Note: Descriptions are shown in the official language in which they were submitted.
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USE OF A PHOSPHOTYROSYL PHOSPHATASE INHIBITOR FOR THE
TREATMENT OF IMMUNOSUPPRESSED PATIENTS
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The present invention relates to a method for
restoring a functional immunity in immunosuppressed
patients. More specifically, the invention relates to
the use of phosphotyrosyl phosphatase inhibitors for
io restoring normal immune functions in individuals
infected with viruses such as the human
immunodeficiency virus (HIV).
(b) Description of Prior Art
T-cell activation results in the induction of
i5 several genes which play crucial roles for the
subsequent functions that these cells accomplish in the
immune system. Different types of transcription factors
are thus induced early on and directly affect
transcription of immune response genes such as
zo inflammatory cytokines. This T-cell activation is of
prime importance for the immune system to mount an
adequate response. One of these transcription factors
is known as the NF-KB complex. Binding sites in the
locus for several T-cell specific genes have been
25 identified for this factor. The NF-KB (Rel) family is
made of several members which either form homo- or
heterodimers. Some of these dimers actually are
involved in higher order transcriptional complexes
which are in return important for the regulation of
3o specific T-cell genes. NF-KB can act in concert with
the transcription factor NFAT to achieve regulation of
the IL-2 and y-interferon genes (Sica et al., J. Biol.
Chem. 272:30412-30420, 1997), as well as the human
immunodeficiency virus type-1 (HIV-1) regulatory
35 elements (LTR) (Kinoshita et al., Immunity 6:235-244,
1997) . -
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NFAT is another family of Rel-related
transcription factors which are activated at early
times after T-cell activation. Like NF-KB, this factor
(at least one member of the family called NFATc or
s NFAT2) is sequestered in the cytoplasm and is
translocated to the nucleus upon increase in
intracellular calcium content. This elevation in
intracellular Ca2+ triggers conformational changes in
calmodulin and increases its binding to the calcineurin
io serine/threonine phosphatase, leading in turn to
activation of this enzyme by displacement of an auto-
inhibitory domain from the catalytic site. The ensuing
dephosphorylation of the NFAT factor by calcineurin
then makes the nuclear localizing sequence accessible
15 and hence allows it to freely migrate into the nucleus.
This mechanistic description of the activation steps of
NFAT has been also well established by the
demonstration that the immunosuppressors cyclosporin A
(CsA) and FK506 were both acting on an upstream
2o component of the NEAT activation cascade which was
resulting in inhibition of the calcineurin phosphatase
activity (Liu et al., Cell 66:807-815, 1991). Upon
reaching the nucleus, NEAT is usually found associated
with the complex AP-1 which is newly synthesized and,
2s after association, acquires high transactivating
potential upon binding to its consensus sequence
5' - (T/A) GGAAA (A/N) (A/T/C) -3' . Certain types of NFAT
binding sites suggested to be cooperative with AP-1 are
known to resemble NF-KB binding sites and include the
3o CD28RE element located in the locus of the IL-2 and GM-
CSF genes. Several members of the NEAT family of
transcription factors are present in human T-cells such
as NFATc (NFAT2), NFAT4/x and NFATp (NFAT1) (Lyakh et
al., Mol. Cell. Biol. 17:2475-2484, 1997). Numerous T-
35 cell expressed genes are regulated by the NFAT
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transcription factors. Indeed, potentially functional
NFAT binding sites have been identified in the promoter
region of the IL-2, IL-3, IL-4 IL-5, GM-CSF, y-
interferon and IL-13 genes, all of which were found to
be sensitive in their transcriptional regulation to
either CsA or FK506. The importance of NFAT members in
T-cell function is thus very clearly demonstrated.
Activation of T-cells is a very complex process
which involves cell-to-cell interactions of several
io cell surface molecules. The most important polypeptide
complex remains the T-cell receptor (TCR). Although by
itself, the TCR/CD3 complex can induce a cascade of
intracellular events, it also needs co-receptors such
as CD4 and CD28 for optimal T-cell activation to
i5 proceed. Several second messengers are orchestrating
the signals induced from the membrane to the nucleus
but one common theme is the increase in the overall
intracellular tyrosine phosphorylation level. Different
studies have shown the importance of this increase in
2o phosphorylation level which is believed to be initially
controlled by two specific protein tyrosine kinases,
p561ck and p59fyn. Other important tyrosine kinases are
involved in the TCR-initiated cascade and include the
syk family member ZAP-70. It is also known that the
z5 protein tyrosine phosphatases (PTPs), enzymes
responsible for the dephosphorylation of proteins on
their tyrosine residues, are very important modulators
of T-cell activation cascade.
Severe immunological abnormalities have been
3o reported to precede the quantitative decline of CD4+ T
cell numbers seen in HIV-1-infected persons. The exact
mechanisms) responsible for this unresponsiveness
(anergy) is (are) still incompletely defined although
in vitro studies have demonstrated that signal
35 transduction of the T-cell activation pathway was
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severely impaired in HIV-1-infected T-cells. It was
first proposed, among several possibilities, that the
reduced proliferative responses were resulting from the
interaction between the CD4 surface molecule and the
external viral envelope glycoprotein gp120 because
inhibition of T-cell receptor-dependent proliferative
response has been observed following gp120 treatment.
The anergic state caused by gp120 treatment was
attributed to inhibition of mRNA IL-2 expression, as
io well as to IL-2 secretion, in CD4+ T lymphocytes since
addition of exogenous IL-2 was able to restore
proliferative responses (Oyaizu et al., Proc. Natl.
Acad. Sci. USA 87:2379-2383, 1990). The molecular
mechanism responsible for the functional impairment of
i5 CD4+ T lymphocytes before CD4+ T-cell depletion seen in
HIV-1-infected persons is still incompletely under
stood. However, for specific antiviral therapies to be
effective in the improvement of seropositive
individuals, immunomodulators should be considered as
2o important additions.
It would be highly desirable to be provided
with a method for activating T-cells, such activation
restoring a functional immunity in immunosuppressed or
immunodeficient patients.
STJNIMARY OF THE INVENTION
One aim of the present invention is to provide
a method for activating T-cells for restoring a
functional immunity in immunosuppressed or
3o immunodeficient patients.
In accordance with the present invention there
is provided a bis-peroxovanadium (bpV) compound for
restoring immune functions and an overall immune
response of a patient suffering from an immune
disorder. The bpV compound of the present invention is
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a phosphotyrosyl phosphatase inhibitor, and is
preferable made of an oxo ligand, two peroxo anions,
and an ancillary ligand located in an inner
coordination sphere of vanadate.
s The compound of the present invention may be
used for treating immune disordes caused for example by
an infection with a virus, preferably a human
immunodeficiency virus.
In accordance with the present invention there
io is also provided a pharmaceutical composition for
restoring immune functions and an overall immune
response of a patient suffering from an immune
disorder. The pharmaceutical composition comprises a
bis-peroxovanadium (bpV) compound and a
is pharmaceutically acceptable carrier.
In accordance with the present invention, the
bpV compound may be used for the manufacture of a
medicament for restoring immune functions and an
overall immune response of a patient suffering from an
2o immune disorder.
The medicament is preferably formulated for
intravenous, subcutaneous, intradermal or topical
application. The medicament may be administered via a
patch, an implant or by inhalation of a powder or an
z5 aerosol spray. Alternatively, the medicament may be
formulated in a liposomal composition suitable for
administration or in a tablet form.
The bpV compound of the present invention may
also be used in combination with an antiviral agent or
3o with another immunomodulator.
Also in accordance with the present invention,
there is provided a method for restoring immune
functions and an overall immune response in a patient
suffering from an immune disorder comprising
3s administering to a patient in need of such a treatment
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a therapeutically effective amount of a bis-
peroxovanadium (bpV) compound.
BRIEF DESCRIPTION OF THE DRAWINGS
s Fig. 1 illustrates a bar chart indicating that
the bpV-mediated induction of HIV-1 LTR is sensitive to
Nasal, FK506 and CsA;
Figs. 2A and 2B illustrate a bar chart
indicating that the induction of wild-type and NF-KB
io mutated HIV-1 LTR by bpV compounds is sensitive to
FK506;
Figs. 3A and 3B show that NFAT-driven
luciferase gene expression is induced by the bpV
molecules and is FK506-sensitive;
i5 Figs. 4A and 4B illustrate a radiographic film
representing the bpV-mediated induction of nuclear
translocation of NFAT;
Fig. 5 illustrates a bar chart showing that
expression of a dominant negative mutant of p2lras
2o blocks NEAT activation by bpV compounds;
Figs. 6A and 6B illustrate bar charts illus-
trating that the presence of p561ck is required for
bpV-mediated activation of NFAT;
Figs. 7A and 7B illustrate a scattergram and
2s bar chart indicating that bpV compounds induce a FK
506-sensitive activation of the human IL-2 promoter
which synergizes with PMA; and
Figs. 8A and 8B illustrate bar charts illus
trating the correlation between IL-2 promoter activity
3o and IL-2 secretion.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with a preferred embodiment, the
present invention relates to the use of bpV compounds,
3s a new class of potent phosphotyrosyl phosphatase
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inhibitors, in the treatment of humans suffering from a
deficient immune system, a pathological condition which
can lead to the development of opportunistic infections
and cancers.
s The present invention comprises a class of
biologically compounds which are acting as potent
protein tyrosine phosphatase inhibitors which are
useful in treating various pathological conditions in
humans accompanying immune suppression. These compounds
io are useful for enhancing the protective response of the
immune system and to restore natural immunity.
bpV compounds are made of an oxo ligand, two
peroxo anions, and an ancillary ligand located in the
inner coordination sphere of vanadate.
i5 Ancillary ligands located in the inner
coordination sphere of the vanadate atom include
bipyridine [bipy]; picolinic acid (i.e. pyridine-2-
carboxylic acid) anion [pic]; S-hydroxypyridine-2-
carboxylic acid anion [HO-pic]; 1,10-phenanthroline
20 [phen]; 4,7-dimethyl-1,10-phenanthroline (Me2phen];
3,4,7,8-tetramethyl-1,10-phenanthroline [Me4phen];
oxalic acid dianion [ox] .
Formulas and abbreviations of a number of
structurally defined bpV compounds are listed herein to
2s illustrate the invention rather than to limit it:
K [V0 (OZ) 2bipy] 'SHzO, bpV [bipy] ; Kz [V0 (OZ) zpic] 'H20, bpV
[pic] ; KZ [V0 (02) 2 (EOpic) ] 'H20, bpV [Hopic] ;
K [V0 (Oz) zphen] '3H20, bpV [phen] ; K [V0 (OZ) 2 (4 , 7-Nezphen) ] ;
bpV [Me2phen] ; K [V0 (OZ) 2 (Me4phen) ] , bpV [Me4phen] ; and
3o K3 [V0 (OZ) 20X] '2H20, bpV [ox] .
Treatment with bpV compounds represent a new
therapeutic avenue to reconstitute and/or enhance
mucosal, humoral and cellular immune responses in
patients suffering from immune response related
3s syndromes.
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In relation to Examples 1 to 7 given
hereinafter, the materials used and the analyses and
assays carried out were as follows:
Cell lines
s The lymphoid T-cell lines used include Jurkat
(clone E6-1), Sup-T1, JCaMl.6 and JCaMI.Tag (Straus et
al., J. Biol. Chem. 271:9976-9981, 1996). JCaMl.6 is a
derivative of the Jurkat leukemic T-cell line that is
deficient in p561ck expression. JCaMI.TAg is a
io derivative of JCaMl.6 cells that stably express the
SV40 large T antigen. The 1G5 T-cell line is a Jurkat
derivative that harbors two stably integrated
constructs constituted of the luciferase gene under the
control of the HIV-1SF2 LTR. The Jurkat derivatives
is deficient in capacitative calcium entry (clones CJ1
through CJ5, plus the parental line CJ) (Fanger et al.,
J. Cell. Biol. 131:655-667, 1995) were also used. These
cells were maintained in complete culture medium made
of RPMI-1640 supplemented with 10% fetal bovine serum,
2o glutamine (2 mM), penicillin G (100 U/ml), and
streptomycin ( 100 ~Cg/ml ) .
Plasmids and antibodies
pLTR-LUC (HIV-1 LTR from strain HXB2) and
pmKBLTR-LUC plasmids were used (Henderson et al., J.
2s Virol. 69:5337-5344, 1995). These molecular constructs
contain the luciferase reporter gene under the control
of wild-type (GGGACTTTCC) or NF-KB-mutated (CTCACTTTCC)
HIV-1 LTR. The pKB-TATA-LUC plasmid contains the
minimal HIV-1 KB region (-105/-70) and a TATA box
3o placed upstream of the luciferase reporter gene (Sun et
al., Mol. Cell. Biol. 16:1058-1065, 1996). pIL-2-LUC,
containing the complete 320-by IL-2 promoter
controlling the luciferase gene, and pNFAT-LUC,
containing the IL-2 minimal promoter with 3 tandem
3s copies of the NFAT-1 binding site were also used
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(Timmerman et al., Nature 383:837-840, 1996). The
pLTR/N17 plasmid codes for a dominant negative of
p2lras under the control of murine leukemia virus LTR
(rasNl7) (Baldari et al., J. Biol. Chem. 268:2693-2698,
s 1993). pEFneoLckWT encodes for the wild type human
p561ck protein and pEFneo is the control plasmid (Liu
et al., Proc. Natl. Acad. Sci. USA 90:8957-8961, 1993).
Preparation of bpV compounds
bpV molecules were prepared as described
io previously (Posner et al., J. Biol. Chem. 269:4596
4604, 1994). Briefly, Vz05 was dissolved in an aqueous
KOH solution and then mixed with 30% H202 and the
respective ancillary ligand in addition to the ethanol
for optimal precipitation. Characterization of the bpV
i5 molecules were carried out by infrared 1H-NMR and
Vanadium-51 (51V) NMR spectroscopy. Stock solutions of
bpV molecules (1 mM in phosphate buffered saline pH
7.4) were kept at -85°C until use.
Transfections
2o Transient transfections were done using the
DEAF-Dextran method, as follow. Cells (5 x 106) were
first washed once in TS buffer (25 mM Tris-HCl [pH
7 . 4 ] , 5 mM KCl , 0 . 6 mM Na2HP04 , 0 . 5 mM MgCl2 , and 0 . 7 mM
CaCl2) and resuspended in 0.5 ml of TS containing 15 to
2s 30 ~.g of the indicated plasmid(s) and 500 ~,g/ml of
DEAE-dextran (final concentration). The
cells/TS/plasmid/DEAE-dextran mix was incubated for 25
min at room temperature. Thereafter, cells were diluted
at a concentration of 1 x 106/m1 using complete culture
3o medium supplemented with 100 ~M of chloroquine and
transferred into 6-well plates. After 45 minutes of
incubation at 37°C, cells were centrifuged, resuspended
in complete culture medium and incubated at 37°C for 24
h. To minimize variations in plasmid transfection
35 efficiencies, transfected cells were pooled 24 h after
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transfection and were next separated into various
treatment groups. Stably transfected Jurkat cells were
obtained by electroporation. Briefly, 10 x 106 cells in
mid-log phase were washed once and resuspended in 400
s ~.1 of complete RPMI medium plus 20 ~,g of either pNFAT-
LUC or pIL-2-LUC plasmid. This mixture was transferred
to a 0.4 cm-gap electroporation cuvette. Cells were
transfected in Bio-Rad apparatus using standard voltage
and capacitance conditions (250 V and 960 ~F). After a
l0 10 min incubation period, transfected cells were
resuspended at a density of 1 x 106/m1 in complete RPMI
medium for 24 h. Cells were then diluted at 5 x 104
cells/ml and 1 mg/ml Geneticin was added. After two
weeks of selection, 6418-resistant T-cells were pooled
15 and identified as either J-NFAT-LUC or J-IL-2-LUC.
Stimulations and luciferase assays
Stably or transiently transfected cells were
seeded at a density of 105 cells per well (100 ~.1) in
96-well flat-bottom plates. Cells were either left
2o unstimulated or were stimulated with the different bpV
molecules at 10 ~,M (bpV [HOpic] , bpV [bipy] , and
bpV[pic]), phytohemagglutinin (PHA-P at 3 ~g/ml),
phorbol 12-myristate 13-acetate (PMA at 20 ng/ml),
Ionomycin (Iono at 1 ~M), anti-CD3 antibody (clone OKT3
25 at 3 ~.g/ml ) , ant i -CD2 8 ant ibody ( clone 9 . 3 at 1 ~g/ml )
in a final volume of 200 ~.1. Next, cells were incubated
at 37°C for 8 h unless otherwise specified. For some
experiments, prior to the addition of activators, cells
were either left untreated or pretreated with FK506 (10
3o ng/ml) or cyclosporin A (CsA at 100 ng/ml) for 15 min
at 37°C or sodium salicylate (Nasal at 2.5 mM) for 1 h
at 37°C. Luciferase activity was determined as follow.
Briefly, following the incubation period, 100 ~.1 of
cell-free supernatant were withdrawn from each well and
35 25 ~,1 of cell culture lysis buffer (25 mM Tris
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phosphate [pH 7.8], 2 mM DTT, 1% Triton X-100', and 10%
glycerol) were added for incubation at room temperature
for 30 min. The extracts (20 ~.l) were analyzed in 96-
well plates using a Dynex MLX luminometer. Each well
was injected with 100 ~l of luciferase assay buffer (20
mM tricine, 1 . 07 mM (MgC03) 4.Mg (OH) z. 5 H20, 2 . 67 mM
MgS04, 0.1 mM EDTA, 270 ~,M coenzyme A, 470 ~.M
luciferin, 530 ~M ATP, and 33.3 mM DTT). Light output
was measured for 20 s with a two second delay. Values
to are expressed as RLU (Relative Light Units) as measured
by the apparatus.
IL-2 production
Following stimulation for 24 h, cell-free
supernatants from either Jurkat or J-IL-2-LUC cells
were removed and quantification of secreted IL-2 was
carried out using a commercial cytokine matched
antibody pair (Endogen). Briefly, polyclonal anti-IL-2
antibody was first diluted to a concentration of 2
~,g/ml in phosphate-buffered saline (PBS) and 100
~.1/well were transferred in the wells of a 96-well
plate and left overnight at room temperature. After
three washes with phosphate-buffered saline (PBS)
containing 0 . 05 % (v/v) Tween-2OT"" (PBST) , 200 ~.l of PBST
plus 1% BSA (w/v) was added per well and incubated for
30 min at 37°C. Following three further washes with
PBST-BSA, 50 ~Cl of a biotinylated anti-IL-2 antibody at
a final concentration of 0.25 ~g/ml in PBST-BSA along
with 50 ~.1 of tested cell supernatant were dispensed in
the wells for another incubation period of 1 h at 37°C.
3o Three washes with PBST-BSA were then performed and 100
~1 of a 1:4000 dilution of the streptavidin-tagged
horseradish peroxidase enzyme (Streptavidin-HRP40,
Research Diagnostics Inc., Flanders, NJ) was added to
the wells and left at room temperature for 30 min.
After another three washes with PBST-BSA, 100 ~.1 of
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TMB-S (Research Diagnostic Inc., Flanders, NJ) were
finally dispensed in each well. After 15 min, the
reaction was stopped by adding 50 ~.1 of 1 M H3P04 and
absorbance was read using a microtiter plate reader
(450 nm filter, reference wavelength 690 nm).
Appropriate dilutions of recombinant human IL-2 were
used to produce standard curves (3.9 to 250 U/ml).
Preparation of nuclear extracts
Jurkat cells were either left untreated or were
io incubated for 1 h at 37°C with the bpV compounds (10
~,M). The incubation of Jurkat cells with the various
stimulating agents was terminated by the addition of
ice-cold PBS and nuclear extracts were prepared as
follow. Sedimented cells were resuspended in 400 ~,1 of
i5 cold buffer A (10 mM N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid [HEPES; pH 7.9], 1.5 mM MgCl2, 10
mM KC1, 0.5 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonyl fluoride). After 10 min on ice,
the lysate was vortexed for 10 s, and samples were
2o centrifuged for 10 s at 12,000 x g. The supernatant
fraction was discarded and the cell pellet was
resuspended in 100 ~,1 of cold buffer C (20 mM HEPES-KOH
[pH 7 . 9 ] , 2 5 % glycerol , 42 0 mM NaCl , 1 . 5 mM MgCl2 , 0 . 2
mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM
z5 phenylmethylsulfonyl fluoride) and incubated on ice for
20 min. Cellular debris were removed by centrifugation
at 12,000 x g for 2 min at 4°C and the supernatant
fraction was stored at -70°C until use.
EMSA
3o Electrophoretic mobility shift assay (EMSA) was
performed with 10 ~g of nuclear extracts. Protein
concentrations were determined by the bicinchoninic
assay with a commercial protein assay reagent (Pierce,
Rockford, IL). Nuclear extracts were incubated for 30
35 min at 23°C in 15 ~1 of buffer (100 mM HEPES [pH 7.9] ,
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40% glycerol, 10% Ficoll, 250 mM KC1, 10 mM
dithiothreitol, 5 mM EDTA, 250 mM NaCl, 2 ~.g poly[dI-
dC], 10 ~g nuclease-free bovine serum albumin fraction
V) containing 0.8 ng of 32P-5'-end-labeled double
stranded DNA (dsDNA) oligonucleotide. Double stranded
DNA (100 ng) was labeled with [y-32P] -ATP and T4
polynucleotide kinase in a kinase buffer (New England
Biolabs, Beverly, MA). This mixture was incubated for
30 min at 37°C and the reaction was stopped with 5 ~1
to of 0.2 M EDTA. The labeled oligonucleotide was
extracted with phenol/chloroform and passed through a
G-50 spin column. The dsDNA oligonucleotide, which was
used as a probe or as a competitor, contained the
consensus NFAT-binding site from the murine IL-2
promoter (5'-TCGAGCCCAA AGAGGAAAAT TTGTTTCATG-3'). A
dsDNA oligonucleotide containing a mutated NFAT-binding
site was also used in competition experiments (5'-
TCGAGCCCAA AGACCTTAAT TTGTTTCATA CAG-3'). Oligonucleo-
tides were purchased from Gibco-BRL. DNA-NFAT complexes
2o were resolved from free labeled DNA by electrophoresis
in native 4% (w/v) polyacrylamide gels containing 0.5X
TBE buffer. The gels were subsequently dried and
autoradiographed. Cold competitor assays were carried
out by adding a 100-fold molar excess of homologous
unlabeled dsDNA NFAT oligonucleotide or of dsDNA NF-KB
oligonucleotide (5'-ATGTGAGGGG ACTTTCCCAG GC-3')
simultaneously with the labeled probe. Supershift
assays were performed by pre-incubation of nuclear
extracts with 1 ~cl of either preimmune serum (control)
or specific antibody in the presence of all the
components of the binding reaction described above for
15 minutes at room temperature prior to the addition of
the probe. The antibody used for the supershift assay
is specific for the NFATc subunit of the NFAT complex.
This antibody along with the NF-KB oligonucleotide were
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purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA) .
Dose Ranges
The therapeutically effective amount of the
s inhibitor of the present invention to be administered
will vary with the particular inhibitor used, the type
or mode of administration, the concurrent use of other
active compounds, host age and size, type, response of
individual patients, and the like. In the case of bpV
io compounds, it will be administered in sufficient doses
for restoring immunity and to obtain an effective peak
or steady-state concentration of about 100 nM to 25 ~.M,
usually about 10 ~.M in plasma as suggested by the
concentrations of bpV compounds tested and found to be
i5 effective in in vitro experiments. An effective dose
amount of the bpV compounds is thus to be determined by
the clinician after a consideration of all the above-
mentioned criteria. The dosage amount of agent
necessary to obtain the desired concentrations in blood
2o can be determined by pharmacokinetic studies, as
described in Marleau et al., J. Immunol. 150: 206,
1993, and Marleau et al, Br. J. Pharmacol. 112: 654,
1994.
Pharmaceutical Compositions
25 Any suitable type or mode of administration may
be employed for providing a mammal, especially a human
with an effective dosage of a bpV compound of the
present invention. For example, oral, parenteral and
topical may be employed. Dosage forms include tablets,
3o capsules, powders, solutions, dispersions, suspensions,
creams, ointments and aerosols.
The pharmaceutical compositions of the present
invention comprise a bpV compound as a phosphotyrosyl
phosphatase inhibitor and as the active ingredient, and
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a pharmaceutically acceptable carrier and optionally
other therapeutic ingredients.
It should be recognized that the bpV compounds
can be used in a variety of ways in vivo. It can be
s formulated into pharmaceutical compositions according
to any known methods of preparing pharmaceutically
useful compositions. In this manner, the bpV compounds
are combined in admixture with a pharmaceutically
acceptable carrier vehicle. Suitable vehicles and
io their formulation, including human proteins, e.g.,
human serum albumin, are described for instance in
Remington's Pharmaceutical Sciences (16th ed. Osol, A.,
ed., Mack, Easton, PA [1980]). In order to form a
pharmaceutically acceptable composition suitable for
i5 effective administration, such compositions will
contain a therapeutically effective amount of the bpV
compound or amount resulting in the restoration of
functional immunity, together with a suitable amount of
carrier vehicle. The amounts required for restoring
2o functional immunity can be determined by in vivo
pharmacological studies.
The bpV compound can be formulated as a sterile
pharmaceutical composition for therapeutic use which is
suitable for intravenous or intraarterial
2s administration. The product may be in a solvent-free
form and ready to be reconstituted for use by the
addition of a suitable carrier or diluent, or
alternatively, it may be in the form of solution which
may be aqueous or organic.
3o For reconstitution of a solvent-free product in
accordance with the present invention, one may employ a
sterile diluent, which may contain materials generally
recognized for approximating physiological conditions.
In this manner, the sterile diluent may contain salts
35 and/or buffering agents to achieve a physiologically
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acceptable tonicity and pH, such as sodium chloride,
phosphate and/or other substances which are
physiologically acceptable and/or safe for use.
When used as an aqueous solution, the
s pharmaceutical composition will for the most part
contain many of the same substances described above for
the reconstitution of a solvent-free product. when
used in solution in an organic solvent, a small volume
of the solution containing the bpV compound will be
to diluted with an aqueous solution that will contain many
of the same substances described above for the
reconstitution of a solvent-free product. The
pharmaceutical composition, for the most part, will
thus contain many of the same substances described
is above for the reconstitution of a solvent-free product.
The bpV compound useful in the methods of the
present invention may be employed in such forms as, for
example, sterile solutions for injection or
encapsulated (for instance in liposomes) or embedded
20 (for example in suppositories) for slower long-lasting
release.
The bpV compound may be used in combination
with other agents including, but not limited to, anti-
viral agents or other immunomodulator.
2s Where the subject bpV compound is to be admin
istered to a host as an inhibitor of phosphotyrosyl
phosphatase, the bpV compound may be administered, for
example, intraarterially, intravenously, intraperito
neally, subcutaneously, intramuscularly, by injection,
3o by suppository, by inhalation, or the like.
The mode of administration by injection
includes continuous infusion as well as single or
multiple boluses. Useful administration type or mode
also includes the use of implantable internal pumps for
3s continuous infusion into a blood vessel or at different
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sites such as the peritoneal cavity or subcutaneously.
Such techniques are disclosed in Cecil's Text Book of
Medicine (chapter 164, 19th Edition, 1992) for the
treatment of hepatic cancers. Transdermal
s administration by means of a patch containing the bpV
compound of the present invention may also be a useful
administration mode.
Additional pharmaceutical methods may be
employed to control the duration of action. For exam
to ple, controlled release preparations may be achieved
through the use of macromolecules to complex or absorb
the bpV compound. The controlled delivery may be
achieved by selecting appropriate macromolecules (for
example, polyesters, polyamino acids, polyvinyl pyrro-
is lidone, ethylene-vinyl acetate, methyl cellulose, car
boxymethyl cellulose, protamine sulfate or serum
albumin, the appropriate concentration of macromole
cules, as well as the methods of incorporation). In
this manner, release of the bpV compound can be
2o controlled.
Another possible method useful in controlling
the duration of action by controlled release
preparations is the incorporation of the bpv compound
into particles of a polymeric material such as
2s polyesters, polyamino acids, hydrogels, poly(lactic
acid), or ethylene-vinyl acetate copolymers.
Instead of incorporating the subject bpV
compound into polymeric particles, it is also possible
to entrap them in microcapsules prepared, for instance,
3o by coacervation techniques or by interfacial
polymerization (for example, hydroxymethyl cellulose or
gelatin microcapsules and polymethyl methacrylate
microcapsules, respectively), in colloidal drug
delivery systems (for example, liposomes, albumin
35 microspheres, microemulsions, nanoparticles and
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nanocapsules) or in macroemulsions. Such techniques
are disclosed in Remington's Pharmaceutical Sciences
(16th ed. Osol, A., ed., Mack, Easton, PA [1980]).
The compositions include compositions suitable
s for oral or parenteral administration. Conveniently
they are presented in unit dosage form and prepared by
any of the methods well-known in the art of pharmacy.
In practical use, the bpV compound can be
combined as the active ingredient in intimate admixture
io with a pharmaceutical carrier according to conventional
pharmaceutical compounding techniques. The carrier may
take a wide variety of forms depending on the form of
preparation desired for administration. In preparing
the compositions for oral dosage form, any of the usual
15 pharmaceutical media may be employed, such as, for
example, water, glycols, oils, alcohols, flavoring
agents, preservatives, coloring agents and the like in
the case of oral liquid preparations, such as, for
example, suspensions; elixirs and solutions; or
2o carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants,
binders, disintegrating agents and the like in the case
of oral solid preparations such as, for example,
powders, capsules and tablets. If desired, tablets may
2s be coated by standard aqueous or nonaqueous techniques.
Pharmaceutical compositions of the present
invention suitable for oral administration may be
presented as discrete units such as capsules, cachets
or tablets each containing a predetermined amount of
3o the bpV compound, as a powder or granules or as a
solution or suspension in an aqueous liquid, a non-
aqueous liquid, an oil-in-water emulsion or a water-in-
oil emulsion. Such compositions may be prepared by any
of the methods of pharmacy such methods including the
35 step of bringing the bpV compound into association with
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the carrier which includes one or more necessary
ingredients. In general, the compositions are prepared
by uniformly and intimately admixing the bpV compound
with liquid carriers or finely divided solid carriers
s or both, and then, if necessary, shaping the product
into the desired presentation. For example, a tablet
may be prepared by compression of molding, optionally
with one or more accessory ingredients. Compressed
tablets may be prepared by compressing in a suitable
to machine, the active ingredient in a free-flowing form
such as powder or granules, optionally mixed with a
binder, lubricant, inert diluent, surface active or
dispersing agent. Molded tablets may be made by
molding in a suitable machine, a mixture of the
15 powdered compound moistened with an inert liquid
diluent.
It will be understood that the bpV compound is
to be administered in pharmacologically or
physiologically acceptable amounts, by which is to be
2o understood amounts not harmful to the patient, or
amounts where any harmful side effects in individual
patients are outweighed by the benefits. Similarly,
the bpV compound is to administered in a
therapeutically effective amount, which is to be
2s understood is an amount meeting the intended
therapeutic objectives, and providing the benefits
available from administration of bpV compound.
The present invention will be more readily
understood by referring to the following examples which
3o are given to illustrate the invention rather than to
limit its scope.
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EXAMPLE 1
bpV induction of HIV-1 LTR is FK-506
and CsA-sensitive
Since NFAT has recently been shown to be of
importance for the regulation of the HIV-1 LTR, it was
first tested whether FK506 and CsA, two known
inhibitors of calcineurin and thus of NFAT activation,
could block the bpV-mediated activation of the HIV-1
LTR. To achieve this goal, the cell line 1G5, a Jurkat
1o derivative containing stably integrated HIV-1 LTR-LUC
constructs, was used. In Fig. 1, 1G5 cells were either
left untreated or were pretreated with Nasal (2.5 mM) ,
FK506 (10 ng/ml), CsA (10 ng/ml) or a combination of
Nasal with either FK506 or CsA for 1 h at 37°C.
i5 Following treatment, cells were either stimulated or
not for 8 h with the following agents: PHA (3 ~,g/ml) ,
bpV [bipy] , bpV [HO-pic] or bpV [pic] (10~.M) . Cells were
then lysed and evaluated for luciferase activity.
Values are the mean of three different measured samples
20 ~ S.D. This is representative of two independent
experiments. The addition of three different bpV
molecules (i . a . bpV [bipy] , bpV [HO-pic] and bpV [pic] )
resulted in a strong induction of HIV-1 LTR-driven
luciferase activity in the cell extract (49.8-, 49.4-,
25 and 48.5-fold increase), which was stronger than with
the mitogenic agent PHA (4.5-fold) (Fig. 1).
In order to further corroborate our initial
observation that a factor other than NF-KB was being
activated by bpV compounds, we initially incubated 1G5
3o cells with sodium salicylate (Nasal), a known inhibitor
of NF-xB translocation. As depicted in Fig. 1, Nasal
led to a significant but incomplete loss of the
induction of HIV-1 LTR-driven luciferase activity by
the various bpV molecules whereas no effect was
35 discernible with PHA stimulation. FK506 and CsA were
both found to-inhibit PHA-mediated HIV-1 activation as
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expected since it is mainly acting through NFAT.
Surprisingly, both NFAT inhibitors also blocked bpV-
mediated induction of luciferase activity. Furthermore,
when either FK506 or CsA were added along with Nasal, a
greater inhibitory action on measured HIV-1 LTR-
dependent luciferase activity was apparent in cells
treated with the three tested bpV molecules. It should
be noted that no detectable toxicity was measured upon
cell incubation with any combination of the inhibitors
io used as determined by the MTS assay. Inability to
completely inhibit bpV-mediated HIV-1 LTR activation by
the FK506/NaSal or CsA/NaSal combination is probably
due to residual NF-KB activity, since FK506 completely
inhibited NFAT activation. Higher concentrations of
i5 Nasal could not be used to achieve a more complete
inhibition of NF-KB due to its intrinsic toxicity at
these doses. The bpV molecule bpV[pic] was used for our
subsequent experiments as it is representative of the
other bpV compounds.
EXAMPLE 2
Induction of wild-type and NF-KB-mutated HIV-1 LTR by
bpV compounds is sensitive to FK506 and CsA
Since several transcription factors binding in
the -105/-70 enhancer region of the HIV-1 LTR have been
found to be of importance in virus gene transcription
(including the classical NF-KB factor), it was next
examined if the bpV-induced signaling component which
is sensitive to FK506 and CsA could equally be detected
3o in the context of the isolated HIV-1 enhancer region or
a NF-KB-mutated HIV-1 LTR. Jurkat cells were hence
transiently transfected with two different constructs,
one containing the -105/-70 region of the HIV-1 LTR
(pxB-TATA-LUC). and the other harboring the HIV-1 LTR
mutated at the NF-KB binding sites (pmKBLTR-LUC), both
of which were placed upstream of the luciferase
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reporter gene. In Figs. 2A and 2B, Jurkat cells were
transiently transfected by DEAF-Dextran with 15 ~.g of
either pKB-TATA-LUC (Fig. 2A) or pmKBLTR-LUC (Fig. 2B).
After 24 h, transfected cells were either left
s untreated (~) or were pretreated with FK506 (10
ng/ml)(~) for 1 h at 37°C. Following treatment, cells
were stimulated for 8 h with the following agents: PHA
(3~.g/ml) , PHA (3~.g/ml) /PMA (20 ng/ml) or
bpV [pic] (10~,M) . Cells were then lysed and evaluated
io for luciferase activity. Values are the mean of three
different measured samples ~ S.D. This is
representative of two independent experiments. When pxB-
TATA-LUC was transfected in the Jurkat cells, strong
induction of luciferase activity was seen upon the
Zs addition of the bpV compound bpV[pic] which was
severely affected upon pretreatment of the transfected
cells with FK506 (Fig. 2A).
As controls, inhibition of PHA and PHA/PMA
induction of luciferase activity was also evaluated in
2o cells pretreated with FK506, PHA/PMA being less
inhibited most likely because of the concomitant strong
activation of NF-KB by PMA (Fig. 2A). Similar
inhibitions for all three tested activators were
observed when CsA was instead used. As depicted in Fig.
2s 2B, the transfection of the pmKBLTR-LUC plasmid in
Jurkat cells led to an induction of luciferase activity
upon the addition of either PHA, PHA/PMA or bpV[pic].
In this case, a lesser difference in terms of HIV-1 LTR
activation was observed between PHA and PHA/PMA because
30 of the mutation of the two NF-KB binding sites on the
HIV-1 LTR. When cells were pretreated with subcytotoxic
concentrations of FK506, a strong down-modulation of
HIV-1 LTR-mediated luciferase activity was again
measured for all types of activators including
35 bpV[pic]. Similar decrease of luciferase activity was
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apparent following treatment with CsA. These results
further suggested that the NF-KB-independent signaling
pathway which is activated by the bpV molecules was
FK506- and CsA-sensitive and that it was acting through
s the -105/-70 HIV-1 enhancer region.
EXAMPLE 3
NFAT-dependent luciferase expression is
markedly stimulated by the bpV compounds
io Based on our previous results and since the
enhancer region (-105/-70) of the HIV-1 LTR is a bind-
ing site for both NF-KB and NFAT transcription factors,
the obvious candidate for the NF-KB-independent tran-
scription factor induced by the bpV is NFAT. In order
i5 to test whether bpV compounds act directly on the NFAT
factor, transient transfections were performed in
Jurkat cells with pNFAT-LUC, a vector made of three
tandem repeats of the human IL-2-derived NFAT binding
site positioned in the context of the human IL-2 mini-
2o mal promoter upstream of the luciferase reporter gene.
Luciferase activity was evaluated at different time
points after initiation of the stimulation by either
the combination of PHA/PMA or the bpV compound
bpV[pic]. In Fig. 3A, Jurkat cells were transiently
25 transfected by DEAE-Dextran with 15 ~.g of pNFAT-LUC.
Following a 24 h incubation period, cells were left
untreated (~) or were stimulated with either PHA
(3~.g/ml) /PMA (20 ng/ml) (~) or bpV [pic] (lO~CM) (~) and
lysed after 4, 6, 8, 12, and 24 h of stimulation. In
3o Fig. 3B, Jurkat cells stably transfected with pNFAT-LUC
were either left untreated (0) or were pretreated with
FK506 (10 ng/ml) (~). After 1 h, cells were stimulated
for 8 h with PHA (3 ~g/ml) , PMA (20 ng/ml) , PHA (3
~,g/ml) /PMA(20 .ng/ml) , bpV [pic] (10 ~.M) or bpV [pic] (10
35 ~.M) /PMA(20 ng/ml) . Values are the mean of three
different measured samples ~ S.D. This is repre-
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sentative of two independent experiments. As expected,
a strong induction of NFAT-dependent luciferase activ-
ity was mediated by the combined action of PHA and PMA
(Fig. 3A).
s Interestingly, a stronger induction of this
same construct was observed upon stimulation of
transfected Jurkat cells with the compound bpV[pic].
Time kinetic analysis showed a transient increase in
luciferase activity which peaked around 6 to 8 hours
to for PHA/PMA (53.5-fold and 61.1-fold increase), while
maximal activation was reached after 8 to 12 hours for
bpV[pic] (92.2-fold and 73.2-fold increase). For the
subsequent experiments, the 8 hour time point was hence
chosen for time of treatment of pNFAT-LUC-transiently
i5 transfected cells. To further demonstrate the ability
of bpV compounds to activate NFAT, stably pNFAT-LUC-
transfected Jurkat cells (J-NFAT-LUC) were derived and
pooled resistant clones were then treated with various
NFAT inducers with or without pre-treatment with FK506.
2o As illustrated in Fig. 3B, J-NEAT-LUC cells were highly
responsive to the inducer PHA added alone or in the
presence of PMA (48.9-and 72.1-fold increase,
respectively), whereas PMA alone had no effect, a
pattern consistent with the need of a calcium-inducing
25 agent for the induction of NFAT. Similarly, however,
the bpV compound bpV[pic] also led to a significant
induction of luciferase activity in J-NFAT-LUC cells
(113.3-fold increase) which was further enhanced in the
presence of PMA (208.1-fold increase). After pre-
3o treatment with FK506, NFAT-driven luciferase induction
by all tested activators including bpV[pic] was totally
abrogated without any measurable effect on cell
viability. These results hence represented the first
indication that PTP inhibitors such as bpV compounds
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were inducing activation of the NFAT transcription
factor.
EXAMPLE 4
s Nuclear translocation of NFAT by the bpV compounds
Electrophoretic Mobility Shift Assays (EMSA)
were next performed to demonstrate the bpV-mediated
nuclear translocation of NFAT. In Fig. 4A, Jurkat cells
were either left untreated or were stimulated with PMA
(20 ng/ml) /Iono (1 mM) (P/I) or bpV [pic] (10 ~.M) for 1 h.
Nuclear extract of bpV-stimulated Jurkat cells (10 ~.g)
were incubated with 32P-labeled murine IL-2 derived NFAT
binding site for 20 min in the absence (lane 2) or
presence of 10-, 100-, 200-fold excess of unlabeled
i5 NFAT oligonucleotide (lanes 4, 5, and 6, respectively)
or 100-fold excess of unlabeled NF-KB oligonucleotide
(lane 7). Labeled NFAT oligonucleotides were incubated
with nuclear extracts from unstimulated Jurkat cells as
a negative control (lane 1) or from PMA/Iono-stimulated
2o Jurkat cells as a positive control (lane 3). In
untreated Jurkat cells, no specific signal was seen
(Fig. 4A, lane 1), while PMA/Ionomycin (P/I), a strong
NFAT-inducing combination, gave a specific signal (lane
2, arrow). Similarly, the bpV compound bpV[pic] also
2s led to the induction of a new signal which migrated at
the same height as the one observed with P/I and which
was competed by increasing concentrations of unlabelled
NFAT oligonucleotide (compare lane 3 with lanes 4 to 6)
but not by 100-fold excess of unlabelled NF-KB
30 oligonucleotide (lane 7). In Fig. 4B, nuclear extracts
from bpV-stimulated Jurkat cells were either untreated
(lane 2) or preincubated with an anti-NFATc polyclonal
antibody (lane 3) or pre-immune serum (lane 4) for 15
min at room temperature. Extracts from unstimulated
35 Jurkat cells were used as a negative control (lane 1) .
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Free probe and the specific signal observed are
indicated at the left of Figs. 4A and 4B.
To identify the composition of this shifted
signal, antibodies were incubated in the presence of
s bpV-treated Jurkat nuclear extracts. As presented in
Fig. 4B, the signal induced by bpV[pic] was completely
abrogated by the addition of anti-NFATc antibodies to
the nuclear extract prior to incubation with the
labeled NFAT oligonucleotide (compare lanes 2 and 3)
to while no such decrease in band intensity was observed
upon pre-treatment of these same extracts with pre-
immune serum (lane 4). The fast migrating band in these
EMSA experiments was not clearly identified but most
likely represents degradation of the NEAT signal. These
15 experiments hence showed that bpV molecules indeed led
to the activation (i.e. the translocation to the
nucleus) of the NFAT transcription factor and that the
major if not only isoform activated in Jurkat T-cells
was the NFATc isoform.
EXAMPLE 5
NFAT activation by bpV compounds is dependent
on p2lras and p561ck
Cooperativity between two different signaling
2s pathways is known to be essential to achieve optimal
transcriptional activation through the NFAT motifs of
the human IL-2 gene. Previous reports have shown that
NFAT activation requires the induction of the
calcineurin pathway leading to dephosphorylation of the
3o NFAT factor and its subsequent translocation and also
necessitates the participation of p2lras. Indeed for
NFAT to become fully transcriptionally active, the
transcription factor AP-1 is often required which
becomes induced by a ras-dependent pathway. It is shown
3s that bpV molecules activate the calcineurin-dependent
pathway as bpV-mediated NFAT activation is blocked by
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FK506 (Figs. 3A and 3B). Jurkat cells were transiently
transfected by DEAF-Dextran with 15 ~g of the pNFAT-LUC
plasmid plus 15 ~.g of either a control vector (0) or
the RasNl7 (~) encoding for the dominant negative
s mutant of p2lras. After 24 h incubation period, cells
were treated or not with PHA ( 3 ~.g/ml ) , PHA ( 3
~.g/ml) /PMA (20 ng/ml) , bpV [pic] (10 ~.M) or bpV [pic] (10
~.M)/PMA (20 ng/ml). Cells were next lysed 8 h post-
stimulation and luciferase activity was monitored.
io Results are the means ~ S.D. for triplicate samples and
are representative of two independent experiments. As
shown in Fig. 5, the expression of the p2lras dominant
negative protein in the Jurkat cells inhibited PHA- and
PHA/PMA-mediated NFAT activation by approximately 60 to
i5 70 percent, confirming p2lras implication in NFAT-based
transcriptional regulation by these agents.
Involvement of p2lras in bpV[pic]-mediated
activation of NFAT was also clearly demonstrated as
expression of the rasNl7 dominant negative mutant
2o reduced NFAT activation by bpV with or without PMA down
to 20 to 30 percent of the level observed in the
absence of the dominant negative mutant.
The implication of the tyrosyl kinase p561ck in
the bpV-induced NFAT activation was also examined since
2s this src family member has been previously demonstrated
to act on the activation of NFAT. The p561ck-deficient
Jurkat-derivative JCaM-TAg was initially tested to see
whether bpV compounds could induce NFAT activation in
this cell setting.
3o In Figs. 6A and 6B, JCaM-TAg cells (p561ck-
negative) were transiently transfected by DEAE-Dextran
with 15 ~g of the pNFAT-LUC plasmid (Fig. 6A) plus 30
~,g of either the control vector pEFneo (~) or the
p561ck-encoding vector pEFneo-Lck (~)(Fig. 6B). After
35 a 24 h incubation, cells were treated or not with PHA
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(3 ~.g/ml) , PMA (20 ng/ml) , Iono (1~CM) , PHA (3
~g/ml) /PMA (20 ng/ml) , bpV [pic] (10~,M) or Iono
(1~M)/PMA (20 ng/ml). Cells were lysed after 8 h post-
stimulation and luciferase activity monitored.
Luciferase activity is presented on a log scale in Fig.
6A. Results are the means ~ S.D. for triplicate samples
and are representative of two independent experiments.
As presented in Fig. 6A, the p561ck-deficient
T-cell line when transfected with the pNFAT-LUC plasmid
io was found to be unresponsive to all activators employed
including the bpV compound bpV[pic] except for the
Ionomycin (Iono)/PMA combination which acts more
downstream than the other agents. Similar observations
were made in parental lck-deficient JCaMl.6 cell line,
which was again found to be unresponsive to NFAT
activation by bpV molecules.
To demonstrate that this lack of bpV-mediated
NFAT induction was due to the absence of p561ck, the
lck-negative JCaM-TAg cell line were cotransfected with
2o the pNFAT-LUC plasmid plus either the p561ck-encoding
vector pEFneo-lck or, as a negative control, the empty
vector pEFneo. As depicted in Fig. 6B, no NFAT
activation was observed with PHA/PMA when co-
transfecting pNFAT-Luc with pEFneo. However, response
to these stimulating agents was restored upon
cotransfection of both pNFAT-Luc and the p561ck-
encoding vector, consistent with the need for the
presence of p561ck for PHA to mediate a signal through
the T-cell receptor. The same p561ck dependency was
observed with the bpV compound bpV[pic], confirming
that p561ck is a mediator of the signal generated by
the bpV PTP inhibitors. No strong differences in
induction were apparent in cells similarly stimulated
with Iono/PMA whether the p561ck expression vector was
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co-transfected or not, due to the downstream action of
this combination.
EXAMPLE 6
s bpV molecules in combination with PMA can activate
transcription from the complete IL-2 promoter in a
FK506-sensitive manner
IL-2 expression is known to be highly regulated
by NFAT binding sites and pervanadate has previously
to been shown to upregulate the expression of IL-2
mediated by agents such PMA, anti-CD3 and anti-CD28
antibodies. Since it is demonstrated that bpV molecules
led to the activation of NFAT, it is next investigated
as to whether the bpV compounds alone or with other
is activators would be able to also activate the IL-2
promoter.
In Fig. 7A, Jurkat cells were transiently
transfected by DEAF-Dextran with 15 ~.g of the pIL-2-LUC
plasmid. After a 24 h incubation, cells were either
20 left untreated (~) or treated with PHA (3 ~.g/ml)/PMA
(20 ng/ml) (O) or bpV [pic] (10~.M) /PMA (20 ng/ml) (~) .
At fixed time after stimulation (0, 4, 6, 8, 12, and 24
h), cells were lysed and luciferase activity was
monitored. In Fig. 7B, J-IL-2-LUC cells were either
2s left untreated (D) or were pretreated (~) for 15 min
with FK506 (10 nM) before being stimulated with PHA (3
~.g/ml) /PMA (20 ng/ml) or bpV [pic] (10 ~.M) /PMA(20
ng/ml). Cells were lysed 8 h post-stimulation and
luciferase was monitored. Results are the means + S.D.
3o for triplicate samples and are representative of two
independent experiments.
As demonstrated in Fig. 7A, PHA/PMA treatment
of Jurkat cells transiently transfected with the pIL-2-
LUC reporter plasmid revealed a maximal induction of
3s luciferase activity at 8 h followed by a gradual
decrease and a return to basal level at 24 h. More
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importantly, the bpV compound bpV [pic] was found to be
a potent activator of IL-2-dependent luciferase
activity in the presence of PMA. Neither PMA nor
bpV[pic] alone results in an increase in IL-2 promoter
activation in this transient transfection system. The
kinetic of luciferase induction observed with the
bpV[pic]/PMA combination is grossly the same as
PHA/PMA, except that the maximal luciferase induction
is higher and that the luciferase activity is reaching
to a plateau between 8 and 12 h returning to backgroung
level after 24h. With the use of stable IL-2-LUC
transfectant Jurkat cells (J-IL-2-LUC), the sensitivity
of bpV[pic]/PMA-mediated activation of the IL-2
promoter to the immunosuppressive drug FK506 was next
tested. Pretreatment of J-IL-2-LUC cells with 10 ng/ml
of FK506 resulted in a 80% inhibition of the IL-2
promoter-driven luciferase activity induced by the
bpV[pic]/PMA combination (Fig. 7B). Similarly, PHA/PMA
stimulation was reduced by more than 75% with FK506.
EXAMPLE 7
Correlation between IL-2 promoter-driven luciferase
expression and IL-2 secretion
In order to look for potential synergy between
2s bpV compounds and other stimuli, various combinations
of activators were tested on J-IL-2-Luc cells and
looked at luciferase activity and IL-2 secretion. In
Fig. 8A, J-IL-2-LUC cells were treated with various
activators (PHA, 3 ~,g/ml; bpV [pic] , 10 ~.M; Iono, 1 ~.M;
3o PMA, 20 ng/ml; anti-CD3 antibody, 3 ~g/ml; anti-CD28
antibody, 1 ~.g/ml) . Cells were lysed after 8 h post-
stimulation and luciferase activity was monitored. In
Fig. 8B, J-IL-2-LUC cells were treated with the same
activators as in Fig. 8A. Cell-free supernatants were
35 collected after 24 h post-stimulation and IL-2 was
quantified by an enzymatic assay. Results for both
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Figs. 8A and 8B are the means ~ S.D. for triplicate
samples and are representative of two independent
experiments. PHA, Iono or PMA alone did not result in
any measurable activation of the IL-2 promoter, while
s bpV[pic] alone only resulted in a modest increase in
promoter activity. However, when combining bpV[pic]
with those activators, activation of the IL-2 promoter
was observed. With the bpV[pic]/PHA combination, a fair
increase in IL-2 promoter was obtained with 21-fold
io increase ( Fig . 8A) .
The bpV[pic]/Iono combination was somewhat
stronger, resulting in a 49-fold increase of luciferase
activity. A more stronger activation was achieved when
combining bpV[pic] and PMA, as luciferase activity was
15 increased from about 143-fold compared to basal level.
However the strongest increase was obtained when
combining bpV[pic] with Iono and PMA, where the
luciferase activity could be induced to a 220-fold,
almost 7-times the value obtained with the Iono/PMA
2o treatment. Importantly, the same potentiation effect of
the bpV compound was observed when using more
physiological stimuli. As previously determined in
Jurkat cells, simultaneous stimulation through TCR
(anti-CD3 antibody) and CD28 resulted in only a modest
2s increase in IL-2 promoter activity, whereas stimulation
through each of the receptor alone did not result in
any detectable activation. However, addition of
bpV[pic] to either anti-CD3, anti-CD28 antibodies or
both resulted in a strong stimulation of IL-2 promoter-
3o driven luciferase activity (36-, 119- and 156-fold,
respectively).
To confirm these results at the protein level,
these IL-2 promoter stimulating agents were added for
24 h to the J-IL-2-LUC and supernatants were tested for
35 IL-2 production by enzymatic assays. Overall, the IL-2
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production in the supernatant fairly reflected the IL-2
promoter activity as measured by luciferase activity
(Fig. 8B). Indeed, a strong correlation between IL-2
promoter activity and IL-2 secretion was noticed when
s IL-2 production was plotted against luciferase activity
(r - 0.968). The only major difference between these
two types of measurements of IL-2 expression is the
treatment PMA/Ionomycin where IL-2 secretion seems to
be more pronounced. Therefore the induction of IL-2
io expression and secretion by bpV molecules when used in
combination with agents mimicking signal transduction
mediated upon antigen recognition (e.g. anti-CD3 and
anti-CD28 antibodies) is demonstrated.
NFAT activation is known to be mediated by
is several different inducers which are also strong
activators of T-cells. In the present application,
phosphotyrosyl phosphatase inhibitors have been shown
to also induce T-cell activation and proliferation.
Using a new set of PTP inhibitors, it is demonstrated
2o for the first time that bpV compounds lead to the
activation of the transcription factor NFAT and are
demonstrated to be much stronger activators than the
PHA/PMA combination. Applicants were also interested in
identifying the other transcription factors) which was
2s positively affecting the regulatory sequences of HIV-1
in conjunction with NF-KB.
The first analysis was aimed at testing the
sensitivity of the HIV-1 LTR expression induced by bpV
to the immunosuppressors FK506 and CsA. It was found
3o that those two molecules were greatly diminishing bpV-
induced activation of HIV-1 LTR transcription. Similar
inhibition was observed with the two immunosuppressors
when the pKB-TATA-LUC construct, which contain only the
tandem KB sites of the HIV-1 LTR (-105/-70), was
3s instead used. Since these calcineurin inhibitors have
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been shown to also affect NF-KB activation in certain
cascade, the inhibitory potential of these same
immunosuppressors on NF-KB-mutated LTR-LUC construct
have been tested and it has been demonstrated that
s inhibition by FK506 and CsA of the bpV-induced HIV-1
LTR-based transcription could also be observed to the
same extent. Two lines of evidence suggest that NFAT
was most likely the other transcription factor
activated by bpV molecules. First, NFAT is greatly
to affected by these two immunosuppressors and, second, it
was recently demonstrated that NFAT is playing an
important role in the regulation of HIV-1 replication.
In the present application, the use of Jurkat T-cells
either transiently or stably transfected with pNFAT-
15 LUC-vector clearly showed a FK506/CsA-sensitive
induction of reporter gene activity by bpV molecules.
It was also determined by shift assays that bpV
compounds led to the accumulation of the NFATc isoform
in the nucleus. Although it seemed that this isoform is
2o most likely the only isoform being activated in Jurkat
T-cells, other NFAT members could also be similarly
activated by bpV, especially in different T-cell types
and also in peripheral blood leukocytes. Based on these
findings, bpV-mediated activation of HIV-1 LTR results
25 from a synergistic interaction between NFAT and NF-KB
which accounts for the important induction observed for
the HIV-1 LTR by bpV molecules (50-fold increase).
The role of p2lras in the activation of the
pNFAT-LUC plasmid is thought to occur via the
3o activation of the AP-1 factor which would act
cooperatively with NFAT as demonstrated in several
examples. AP-1 activation by pervanadate, another PTP
inhibitor, has been described and, based on preliminary
results from the inventors, it seems clear that AP-1
3s can be equally activated by bpV molecules. The p561ck
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dependence of NFAT activation by bpV molecules is an
attribute of a likely membrane-initiated signaling
pathway. However, the importance of the TCR in the bpV-
mediated induction of NFAT is not clear as the TCR-
negative T-cell line Sup-T1 demonstrated reproducible,
albeit lower, bpV-induced gene expression of NFAT-
regulated plasmids.
The results in the present application have
also demonstrated that bpV led to the activation of the
to IL-2 promoter when added along with PMA. Similar
observations have already been noticed when using the
PTP inhibitor pervanadate along with PMA, anti-CD3 or
anti-CD28 antibodies (Imbert et al., J. Inflammation
46:65-77, 1996; O'Shea et al., Proc. Natl. Acad. Sci.
USA 89:10306-10310, 1992; and Secrist et al., J. Biol.
Chem. 268:5886-5893, 1993). The results suggest that
the multifactorial requirement for IL-2 expression is
in part greatly helped by the bpV PTP inhibitor. In
fact, in a correlative fashion, when measuring both
luciferase-based IL-2 promoter activity and secreted
IL-2, it is found that bpV compounds are acting as
complementing agents leading to the production of IL-2
induced by several agents such as ionomycin, PMA, PHA,
anti-CD3 and anti-CD28 antibodies. This is most likely
2s due to the fact that bpV induces NF-KB, NFAT and most
probably AP-l, all of these considered as transcription
factors known to be important in IL-2 regulation. More
importantly, the powerful combination ionomycin/PMA was
observed to be much more fully potentialised in the
3o presence of bpV. This bpV-dependent potentiating effect
was also illustrated in a more physiological setting
for T-cell stimulation involving anti-CD3 and anti-CD28
antibodies where superinduction of IL-2-regulated
luciferase expression and IL-2 secretion was noticed.
35 It is believed that bpV molecules are acting primarily
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via calcium-dependent signaling pathways) since they
are strongly potentiating agents known to initiate
calcium-independent cascade such the one initiated by
PMA or anti-CD28 antibodies. In fact, it is observed
s that activation of NEAT by bpV was severely hampered in
calcium-deficient Jurkat-derived cell lines, which thus
gives an importance of the calcium flux in the
mechanistic action of bpV leading to NFAT activation.
Other groups have previously demonstrated that
io pervanadate PTP inhibitor was generating an increase in
intracellular calcium levels in Jurkat T-cells (Imbert
et al., J. Inflammation 46:65-77, 1996; and O'Shea et
al., Proc. Natl. Acad. Sci. USA 89:10306-10310, 1992).
It is hence demonstrated that the activation of IL-2
15 expression by bpV PTP inhibitors is likely to occur
through concomitant activation of the NF-KB, NFAT and
AP-1 transcription factors and has a dependency toward
calcium flux.
The results presented in the present
2o application show that bpV PTP inhibitors activate NFAT.
It is hereby demonstrated for the first time that
blocking of PTP activity is resulting in an activation
of NFAT. These results suggest that PTP activity acts
by attenuating a constitutive kinase activity that
2s otherwise would lead to a constant NFAT activation. The
strong induction of IL-2 expression by bpV in
conjunction with PMA or the more physiological stimuli
such as anti-CD3/anti-CD28 antibodies suggest that bpV
compounds, which are more stable PTP inhibitors than
3o pervanadate, might be acting through an alternative
complementary pathway to TCR-mediated T-cell signaling.
In conclusion, bpV molecules have therapeutical
values in inducing IL-2 expression in immunosuppressed
individuals, including AIDS patients. It should be
35 stressed out that bpVs by themselves do not induce IL-2
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production. Hence, these compounds could help
specifically prime the cells which have already been in
contact with nominal antigen and partially alleviate
the unresponsive cellular state which is a feature of
s the immune cells of these persons.
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
io tions, uses, or adaptations of the invention following,
in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
i5 applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
