EXTRACELLULAR VESICLES COMPRISING MEMBRANE-TETHERED TGF-BETA, COMPOSITIONS AND METHODS OF USE THEREOF
20200392219 · 2020-12-17
Assignee
Inventors
Cpc classification
A61K35/51
HUMAN NECESSITIES
C12N5/0665
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K14/70596
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
G01N33/5076
PHYSICS
C07K14/70578
CHEMISTRY; METALLURGY
International classification
C07K16/22
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
C12N5/00
CHEMISTRY; METALLURGY
Abstract
Provided are mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) having tethered (membrane-bound) TGF- (MSC-derived membrane-tethered TGF- EV), and compositions containing such EV for use as therapeutics and immunomodulatory agents. Provided also are diagnostic methods and methods of assessing or monitoring disease status and/or progression in patients using membrane-tethered TGF- derived from a variety of cell sources that serve as detectable, quantifiable biomarkers in biological samples. The MSC-derived membrane-tethered TGF- EV can also be used to deliver various bioactive agents to a target cell or tissue for treating various diseases. The level of TGF- tethered to the membrane of the EV can also be modified or manipulated in vitro or ex vivo. Such modified MSC-derived membrane-tethered TGF- EV are useful as immunotherapeutic agents in the treatment or management of certain diseases, particularly those involving inflammation, autoimmunity, transplant rejection and cancer.
Claims
1.-98. (canceled)
99. An isolated extracellular vesicle (EV) comprising transforming growth factor-beta (TGF-13) or an isoform thereof tethered to the membrane surface, wherein the EV is produced by an immortalized cell.
100. The extracellular vesicle (EV) according to claim 1, wherein the immortalized cell is an immune privileged cell selected from the group consisting of umbilical cord, placenta, fetus, testes and articular cartilage.
101. The extracellular vesicle (EV) according to claim 1, wherein the immortalized cell is derived from a stromal cell, stem cell, stromal stem cell, mesenchymal stromal cell (MSC), cancer-associated cell, or fibroblast-like cell.
102. The extracellular vesicle (EV) according to claim 1, wherein the TGF-I3 or isoform thereof is tethered to the membrane of the EV via attachment to one or more of a glycoprotein, P-glycan, or heparin.
102. The extracellular vesicle (EV) according to claim 1, wherein the tethered TGF-I3 is TGF-I31, TGF-r32, TGF-I33, TGF-I34, or a latent form thereof.
104. The extracellular vesicle (EV) according to claim 1, wherein the EV comprises tethered TGF-(3 and at least one other tethered immunomodulatory molecule.
105. The extracellular vesicle (EV) according to claim 6, wherein the at least one other tethered immunomodulatory molecule is selected from PD-1, PD-LI, B7-H4, B7-H5, CTLA-4, 4-1-BB, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, GITR, LAG-3, OX40, TIM3, TIM4, CEA, TLR, TLR2, etc., or cytokines, e.g., IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IFN-, Flt3, BLys, chemokines, e.g., CCL21, or Galectin-1.
106. The extracellular vesicle (EV) according to claim 1, wherein the EV comprises an exogenous agent.
107. The extracellular vesicle (EV) according to claim 8, wherein the exogenous agent is a polypeptide, polynucleotide, or small molecule.
108. A method of isolating mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) having membrane-tethered TGF-I3 (MSC-derived, membrane-tethered TGF-I3 EV), the method comprising: culturing MSC, or a cell or tissue source of MSC, in cell culture or conditioned medium; isolating the MSC-derived, membrane-tethered TGF-I3 EV from the cell culture or conditioned medium; and optionally, quantifying the amount of MSC-derived, membrane-tethered TGF-I3 EV from the cell or tissue source.
109. The method according to claim 10, wherein cell or tissue source is selected from a biological fluid, umbilical cord tissue, placental tissue, fat, or bone marrow.
110. The method according to claim 10, wherein the MSC are cultured in culture medium for from about 1 day to about 20 days.
11. The method according to claim 10, wherein the culture or conditioned medium is a serum free chemically defined buffered medium, or medium comprised of autologous serum and defined constituents.
112. The method according to claim 10, wherein the MSC-derived, membrane-tethered TGF-f3 EV are isolated by one or more of affinity column chromatography, immune affinity capture, tangential flow filtration, precipitation, differential ultracentrifugation, density gradient centrifugation, or size exclusion chromatography.
113. The method according to claim 10, further comprising quantifying the amount of TGF-P, or a latent form thereof, tethered to the isolated EV having membrane tethered TGF-f3.
114. The method according to claim 15, wherein membrane tethered TGF-f3 is quantified by single vesicle nanoparticle tracking assay, vesiculometry, interferometry, or flow cytometry.
115. A composition for imaging cells or tissue, the composition comprising an extracellular vesicle (EV) according to claim 1, containing an imaging agent.
116. The composition according to claim 17, wherein the imaging agent is a nanoparticle, magnetite, nanoparticle, paramagnetic particle, microsphere, nanosphere, and is selectively targeted to cancer cells.
117. A kit for providing to a subject an extracellular vesicle (EV) derived from mesenchymal stromal cells (MSC) and comprising membrane-tethered TGF-r3 or an isoform thereof (MSC-derived, membrane-tethered TGF-13 EV) as a therapeutic agent, the kit comprising MSC-derived, membrane-tethered TGF-13 EV isolated from MSC.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
[0105]
[0106]
[0107]
[0108]
DETAILED DESCRIPTION OF THE INVENTION
[0109] The invention provides methods for disease assessment, evaluation, diagnosis and monitoring involving extracellular vesicles (EV) derived from a cell source, such as, without limitation, a cancer cell, cancer associated cell (e.g., fibroblast-like cell, cancer-associated fibroblast), a dendritic cell, stromal cell, or stromal stem cell, having tethered to the membrane (i.e., membrane bound) an immunomodulatory molecule (e.g., polypeptide, polynucleotide, small molecule). In a particular embodiment, the cell source is a mesenchymal stromal cell or MSC (also called a meschenchymal stem cell). In another particular embodiment, the membrane-tethered molecule is TGF- or an isoform thereof, e.g., TGF-1-4. In another particular embodiment, the cell source is an MSC and the extracellular vesicles (EV) are MSC-derived and comprise TGF- or an isoform thereof, e.g., TGF-1, tethered to the membrane surface.
[0110] The present invention is based, at least in part, on the finding that TGF- is tethered (i.e. membrane bound) to the membrane of extracellular vesicles (EV) derived from a number of different cell types, including, by way of example, cancer cells, cancer associated cells (e.g., fibroblast-like cells, cancer-associated fibroblasts), dendritic cells, stromal cells, e.g., mesenchymal stromal cells (MSC), or stromal stem cells, and the recognition that MSC-derived EV expressing membrane-tethered TGF- play a critical role in immunomodulation of diseases and conditions, and immunomodulatory activity, and therefore, also play a critical role in the potency of cells such as MSC. For example, EV having membrane-tethered TGF-, such as MSC-derived TGF- having membrane-tethered TGF-, can have profound immunosuppression and anti-inflammatory actions, particularly in subjects having a disease or condition, such as an inflammatory disease, an autoimmune disease, transplant rejection, or cancer. Such TGF--tethered MSC EV can exert an immunosuppression effect that is counterproductive in an individual's defense against disease or the treatment thereof, for example, cancer or cancer treatment, as well as in the effectiveness of EV-based vaccines.
[0111] Accordingly, provided by the invention are extracellular vesicles (EV) derived from a cell type (e.g., cancer associated cells, fibroblast-like cells, stromal cells, stromal stem cells, dendritic cells, cancer cells, MSCs, or other cells that originate from a site of immune privilege), that express TGF- or an isoform thereof (e.g., TGF-1, TGF-2, TGF-3, or TGF-4) tethered to the membrane of the EV, and methods in which such TGF--tethered EV are used in diagnosis, determining immune status, monitoring therapy or treatment of disease, or assessing, evaluating or diagnosing a disease or condition in a subject or in a patient population. In other embodiments, another immunomodulatory agent, (e.g., polypeptide, polynucleotide, small molecule), in addition to or instead of TGF- is tethered to the membrane of the EV. Methods are also provided to quantify TGF- on EV as a benchmark of disease activity (e.g., a standard against which disease activity is measured, evaluated, or compared) in a subject or in a patient population, e.g., a subject or patient population undergoing treatment or therapy for a disease, e.g., an inflammatory disease, an autoimmune disease, transplant rejection, heart disease, or cancer. In addition, methods of altering or controlling the amount, level, or density of TGF- tethered to the EV membrane are encompassed by the invention, as described herein. Also encompassed by the invention are the resulting membrane-tethered TGF- EV, which can be used as therapeutic products in patients with a number of diseases and conditions.
[0112] In accordance with the invention, the measurement of tethered TGF- on EV membrane provides an advantageous approach to assessing, monitoring, or benchmarking the immune status of human or animal (veterinary) patients. As used herein, benchmarking refers to methods and approaches for comparing to best practices, novel technologies, or other gold standard technologies or assays for assessing immune status. The accurate quantification of membrane-tethered TGF- (or other immunomodulatory proteins) on the EV in a biofluid obtained from a subject (patient) provides an improved index or determination of disease activity, aggressiveness, prognosis, and/or response to therapy, or other aspects of the natural history or the course of a disease.
[0113] In addition, because extracellular vesicles (EV) having membrane-tethered TGF- are biologically active, they provide an actionable target which may be useful for increasing or restricting activity related to a disease. For example, in instances in which the fraction of EV with membrane-tethered TGF-, or the level of membrane-tethered TGF- expression per EV, in a subject is lower than expected based on quantification as described herein and comparison with a control or reference (e.g., reference ranges for a particular cohort), reduced immunosuppression (e.g. in autoimmune disease, chronic inflammation, allergy) may be indicated, thereby leading to the supplementation of EV having membrane-tethered with TGF- in the subject. In an embodiment, the EV having membrane-tethered TGF- can be selected or isolated from total EV derived from MSC as described herein. Conversely, a finding of an excessive level of MSC-derived EV having membrane-tethered TGF- in a subject (e.g., a subject having cancer) as quantified by the methods described herein can prompt interventions that neutralize the tethered TGF- in the subject, thus specifically restoring immune activity. Nonlimiting examples of auto-immune diseases and disorders associated with a low concentration, amount, or level of membrane-tethered TGF- on EV include asthma, atopic dermatitis, inflammatory bowel diseases, psoriasis, multiple sclerosis, or lupus. Nonlimiting examples of immunosuppression diseases and conditions that are associated with a high concentration, amount, or level of membrane-tethered TGF- on EV include cancer or drug induced (chemotherapeutic) or radiation induced immunosuppression.
[0114] Pertinent to therapeutics and the methods described herein, the accurate quantification of immunomodulatory protein tethered to the membrane of EV produced by immunotherapeutic MSC or other cell types, e.g., membrane-tethered TGF- EV derived from MSC, allows for improved selection criteria for EV potency to improve patient therapy or treatment. In an embodiment, membrane-tethered TGF- provides a target for direct isolation of a specific subset of EV with high levels of membrane-tethered TGF- (or other immunomodulatory molecules). Such EV comprising membrane-tethered TGF- can be used as immunosuppressive agents, for example, if the isolated membrane-tethered TGF- EV are administered to a subject or to a cell culture. In another embodiment, membrane-tethered TGF- provides a target for direct isolation of a specific subset of EV with high levels of membrane-tethered TGF- (or other immunomodulatory molecules) from a biological sample or cell culture, so as to deplete EV with membrane-tethered TGF- (or other immunomodulatory molecules) from the sample, Accordingly, EV obtained from a sample depleted of EV with a high level of membrane-tethered TGF- can be used as a therapeutic in a subject or cell culture, for example, to reduce the immunosuppressive effect of EV having high level of membrane-tethered TGF-. In another embodiment, EV having membrane-tethered TGF- provide a target for direct isolation of a specific subset of EV with low levels of membrane-tethered TGF- (or other immunomodulatory molecules).
Extracellular Vesicles with Membrane-Tethered (Membrane Bound) TGF-
[0115] Extracellular vesicles (EV) are nanoscale membrane-bound structures originating from early endosomes, which are released by all cells as part of the paracrine system of intercellular communication (Yanez-Mo, M. et al., 2015, J. Extracell. Vesicles, Vol. 4:27066). The membranous and internal compartment of EV contains bioactive molecules including RNA, DNA, protein, and lipids. EV from dendritic cells, T regulatory cells, tumor cells, tumor stromal cells, and mesenchymal stem cells impart immunosuppressive effects on recipient cells (Zhang, B. et al., 2014, Front. Immunol., Vol 5:518). The immunotherapeutic potential possessed by mesenchymal stem cells (MSC) and extracellular vesicles (EV) derived from MSC may be associated with their expression of various proteins, including TGF-, PD-LI, Galectin 1, PGE2, CD73 and CD39, IDO, and IL-10 (Bruno, S. et al., 2015, Immunol. Lett., Vol. 168(2):154-158). As embraced by the invention and as will be appreciated by the skilled practitioner, TGF- represents a class of immunomodulatory molecules that can be tethered to the membrane of extracellular vesicles (EV), alone or in combination with other molecules, for example and without limitation, PD-L1, FasL, and Galectin-1.
[0116] While soluble TGF- protein present in plasma or serum may indicate chronic inflammatory or autoimmune diseases (e.g., psoriasis) or fibrotic conditions (e.g. interstitial pneumonia), it is believed that the use of TGF- tethered to the EV membrane was not known or considered to be an effective, quantifiable molecule or biomarker for any disease or condition until the present invention. Tethered TGF- comprises only a fraction of the total TGF- in biofluids or cell culture supernatants; yet TGF- tethered to the EV membrane is more bioactive than soluble TGF-. By way of example, membranous TGF- on EV from tumor cells induced a cancer-like phenotype (e.g. pro-angiogenic, tumor promoting) in myofibroblasts, while soluble TGF- did not (Webber, J. P. et al., 2015, Oncogene, Vol. 34(3):290-302). Thus, the invention provides advantageous methods involving quantification of TGF- tethered to the membrane of extracellular vesicles (EV) as a more bioactive form of TGF- for the assessment and evaluation of disease status or for the provision of disease therapy or treatment in a subject who is afflicted with a disease. In embodiments, the various isoforms of TGF- tethered to the EV membrane, namely, TGF-1, TGF-2, TGF-3 or TGF-4, can be quantified and assessed or evaluated as a disease biomarker in accordance with the described methods, which provide an improvement on current processes that consider only total serum or plasma levels of soluble TGF-. In a particular embodiment, EV having membrane-tethered TGF-1 serves as a biomarker that may be quantified and assessed as correlating with a number of diseases, or the status thereof, including cancer and non-cancer diseases and conditions, for example, cancers of the types described herein, as well as autoimmune diseases (e.g., psoriasis, arthritis, multiple sclerosis, system sclerosis, amyotrophic lateral sclerosis (ALS)), inflammatory diseases, transplantation rejection, myocardial infarction; and coronary disease. In another embodiment, the quantification of TGF- tethered to EV from MSC can be used to determine the potency of MSC cell lines, thereby enabling the selection or isolation of MSC cell lines that are more immunosuppressive by virtue of their production of membrane-tethered TGF- EV that are determined to have a higher concentration of tethered TGF-. In another embodiment, tethered TGF- EV produced by such MSC can be isolated and administered as a therapeutic having immunosuppressive function in disease treatment for a subject in need.
Extracellular Vesicles (EV) Comprising Membrane-Tethered TGF-Methods of Detection, Isolation and Use
[0117] TGF- tethered to the membrane of a variety of cell types, and particularly, TGF- tethered to the membrane of extracellular vesicles (EV) derived from these cell types, e.g., cancer-associated fibroblasts, stromal cells, dendritic cells, mesenchymal stromal cells, and cells obtained from sites of immune privilege as described herein by way of nonlimiting example, provides a measurable and specific, disease-associated target for use in the methods described and provided herein. In a certain embodiment, isolated EV derived from mesenchymal stromal cells (MSC) and comprising membrane tethered TGF- are particularly suitable for the uses as described herein. In another embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes on their surfaces during the isolation process, for example, centrifuged or sedimented EV may be treated with hyaluronidase at the time of resuspending the centrifuged pellet containing EV (See, e.g., Example 5, infra).
[0118] In embodiments of the methods, TGF- (including isoforms TGF-1, TGF-2, TGF-3 and/or TGF-4) on the surface of extracellular vesicles (EV), e.g., tethered to the EV surface membrane via beta-glycan, (also referred to as TGF-R3), can be quantified (measured) in a biological sample from a subject or in cell culture using several different methods. In an embodiment, single vesicle nanoparticle tracking analysis can be used in which TGF- is immunolabeled by QDOT conjugated antibody or indirect labeling (e.g. biotin-antibody, streptavidin-QDOT) is used to quantify TGF- tethered to the membrane of EV. According to this method, membrane-tethered TGF- EV are labeled using a biotinylated primary antibody (e.g., anti-human TGF-/LAP, clone: CH6-17E5.1 (Miltenyi Biotec Inc., San Diego, Calif.), incubating at 4 C. for from 10-60 minutes, or for 20-45 minutes, or for at least 30 minutes, or for 30 minutes, washing by ultracentrifugation (100,000G, 120 min, 70Ti rotor) to remove unbound antibody, filtering with 0.22 um filter to remove aggregates if necessary, and labeled secondarily with streptavidin conjugated quantum dots (655 nm emission, QDOT, ThermoFisher, Waltham, Mass.). Unbound streptavidin-QDOTS (20 nm diameter) are separated from labeled EV using size exclusion chromatography (HPLC, e.g. Agilent 1100, column: AdvanceBio SEC-5, 300 {dot over (A)}, 2.7 um, 7.8300 mm, mobile phase pH 7.4 PBS, flow rate 0.5 ml-1.0 ml/min), based on UV absorbance at 220 nm or 280 nm. QDOT separation is confirmed using fluorescence detection (488 nm excitation/655 nm emission) coupled to the HPLC instrument, The percentage of membrane-tethered TGF- EV that are QDOT labeled (TGF-.sup.pos) is quantified. The intensity of TGF- expression on EV of different sizes can also be evaluated. Mathematical corrections for subdiffusion may be necessary to obtain accurate particle size distribution; however, this does not interfere with the measurement of single vesicle expression of TGF-. Total EV in a sample used as the denominator, can be identified (and distinguished from protein complexes including lipoproteins) based on staining with EV-specific or EV-enriched surface markers such as CD9, CD63, CD81, LAMP-2, heat shock proteins, Alix, synectin, or flotillin, or by the use of fluorogenic dyes, or molecular beacons, or by staining the nucleic acid cargo of the EV.
[0119] In another embodiment, vesiculometry employing fluorescence detection of immunolabeled EV or those absorbed to beads can be used to quantify membrane-tethered TGF- EV. In another embodiment, TGF- and isoforms thereof tethered to the membrane of EV, such as EV derived from MSC, can be quantified in a subject's biological sample, e.g., blood or plasma, or in a cell culture by a method, e.g., an interferometry method, that does not require the isolation of EV from the sample or culture.
[0120] Interferometry is an example of a system used by the skilled practitioner that utilizes fluidics to analyze very small volumes of plasma or serum. In general, antibodies that bind to TGF- (e.g. anti-TGF- antibodies) are used to capture and immobilize EV with membrane-tethered TGF-, e.g., in a well of an assay plate; this binding interferes with the transmission of light at that location in the well (Daaboul, G. G. et al., 2016, Scientific Reports, 6, Article number:37246). In an embodiment, a direct immunocapture method can be utilized for the enumeration of EV with membrane-tethered TGF- in which specific antibodies, e.g., anti-TGF- antibodies (anti-TGF- capture antibodies) are attached to a substrate or solid phase, e.g., a chip, membrane, or film. A biological sample or culture supernatant (culture medium) is contacted with the substrate, and TGF- tethered to the EV membrane binds directly to the capture antibodies. For example, the sample or supernatant can be contacted with the substrate and capture antibodies for 10-60 minutes, for 20-45 minutes, for at least 30 minutes, or for 30 minutes, at room temperature or at 4 C. Thereafter, light interference measurement is used to determine the amount of TGF- (membrane-tethered TGF-) that is bound to the anti-TGF- antibodies on the film (e.g., NANO-VIEW nano- and micro-positioners, MCL, Madison, Wis.). The use of anti-TGF- antibodies to capture EV comprising membrane-tethered TGF- allows for direct assay of EV with TGF- tethered to the membrane surface, including all TGF- isoforms. The technique of interferometry (e.g., attachment to a chip or membrane) can be used to quantify the frequency with which an epitope, such as membrane-tethered TGF-, can be found on EV. The membrane-tethered form of TGF- can be distinguished from soluble non-membrane-tethered TGF-, because the soluble form does not interfere with light sufficiently to be counted as an EV having membrane-tethered TGF-, i.e., it is undetected by the system.
[0121] Accordingly, interferometry, vesiculometry (flow cytometry technology adapted for nanoparticle assessment), nanoparticle tracking analysis fluorescence or any method that assesses, determines, or benchmarks the quantity, phenotype and size distribution of EV with membrane-tethered TGF- may be used to quantify TGF- tethered to the membrane of EV. The measurement data are quantified relative to total EV, total protein, or total EV proteins (e.g. CD9, CD63, CD81, TSG101, flotillin, synectin, LAMP-2, or Alix), nucleic acids, lipids, or other constituents that represent the total EV population in a sample. In addition, the data may be used to stratify patient status by stage, aggressiveness, prognosis, resistance to therapy, or any aspect of disease status.
[0122] In an embodiment, membrane-tethered TGF- EV are isolated or enriched from a subject's biological sample or from cell culture medium or supernatant. By way of example, biofluids samples such as blood, urine, cerebrospinal fluid, or saliva obtained from subjects (patients), or cell culture supernatants, are cleared of cells, platelets, apoptotic bodies, cell debris, protein aggregates, and other particulates that are not extracellular vesicles. This can be achieved by differential centrifugation (e.g., 1300g for 10 minutes to remove cells and platelets, 2000g for 10 minutes to remove apoptotic bodies, and 10,000g for 30 minutes to remove microvesicles), or by sequential filtration after clarification of cells and apoptotic bodies using a 200 nm filter.
[0123] The measured levels of EV with membrane-tethered TGF- isolated from a biological sample should fall within a reference range, above or below which is interpretable as active disease or abnormal levels. The reference range can be determined from a subject having no disease (healthy or normal subject), from a subject having non-active disease, or from a sample obtained from the same subject at a first time point, or at an earlier, or pre-active disease, time point. In addition, a suitable reference or control can be the amount of membrane-tethered-TGF- EV relative to total protein, EV specific proteins, or total EV numbers in the sample undergoing analysis. By way of example, a greater than or equal to 1.5 fold change, or an at least 1.5-fold change, in the level of membrane-tethered TGF- EV in a sample undergoing testing or quantification and the reference (or control) sample, is indicative of a biologically relevant or significant difference in the quantification analysis. In embodiments, a change in the level of membrane-tethered TGF- EV in a sample relative to a reference or control level of at least or equal to 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 10.5-fold, 11-fold, 12-fold, 15-fold, 20-fold, 25-fold, or greater, including values therebetween, is indicative of a biologically relevant or significant difference in the quantification analysis. Active disease is defined as disease with an abnormal, i.e., high, elevated, or low, level or amount of membrane-tethered-TGF- EV or progression of this trend in a single patient. In an embodiment, the level or amount of membrane-tethered-TGF- EV is increased or elevated relative to a control level or amount. Inactive disease implies normal membrane-tethered-TGF- EV and normal immune function associated with this molecule. In an embodiment, an increased amount of membrane-tethered-TGF- EV derived from MSC indicates superior immunosuppression potential of such membrane-tethered TGF- EV as a therapeutic. Decreased amounts of membrane-tethered-TGF- EV or on parent MSC indicates that the membrane-tethered TGF- EV have low potential or low potency potential as a therapeutic. Hence, membrane-tethered-TGF- EV, such as MSC-derived membrane-tethered TGF- EV, can be used to select superior cells, e.g., MSC, for producing membrane-tethered TGF- EV of high potency as a therapeutic. Alternatively, EV with decreased amounts of membrane-tethered-TGF-, e.g., derived from MSC, or EV without any membrane-tethered-TGF-, may be applicable for use, e.g., as a therapeutic, in immunosuppressive states to avoid compounding the native immunosuppressive state of the patient.
[0124] In an embodiment, membrane-tethered TGF- EV are isolated or enriched from cultured cells, e.g., MSC. MSC isolated from biological samples are typically maintained in culture for 1 to 20 days, or for 48 hours, for collection of EV having membrane-tethered TGF-. In general, MSC are maintained under standard culture conditions for cell growth, and are washed and transferred to serum free defined chemical medium, e.g. DMEM, L-glutamine (1 mM), and 5 ng/ml FGF2 and 5 ng/ml PDGF-AB glycoprotein (platelet derived growth factor-AB). Buffer (HEPES) is added to stabilize pH, e.g., pH 5-8.5, or pH 5.5-7.5, or pH 6-7.5, or pH 6-7. Other growth factors (e.g., EGF, 5 ng/ml) can be used to enhance the production and release of EV in certain cell lines. Immortalized MSC may be maintained in culture for 1-20 days and longer, e.g., for weeks or months with appropriate cell culture techniques. For cell culture supernatants, initial steps of concentration of EV are necessary, for example filtration (e.g., 200 nm pore size) to remove cells and cell debris, followed by tangential flow filtration (e.g. 50,000-300,000 kDa molecular weight cutoff (MWCO)). Extracellular vesicles are isolated from the clarified sample (e.g. plasma, serum, cell culture supernatant), for example, either by affinity column chromatography, tangential flow filtration (e.g., >50 kDa MWCO filter), precipitation (e.g., using PEG or ExoQuick, a polymer that gently precipitates extracellular vesicles, System Biosciences (SBI), Palo Alto, Calif.), differential ultracentrifugation (e.g., 100,000g for 70 min using 70Ti rotor to sediment EV), density gradient centrifugation, size exclusion chromatography (e.g. 30-45 nm pore size), or other methods practiced in the art for the isolation and concentration of EV. In an embodiment, the EV, e.g., centrifuged or sedimented preparations of EV, are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes on their surfaces.
[0125] In an embodiment, the biological activity of isolated EV comprising membrane-tethered TGF- can be assayed. Assay of biological activity of TGF- tethered to the EV membrane is achieved by any number of immunoassays (assays using immune cells), including assessment of the degree of suppression of (i) mitogen-induced (e.g., Concanavalin A, 5 ng/ml for 72 hr) T cell proliferation; (ii) CD3/CD28-induced T cell proliferation; (iii) T cell production of IFN or IL-17; (iv) CD69 expression by activated T cells; (v) differentiation or expansion of a T regulator cell subset; (vi) natural killer (NK) cell differentiation or activation; or (vii) maturation of dendritic cells (e.g., CD1a, MHCII, CD80, CD86 expression), using methods practiced by one skilled in the art. Tethered-TGF- EV isolated from cell culture supernatant or patient samples (e.g., blood) can be assayed biologically to evaluate immunosuppressive function.
Therapeutic Applications Involving TGF- Tethered EV and Modified Forms Thereof
[0126] Quantification of the levels or amount of endogenous EV having membrane-tethered TGF- relative to the total EV present in a biological sample obtained from a subject having a disease can be used by a physician or medical practitioner to decide on a specific therapy or treatment for the subject, in particular, a subject who is suffering from immunosuppression, e.g., during or as a result of treatment or therapy for a disease or condition. In an embodiment, patient status is defined as the level or amount of endogenous EV having membrane-tethered TGF- relative to the total EV present in the patient's biological sample, and can be assessed, evaluated, identified and monitored via the methods involving membrane-tethered TGF- EV as described herein. In an embodiment, a specific therapy directed by patient status includes a specific total dose (e.g., total number, concentration, route of administration) of EV expressing membrane-tethered TGF- provided to the patient to achieve a desired outcome in the treatment of the patient's disease.
[0127] Examples of autoimmunity diseases, conditions, or disorders that are associated with low measured levels of TGF- tethered to the EV membrane include asthma, atopic dermatitis, inflammatory bowel diseases, psoriasis, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, graft versus host disease, sarcoidosis, Sjogren's Disease, or lupus. Low or decreased levels of membrane-tethered TGF- EV may also be induced by drugs, radiation, trauma, or stress. In an embodiment, subjects (patients) who have or are determined to have low or decreased levels or amounts of membrane-tethered TGF- EV are administered EV comprising membrane-tethered TGF- to provide an immunosuppressive effect in the subject, for example, by direct intravenous, intra-spinal, intra-coronary, intra-lesional, topical, or other appropriate route of administration. The levels of exogenously supplied EV with membrane-tethered TGF- are expected to rise transiently (e.g., for <24 hours), followed by a longer increase of EV with membrane-tethered TGF- in circulating and tissue by recruitment of T regulatory cells and other cells that synthesize EV with membrane-tethered TGF-.
[0128] While a primary cause of immunosuppression in patients with low levels of EV with membrane-tethered TGF- (e.g., low levels in circulation or other biofluids) is cancer, there are also many examples of immunosuppression that is induced by other causes, such as viral infection, drugs, stress, trauma, degeneration, other infections. Without wishing to be bound by theory, in patients who are afflicted with diseases or conditions of these types, membrane-tethered TGF- EV are expected to be disadvantageous; hence, interventions to reduce membrane-tethered TGF- EV are preferred. Methods for reducing EV with membrane-tethered TGF- or activity include, without limitation, serum neutralizing antibodies or molecularly-engineered or recombinantly produced antibodies, oligonucleotides (RNAi, anti-sense) to knock down membranous TGF-, gene therapy, or pharmacological inhibitors of TGF-, e.g., Smad inhibitors (e.g., SIS3, which inhibits Smad3), pirfenidone, or other commercially available TGF- inhibitors. By way of example, Smad refers to a family of eight proteins that participate in tumor suppression in conjunction with TGF-. Smad 1,2,3,5 and 8 are receptor-activated; Smad 4 is a co-mediator; and Smad 6 and 7 are inhibitory. The term Smad is derived from the homology of these proteins to the Sma protein of Caenorhabditis elegans and the MAD proteins of Drosophila. Alternatively, the cell sources for membrane-tethered TGF- EV can be reduced or eliminated by a method such as, for example, aphaeresis, targeted cytotoxicity, or chemotherapeutic agent treatment as known by the skilled practitioner. Reduction of EV with membrane-tethered TGF- may be combined with approaches that reduce tumor or cancer cell burden. The recurrence of the primary source of EV with membrane-tethered TGF- is reflected in rising levels of EV with membrane-tethered TGF- in biofluid samples of subjects, e.g., patients who are being treated for cancer or who have been treated for cancer. Thus, EV with membrane-tethered TGF- is a useful biomarker of residual cancer burden, recurrence, failure of cancer therapy, or resistance to therapy. Rising levels or amounts of membrane-tethered TGF- EV is an indicator or biomarker of prognosis in cancer, with rising levels indicating or correlating with poorer prognosis or increased risk for or presence of malignancy or metastases.
[0129] In an embodiment, extracellular vesicles (EV) having membrane-tethered TGF- are isolated, e.g., from primary or immortalized MSC in cell culture, and TGF- tethered to the EV membrane may be substituted with one more different immunomodulatory molecules using routine molecular biological techniques. In an embodiment, one or more different immunomodulatory molecules may be tethered to the EV membrane in addition to tethered TGF-. In another embodiment, an extracellular vesicle (EV) which has one of several immunomodulatory molecules tethered to the membrane can be produced by common molecular biological methods. Illustrative, yet nonlimiting examples of such immunomodulatory molecules that can be tethered with (or instead of) TGF- on the membrane of EV include PD-1, PD-LI, B7-H4, B7-H5, CTLA-4, 4-1-BB, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, GITR, LAG-3, OX40, TIM3, TIM4, CEA, TLR, TLR2, etc., or cytokines, e.g., IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IFN-, Flt3, BLys, chemokines, e.g., CCL21, or Galectin-1. By way of example, such additional molecules may enhance the activity of tethered TGF- on MSC-derived EV having membrane tethered TGF- when such EV are used as a therapeutic. Moreover, should such immunomodulatory molecules be tethered to the membrane of EV, in addition to membrane tethered TGF-, they may be used as further markers in the detection or diagnostic the methods described herein.
[0130] In another embodiment, EV comprising membrane-tethered TGF- (e.g., mesenchymal stromal cell (MSC)-derived or dendritic cell-derived EV) are isolated and TGF- removed from the EV membrane using proteinases, glycanases, or heparinases. By way of example, dendritic-cell-derived EV lacking TGF- can be used as antigen-presenting agents administered to a subject in need, e.g., as tumor vaccines, thereby alleviating or substantially alleviating immunosuppression associated with membrane-tethered TGF-. In an embodiment, such dendritic cells, and, in turn, the EV derived therefrom, can be recombinantly modified to express certain tumor associated antigens to enhance immune cell response against tumors. In another embodiment, MSC-derived EV which lack membrane-tethered TGF-, for example, the EV lacking membrane-tethered TGF- can be negatively selected by immune affinity techniques. By way of example, this can be accomplished by depletion of membrane-tethered TGF- EV from a mixture of EV, in which the mixture contains EV that have membrane-tethered TGF- as well as EV that lack membrane-tethered TGF-, using magnetic sorting (e.g., EV are incubated with anti-TGF- antibodies conjugated to biotin, which is then incubated with secondary streptavidin bound to magnetic beads, which is used to remove EV with TGF- tethered on the membrane surface magnetically (e.g. using an AUTO-MACS cell separation device, Miltenyi Biotec Inc., San Diego, Calif.), while leaving EV that do not contain measurable tethered TGF- in the depleted fraction for utility. Such EV depleted of membrane-tethered TGF- could be advantageously loaded with a bioactive agent or cargo (e.g., polypeptides or polynucleotides (RNA, miRNA) and would not possess the biological activity of TGF-. Other methods for EV sorting on the basis of membrane-tethered TGF- would serve the same purpose. By way of example, TGF- negative EV therapy may be more effective than EV with membrane-tethered TGF- as treatment or therapy for particular diseases or conditions, such as pro-fibrotic states, e.g., the fibrotic phase after myocardial infarction.
[0131] In an embodiment, TGF- can be removed from isolated EV having membrane-tethered TGF- by enzymatic digestion, e.g., with proteases, glycanases, or heparinases. In an embodiment, EV having membrane tethered TGF- of EV with membrane tethered EV removed may be loaded with a bioactive agent as described herein and employed as an immunogenic vector. By way of example, a dendritic cell-derived extracellular vesicles (EV) in which membrane-tethered TGF- is removed can be genetically engineered to contain an antigen, e.g., protein or peptide, that is presented by the dendritic EV to immune cells can serve as a cancer vaccine which lacks immunosuppressive activity. Accordingly, the removal of membrane tethered TGF- from EV may enhance the anti-tumor effectiveness of EV used as tumor vaccines, or may allow a reduced amount of such EV to be administered to a subject in need.
[0132] In another embodiment, the determination of disease status in a patient may be used to implement indirect therapy, aimed at augmenting endogenous membrane-tethered forms of TGF-, without directly resupplying membrane-tethered TGF- EV to the patient, for example, gene therapy, oligonucleotides (RNAi, antisense), nano-pharmaceuticals, artificial EV comprising excess membrane-tethered TGF-, or inducers of downstream signals to TGF- (e.g., SMADs, which are transcription factors that transduce extracellular TGF- superfamily ligand signaling from membrane bound TGF- receptors into the nucleus (following phosphorylation at their carboxy termini by the activated receptors where they activate the transcription of TGF- target genes.).
[0133] In another embodiment, the surface membrane-expression of TGF- may be enhanced or augmented in EV (e.g., derived from MSC) by pre-conditioning donor cells or cell lines, in particular, MSC, in culture (e.g. by treatment of the cell lines with hypoxia as described infra, or by exposure to inflammatory mediators such as IFN, TNF, LPS, or IL-17, 5-50 ng/ml); by overexpressing tethered TGF- using direct conjugation of TGF- plus beta-glycan complexes; or by synthesis of artificial EV which are chemically decorated with a high density of tethered TGF-. In a particular embodiment, EV derived from MSC are employed in the biomanufacture of MSC-EV with membrane-tethered TGF-. In other embodiments, cells lines that are immune privileged, e.g., cells obtained from umbilical cord, placenta, fetal tissue, testes, etc., are also useful for production of membrane-tethered TGF- EV. In another embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes on their surfaces.
[0134] In an embodiment, the invention provides extracellular vesicles (EV) which express TGF- or an isoform thereof tethered to the vesicle membrane, in which the TGF- is recombinantly expressed and produced using the techniques of molecular biology as defined and described herein. In an embodiment, the EV comprising recombinant TGF- tethered to the membrane are expressed in immune privileged cells, such as mesenchymal stromal cells or they are expressed in dendritic cells. By way of example, cells employed for EV production (e.g., MSC or immortalized MSC) can be transfected with a lentivirus or adeno-associated virus vector or transduced with a plasmid vector for selection and overexpression of encoded tethered TGF- or a fusion protein (e.g., TGF- fused to an EV membrane protein such as LAMP-2 or CD29). Following stable transduction, biogenesis of EV from transduced cells that overexpress TGF- (or fusion protein) will increase the quantity of TGF- that is decorated (expressed) on the surface of EV generated by those cell lines. Extracellular vesicles (EV) with membrane-tethered TGF- produced and expressed in this way can have increased immunosuppressive effects which are therapeutically advantageous.
[0135] By way of example, a nonlimiting process for the production of extracellular vesicles (EV) comprising recombinant TGF- tethered to the membrane can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses TGF- protein, e.g. TGF-1, TGF-2, TGF-3, TGF-4, or a combination thereof, thereby producing a vector for TGF- expression; b) transferring or delivering the expression vector to a host cell by conventional molecular biology methods to produce a transfected host cell which expresses TGF- in the membrane; and c) culturing the transfected (or transformed) host cell by conventional cell culture techniques so as to produce cells that produce and shed extracellular vesicles (EV) comprising recombinant TGF- tethered to the membrane. The host cell used to express the recombinant TGF- is preferably a eukaryotic cell (e.g., an MSC or another cell type, such as Chinese hamster ovary (CHO) cell). The choice of expression vector is dependent upon the choice of the host cell and may be selected so as to have the desired expression and regulatory characteristics in the selected host cell. In an embodiment, the host cell is a mesenchymal stromal cell (MSC), in particular, immortalized MSC.
[0136] In an embodiment, the amount of TGF- tethered to the membrane of the extracellular vesicles (EV) may be modified, e.g., reduced or increased, by culturing mesenchymal stromal cells (MSC) expressing the EV having membrane tethered TGF- in medium comprising or conditioned with certain additives, such as cytokines, factors, or agents, for example, interferon-gamma (IFN-), tumor necrosis factor (TNF), interleukins, such as IL-17, or lipopolysaccharide (LPS), or by modifying the pH or microenvironment (e.g., employing 3D-cultures , spheroids, or similar structures instead of 2D cultures). In an embodiment, the enhancement of or increase in the amount of extracellular vesicles (EV) having membrane tethered TGF- expressed by mesenchymal stromal cells (MSC) can be achieved by culturing the MSC under hypoxic or oxygen-glycose-deprived conditions. For example, MSC in culture medium are exposed to hypoxic conditions (e.g., 1-5% O.sub.2 for 24 hours), are deprived of oxygen, or are deprived of oxygen and glucose for 1-12 hours at 37 C., 100% humidity, and 5% CO.sub.2 Under such conditions, an increase of membrane-tethered TGF- EV produced by MSC may be from about 1.5-fold to 4-fold relative to MSC not subject to such conditions. In embodiment, an increase of membrane-tethered TGF- EV produced by MSC may be from least about or equal to 1.5-fold to 25-fold, or at least about or equal to 1.5-fold to 15-fold, or at least about or equal to 1.5-fold to 10-fold, or at least about or equal to 1.5-fold to 5-fold, including values therebetween relative to MSC not subject to such conditions.
[0137] In another embodiment, decreased levels of TGF- tethered to the EV membrane can be achieved by altering gene expression (siRNA, miRNA or miRNA mimics, oligonucleotides) in the vesicles. In another embodiment, decreased levels of TGF- tethered to EV can be achieved by disrupting the beta-glycan structure that tethers TGF- to the membrane by exposing the membrane-tethered TGF- EV to heparinases, betaglycanases (pervanadate), or other methods of enzymatic digestion, or by acid treatment. In another embodiment, mesenchymal stromal cells (MSC) that have been immortalized (e.g., using transduction with hTERT or SV40T) can be used to express and produce extracellular vesicles (EV) having increased levels of membrane-tethered TGF- on EV as described supra (e.g., using plasmid transduction of TGF- or TGF-(3-fusion protein).
[0138] In another embodiment, extracellular vesicles (EV) comprising TGF- or an isoform thereof tethered to the membrane surface can be synthetically produced using techniques, e.g., phospholipid chemistry techniques, known in the art. For example, techniques routinely practiced in the art of liposome production may be used. (Alving, C. R., 1991, J. Immunol. Methods., 140:1-13; Wagner, A. et al., 2011, J. Drug Delivery, Vol. 2011, Article ID 591325, 9 pages). As would be appreciated by the skilled practitioner, liposomes are vesicles comprised of concentrically ordered phopsholipid bilayers, which encapsulate an aqueous phase. Liposomes typically comprise various types of lipids, phospholipids, and/or surfactants. The components of liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes (cell or extracellular vesicle (EV) membranes). Methods for preparation of liposomes are known in the art, for example, as provided by Epstein et al, 1985, Proc. Natl. Acad. Set USA, 82:3688; Hwang et al, 1980, Proc. Natl. Acad. Sci. USA, 77:4030-4; and U.S. Pat. Nos. 4,485,045 and 4,544,545. In addition, vesicle forming lipids can be used to formulate liposomes. Such lipids typically comprise with two hydrocarbon chains, such as acyl chains and a polar head group. Examples of vesicle forming lipids include phospholipids, e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin and glycolipids, e.g., cerebrosides, gangliosides. In some embodiments, the liposomes or liposomal compositions further comprise a hydrophilic polymer, e.g., polyethylene glycol and ganglioside GM1, which increases the serum half-life of the liposome.
[0139] Also envisioned by the invention are sterically stabilized liposomes, which comprise membrane-tethered TGF- and can be prepared using common methods known to the skilled practitioner. In general, sterically stabilized liposomes contain lipid components with bulky and highly flexible hydrophilic moieties that reduce the reaction of liposomes with serum proteins, reduce oposonization with serum components and reduce recognition by mononuclear phagocytic cells. Sterically stabilized liposomes can be prepared using polyethylene glycol. Liposomes and sterically stabilized liposomes can be prepared, for example, as reported in Bendas et al., 2001, BioDrugs, 15(4):215-224; Allen et al., 1987, FEBS Lett. 223:42-6; Klibanov et al, 1990, FEBS Lett, 268:235-7; Blum et al, 1990, Biochim. Biophys. Acta., 1029: 1-7; Torchilin et al, 1996, J. Liposome Res. 6:99-116; Litzinger et al, 1994, Biochim. Biophys. Acta, 1190:99-107; Maruyama et al, 1991, Chem. Pharm. Bull, 39:1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062;142-8; Allen et al, 1994, Adv. Drug Deliv. Rev, 13:285-309. Liposomes that are adapted for specific organ targeting (U.S. Pat. No. 4,544,545), or specific cell targeting can also be used. Liposomes can be generated by a reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. In an embodiment, artificial EV with membrane-tethered TGF- can be synthesized as liposomes or exosome-liposome fusions in the size range (e.g., diameter) of EV and composed of phospholipids that closely resemble EV (e.g., ceramide, sphingomyelin). Such synthetic EV are decorated covalently with the TGF--beta-glycan complex (i.e., express a plurality of TGF- proteins covalently attached to beta-glycans on the surface of the vesicles). In an embodiment, the expression of TGF- on the EV membrane can be controlled using conventionally employed conditional expression systems, e.g., inducers or repressors that activate or inhibit, respectively, a response gene (e.g., TetON or TetOFF) that controls the promotor region of a gene transduced into a host cell, such as MSC. Fine tuning can be achieved by exposing the MSC or another cell line producing EV with specific levels of inducers or repressors, by methods practiced in the art, for example, Goverdhana, S. et al., 2005, Mol. Ther., 12(2):189-211.
[0140] In other embodiments, for therapeutic applications, MSC-derived EV with membrane-tethered TGF- can be separated from the total EV population using immune affinity techniques, e.g., affinity chromatography (also called immune affinity capture herein), as described supra. By way of example, affinity chromatography can separate EV having membrane-tethered TGF- based on the specificity of the interaction between TGF- and a cognate molecule with which TGF- has specificity, such as an anti-TGF- antibody or a receptor ligand, e.g., through interactions such as hydrogen bonding, ionic interactions, disulfide bridges, hydrophobic interactions, etc. The high selectivity of affinity chromatography (e.g., immune affinity chromatography, which involves antibody-ligand binding interaction) results from the interaction of a desired molecule, e.g., TGF-, with a specific ligand attached to the stationary phase, matrix, or medium of a chromatography column (e.g., a gel matrix which can be a polysaccharide polymer material typically derived from seaweed, e.g., agarose (a crosslinked, beaded form of agarose, e.g., Sepharose), such that the desired molecules becomes trapped within the column and can then be separated from non-specific or unwanted materials and components which do not interact (bind) to the ligand on the column and elute from the column in the mobile phase. The desired molecule, e.g., TGF-, can be removed from the stationary phase by elution with an appropriate buffer solution, typically by changing the salt concentration, pH, pI, charge or ionic strength, as routinely practiced in the art. In addition to the foregoing, other types of affinity chromatography columns are also envisioned for use to separate and isolate extracellular vesicles (EV) having membrane-tethered TGF-. In an embodiment, the MSC-derived EV with membrane-tethered TGF- for therapeutic use are isolated from cell cultures of MSC or immortalized MSC as described herein.
[0141] In another embodiment, magnetic beads having bound anti-TGF- (and/or antibodies to TGF- isoforms, variants, signaling peptides, non-signaling peptides, or tethering proteins or side chains for example) can be used to separate EV having membrane-tethered TGF- from the total population of EV in a sample. In an embodiment, EV with membrane-tethered TGF- can be precipitated using magnetic columns (e.g. using AUTO-MACS, Miltenyi Biotech, Inc., San Diego, Calif.). The purity of the selected EV having membrane-tethered TGF- can be evaluated using methods described herein (e.g., nanoparticle tracking analysis (NTA), vesiculometry, or interferometry) or other methods that quantify or measure the abundance of EV with membrane-tethered TGF-. Armed with knowledge of the quantity and purity of EV with membrane-tethered TGF-, the skilled practitioner can derive and determine a dosage and formulation of TGF- membrane-tethered EV for therapeutic purposes.
[0142] Results provided infra (see, e.g., Examples 2 and 3, infra) show that MSC-derived EV with membrane-tethered TGF- have immunomodulatory effects on T cells, i.e., suppression of proliferation stimulated by mitogen. The invention provides methods for using MSC-derived EV with membrane tethered TGF-, for example, those whose levels of membrane-tethered TGF- have been manipulated so as to decrease or increase the levels of membrane-tethered TGF-, for affecting changes in disease status or treatment, e.g., decrease immunosuppression of immune cell response or inhibition of cancer cell growth in vitro and in vivo. In an embodiment, EV comprising membrane-tethered TGF- and derived from a given cell source, e.g., stromal cell, cancer-associated cells, mesenchymal stromal cells, can be engineered to contain and specifically deliver therapeutic agents for disease treatment or improved disease treatment, particularly, in vivo. In another embodiment, the EV comprising membrane-tethered TGF-, e.g., MSC-derived, membrane-tethered TGF- EV, can be engineered to express increased or decreased amounts of TGF- in the membrane as described herein. In an embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes on their surfaces.
[0143] Disease treatment and therapy e.g., treatment or therapy for autoimmune disease, inflammatory disease, cardiac disease, cancer, may be provided wherever disease treatment or therapy is performed, including a doctor's office, a clinic, a health or critical care facility, a hospital, a hospital's outpatient department, or at home. Treatment generally begins in a hospital or clinic so that the doctor or medical practitioner can observe the treatment's/therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the kind of disease being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient's body responds to the treatment. The administration of a treatment product or drug may be performed at different intervals (e.g., daily, weekly, or monthly) and may be repeated over time. For example, therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells, exhibit a response and regain its strength.
[0144] Depending on the type of disease and its stage of development, the therapy can be used to reduce, abrogate, abate, diminish, ameliorate, or eliminate the disease or the symptoms or effects of the disease in a patient undergoing treatment. By way of example, the therapy can be used to slow the spreading of a cancer, to slow the cancer's growth, to kill or arrest cancer cells that may have spread to other parts of the body from the original tumor, to relieve symptoms caused by the cancer, or to prevent cancer in the first place. In addition, the therapy can be used to reduce the immunosuppression of immune cells involved in combatting the disease, to reduce inflammation, or to augment an immune response by immune cells. As described above, if desired, treatment with an agent, such as MSC-derived extracellular vesicles (EV) expressing membrane-tethered TGF- as described herein, may be combined with conventional therapies, including therapies for the treatment of proliferative disease (e.g., disease-specific drugs and therapeutic compounds, radiotherapy, surgery, or chemotherapy). For any of the methods of application described above, an MSC-derived EV comprising membrane-tethered TGF- of the invention is desirably administered intravenously or is applied to the site of neoplasia (e.g., by injection). Other modes of administration are also encompassed, including, without limitation, subcutaneous, intraperitoneal, intramuscular, intravaginal, intrathecal, bucal, rectal, intradermal, modes of administration.
[0145] In an embodiment, MSC-derived extracellular vesicles (EV) expressing membrane-tethered TGF- can be administered or used to deliver therapeutic agents in vivo, without adverse reaction and without the development of a cellular inflammatory reaction. In a particular embodiment, MSC-derived EV comprising membrane-tethered TGF- can be used as specific therapeutic agents. By way of example, MSC-derived EV comprising membrane-tethered TGF- can be derived from immune privileged cells, i.e., those that do not elicit an inflammatory immune response (e.g., from immune privileged sites such as the umbilical cord, placenta, fetus, testes, articular cartilage), and can be administered to, or transplanted into, a subject having a disease and who is in need. MSC-derived EV comprising membrane-tethered TGF- can contribute to modulation of cellular, e.g., immune cell, responses during disease. In addition, MSC-derived EV comprising membrane-tethered TGF- can be used to exert an immunosuppressive effect in chronic inflammatory or auto-immune disease, or conditions leading to fibrosis, or those diseases and conditions that are preceded by inflammation (e.g., wound healing, arthritis, inflammatory bowel disease). In an embodiment, such MSC-derived EV comprising membrane-tethered TGF- provide a therapeutic which has anti-immunosuppressive and anti-proliferative properties, as well as disease mitigating and survival-extending properties in vivo. In aspects of each of the above embodiments, the MSC-derived EV comprising membrane-tethered TGF- can be loaded with one or more bioactive agents, e.g., a polypeptide, polynucleotide, or small molecule, as described herein below for use in disease treatment or therapy. In an embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes.
[0146] In other aspects, the invention provides extracellular vesicles (EV) comprising membrane-tethered TGF-, in particular, EV comprising membrane-tethered TGF- isolated from mesenchymal stromal cells (MSC), for treating a disease, condition, pathology, or for diagnosing a disease, condition, or pathology (e.g., assessing or evaluating the status or progression of a disease, condition, or pathology, such as post-treatment or therapy monitoring), in which the disease is an autoimmune disease, transplant rejection, or other inflammatory disease, condition, or pathology. In accordance with aspects of the invention, MSC-derived extracellular vesicles (EV) expressing membrane-tethered TGF-, or MSC-derived extracellular vesicles (EV) expressing modified (e.g., increased or reduced) amounts of membrane-tethered TGF-, or MSC-derived extracellular vesicles (EV) expressing membrane-tethered TGF- loaded with a bioactive agent, have utility in the treatment of inflammatory disease, autoimmune disease, or transplant rejection. In particular, such EV are capable of down-modulating the immune system of a subject, e.g., impairing the function or suppressing the proliferation and/or activity of immune cells such as CD4+ or CD8+ T cells, or other immune cells, e.g., natural killer (NK) cells, in vivo or in vitro. In another embodiment, the above-described MSC-derived membrane-tethered TGF- EV have utility as a therapeutic in the treatment of various types of cancer.
[0147] Such down-modulation of the immune system and suppression of immune cell activity is typically desirable in the treatment and therapy of inflammatory and autoimmune diseases and in transplant rejection. Nonlimiting examples of autoimmune disorders that may be treated or managed by administering the extracellular vesicles (EV) comprising membrane-tethered TGF-, particularly MSC-derived EV having membrane tethered TGF-, of the present invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Bechet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, neuromyelitis optica (NMO), type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogrens' syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, e.g., dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
[0148] Nonlimiting examples of inflammatory diseases that can be treated or managed with the extracellular vesicles (EV) comprising membrane-tethered TGF-, particularly MSC-derived EV having membrane tethered TGF-, of the present invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral infection or bacterial infection.
Companion Diagnostics
[0149] In an aspect of the invention, TGF- tethered to EV derived from a given cell type, e.g., a mesenchymal stromal cell, can be used as a biomarker of disease in a companion diagnostic method. As appreciated by the skilled practitioner, companion diagnostics are bioanalytical methods (diagnostic tests) designed to assess whether a patient having a disease will respond or has responded favorably to a specific medical treatment or therapy for the disease. A biomarker (e.g., levels or characteristics of a biomarker relative to those of a control) in a patient's biological sample undergoing testing is typically assessed in the companion diagnostic method. The linkage between the therapeutic treatment and biomarker levels could be important in the therapeutic application and clinical outcome of the use of a drug or therapeutic regimen in the patient (personalized medicine), or an important component of the drug development process. In addition, biomarker(s) used in the specific context of disease being treated provide(s) biological and/or clinical information that enables better decision making by the medical and clinical practitioner (and sometimes by the patient) about the course of present and future treatment of the patient's disease, as well as the development and use of other treatments or other potential drug therapy. The practice of a companion diagnostic method can be applied anywhere along the preclinical, clinical and post-product launch of a drug or therapy for a disease.
[0150] Accordingly, extracellular vesicles (EV) comprising membrane-tethered TGF- can be used for diagnostic purposes, such as to detect, diagnose, or monitor diseases, disorders or infections. By way of example, the detection or diagnosis of a disease, disorder or infection, particularly an autoimmune disease comprises: (a) assaying the level of extracellular vesicles (EV) comprising membrane-tethered TGF- in a biological sample obtained from a subject having a disease, disorder, or infection using one or more antibodies (or fragments thereof) that immunospecifically bind to the tethered TGF-; and (b) comparing the level of the tethered TGF- with a control level, e.g., levels in normal (non-diseased or healthy) subjects' samples, e.g., those who do not have, or who do not have detectable amounts of, membrane tethered-TGF- EV, wherein an increase or decrease in the assayed level of tethered TGF- compared to the control level of tethered TGF- is indicative of the disease, disorder or infection. Such assays may include, without limitation, immunoassays, such as the enzyme linked immunosorbent assay (ELISA), radioimmunoassay (MA), fluorescence-activated cell sorting (FACS), and flow cytometry assays. In an embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes. In another embodiment, the TGF- tethered to the EV is in a latent form.
[0151] Patient status in response to therapy comprising MSC-derived membrane-tethered TGF- EV (or other therapies) can be determined and monitored in a biofluid sample obtained from a subject using EV with tethered TGF- (including TGF- isoforms, mutant or variant forms of TGF-, latency versus active forms of TGF-, or quantified in relation to more traditional markers), thus constituting a companion diagnostic. Thus, the level or amount of endogenous EV having membrane tethered TGF- as detected by the methods described herein can guide the use of MSC-derived EV with membrane-tethered TGF- as a therapeutic. For patient screening or monitoring, EV expressing tethered TGF- in biological samples can be quantified, and/or these EV can be analyzed further for the presence of particular isoforms or variant forms of TGF- (or secondary molecules as markers or molecular signatures) in a test population, and can be compared, for example, to a control which is a sample having tethered TGF- negative (or low expressing) EV, which can be used to refine the diagnostic accuracy of the findings. Also, the observed differences in types or forms of the tethered TGF- in patients identified as having high levels of TGF- tethered to EV versus those in patients identified as having low/negative levels of TGF- tethered to EV may be exploited as a biomarker or signature of patient status leading to decisions on disease treatment and management in patients. Examples of the abovementioned secondary molecular signatures include RNA species, DNA, bioactive lipids, proteins, and metabolites such as adenosine. These biomarkers can also be assessed in patient populations to evaluate safety, activity, efficacy, or clinical effectiveness of an intervention in a clinical setting.
[0152] In some instances, MSC-derived EV with membrane-tethered TGF- may be employed as a treatment agent or therapeutic without guidance from patient status (endogenous EV with membrane-tethered TGF-), for example, to exert an immunosuppressive effect in chronic inflammatory or autoimmune disease, or in conditions leading to fibrosis, or those that are preceded by inflammation (e.g. wound healing). In other instances, patient status can be employed to understand the immunologic status of the patient without the use of specific therapy, such as therapy involving MSC derived EV with membrane-tethered TGF-.
TGF- Tethered EV Containing Proteins, Polypeptides, or Peptides
[0153] The EV comprising membrane-tethered TGF-, in particular, MSC-derived EV expressing membrane-tethered TGF- as described herein, may contain proteins, polypeptides, or peptides, particularly for therapeutic or treatment purposes as described supra. In an embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes. In a specific embodiment, the EV expressing membrane-tethered TGF- can contain (and deliver to a cell or tissue, or to a cell or tissue in a subject) an agent, e.g., a protein, that increases or decreases an immune response by (an) immune cell(s), corrects a deficiency of the cell or subject, or induces the death of infected or deficient cells. Recombinant polypeptides are produced using virtually any method known to the skilled practitioner. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.
[0154] Those skilled in the field of molecular biology will appreciate that any of a wide variety of expression systems may be used to provide a recombinant protein. The precise host cell used is not critical to the invention. A polypeptide for use may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells or CHO cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Manassas, Md.; also, see, e.g., Ausubel et al., Current Protocols in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., supra; expression vehicles can be from among those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).
[0155] A variety of expression systems exist for the production of the polypeptides that can be used in conjunction with the membrane-tethered TGF- EV described herein. Membrane-tethered TGF- EV derived from a given cell type, e.g., MSC or fibroblast-like cells, can be loaded with one or more expression vectors or with the polypeptides generated using such vectors. Nonlimiting examples of expression vectors useful for producing polypeptides include chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
[0156] A particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis.). In this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically accomplished using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
[0157] Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids can be cleaved with thrombin; those expressed in pGEX-3X plasmids can be cleaved with Factor Xa.
[0158] Alternatively, recombinant polypeptides may be expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is encoded by the AOX1 gene is induced by methanol. The AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.
[0159] Once a given recombinant polypeptide is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody raised against the polypeptide may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column.
[0160] Once isolated, the recombinant protein can, if desired, be further purified by methods known and practiced in the art, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs.
[0161] The isolated polypeptides or fragments can be loaded into TGF- tethered EV using methods practiced in the art.
TGF- Tethered EV Containing Polynucleotides
[0162] The EV comprising membrane-tethered TGF-, in particular, MSC-derived EV expressing membrane-tethered TGF- as described herein may contain one or more polynucleotides, particularly for therapeutic or treatment purposes as described supra. In a specific embodiment, the EV expressing membrane-tethered TGF- can contain (and deliver in a subject) a polynucleotide, that corrects a deficiency of the cell or subject, or induces the death of infected or deficient cells. In an embodiment, the polynucleotide encodes a protein product that corrects a deficiency of the cell or subject, or induces the death of infected or deficient cells. Nonlimiting examples of polynucleotides include RNA, DNA, an antisense oligonucleotide, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), plasmid DNA polynucleotides and modified oligonucleotides.
[0163] Membrane-tethered TGF- EV may also be molecularly engineered to contain expression vectors harboring a polynucleotide with therapeutic function. In an embodiment, MSC-derived membrane-tethered TGF- EV may be administered to a subject having a disease, e.g., inflammation or autoimmune disease or cancer, for delivery to the subject's cells. In an embodiment, the DNA encodes a protein with a specific diagnostic or therapeutic function. Membrane-tethered TGF- EV, particularly, MSC-derived membrane-tethered TGF- EV, comprising nucleic acid molecules are selectively delivered to target cells of a subject (e.g., cancer cells) in a form in which they are taken up and are advantageously expressed so that therapeutically effective levels can be achieved.
[0164] An isolated nucleic acid molecule can be manipulated using recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5 and 3 restriction sites are known, or for which polymerase chain reaction (PCR) primer sequences have been disclosed, is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid molecule that is isolated within a cloning or expression vector may comprise only a small percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein, because it can be manipulated using standard techniques known to those of ordinary skill in the art.
[0165] Transducing viral (e.g., retroviral, adenoviral, lentiviral and adeno-associated viral) vectors can be used to deliver polynucleotides to/into cells (as well as TGF- tethered EV) and for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy, 8:423-430, 1997; Kido et al., Current Eye Research, 15:833-844, 1996; Bloomer et al., J. Virology, 71:6641-6649, 1997; Naldini et al., Science, 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A., 94:10319, 1997). By way of nonlimiting example, a polynucleotide can be cloned into a retroviral or other vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other useful viral vectors include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors reported by Miller, Human Gene Therapy, 15-14, 1990; Friedman, Science, 244:1275-1281, 1989; Eglitis et al., BioTechniques, 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology, 1:55-61, 1990; Sharp, The Lancet, 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology, 36:311-322, 1987; Anderson, Science, 226:401-409, 1984; Moen, Blood Cells, 17:407-416, 1991; Miller et al., Biotechnology, 7:980-990, 1989; Le Gal La Salle et al., Science, 259:988-990, 1993; and Johnson, Chest, 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med., 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346).
[0166] Polynucleotide expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. Such enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.
[0167] MSC-derived membrane-tethered TGF- EV can contain and deliver nucleic acid molecules comprising a modified nucleic acid. Nucleic acid molecules include nucleobase oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers. Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.
[0168] Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or by one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
[0169] Nucleobase oligomers may also contain one or more substituted sugar moieties. Such modifications include 2-O-methyl and 2-methoxyethoxy modifications. Another desirable modification is 2-dimethylaminooxyethoxy, 2-aminopropoxy and 2-fluoro. Similar modifications may also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3 position of the sugar on the 3 terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.
[0170] In other nucleobase oligomers, both the sugar and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. Methods for making and using these nucleobase oligomers are described, for example, in Peptide Nucleic Acids (PNA): Protocols and Applications, Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. PNA compounds are also reported by Nielsen et al., Science, 1991, 254, 1497-1500.
TGF- Tethered EV and Imaging Agents
[0171] The EV comprising membrane-tethered TGF- as described herein may contain a detectable agent useful for imaging studies. The invention provides TGF- tethered EV comprising any one of the following exemplary small molecules useful in imaging: carbocyanine, indocarbocyanine, oxacarbocyanine, thilicarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, borondipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800R5, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
[0172] In other embodiments, the TGF- tethered EV comprise a nanoparticle useful in imaging studies. In one embodiment, nanoparticles are synthesized using a biodegradable shell known in the art. In one embodiment, a polymer, such as poly (lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used. Such polymers are biocompatible and biodegradable, and are subject to modifications that desirably increase the circulation lifetime of the nanoparticle. In one embodiment, nanoparticles are modified with polyethylene glycol (PEG), which increases the half-life and stability of the particles in circulation (Gref et al., Science, 263(5153): 1600-1603, 1994). In an embodiment, the EV are treated with hyaluronidase to remove hyaluronic acid/proteoglycan complexes.
[0173] Biocompatible polymers useful in the compositions and methods described herein include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetage phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methylmethacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutylacrylate), poly(octadecylacrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene, polyvinylpryrrolidone, polyhyaluronic acids, casein, gelatin, gluten, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecl acrylate) and combinations of any of these. In one embodiment, the nanoparticles of the invention include PEG-PLGA polymers.
[0174] In response to the growing need for encapsulation materials, several different approaches to producing hollow polymeric capsules are available. In one example, the shell is composed of dendrimers (Zhao, M., et al., 1998, J Am. Chem. Soc., 120:4877). A dendrimer is an artificially manufactured or synthesized large molecule comprised of many smaller ones linked togetherbuilt up from branched units called monomers. Technically, dendrimers are a unique class of a polymer, about the size of an average protein, with a compact, tree-like molecular structure, which provides a high degree of surface functionality and versatility. Their shape gives them vast amounts of surface area, making them useful building blocks and carrier molecules at the nanoscale level; they are available in a variety of forms, with different physical (including optical, electrical and chemical) properties. In other embodiments, the shell comprises block copolymers (Thurmond, K. B., II, et al., 1997, J. Am. Chem. Soc., 119:6656; MacKnight, W. J., et al., 1998, Acc. Chem. Res., 31:781; Harada, A. and Kataoka, K., 1999, Science, 283:65), vesicles (Discher, B. M., et al., 1999, Science, 284:1143), hydrogels (Kataoka, K. et al., 1998, J. Am. Chem. Soc., 120:12694) and template-synthesized microtubules (Martin, C. R. and Parthasarathy, R. V., 1995, Adv. Mater., 7:487) that are capable of encapsulating a photosensitizer.
[0175] In another embodiment, a TGF- tethered EV of the invention comprises an isotopic label for positron or scintillation or SPECT imaging. In another embodiment, a TGF- tethered EV of the invention comprises a magnetic nanoparticle that has a high magnetic moment to enhance the selectivity of the nanoparticle for detection. In another embodiment, a magnetic nanoparticle includes a magnetic core and a biocompatible outer shell, in which the outer shell both protects the core from oxidation and enhances magnetic properties of the nanoparticle. The enhanced magnetic properties can include increased magnetization and reduced coercivity of the magnetic core, allowing for highly sensitive detection as well as diminished non-specific aggregation of nanoparticles. By forming biocompatible nanoparticles having enhanced magnetic properties, detection of specific target proteins and cells is provided. In one embodiment, a nanoparticle core is formed from ferromagnetic materials that are crystalline, poly-crystalline, or amorphous in structure. For example, the nanoparticle core can include materials such as, but not limited to, Fe, Co, Ni, FeOFe.sub.2O.sub.3, Ni O Fe.sub.2 O.sub.3, CUOFe.sub.2 O.sub.3, MgOFe.sub.2 O.sub.3, MnBi, MnSb, MnOFe.sub.20.sub.3, Y3Fe.sub.5 O i.sub.2, Cr O.sub.2, MnAs, SmCo, FePt, or combinations thereof.
[0176] In another embodiment, the outer shell of the magnetic nanoparticle partially or entirely surrounds the nanoparticle core. In some implementations, the shell is formed from a superparamagnetic material that is crystalline, poly-crystalline, or amorphous in structure. In some cases, the material used to form the shell is biocompatible, i.e., the shell material elicits little or no adverse biological/immune response in a given organism and/or is nontoxic to cells and organs. Exemplary materials that can be used for the shell include, but are not limited to, metal oxides, e.g., ferrite (Fe.sub.3C4), FeO, Fe203, CoFe.sub.204, MnFe.sub.204, NiFe.sub.204, ZnMnFe.sub.204, or combinations thereof.
[0177] Methods of making and delivering nanoparticles are known in the art and described, for example, in the following US Patent Publications: 20150258222, 20140303022, 20130309170, and 20130195767.
TGF- Tethered Extracellular Vesicle (EV) Isolation, Loading, and Targeting
[0178] TGF- tethered EV as described herein are generated as described herein below. In general, the EV expressing membrane tethered TGF- are released by cells (e.g., stromal cells, stromal-stem cells, mesenchymal stromal cells, cancer-associated fibroblasts, fibroblast-like cells) into the extracellular environment. TGF- tethered EV can be isolated from a variety of biological fluids (biofluids), including, but not limited to, blood, plasma, serum, saliva, sputum, urine, stool, semen, cerebrospinal fluid, prostate fluid, lymphatic drainage, bile fluid, and pancreatic secretions. The TGF- tethered EV can be separated or isolated using routine methods known in the art. In an embodiment, TGF- tethered EV are isolated from the supernatants of cultured cells using differential ultracentrifugation. In another embodiment, TGF- tethered EV are separated from nonmembranous particles, using their relatively low buoyant density (Raposo, G. et al., 1996, JEM, 183(3):1161; Raposo, G. et al., 2013, J. Cell Biol., 200(4):373-383); Escola, J. M. et al., 1998, J. Biol. Chem., 273(32):20121-7; van Niel, G. et al., 2003, Gut, 52(12):1690-7); Wubbolts, R. et al., 2003, J. Biol. Chem., 278:10963-10972). Kits for such isolation are commercially available, for example, from Qiagen, InVitrogen and SBI.
[0179] Methods for loading EV, in particular, TGF- tethered EV, with an agent of interest, such as a bioactive agent: polypeptide or polynucleotide (cargo), are known in the art and include lipofection, electroporation, calcium chloride precipitation, as well as any standard transfection method.
[0180] In one embodiment, the TGF- tethered EV comprising a polynucleotide or polypeptide, or small molecule of interest are obtained by over-expressing the polynucleotide or polypeptide or loading the cells with the small molecule in culture and subsequently isolating indirectly modified TGF- tethered EV from the cultured cells. In another embodiment, TGF- tethered EV comprising a polynucleotide or polypeptide or small molecule of interest are generated by loading previously purified TGF- tethered EV with the molecule(s) of interest into/onto the TGF- tethered EV by electroporation (polynucleotide or polypeptide), covalent or non-covalent coupling to the EV surface (polynucleotide or polypeptide or small molecule) or simple co-incubation (polynucleotide or polypeptide or small molecule).
Pharmaceutical Compositions
[0181] Provided in another aspect are MSC-derived membrane-tethered TGF- EV for as a therapeutic and MSC-derived membrane-tethered TGF- EV for delivering an agent (e.g., a bioactive agent such as a polynucleotide, polypeptide, or small molecule) for the treatment of disease. In an embodiment, the present invention provides a pharmaceutical composition comprising MSC-derived membrane-tethered TGF- EV as a therapeutic. In another embodiment, a pharmaceutical composition comprising MSC-derived membrane-tethered TGF- EV for delivery of an agent (e.g., polynucleotide, polypeptide, or small molecule) is provided. In embodiments, the TGF- tethered EV is derived from a stromal cell, a stromal stem cell, a mesenchymal stromal cell (MSC), a cancer-associated fibroblast, or a fibroblast-like cell. In a particular embodiment, the EV expressing membrane tethered TGF- is derived from MSC. The MSC-derived membrane-tethered TGF- EV of the invention may be administered as part of a pharmaceutical composition. In general, the MSC-derived membrane-tethered TGF- EV are provided in a physiologically balanced saline solution. The solution comprising the MSC-derived membrane-tethered TGF- EV may be stored at room temperature for up to about 24 hours, for longer than twenty four hours; such solutions can also be stored at about 4 C. for days, weeks, or months. MSC-derived membrane-tethered TGF- EV may be frozen for long term storage, e.g., for up to 10 years. The compositions should be sterile and contain a therapeutically effective amount of the MSC-derived membrane-tethered TGF- EV in a unit of weight or volume suitable for administration to a subject.
[0182] MSC-derived membrane-tethered TGF- EV of the invention may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease (e.g., cardiac disease, cancer). Administration may begin before the patient is symptomatic.
[0183] Any appropriate route of administration may be employed. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, i.e., any mode that produces effective levels of the TGF- tethered EV (as active) without causing clinically unacceptable, adverse effects. By way of nonlimiting example, modes and routes of administration may include parenteral, bucal, intravenous, intra-arterial, subcutaneous, intratumoral, intramuscular, intracranial, intracerebroventricular, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intratracheal, aerosol, topical, transdermal, intravaginal, rectal (suppository), oral administration, or within/on implants, e.g., fibers such as collagen, osmotic pumps, or tissue or synthetic grafts comprising appropriately transformed cells, etc. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
[0184] Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000, and updates thereof. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the TGF- tethered EV include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
[0185] The formulations can be administered to human patients in therapeutically effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. The preferred dosage of a TGF- tethered EV of the invention is likely to depend on such variables as the type and extent of the disease or disorder, the overall health status and condition of the particular patient, the formulation of the excipients, and its route of administration.
[0186] With respect to a subject having a neoplastic disease or disorder, an effective amount is sufficient to stabilize, slow, or reduce the proliferation of the neoplasm. Generally, doses of TGF- tethered EV or compositions thereof of the present invention would be from about 0.01 mg/kg per day to about 1000 mg/kg per day. It is expected that doses ranging from about 50 to about 2000 mg/kg will be suitable. Lower doses will result from certain forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of TGF- tethered EV.
Kits
[0187] Kits provided by the invention include MSC-derived membrane-tethered TGF- EV or a composition thereof; or MSC-derived membrane-tethered TGF- EV containing an agent formulated for delivery to a cell in vitro or in vivo, or a composition thereof. In an embodiment, a kit contains MSC-derived EV that have been modified to contain a reduced level of TGF- tethered to the membrane or MSC-derived EV that have been modified so as to remove TGF- tethered to the membrane as described supra. Optionally, the kit includes directions for administering or delivering the MSC-derived membrane-tethered TGF- EV (or modified EV) to a subject. In other embodiments, the kit comprises a sterile container which contains the MSC-derived membrane-tethered TGF- EV or composition thereof; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the MSC-derived membrane-tethered TGF- EV or a composition thereof. The instructions will generally include information about the use of the MSC-derived membrane-tethered TGF- EV. In other embodiments, the instructions include at least one of the following: description of the MSC-derived membrane-tethered TGF- EV; methods for using the enclosed materials for the treatment of a disease; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
[0188] It is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and are not intended to limit the remainder of the disclosure in any way.
EXAMPLES
[0189] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and not to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
Example 1
Isolation of Immunomodulatory Extracellular Vesicles (EV) with Tethered TGF-
[0190] In accordance with the present invention, the measurement of TGF- tethered to the membrane of extracellular vesicles (EV) derived from a variety of cell, tissue, or organ types can be used for the assessment of the immune status of a subject, e.g., human and veterinary subjects, in need. The accurate quantification of TGF- (or other immunomodulatory proteins) tethered to EV in subjects' biofluids provides an improved index of disease activity, aggressiveness, prognosis, and/or response to therapy, as well as other aspects related to the natural history of a subject's disease (e.g., status at a given time, progression, remission, regression, refraction, and the like) as described hereinabove.
[0191] Biofluid samples, such as blood, urine, cerebrospinal fluid, or saliva, obtained from patients, or cell culture supernatants, were cleared of cells, platelets, apoptotic bodies, cell debris, protein aggregates, and other particulates that were not extracellular vesicles (EV). This was achieved by differential centrifugation (1300g for 10 minutes to remove cells and platelets; 2000g for 10 minutes to remove apoptotic bodies; and 10,000g for 30 minutes to remove microvesicles), or by sequential filtration after clarification of cells and apoptotic bodies using a 200 nm filter. For cell culture supernatants, initial concentration of EV was carried out by filtration to remove cells and cell debris (200 nm pore size), followed by tangential flow filtration (50,000-300,000 kDa molecular weight cutoff).
[0192] Extracellular vesicles (EV) were isolated from the clarified sample (e.g. plasma, serum, cell culture supernatant) by either affinity column, tangential flow filtration (e.g., >50 kDa molecular weight cut off filter), precipitation (e.g., using PEG, ExoQuik), differential ultracentrifugation (e.g., 100,000g for 70 minutes using a 70Ti rotor to sediment EV), density gradient centrifugation, or size exclusion chromatography (e.g., 30-45 nm pore size). Other conventionally used methods for isolation and concentration of EV can also be used. Alternatively EV were isolated from clarified samples using a non-affinity (i.e., negatively charged like EV which have negative zeta potential) spin column that retained EV and could be washed to remove non-EV constituents without the loss of EV in the column.
[0193] The fraction of EV expressing TGF- (including isoforms TGF-1, TGF-2, TGF-3, and/or TGF-4) on the surface, i.e. tethered to the EV membrane via beta glycan, (also referred to as TGF-R3), was measured using a number of quantitative methods. Such methods include single vesicle nanoparticle tracking analysis by immunolabeling (e.g., fluorescent labeling) of TGF- by QDOT(ThermoFisher Scientific, Waltham, Mass.) conjugated antibody (anti-TGF- antibody) or by indirect labeling (e.g. biotin-conjugated antibody, streptavidin-QDOT) (Thane, K. E. et al., 2017, J. Extracell. Vesicles, in review); vesiculometry employing fluorescence detection of immunolabeled EV (Enjeti, A. K., et al., 2016, Thromb. Res., V. 145:18-23) or EV absorbed to beads; or interferometry (Daaboul, G. G., et al., 2016, Sci. Rep., Vol. 6:37246), as described hereinabove. As will be appreciated by the skilled practitioner, QDOT nanocrystal labelled-antibody conjugates provide both single and multicolor, multiplexed fluorescence detection using an excitation source, such as a 405 nm violet laser, particularly for low abundance molecules (antigens) with minimal photobleaching.
[0194] The above methods (and other suitable methods) assess or benchmark the quantity, phenotype and size distribution of EV with membrane-tethered TGF- as biomarker. Data are quantified relative to total EV, total protein, or total EV-expressed proteins (e.g., CD9, CD63, CD81, TSG101, flotillin, synectin, LAMP-2, Alix), nucleic acids, lipids, or other constituents that represent the total EV population in a sample. The biomarker data were used to stratify patient status by stage, aggressiveness, prognosis, resistance to therapy, or any aspect of disease status. Stratification may involve (1) measuring membrane-tethered TGF- on EV obtained from patients, (2) monitoring the levels (high and low) of TGF- tethered to EV obtained from patients at different time points; or (3) treating patients presenting with high versus low TGF- tethered to EV with different therapies, treatment regimens, monitoring schedules, drugs, adjuvant treatments, etc., for example.
Example 2
TGF- Tethered to Exosome Extracellular Vesicles from Mesenchymal Stem-Stromal Cells (MSC) Suppress T-Helper Cell Division
[0195] Mesenchymal stem-stromal cells (MSC) suppress activation and proliferation of CD4+ T cells, and soluble transforming growth factor beta (), (TGF-) plays an important role in that mechanism. Immune suppression by membrane bound TGF- is recognized in a dendritic cell and cancer associated fibroblast extracellular vesicles (EV), but this mechanism has not been documented for MSC-EV. It was hypothesized that EV membrane bound TGF- (i.e., membrane-tethered TGF-) is central to the immunomodulatory mechanism of MSC.
[0196] Serum-free culture medium from canine Wharton's Jelly mesenchymal stem cells (WJ-MSC: CD44.sup.+, CD90.sup.+, CD34.sup., CD45.sup., MHCII.sup., n=6 cell lines) was collected after 48 hours, and extracellular vesicles (WJ-EV) were isolated by differential centrifugation. WJ-EV output was assessed using single vesicle nanoparticle tracking analysis (NTA). CFSE-stained peripheral blood mononuclear cells (PBMC) were collected from healthy dogs (n=8), exposed to Concanavalin A mitogen (ConA; 5 g/ml) and co-incubated with WJ-MSC (1:10) across transwell membrane (0.4 m pore size) or with WJ-MSC EV (1:10.sup.4)10 M SB431542 (TGFR1 inhibitor) or TGF- neutralizing (e.g., inhibiting) antibody (Ab) for 72 hours. Analysis of CFSE fluorescence using FlowJo (v7.6.5) yielded % CD4.sup.+ cells that had undergone division (T-cell proliferation).
[0197] An average of 832% of the particle count from WJ-MSC conditioned medium were in the exosome size range (30-200 nm) based on NTA. The % CD4.sup.+ division in response to ConA alone (6017%) was significantly higher than that observed in ConA+WJ-MSC (2511%, P<0.01), ConA+WJ-EV (2313%, P<0.01), or soluble TGF-1 alone (2110%, P<0.01). The addition of the TGFR1 inhibitor SB431542 to ConA+WJ-EV increased CD4.sup.30 division to 5217% (P<0.01 vs ConA+WJ-EV). The addition of TGF- Ab to ConA+WJ-EV at 0.1, 1, or 10 g/ml resulted in CD4.sup.+ division of 5814%, 6016% and 5810% (P<0.01 vs ConA+WJ-EV), respectively. (See, e.g.,
[0198] The experimental data in this Example demonstrate that mitogen-induced T-cell proliferation, which is markedly suppressed by WJ-EV, is mediated in part by membrane-bound TGF-. The suppression of T cell proliferation by EV isolated from MSC (WJ-EV) is antagonized by a TGFR1 inhibitor or TGF- neutralizing antibody. It is possible to measure TGF- tethered to EV alone, or in relation to other cytokines, e.g., IL-6, as a biomarker of various diseases and conditions.
Example 3
Canine Wharton's Jelly Mesenchymal Stem Cells (WJ-MSC) Regulate T Helper Cell Suppression Using Extracellular Vesicle Associated Transforming Growth Factor Beta (TGF-) and Adenosine
[0199] Wharton's Jelly has emerged as a source of mesenchymal stem cells (MSC) in regenerative medicine. Wharton's Jelly MSC (WJ-MSC) are readily isolated from multiple regions of the umbilical cord, yielding greater numbers of MSC per gram of tissue than fat or bone marrow, for extended periods after discard and from cords harvested at multiple stages in gestation. WJ-MSC derived from extra-embryonic fetal tissue exhibit youthful properties, such as Oct4 and Nanog expression, over several passages. This is in contrast to bone marrow MSC which demonstrate substantial donor age effects that reduce colony formation, cell expansion, and differentiation potential.
[0200] The immunomodulatory capacity of WJ-MSC has served as a rationale for the development of WJ-MSC for cell therapy (M. Rizk et al., 2017, Biol. Blood Marrow Transplant., 23(10):1607-1613). Some studies have reported that WJ-MSC exhibit comparable or superior immunomodulatory potential to that of adipose tissue derived MSC (AT-MSC) and bone marrow MSC (BM-MSC). WJ-MSC have also been reported to be less immunogenic than MSC from other sources (R. N. Barcia et al., 2017, Cytotherapy, 19(3):360-370).
[0201] A wide range of immunologic process are mitigated by WJ-MSC, including, but not limited to, suppression of T cell proliferation, promotion of a T regulatory cell phenotype, and polarization of macrophages toward an anti-inflammatory M2 phenotype in vitro. In one report, WJ-MSC failed to mitigate NK or B cell activation (Ribeiro, A. et al., 2013, Stem Cell Res Ther, 4(5):125), suggesting that monocytes and T cells are major targets. Immune modulation has been observed with or without contact between MSC and immune effector cells. In studies, contact between WJ-MSC and lymphocytes has shown either increased or decreased biological activity. Hence, MSC-immune effector cell interactions are complex and include reciprocal processes that may impact either cell type positively or negatively. The adverse effects of immune effector cells on MSC has ignited interest in their secretome, and a search for acellular MSC based products that may be more stable in the host microenvironment.
[0202] Within the MSC secretome are extracellular vesicles (EV), nanoscale cellular products that contain RNA, protein, and lipids that recapitulate many biological properties previously attributed to parent cells or their soluble secretions. MSC EV may have potential for use as therapeutic agents or vectors, including a clinical application of MSC EV for treatment of severe graft-versus-host disease (Kordelas, L. et al., 2014, Leukemia, 28(4):970-973). However, there is insufficient knowledge about the molecular, as well as biochemical and genomic, mechanisms by which MSC EV exert putative immune modulation, in contrast to tumor or tumor stroma derived EV, which have been reported to possess a number of immunosuppressive protein ligands (e.g., PD-L1, PD-L2, PD-1, FasL, TGF-1, CD39, CD73, Galectin-1, CTL4). Certain ligands (PD-L1, TGF-1, and Galectin) can be transferred by murine MSC derived EV to lymphocytes, inducing their autocrine production of IL-10 and TGF-1. Similarly, immune modulatory proteins typically associated with paracrine signaling in MSC are also carried by MSC EV (e.g., DO, NO, PGE2, TGF-1, adenosine, IL-10), although their functional relevance in association with EV is unclear.
[0203] In experiments similar to those described in Example 2, this Example describes experiments in which extracellular vesicles (EV) isolated from canine Wharton's Jelly derived MSC (WJ-MSC) were assayed for their ability to suppress peripheral blood CD4+ T lymphocyte proliferation through a transforming growth factor- (TGF-) signaling mechanism. The experiments were further designed to assess whether WJ-MSC EV can suppress CD4.sup.+ T helper cells within PBMC in a manner that is consistent with the effects of the parent WJ-MSC.
[0204] Materials and Methods
[0205] Wharton's Jelly MSC
[0206] Animals, tissue collections, and WJ-MSC isolation: The study described in this Example received prior approval by the Institutional Animal and Care Usage Committee of Tufts University. Privately owned healthy donor dams from various breeds, e.g., Corgi, American Staffordshire Terrier, Labrador Retriever, Golden Retriever, Rottweiler and German Shepherd, between 1-10 years old participated under owner consent at the time of elective Cesarean section. Donors (adult females) were tested negative for Brucella canis, Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum antigens prior to breeding. All puppies removed by Cesarean section received standard of care and were returned to their owners. Fresh placental tissue was collected under aseptic conditions and were processed within 24 hours. Wharton's Jelly (WJ) tissue was dissected away from the umbilical artery and vein, placed in cold phosphate buffered saline (PBS), and minced with a scalpel. Explanted tissue fragments were washed three times with PBS through a 100 m filter, and incubated in 3 mg/ml collagenase/dispase (Sigma-Aldrich, St Louis Mo.) at 37 C. for one hour using a procedure modified from Lee, K. S et al., 2013, Res Vet Sci, 94(1):144-151). The tissue digest was filtered through a 100 m filter, and cells were plated at low density (passage 0, approximately 210.sup.3 cells/cm.sup.3) in Alpha-MEM (Sigma-Aldrich, St Louis, Mo.), supplemented with 15% fetal bovine serum (Hyclone, GE Life Sciences, Little Chalfont, UK), 10,000 U/ml penicillin-streptomycin, and 2 mM L-glutamine (Life Technologies, Carlsbad, Calif.), called cAlpha-MEM. Cells adhered to culture plates for 48 hours prior to changing the medium every 48-72 hours thereafter. Cells were routinely passaged using 0.25% trypsin with EDTA (HyClone), washed, and cryopreserved (160 C.) at passage 1 in 60% FBS, 30% cAlpha-MEM, and 10% DMSO (10%) until further use.
[0207] Flow cytometry: Cells were incubated with primary antibodies in 5% FBS for 30 minutes on ice, including anti-CD34-PE (AbD Serotec, mouse anti-dog clone 1H6), CD44-APC (AbD Serotec, rat anti-dog clone YKIX337.8.7), CD45-APC (AbD Serotec, rat anti-dog clone YKIX716.13), MHCII-FITC (AbD Serotec, rat anti-dog clone YKIX334.2), CD90-APC (eBioscience, rat anti-dog clone YKIX337.217). A viability marker (7AAD) was applied to all samples for gating of viable cells. After gating on the viable cells, cell phenotype was determined by comparing histograms of the stained samples to the isotype control. Samples were evaluated using an Accuri C4 (Accuri Cytometers Inc), with a minimum of 100,000 events analyzed using CFlow Plus v. 1.0.208.2.
[0208] Trilineage differentiation: All cells were differentiated at passage 3 and were plated in 6 well plates using 1.410.sup.5 cells per well in aMEM containing 1 L-glutamine, 100 U/mL penicillin/streptomycin, and 15% FBS. Cells were changed to differentiation or control medium upon reaching 80% confluence. DMEM low glucose with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 5-10% FBS was used as control medium for all three lineages. To induce adipogenesis, cells were incubated for 12 days in DMEM (Gibco, 31053) containing 10% rabbit serum (Sigma, R4505), 1 M dexamethasone (Sigma, D2915), 10 M insulin (Humulin N U-100, Lilly), and 200 M indomethacin (Sigma, I7378). Adipogenesis was assessed by Oil Red O staining.
[0209] To induce osteogenesis, cells were incubated for 21 days in DMEM (Gibco, 31053) containing 10% FBS (Hyclone, sh3007003), 100 nM dexamethasone (Sigma, D2915), 10 mM b-glycerophosphate (Sigma, G5422), and 50 M L-ascorbic-acid-2-phosphate (Sigma, A8960), and 2 mM L-glutamine. Osteogenesis was assessed using the StemPro Osteogenic Kit staining protocol using Alizarin Red. To induce chondrogenesis, cells were incubated for 21 days in DMEM (Gibco, 31053) containing 1 mM sodium pyruvate (Gibco, 11360-070), 100 nM dexamethasone (Sigma, D2915), 50 M L-ascorbic-acid-2-phosphate (Sigma, A8960), 40 g/mL L-proline (Sigma, P5607), 1% ITS (Lonza, 17-838Z), 50 ng/mL BMP-2 (Millipore, GF166), and 50 ng/mL TGFb1 (Cell Signaling, 8915LC). Chondrogenesis was assessed using Alcian Blue staining.
[0210] Quantitative real-time PCR: Total RNA was isolated from WJ-MSC using the RNAeasy kit (Qiagen, Hilden, Germany) as per the manufacturer's instructions. RNA concentrations and quality were determined with the RNA 6000 Nano Assay Kit and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif.). All RNA samples had RNA integrity numbers>8. Complimentary DNA was generated by using the RT.sup.2 First Strand Synthesis kit (Qiagen), and heat cycling at 42 C. for 15 minutes followed by 95 C. for 5 minutes prior to placing on ice. 5 l of cDNA was mixed with RT2 SYBER Green Mastermix, RNase free water, and 10 M primer. mRNA expression of CD73 (Qiagen, PPF01104A), CD44 (Qiagen, PPF00491A), MCHII (Qiagen, PPF01028A), CD45 (Qiagen, PPF10210A), CD34 (Qiagen, PPF00586A), and CD90 and CD105 (Invitrogen) was measured. HPRT and RP519 were used as housekeeping genes for normalization of Ct data.
[0211] WJ-MSC Extracellular Vesicles: Isolation and Characterization
[0212] Serum-free culture and stepwise ultracentrifugation: WJ-MSC were thawed and seeded at low density (6000 cells/cm.sup.2) in cAlpha-MEM. Once 70% confluent, cells were transitioned to serum-free defined chemical medium (DCM) modified from Lai et al. (2011, Regen Med., 6(4):481-492), which contained DMEM (Life Technologies) supplemented with 25 M HEPES (Life Technologies), 1 penicillin-streptomycin and L-glutamine (Life Technologies), Insulin-Transferrin-Selenium premix (Gibco), 5 ng/ml recombinant human fibroblast growth factor 2 (Invitrogen, Carlsbad, Calif.), and 5 ng/ml recombinant human platelet-derived growth factor AB (also Invitrogen). Cells were transferred from serum containing medium to 50% DCM plus 50% cAlpha-MEM for 24 hours, washed with PBS, and then the medium was replaced with 100% DCM for 48 hours. Conditioned medium was collected after 48 hours. Supernatant was collected after each of the following steps in centrifugation: 300g for 10 minutes, 2,000g for 10 minutes, and 10,000g for 30 minutes (Eppendorf 5810). The remaining supernatant was then diluted 1:1 with PBS and ultracentrifuged at 100,000g for 70 minutes. (Beckman Coulter Optima L-90K Ultracentrifuge, Brea, Calif.) using a 45Ti rotor (k-factor 133). The pellet was then resuspended in 1 ml PBS for downstream applications.
[0213] Particle size distribution using nanoparticle tracking analysis (NTA): Samples were analyzed using a NanoSight N300 unit (Malvern) equipped with a 488 nm (blue) laser module and Nanoparticle Tracking Analysis 3.0 software. All samples were diluted in sterile PBS to a concentration of 1-1010.sup.8 particles/mL for analysis. Specific NTA settings were optimized for each sample, with fixed settings of temperature (23 C.), screen gain (1.0), infusion flow rate (5 L/min), and camera level set at 12-14 depending on sample characteristics. Five videos were recorded for each sample (30-120 s video length) with all settings remaining constant within each sample source to minimize variation. The detection threshold was set to 5 using auto blur and auto max jump distance settings, with a minimum analysis of 200 valid tracks per video and a minimum of 1000 valid tracks per sample. The NTA unit was periodically evaluated for accuracy of size determination using polystyrene beads of known size (100 and 200 nm).
[0214] Density gradient separation of WJ-MSC EV samplesbuoyancy measurements based on TSG101 expression: Gradients were constructed with iodixanol (OptiPrep Density Gradient Medium, 60% aqueous preparation, Sigma) diluted in gradient buffer containing 0.25 M sucrose, 10 mM Tris and 1 mM EDTA, at pH 7.4. A concentrated EV sample in a volume of 500 l PBS was supplemented with 0.25 M sucrose and 1 mM EDTA and was mixed with 1 ml of 60% iodixanol to give 1.5 ml of sample in 40% iodixanol. The sample was loaded in the bottom of Ultra-Clear by 2 inch centrifuge tubes (Beckman Coulter). Iodixanol solutions were layered on top as follows: 1.2 ml of 30%, 1.2 ml of 20%, 1.4 ml of 10%. A control gradient prepared in the same manner, minus the EV sample, was performed simultaneously. Tubes were placed in an SW55Ti rotor and subjected to 2 hours of 350,000g at 4 C. in an Optima L-90K ultracentrifuge (Beckman Coulter). Following centrifugation, 8 fractions of 625 l were removed from the top of the tube, leaving a small amount of residual volume or 9.sup.th fraction). Fraction density was measured by adding 20 l of each fraction with 80 l water into duplicate wells of 96 well plate and measuring absorbance at 340 nm in a plate reader compared to a linear standard curve of 0, 10, 20, 30, 40 and 60% iodixanol, also diluted 1:4 in water. Expected density of iodixanol in 0.25 M sucrose buffer was taken from the Axis-Shield OptiPrep application sheet from the manufacturer, and density of the collected fractions was calculated from the standard curve. NTA was performed on collected fractions. Fractions were then concentrated to a volume of 175 l with ULTRA-10K regenerated cellulose 10,000 MWCO centrifugal filters (Amicon Ultra 0.5 ml). The BCA assay was performed on the concentrated fractions for protein quantification, NTA for particle count, and immunoblot for TSG101 (Tumor Susceptibility Gene 101) as described for Western Blots.
[0215] Transmission electron microscopy (TEM): EV were diluted in PBS and adhered to copper mesh SPI 200 SuperGrids (2620C, West Chester, Pa.). Uranyl acetate (1%) in deionized water was used for negative staining. Images were obtained at 3870 and 4135 magnification using an FEI Tecnai Spirit 12 electron microscope.
[0216] Peripheral Blood Mononuclear Cell (PBMC) Suppression Assays
[0217] PBMC responder assay: Twelve healthy adult purpose-bread Beagle dogs (5 neutered males, 7 spayed females) housed at the Laboratory Animal Medicine department at the Cummings School of Veterinary Medicine at Tufts University served as blood donors under an approved protocol. WJ-MSC or WJ-MSC were tested in triplicate against a minimum of 3 different PBMC donor cell samples (see Figure legends for n of each experiment). Healthy status was confirmed by clinical examination, and hematological and serum biochemical testing within six months of blood sampling. All dogs were fasted for 12 hours prior to peripheral blood sampling. Peripheral blood samples were taken via jugular venipuncture using a 21 gauge needle, and blood was immediately placed into an EDTA collection tube and rotated 1-2 times gently. Blood was diluted 1:1 with cold PBS or centrifuged (1300g, 10 min) prior to dilution 1:2 in PBS, followed by density-gradient centrifugation using Ficoll-Paque (density 1.077; GE Healthcare Life Sciences) to harvest peripheral blood mononuclear cells (PBMC). PBMC were washed in RPMI (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 25 M HEPES (Life Technologies), 100 M -mercaptoethanol (Sigma-Aldrich), 10,000 U/ml penicillin-streptomycin, 2 mM L-glutamine (Life Technologies), and 2.2 mg/mL of sodium bicarbonate (Thermo Fisher Scientific) (complete RPMI, cRPMI). For mitogen induced lymphocytes proliferation assays, PBMC were suspended in 1 ml cRPMI and 0.5 of 10 mM carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific) and were incubated (10 min). For initial characterization of WJ-MSC effects on responder PBMC, PBMC were plated on a 24-well plate on the top of a 0.4 m Transwell (Corning) across from the WJ-MSC in a 10:1 ratio (PBMC:MSC) in a total volume of 1 ml medium (n=3 technical replicates/sample). All plates were incubated at 37 C. in the dark for 72 hours prior to analysis.
[0218] Dose-responses of TV MSC-EV: To investigate the dose response of PBMC to WJ-MSC EV, the WJ-MSC were plated on a 96-well plate in cRPMI with addition of 1 mM ATP, with or without 5 g/ml Concanavalin A (ConA, Sigma-Aldrich). Responder PBMC were incubated with WJ-MSC EV in a total volume of 200 L medium (n=3 technical replicates per sample). PBMC were incubated with WJ-MSC EV in a 1:10.sup.2, 1:10.sup.3, or 1:10.sup.4 (PBMC:EV) ratio. After 72 hours of incubation, the PBMC were collected and surface stained for CD4 (rat anti-canine CD4-Alexa 647, clone YKIX302.9, AbD Serotec). Cells were stained for 30 minutes, washed twice with cold PBS and 5% FBS, and resuspended in 200 l PBS and 5% FBS with 5 l 7AAD (Becton Dickinson) for analysis by flow cytometry (Accuri C6, Becton Dickinson). The effect of WJ-MSC or WJ-MSC EV on CD4-positive (CD4.sup.+) T cell (i.e., T helper cell) proliferation (percentage of cells proliferating, number of divisions for proliferating cells) was measured by evaluating CFSE fluorescence utilizing the FlowJo proliferation platform (FlowJo, V10, Ashland, Oreg.) after gating on the viable CD4.sup.+ lymphocyte population.
[0219] EV depletion and enzymatic digestion experiments: EV were isolated as described, resuspended in 5 ml PBS, and divided into 5 fractions of 1 ml each. The fractions were either left untreated, or were treated with 0.1% Triton-X, 2 g/ml RNAse A, or both RNAse A and proteinase K for 30 minutes at 37 C. The samples were then diluted with PBS and centrifuged at 100,000g for 70 minutes. The EV sediment was resuspended in PBS and particle numbers were quantified using nanoparticle tracking analysis (NTA) as described supra. A ratio of 1 PBMC to 10.sup.4 EV was cultured in 200 l of cRPMI with 1 mM ATP, with or without 5 g/ml ConA for 72 hours (n=3 technical replicates per sample). WJ-MSC EV depleted controls: following exosome isolation, the sediment was resuspended in 1 ml PBS and filtered through a 10 kDa (Amicon Ultracel, Sigma) or 50 kDa (Sartorius Vivaspin 4, Gottingen, Germany) molecular weight cutoff filter (MWCO) and centrifuged (4,000g) for 15 minutes. The filtrate was collected. The retentate was resuspended in 1 ml PBS, and particle content was evaluated by NTA. A volume of EV from retentate to generate a ratio of 10.sup.4 EV:1 PBMC, or an equivalent volume of filtrate, were cocultured with PBMC as described. In addition, the supernatant generated by stepwise ultracentrifugation (100,000g) was employed as an EV depleted sample. Chemical inhibition of EV biogenesis was achieved using GW4869 (6 M), an inhibitor of neutral sphingomyelinase (Guo, Bellingham et al. 2015) for 48 hours, then re-plating GW4869-treated or non-treated WJ-MSC across 0.4 m Transwell from PBMC in a 1:10 ratio for 72 hours prior to collection of PBMC and evaluation of lymphocyte suppression. As well, WJ-MSC EV, EV (10.sup.4:1 PBMC) from conditioned medium of non-MSC fibroblasts (canine left ventricular cardiac fibroblasts) were evaluated for suppression of PBMC.
[0220] Functional evaluation of WJ-MSC EV TGF-: Ten M of SB4312 (Tocris, Bristol, UK), a specific TGF-RI inhibitor (Hasan, Neumann et al. 2015), 50 M of ZM241385 (Tocris), a specific adenosine2A receptor inhibitor, or 0.1 g/ml of TGF- 1,2,3 neutralizing antibody (R&D Systems, Minneapolis, Minn.) was added to PBMC plus WJ-MSC EV or PBMC in coculture with WJ-MSC at the beginning of the 72-hour incubation period. Alternatively, 5, 10, or 50 ng/ml of recombinant human TGF-1 (R&D Systems) or TGF-3 (Sigma) were added to ConA stimulated PBMC cultures. For disruption of the heparin sulfate side chains on TGFRIII (beta-glycan), WJ-MSC EV were incubated with heparinase III (Sigma) or heat-inactivated heparinase (control) at 0.006 U/ml for 3 hours at 37 C. according to the method of Webber et al. (2015, Oncogene, 34(3):290-302). The WJ-MSC EV were washed by resuspending in PBS and ultracentrifugation at 100,000g for 70 minutes prior to application in PBMC coculture.
[0221] Bead-assisted flow cytometry of TGF- on WJ-MSC EV. A total volume of 110.sup.10 EV was incubated with 10 l (1.210.sup.7) 3.9 m latex beads for 15 minutes at room temperature. The sample was diluted to a volume of 1 ml with PBS, and the sample was incubated overnight on a tube rotator at room temperature. The bead-EV sample was pelleted by centrifugation for 3 minutes at 1500g. The supernatant was removed, and 1% BSA was added to a total volume of 500 l for 30 minutes. The sample was pelleted again, the supernatant removed, and the sample resuspended in a final volume of 100 l 0.1% BSA. Samples were incubated for 30 minutes with 1 g of mouse anti-TGF-1, 2, 3 (R&D Systems), isotype, or secondary antibody alone; 10 l of sample was used incubation with each antibody. Samples were washed, then incubated with secondary antibody for 30 minutes prior to washing and evaluating by flow cytometry.
[0222] Enzyme linked immunosorbent assay (ELISA) to evaluate content of latent versus mature TGF- on WJ-MSC EV: ELISA immunoassay was performed using the TGF-1 Quantikine ELISA kit (R & D Systems) on WJ-MSC EV sediments, as per manufacturer's instructions, with exception that for some samples, pretreatment with acid was omitted (in order to measure the quantity of native active TGF- form only). Samples were diluted 1:4 for ELISA analysis prior to acid activation where appropriate and absorbance was measured at 450 nm. (e.g.,
[0223] Western Blots: Protein was solubilized from PBMC, CD4.sup.+ T cells, or WJ-MSC using m-PER (Thermo Scientific). Protein obtained from cell extracts or EV was quantified by a bicinchoninic acid kit (Pierce BCA Protein Kit, Thermo Scientific). Western blotting was performed using the iBLOT kit (Becton Dickinson) according to manufacturer's instructions. Equal amounts of protein were loaded into each lane of Bolt 4-12% Bis-Tris gels (Invitrogen), resolved in Bolt MES SDS running buffer (Invitrogen), and electroblotted onto nitrocellulose membranes. The iBIND Flex kit was used for blocking and antibody application. Antibodies used included anti-TGFRI antibody (Abcam, clone ab125310) at 1:500, anti-TGF-1 antibody (Abcam, ab190503) at 1:500 dilution, anti-TSG101 (BD Biosciences 612696) at 1:1000 dilution, anti-PDC6I (Alix) (Abcam ab76608) at 1:1000 dilution, and anti-calnexin (Abcam ab75801) at 1:1000 dilution. Biotinylated conjugated horse anti-mouse antibody (BA-2000, Vector Laboratories) or biotinylated goat anti-rabbit (BA-1000) at 1:40 dilution was used as a secondary antibody, and detection was performed using the Vectastain ABC Kit (Vector Laboratories), followed by use of the Peroxidase DAB substrate kit (Vector Laboratories). Control cells included HeLa cells, Mardin-Darby Canine Kidney (MDCK) cells and EV, and dog brain cells isolated from donated tissue after client approval from euthanized animals.
[0224] Statistical Analysis: The distributions of percent dividing CD4.sup.+ cells were explored for normality through descriptive statistics, and pairwise statistical comparisons were performed using a paired-sample t-test. Comparisons between 3 or more groups were made using ANOVA, followed by Tukey multiple means comparison post-test. Analyses were performed using SPSS (Version 24, IBM). Pearson's correlation coefficients were performed in Microsoft Excel (Version 15.24). For all analyses, statistical significance was set at p<0.05. Values are expressed as mean standard deviation.
[0225] The findings and results of the experiments described in this Example are further described hereinbelow.
Wharton's Jelly-MSC (WJ-MSC) Exhibited MSC Phenotype and Differentiation Capacity
[0226] The WJ-MSC isolated from canine Wharton's Jelly exhibited plastic adherence colony formation, surface phenotype, and trilineage differentiation (
WJ-MSC EV Size Distribution and Morphology was Consistent with Small EV
[0227] The mode and mean particle size derived from the composite data of 5 WJ-MSC EV lines was 125 nm and 199 nm, respectively, with 76% of all EV ranging from 50-250 nm, consistent with the size characteristics of small EV (
WJ-MSC or WJ-MSC EV Suppress CD4.SUP.+ T Cell Proliferation
[0228] The dose-response of WJ-MSC EV immunomodulatory capacity was evaluated through coculture of EV with ConA-stimulated PBMC. This showed that WJ-MSC EV mediated suppression of ConA stimulated CD4.sup.pos T cell proliferation was dose dependent (
Depletion of EV Ameliorated Lymphocyte Suppression
[0229] The neutral sphingomyelinase (NSMase) inhibitor, GW4869, was employed to assess exosome output from MSC. Increasing doses GW4869 were applied to the WJ-MSC to assess exosome output that resulted in maximal suppression without an unacceptable loss of viability at 5-10 M (
Reproducibility of WJ-MSC and WJ-MSC EV Activity
[0230] Across all cells lines, WJ-MSC and WJ-MSC EV consistently suppressed ConA stimulated T cell proliferation (
TABLE-US-00001 WJ Line Number of Cells Number of EV/mL EV/Cell 12 2.20E+07 2.20E+011 10000.0 13 1.07E+07 6.20E+10 5794.4 34 3.00E+07 4.12E+11 13733.3 34 6.00E+06 5.10E+10 8500.0 49 1.70E+07 1.49E+11 8764.7 49 4.40E+07 2.00E+11 4545.5 49 6.28E+07 1.95E+11 3105.1 52 5.80E+06 2.40E+10 4137.9 69 2.40E+07 9.20E+10 3833.3 76 9.00E+06 5.90E+10 6555.6 85 6.70E+07 2.80E+11 4179.1 85 6.60E+07 5.80E+10 878.8 85 1.07E+09 3.60E+11 336.4 85 8.00E+07 3.90E+11 4875.0 96 3.00E+07 1.80E+11 6000.0 96 5.00E+07 3.50E+11 7000.0 111 1.80E+07 1.51E+11 8388.9 157 4.40E+07 1.50E+11 3409.1
IFN- Pre-Conditioning of WJ-MSC Did Not Increase Suppression of T Cell Division by WJ-MSC or WJ-MSC EV
[0231] Pretreatment of WJ-MSC with 500 ng IFN- for 48 hours prior to collection of WJ-MSC EV was performed to determine if such pretreatment would augment either WJ-MSC (across transwell) or WJ-MSC EV activity. While there were trends in further suppression by WJ-MSC or WJ-MSC EV following pre-conditioning of WJ-MSC, these effects were not statistically significant. (
TGF- and Adenosine Signaling are Mechanisms of WJ-MSC EV Mediated Suppression of CD4.SUP.+ T Cells
[0232] To determine if TGF-1 or adenosine contribute to EV induced immune modulation, WJ-MSC EV and PBMC were cocultured with TGF- (1, 2, and 3) neutralizing antibody or with pharmacological inhibitors of TGFRI, adenosine 2A receptors, or both. Neutralization of TGF- (1, 2, and 3) with functional antibody significantly reduced suppression of CD4+ (CD4.sup.pos) cell division (
Exogenous Soluble TGF- Suppresses EV
[0233] The presence of TGFRI on both PBMC and isolated CD4.sup.+ T cells was demonstrated by Western blot (
[0234] In this Example, the data and results demonstrate that EV from canine WJ-MSC significantly disrupted mitogen (ConA)-activated T cell proliferation through biochemical signaling pathways. The data and results described supra suggest that a substantial fraction of the effects of MSC in PBMC assays may arise from insoluble EV-associated factors such as TGF- and adenosine.
Wharton's Jelly-MSC (WJ-MSC) Phenotype
[0235] The cells isolated from Wharton's Jelly (WJ-MSC) and employed in this Example exhibited surface markers and gene expression that are typical for MSC, such as canine WJ-MSC, including CD44, CD90, and CD105 and the absence of CD45, CD34, and MHCII. Discordance for CD73 and CD34 was observed between gene expression (positive) and protein expression (negative), which may relate to technical issues with antibody reactivity (CD73) and post-transcriptional silencing (CD34). To this point, the collagenase digestion method of WJ-MSC isolation may not have been ideal for isolation of WJ-MSC, since the explant method yields more MSC with greater expansion potential and retention of MSC markers.
WJ-MSC EV Size Distribution and Morphology is Consistent with Small EV
[0236] Minimal criteria for characterization of EV were put forth by a consortium from the International Society of Extracellular Vesicles in 2014 (MISEV), (Lotvall, J. et al., 2014, J. Extracell Vesicles, 3:26913). In the experiments described in this Example, such guidelines were adhered to by detailing EV isolation methods and performing general characterizations (EV-specific and non-EV cellular proteins by Western blot, buoyancy measurements by density gradient and Western blot for TSG101), single vesicle characterization using two methods (TEM and NTA), and functional assays including dose-response, response controls to exclude non-EV (by EV depletions four different ways), and controls for the source of EV (using non-MSC EV). The data and results have rigorously demonstrated that the biological activity measured in PBMC responder assays is based on the use of WJ-MSC EV.
Interpretation of WJ-MSC EV Phenotype
[0237] The particle size distribution and ultrastructural morphology of WJ-MSC EV was consistent with small EV (range 50 to 100 nm), but included smaller vesicle-like structures that were not identified. The wide range of EV sizes detected by NTA and TEM in this Example is consistent with the diverse repertoire of vesicles from a single source (Zabeo, D., 2017, J. Extracell. Vesicles, 6(1):1329476). The low buoyancy and detection of TSG101 and Alix implied that canine WJ-MSC EV as isolated for these experiments consisted mainly of small EV (Kourembanas, S., 2015, Annu Rev Physiol., 77:13-27), although canine WJ-MSC EV were larger exosomes isolated from human WJ-MSC that expressed both TSG101 and Alix (Willis, G. R. et al., 2017, Front Cardiovasc Med, 4:63). Differences in isolation and size measurements can make comparisons among studies difficult. Notwithstanding, the method of stepwise ultracentrifugation yielded an EV enriched population of particles for the studies described in this Example.
WJ-MSC EV Suppress CD4.SUP.+ T Cell Proliferation
[0238] Striking T helper cell suppression was observed as a function of EV dosage, a finding that was absent in EV-depleted fractions or in EV from non-MSC fibroblasts. Similarly, MSC EV, including WJ-MSC EV, were strongly suppressive of mitogen stimulated CD4.sup.+ T cells, but not of those stimulated by mixed lymphocyte reaction (MLR), which led to the use of the mitogen stimulation assay in the experiments described supra. While some have reported that isolated MSC EV are not immunosuppressive in mitogen stimulation assays, even at 10 fold higher concentrations than employed in the assays described here, subtle variations in experimental protocol, as well as other factors (e.g., isolation, purification, handling), may contribute to differences.
[0239] The specific observation that canine WJ-MSC EV (or parent WJ-MSC) suppressed the percentage of dividing CD4.sup.+ T helper cells (responders), but not the number of cell divisions of responders, is consistent with arrest at G0/G1 for suppressed T cells (Hosseinikia, R. et al., 2017, Int J Hematol Oncol Stem Cell Res, 11(1):63-77). That WJ-MSC EV affected CD4.sup.+ cells to a greater extent than CD4.sup. (CD4.sup.neg) cells is a reflection of greater proportions of CD4.sup.+ than CD4.sup.neg mitogen responders in the assays described herein, and not necessarily the specific impact on CD4.sup.neg cells. According to the data presented here, the number of EV produced by each MSC is on average approximately 5,000 (510.sup.3), and the total EV introduced into each PBMC responder well containing 510.sup.5 PBMC is 510.sup.9, or the equivalent EV from 10.sup.6 MSC. Commonly employed doses of MSC in vivo (210.sup.6/kg or 15010.sup.6) would effectively suppress T cells within 75 million PBMC. Whether this has any relevance to dose equivalence of biological activity can readily be evaluated by concurrent in vitro and in vivo studies.
[0240] Intrinsic variation in the effectiveness (i.e., potency) of WJ-MSC to modulate immune effector cells is a confounding aspect of MSC therapies in regenerative medicine. The range of responses observed across 11 cell lines (only 2 pairs were littermate donors) was explored here. Overall, the responses ranged from 9-92% mean suppression of CD4+ T cells across all WJ-MSC EV lines tested. The major variation was due to two cell lines (9.1%, 14.2% suppression) versus the range across the other 9 cell lines (48-92% suppression). The magnitude observed, under the conditions used, namely, 1 WJ-MSC to 10 PBMC across a transwell, is consistent with reports in the literature (e.g., Barcia, R. N. et al., 2017, Cytotherapy, 19(3):360-370).
[0241] Priming MSC with a pro-inflammatory stimulus has been shown in various studies to increase their immunosuppressive capacity, and even to increase the capacity of MSC EV to suppress T cells, B cells, and NK cells. However, pretreatment of WJ-MSC with IFN over 3 days prior to introduction into transwell assays did not enhance the anti-proliferative activity of WJ-MSC or WJ-MSC EV in the experiments described herein. Similarly, immunosuppression by umbilical cord matrix MSC (human) was not enhanced by IFN (Barcia, R. N. et al., 2017, Cytotherapy, 19(3):360-370). Thus, without being bound by theory, IFN priming may not be effective in this cell type or in the canine species.
[0242] The magnitude of suppression by WJ-MSC EV (50-75%) under the conditions used in the methods described in this Example is comparable to studies using canine AD-MSC or BM-MSC types (see, e.g., Clark, K. et al., 2016, Stem Cell Rev, 12(2):245-256), although direct comparisons are affected by different subsets of lymphocytes stained in these studies.
[0243] The presence of TGFRI on both PBMC and isolated CD4.sup.+ T cells was demonstrated by Western blot, demonstrating that direct interaction of EV-associated TGF- with this receptor on CD4.sup.+ T cells is a plausible mode of action as detected in this assay. In support of this, the addition of 10 or 50 ng/ml, but not 5 ng/ml, TGF-1 or TGF-3 suppressed mitogen driven T cell division. Of note, the amount of EV-derived TGF-1 needed to achieve suppression of CD4.sup.+ T cell division was approximately 10% of the amount of the soluble form required to produce an equivalent effect (in the assay using 10 ng/mL of recombinant human TGF-1 (rhTGF-1). This suggests that EV-associated TGF-1 may have a heightened, or a qualitatively different, biological activity when compared to soluble TGF-1. This may be similar to comparisons of EV membrane-associated TGF-1 derived from cancer-associated fibroblast and dendritic cells versus soluble TGF-1 (Clayton, A., 2007, Cancer Res, 67(15):7458-7466; Yu, L. et al., 2013, Eur J Immunol, 43(9):2461-2472). The findings that EV associated adenosine had similar effects supports the notion that WJ-MSC EV may introduce a number of mechanisms, similar to the repertoire of growth factors attributed to parent MSC.
[0244] The participation of the TGFIII receptor (betaglycan) was explored, given its role as a co-receptor for TGF- signaling. Betaglycan can increase affinity for TGF- to its receptors TGF-RI and TGF-RII. TGF--induced fibroblast differentiation and angiogenesis by cancer EV require the interaction of betaglycan with TGF-1 (Webber, J. P. et al., 2015, Oncogene, Vol. 34(3):290-302). The TGF-1 and betaglycan interaction requires heparin sulfate side chains. In order to determine if a betaglycan interaction capacitated the anti-proliferative effect of WJ-MSC EV on T cells, EV were treated with heparinase as described supra and shown in
[0245] The latent form of TGF-1 was activated to release the mature form of TGF-1 in the PBMC assay described here. It may also be that EV-derived TGF-1 is not the major active form in this assay; but rather, non-EV derived sources of TGF-1, along with other soluble mediators (e.g. IL-10), might be produced de novo in the assay following EV interactions with monocytes or T cells, as an autocrine loop. Knocking down specific cell types, e.g., monocytes, in this assay may resolve the interdependence of cells within the PBMC that are associated with the effect of TGF-1 and adenosine on T helper cells (CD4+ T cells).
[0246] The experiments described in this Example have demonstrated that WJ-MSC EV possess intrinsic mechanisms previously attributed mainly to soluble factors that suppress the proliferation of CD4.sup.+ T helper cells. As described supra, it was found that WJ-MSC and EV consistently suppressed ConA-induced CD4.sup.+ T cell proliferation in a dose-dependent fashion, and that the effect was abolished in EV-depleted samples including ultracentrifuge supernatants, EV filtrates, EV from GW4869-pretreated MSC, and Triton-X exposed EV samples. Non-MSC EV did not show lymphocyte suppression. Blockade of TGF-1 signaling by pretreatment of PBMC with a TGF-RI antagonist (SB431542) or with neutralizing antibodies to TGF-1 significantly reduced the anti-proliferative effect of the WJ-MSC EV. Western blotting and ELISA analyses showed that TGF- was present on WJ-MSC EV in the large latent and pro-form complexes. These data demonstrate that canine WJ-MSC EV are immune modulatory through TGF- signaling, which may be employed in a method of evaluating the biological potency of cell lines.
Example 4
TGF- in Latent Form on Native WJ-MSC EV
[0247] TGF- was found in a latent form in EV derived from MSC, e.g., WJ-MSC EV. To detect active/mature TGF-, it was necessary to pre-treat the EV with acid, as shown by ELISA analysis (
[0248] More specifically, using ELISA analysis, it was determined that the amount of TGF-1 ranged from 0.1-1.0 ng per 510.sup.9 EV (the number of EV used in each standard PBMC assay well for a 10.sup.4 EV:PBMC ratio). Some TGF-1 was detected only after acid activation, consistent with the bulk of TGF-1 in the latent form associated with EV (
Example 5
Hyaluronic Acid/Proteoglycan Complexed to Extracellular Vesicles (EV) Derived from Different Sources and Cell Types and Removed from EV by Treatment with Hyaluronidase
[0249] This Example describes the unexpected finding that some EV, e.g., umbilical cord-derived EV, placental-derived EV, or other mesenchymal stem cell-derived EV (or other cell types producing EV) are covered with hyaluronic acid/proteoglycan complexes (or are coated with complexes of hyaluronic acid/proteoglycan). Pre-treatment of such EV with hyaluronidase completely removed those complexes, reducing aggregation and doubling the number of single EV which were available for analysis or treatment, and exposing epitopes including TGF-. Based on this finding, hyaluronidase is effectively used as a pretreatment of EV preparations, particularly when centrifuged (ultracentrifuged) or concentrated EV preparations are resuspended, e.g., during isolation and manufacture.
[0250] Hyaluronidase also dissolves hyaluronic acid in unfractionated synovial fluid, which is too viscous for centrifugation and concentration of EV from that fluid. Amounts of hyaluronidase used to treat synovial fluid, e.g., as provided in Boere, J. et al., 2016, J. Extracell Vesicles, 5:31751, may be used for directly treating EVs covered with hyaluronic acid/proteoglycan complexes. By way of example, an EV preparation may be incubated with 40 l hyaluronidase (1500 U/mL) for 15 minutes at 37 C. (or room temperature for a longer time period). Unlike the treatment of unfractionated synovial fluid, concentrated, semi-purified or purified EV were optimally treated directly with hyaluronidase as a pretreatment.
[0251]
Other Embodiments
[0252] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0253] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0254] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.