .+-. 0.16.sup.1 20% 300 .mu.g
(n = 17) Methylprednisolone acetate 1.00 .+-. 0.00 1.50 .+-. 0.22
33% (n = 6) .sup.1P < 0.05 (vs. saline; Mann Whitney Test)
[0162] 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).
[0163] Histology
[0164] 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. Sagittal 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.
7TABLE 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%).sup.1 Control
IgG1, 100 .mu.g 6/6 (100%) Anti-TNF mAb, 300 .mu.g 7/17 (41%).sup.2
Methylprednisolone acetate 4/6 (67%) .sup.1P < 0.05 (vs.
saline). .sup.2P < 0.01 (vs. saline). Data were compared by
Chi-square analysis.
[0165] 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.
[0166] Anti-collagen Antibody Levels
[0167] Anti-collagen IgG levels on day 10 were measured by ELISA as
described (Williams, R. O. et al., Proc. Natl. Acad. Sci. USA 89:
9784-9788 (1992)). Microtitre plates were sensitized with type II
collagen, then incubated with serially-diluted test sera. Bound IgG
was detected using alkaline phosphatase-conjugated goat anti-mouse
IgG, followed by substrate (dinitrophenol phosphate). Optical
densities were read at 405 nm. No differences between any of the
treatment groups were detected, suggesting that the therapeutic
effect of p55-sf2 is not due to a generalized immunosuppressive
effect.
[0168] C. Effect of Treatment With p55-sf2 in Conjunction With
Anti-CD4 Antibody
[0169] In view of the high titres of antibodies to p55-sf2 that
were detected in mice treated with the fusion protein, an
experiment was carried out to determine whether concurrent
administration of anti-CD4 monoclonal antibody (mAb) could enhance
the therapeutic effects of p55-sf2. Using the Experimental
Procedure described above, a comparison was made of three different
treatment regimes: anti-CD4 mAb alone (200 .mu.g), p55-sf2 alone
(100 .mu.g) or anti-CD4 mAb (200 tg) plus p55-sf2 (100 .mu.g). A
fourth group consisted of untreated control mice. The
cell-depleting anti-CD4 mAb (rat IgG2b) consisted of a 1:1 mixture
of YTS 191.1.2 and YTA 3.1.2, provided by H. : Waldmann (University
of Cambridge, UK) (Galfre, G. et al., Nature 277: 131-133 (1979);
Cobbold, S. P. et al., Nature 312: 548-551 (1984); Qin, S. et al.,
European J. Immunology 17:1159-1165 (1987)). p55-sf2 is described
above.
[0170] Paw-swelling
[0171] Treatment with p55-sf2 alone resulted in a marked inhibition
of paw-swelling, but the synergistic inhibitory effect of anti-CD4
mAb in combination with p55-sf2 was remarkable. In contrast,
anti-CD4 mAb treatment alone had very little effect on
paw-swelling.
[0172] Limb Involvement
[0173] As before, the progressive involvement of additional limbs
following the initial appearance of arthritis was studied. Results
are shown in Table 7.
8TABLE 7 Anti-CD4 Antibody and p55-sf2 Prevent New Limb Recruitment
Limbs Affected Treatment (number of (mean .+-. SEM) Increase
animals) Day 1 Day 10 (%) Control (n = 6) 1.17 .+-. 0.17 2.00 .+-.
0.26 71% Anti-CD4 mAb 1.17 .+-. 0.17 1.83 .+-. 0.31 56% (n = 6)
p55-sf2 (n = 7) 1.43 .+-. 0.20 1.71 .+-. 0.18 19% Anti-CD4 mAb/
1.33 .+-. 0.21 1.33 .+-. 0.21.sup.1 0% p55-sf2 (n = 6) .sup.1P <
0.05 (vs. controls; Mann Whitney test).
[0174] There was a mean increase in limb involvement of 71% in the
control group, which was reduced to 56% in the group given anti-CD4
mAb alone, and only 19% in the group given p55-sf2. However, in the
group given anti-CD4 mAb plus p55-sf2, the increase in limb
involvement was 0%, a statistically significant difference.
[0175] Histology
[0176] Histological analysis of PIP joints of treated mice was
carried out as described above. Results are shown in Table 8.
9TABLE 8 Effects of Anti-CD4 mAb and p55-sf2 in the Prevention of
Joint Erosion Treatment PIP Joints with Erosions Control 6/6 (100%)
Anti-CD4 mAb 6/6 (100%) p55-sf2 2/6 (33%).sup.1 Anti-CD4 mAb plus
p55-sf2 1/6 (17%).sup.2 .sup.1P = 0.06 (vs. control) .sup.2P <
0.05 (vs. control) Data were compared by the Fisher exact test.
[0177] The control group and the group given anti-CD4 mAb alone
gave identical results, with {fraction (6/6)} (100%) of PIP joints
in both groups showing significant erosions. However, in the group
given p55-sf2 alone, only {fraction (2/6)} (33%) of PIP joints
showed erosions. Only 1/6 (17%) of joints showed erosions in the
group given anti-CD4 plus p55-sf2.
[0178] Antibody Responses to p55-sf2
[0179] The IgM/IgG responses to injected p55-sf2 were measured by
ELISA at the end of the treatment period (day 10). Microtitre
plates were coated with p55-sf2 (5 .mu.g/ml), blocked, then
incubated with serially diluted test sera. Negative controls
consisted of sera from saline-treated mice. Bound IgM or IgG were
detected by the appropriate goat anti-mouse Ig-alkaline phosphatase
conjugate, followed by substrate. Results are shown in Table 9.
10TABLE 9 Anti-p55-sf2 Responses and Levels of Free p55-sf2 in Sera
of Mice Treated with p55-sf2 Alone or in Combination with Anti-CD4
mAb Anti-p55-sf2 Response (titres) Treatment IgM IgG p55-sf2 Level
Experiment 1 saline 1:20 1:35 -- p55-sf2, 1:50 1:590 <0.2
.mu.g/ml 2 .mu.g p55-sf2, 1:232 1:3924 <0.2 .mu.g/ml 20 .mu.g
p55-sf2, 1:256 1:5280 <0.2 .mu.g/ml 100 .mu.g Experiment 2
p55-sf2, 1:336 1:5100 <0.2 .mu.g/ml 100 .mu.g p55-sf2, 1:15
1:200 12.3 .+-. 1.1 .mu.g/ml 100 .mu.g, plus anti- CD4 mAb
[0180] High titres of both IgM and IgG antibodies to p55-sf2 were
detected in treated mice, with the highest titres being found in
the mice given the 100 .mu.g dose. These results indicate that
p55-sf2, which is derived from human proteins, is highly
immunogenic in mice. This may account for the slightly greater
efficacy of anti-TNF mAb in vivo described in Section B above,
despite the higher neutralizing titre of the fusion protein in
vitro. Anti-CD4 nAb treatment was found to block almost completely
the formation of both IgM and IgG antibodies to p55-sf2.
[0181] Serum Levels of Free p55-sf2
[0182] Microtitre plates were coated with recombinant murine
TNF.alpha. (Genentech Inc., South San Francisco, Calif.), blocked,
then incubated with test sera. Goat anti-human IgG-alkaline
phosphatase conjugate was then applied followed by substrate.
Quantitation was by reference to a sample of known concentration of
p55-sf2.
[0183] The inhibition of the antibody response was associated with
pronounced differences in the circulating levels of p55-sf2 in
treated mice. Thus, free p55-sf2 was undetectable in mice given the
fusion protein alone, whereas in the mice given anti-CD4 mAb plus
p55-sf2, the mean serum level of p55-sf2 was 12.3 .mu.g/ml.
Example 3
Combined Therapeutic Effect of TNF Receptor/IgG Fusion Protein and
Anti-CD4 Antibody at Various Doses in the Treatment of Induced
Arthritis in a Murine Model
[0184] The murine model of collagen type I 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, for the ability to modulate the severity of
joint disease in collagen-induced arthritis. A comparison was made
between the efficacy of treatment with TNF receptor/IgG fusion
protein in combination with anti-CD4 antibody at various
dosages.
[0185] Experimental Procedure
[0186] 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 (6 mice per group) were
subjected to treatment with one of the following therapies: 100
.mu.g TNF receptor/IgG protein (p55-sf2; provided by Centocor,
Inc., Malvern Pa.), injected intra-peritoneally on day one; or 100
.mu.g TNF receptor/IgG protein, injected intra-peritoneally in
conjunction with either 6 .mu.g, 25 .mu.g, 100 .mu.g, or 400 .mu.g
anti-CD4 antibody (rat IgG2b) (1:1 mixture of YTS 191.1.2 and YTA
3.1.2; provided by H. Waldmann, University of Cambridge, UK),
injected intra-peritoneally, on day one. The TNF receptor/IgG
fusion protein, herein referred to as p55-s2, and the anti-CD4
antibody are described in Example 2. Arthritis was monitored for
paw-swelling (measured with calipers) for 10 days, after which the
mice were sacrificed and joints were processed for histology.
[0187] Clinical Score
[0188] 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.
[0189] The results are presented in FIG. 3 and show that TNF
receptor/IgG fusion protein, administered alone, reduced the
severity of arthritis. However, when anti-CD4 antibody was
administered in combination with TNF receptor/IgG fusion protein,
greater and prolonged protection was provided. The results of this
experiment also show that the duration of the synergistic
ameliorative effect (therapeutic effect) between TNF receptor/IgG
fusion protein and anti-CD4 antibody is dependent on the dosage of
anti-CD4 antibody administered (Williams et al., Immunology
84:433-439 (1995)).
Example 4
Combined Therapeutic Effect of Sub-optimal Doses of Cyclosporin A
and Anti-TNF Monoclonal Antibody in the Treatment of Induced
Arthritis in a Murine Model
[0190] The murine model of collagen type II induced arthritis,
described above, was used to investigate the efficacy of
co-administering a sub-optimal dose of the CD4+ T cell inhibiting
agent cyclosporin A and a sub-optimal dose of 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 a sub-optimal dose of
anti-TNF antibody alone, a sub-optimal dose of CsA alone, and a
combination of sub-optimal doses of CsA and anti-TNF antibody.
[0191] Experimental Procedure
[0192] 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 mice per group) were
subjected to treatment with one of the following therapies: 50
.mu.g (2 mg/kg) L2 (the isotype control for anti-TNF antibody),
injected intra-peritoneally once every three days (days 1, 4 and
7); 250 .mu.g (10 mg/kg) cyclosporin A (SANDIMMUNE.RTM., Sandoz
Pharmaceuticals, East Hanover, N.J.), injected intra-peritoneally
daily; 50 .mu.g (2 mg/kg) anti-TNF mAb TN3-19.12, injected
intra-peritoneally once every three days (days 1, 4 and 7); 250
.mu.g cyclosporin A, injected intra-peritoneally daily in
conjunction with 50 .mu.g anti-TNF mAb, injected intra-peritoneally
once every three days; or phosphate-buffered saline (PBS), injected
intra-peritoneally daily. The doses of CsA and anti-TNF mAb used in
this experiment had in previous studies been shown to be
sub-optimal, i.e., neither reagent alone had any significant effect
on the severity of 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.
[0193] Paw-swelling
[0194] Paw-swelling was monitored throughout the treatment period
by measuring the thickness of each affected hind paw with calipers.
The results are expressed in paw thickness (mm).
[0195] Treatment with a sub-optimal dose of cyclosporin A in
conjunction with a sub-optimal dose of 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.
4.
[0196] Clinical Score
[0197] 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.
[0198] The results are presented in FIG. 5 and confirm that at
sub-optimal doses neither CsA nor anti-TNF mAb, administered alone,
significantly ameliorated disease. However, when the two reagents
were given together, there was a highly significant reduction in
the severity of arthritis. P<0.05 and relates to differences
between the PBS treated group (Mann-Whitney U test). The results of
this experiment show that there is an additive or synergistic
ameliorative effect between CsA and anti-TNF antibody administered
at sub-optimal doses.
[0199] Limb Involvement
[0200] In collagen-induced arthritis, as in RA, it is usual 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
treatment with a sub-optimal dose of cyclosporin A in conjunction
with a sub-optimal dose of anti-TNF mAb 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
limbs with arthritis before treatment. Results are shown in Table
10.
11TABLE 10 Anti-CD4 Antibody and Cyclosporin A Prevent New Limb
Recruitment Limbs Affected (mean .+-. SEM) Increase Treatment Day 1
Day 10 (%) 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 1.54
.+-. 0.20.sup.1 21.0% .sup.1P = 0.03 (vs. control).
[0201] Treatment with a sub-optimal dose of cyclosporin A in
conjunction with a sub-optimal dose of 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 cyclosporin
A to prolong the therapeutic effect of a single injection of
anti-TNF antibody to modulate the severity of joint 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.
[0203] Experimental Procedure
[0204] 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) cyclosporin A (SANDIMMUNE .RTM., Sandoz
Pharmaceuticals, East Hanover, N.J.), injected intra-peritoneally
in conjunction with 300 .mu.g (12 mg/kg) L2 (the isotype control
for anti-TNF antibody), injected intra-peritoneally, on day one;
250 .mu.g cyclosporin A, 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.
[0205] Paw-swelling
[0206] Paw-swelling was monitored as described in Example 4.
Treatment with a sub-optimal dose of cyclosporin 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.
[0207] Histology
[0208] 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.
12TABLE 11 PIP Joint Erosions Treatment Incidence of Erosions
L2/CsA 8/10 (80%) TN3 alone 8/9 (89%) CsA/TN3 6/10 (60%)
[0209] 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
[0210] 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 alone, and
a combination of CsA and anti-TNF antibody, for the ability to
modulate the severity of joint disease in collagen-induced
arthritis.
[0211] Experimental Procedure
[0212] 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 cyclosporin 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.
[0213] Clinical Score
[0214] 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.
[0215] The results are presented in FIG. 7 and show that treatment
with 500 .mu.g cyclosporin 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 or 500
.mu.g cyclosporin A alone also reduced the severity of arthritis.
(P<0.05 and relates to differences between the PBS treated group
(Mann-Whitney U test)).
[0216] Histology
[0217] For histology, the mice were sacrificed after 10 days and
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 haematoxylon and
eosin. A sagittal 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 PIP 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.
13TABLE 13 Therapeutic Effects of Cyclosporin A and Anti-TNF
Monoclonal Antibody in Established Collagen-Induced Arthritis
Histology: proportion No. mice of PIP joints with Treatment per
group 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 anti-TNF mAb 11 1/11 (9%) (10 mg/kg) (P <
0.001)
[0218] P values refer to comparisons with the PBS-treated
group.
[0219] Treatment with cyclosporin 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
cyclosporin A and anti-TNF antibody.
Example 7
Treatment of Induced Arthritis in a Murine Model Using Rolipram and
Anti-CD4 Antibody
[0220] The murine model of collagen type II induced arthritis,
described above, was used to investigate the efficacy of the TNF
antagonist rolipram in conjunction with anti-TNF monoclonal
antibody or anti-CD4 monoclonal antibody, for the ability to
modulate the severity of joint disease in collagen-induced
arthritis. First, a comparison was made between the efficacy of
rolipram treatment and anti-TNF monoclonal antibody (mAb)
treatment. Second, therapy with rolipram in conjunction with
anti-TNF mAb was investigated. Third, therapy with rolipram in
conjunction with anti-CD4 mAb was investigated. Rolipram is a type
IV phosphodiesterase (PDE IV) inhibitor that has been reported to
suppress TNF.alpha. production via a cyclic 3',5'-adenosine
monophosphate (cAMP) dependent mechanism.
[0221] A. Experimental Procedure
[0222] 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), mice were injected
intra-peritoneally with therapeutic agents. Arthritis was monitored
for clinical score and paw swelling (measured with calipers) for 10
days. The therapeutic agents included rolipram, anti-TNF antibody,
and anti-CD4 antibody.
[0223] B. Comparison of Treatment With Rolipram or Anti-TNF
Antibody
[0224] Using the Experimental Procedure described above, three
groups of mice (6 mice per group) were subjected to treatment with
one of the following therapies: Cremophor ELS (control; Sigma),
injected intra-peritoneally twice daily; either 3 mg/kg body
weight, 5 mg/kg body weight or 10 mg/kg body weight of rolipram
(Schering AG; Berlin, Germany) dissolved in Cremophor EL.RTM.,
injected intra-peritoneally twice daily; or 300 .mu.g (12 mg/kg)
anti-TNF antibody TN3-19.12, injected intra-peritoneally once every
three days (days 1, 4 and 7).
[0225] Clinical Score
[0226] 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.
[0227] The results are presented in FIG. 8 and show that treatment
with rolipram resulted in a significant reduction in the severity
of arthritis over the treatment period, relative to mice treated
with Cremophor EL.RTM.. For example, the mean clinical score on day
10 (.+-.SE) was 1.0.+-.0.3 for mice treated with 3 mg/kg rolipram
(P<0.05 (vs. control)), 2.0.+-.0.4 for mice treated with
anti-TNF mAb, and 3.7.+-.0.7 for mice treated with Cremophor
EL.RTM. (control arthritic mice), indicating that the magnitude of
the therapeutic effect of rolipram treatment was comparable to, or
greater than anti-TNF antibody treatment. Preliminary
immunohistochemical studies designed to elucidate the mechanism by
which rolipram ameliorates disease in this murine model of collagen
type II induced arthritis suggest that rolipram treatment results
in down-regulation of TNF expression in the joints of mice with
collagen-induced arthritis. These findings indicate that rolipram
is effective in established collagen-induced arthritis and may
therefore be useful in the treatment of RA.
[0228] C. Effect of Treatment With Rolipram in Conjunction With
Anti-TNF Antibody
[0229] Using the Experimental Procedure described above, 10 mice
were subjected to treatment with one of the following therapies:
Cremophor EL.RTM. (control), injected intra-peritoneally twice
daily; rolipram (either 0.5 mg/kg body weight, 3 mg/kg body weight
or 5 mg/kg body weight) dissolved in Cremophor EL.RTM., injected
intra-peritoneally twice daily; 300 .mu.g (12 mg/kg) anti-TNF
antibody TN3-19.12, injected intra-peritoneally once every three
days (days 1, 4 and 7); or 300 .mu.g anti-TNF antibody TN3-19.12,
injected intra-peritoneally once every three days, in conjunction
with rolipram (5 mg/kg body weight) dissolved in Cremophor EL.RTM.,
injected intra-peritoneally twice daily.
[0230] Clinical Score
[0231] 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.
[0232] The results are presented in FIG. 9 and show that
co-administration of, for example, two different TNF antagonists
provides a more complete therapeutic effect than administration of
either antagonist alone in modulating the severity of joint disease
in collagen-induced arthritis. This suggests that there is an
additive or synergistic ameliorative effect between different TNF
antagonists.
[0233] D. Effect of Treatment With Rolipram in Conjunction With
Anti-CD4 Antibody
[0234] Using the Experimental Procedure described above, 9 mice
were subjected to treatment with one of the following therapies:
Cremophor EL.RTM., injected intra-peritoneally twice daily;
rolipram (5 mg/kg body weight) dissolved in Cremophor EL.RTM.,
injected intra-peritoneally twice daily; 50 .mu.g anti-CD4 antibody
(rat IgG2b) (1:1 mixture of YTS 191.1.2 and YTA 3.1.2), injected
intra-peritoneally once every three days (days 1, 4 and 7); or 50
.mu.g anti-CD4 antibody, injected intra-peritoneally once every
three days, in conjunction with rolipram (5 mg/kg body weight)
dissolved in Cremophor EL.RTM., injected intra-peritoneally twice
daily.
[0235] Clinical Score
[0236] 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.
[0237] The results are presented in FIG. 10A and show that when
rolipram and anti-CD4 antibody were co-administered, there was a
highly significant reduction in the severity of arthritis over the
treatment period. The results of this experiment show that there is
an additive or synergistic ameliorative effect between rolipram and
anti-CD4 antibody.
[0238] Paw-swelling
[0239] Paw-swelling was monitored throughout the treatment period
by measuring the thickness of each affected hind paw with calipers.
The results are expressed as paw thickness (mm).
[0240] Treatment with rolipram in conjunction with anti-CD4 mAb
resulted in a significant reduction in paw-swelling over the
treatment period, relative to mice treated with rolipram alone or
mice treated with anti-CD4 mAb alone. Results are shown in FIG.
10B. The results of this experiment show that there is an additive
or synergistic ameliorative effect between rolipram and anti-CD4
antibody.
[0241] Equivalents
[0242] While this invention has been particularly shown and
described with references to preferred embodiments thereof it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
Sequence CWU 1
1
2 1 22 PRT Homo sapiens 1 Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly
Cys Pro Ser Thr His Val 1 5 10 15 Leu Leu Thr His Thr Ile 20 2 22
PRT Homo sapiens 2 Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys
Ser Pro Cys Gln 1 5 10 15 Arg Glu Thr Pro Glu Gly 20
* * * * *