Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TRAIL: An Inhibitor of Autoimmune Inflammation and Cell Cycle Progression
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application 60/157,222,
filed
September 30, 1999.
GOVERNMENT SUPPORT
This work was supported in part by grants from the National Institutes of
Health,
grant numbers AR44914, NS40188, NS36581 and NS40447.
to
FIELD OF THE INVENTION
The present invention is related to the genetic regulation of cellular
apoptosis in
nontransformed tissues, specifically the effect of Tumor necrosis factor (TNF)-
Related
Apoptosis-Inducing Ligand (TRAIL).
BACKGROUND OF THE INVENTION
In view of the potent effects of cellular apoptosis on growth, development,
and
homeostasis, it is not surprising to find that the initiation of apoptosis is
tightly regulated,
especially in normal cells.
2o TRAIL, the Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand,
is
a type II membrane protein of the TNF superfamily (Wiley et al, Immunity
3(6):673-682
(1995)). Some members of the TNF family are capable of inducing apoptosis of
normal
and/or tumor cells. Both TNF and CD95L have been shown to mediate activation-
induced cell death (AICD) of lymphocytes (Ju et al., Nature 373:444-448
(1995); Dhein
et al., Nature 373:438-441 (1995); Brunner et al., Nature 373:441-444 (1995)).
However, unlike other members of the TNF superfamily that interact with one or
two
specific receptors, TRAIL can potentially interact with five different
receptors. These
include Death Receptor 4 (DR4, TRAIL-Rl), Death Receptor 5 (DRS, TRAIL-R2),
Decoy Receptor 1 (DcRI, TRAIL-R3, TRID), Decoy Receptor 2 (DcR2, TRAIL-R4,
3o TRUNDD) (Pan et al., Science 277(5327):815-818 (1997); Pan et al.,
FEBSLett. 424(1
2):41-45 (1998); Schneider et al., FEBSLett. 416(3):329-334 (1997); Sheikh et
al.,
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Cancer Research 58(8):1593-1598 (1998); Sheridan et al., Science 277(5327):818-
821
(1997); Walczak et al., EMBO J. 16(17):5386-5397 (1997); Screaton et al.,
Curr. Biol.
7(9):693-696 (1997)), and a soluble receptor called osteoprotegerin (Emery et
al.,
J.Biol.Chem. 273(23): 14363-14367 (1998)).
While the presence of multiple TRAIL receptors strongly suggests that TRAIL
could be involved in multiple processes, the precise roles of TRAIL in health
and disease
have, to date, been unknown. In vitro studies have shown that TRAIL induces
apoptosis
of some, but not all, tumor cell lines (Pan et al., 1997; Sheridan et al.,
1997). This
appears to be mediated by the death receptors, DR4 and DRS, which are capable
of
to activating the caspase cascade.
The presence of DcRl and DcR2 which possess similar intracellular death
domains as TNF receptors and CD95 (Fas/Apo-1), and are capable of activating
the
caspase cascade. The presence of decoy receptors, DcRl and DcR2, which do not
contain functional death domains, blocks TRAIL-induced apoptosis (Pan et al.,
1997;
Sheridan et al., 1997).
Although both TRAIL and TRAIL receptors are constitutively expressed in
various tissues and in a variety of cell types, including lymphocytes, natural
killer cells,
and neural cells (Whey et al., 1995; Pan et al., 1997; Schneider et al, 1997;
Rieger et al.,
FEBSLett. 427:124 (1998); Kayagaki et al., J. Immunol. 163:1906 1999); Bretz
et al., J.
2o D., J. Biol. Chem. 274:23627 (1999); Frank et al., Biochem. Biophys.
Research Comm.
257:454 (1999); Sedger et al., J. Immunol. 163:920 (1999); Wu et al., Cancer
Research
59(12):2770-2775 (1999)), and are upregulated upon cell activation (Sheikh et
al., 1998;
Mariani et al., European J. Immunol. 28(5):1492-1498 (1998); Jeremias et al.,
European
.I. Immunol. 28(1):143-152 (1998)), TRAIL may not induce apoptosis of most non-
transformed cells (Pan et al., 1997; Sheridan et al., 1997). In vivo
administration of
recombinant TRAIL selectively kills tumor cells, but not normal cells, leaving
the host
organ systems unharmed (Walczak et al., Nature Med. 5(2):157-163 (1999);
Ashkenazi et
al., J. Clin. Invest. 104(2):155-162 (1999)).
While this fording has generated tremendous interest in using recombinant
TRAIL
3o for cancer therapy, it has also raised fundamental questions regarding the
roles of TRAIL
in normal non-transformed tissues. Thus, there has been a need in the art to
understand
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TRAIL as a member of the TNF family, and as a potent inhibitor of autoimmune
inflammation and cell cycle progression.
SUMMARY OF THE INVENTION
TRAIL, the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, induces
apoptosis of tumor cells, but not normal cells; yet its role in normal non-
transformed
tissues was, until the present invention, unknown. The present invention
demonstrates
that chronic blockade of TRAIL in mice exacerbates autoimmune rheumatoid
arthritis in
a representative animal model, while intra-articular TRAIL gene transfer
ameliorates the
to disease. In vivo, TRAIL-blockade led to profound hyper-proliferation of
synovial cells
and arthritogenic lymphocytes, and heightened the production of cytokines and
autoantibodies. In vitro, TRAIL inhibited DNA synthesis and prevented cell
cycle
progression of lymphocytes.
However, TRAIL had no effect on apoptosis of inflammatory cells either in vivo
or in vitro. Thus, unlike Fas ligand or other members of the tumor necrosis
factor
superfamily, TRAIL is a prototype inhibitor protein that inhibits autoimmune
inflammation by blocking cell cycle progression of T cells and inhibited their
differentiation into effector cells, but it does not mediate activation-
induced cell death of
T lymphocytes.
2o The present invention also, for the first time, demonstrates the
consequences of
TRAIL-blockade in an animal model of multiple sclerosis. Indeed, confirming
the effect
in autoimmune diseases, as shown in arthritis, chronic TRAIL-blockade in mice
exacerbated experimental autoimmune encephalomyelitis (EAE) induced by myelin
oligodendrocyte glycoprotein (MOG). The exacerbation was evidenced primarily
by
increases in disease score and degree of inflammation in the central nervous
system
(CNS). Interestingly, the degree of apoptosis of inflammatory cells in the CNS
was not
affected by TRAIL-blockade, suggesting that TRAIL may not regulate apoptosis
of
inflammatory cells in EAE.
By contrast, MOG-specific TH1 and TH2 cell responses were significantly
3o enhanced in animals treated with the soluble TRAIL receptor. Thus, unlike
TNF, which
promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis
and
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prevents activation of autoreactive T cells. Accordingly, unlike many other
members of
the TNF family that promote autoimmune encephalomyelitis, TRAIL inhibits EAE
and
prevents activation of encephalitogenic T cells.
The present invention provides a method for achieving normal levels of
cellular
apoptosis in non-tranformed cells of a patient by administering to the patient
a
therapeutically effective amount of purified TRAIL ligand or active fragment
thereof. In
particular, the present invention provides such method to a patient suffering
from an
autoimmune disease or condition.
The method is also provided in which normal levels of cellular apoptosis is
1o achieved in non-tranformed cells of a patient by administering to the
patient a
therapeutically effective amount of an isolated nucleic acid sequence encoding
TRAIL or
active fragment thereof.
The present invention further provides a method of blocking the activity of an
endogenous TRAIL receptor or inhibitor in a patient by administering to the
patient a
therapeutically effective amount of a purified TRAIL agonist, in an amount
sufficient to
enhance the patient's level of TRAIL ligand. In particular, the present
invention provides
such method to enhance ameliorate or restore cellular apoptosis in non-
tranformed cells
of the patient, by administering to the patient a therapeutically effective
amount of an
isolated nucleic acid sequence encoding said TRAIL agonist, in an amount
sufficient to
enhance the patient's level of TRAIL ligand.
The method is also provided in which normal levels of cellular apoptosis is
achieved in non-tranformed cells of a patient by administering to the patient
a
therapeutically effective amount of an isolated nucleic acid sequence encoding
a TRAIL
agonist. Such TRAIL agonist is a receptor or inhibitor of a TRAIL receptor or
inhibitor,
wherein the agonist is selected from the group consisting of antibodies to
TRAIL
receptors or inhibitors, antisense molecules complimentary to TRAIL receptors
or
inhibitors, and any molecule which binds to or blocks TRAIL receptors or
inhibitors.
It is an object of the foregoing methods to prevent, inhibit or decrease
inflammation, tissue damage or injury related to an autoimmune disease or
condition in a
patient. Such autoimmune diseases or conditions include arthritis, autoimmune
encephalomyelitis, insulin-dependent diabetes mellitus, hemolytic anemias,
rheumatic
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fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis,
autoimmune
hepatitis, multiple sclerosis, systemic lupus erythematosus, ankylosing
spondylitis, and
others. The methods are also useful for the treatment of autoimmune disease or
conditions resulting from allogeneic tissue or organ transplant or graft-
versus-host
disease. In particular, the present methods are useful for the treatment or
prevention of
symptoms associated with arthritis, including induced, rheumatoid or chronic
arthritis, or
autoimmune encephalomyelitis or multiple sclerosis. The affected non-
transformed cells
include, but are not limited to, lymphocytes, including autoreactive
lymphocytes,
cytokines and synovial cells.
Additional objects, advantages and novel features of the invention will be set
forth in part in the description, examples and figures which follow, and in
part will
become apparent to those skilled in the art on examination of the following,
or may be
learned by practice of the invention.
DESCRIPTION OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the
invention, will be better understood when read in conjunction with the
appended
drawings. For the purpose of illustrating the invention, there are shown in
the drawings,
to certain embodiments) which are presently preferred. It should be
understood, however,
that the invention is not limited to the precise arrangements and
instrumentalities shown.
Figures lA-1D are histograms depicting the blocking effect of recombinant sDRS
on TRAIL-induced apoptosis of Jurkat T leukemia tumor cells and K562 B cells.
The
Jurkat T leukemia cells were treated with the following: 5 ~g/ml of BSA
(Figure 1A),
1s 100 ng/ml of TRAIL (Figure 1B), 5 ~,g/ml of sDRS (Figure 1C), or 100 ng/ml
of TRAIL
plus S ~g/ml of sDRS (Figure 1D). Each histogram represents 10,000 events,
with the
apoptotic cells gated. Figure 1E graphically depicts dose-dependent killing of
B
lymphoma cells by TRAIL, with (open circle) or without (filled square) 5 ~g/ml
of sDRS.
Figures 2A and 2B graphically depict the effect of TRAIL-blockade in vivo in a
2o mouse model of rheumatoid arthritis. Figure 2A depicts the disease course
in mice
treated with BSA (open square) or sDRS (filled circle). Each data point
represents a
mean ~ standard deviation (SD) from a total of 5 mice (for sDRS-treated group)
or 6 mice
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(for BSA-treated group). Figure 2B illustrates the dose-dependent effect of
sDRS on
arthritis as judged by disease scores of individual feet taken 12 days after
the second
immunization. Four groups of mice are shown: one was treated with 100 ~g BSA,
while
the other three were treated with 50-300 ~g sDRS. Each data point represents
an
individual foot, with 16 to 24 feet per group. P<0.05 for mice treated with SO
~g sDRS
and p<0.01 for mice treated with 100 or 300 ~g sDRS.
Figure 3 graphically depicts the ability various concentrations of recombinant
TRAIL virus on arthritis following infra-articular injection, and the ability
of sDRS to
neutralize the arthritis-ameliorating effect as measured by the return of the
disease.
1o Figures 4A-4H depict the histochemical profiles of arthritic joints of mice
treated
as in Figure 2. Figure 4A depicts ankle joint of BSA-treated mouse with a
pathology
score of 2 (HE staining, original magnification x 20). Arrow indicates signs
of synovitis.
Figure 4B depicts ankle joint of sDRS-treated mouse with a pathology score of
4 (HE
staining, original magnification x20). Arrows indicate severe synovitis,
hyperplasia,
cartilage and bone destruction. Figure 4C depicts ankle joint of BSA-treated
mouse with
a pathology score of 1 (HE staining, original magnification x100). Arrow
indicates signs
of synovitis. Figure 4D depicts ankle joint of sDRS-treated mouse with a
pathology score
of 4 (HE staining, original magnification x100). Arrows indicate severe
synovitis,
hyperplasia, cartilage and bone destruction. Figure 4E depicts ankle joint of
BSA-treated
2o mouse with a disease score of 2 (BrdU staining, original magnification
x400). Arrows
indicate BrdU+ nuclei. Figure 4F depicts ankle joint of sDRS-treated mouse
with a
disease score of 3 (BrdU staining, original magnification x 400). Figure 4G
depicts ankle
joint of BSA-treated mouse with a pathology score of 4 (apoptotic staining,
original
magnification x200). Arrows indicate apoptotic cells. Figure 4H depicts ankle
joint of
sDRS-treated mouse with a pathology score of 4 (apoptotic staining, original
magnification x 200).
Figures 5A and SB graphically depict the quantitative analysis corresponding
to
the histochemical data displayed in Figure 4, comparing an sDRS-treated group
of mice
with a BSA-treated group. Figure 4A presents the pathology scores revealed
significant
3o differences between the two groups, as determined by ANOVA (p<0.01). Figure
4B
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presents the apoptotic index, wherein the differences between the two groups
are not
statistically significant, as determined by ANOVA (p = 0.21).
Figures 6A-6E graphically depict the effect of TRAIL-blockade on anti-collagen
immune responses in mice treated as in Figure 2A. Figure 6A depicts lymphocyte
proliferative responses as determined by 3H-thymidine incorporation. Figure 6B
depicts
IL-2 production; Figure 6C depicts IFN-y production. Each data point
represents a mean
~ SD from 5 (for sDRS-treated group) or 6 (for BSA group) mice. Radioactivity
is
presented as counts per minute (CPM). To test humoral immune responses, anti-
collagen
IgG2a antibodies (Figure 6D) and IgGI antibodies (Figure 6E) were determined
by
to ELISA using chicken type II collagen as antigen. The experiments were
repeated three
times with similar results. Open bar = mice treated with BSA; filled bar =
mice treated
with sDRS.
Figures 7A-7D graphically depict inhibition of DNA synthesis and cell cycle
progression by TRAIL on splenocytes prepared from BALB/c mice. Purified live
cells
were cultured with or without the following reagents: 1) 100 ng/ml of TRAIL;
2) 5 ~g/ml
of sDRS; 3) 5 ~g/ml of anti-CD95L mAb (MFL-3), and 4) anti-mouse CD3 mAb.
Figure
7A shows the percentage of apoptotic cells as determined by flow cytometry.
The
differences between anti-CD95L mAb treated culture and all other cultures are
statistically significant as determined by ANOVA (p<0.0001). Figure 7B depicts
the
2o number of S-G2/M cells/well, as determined by flow cytometry. The total
numbers of
live cells/well recovered from each group were as follows: cultures with anti-
CD3 mAb
alone, 0.3 x 105; cultures with anti-CD3 mAb + sDRS, 1.6 x 105; cultures with
anti-CD3
mAb + anti-CD95L mAb, 1.1 x 105; cultures with anti-CD3 mAb + sDRS + TRAIL,
0.6
x 105. The differences between all four groups are statistically significant
as determined
by ANOVA (p<0.01 ). Figures 7C and 7D depict DNA synthesis as determined by 3H-
thymidine incorporation. For cultures containing anti-CD3 mAb, the differences
between
the two groups are statistically significant as determined by ANOVA (p<0.01).
Figure 8 graphically depicts the blocked TRAIL-induced apoptosis of mouse
L929 cells by recombinant sDRS. L929 cells were treated with different
concentrations
of recombinant TRAIL with or without sDRS.
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Figures 9A and 9B graphically depict exacerbation of EAE by TRAIL-blockade.
In Figure 9A, the sDRS injections were performed 7 days after disease onset
until the end
of the experiment; whereas in Figure 9B, the sDRS injections were performed
from the
day of immunization to the day of disease onset (17 days after immunization).
Only mice
that developed EAE were included for calculating mean clinical scores. The
differences
between the two groups are statistically significant as determined by Mann-
Whitney test
(P<0.01 ) for panel A, but not for B.
Figures l0A-lOD depict inflammation and apoptosis in the spinal cord. Mice
were treated as in Figure 9A and sacrificed 42 days after immunization. Spinal
cord was
to treated and examined for histology and apoptosis. Figures 10A and lOB
depict Luxol
fast blue staining of spinal cords from sDRS (Figure 10A) and BSA (Figure 10B)
treated
mice (original magnification x 100). Figures lOC and lOD depict TUNEL staining
of
spinal cords from sDRS (Figure 10C) and BSA (Figure 10D) treated mice
(original
magnification x 200). Arrows indicates apoptotic nuclei.
Figure 11 graphically depicts quantitative analysis of the degree of
inflammation
in the CNS. Spinal cord was treated and examined for histology. Each data
point
represents a percentage of spinal cord section that is inflamed. The
horizontal bars
represent the means of respective groups. The differences between the two
groups are
statistically significant as determined by Student t test (p<0.05). Filled
squares = mice
2o treated with HSA. Open circles = mice treated with sDRS.
Figure 12 graphically depicts quantitative analysis of the degree of apoptosis
in
the CNS. Spinal cord was treated and examined for apoptosis. The numbers of
apoptotic
nuclei per mm2 of inflamed tissue were counted and plotted against the
percentages of the
corresponding spinal cord sections that were inflamed. Filled squares = mice
treated with
HSA. Open circles = mice treated with sDRS.
Figure 13 depicts MOG-specific proliferation and cytokine production in vitro.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The TNF family of proteins play crucial roles in a number of biological
processes
3o including apoptosis, immunity, inflammation and development. TRAIL is a
newly
identified member of the TNF family. Although it has been established that
TRAIL,
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unlike TNF, induces apoptosis of tumor cells, but not normal cells, the roles
of TRAIL in
health and disease have, to date, been virtually unknown. However, both TRAIL
and
TRAIL receptors are expressed in normal non-transformed tissues.
As is known to those of skill in the art, apoptosis is an active process of
gene-
directed cellular self destruction. The present invention provides
compositions and
methods for controlling or enhancing normal levels of apoptosis in a suitable
cell or a
population of suitable cells by introducing into the cell or cells an
effective amount of a
nucleic acid molecule coding for a gene product having TRAIL biological
activity, or for
inhibiting or reducing TRAIL receptor or inhibitor activity. The method also
may be
1o practiced using the gene product itself. Accordingly, this method provides
an
improvement over prior art methods, wherein apoptosis can be controlled or
enhanced by
affecting the induction pathway at the level of ligand induction, such as by
blocking or
inhibiting TRAIL receptors or inhibitors, including antibodies or anti-ligand
antibodies
that interfere with the binding of the ligand to its cell surface receptor.
Moreover, this
invention can be combined with the use of prior art methods, known to affect
apoptosis.
The present invention shows that TRAIL is a potent inhibitor of autoimmune
conditions, such as arthritis or multiple sclerosis or autoimmune inflammation
in the
CNS. Blocking endogenous TRAIL with sDRS eliminates this inhibition, and
exacerbates autoimmune arthritis or encephalomyelitis. Furthermore, TRAIL
apparently
2o inhibits activation of autoreactive T cells that initiate autoimmune
inflammation. The
inhibitory effect of TRAIL on arthritis appears to result from inhibition of
cell cycle
progression and/or cytokine production. Blocking endogenous TRAIL with sDRS
eliminates this inhibition, and enhances proliferation of autoreactive
lymphocytes (as
shown in Figure 6A) or synovial cells (as shown in Figure 4). This may in turn
contribute to the exacerbation of arthritic inflammation and joint tissue
destruction.
Thus, one of the functions of TRAIL in vivo is to maintain immune homeostasis
and to down-regulate immune responses including autoimmune responses. This
discovery of the present invention is a startling contrast to TNF, which
initiates and
exacerbates autoimmune diseases. In fact, anti-TNF therapy is effective in
preventing
3o arthritic inflammation both in humans and animals (Maim et al., Immunol.
Rev. 144:195-
223 (1995); Joosten et al., Arthritis & Rheumatism 39(5):797-809 (1996);
Williams et al.,
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Proc. Natl. Acad. Sci. USA 89(20):9784-9788 (1992); Eliaz et al., Cytokine
8(6):482-487
(1996)). Other members of the TNF family that have been reported to inhibit
autoimmune inflammation are CD95 ligand (CD95L, Fas) and CD30 ligand (CD30L).
The findings reported in the present invention indicate that the role of TRAIL
in
autoimmunity may be more closely related to that of CD95L or CD30 ligand
(CD30L).
Mutations in CD95/CD95L genes lead to the development of systemic autoimmune
diseases in both humans (Bettinardi et al., Blood 89(3):902-909 (1997);
Sneller et al.,
Blood 89(4):1341-1348 (1997)) and mice (Nagata et al., Science 267:1449-1456
(1995);
Cohen et al., Immunol. Today 13(11):427-428 (1992); Nagata et al., Immunol.
Today
l0 16(1):39-43 (1995); Mountz et al., Int'l. Rev. Immunol. 11(4):321-342
(1994)), although
paradoxically they also prevent several organ-specific autoimmune diseases
(Waldner et
al., J. Immunol.159:3100-3103 (1997); Sabelko et al., J. Immunol. 159:3096-
3099
(1997); Giordano et al., Science 275(5302):960-963 (1997); Chervonsky et al.,
Cell
89(1):17-24 (1997); Kang et al., Nature Medicine 3(7):738-743 (1997); Muruve
et al.,
Human Gene Therapy 8(8):955-963 (1997); Itoh et al., J. Exp. Med. 186(4):613-
618
(1997)).
Up-regulating CD95 or CD95L function in synovial joints has been reported to
ameliorate autoimmune arthritis (Zhang et al, 1997; Fujisawa et al., J. Clin.
Invest.
98(2):271-278 (1996)). Similarly, CD30L may also play an anti-inflammatory
role in
autoimmune diseases (Kurts et al., Nature 398(6725):341-344 (1999)).
Autoreactive
CD8+ T cells deficient in CD30L elicits more severe autoimmune insulitis in
mice (Kurts
et al., 1999). Thus, unlike TNF, but similar to CD95L and CD30L, TRAIL appears
to be
a member of an inhibitor protein subfamily that prevents autoimmune diseases
by
downregulating immune responses.
However, the present findings indicate that the mechanism of TRAIL action in
vivo is different from that of CD95L. While CD95L induces apoptosis of
activated T
cells, TRAIL appears to inhibit their proliferation without eliminating them
through
apoptosis. This finding is consistent with recent reports that, unlike CD95L,
TRAIL
induces apoptosis of tumor cells but not normal cells (Pan et al., 1997;
Sheridan et al.,
1997). Systemic administration of recombinant TRAIL, but not CD95L,
selectively kills
tumor cells while sparing normal host cells Walczak et al., 1999; Ashkenazi et
al., 1999).
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Thus, the observation in the present invention that TRAIL can inhibit DNA
synthesis
provides direct evidence that TRAIL can prevent Gl-to-S phase progression of
lymphocytes. Therefore, unlike TNF or CD95L, TRAIL inhibits activation and
expansion of lymphocytes in vivo, but does not delete them from the system.
The present invention further demonstrates that autoreactive T cell activation
was
enhanced in mice treated with sDRS, suggesting that TRAIL may inhibit
functions of
autoreactive T cells. Since EAE is a T cell mediated autoimmune disease,
inhibiting T
cell function may diminish the disease. However, it should also be pointed out
that
enhancing T cell function alone during the inductive phase of EAE may not be
sufficient
l0 to exacerbate the disease, since treating mice with sDRS before the onset
of the disease
failed to significantly affect EAE (Figure 9B). A similar effect of TRAIL was
seen in
another model of autoimmunity, i.e., collagen-induced arthritis in DBA/1 mice
(Song et
al., 2000). As shown in the examples that follow, TRAIL-blockade during the
effector
phase of the disease enhanced, for example, arthritic inflammation.
15 In an alternative mechanism, TRAIL may inhibit autoimmune inflammation by
inducing apoptosis of inflammatory cells. Although it has been shown that
TRAIL does
not induce apoptosis of most non-transformed cell, there is evidence to
suggest that
dendritic cells and some T cells may be susceptible to TRAIL-induced apoptosis
in vitro
(Jeremias et al., European J. Immunol. 28:143 (1998); Wang et al., Cell 98:47
(1999)).
20 The demonstration in the present invention that the degrees of apoptosis in
the CNS and
arthritic joints were not affected by TRAIL-blockade suggests that TRAIL may
not
regulate apoptosis of inflammatory cells in these systems.
Thus, by in vivo TRAIL-blockade, it is clearly established in the present
invention
that, unlike TNF, TRAIL inhibits autoimmune encephalomyelitis and prevents
activation
25 of autoreactive T cells. Since EAE is an animal model for human multiple
sclerosis and
since TRAIL and its receptors are also expressed by human cells, the present
results are
important, not only for understanding the pathogenesis of EAE, but also for
designing
therapeutic strategies for the treatment of autoimmune diseases such as
multiple sclerosis.
It has been reported that some of the TRAIL receptors can activate both
caspase
30 (through FADD/TRADD) and NF-kappaB pathways in tumor cells (Schneider et
al.,
Immunity 7(6):831-836 (1997); Degli-Esposti et al., Immunity 7(6):813-820
(1997);
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Chaudhary et al., Immunity 7(6):821-830 (1997)). It is, therefore, necessary
to determine
whether this also occurs in normal cells, and if so, whether this is
responsible for the cell
cycle arresting effect reported in the present invention. It is to be noted
that NF-kappaB
activation occurs in collagen-induced arthritis, and that inhibition of NF-
kappaB
activation in T cells prevents the disease (Seetharaman et al., J. Immunol.
163(3):1577-
1583 (1999)). On the other hand, various signal transduction pathways of TRAIL
receptors are yet to be defined. Novel unidentified pathways may be
responsible for the
inhibitory effect of TRAIL in inflammation and cell cycle progression.
In sum, the regulation of programmed cell death is vital for normal
functioning of
the immune system. For example, T cells that recognize self antigens are
destroyed
through the apoptotic process during maturation of T-cells in the thymus,
whereas other T
cells are positively selected. Insufficient apoptosis has been implicated in
certain
conditions, while elevated levels of apoptotic cell death have been associated
with other
diseases. The desirability of identifying and using agents that regulate
apoptosis in
treating such disorders is recognized (Kromer, Adv. Immunol., 58:211 (1995).
Abnormal resistance of T cells toward apoptosis has been linked to
lymphocytosis, lymphadenopathy, splenomegaly, accumulation of self reactive T
cells,
autoimmune disease, development of leukemia, and development of lymphoma
(Kromer,
1995). Conversely, excessive apoptosis of T cells has been suggested to play a
role in
lymphopenia, systemic immunodeficiency, and specific immunodeficiency, with
specific
examples including
virus-induced immunodeficient states associated with infectious mononucleosis
and
cytomegalovirus infection, and tumor-mediated immunosuppression (Kromer,
1995).
Since TRAIL binds and kills leukemia cells (the Jurkat cell line), TRAIL also
may be useful in treating leukemia. A therapeutic method involves contacting
leukemia
cells with an effective amount of TRAIL. In one embodiment, a leukemia
patient's blood
is contacted ex vivo with an TRAIL polypeptide. The TRAIL may be immobilized
on a
suitable matrix. TRAIL binds the leukemia cells, thus removing them from the
patient's
blood before the blood is returned into the patient. Acted with an amount of
TRAIL
effective in inducing death of leukemia cells in the bone marrow. Following
use of
TRAIL to purge leukemia cells, the thus-treated marrow is returned to the
patient.
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TRAIL also binds to, and induces apoptosis of, lymphoma and melanoma cells
(US Patent No. 5,763223, hence therapeutic approaches using TRAIL may be
applicable
to lymphoma and melanoma cells. TRAIL polypeptides may be employed in treating
cancer, including, but not limited to, leukemia, lymphoma, and melanoma.
In the alternative, rather than utilizing an effective amount of TRAIL,
endogenous
levels of TRAIL may be restored in the patient, presumably therapeutically
effective
levels, by binding or inhibiting TRAIL receptors or inhibitors.
TRAIL polypeptides also find use in treating viral infections. Contact with
TRAIL has been shown to cause death of cells infected with cytomegalovirus,
but not of
the same cell type when uninfected. Such viruses include, but are not limited
to,
encephalomyocarditis virus, Newcastle disease virus, vesicular stomatitis
virus, herpes
simplex virus, adenovirus-2, bovine viral diarrhea virus, HIV, and Epstein-
Barr virus.
An effective amount of TRAIL is administered to a mammal, including a human,
afflicted with a viral infection. In one embodiment, TRAIL is employed in
conjunction
with interferon to treat a viral infection, such as y-interferon pretreatment
of CMV-
infected cells to enhance the level of killing of the infected cells that was
mediated by
TRAIL. TRAIL may be administered in conjunction with other agents that exert a
cytotoxic effect on cancer cells or virus-infected cells.
In another embodiment, TRAIL is used to kill virally infected cells in cell
2o preparations, tissues, or organs that are to be transplanted. For example,
bone marrow
may be contacted with TRAIL to kill virus-infected cells that may be present
therein,
before the bone marrow is transplanted into the recipient.
The TRAIL of the present invention may be used in developing treatments for
any
disorder mediated (directly or indirectly) by defective or insufficient
amounts of TRAIL.
z5 A therapeutically effective amount of purified TRAIL protein is
administered to a patient
afflicted with such a disorder. Alternatively, TRAIL DNA sequences may be
employed
in developing a gene therapy approach to treating such disorders. Disclosure
herein of
native TRAIL nucleotide sequences permits the detection of defective TRAIL
genes, and
the replacement thereof with normal TRAIL-encoding genes. Defective genes may
be
30 detected by in vitro diagnostic assays, and by comparision of the native
TRAIL
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nucleotide sequence disclosed herein with that of a TRAIL gene derived from a
person
suspected of harboring a defect in this gene.
TRAIL DNA and polypeptides encoded thereby may be used in developing
treatments for any disorder mediated (directly or indirectly) by defective, or
insufficient
amounts of, TRAIL. TRAIL polypeptides may be administered to a mammal
afflicted
with such a disorder. Alternatively, a gene therapy approach may be taken.
The TRAIL protein employed in the pharmaceutical compositions preferably is
purified, meaning that the TRAIL protein is substantially free of other
proteins of natural
or endogenous origin, desirably containing less than about 20%, preferably
less than
l0 10%, more preferably less than 5%, most preferably less than 1 % by mass of
protein
contaminants residual of production processes. Such compositions, however, can
contain
other proteins added as stabilizers, Garners, excipients or co-therapeutics.
Another use of the findings of the present invention is as a research tool for
studying the biological effects that result from inhibiting TRAIL/TRAIL
blockade
15 interactions on different cell types. TRAIL blockade in vitro or in vivo
may be achieved
by a variety of known procedures in the art. For example, a purified TRAIL
receptor
polypeptide, such as that taught by US Patent No. 6,072,047, may be used to
inhibit
binding of TRAIL to endogenous cell surface TRAIL receptors. Biological
effects that
result from the binding of TRAIL to endogenous receptors thus are inhibited.
Various
20 forms of TRAIL receptors may be employed, including, for example, the TRAIL
receptor
fragments, oligomers, derivatives, and variants that are capable of binding
TRAIL,
including soluble TRAIL receptor used to inhibit a biological activity of
TRAIL, e.g., to
inhibit TRAIL-mediated apoptosis of particular cells.
Blocking TRAIL receptors or inhibitors or restoring normal levels of TRAIL to
a
25 mammal may mediate TRAIL-deficit disorders. Such TRAIL-deficit disorders
include
conditions caused (directly or indirectly) or exacerbated by blockage of,
removal of, or
significantly diminished levels of TRAIL expression in the patient. In the
alternative,
soluble TRAIL may be used to treat those patients suffering from an
insufficiency of
normal levels of the TRAIL polypeptide, necessary to maintain normal levels of
3o apoptosis needed to minimize inflammation in autoimmune conditions.
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TRAIL, including soluble TRAIL may be employed in conjunction with other
agents useful in treating a particular disorder. Normal levels of TRAIL-
mediated cell
death may be restored by conventional procedures, including T cell death,
believed to
occur through the mechanism known as activation-induced cell death (AICD).
Autoimmune diseases and conditions in which TRAIL-mediated cell death plays a
significant role include, but are not limited to, e.g., rheumatoid arthritis,
insulin-
dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis,
Crohn's
disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple
sclerosis,
systemic lupus erythematosus, ankylosing spondylitis and others. The present
invention
1o is also applicable to patients who have received, or who are about to
receive, an
allogeneic tissue or organ transplant, such as an allogeneic kidney, liver,
heart, skin, bone
marrow, as well as those experiencing graft-versus-host disease. Treatment of
a patient
at an early stage of an autoimmune disease will minimize or eliminate
deterioration of the
disease state into a more serious condition.
15 In one embodiment, a patient's blood or plasma is contacted with TRAIL ex
vivo
to remove TRAIL receptors or inhibitors. The TRAIL may be bound to a suitable
chromatography matrix by conventional procedures. The patient's blood or
plasma flows
through a chromatography column containing TRAIL bound to the matrix, before
being
returned to the patient. The immobilized TRAIL binds TRAIL receptors or
inhibitors,
2o thus removing TRAIL receptor or inhibitors from the patient's blood to
restore normal
levels of controlled cell apoptosis, for example to reduce inflammation in
autoimmune
disease patients.
In treating patients to restore normal levels of controlled cell apoptosis,
TRAIL may be
employed in combination with other effectors of T cell apoptosis, or
inhibitors of TRAIL
25 receptors or inhibitor. Such inhibitors of TRAIL receptors or inhibitors
could include
antibodies to TRAIL receptors or inhibitors, or antisense molecules capable of
binding
same.
Compositions comprising an effective amount of a TRAIL polypeptide of the
present invention, in combination with other components such as a
physiologically
3o acceptable diluent, earner, or excipient, are provided herein. TRAIL can be
formulated
according to known methods used to prepare pharmaceutically useful
compositions.
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TRAIL can be combined in admixture, either as the sole active material or with
other
known active materials suitable for a given indication, with pharmaceutically
acceptable
diluents (e.g., saline, Tris-HCI, acetate, and phosphate buffered solutions),
preservatives
(e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,
adjuvants and/or
Garners. Suitable formulations for pharmaceutical compositions include those
described
in Remington's Pharmaceutical Sciences.
In addition, such compositions can contain TRAIL complexed with polyethylene
glycol (PEG), metal ions, or incorporated into polymeric compounds such as
polyacetic
acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into
liposomes,
1o microemulsions, micelles, unilamellar or multilamellar vesicles,
erythrocyte ghosts or
spheroblasts. Such compositions will influence the physical state, solubility,
stability,
rate of in vivo release, and rate of in vivo clearance of TRAIL, and are thus
chosen
according to the intended application. TRAIL expressed on the surface of a
cell may find
use, as well.
is Compositions of the present invention may contain a TRAIL polypeptide in
any
form described herein, such as native proteins, variants, derivatives,
oligomers, and
biologically active fragments. In particular embodiments, the composition
comprises a
soluble TRAIL polypeptide or an oligomer comprising soluble TRAIL
polypeptides.
In the alternative, the gene or an active fragment of the gene encoding the
TRAIL
2o polypeptide can be introduced into the patient, thereby permitting TRAIL to
be expressed
by, or enhancing the expression of, TRAIL by the patient, at a level not
possible prior to
introduction of the gene or gene fragment. The gene may be introduced by any
known
method, including gene therapy. As used herein, the phrase "gene therapy"
refers to the
transfer of genetic material (e.g., DNA or RNA) of interest into a host to
treat or prevent
25 a genetic or acquired disease or condition. The genetic material of
interest encodes a
product (e.g., the TRAIL protein, polypeptide, peptide or functional RNA)
whose
production in vivo is desired.
As used herein, the term "introduce" or "introducing" in relation to nucleic
acid
encompasses any method of inserting a heterologous or exogenous nucleic acid
molecule,
3o such as the molecule encoding TRAIL, into a cell and includes, but is not
limited to
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transduction, transfection, microinjection and viral infection of host cells.
Methods of
carrying out these procedures are well known to those of skill in the art.
A "gene" is defined as any nucleic acid sequence that encodes an active or
functional molecule. As used herein, the term "nucleic acid" means DNA,
including
cDNA, or RNA. A "cDNA clone" refers to a clone containing a DNA insert that
was
synthesized from mRNA and does not contain introns. The gene may encode
therapeutic
molecules including TRAIL, antisense or ribozyme RNAs, a gene encoding an
enzyme, a
gene encoding a cytokine or other immune modulating macromolecule, a gene
encoding
a recombinant antibody, a gene encoding a vaccine antigen, a gene encoding a
to macromolecule which complements genetic defects in somatic cells, and the
like.
A "recombinant" DNA molecule or a "hybrid" DNA refers to a molecule
consisting of segments of DNA from different genomes, which have been joined
end-to
end outside of living cells, and which can be maintained in living cells.
Isolated nucleic acids useful in this invention are those that contain
substantially
no additional nucleic acids, and encode a polypeptide functionally equivalent
to a
polypeptide encoded by the isolated TRAIL gene when expressed under the
control of
necessary expression control sequences, of the types well known in the art of
recombinant genetic technology.
"Transcription" refers to the process of producing mRNA from a gene or DNA
sequence. "Translation" refers to the process of producing a polypeptide from
mRNA.
"Expression" refers to the process undergone by a gene or DNA sequence to
produce a
polypeptide, meaning a combination of transcription and translation.
"Expression control
sequences" are those nucleotide sequences that control and regulate expression
of genes
when operatively linked to those genes, such as promoters, enhancers and the
like.
Various expression control sequences may also be chosen to effect the
expression
of the DNA sequences of this invention. These expression control sequences
include
those listed above and other sequences known to control the expression of
genes of
prokaryotic or eukaryotic cells or their viruses and various combinations
thereof. For
expression of the DNA sequences of the present invention, these DNA sequences
are
3o operatively-linked to one or more of the above-described expression control
sequences in
the expression vector. Such operative linking, which may be effected before or
after the
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WO 01/22987 PCT/US00/26862
chosen polypeptide DNA sequence is inserted into a cloning vehicle, enables
the
expression control sequences to control and promote the expression of the DNA
sequence.
As used herein the term "functionally equivalent nucleotide sequence" is
intended
to cover minor variations in the viral vector sequence which, due to
degeneracy in the
DNA code, does not result in a peptide having substantially different
biological activities
from the native TRAIL peptide. The encoded TRAIL proteins can have an amino
acid
sequence, which is at least in part different from the native virus sequences,
but which
should retain substantially the same biological activities as the native
TRAIL. This may
1o be achieved by various changes in the sequence, such as insertions,
deletions and
substitutions, either conservative or non-conservative, where such changes do
not
substantially alter the peptide produce.
TRAIL or active fragments or derivatives thereof, or the gene or gene fragment
encoding TRAIL, can be administered to a patient in a therapeutically
effective amount
by any suitable manner, e.g., topically, parenterally, or by inhalation. The
term
"parenteral" includes injection, e.g., by subcutaneous, intravenous, or
intramuscular
routes, also including localized administration, e.g., at a site of disease or
injury.
Sustained release from implants is also contemplated. One skilled in the
pertinent art will
recognize that suitable dosages will vary, depending upon such factors as the
nature of
2o the disorder to be treated, the patient's body weight, age, and general
condition, and the
route of administration. Preliminary doses can be determined according to
animal tests,
and the scaling of dosages for human administration are performed according to
art-
accepted practices. Compositions comprising TRAIL nucleic acids in
physiologically
acceptable formulations are also contemplated. TRAIL DNA may be formulated for
inj ection, for example.
By "therapeutically effective" as used herein, is meant that amount of
composition or expression product that is of sufficient quantity to enhance or
ameliorate
cellular apoptosis or to restore it to normal levels, particularly in
nontransformed cells
and/or tissues in vivo, preferably by modulating the level of TRAIL available
to the cell
3o as described above. The effect may be seen as a change in immunity,
inflammation,
particularly autoimmune inflammation, or development. By "ameliorate" is meant
a
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WO 01/22987 PCT/US00/26862
lessening or reduction or prophylactic prevention of the detrimental effect of
the disorder
in the patient receiving the therapy.
The terms "preventing" or "inhibiting" are intended to mean a reduction in
effect
on TRAIL, such as inhibiting a TRAIL receptor, thereby permitting endogenous
or
introduced TRAIL to reach normal levels in the patient. They also are intended
to mean
a diminution in the effect in a non-transformed cell of the morphological
and/or
biochemical changes normally associated with necessary and desirable levels of
apoptosis, particularly in autoimmune diseases and conditions. Thus, this
invention
provides compositions and methods to modulate survival time and/or survival
rate of a
to cell or population of cells which, absent the use of the method, will die,
but would not be
removed by controlled apoptosis. Accordingly, it also provides compositions
and
methods to prevent or treat diseases or pathological conditions associated
with
uncontrolled cell death in a subject, or to block TRAIL receptors or
inhibitors that would
otherwise preclude normal or desirable expression of the TRAIL peptide, which
has been
found related to normal levels of apoptosis in non-transformed cells, and
which controls
excessive inflammation, tissue injury and the like.
The method is provided in which normal levels of cellular apoptosis is
achieved
in non-tranformed cells of a patient by administering to the patient a
therapeutically
effective amount of an isolated nucleic acid sequence encoding a TRAIL
agonist. Such
TRAIL "agonist" is a receptor or inhibitor of a TRAIL receptor or inhibitor,
wherein the
agonist is selected from the group consisting of antibodies to TRAIL receptors
or
inhibitors, antisense molecules complimentary to TRAIL receptors or
inhibitors, and any
molecule which binds to or blocks TRAIL receptors or inhibitors.
The subject of the invention is preferably a human, however, it can be
envisioned
that any bird, animal, fish or the like, with a autoimmune or apoptotic
condition can be
treated by the method of the present invention.
Antibodies that are immunoreactive with TRAIL receptor or inhibitor
polypeptides are also provided. Such antibodies specifically bind TRAIL
receptors or
inhibitors, in that the antibodies bind to TRAIL receptors or inhibitors via
the antigen-
binding sites of the antibody (as opposed to non-specific binding).
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TRAIL receptors or inhibitors may be employed as an immunogen in producing
antibodies immunoreactive therewith. Alternatively, another form of TRAIL
receptor or
inhibitor, such as a fragment or fusion protein, is employed as the immunogen.
Polyclonal and monoclonal antibodies may be prepared by conventional
techniques.
Antigen-binding fragments of such antibodies, which may be produced by
conventional
techniques, are also encompassed by the present invention. Examples of such
fragments
include, but are not limited to, Fab and F(ab')Z fragments. Antibody fragments
and
derivatives produced by genetic engineering techniques are also provided.
1o The monoclonal antibodies of the present invention include chimeric
antibodies,
e.g., humanized versions of marine monoclonal antibodies. Such humanized
antibodies
may be prepared by known techniques, and offer the advantage of reduced
immunogenicity when the antibodies are administered to humans. In one
embodiment, a
humanized monoclonal antibody comprises the variable region of a marine
antibody (or
just the antigen binding site thereof) and a constant region derived from a
human
antibody. Alternatively, a humanized antibody fragment may comprise the
antigen
binding site of a marine monoclonal antibody and a variable region fragment
(lacking the
antigen-binding site) derived from a human antibody. Procedures for the
production of
chimeric and further engineered monoclonal antibodies include those described
in
2o Riechmann et al. (Nature 332:323 (1988)), Liu et al., (Proc. Nat'l. Acad.
Sci. 84:3439
(1987)), Larrick et al. (BiolTechnology 7:934 (1989)), and Winter et al. (TIPS
14:139
(1993)).
Among the uses of the antibodies is use in assays to detect the presence of
TRAIL, or of TRAIL receptor or inhibitor polypeptides, either in vitro or in
vivo. The
antibodies also may be employed in purifying TRAIL, or TRAIL receptor or
inhibitor,
proteins by immunoaffinity chromatography.
Those antibodies that additionally can block the binding of TRAIL may be used
to inhibit a biological activity that results from such binding. Such blocking
antibodies to
the TRAIL receptor or inhibitor may be identified using any suitable assay
procedure,
3o such as by testing antibodies for the ability to inhibit binding of TRAIL
receptors or
inhibitors. Examples of such cells are the Jurkat cells and PSI cells, and
others.
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Alternatively, blocking antibodies may be identified in assays for the ability
to inhibit a
biological effect that results from binding of TRAIL receptors or inhibitors
to target cells.
Antibodies may be assayed for the ability to inhibit TRAIL-mediated lysis of
Jurkat cells,
for example.
Such an antibody may be employed in an in vitro procedure, or administered in
vivo to inhibit a TRAIL receptor or inhibitor-mediated biological activity.
Disorders
caused or exacerbated (directly or indirectly) by the interaction of TRAIL
receptors or
inhibitors with cell surface TRAIL, thus may be treated. A therapeutic method
involves
in vivo administration of a blocking antibody to a mammal in an amount
effective in
to inhibiting a TRAIL-deficit biological activity. Disorders caused or
exacerbated by
TRAIL, directly or indirectly, are thus treated. Monoclonal antibodies are
generally
preferred for use in such therapeutic methods. In one embodiment, an antigen-
binding
antibody fragment is employed.
Antibodies raised against a TRAIL receptor, for example, may be screened for
15 agonistic (i.e., ligand-mimicking) properties. Such antibodies, upon
binding to cell
surface TRAIL receptor, induce biological effects (e.g., transduction of
biological
signals) similar to the biological effects induced when TRAIL receptor binds
to cell
surface TRAIL.
Compositions comprising an antibody that is directed against TRAIL receptors
or
2o inhibitors, and a physiologically acceptable diluent, excipient, or Garner,
are provided
herein. Suitable components of such compositions are as described above for
compositions containing TRAIL proteins. Also provided herein are conjugates
comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to
an antibody
directed against TRAIL.
25 In addition, the present invention provides other useful compositions for
binding
TRAIL receptor or inhibitor nucleic acids, including antisense or sense
oligonucleotides
comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable
of
binding to target TRAIL receptor mRNA (sense) or TRAIL receptor DNA
(antisense)
sequences. Antisense or sense oligonucleotides, according to the present
invention,
3o comprise a fragment of the coding region of, for example, a TRAIL receptor
DNA. Such
a fragment generally comprises at least about 14 nucleotides, preferably from
about 14 to
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WO 01/22987 PCT/CTS00/26862
about 30 nucleotides. The ability to derive an antisense or a sense
oligonucleotide, based
upon a cDNA sequence encoding a given protein, is known in the art
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences
results in the formation of duplexes that block transcription or translation
of the target
sequence by one of several means, including enhanced degradation of the
duplexes,
premature termination of transcription or translation, or by other means. The
antisense
oligonucleotides thus may be used to block expression of TRAIL receptor
proteins,
permitting normal levels of TRAIL to be restored. Antisense or sense
oligonucleotides
further comprise oligonucleotides having modified sugar-phosphodiester
backbones (or
other sugar linkages) and wherein such sugar linkages are resistant to
endogenous
nucleases. Such oligonucleotides with resistant sugar linkages are stable in
vivo (i.e.,
capable of resisting enzymatic degradation), but retain sequence specificity
to be able to
bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are covalently linked to organic moieties, or other
moieties that
increases affinity of the oligonucleotide for a target nucleic acid sequence,
such as poly-
(L-lysine). Further still, intercalating agents, such as ellipticine, and
alkylating agents or
metal complexes may be attached to sense or antisense oligonucleotides to
modify
binding specificities of the antisense or sense oligonucleotide for the target
nucleotide
2o sequence.
Antisense or sense oligonucleotides may be introduced into a cell containing
the
target nucleic acid sequence by any gene transfer method, including, for
example,
CaP04-mediated DNA transfection, electroporation, or by using gene transfer
vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense or sense
oligonucleotide is inserted into a suitable retroviral vector. A cell
containing the target
nucleic acid sequence is contacted with the recombinant retroviral vector,
either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited to, those
derived from the
murine retrovirus M-MuLV.
Sense or antisense oligonucleotides also may be introduced into a cell
containing
the target nucleotide sequence by formation of a conjugate with a ligand
binding
molecule. Suitable ligand binding molecules include, but are not limited to,
cell surface
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WO 01/22987 PCT/US00/26862
receptors, growth factors, other cytokines, or other ligands that bind to cell
surface
receptors. Preferably, conjugation of the ligand binding molecule does not
substantially
interfere with the ability of the ligand binding molecule to bind to its
corresponding
molecule or receptor, or block entry of the sense or antisense oligonucleotide
or its
conjugated version into the cell. Alternately, a sense or an antisense
oligonucleotide may
be introduced into a cell containing the target nucleic acid sequence by
formation of an
oligonucleotide-lipid complex.
In further embodiments of the present invention, therapeutically effective
amounts
of TRAIL receptor or inhibitor binding proteins are administered as described
above for
1 o TRAIL.
EXAMPLES
The present invention is further described in the following examples. These
examples are provided for purposes of illustration only, and are not intended
to be
limiting unless otherwise specified. The autoimmune scenarios are relevant for
many
practical situations, and are intended to be merely exemplary to those skilled
in the art.
These examples are not to be construed as limiting the scope of the appended
claims.
Thus, the invention should in no way be construed as being limited to the
following
example, but rather, should be construed to encompass any and all variations
which
2o become evident as a result of the teaching provided herein.
Examples 1-4 demonstrate the effect of chronic TRAIL blockade in an animal
model of rheumatoid arthritis. Examples 5-9 demonstrate the effect of chronic
TRAIL
blockade in an animal model of multiple sclerosis as determined by its effect
on
experimental autoimmune encephalomyelitis (EAE) and encephalitogenic T cells.
Example 1 - Recombinant soluble DRS blocks TRAIL-induced apoptosis of tumor
cells.
In order to determine the biological roles of TRAIL in vivo and in vitro,
large
quantities of soluble DR5 (sDRS) were produced using the yeast Pichia pastoris
system
(Higgins, In Current protocols in protein science, Coligan, ed., J. Wiley &
Sons, New
York, 1998). To generate recombinant soluble DRS, the cDNA that contained the
full-
length extracellular domain of the human DR5 (Wu et al., Nature Genetics 12
(2):141-
143 (1997) was cloned into pPIC9K that contains a PAOx~ promotor and a six-
histidine
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WO 01/22987 PCT/US00/26862
tag. (Note that pPIC9K is essentially the same as pGAPZa (Invitrogen,
Carlsbad, CA) for
the purposes of this invention, and the two plasmids are to be considered to
be
interchangable.) Several recombinant Pichia pastoris clones were generated via
homologous recombination (Higgins, 1998), which secreted high levels of sDRS
(up to
25 mg per liter of yeast culture). The sDRS used in this study was purified
through a
nickel ion column, then depleted of lipopolysaccharides (LPS) by incubation
with
polymyxin B agarose (Sigma, St. Louis, MO). _
The sDRS consists of only the extracellular domain of the human TRAIL receptor
DRS, and effectively blocks TRAIL-induced apoptosis of tumor cells (Figure 1).
The
to purity of the sDRS was confirmed by polyacrylamide gel electrophoresis
(PAGE). The
sDRS, which had a molecular weight of 26 kD, was the only protein band
present. The
purified sDRS contained 1-2ng LPS per mg of protein as determined by Limulus
amebocyte lysate (LAL) assay. This is comparable to the LPS level in bovine
serum
albumin (BSA) or human serum albumin (HSA) used in this study, which is 1-4 ng
per
mg of protein. In previous experiments, it had been determined that neither
BSA or HSA
had any effect on collagen-induced arthritis using LPS-free PBS as control
(Zhang et al.,
J. Clinicallnvestigation 100:1951-1957 (1997)).
In vitro, purified sDRS did not induce proliferation of lymphocytes regardless
of
its concentration in the culture (1-100 pg/ml) (unpublished data).
2o A recombinant adenovirus carrying the murine TRAIL gene (Ad-TRAIL) was
generated by inserting the cloned TRAIL full-length cDNA into the plasmid pAd-
tet,
followed by homologous recombination with human adenoviral DNA (Zhang et al.,
1997). Briefly, the murine full-length TRAIL cDNA was generated from mouse
spleen
by PCR using specific primers corresponding to the 5' and 3' ends of the
coding regions
of the TRAIL gene. After adding A-overhangs by incubation with Taq polymerase,
the
PCR fragment was inserted into the expression vector pCRII-TOPO, which
possesses T-
overhangs, to create pCRII-TOPO-TRAIL. After amplification of the pCRII-TOPO-
TRAIL vector DNA, the TRAIL gene was cut out with Not I, and inserted into the
Not I
site of the vector pAdtet to create pAdtet-TRAIL.
3o The CIaI-digested Ad5 genomic DNA was then prepared from
HS.OOOCMVEGFP, a recombinant adenovirus that is depleted of Ela, Elb and a
portion
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WO 01/22987 PCT/US00/26862
of E3 region, but which contains the green fluorescent protein (GFP) cDNA. The
recombinant virus was produced by co-transfecting 293 cells with pAdtet-TRAIL
and
CIaI-digested Ad5 genomic DNA. White plaques of recombinant viruses were
expanded
and screened by PCR using TRAIL-specific primers. The recombinant virus-
carrying
TRAIL gene was then propagated, purified through a cesium chloride gradient,
and de-
salted on Econo-Pac l ODG column (Bio-Rad, Hercules, CA).
Human Jurkat T cells (clone E6-1) 7500 cells/ml, and K562 B cells, 1 x 106/m1,
were
cultured in RPMI 1640 medium containing various concentrations of TRAIL
(Biomol
Research Laboratory, Plymouth Meeting, PA), with or without sDRS. The cells
were
to treated as follows: A) 5 pg/ml of BSA; B) 100 ng/ml of TRAIL; C) 5 p.g/ml
of sDRS or
D) 100 ng/ml of TRAIL + 5 pg/ml of sDRS. Three days later, 2 mg/ml 3-4.5
dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) was added to the
culture, and
cell viability was determined by spectrometry at 575 nm wavelength.
The percentage of apoptotic cells was determined as shown in Figures lA-1D.
Apoptosis was determined after eighteen (18) hours by flow cytometry using
Annexin-V-
FITC per manufacturer's instructions (Pharmingen, San Diego, CA). The
percentage of
live cells was calculated by assuming the survival rate of untreated cells as
100%.
Although the data is not shown, tests were also conducted in parallel
experiments
demonstrating that sDRS had no effect on either Fas- or ultraviolet-induced
apoptosis of
Jurkat cells. In vitro, Ad-TRAIL induced apoptosis of a number of tumor cell
lines
including Rat-1 cells and 293 cells; addition of sDRS to the culture prevented
Ad-
TRAIL-induced apoptosis (Goke and Chen, unpublished).
K562 cells were also treated with various concentrations of TRAIL with (open
circle) or without (filled square) 5 pg/ml of sDRS. The results are shown in
Figure 1E.
Example 2 - Roles of TRAIL in autoimmune arthritis.
The effect of TRAIL-blockade in vivo was first examined in a mouse model of
rheumatoid arthritis (Figure 2). Arthritis was monitored by both clinical
examination and
histochemistry.
Two groups of six- to eight-week old male DBA/1 mice (Jackson Laboratory, Bar
3o Harbor, ME), 4-6 mice per group, were immunized on days 0 and 21 with
chicken type II
collagen (Sigma, St Louis, MO). The mice were immunized by multiple
intradermal
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injections of 100 ~g chicken type II collagen in 100 ~l phosphate buffered
saline (PBS)
emulsified in an equal volume of complete Freund's adjuvant containing lmg/ml
of
Mycobacterium tuberculosis H37 RA (Difco, St. Louis, MO). Mice were
rechallenged
with the same antigen preparation subcutaneously on the flanks 21 days later.
Starting from the day of second immunization (at day 21 ), mice received daily
intraperitoneal injections of 50-300 ~g sDRS or 100 ~.g bovine serum albumin
(BSA) in
0.5 ml PBS for a total of 21 days (Figure 2A). Mice were examined daily for
signs of
joint inflammation, and scored as follows:
0 = Normal;
1 = Erythema and mild swelling confined to the ankle joint or mid-foot;
2 = Erythema and mild swelling extending from the ankle to the mid-foot;
3 = Erythema and moderate swelling extending from the ankle to the metatarsal
joints; or
4 = Erythema and severe swelling extending from the ankle to the digits.
The maximal disease score per foot was 4, and the maximal disease score per
mouse was 16. The mean disease score per group was calculated as: total
disease scores
from all animals in the group / the number of animals in the group. (In
parallel
experiments, other control proteins, such as human or bovine serum albumin,
were also
used. Similar results to those reported were observed.)
Figure 2A depicts the disease courses in mice treated with of BSA (open
square)
or sDRS (filled circle). Each data point represents a mean ~ standard
deviation (SD)
from a total of 5 mice (for sDRS-treated group) or 6 mice (for BSA-treated
group). The
experiments were repeated five times with similar results.
The differences between the two groups were found to be statistically
significant
(P<0.001 ) as determined by Mann-Whitney test. As shown in Figure 2A, mice
that
received the control protein developed typical arthritis, which started
approximately 5-10
days after the second immunization, and reached a maximal disease score of 7.8
by day
33. By contrast, in mice treated with sDRS, arthritis was significantly
exacerbated. The
mean day of onset in the sDRS-treated group was 7.5 ~ 1.9 (days post second
3o immunization), as compared to 14.5 ~ 1.8 in the control group (P<0.01 as
determined by
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ANOVA). The maximal disease score was increased from 7.8 in the control group
to
12.4 in the sDRS-treated group.
Figure 2B depicts the dose-dependent effect of sDRS on arthritis as judged by
disease scores of individual feet taken 12 days after the second immunization.
It was
apparent that sDRS enhanced arthritic inflammation in most feet in a dose-
dependent
manner, indicating that TRAIL is an inhibitor of autoimmune arthritis. A total
of 4
groups of mice are shown: one was treated with 100 ~g BSA, while the other
three were
treated with 50-300 ~g sDRS. Each data point represents an individual foot,
with 16 to
24 feet per group. The differences between BSA- and sDRS-treated groups were
found
1o to be statistically significant as determined by ANOVA (P<0.05 for mice
treated with
SO~g sDRS and p<0.01 for mice treated with 100 or 300 pg sDRS).
To directly test TRAIL's ability to inhibit or ameliorate autoimmune
arthritis,
intra-articular TRAIL gene transfer was performed using a replication-
defective
adenovirus carrying the mouse TRAIL gene. The virus was inj ected directly
into arthritic
joints 6 days after disease onset, as previously described (Zhang et al.,
1997). As shown
in Figure 3, arthritis was dramatically ameliorated by TRAIL gene transfer
following
intra-articular injection of 101°recombinant TRAIL viruses, while
injection of 109
recombinant TRAIL viruses had only a mild effect.
DBA/1 mice, 4 mice per group, were immunized with type II collagen as
2o described above. Six (6) days after disease onset (10 days after the second
immunization), mice were injected intra-articularly and periarticularly to the
ankle and
tarsal joints of the hind feet as follows: 1) 10 ~l PBS (control); 2)
101° particles of
adenovirus (Ad) vector; 3) 109 particles of TRAIL virus; or 4) 101°
particles of TRAIL
virus in 10 ~1 of PBS as described by Zhang et al., 1987. (The Ad vector
contains no
TRAIL gene, but is otherwise identical to the TRAIL virus.) Starting from the
first day
of virus injection, two groups of mice, one non-treated and the other injected
with lOlo
particles of TRAIL virus, were subjected to daily intraperitoneal injections
of 100 ~,g
sDRS for a total of 14 days. The data presented represent disease scores of
individual
hind feet, 6 days after viral injection. A total of 8 hind feet per group are
shown.
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Only mice receiving 10'° particles of TRAIL virus showed
significant
improvement (P<0.001, as determined by ANOVA). Results are representative of
two
experiments.
The effect of the treatment was TRAIL-specific, since it could be neutralized
by
sDRS (Figure 3). The therapeutic effect of the TRAIL virus lasted about 10
days, and
arthritis returned approximately 2 weeks after TRAIL virus injection. As
expected,
injection of sDRS after the disease onset also exacerbated the inflammation
(Figure 3).
Histochemical analysis of the mice treated in Figure 2 revealed dramatic
differences between the groups. DBA/1 mice, 8-9 mice per group, were immunized
for
1o arthritis and treated with sDRS or BSA as shown in Figure 2A. The mice
depicted in
Figures 4A-4D, 4G and 4H were sacrificed 32 days after the second
immunization, and
their ankle joints were analyzed for histology and apoptosis.
In the histochemical analysis, the test animals were sacrificed and paws were
first
fixed in 10% formalin, decalcified in hydrochloric acid, then embedded in
paraffin. Joint
sections (6 Vim) were then prepared and stained with hematoxylin and eosin
(HE). The
degree of arthritic inflammation was scored as follows:
0 = no signs of inflammation;
1 = mild synovitis;
2 = severe synovitis;
3 = severe synovitis with mild cartilage and bone destruction; or
4 = severe synovitis with severe cartilage and bone destruction.
For detection of apoptotic cells, ApopTag system was used (Oncor,
Gaithersburg,
MD). Briefly, synovial tissues were snap-frozen and cryosectioned (6 Vim). The
3'-OH
ends of fragmented DNA were labeled with digoxigenin (DIG)-conjugated
nucleotide
using terminal deoxynucleotidyl transferase. The randomly incorporated
nucleotide
polymers were then detected by peroxidase-labeled anti-DIG antibody and
chromogen
diaminobenzidin. Counterstaining was performed with methyl green. The
apoptotic
index was recorded as follows:
0 = < 1 % of cells in the synovium are apoptotic;
3o 1 = 1-3% of cells in the synovium are apoptotic;
2 = 3.1-5% of cells in the synovium are apoptotic; or
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3 = >5% of cells in the synovium are apoptotic.
By comparison, proliferating cells were labeled in vivo in the mice depicted
in
Figures 4E and 4F with bromodeoxyuridine (BrdU). Mice were immunized twice
with
chicken type II collagen as described above, but starting from the second
immunization,
mice received daily intraperitoneal injections of 0.8 mg BrdU in 0.5 ml PBS.
Mice were
sacrificed 21 days after the second immunization, and their synovial joints
collected and
embedded in paraffin. Synovial sections (6 Vim) were then stained with rat
anti-BrdU
antibody and peroxidase-labeled goat anti-rat IgG as described (Wilson, In
Immunochemical protocols, vol. 80, Pound (ed.), Humana press, 251-255 (1998).
to Control antibodies and tissues were routinely used to exclude non-specific
staining.
In the mice treated with the control protein, arthritis was characterized by
leukocyte infiltration, mild synovitis and pannus formation (Figures 4A, 4C);
cartilage
destruction and bone erosion occurred only in a small number of synovial
joints. Figure
4A depicts an ankle joint of a BSA-treated mouse with a pathology score of 2
(HE
staining, original magnification x20). An arrow indicates signs of synovitis
in the joint.
Figure 4C depicts an ankle joint of a BSA-treated mouse with a pathology score
of 1 (HE
staining, original magnification x 100). An arrow indicates signs of
synovitis.
By contrast, in mice treated with sDRS, severe synovitis, hyperplasia of
synovial
2o membrane and cartilage/bone destruction were observed in most synovial
joints of the
feet (Figures 4B, 4D), which lasted for more than 3 weeks, with little signs
of remodeling
or fibrosis. Figure 4B depicts an ankle joint of a sDRS-treated mouse with a
pathology
score of 4 (HE staining, original magnification x20). Arrows indicate severe
synovitis in
the joint and hyperplasia in the joint, as well as cartilage and bone
destruction. Figure 4D
depicts an ankle joint of a sDRS-treated mouse with a pathology score of 4 (HE
staining,
original magnification x100). Arrows indicate severe synovitis and hyperplasia
in the
joint, as well as cartilage and bone destruction.
To directly label proliferating cells in the joint, the mouse models were
treated
with nucleotide analogue bromodeoxyuridine (BrdU), then BrdU incorporation was
3o examined by immunohistochemistry. As shown in Figures 4E and 4F, BrdU+
cells were
detected in both BSA- and sDRS-treated mice. Figure 4E depicts an ankle joint
of a
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BSA-treated mouse with a disease score of 2 (BrdU staining, original
magnification
x400). Arrows indicate BrdU+ nuclei. Figure 4F depicts an ankle joint of a
sDRS-treated
mouse with a disease score of 3 (BrdU staining, original magnification x400).
However,
the number of BrdU+ cells in sDRS-treated mice was markedly increased as
compared
those in BSA-treated mice.
Figure 4G depicts an ankle joint of a BSA-treated mouse with a pathology score
of 4 (apoptotic staining, original magnification x200). Arrows indicate
apoptotic cells.
Figure 4H depicts an ankle joint of a sDRS-treated mouse with a pathology
score of 4
(apoptotic staining, original magnification x200).
1o Mice were treated and sacrificed, corresponding to the treatment in Figure
4.
Their paws examined for the degrees of inflammation and apoptosis as described
above.
A minimum of 3 comparable synovial sections were analyzed per mouse. A total
of 8
mice (for sDRS-treated group) and 9 mice (for BSA-treated group) were used.
Quantitative analysis of the histochemical data revealed substantial
differences between
sDRS- and BSA-treated groups (pathology score 3.9 vs. 2.8, Figure 5A).
In determining the pathology scores (Figure SA), the differences between the
two
groups were found to be statistically significant, as determined by ANOVA
(p<0.01).
For measuring the apoptotic index (Figure SB), the differences between the two
groups
were not statistically significant, as determined by ANOVA (p = 0.21).
2o As shown in Figure 4G and 4H, apoptotic cells were readily detectable in
arthritic
synovia of both control and sDRS-treated mice 32 days after the second
immunization.
However, no statistically significant differences between the two groups were
observed
when the degrees of apoptosis were compared (Figure SB). In parallel
experiments, mice
were also sacrificed 14 and 21 days after the second immunization, and
examined for
apoptosis as in Figures 4G and 4H. Again, no significant differences in the
degree of
apoptosis were observed between control and sDRS-treated groups (data not
shown).
Example 3 - Roles of TRAIL in autoimmune T and B cell responses in vivo.
Collagen-induced arthritis is initiated by collagen-specific lymphocytes. To
determine whether exacerbation of arthritis by sDRS is associated with
functional
3o alterations of collagen-specific lymphocytes, both cellular and humoral
anti-collagen
immune responses were examined.
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DBA/1 mice, 5-6 mice per group, were treated as in Figure 2A. To test cellular
immune responses (Figures 6A-6C), mice were sacrificed 32 days after the
second
immunization and their inguinal lymph nodes collected. Notably, the short-term
TRAIL-
blockade in DBA/1 mice did not alter the structure or cellular composition of
lymphoid
organs as judged by histochemistry and flow cytometry (unpublished data).
For cytokine assays, lymph node cells, 1.5 x 106/well, were cultured in 0.2 ml
of
serum-free medium (X-vivo 20, Biowhittacker, Walkersville, MD), with or
without 10-50
pg/ml of chicken type II collagen (CII) or 1 ~g/ml of Con-A. Concanavalin
(Con)-A-
treated cultures were included as positive controls to illustrate the levels
of polyclonal T
to cell responses. Culture supernatants were collected 40 hours later, and IL-
2/IFN-y
concentrations were determined by sandwich ELISA, as described by Chen et al.,
J.
Immunol.155:910-916 (1995).
To test lymphocyte proliferation (Figure 6A), lymph node cells, 0.5 x 106
cells/well, were first cultured for 72 hours, and then pulsed with 3H-
thymidine for an
is additional 16 hours. Radioactivity [presented as counts per minute (CPM)]
was
determined using a Wallac beta-plate counter. Figure 6B depicts IL-2
production, while
Figure 6C depicts IFN-y production. Remarkably, both lymphocyte proliferation
and
cytokine production were enhanced in mice treated with sDRS
In parallel experiments, to test humoral immune responses (Figures 6D-6E),
mice
2o were bled retroorbitally on days 14 and 32 after the second immunization,
and tested as in
the Figures SA-6C. Anti-collagen antibody responses were determined by ELISA,
using
chicken type II collagen as antigen. Figure 6D depicts anti-collagen IgG2a
titers; Figure
SE depicts anti-collagen IgGl titers. Each data point represents a mean ~ SD
from 5
mice (for sDRS-treated group) or 6 mice (for BSA group). The experiments were
25 repeated three times with similar results.
In the collagen-specific analyses, anti-collagen IgG2a was seen to be
dramatically
increased in mice treated with sDRS, whereas anti-collagen IgGl was only
moderately
increased on day 14. Similar differences between control and sDRS-treated mice
were
observed when a similar ELISA was performed using sDRS as detecting antigen;
no anti-
3o sDRS antibodies were detected (data not shown). Additionally, sDRS had no
effect on
LPS-induced proliferation of splenocytes in vitro (unpublished data). These
results
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indicate that chronic TRAIL-blockade in mice enhanced both cellular and
humoral
immune responses, which could, in turn, exacerbate autoimmune arthritis.
Example 4 - Roles of TRAIL in apoptosis and cell cycle progression.
TRAIL is unique in that it has been shown to induce apoptosis of some, but not
all, tumor cell lines. It is believed that the effect of sDRS on arthritis can
be explained by
its blockade of TRAIL-induced apoptosis of inflammatory cells. To test this
theory, the
effect of TRAIL-blockade on apoptosis of synovial cells was examined in vivo,
i. e., the
inhibition of DNA synthesis and cell cycle progression by TRAIL was evaluated.
Splenocytes were prepared from 6-8 week old BALB/c mice (Jackson
to Laboratory), and cultured in DMEM for 3 days in the presence of 10% fetal
bovine serum
(FBS), activated with 2.5 pg/ml of Con-A. Live cells were then purified
through a Ficoll
gradient, and cultured in 96-well plates at 3 x 105/well in 200 ~1 of DMEM
containing
10% FBS, with or without the following reagents: 1) 100 ng/ml of TRAIL; 2) 5
pg/ml of
sDRS; 3) 5 ~g/ml of anti-CD95L monoclonal antibody (mAb) (MFL-3); and 4) anti-
mouse CD3 mAb (which was coated on the plate by pre-incubating the plate with
10
p,g/ml of the antibody at 4°C for 16 hr).
Apoptosis and cell cycle progression were analyzed by flow cytometry using the
DNA dye propidium iodide (Noguchi, P.D., In Current Protocols in Immunology.
J.E.
Coligan, ed., Sarah Greene, New York (1993)). For apoptosis and cell cycle
analyses
(Figures 7A-7B), cells were cultured for a total of 24 hours, fixed in 70%
ethanol, and
stained with 50 ~g/ml of PI. For thymidine incorporation assays (Figures 7C-
7D), cells
were cultured for 24 hr, pulsed with 10 p,Ci/ml of 3H-thymidine for an
additional 16 hr.
Cells were then harvested and radioactivity determined using a Wallac beta-
plate counter.
Figure 7A shows the percentage of apoptotic cells as determined by flow
cytometry. As shown, anti-CD3 mAb induced apoptosis of approximately 16% of
the
cells. This was completely prevented by anti-CD95L mAb, confirming an
essential role
for CD95L in activation-induced cell death (AICD) (Ju et al., Nature 373:444-
448
(1995); Dhein et al., Nature 373:438-441 (1995); Brunner et al., Nature
373:441-444
(1995)). By contrast, sDRS moderately increased AICD induced by anti-CD3 mAb
(Figure 7A).
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The spontaneous apoptotic rate in cultures containing no anti-CD3 mAb was 12%,
which was subtracted from the present data. For cultures that contained TRAIL
and anti-
CD3 mAb, the percentage of anti-CD3-induced apoptosis was 16 ~1 %, which was
comparable to that of the cultures treated with anti-CD3 mAb alone. The
differences
between anti-CD95L mAb treated culture and all other cultures are
statistically
significant as determined by ANOVA (p<0.0001). Unexpectedly, the total number
of
cells, especially cells in the S-G2/M phases of the cell cycle, were
dramatically increased
in cultures containing sDRS. This increase in S-G2/M cells was TRAIL-specific
since it
was partially blocked by recombinant TRAIL.
to Figure 7B depicts the number of S-G2/M cells/well, as determined by flow
cytometry. For cultures that contained TRAIL and anti-CD3 mAb, the number of
cells in
the S-G2/M phases was 70-90% of that of the anti-CD3 mAb treated culture. The
total
numbers of live cells/well recovered from each group were as follows: cultures
with anti-
CD3 mAb alone, 0.3 x 105; cultures with anti-CD3 mAb + sDRS, 1.6 x 105;
cultures with
anti-CD3 mAb + anti-CD95L mAb, 1.1 x 105; cultures with anti-CD3 mAb + sDRS +
TRAIL, 0.6 x 105. The differences between all four groups are statistically
significant as
determined by ANOVA (p<0.01).
These results strongly suggest that although TRAIL may not induce apoptosis of
lymphocytes, it does play important roles in regulating cell cycle
progression. To directly
2o test this theory, the effect of recombinant TRAIL and sDRS was examined on
cell cycle
progression of lymphocytes. T lymphocytes were stimulated with anti-CD3 mAb as
in
Figure 7A, and G1-to-S phase progression (DNA synthesis) was determined by 3H-
thymidine incorporation (cells entering the S phase of the cell cycle
synthesize DNA and
take up thymidine) (Figures 7C and 7D).
The data presented in Figures 7C and 7D represent the means ~ SDs of CPM of
triplicate cultures. For cultures containing anti-CD3 mAb, the differences
between the
two groups are statistically significant as determined by ANOVA (p<0.01). The
experiments were repeated twice with similar results. The concentrations of
TRAIL and
sDRS used in these experiments were selected based on previously performed
dose-
3o dependency studies. When 0.2-1 ~g/ml of sDRS was used, less significant
effects on cell
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cycle progression were observed. Similarly, when 50-1 SO ng/ml of TRAIL was
tested,
much less significant effects on cell cycle were detected (data not shown).
As shown in Figures 7C and 7D, anti-CD3 mAb induced a marked increase in
thymidine uptake. This was significantly inhibited by TRAIL (Figure 7C), but
enhanced
by sDRS (Figure 7D). Again, neither TRAIL nor sDRS affected apoptosis of cells
in the
culture (unpublished data). Since AICD is regulated by cell cycle progression,
inhibition
of cell cycle by TRAIL may modulate the sensitivity of cells to AICD as shown
in Figure
7A.
Example 5 - Generation of a recombinant soluble TRAIL receptor that blocks
TRAIL
l0 function.
The recombinant TRAIL receptor used in this study was the soluble DRS (sDRS)
produced in the yeast Pichia pastoris system, as described in Example 1. The
cDNA
containing the full-length extra-cellular domain of the human DRS (Wu et al.,
1997) was
cloned into pGAPZa (Invitrogen, Carlsbad, CA) that contains a PAOxi promoter
and a six
histidine tag as well as a zeocin resistance gene. Several recombinant Pichia
pastoris
clones with zeocin resistance were generated, which secreted up to 25 mg sDRS
per liter
of yeast culture.
The recombinant protein was purified by Ni ion column chromatography and
treated with polymyxin B agarose, as in Example 1. The purity of the sDRS was
confirmed by polyacrylamide gel electrophoresis and Coomasie Blue staining. As
above,
sDRS, at 26 kD, was the only protein band present. The purified sDRS contains
1-2 ng of
LPS per mg of protein as determined by LAL assay. In previous experiments, it
had been
determined that this level of LPS had no effect on the development of EAE or
myelin
oligodendrocyte glycoprotein (MOG)-specific immune responses using LPS-free
PBS as
a control.
To assess the biological activities of recombinant sDRS, the TRAIL-induced
apoptosis of mouse L929 cells was studied. As shown in Figure 8, TRAIL induced
apoptosis of L929 cells in a dose-dependent manner.
By the method of Song et al., 1997, L929 cells were first cultured in flat-
bottom
96-well plate, at 2 x 104 cells/well, in 100 p,1 AIM-V medium (Gibco BRL).
Sixteen
hours later, actinomycin D was added to the culture at 1 pg/well, and cells
were cultured
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for another 2 hours. Recombinant TRAIL (Biomol Research Laboratory, Plymouth
Meeting, PA) was then added with or without 5 ~g/ml of sDRS, and culture was
continued for an additional 5 hours. 3-4.5 dimethylthiazol-2,5 diphenyl
tetrazolium
bromide (MTT) was added only for the last hour of the culture to determine the
percentage of dead cells.
At the end of the culture, medium was removed and DMSO (100 ~1/well) was
added, and absorbance was determined at 595 nm. The percentage of dead cells
was
calculated using untreated cells as control. The survival rate of untreated
cells was
assumed to be 100%.
1o As seen in Figure 8, TRAIL induced apoptosis in the L929 cells was
completely
blocked by addition of recombinant sDRS. Thus, as demonstrated in the parallel
experiments in Example 1 in other tumor cell lines, including human Jurkat
cells and
K562 cells, the recombinant sDRS is biologically active and can be used to
block TRAIL
function in vitro.
Example 6 -TRAIL-blockade exacerbates MOG-induced autoimmune
encephalomyelitis.
To investigate the roles of TRAIL in vivo, the consequences of TRAIL-blockade
in an animal model of multiple sclerosis were examined. In this example,
female
C57BL/6 mice, 4-6 week old, were purchased from Jackson Laboratory (Bar
Harbor,
ME). Mice were housed in the University of Pennsylvania animal care facilities
and
2o were acclimated for 5-7 days before being used for experiments.
Mouse MOG38-50 (MOG, peptides 38-50) peptide was synthesized using Fmoc
solid phase methods and purified through HPLC by Research Genetics
(Huntsville, AL).
Pertussis toxin was purchased from List Biological Laboratories (Campbell,
CA). The
following reagents were purchased from PharMingen (San Diego, CA): rat anti-
mouse
IL-2 (clone JES6-1A12), IL-4 (BVD4-1D11), and IFN-y (R4-6A2) mAb; biotin-
labeled
rat anti-mouse IL-2 (clone JES6-5H6), IL-4 (BVD6-24G2), and IFN-y (XMG112D)
mAb; recombinant mouse IL-2, IL-4, IL-10 and IFN-y. Quantitative enzyme-linked
immunosorbent assay (ELISA) for IL-2, IL-4, and IFN-y was performed as per
manufacturer's recommendations.
3o Groups of C57BL/6 mice (6-8 mice per group) were immunized by subcutaneous
injection with 400 pg of MOG 38-50 peptide to induce experimental autoimmune
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encephalomyelitis (EAE). Prior to administration, the MOG 38-50 peptide was
emulsified in complete Freund's adjuvant (CFA) containing 100 p.g of
Mycobacterium
tuberculosis H37 RA (Difco, Detroit, MI). Pertussis toxin, 250 ng per mouse,
was
injected intravenously on the day of immunization, and again 48 hours later.
Mice were
evaluated daily and scored for (EAE) as follows (Chen et al., Science
265:1237(1994).
0 = no disease;
1 = tail paralysis;
2 = hind limb weakness;
3 = hind limb paralysis;
l0 4 = hind limb paralysis plus forelimb paralysis; or
5 = moribund or dead.
The incidence of the disease was more than 90% for all the experiments. Only
mice that developed EAE were included for calculating mean clinical scores.
Eight days after disease onset (when approximately 90% of the mice had
developed signs of EAE), mice were injected intraperitoneally with either 200
~g of
sDRS or a control protein (HAS), 200 ~g/mouse, was injected intraperitoneally
once
every other day for a total of 17 days. The presented data are representative
of three
experiments.
As shown in Figure 9A, C57BL/6 mice developed typical EAE, starting
2o approximately 18 days after immunization. Injection of sDRS, performed 7
days after
disease onset until the end of the experiment, shown as days 25 to 42,
significantly
exacerbated the disease. The mean maximal disease score in the control group
was
2.0+0.5. This was increased to 3.3+0.4 in the sDRS treated group. One out of
six mice
died from EAE in the sDRS treated group, whereas none died in the control
group.
To determine whether the effect of TRAIL-blockade is limited to the effector
phase of EAE, the consequences of TRAIL-blockade were also investigated during
the
inductive phase (days 0-16) of the disease. Thus, MOG-immunized mice were
treated
with sDRS or a control protein from the day of immunization until the day of
disease
onset. One mouse out of 12 developed signs of EAE.
3o As shown in Figure 9B, no significant differences between control and sDRS
treated groups were observed with respect to disease onset or severity. Thus,
the
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differences between the two groups are statistically significant as determined
by Mann-
Whitney test (P<0.01) for panel A, but not for B. This suggests that TRAIL-
blockade
during the inductive phase of EAE alone may not be sufficient to affect the
disease
course.
Example 7 - TRAIL-blockade enhances the formation of inflammatory lesions in
the
CNS.
To investigate the effect of TRAIL-blockade on the formation of inflammatory
lesions in the central nervous system (CNS), quantitative histopathological
studies were
conducted to determine inflammation and apoptosis of spinal cords.
1o Mice were treated as in Example 6, and sacrificed 42 days after
immunization.
The mice were then perfused with PBS and 10% formalin phosphate. Spinal cords
were
first embedded in paraffin, cut into 5 pieces, and then sectioned at 5 ~m and
stained with
luxol fast blue and cresyl violet (Moore et al., Laboratory Invest. 51:416
(1984).
The total area of tissue section and the area of inflammation were measured
using
the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) in a
blinded
manner. The percentage of the spinal cord with inflammation was calculated as
follows:
(area of the spinal cord that is infiltrated by inflammatory cells / total
area of the spinal
cord sections measured) x 100. A total of ten (10) tissue sections from
cervical, thoracic,
lumbar and sacral spinal cord were analyzed for each animal.
2o As shown in Figures 10A (sDRS treated mice) and lOB (control; BSA treated
mice), inflammatory lesions were readily detectable.
Apoptosis was determined by TUNEL staining of spinal cords from sDRS (Figure
10C) and BSA (Figure 10D) treated mice (original magnification x 200). TUNEL
staining was performed on paraffin-embedded formalin-fixed spinal cord
sections, as
described by Chen et al., J. Neuroimmunol. 82:149 (1998). Briefly, sections
were de-
waxed in xylene, hydrated in water/ethanol and washed in PBS. Endogenous
peroxidase
activity was inactivated by incubating the tissue in 3% H20z. Fragmented DNA
in
apoptotic cells was labeled with digoxigenin-conjugated dUTP (Roche Molecular
Biochemicals, Indianopolis, IN) using TdT enzyme (Clontech, Palo Alto, CA).
The
labeled DNA was then detected by peroxidase-conjugated anti-digoxigenin
antibody
(Roche) using diaminobenzidine as substrate. Counterstaining was performed
with
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methyl green. The number of apoptotic cells in the lesions was determined by
light
microscopy. Arrows in Figures l OC and l OD indicate apoptotic nuclei.
The inflammatory lesions consisted mostly of lymphocytes and macrophages as
well granulocytes and microglial cells. To quantify the degree of
inflammation, the area
of spinal cord sections that showed signs of infiltration, as well as the
total area of each
spinal cord, sections were measured using the Image-Pro Plus software. The
degree of
inflammation was then evaluated based on the percentage of the spinal cord
areas that
show signs of inflammation. As shown in Figure 1 l, the inflammation in the
mice
treated with sDRS was significantly more severe, than that seen in the control
mice. Each
to data point represents a percentage of spinal cord section that was
inflamed. The
horizontal bars represent the means of respective groups. The differences
between the
two groups are statistically significant as determined by Student t test
(p<0.05). Filled
squares represent mice treated with HSA. Open circles represent mice treated
with sDRS.
The extent of demyelination correlated well with the degree of inflammation
and
clinical score. Mice with similar disease scores exhibited similar degrees of
demyelination and inflammation, regardless of treatment groups (data not
shown).
Example 8 - TRAIL-blockade does not affect apoptosis of inflammatory cells in
the CNS.
To test whether the effect of sDRS on EAE can be explained by its blockade of
"TRAIL-induced apoptosis" of inflammatory cells, the effect of TRAIL-blockade
on
apoptosis of inflammatory cells in the CNS was tested. As shown in Figures l
OC and
l OD, apoptotic cells were readily detectable in spinal cords of both control
and sDRS
treated mice. The vast majority of apoptotic cells were localized within the
inflammatory
lesions, with a few detected outside the inflamed area. In sections that did
not contain
inflammatory lesions, no apoptotic cells were detected.
To quantitatively compare the degree of apoptosis between control and sDRS
treated groups in the CNS, the number of apoptotic cells per mm2 were
calculated in
spinal cord that showed signs of inflammation. Groups of C57BL/6 mice, 4 mice
per
group, were treated as in Example 6, and sacrificed 42 days after
immunization. Spinal
cord was treated and examined for apoptosis as previously described. The
numbers of
apoptotic nuclei per mm2 of inflamed tissue were counted and plotted against
the
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percentages of the corresponding spinal cord sections that were inflamed
(degree of
inflammation) in the same section.
As shown in Figure 12, no statistically significant differences in the degree
of
apoptosis were observed between control and sDRS treated groups as determined
by
Mann-Whitney test (p>0.05). Filled squares represented mice treated with HSA.
Open
circles represented mice treated with sDRS. These results suggest that TRAIL
may not
regulate apoptosis of inflammatory cells in the CNS.
Example 9 - TRAIL-blockade enhances anti-MOG T cell responses.
EAE is a T cell-mediated autoimmune disease. To determine whether TRAIL-
1 o blockade affected the functions of encephalitogenic T cells, anti-MOG T
cell responses
were studied in mice following TRAIL-blockade.
Groups of C57BL/6 mice, 6 mice per group, were immunized with MOG peptide
to induce EAE. Recombinant sDRS or HSA, 200 ~g/mouse, was injected
intraperitoneally once every other day for a total of 16 days as in Example 6.
Mice were
sacrificed 10 days after the last injection of sDRS, and splenocytes were
tested for anti-
MOG proliferative and cytokine responses.
For cytokine assays, splenocytes were cultured at 1.5 x 106 cells/well in 0.2
ml of
DMEM (Gibco BRL Life Technologies, Grand Island, NY) containing 10% fetal
bovine
serum and various amounts of MOG38-50 peptide. Culture supernatants were
collected
40 hours later and cytokine concentrations determined by ELISA. For
proliferation
assays, 0.5 x 106 cells/well were used. 3H-thymidine was added to the culture
at 48 hr,
and cells were harvested 16 hours later. Radioactivity was determined using a
flatbed
beta counter (Wallac, Gaithersburg,'MD).
The data presented are representative of two experiments. Mice were immunized
with MOG peptide to induce EAE and treated with either sDRS or BSA for a total
of 16
days. Anti-MOG T cell responses were determined ex vivo 10 days after the last
injection
of sDRS. As shown in Figure 13, splenocytes of BSA-treated mice produced
primarily
THl type cytokines (i.e., IL-2 and IFN-y) in response to MOG peptide. This was
significantly increased in mice treated with sDRS. A small but significant
amount of IL-
4 was also detected in sDRS-treated group. Interestingly, lymphocyte
proliferative
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responses were comparable between BSA- and sDRS-treated groups. These results
suggest that TRAIL-blockade enhances functions of both TH1 and TH2 type cells
in vivo.
In summary, the present findings establish that 1 ) unlike its apoptotic
effect on
tumor cells, TRAIL inhibits cell cycle progression of non-transformed
lymphocytes, and
2) unlike TNF that promotes inflammation, TRAIL inhibits autoimmune
inflammation
and prevents self tissue destruction.
Each and every patent, patent application and publication that is cited in the
foregoing specification is herein incorporated by reference in its entirety.
While the foregoing specification has been described with regard to certain
to preferred embodiments, and many details have been set forth for the purpose
of
illustration, it will be apparent to those skilled in the art that the
invention may be subject
to various modifications and additional embodiments, and that certain of the
details
described herein can be varied considerably without departing from the spirit
and scope
of the invention. Such modifications, equivalent variations and additional
embodiments
15 are also intended to fall within the scope of the appended claims.
