CA2453198A1 - Quantification and generation of immune suppressive exosomes - Google Patents
Quantification and generation of immune suppressive exosomes Download PDFInfo
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- CA2453198A1 CA2453198A1 CA 2453198 CA2453198A CA2453198A1 CA 2453198 A1 CA2453198 A1 CA 2453198A1 CA 2453198 CA2453198 CA 2453198 CA 2453198 A CA2453198 A CA 2453198A CA 2453198 A1 CA2453198 A1 CA 2453198A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70539—MHC-molecules, e.g. HLA-molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
Abstract
Methods of identifying and quantifying immune suppressive factors in biological fluids of cancer patients comprising purification and assessment of small (60-250nm) microvesicles are disclosed. Said microvesicles can serve alone, or in combination with other immunological assays as a prognastic indicator for a cancer patient, or alternatively as a determinant for various immunological and non-immunological therapeutic interventions. Methods of generating immune suppressive exosomes are also described.
Description
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.
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 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.
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.
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 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.
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 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 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.
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.
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 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 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 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.
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 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 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.
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.
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 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 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.
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.
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 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 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 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.
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 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 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.
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Claims (29)
1. A method of detecting microvesicular particles hereafter referred to as exosomes comprising the steps of:
a) Attaching an antibody that binds specifically to an epitope of a known protein found on the exosome whose detection is sought, to a surface;
b) Performing a washing step to remove antibody that is not attached to the said surface;
c) Adding to the said antibody a fluid in which exosomes are believed to be found;
d) Performing a washing step to remove matter that is unbound to the said surface-bound antibody;
e) Adding a secondary antibody, that binds specifically to an epitope found on the exosome, said secondary antibody labelled with a detection marker;
f) Performing a washing step in order to remove secondary antibody that is not bound to the exosome;
g) Adding a chemical composition to the said mixture of antibody, exosome containing fluid and secondary antibody, in order to indicate amount of said secondary antibody bound to the exosome.
a) Attaching an antibody that binds specifically to an epitope of a known protein found on the exosome whose detection is sought, to a surface;
b) Performing a washing step to remove antibody that is not attached to the said surface;
c) Adding to the said antibody a fluid in which exosomes are believed to be found;
d) Performing a washing step to remove matter that is unbound to the said surface-bound antibody;
e) Adding a secondary antibody, that binds specifically to an epitope found on the exosome, said secondary antibody labelled with a detection marker;
f) Performing a washing step in order to remove secondary antibody that is not bound to the exosome;
g) Adding a chemical composition to the said mixture of antibody, exosome containing fluid and secondary antibody, in order to indicate amount of said secondary antibody bound to the exosome.
2. The method of claim 1 wherein the plate-bound antibody detects epitopes selected from a group of proteins comprising MHC I, MHC II, HLA-G, CD9, CD81, CD82, or FasL.
3. The method of claim 1 wherein the washing step is performed with physiological saline constituted with an agent for maintaining protein shape and structure.
4. The method of claim 3 wherein said agent is albumin.
5. The method of claim 1 wherein the fluid assessed for exosomal content is selected from a group comprised of: blood, plasma, serum, urine, lacrimal secretions, sweat, or saliva.
6. The method of claim 1 wherein the secondary antibody recognizes a different epitope found on the exosome than the first, plate bound antibody, said different epitope being chosen from a group of proteins comprising MHC I, MHC II, HLA-G, CD9, CD81, CD82, or FasL.
7. A method of assessing the relative amount of cancer-induced immune suppression through quantification of FasL content on exosomes derived from biological fluids of cancer patients.
8. A method of assessing the relative amount of cancer-induced immune suppression through quantification of HLA-G content on exosomes derived from biological fluids of cancer patients.
9. A method of assessing the relative amount of cancer induced immune suppression through co-culture of cancer-derived exosomes with activated lymphocytes and detecting the amount of exosome-induced apoptosis, and exosome-induced suppresser cytokine production.
10. The method of claim 9 wherein T-cell apoptosis is detected by decreased mitochondrial potential, increased stain with Annexin-V or alterations in T
cell morphology.
cell morphology.
11. The method of claim 9 wherein suppresser cytokine production is assessed by relative levels of interleukin-4, 10, 13, or 20, transforming growth factor-beta, and other soluble agents that inhibit the proliferation of T cells.
12. A method of identifying immune-suppressive exosomes from the serum of a cancer patient comprising of:
a) Extracting a sample of patient blood;
b) Purifying plasma or serum from said patient blood;
c) Staining with an antibody directed against a surface molecule found on exosomes;
d) Staining with another antibody directed against an immune suppressive molecule found on the patient exosome;
e) Analyzing said stained exosomes through flow cytometry for expression of both the exosome marker and the immune suppressive molecule.
a) Extracting a sample of patient blood;
b) Purifying plasma or serum from said patient blood;
c) Staining with an antibody directed against a surface molecule found on exosomes;
d) Staining with another antibody directed against an immune suppressive molecule found on the patient exosome;
e) Analyzing said stained exosomes through flow cytometry for expression of both the exosome marker and the immune suppressive molecule.
13. The method of claim 12 wherein said exosome-staining antibody is directed against MHC I.
14. The method of claim 12 wherein said immune suppressive molecule on exosomes is Fas ligand.
15. The method of claim 12 wherein said immune suppressive molecule on exosomes is HLA-G.
16. A method for suppressing immune responses in an antigen-specific manner.
17. The method of claim 16, wherein immune responses refers to activation of T
cells, said T cells comprising CD8+, CD4+, or NKT cells
cells, said T cells comprising CD8+, CD4+, or NKT cells
18. The method of claim 16, wherein activation refers to proliferation, cytokine secretion, and direct or indirect cytotoxic and cytolytic activities.
19. The method of claim 16 wherein antigen-specific means the immune response directed against a certain chemical entity, or class of chemical entities.
20. The method of claim 16 wherein where the method of suppressing immune responses involves:
a) The extraction of antigen presenting cells with suppressive properties from a mammal and artificially endowing a population of antigen presenting cells with suppressive properties through manipulation;
b) Purification of exosome vesicles released from the suppressive cell population;
c) Administration of the exosome vesicles to the mammal in which immune suppression is desired.
a) The extraction of antigen presenting cells with suppressive properties from a mammal and artificially endowing a population of antigen presenting cells with suppressive properties through manipulation;
b) Purification of exosome vesicles released from the suppressive cell population;
c) Administration of the exosome vesicles to the mammal in which immune suppression is desired.
21. The method of claim 20 wherein antigen presenting cells can include B
cells, macrophages, and dendritic cells.
cells, macrophages, and dendritic cells.
22. The method of claim 20 wherein the exosome is prepared by extracted supernatant from antigen presenting cells and centrifuging at 300 g for 5 minutes, then 1200 g for 20 minutes, then 10000 g for 30 minutes, and 60 minutes at 110 000 g, subsequently to which the remaining pellet is resuspended in a physiological mixture and used as a source of exosomes.
23. The method of claim 20 wherein extraction of antigen presenting cells with suppressive properties is accomplished by purification with antibody based approaches.
24. The method of claim 20 wherein antibody-based approaches include purification though antibody-coated magnetic beads using antibodies for markers found on immature or T cell suppressing DC
25. The method of claim 24 wherein said antibody is selected from a group targeting DEC-205, CD123, PD-1 ligand, or TGF-b.
26. The method of claim 20 wherein the induction of suppressive properties in the antigen presenting cell is accomplished by treatment of the cells with cytokines selected from a group comprising: IL-4, IL-10, VEGF, TGF-b or IL-13.
27. The method of claim 20 wherein the induction of suppressive properties in the antigen presenting cell is accomplished by transfection of the antigen presenting cell with molecules which will suppress T cell activation such as IL-4, IL-10, VEGF, TGF-b or IL-13 or indoleamine-2,3-deoxygenase.
28. The method of claim 20 wherein the induction of suppressive properties in the antigen presenting cell is accomplished by transfection of the antigen presenting cell with molecules that inhibit T cell viability such as Fas ligand.
29. The method of claim 20 wherein the induction of suppressive properties in the antigen presenting cell is accomplished by transfection of the antigen presenting cell with agents that specifically inhibit expression of immune stimulatory molecules such as short interfering RNA targeting CD40, CD80, CD86, Rel-b, Rel-c, NF-kB p50, NF-kB
p65, STAT4 or T-bet.
p65, STAT4 or T-bet.
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