Note: Descriptions are shown in the official language in which they were submitted.
CA 02453198 2004-O1-07
I~ISCLOSUI~
Field of The Invention
The present invention relates to the field of immune modulatory factors, more
specifically, it relates to the detection and synthesis of immune suppressive
exosomes.
Background of Invention
At present, little therapeutic options are available for patients with late
stage cancer. This
demands more sensitive diagnostic assays, as well as improvements in treatment
available. An appealing method of treating cancer is through harnessing the
power of the
host's immune system. Although great progress has been made in the development
of
immune therapies, little clinical success has been achieved. A reason for this
may be the
profound immune defects found in advanced cancer patients. Soluble inhibitory
molecules such as TGF-(3, IL-lU, and VEGF [1] and surface-bound T cell killing
molecules such as Fas ligand (Fast) have been reported in a wide variety of
cancers [2].
Additionally, circulating Fast has been described as a T-cell apoptosis-
inducing factor
found in the plasma of cancer patients, being correlated with poor prognosis
[2].
Although this type of immune suppression is antigen-nonspecific, specific T
cell deletion
is also seen in patients (reviewed in [3, 4]. Antigen specific deletion
implies 2 things: a)
that the T cell receptor interacts with tumor MHC and tumor antigen, and b)
the tumor
provides a death signal to the T cell. Such a signal could be an active death
signal, such
as tumor-expressed Fast, or absence of a survival signal, such as lack of
costimulation
[5, 6]. In light of a recent report that serum from tumor-bearing patients
induces T
apoptosis in TCR-specific manner [7], we questioned whether MHC-dependent T
cell-
killing factors may be circulating in the plasma of patients with advanced
cancer.
CA 02453198 2004-O1-07
2
It is known in the art that microvesicles termed "exosomes" possess powerful
immune
stimulatory functions both in vitro and in vivo [8]. Dendritic cell (DC) and B
cell-
derived exosomes contain high quantities of MHC I, MHC II, and CD$6, allowing
potent
activation of T cells [9, 10]. Administration of exosomes from DC pulsed with
tumor
antigen into cancer-bearing mice induces antitumor T cell responses and tumor
regression. Tumor cells have been reported to secrete exosomes which possess
MHC I
and tumor antigen, thus allowing possible vaccination with these entities [ 11
].
It is the purpose of this disclosure to describe a novel biological function
of exosomes.
This being immune suppression. With the exception of a publication by Karlsson
et' ad,
who reported intestinal-derived exosome-like particles termed "tolerosomes"
[12], there
has not been indication in the literature that exosomes may serve
physiological immune
suppressive functions. Prolongation of allograft survival using donor DC-
derived
exosomes was reported in a rat allogeneic cardiac transplant model [13].
Prolongation
was associated with decreased interferon-gamma secretion and inhibited number
of graft-
infiltrating lymphocytes. Interestingly, alto-antibody responses were
increased after
exosome-treatment, indicating that true tolerance was not induced. It has
however been
observed that melanoma [ 14] and leukemic [ I S] exosomes express bioactive
Fast
suggest that a two step interaction may be occurring between the tumor exosome
and the
T cell in which tumor antigen in MHC I induce a T cell activation event
causing
upregulation of Fas on the T cell. However before this disclosure, such an
interaction
was never demonstrated clinically.
Since the tumor exosomes expresses Fast, subsequent T cell apoptosis should
occur.
Another putative method by which tumor-derived exosmes rnay possess an
immunoregulatory function is suggested in a report that melanoma-derived cell
lines
secrete exosomes expressing the immune inhibitory molecule HLA-G [16]. However
no
evidence of immune suppressive activities was demonstrated. With exception to
the
CA 02453198 2004-O1-07
3
present disclosure, this model of antigen-specific immune suppression by tumor
exosomes has never been demonstrated
Methods of generating exosomes from natural sources and synthetically have
been
previously described in Canadian Patent Application # 2296150. ~~one of these
methods
describe generation of immune suppressive exosomes.
Brief Description of the Invention
The basis of the present invention is that exosomes from cancer patients
possess FaslJ or
other immune suppressive molecules that contribute to specific
killing/inactivation of
tumor-reactive T cells. Through quantification of these immune suppressive
exosomes,
as well as qualitatively assessing their functional activity, one can assess
the
immunological status of a patient suffering from an immunological deficient
state such as
cancer.
Alternatively, immune suppressive exosomes can be used therapeutically to
treat
conditions associated with immune hyperactivation, or inapxsropriate
activation. Said
conditions can include, but are not Limited to autoimmunity, transplant
rejection, or
immune mediated pathology such as septic shock.
Using the immune suppressive properties of exosomes as a basis for
immunotherapy,
exosome generated from cancer cells, or dendritic cells (DC~ that have been
manipulated
to possess immune suppressive properties can be applied therapeutically for
treatment of
pathologies associated with hyperactivation of immune responses. The ability
t~ antigen-
specifically pulse DC such that said antigen becomes attached to exosomes
allows the
utilization of suppressive-DC exosomes as a treatment for specific autoimmune
diseases.
CA 02453198 2004-O1-07
4
Detailed Description of the Invention
A novel teaching of this invention is to use patient-derived exosomes as a
measure of
immune suppression. Said immune suppression could consist of, but is not
limited to: T
cell dysfunction, decreased ability of T cells to proliferation, decreased
ability of T cells
to secrete tumor inhibitory cytokines, suppressed ability of T' cells to
produce
antiangiogenic effects, and upregulation of T cell immune-inhibitory
activities either
through contact-dependent or independent mechanisms. Assessing the ability of
a tumor,
or another immunosuppressive pathology would be useful in evaluating the
eligibility of
patients for immunotherapy. Another impetus for identifyin;~ the state of a
patient's
immune suppression is to guide the amount, and/or frequency of immune
stimulatory
agents the patient should receive.
In one embodiment of the present invention, serum is extracted from a patient
in wham
immune suppressive activity is being assessed. Exosomes can be purified using
a variety
of methods well known in the art, such methods are hereby i:r~cluded for
reference: 1) 10
ml of patient blood is extracted using a Vacutainer or similat°
apparatus. Plasma is
separated by centrifugation for a period of time sufficient to allow
fractionation of the
cellular particulates from the plasma. A suitable centrifugation time is 30
min at 10008;
2) Cell-free plasma is subsequently centrifuged at for a period of 4-24 hours
at 40,000 to
100,0008; 3) Collected pellets are subsequently washed in saline or a
physiological
solution by centrifugation at 4-24 hours under a force of 40,(100 to 100,0008;
and 4)
Protein content of the collected exosomes is assessed by Bradfort assay.
Variations of
exosome purification procedures are well-described in the art. Said methods
have been
optimized for purifying exosomes from cord-blood [ 17], ascites fluid [ 18],
tissue culture
[19], and bronchoalveolar lavage fluid [20].
Upon purification and standardization of exosomal protein content immune
suppressive
activity can be measured using a variety of assays well known to the skilled
artisan. lior
assessment of direct T cell apoptosis-inducing ability, exoso~nes ,are co-
cultured with
CA 02453198 2004-O1-07
allogeneic PHA-activated T cells for a period sufficient to induce apoptosis
if the
exosomes contained Fas-L. Such a co-culture can last from 4-72 hours depending
on the
type of patient, the purity of the exosome preparation, and th.e method of
evaluating
apoptosis induction. In one method a concentration of 0.1~~;-20pg of exosomes
are
purified from patient plasma and incubated with PHA activated (10~g/ml for 24
hours)
peripheral blood mononuclear cells from healthy person. The exosome-PBMC
combination is incubated for 72 hours at 37 Celsius and apoptosis of the PBMC
is
quantified using Annexin-V staining and analyzed by flow cytometry. Using this
system
an increased level of PBMC apoptosis is indicative of patient immune
suppression
through induction of immune cell apoptosis by circulating exosomes.
Many variations of this technique can be performed by a person skilled in the
art without
departing from the spirit or essence of this invention. For example, instead
of using
patient plasma as an initial source of exosomes one could utilize various
biological fluids
such as ascites fluid, bronchoalveolar lavage, or intratumora'.'l secretions.
Alternatively, primary tumour tissue can be extracted by biopsy, cultured in
vitro, and
exosomes from culture supernatant can be purified and admixed with PBMC in
order to
assess apoptosis-inducing capacity
A potential drawback of using healthy volunteer PBMC as the targets for
exosome-
induced apoptosis is variability of healthy volunteer PBMC populations, and
the fact that
PBMC constitute a plethora of cell types. In order to rule out such
variability as
standardized cellular target population can be employed. More specifically,
exosomes
purified from the cancer patient can be co-cultured with a standard cell line
sensitive to
exosome-induced killing. One such cell line is the human T cell leukemia cell
line
Jurkat. It is known in the art that exosomes released by activated T cells can
induce
apoptosis of the Jurkat cell line [2I]. This is believed to be clue to the
expression of
bioactive Fas receptor on this cell line. In agreement with our data that
exosomes derived
CA 02453198 2004-O1-07
6
from prostate cancer patients express Fast, the utilization of Turkats as a
standard target
cell Line for patient-exosome imrnunosuppressive activity is feasible.
Within the scope of the present invention lies the utilization of purified sub-
population of
healthy volunteer cells as a target for exosorne-mediated killing. For
example, some
types of cancers may be more immunosuppressive to CD4, C:DB, or CD56 immune
cells.
Thus purified exosomes from plasma of cancer-bearing patients can be co-
cultured with
sub-populations of immunological cells and apoptosis of said cells can be
assessed.
Methods of purifying subpopulations of immune cells are well-known in the art
and
include flow sorting, magnetic activated cell sorting, cell panning, and
purification using
column methods.
The immunosuppressive aspects of cancer-derived exosomes do not have to be
limited to
induction of apoptosis but may also induce cellular changes that render said
immune cell
unresponsive or hyporesponsive to further immunological stimuli. Such a state,
termed
"energy" can result from inappropriate activation of the T cell receptor
(TCR), activation
of the TCR in absence of co-stimulatory molecules, and presentation to the T
cell of
inhibitory molecules such as HLA-G. The observation that exosomes possess IILA-
G
X16] suggests that cancer-immune evasion via exosomes does not have to
strictly occur
through induction of apoptosis but could also function via ar.~ergy induction.
Thus, within
the scope of the disclosed invention, patient-purified exosornes can be
admixed with a
population of immunological cells derived from a healthy volunteer and various
functional aspects of the immune cells can be assessed instead of, or in
conjunction with
apoptosis evaluation. For example, exosomes from a cancer patient can be mixed
with T
cells purified from a cancer patient and stimulated with anti-CD3. A reduction
in the
proliferation, cytokine production, or upregulation of immune suppressive
cytokines,
would be indicative of the exosomes possessing immunosuphressive activity.
Cytokines
known in the art to be reduced in cancer patients include IFN--y, IL-~, IL-12,
and TNF-a.
In contrast, cytokines that are associated with cancer-induced immune
suppression
include TGF-j3, IL-10, VEGF, and IL-13.
CA 02453198 2004-O1-07
Utilization of healthy volunteer cells as an immunological target for
assessing
suppressive capabilities of cancer-derived exosomes is performed since the
immunological cells of the cancer patient are often suppressed as a result of
past
treatments (ie chemotherapy, radiation, surgery), or as a result of the cancer
itself. It may
be, however, desired by the practitioner of the invention to determine the
extent to which
exosome-derived immune suppression is responsible for the overall decrease in
the
patient's immune response. In this situation the practitioner of the invention
may choose
to utilize autologous immune cells as the source of exosome-targets. In this
situation, an
antigen-specific approach may be used. Patient 'T cell lines can be
established in vitro
using commonly known antigens such as KLH or ovalbumin.. These cell lines can
then
be admixed with patient exosomes and inhibition of viability or immunological
functions
can be assessed.
In addition to determining the extent of immune suppression in a patient by
assessing
exosomal immune activity, the therapeutic utilization of immune suppressive
exosomes is
also possible. Generation of exosomes with ability to kill T cells can be
performed by
transfection of the DC with various membrane-bound killer molecules such as
Fast.
Within the disclosed invention are artificially generated exos~ornes that
possess the ability
to induce apoptosis of T cells in an antigen-specific manner. Such exosomes
can be used
therapeutically for silencing antigen-specific immune responses. Within the
present
disclosure we demonstrate that administration of exosomes from DC that are
transfected
with Fast and pulsed with the antigen KLH can inhibit the KLH-specific but not
3rd party
responses (ie anti-ovalbumin) responses.
CA 02453198 2004-O1-07
g
Examples
1. Time-dependent Increase In Exosome Production by Human Prostate Cancer
Cell Lines
The human prostate cancer cell lines DU-145, PC-3 and LNCaP were allowed to
grow to
50% confluence in OptiMEM media supplemented with 10°/~ fetal calf
serum. 10 ml
samples of media were extracted at the indicated timepoints. Exosomes were
collected
by centrifugation at 7,000 g for 30 minutes, followed by a second
centrifugation at
100,000g for 3 hours.. Exosomal proteins were assessed using the Bradford
Assay
(Biorad). A time-dependent increase in the production of exosomal proteins
indicates
exosornes were actively produced by the cell lines and rules out the
possibility that non-
specific exosoma.l contamination was being detected (Figure 1).
2. Detection of PSMA Expressing Exosomes From LNCaP Cells
Exosomes were purified as described above from LNCaP cells at 75% confluency.
The
anti-prostate specific membrane antigen (PSMA) antibody clone 1G3, mouse IgG2a
(Northwest Biotherapeutics, Seattle) was used for staining exosomes in
combination with
a FITC labelled secondary antibody. This antibody targets the extracellular
domain of
PSMA, increasing the likelihood of staining exosomes. As can be seen, a
distinct
population of LNCaP-derived exosomes specifically stains positive for
expression of this
tumor antigen (Figure 2).
3. Cancer Cell line Exosome-Induced Apoptosis is Fast- and M(HC I-Dependent
Exosome-induced apoptosis is Fast-dependent. PC-3 human prostate cancer cell
exosomes were purified by ultracentrifugation. Exosomes (10~.g/ml) were added
to PHA
(10~g/ml)-activated T cells for 48 hours in the presence of the indicated
concentration of
anti- HLA class-I antibody, w6/32. Apoptosis was assessed using annexin-V
staining and
CA 02453198 2004-O1-07
9
analyzed by flow cytometry. As seen in Figure 3A, cancer-ex:oso~ne induced
apoptosis
was inhibited by addition of anti-MHC I antibody. Exosome-induced apoptosis is
FasL-
dependent. Increasing concentrations of the anti--human Fas ligand
antagonistic antibody,
NOK-2 (Pharmingen, San Diego, CA, USA) were added to a. 72 hour coculture of
PC-3
exosomes and activated T cells as described above. Apoptosis was assessed
using
annexin-V staining and analyzed by flow cytornetry. As seen in Figure 3B,
cancer-
exosome induced apoptosis was significantly inhibited by addition of anti-Fast
antibody.
4. Prostate Cancer Patient Exosomes Induce Apoptosis Through a Fasl,- and MHC
I-Dependent Mechanism
Apoptosis induced by exosomes isolated from prostate cancer patients. Plasma
was
purified from peripheral blood of healthy controls or 3 prostate cancer
patients by
centrifugation at 500g for half hour. Separation of cellular debris was
performed by
centrifugation at 7,000 g for 1/z hour followed by pelleting of the exosome
through
centrifugation at 100,000g for 3 hours, followed by one wash in I'BS. Protein
concentration of exosomes was detected by the Bradford assay (Biorad).
Exosomes were
added at the indicated concentrations to PHA (10~g/ml)-activated T cells for
48 hours.
Apoptosis was assessed using annexin-V staining and analyzed by flow
cytometry. This
figure is representative of 3 other prostate cancer patients whose exosomes
were
analyzed. Has seen in Figure 4A exosomes purified from plasma of cancer
patients but
not healthy controls induced a dose-dependent apoptosis in healthy T cells.
Prostate
cancer patient derived exosomes induced apoptosis is MHC l: and. Fast-
dependent.
Exosomes were purified from prostate cancer patient M142 a.s described in
Figure 4.
Increasing concentrations of anti- HLA class-I antibody, w6/:32, or anti-human
Fas ligand
antagonistic antibody, NOK-2 (Pharmingen, San Diego, CA, USA) were added to a
48
hour coculture of exosomes (5 ~.g/ml) and activated T cells (l?Hl~. IOpg/ml).
Apoptosis
was assessed using annexin-V staining and analyzed by flow cytometry. As seen
in
Figure 4B apoptosis was dependent on MHC I and Fast.
CA 02453198 2004-O1-07
S. Detection of Fast-bearing Exosomes From Plasma of :E~rostate Cancer
Patients
Using Cancer-Exosome ELISA
ELISA plates were coated with human anti-MHC I monoclonal antibodies (w6/32)
and
incubated overnight. Serum was collected from healthy controls and advanced
prostate
cancer patients and added to the coated plates. After washin,j, biotinylated
anti-human
Fas ligand antibody (NOK-2) was added to the plates. Following an additional
wash.,
Streptavidin-conjugated Horse Radish Peroxidase was added and after a half
hour
incubation ABTS (3-ethylbenzthiazoline-6-sulfonic acid was used as a
developing agent.
The reaction was stopped with HZOZ and fluorescence was a~>sessed by
spectospectometry. A dose-dependent increase in fluorescence was observed in
serum
samples from prostate cancer patients but not controls.
6. Induction of Antigen-Specific Immune lO~Iodulation Using Fast-bearing
Exosomes
DC were generated as follows: bone marrow cells were flushed from femurs and
tibias of
C57BL/6 mice, washed, and cult~.~red in 6-well plates (2 x 106/ml) in 4 ml
RPMI 1640
containing rGM-CSF (10 ng/ml; Peprotech, Rocky Hill, NJ) and mouse rIL-4 (10
ng/ml;
Peprotech). Nonadherent granulocytes were removed after 48 h of culture, and
fresh
media added every 48 h. By day 4 to 6 of culture, proliferating clusters of
cells with
typical dendritic morphology were seen, and by day 7 to 9 more than 90% of the
cells
expressed the DC cell surface marker CD 11 c. On day 6 of culture DC were
pulsed with
50 ug/ml of KLH, ovalbumin, or left untreated. On day 7, DCJ were transfected
with the
pBK-CMV phagemid vector (2 ~,g) containing full-length human Fast cDNA or
empty
control vector. Transfection reagent was prepared by incubating 8 lal
Lipofectin (Life
Technologies, Gaithersburg, MD) in a volume of 100 ul of P:~S at room
temperature for
45 min with the plasmid DNA. This mixture was added to 7-day cultured DC in a
final
volume of 1 ml of serum-free medium. After 4-h incubation at 37°C with
5% CO2, the
cells were washed and cultured in RPMI 1640 with 10% FCS for 48 h. Subsequent
to
CA 02453198 2004-O1-07
11
transfection, supernatants were collected at 48 hours and exosomes were
purified by
centrifugation at 7,000 g for'/a hour, pelleting, and then centrifuging at
100,000g for 3
hours, followed by one wash in PBS. Protein concentration of exosomes was
determined
by Bradfort assay. In order to test the ability of Fast-transfected and
antigen-pulsed
exosomes to suppress immune response in an antigen-specif c manner we
immunized
adult C57/BL6 mice with SOp,g/ml KLH in complete Freund's adjuvant. At the
same
time mice were administered various types of exosomes. 10 pg of exosomes per
mouse
where injected intravenously using the following groups of C57/BL6 mice: 1).
Exosomes from untransfected DC; 2). Exosomes from Fast-Transfected DC with no
antigen pulse; 3) Exosomes from Fast-transfected DC pulsed with ovalbumin
(OVA);
and 4) Exosomes from Fast-transfected DC pulsed with KLH. After a period of 2
weeks
lymph node cells were purified and stimulated in vitro with l0p,glml KLH for
72 hours
with a pulse of tritiated thymidine (1 pCulwell) for the last 16 hours of
culture.
Proliferation was assessed by scintillation counting. As seen in Figure 5 only
the KLH-
FasL-exosome caused a specific inhibition of KLH-reactive responses. In
contrast the
co-administration of Fast exosomes, OVA-pulsed-Fast-exc~somes,, or non-
transfected,
non-antigen-pulsed exosomes did not affect the KLH-specific response.
References:
1. Ohm, J.E. and D.P. Carbone, VEGF as a mediator of tumor-associated
immunodeficiency. Immunol Res, 2001. 23(2-3): p. 2:63-72.
2. Whiteside, T.L., Tumor-induced death of immune ce,~ls: its mechanisms and
consequences. Semin Cancer Biol, 2002. 12(1): p. 43-50.
3. De Visser, K.E., T.N. Schumacher, and A.M. Kruisbeek, C:,78+ T Cell
Tolerance
and Cancerlmmunotherapy. J Immunother, 2003. 26(1): p. 1-11.
4. Parmiani, G., Tumor immunity as autoimmunity: turraor antigens include
normal
self proteins which stimulate anergic peripheral T cells. Imrnunol Today,
1993.
14(11): p. 536-8.
CA 02453198 2004-O1-07
12
5. Johnson, J.G. and M.K. Jenkins, Accessory cell-derived signals required for
T cell
activation. Immunol Res, 1993. 12(1): p. 48-64.
6. Antonia, S.J., M. Extermann, and R.A. Flavell, Immunolagic
nanresponsiveness
to tumors. Crit Rev Oncog, 1998. 9(1): p. 35-41.
7. Maccalli, C., et al., Differential loss of T cell signaling molecules in
metastatic
melanoma patients' T lymphocyte subsets expressing distinct TCR variable
regions. J Immunol, 1999. 163(12): p. 6912-23.
8. Zitvogel, L., et al., Eradication of established murine~ tumors using a
novel cell-
free vaccine: dendritic cell-derived exosames. Nat Med, 1998. 4(5): p. 594-
600.
9. Wubbolts, R.W., et al., Proteomic and biochemical analyses of human B cell-
derived exosomes: potential implications for their functian and multivesicular
body formation. J Biol Chem, 2003.
10. Lamparski, H.G., et al., Production and characterization of clinical grade
exosomes derived from dendritic cells. J lmrnunol Methods, 2002. 270(2): p.
211-
26.
11. Wolfers, J., et al., Tumor-derived exosomes are a so~!rce of shared tumor
rejection antigens for CTL cross priming. Nat Med, :?001. T(3): p. 297-303.
12. Karlsson, M., et al., "Tolerosomes °' are produced by intestinal
epithelial cells. Eur
J Immunol, 2001. 31(10): p. 2892-900.
13. Peche, H., et al., Presentation of donor major histocampatibility complex
antigens
by bone marrow dendritic cell-derived exosomes modulates allograft rejection.
Transplantation, 2003. 76(10): p. 1503-10.
14. Andreola, G., et al., Induction of lymphocyte apoptosis by tumor cell
secretion of
Fast-bearing microvesicles. J Exp Med, 2002. 195(10): p. 1303-16.
15. Monleon, L, et al., Differential secretion of Fas ligand- or AP02
ligandlTNF
related apoptosis-inducing ligand-carrying microvesicles during activation-
induced death of human T cells. J lmmunol, 2001. 167(12): p. 6736-44.
16. Riteau, B., et al., Exosomes bearing HLA-G are redeG!sed by melanoma
cells. Hum
Immunol, 2003. 64(11): p. 1064-72.
CA 02453198 2004-O1-07
13
17. Gansuvd, B., et al., Umbilical cord blood dendritic cells are a rich
source of
soluble HLA-DR: synergistic effect of exosomes and dendritic cells on
autologous
or allogeneic T Cell proliferation. Hum Immunol, 2003. 64(4): p. 427-39.
18. Andre, F., et al., Malignant effusions and immunogenic tumour-derived
exosomes.
Lancet, 2002. 360(9329): p. 295-305.
19. de (~assart, A., et al., Lipid raft-associated protein sorting in
exosomes. Blood,
2003. 102(13): p. 4336-4~.
20. Admyre, C., et al., Exosomes with major laistocompat'ibility complex class
II and
co-stimulatory molecules are present in human BAL J~luid. Eur Respir J, 2003.
22(4): p. 578-83.
21. Blanchard, N., et al., TCR activation of human T cells induces the
production of
exosomes bearing the TCRlCD3/zeta complex. J Imrnunol, 2002. 16(7): p. 3235-
41.
