Note: Descriptions are shown in the official language in which they were submitted.
CA 02431080 2003-06-02
DESCRIPTION
Field of the Invention
The invention disclosed relates to the field of methods of treating cancers.
More
specifically, the invention pertains to the field of cancer irrununotherapy.
Background of Invention
It is established that tumor cells evade the immune system of the patient.
This
contributes to the general failure of immunotherapeutic approaches [1-3].
Secretion of
soluble factors by tumors, such as IL-10 [4-6], free TGF-(~ [7], TGF-(3 bound
to
immunologlobulin [8), prostaglandins [9], low molecular weight retroviral plSE-
like
factors [ 10], immunosuppressive acidic protein [ 11 ], soluble MUC- i [ 12],
and tumor-
shed gangliosides [13) contribute to suppression of various aspects of the
immune
responses. Tumors also protect themselves from immune attack by coercing cells
of the
immune system to inhibit other cells of the immune system.
CD4 T cells have the ability to differentiate along the Thl or Th2 pathway,
which in turn
stimulate cell-mediated, or antibody-mediated immune responses [14]. In order
to
protect the host from autoimmunity, CD4 T cells also possess the ability to
differentiate
into T regulatory (Treg) cells that have the ability to inhibit activated T
cells regardless
whether they are Thl or Th2 [15]. A specific type of Treg possesses the
phenotype of
CD4+ CD25+. These cells are critical for protection against autoimmunity, as
demonstrated by experiments in which depletion of this cell population
resulted in
accelerated diabetes [16], or organ-specific autoimmune diseases [17].
Furthermore,
transfer of Treg cells into animals prone to autoimmunity can delay
progression or
outright block disease occurrence [18].
Thus while Treg cells naturally possess the function of protecting the body
against
harmful autoimmune attacks, it is these same Treg cells that cancer uses to
further shield
itself from immune attack. For example, as stated above, tumor veils secrete
various
immune-inhibitory factors. These factors possess the property of not only
inhibiting
CA 02431080 2003-06-02
2
immune attack, but also programming the differentiation of T cells into Treg
cells.
CD4+ CD25+ Treg cells have been found to circulate in higher numbers in cancer
patients, where they inhibit T cell and NK cell responses [19]. Depletion of
Treg cells
allows for greater efficacy of cancer-specific vaccines in marine models [20,
21].
Another type of immunological cell that cancer subverts for its own means is
the
macrophage. Classically, macrophages are known to possess anti-cancer
properties [22],
and have been described to spontaneously lyse or arrest proliferation of tumor
cells [23,
24]. However evidence is accumulating the macrophages can actually be involved
in the
stimulation of tumor growth. One suggestion of this was the observation that
mice
lacking mature macrophages (op/op mice) due to congenital absence of M-CSF are
actually more resistant to growth of implanted tumors when compared to wild-
type mice
[25J. Further investigation found that cancer patients possessing high number
of
macrophages infiltrating the tumor had a poorer prognosis than patients with
low
numbers of tumor-infiltrating macrophages [26]. It was thought the mechanism
by which
the tumor-infiltrating macrophages stimulated an aggressive cancer phenotype
was
through increasing angiogenesis via secretion of endothelial-promoting factors
such as
VEGF [27].
To parallel the example with Treg cells, macrophages with immune inhibiting
fianction,
also termed M-2 or alternatively activated macrophages, are found in the
normal life of a
mammal. The normal function of these alternatively activated macrophages is
thought to
be promotion of wound healing [28]. Alternatively activated macrophages
possess
distinct molecular markers such as the genes Ym1 and Fizzl [29]. However, as
is the
case for Treg, tumors can induce alternatively activated macrophages in the
tumor
environment [30J. These cells inhibit local immune responses, and are
themselves
involved in the generation of Treg cells [31 ].
Tumors themselves and alternatively activated macrophages express high levels
of the
arginase, an enzyme that breaks down arginine into L-ornithine and urea [32,
33]. L-
ornithine is further degraded into polyamines such as spermine and spermidine
by the
CA 02431080 2003-06-02
3
enzyme ornithine decarboxylase. Tmmune suppression induced by arginase
overexpression can occur through several pathways: a) arginine-depletion by
the tumor
results in destruction of important T cell signaiing molecules such as the TCR-
zeta chain
[34]; b) polyamines suppress T cell activation [35]; c) polyamines suppress
the
proinflammatory function of macrophages [36]; and d) polyamines suppress
activity of
NK cells [37].
Macrophages from prototypical Thl strains (C57BL/6, BlOD2) are more easily
activated
to produce NO with either IFN-gamma or LPS than macrophages from Th2 strains
(BALB/c, DBAI2). In marked contrast, LPS stimulates 'Th2, but not 'Thl,
macrophages to
increase arginine metabolism to ornithine [38]. In the context. of tumors, it
is known that
Th2 cytokines predispose to increased tumor aggressiveness and promote immune
evasion. Th2 cytokines are also known to stimulate arginase production. In
macrophages incubated with T cells: Thl T cells lead to an exclusive induction
of iN~S,
whereas Th2 T cells up-regulated macrophage arginase without inducing iNOS. Ab
blocking experiments revealed the critical importance of IL-4 and IL-10 for
arginase up-
regulation [39]. Furthermore, the component of macrophage supernatant that
stimulated
tumor growth was substantially correlated with arginase [40].
As stated above, a characteristic feature of tumors is high production of the
enzyme
arginase. Serum arginase is a good marker for colorectal carcinoma progression
[41-43].
Values of serum arginase activity in patients with breast carcinoma were up to
4-fold
higher than those found in healthy women [44]. In prostate cancer tissue
arginase
expression correlates with severity of disease. A study evaluating arginase
activities in
prostate tissues of 50 patients with benign prostatic hyperplasia and in 23
patients with
prostatic carcinoma found that arginase was elevated in cancer tissues as
compared to
benign prostatic hyperplasia (fivefold; P < .001). A positive correlation
between arginase
activity and Gleason grade of the tumors was also found [45].
Although tumor-derived arginase and macrophage argina.se are believed to be
associated
with immune suppression in cancer, a substantial question remains: tumors
themselves
CA 02431080 2003-06-02
4
are poor T cell stimulators, similarly macrophages can stimulate T cells but
are not
optimal, since dendritic cells (DC) are the most potent stimulators of T
cells, do they
express arginase? Is tumor-induced arginase expression a mechanism by which
cancer
cells block T cell activation?
Although inhibitors of arginase [46], and inhibitors of enzymes downstream of
arginase
can block tumor growth and progression, the effects of these inhibitors is
still
therapeutically limited. The present invention is based on our findings that
1) DC
expression of arginase causes inhibition of immune function, and 2) reversal
of arginase
expression on DC can over-come the inhibitory effects of the tumor on DC
function,
those stimulating antitumor immunity.
This background information is provided for the purpose of making known
information
believed by the applicant to be of possible relevance to the present
invention. hto
admission is necessarily intended, nor should be construed, that any of the
proceding
information constitutes prior art against the present invention.
I3escription of the Invention
Disclosed are methods rendering dendritic cells potent stimulators of immune
activation
in such a manner that tumor-secreted immune suppressants do not inhibit their
function.
A specific aspect of the invention relates to treating dendritic cells with a
pharmacological inhibitor of the enzyme arginase such as l~(omega)-hydroxy-L-
arginine
or N(omega)-hydroxy-nor-L-arginine. This treatment wot~.ld inhibit the ability
of the
dendritic cell to react to a variety of tumor-secreted immune suppressants, in
particular
cytokines such as IL-10. In the same spirit, the dendritic cell may be
modulated through
the use of other known inhibitors that block the function of the enzyme
arginase. Said
inhibitors may be small molecular peptides, chemical analogues or suicide
substrates.
In another aspect, dendritic cell expression of the enzyme arginase is
suppressed using
known genetic inhibitory techniques such as siRlVA, antisense
oligonucleotides,
CA 02431080 2003-06-02
hammerhead ribozymes or morpholinos. The suppression of arginase may be
performed
in an ex vivo manner, wherein the dendritic cells, or their progenitors are
extracted from
the blood or bone marrow of the patient, cultured and transfected outside of
the body, and
subsequently reintroduced into the patient in such a manner as to stimulate
immune
response against the tumor. In this aspect, the dendritic cells are either
pulsed with
tumor-derived antigen, or alternatively, are allowed to naturally engulf tumor
antigens
from the tumor it self if the dendritic cells are injected intratumorally.
Another aspect of the invention is that dendritic cells whose ornithine
decarboxylase is
inhibited can be used for stimulating anti-tumor responses by virtue of the
fact that they
are not prone to tumor-induced immune suppression. Bath chemical and/or
genetic (ie
siRNA, antisense oligoriucleotides, hammerhead ribozymes or morpholinos)
inhibition of
omithine decarboxylase will cause the formation of potent immune stimulatory
dendritic
cells that can prime anti-tumor responses more effectively than unmanipulated
dendritic
cells.
Another aspect of the invention is that inhibition of both arginase and
ornithine
decarboxylase endow the dendritic cell with protection from tumor-induced
apoptosis.
Inhibition of expression and/or function of arginase and/or ornithine
decarboxylase
allows for more effective immune stimulation by increasing the amount of time
that a
dendritic cell can live in the context of the tumor-secreted, and surface
bound apoptotic
factors.
Another aspect of the invention is that manipulation of arginase and ornithine
activity in
dendritic cells can be achieved by systemic manipulation through dietary
restriction or
through systemically administered drugs such as DFMO. While this approach may
be
less efficacious than ex vivo manipulation, it is more convenient in
situations were ex
vivo culture and manipulation of dendritic cells is impossible.
CA 02431080 2003-06-02
6
Description of Figures
Figure 1 illustrates that arginase inhibition blocks IL-10-induced suppression
of DC IL-
12 production.
Figure 2 illustrations that arginase inhibition renders DC resistant to IL-10
mediated
suppression of allostimulatory capacity.
Figure 3 illustrates that arginase inhibition blocks tumor supernatant-induced
suppression
of DC IL-12 production.
Figure 4 illustrates that arginase inhibition renders DC resistant to tumor
supernatant
mediated suppression of allostimulatory capacity.
Figure 5 illustrates that arginase inhibition renders DC resistant to tumor
supernatant-
induced apoptosis.
Figure 6 illustrates that ODC inhibition renders DC resistant to tumor
supernatant-
mediated suppression of IL-12 production.
Figure 7 illustrates that ODC inhibition renders DC resistant to tumor
supernatant-
mediated suppression of allostimulatory function.
Figure 8 illustrates that ODC inhibition renders DC resistant to tumor
supernatant-
induced apoptosis.
Figure 9 illustrates that arginase and ODC-inhibited DC, but not untreated DC,
are able to
induce tumor suppression in vivo.
CA 02431080 2003-06-02
7
I9etailed Description of Invention
~ne embodiment of the disclosed invention teaches methods of preparing a
cancer
vaccine using dendritic cells that are resistant to cancer-induced immune
suppression. As
stated in the Background section, the arginase pathway of metabolism is
upregulated in
cancer cells and cancer-secreted metabolites of this pathway are involved in
immune
suppression. However, part of the novelty of this invention resides in our
findings that
tumor-secreted immune suppressive factors, such as IL-10, are also potent
inducers of the
arginase pathway in dendritic cells. Since T cells are more potently
stimulated or
inhibited during their interaction with dendritic cells, as compared to their
interaction
with tumor cells, it would be more likely that the arginase upregulation in
the dendritic
cell is more important for tumor-immune suppression than arginase upregulation
in the
tumor itself. Furthermore, arginase pathway activation is involved in the
induction of
dendritic cell apoptosis. The ability of tumors to secrete cytokines such as
IL-I0 which
upregulate arginase activity in dendritic cells, could be a mechanism by which
tumor
cells induce apoptosis of dendritic cells. Therefore the inhibition of the
arginase pathway
in tumor cells would not only increase the resistance of dendritic cells to
tumor-secreted
immune suppressive factors but also protect dendritic cells from apoptosis.
For manufacturing a cancer vaccine using dendritic cells, blockade of the
arginase
pathway could be achieved through: a) inhibition ~f argin.ase enzymatic
activity, b)
inhibition of arginase gene expression, c) inhibition of onuthine
decarboxylase enzymatic
activity, or d) inhibition of omithine decarboxylase gene expression.
In one cancer vaccine embodiment, dendritic cells are generated from
peripheral blood
monoclear cells purified by density gradient. Monocytic cells are purified by
adherence
to plastic and cultured for 7-days in GM-CSF and IL-4 at a concentration
between 20-
100ng/ml, preferentially, 50 ng/ml. A concentration of 200 ~,Mol 1VOAH is
added to the
culture every time the cells are passaged. Tissue culture media is changed
once every
two days. An alternative modification is that ornithine-decarboxylase can be
inhibited in
CA 02431080 2003-06-02
the DC culture by addition of the ornithine-decarboxylase inhibitor DFM~.
Although
several concentrations are useful, 3mM seems ideal based on our studies. These
arginase/ornithine decarboxylase-inhibited DC are subsequently injected infra-
tumorally,
preferably at a concentration of 2xI09 cells. Based on the endogenous ability
of DC to
uptake tumor antigens, these cells provide an autologous "natural" vaccine
that stimulates
T cell responses against the tumor antigens.
Examples
Example 1: Generation of dendritic cells resistant to the tumor-secreted
cytoldne
IL-10
This example illustrates the generation of dendritic cells that are resistant
to the immune-
inhibitory activities of IL-10 by pretreating said dendritic cells with the
arginase inhibitor
N hydroxy-L-arginine (NOHA).
DC were generated from bone marrow progenitor cells as previously described
[47).
Briefly; bone marrow cells Were flushed from the femurs and tibias of C57BL/6
mice
(Jackson Labs, Bar Harbor ME), washed and cultured in ?4-well plates (2 x 106
cells/ml)
in 2 ml of complete medium (RPMI-1640 supplemented with 2rnM L-glutamine, 100
U/ml of penicillin, 100 ~.g of streptomycin, 50 ~.M 2-mercaptoethanol, and 10
% fetal
calf serum (all from Gibco RBL)) supplemented with recombinant GM-CSF (10
ng/ml;
Peprotech, Rocky HiII, NJ) and recombinant mouse IL-4 (10 nglml; Peprotech).
All
cultures were incubated at 37°C in 5% humidified C~2. Non-adherent
granulocytes were
removed after 48 hours of culture and fresh medium was added.
4 experimental groups were used:
a) Unmanipulated DC cultures
b) DC cultured in the presence of IL-10
c) DC cultured in the presence of arginase inhibition
d) DC cultured with IL-10 and arginase inhibition
CA 02431080 2003-06-02
In group (b) IL-10 was added throughout the culture period at a final
concentration of 50
ng/ml. In group (c) a 200 ~.M concentration of the arginase inhibitor NOHA
(Sigma-
Aldrich, Rockville IL) was added for the whole culture period. In group (d),
the
combination IL-10 and the arginase inhibitor NOHA were added as described in
groups
(b and c).
After 7 days of culture >90°/~ of the cells expressed the
characteristic DC-specific marker
CDl lc as determined by FRCS. DC were washed in phosphate buffered saline
(PBS)
and plated in 24-well plates at a concentration of 2 x 105 cells per well.
Cells were
activated for 48 hours with LPS (10 ng/ml, Sigma Aldrich; St Louis, MO) + TNF-
oc (10
ng/rnl, Peprotech). Supernatants were harvested and production of the tumor-
inhibitory
and Thl-activating cytokine IL-12 was assessed by sandwich ELISA (R&D Systems,
Minneapolis, MN). As illustrated in Figure 1, DC generated in absence of IL-10
or
arginase inhibition possessed strong IL-12 producing ability (a). Addition of
IL-10 in the
culture media resulted in the inhibition of IL-I2 production (b). Inhibition
of arginase
activity induced a slight increase in activity production of IL-I2(c).
Surprisingly,
arginase inhibition spared the DC of IL-10-mediated downregulation of IL-12
production.
Additionally, mixed lymphocyte reaction (MLR) was performed to assess
functional
allostimulatory capacity of the DC in groups a-d. For MLR, T cells were
purified from
BALB/c splenocytes using nylon wool columns and were used as responders
(1x106~well). DC from groups a-d (5-40x103, C57BL6 origin) were used as
stimulators.
72 hour MLR was performed and the cells were pulsed with 1 ~,Ci [3H]-thymidine
for the
last 18 hours. The cultures were harvested on to glass fiber filters (Wallac,
Turku,
Finland). Radioactivity was counted using a Wallac 1450 Microbeta liquid
scintillation
counter and the data were analyzed with IJltraTerm 3 software. As seen in
Figure 2, DC
generated in the presence of IL-10 were poor stimulators of allogeneic T cell
proliferation. DC raised in the presence of arginase inhibition possessed a
similar
allostimulatory capacity as DC raised under control conditions. When DC were
CA 02431080 2003-06-02
generated in the presence of IL-10 under conditions of arginase inhibition,
the
allostimulatory effects were preserved. This suggests that arginase inhibition
can act at
the level of the dendritic cell to protect from immune suppressive activities
of IL-10.
Example 2: Arginase inhibition allows normal DC maturation despite presence of
tumor-secreted inhibitory factors.
It is reported that generating DC in the presence of supernatants from a
variety of tumors
results in the production of immature DC with poor allostimulatory capacity.
t~lthough
IL-10 is a known tumor-secreted factor responsible for the inhibition of DC
maturation,
other factors such as ceramides, gangliosides, TGF-b and prostaglandins have
also been
reported. We therefore assessed whether inhibition of arginase activity could
render DC
resistant to the inhibitory effects of tumor -supernatant in a manner similar
to IL-10.
DC were generated as described in Example 1 with the modification that IL-10
was
replaced with 20% volume of day-2 supernatant from the marine Lewis lung
carcinoma
(3LL) cell line (American Type Culture Collection, lVlanassas VA). Arginase
inhibition
was performed by supplementation of the cell culture media with a 200 p.M
concentration
of the arginase inhibitor NOHA as in Example 1.
Experimental groups consisted of
a) Unmanipulated DC cultures
b) DC cultured in the presence of 20% 3LL supernatant
c) DC cultured in the presence of arginase inhibition
d) DC cultured with 20% 3LL supernatant and arginase inhibition
Using the experimental conditions of Example 1, it was demonstrated that
arginase
inhibition was able to block the 3LL-induced inhibition of IL-12 production
from DC
(Figure 3).
CA 02431080 2003-06-02
11
The conditions of Example 1 were also used to assess whether arginase
inhibition could
block 3LL-induced suppression of DC allostirnulatory capacity. As seen in
Figure 4,
arginase inhibition was successful at preserving DC allostimulatory ability
despite the
presence of 3LL supernatant.
Example 3 Arginase inhibition protects DC from tumor-induced apoptosis
It is reported that supernatants from a variety of tumor cells can induce
apoptosis in DC.
A method of protecting the DC from such apoptosis would theoretically result
in a more
efficacious tumor vaccine, as well as protect the immune responsive ability of
the cancer
patient.
Dendritic cells were generated as described in Example 1. At day 7 of culture
the cells
were divided into 4 groups
a) Control: media from the 3T3 fibroblast cell line was added.
b) Arginase inhibition: a 200 ~,1~ concentration of the arginase inhibitor
N~HA was
added.
c) Tumor supernatant: B-16 supernatant was added at 20% of tissue culture
volume.
d) Arginase inhibition in the presence of tumor supernatant. Both b and c were
added.
At day 9 apoptosis was assessed using Annexin-V staining and analyzed by flow
cytometry. As indicated in Figure 5 apoptosis was induced by the presence of
tumor-
supernatant while arginase inhibition was able to reduce this effect.
CA 02431080 2003-06-02
12
Example 4 Inhibition of ornithine decarboxylase (ODC) protects DC from tumor-
induced immune suppression
DC were cultured as described in Example 1. In some groups media was replaced
with
~0% fresh media and 20% volume of day-2 culture supernatant from the marine
Lewis
lung carcinoma (3LL) cell line.
Experimental groups consisted of
a) Unmanipulated DC cultures
b) DC cultured in the presence of 20°/~ 3LL supernatant
c) DC cultured in the presence of ODC inhibition
d) DC cultured with 20% 3LL supernatant and ODC inhibition
ODC inhibition was achieved by administration of 3 mM DL-alpha-
difluoromethylornithine (DFMO) into the culture media for the whole Culture
period.
Using the experimental conditions of Example 1, it was demonstrated that ODC
inhibition was able to block the 3LL-induced inhibition of IL-12 production
from DC
(Figure 6).
The conditions of Example 1 were also used to assess whether ODC inhibition
could
block 3LL-induced suppression of DC allostimulatory capacity. As seen in
Figure 7,
ODC inhibition was successful at preserving DC allostimulatory ability despite
the
presence of 3LL supernatant.
Example 5 ~DC inhibition protects DC from tumor-induced apoptosis
Dendritic cells were generated as described in Example 1. At day 7 of culture
the cells
were divided into 4 groups
CA 02431080 2003-06-02
13
a) Control: media from the 3T3 fibroblast cell line was added.
b) ODC inhibition: a concentration of 3 mM DL-alpha-difluoromethylornithine
(DFMO) was added to the culture media.
c) Tumor supernatant: B-16 supernatant was added at 20% of tissue culture
volume.
d) ODC inhibition in the presence of tumor supernatant. Both b and c were
added.
At day 9 apoptosis was assessed using Annexin-V staining and analyzed by flow
cytometry. As indicated in Figure 8 apoptosis was induced by the presence of
tumor-'
supernatant while arginase inhibition was able to reduce this effect.
Example 6 Arginase-inhibited and ODC-inhibited DC induce ante-tumor responses
in vivo
Male 6-8 week old C57BL/6 mice were injected with RM-I marine prostate tumor
cells
(20,000 cells/100 ~,l) in the right flank. Tumor size was determined using
Vernier
caliper. DC (20,000/100 ~ul) raised using the conditions below were co-
injected with
tumor cells or tumor cells were injected alone:
DC generated as described in Example i in and raised under the following
conditions:
a) Fed every other day control media as described in Example 1
b) Arginase was inhibited by addition of 200 p,M NOAH
c) ODC was inhibited by addition of 3 mM DMFO
mice/group were used. Tumor size was evaluated on day 20 was evaluated. As
seen in
Figure 9, tumor cells injected alone, or with control DC grew to a much
greater extend
than tumors injected with DC raised in the presence of arginase or ODC
inhibition. As
repetition of this experiment in nu/nu on C57~L6 backgrounds resulted in
equivalent
tumor growth in all groups (data not shown), we postulate that relieving the
DC of tumor-
induced immune suppression by arginase inhibition or ODC inhibition, is a
valuable
strategy for induction of antitumor responses.
CA 02431080 2003-06-02
14
The foregoing invention has been described in such a manner to enable one
skilled in the
art to practice it. However, it will be readily apparent to one skilled in the
art that after
reading the teachings of this invention certain changes and modifications may
be made
without departing from the spirit or scope of the appended claims.
Furthermore, the
examples illustrated are by no means binding but provide a guide for
practicing certain
embodiments and aspects of the invention disclosed.
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