WO2016168680A1

WO2016168680A1 - Method for developing exosome-based vaccines

Info

Publication number: WO2016168680A1
Application number: PCT/US2016/027872
Authority: WO
Inventors: John W. Holaday; Janette DIXON; Roland Probst
Original assignee: Exocyte Therapeutics Pte Ltd.
Priority date: 2015-04-17
Filing date: 2016-04-15
Publication date: 2016-10-20

Abstract

The embodiments disclosed herein are directed to methods for making immunogenic compositions and methods of using such immunogenic compositions to treat various diseases. The methods provide improved methods for priming immune cells by delivering to the immune cells extracellular vesicles obtained from diseased cells representative of the disease condition to be treated. In certain example embodiments, the immune cells and extracellular vesicles are obtained from the subject to be treated. The isolated extracellular vesicles are then delivered ex vivo to the isolated immune cells. Primed immune cells may then be re¬ introduced to the subject to illicit an immune response directed to the diseased tissue of interest.

Description

METHOD FOR DEVELOPING EXOSOME-BASED VACCINES

TECHNICAL FIELD

[0001] The subject matter disclosed herein is generally directed to live cell vaccine compositions. Specifically, the subject matter is directed to live cell vaccines comprising immune cells derived from a subject and primed with extracellular vesicle material from diseased cells or tissues to be treated

BACKGROUND

[0002] A rapidly growing field of cancer research is immunotherapy - harnessing of the body's own immune system to seek out and kill cancer cells. Early cancer vaccines demonstrated the need to overcome the following three key challenges: clinically useful tumor vaccines must target an immune response against multiple cancer genes and proteins that drive tumor growth and progression; substantial genetic variation exists between patients with the same type of cancer, within a single primary tumor and between metastatic tumors, so clinically useful tumor vaccines need to account for a high level of tumor heterogeneity; and overcoming immunosuppressive effects exerted by tumors.

[0003] Exosomes are a multi-valent source of tumor antigens that may be used to address these challenges. Exosomes are "nanoballs," between 30-100 nanometers in size and surrounded by a protective lipid bilayer. They are shed into the blood, urine, cerebrospinal fluid and other bodily fluids from tumors and other cells, and contain the protein and functional ribonucleic acid (RNA) molecules from their cells of origin. Specifically, tumor exosomes contain the genetic and protein fingerprints of the cancer they are shed from, which means they include all, or the majority of, the mutations and markers that are specific to the malignancy. In addition, they contain factors that help the tumor grow and metastasize.

[0004] The problem of existing methods of using tumor exosomes to educate the immune system is that they need to be inserted into the immune cells in an efficient manner. Co-incubation, otherwise known as co-culture, or "pulsing", is the standard method used for educating dendritic cells with exosomes. Dendritic cells take up exosomes in small numbers by pinocytosis. U.S. Patent No. 9,107,878 to Holaday previously proposed electroporation as an alternative to co-incubation as a method for administering tumor exosomes and/or their contents into dendritic cells.

[0005] Electroporation has been shown to be more highly effective than co- incubation in loading dendritic cells with antigen as recognized by Wolfraim et al. Int. Immunopharmacol 2013, 15(3):488-97. However, electroporation may not be the most efficient method for loading dendritic cells with either intact exosomes or the contents of lysed exosomes, which are comprised of a mixture of nucleic acids and proteins.

SUMMARY

[0006] A method for generating a therapeutic vaccine for treating diseases comprises delivering, ex vivo, isolated extracellular vesicle material obtained from diseased cells or tissues into immune cells isolated. The extracellular vesicle material may be delivered into the isolated immune cells by physical or non-physical delivery methods. Example physical methods include shear induced loading, shear and squeeze induced loading, use of electronic ejector microarrays, sonoporation, magnetoporation, photothermal poration (gold particle mediated poration and vapor nanobubbles mediated), jet injection, use of massively parallelized nanoneedles, filtroporation, carbonate apatite nano-complexe mediated delivery, or a combination thereof. Example non-physical delivery methods include delivery using virus or virus like synthetic nanoparticles, delivery using cationic coatings, delivery using cell-permeating peptides, and ligand-mediated internalizations. The extracellular vesicle material may comprise whole exosomes, whole microvesicles, and/or lysates derived therefrom. In certain example embodiments, the extracellular vesicle material may include RNA, DNA, proteins, or a combination thereof isolated from the extracellular vesicles. In certain example embodiments, the immune cell is an antigen presenting cell. In certain other example embodiments, the antigen-presenting cell is a dendritic cell. The primed immune cells may then be re-introduced to the subject to elicit an immune response against the diseased tissue.

[0007] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of illustrated example embodiments.

DETAILED DESCRIPTION

[0008] The embodiments disclosed herein are directed to methods for making immunogenic compositions and methods of using such immunogenic compositions to treat various diseases. The methods provide improved methods for priming immune cells by delivering to the immune cells extracellular vesicle material obtained from diseased cells representative of the disease condition to be treated. In certain example embodiments, the immune cells and extracellular vesicles are obtained from the subject to be treated. The isolated extracellular vesicles are then delivered ex vivo to the isolated immune cells. Primed immune cells may then be re-introduced to the subject to illicit an immune response directed to the diseased tissue of interest. The use of subject-derived extracellular vesicles material - or extracellular vesicle material derived from a similar diseased tissue - provides a source of current protein and genetic disease markers to arm subject-derived immune cells. In this way the vaccine compositions prepared using the methods disclosed herein enable therapeutic vaccine treatment protocols that can address changes in genomic expression by diseased cells over time.

[0009] In certain example embodiments, the whole extracellular vesicle is delivered to an immune cell. In certain other example embodiments, the extracellular vesicle is lysed and the extracellular vesicle lysate is delivered to the antigen presenting cells. In certain other example embodiments, proteins, RNA, DNA, or combination thereof is isolated from the lysate and delivered to the antigen presenting cells. In certain example embodiments, the RNA and/or DNA may first be amplified prior to delivery to the antigen presenting cells. As used herein, the term "extracellular vesicle material" includes whole extracellular vesicles, extracellular lysate, nucleic acids - both amplified and un-amplified - and proteins isolated from extracellular lysate. Extracellular vesicles that may be used in the present invention include oncosomes, apoptotic bodies, microvesicles, exosomes, and virus like particles.

[0010] In certain example embodiments, the extracellular vesicles may be isolated directly from a patient sample containing diseased cells, such as a biopsy sample, or a sample of biological fluid such as, but not limited to, blood, urine, pleural fluid, ascites, cerebrospinal fluid, lymph, or saliva. The extracellular vesicles are then isolated from the sample using known methods in the art, such as differential centrifugation and/or ultrafiltation. In certain other example embodiments, the diseased cells may be obtained from a biological sample as discussed above and cultured prior to isolating the extracellular vesicles, or the diseased cells may be obtained from a non-autologous cell line or sample. The extracellular vesicles may be isolated directly from the cell culture medium or by first lysing the cells and isolating the extracellular vesicles from both the culture medium and the lysed cells.

[0011] In certain example embodiments, the immune cell is an antigen presenting cell.

As used herein, 'antigen presenting cell" or "APC" means a cell capable of presenting an antigen to a lymphocyte. Examples of APCs include, but are not limited to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B cell, monocytes, fibroblasts and fibrocytes. In one example embodiment, the antigen presenting cell is a dendritic cell.

[0012] The immune cells may be isolated from the subject to be treated using known methods in the art. For example, the immune cells, or corresponding progenitor cells, may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissue- infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. Progenitor cells may be isolated and then differentiated into the desired immune cell type by culturing the isolated progenitor cell in the presence of growth factors and/or cytokines known to induce differentiation in to the desired cell type. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13, IL-15 and/or TNFa to cultures of isolated monocytes harvested from peripheral blood. Also, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compounds that induce maturation and proliferation of dendritic cells.

[0013] The present invention provides methods for extracellular vesicle delivery into immune cells, in particular dendritic cells. In certain example embodiments, the methods disclosed herein use membrane disruption of dendritic cells by mechanical deformation as they pass through a microfluidic constriction to induce the uptake of extracellular vesicles or the contents of lysed extracellular vesicles (i.e. proteins, peptides, RNA, miRNA, DNA and any other components of exosomes and microvesicles).

[0014] In one example embodiment, the method comprises the use of shear induced loading to deliver the extracellular vesicle material to the cells. Shear induced methods involve the co-placement of cells in suspension with extracellular vesicle material, for example in a microfluidic device. The suspension is then passed through channels that expose the cells to shear forces which cause transient pores to form in the cell membrane allowing uptake of the extracellular vesicle material. In certain example embodiments, mechanical deformation is used to introduce extracellular vesicles into the immune cells. Mechanical deformation of immune cells may be used to induce membrane disruption that results in the formation of pores greater than 200 nm in size. Isolated immune cells are placed in suspension with the isolated extracellular vesicle material. The suspension is then passed under pressure through constricted channels having a diameter smaller than the diameter of the cells. As the cells pass through the constrictions the cells are squeezed. Due to this gentle squeezing, transient pores open up in the membranes of the cells allowing the extracellular vesicle material to diffuse into the dendritic cells. After passing through the constricted channels, the immune cell membranes close completing delivery of the extracellular vesicle. Devices and methods for generating pores in cells using the above "shear and squeeze" mechanism are known in the art. See e.g. Shari et al. Integr Biol (Camb), 2014, 6(4):470-475. As extracellular vesicles are typically 30 nm to 100 nm in diameter, the extracellular vesicle may be taken up by the cells in greater numbers than they would be by co-incubation or electroporation, leading to a stronger immune response. Mechanical deformation of cells in the presence of exosome lysate may result in substantial uptake of large proteins in addition to smaller proteins, peptides and nucleic acids, which may also lead to a stronger immune response that could be obtained through the use of co-incubation or electroporation. Alternatively, the use of mechanical deformation for loading of cells may allow a much smaller starting sample of exosomes than is required for loading by electroporation. This is important because certain subject derived extracellular vesicles, such as tumor-derived exosomes, may only be available in limited quantities.

[0015] In one example embodiment, the methods disclosed herein comprise use of electronic ejector microarrays or ultrasonic atomizers to achieve delivery of the extracellular vesicle material to immune cells. Extracellular vesicles and the target immune cells are loaded onto a microelectromechanical system (MEMS) device that ejects a sample of the cells through microscopic nozzles with incorporated electroporation electrodes, which cause the cell membrane to become transiently permeable to the uptake of the extracellular vesicle material. Extracellular vesicle material may be passed through the nozzles along with the cells, or may be incubated with the cells immediately after poration.

[0016] In another example embodiment, the methods disclosed herein comprise use of sonoporation to deliver extracellular vesicle material into the immune cells. In sonoporation, ultrasound waves are applied to the immune cells which causes a perturbation of the cell membrane through the interaction of cavitation bubbles with the cell membrane. Extracellular vesicle material to be delivered to the immune cells may be placed in suspension with immune cells prior to or after application of the ultrasound. [0017] In another example embodiment, the methods disclosed herein comprise use of magnetoporation, or magnetotransfection, to deliver extracellular vesicle material into cells. An external magnetic gradient is applied to the immune cells in the presence of a solution comprising the extracellular vesicle material. The extracellular vesicle material in turn are associated with magnetic particle-vector complexes. The external magnetic gradient field pulls the extracellular vesicle material loaded magnetic particle-vector complexes toward the cells to be transfected. Thus, in this embodiment, the extracellular vesicle material to be delivered are first associated with a magnetic material. For example, a magnetic nanoparticle may be delivered into or bound to the extracellular vesicle material prior to placing the extracellular vesicle material in suspension with the immune cells and application of the magnetic field. In an alternative embodiment, one or more extracellular vesicle material may be bound to a magnetic carrier known in the art, for example a magnetic bead.

[0018] In another example embodiment, the methods disclosed herein comprise use of photothermal poration to deliver extracellular vesicle material into immune cells. In one example embodiment, the method comprises exposing the cell surface to an array of nanoblades. Nanoblades are metallic nanostructures that harvest a short laser pulse of energy and convert it into a highly localized vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induces transient shear stress. The cavitation bubble is controlled by the metallic structure configuration and laser pulse duration and energy. The nanoblade generates a micrometer-sized hole in the cell membrane facilitating delivery of extracellular vesicle material into the cell. In another example embodiment, the methods disclosed herein comprise the use of photoporation to deliver extracellular vesicle material into cells. In one-example embodiment, nano-sized membrane pores are created in the cell membrane by laser illumination and heating of gold nanoparticles on the cell membrane surface, which causes pores to form in the cell membrane and extracellular vesicle material to be passively taken up by the porated cells. In another example embodiment, the pores are formed by localized heating of the cell membrane or application of vapor nanobubbles (V Bs).

[0019] In another example embodiment, the methods disclosed herein comprise use of massively-parallelized nanoneedles to achieve delivery of extracellular vesicle material via physical puncturing of the immune cell membrane. Immune cells are loaded along with extracellular vesicle material to be delivered onto a MEMS-based or microfluidic device with an array of nanoneedles. The device causes contact between the cells and the array of nanoneedles resulting in mechanical puncture of the cell membranes. The punctured immune cells may then passively uptake extracellular vesicle material.

[0020] In another example embodiment, the methods disclosed herein comprise the use of jet injection to deliver extracellular vesicle material into immune cells. The cells are loaded into a microfluidic device configured to deliver a macromolecular solution comprising the extracellular vesicle material to the immune cells via a high-velocity, ultra-fine stream of the macromolecular solution onto the cell surface to penetrate the cell membrane.

[0021] In another example embodiment, the methods disclosed herein comprise the use of filtroporation. Immune cell are porated by forcing the cells through uniformly sized micropores of filter membranes. The porated cells are then place in suspension with extracellular vesicle material and allowed to passively uptake the extracellular vesicle material.

[0022] In another example embodiment, the methods discloser herein comprise the use of carbon apatite-exosome complexes to deliver exosomes to cells. Extracellular vesicles are associated with carbonate apatite prior to delivery into the cells. Carbonate apatite is stable under typical physiological pH but is easily dissolved in the acidic environment of cellular vesicles such as endosomes. The carbonate apatite-exosome complexes are then incubated in the presences of the cell, which then take the complexes up by endocytosis and/or phagocytosis.

[0023] Immune cells primed by delivering of extracellular vesicle material using one of the above described methods, may then be formulated in compositions suitable for delivery of live immune cells to a subject. The compositions may further comprise various agents to reverse immunosuppression or adjuvants to help increase the immune response elicited by the primed immune cells. In certain example embodiments, agents that reverse immunosuppression may include checkpoint inhibitors to CTLA-4, PD-1, PDL-1, and other similar immune checkpoints. In certain example embodiments, the adjuvant may comprise agonists for TLR3, TLR7, TLR8 or TLR9 and other similar immunostimulatory agonists.

[0024] In certain example embodiments, the subject to be treated suffers from pathological indications including, but not limited to, cancer, infectious disease, autoimmune disease, metabolic disease, or a cardiovascular disease wherein eliciting a response using the compositions described herein may be useful to treat the disease or an underlying symptom of the disease. The term "treating", "treat" or "to treat" as used herein means the prevention, reduction, partial, or complete alleviation or cure of a disease or symptom thereof. [0025] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example I. Purification of Exosomes from Cancer Cells

[0026] Samples are obtained after informed consent from a cancer cell sample from the individual such a s tumor biopsy, or a sample of a biological fluid from the individual such as blood, urine, pleural effusion, ascites, lymph, or saliva. The extracellular vesicle fraction is prepared by differential centrifugation. First, cells are pelleted at 500 g for 20 min at IOC and discarded, and then additional cellular debris is removed by centrifugation at 16 000 g for 20 min at IOC, followed by filtration through a 0.45 mm filter device (Millipore). The extracellular vesicles in the filtrate are then pelleted by ultracentrifugation (Beckman Ti70 rotor) at 100 000 g for 90 min at IOC. For electron microscopic studies, the extracellular vesicles are additionally purified by ultracentrifugation in a 20 and 40% sucrose gradient and then washed with filtered phosphate-buffered saline (PBS). Alternative methods for isolating exosomes include isolation using size-exclusion chromatography as described in Hong et al. (Journal of Immunological Methods (2014), 411 :55-56), and isolation using field flow fractionation as described in Petersen, (Analytical and Bioanalytical Chemistry, (2014), 406(30)).

Example II: Purification of Cancer Cell Exosomes and Transfection of Dendritic Cells

[0027] Cancer cells at passage 1-15 are cultured in microvesicle-free medium

(DMEM containing 5% dFBS) and conditioned medium from 4x 10⁷ cells is collected after 48 h. Extracellular vesicles are purified by differential centrifugation. In brief, cancer cell- conditioned medium is centrifuged for 10 min at 100g to eliminate cell contamination. Supernatants are further centrifuged for 20 min at 16,500g- and filtered through a 0.22 μπι filter. Extracellular vesicles are pelleted by ultracentrifugation at 110,000g- for 70 min. The extracellular vesicle pellets are washed in 13 ml PBS, pelleted again and resuspended in PBS. Extracellular vesicles are measured for their protein content using the DC protein assay (Bio- Rad). Serum extracellular vesicles from healthy controls and cancer patients are diluted to 13 ml in PBS and sterile-filtered before centrifugation.

[0028] Bone marrow-derived DCs are generated following the protocol described by

Lutz et al. Briefly, bone marrow cells are isolated from the hind limbs and treated with red blood cell lysis buffer. The cells are plated in a 10-cm bacteriological Petri dish (Falcon- Becton Dickinson, Erembodegem, Belgium) at 2x 10⁶ cells in 10 mL of complete medium (DMEM supplemented with 5% heat - inactivated FCS, 2 mM glutamine, 50 M 2-ME, 100 U/mL penicillin, 100 g/mL streptomycin, and 20 ng/mL rrao GM-CSF). On day 3 of culture, 10 mL of culture medium containing 20 ng/mL rmo GM-CSF is added. On day 5, 50% of the medium is refreshed with culture medium containing 20 ng/mL rmo GM-CSF. On day 7, cells are used for mRNA electroporation and according to the experimental set -up DCs are further matured with LPS derived from Escherichia coli serotype 055 :B5 (Sigma- Aldrich, Bornem, Belgium) at a concentration of 0.1 g/mL.

[0029] Immediately before transfection, dendritic cells are washed twice in PBS

(Invitrogen -Life Technologies ) and collected by centrifugation for 10 minutes at 1500 rpm. The cells are resuspended in Opti-MEM (Invitrogen -Life Technologies ) to a final concentration of 20 xlO⁶ cells /mL. Cells are transfected using one of the methods described above. After transfection, cells are immediately re-suspended in fresh complete medium and further incubated at 37C in a humidified atmosphere supplemented with 7% C02.

[0030] Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

CLAIMS We claim:

1. A method of generating a therapeutic vaccine for treating diseases, comprising:

delivering, ex vivo, isolated extracellular vesicle material obtained from diseased cells or tissues into isolated immune cells to generate primed immune cells, wherein the extracellular vesicle material is delivered to the immune cells by shear induced loading, shear and squeeze induced loading, use of electronic ejector microarrays, sonoporation, magnetoporation, photothermal poration, jet injection, use of massively parallelized nanoneedles, filtroporation, carbonate apatite nano-complexe mediated delivery, or a combination thereof; and

preparing a composition comprising said primed immune cells to form a therapeutic vaccine

2. The method of claim 1, wherein the extracellular vesicle material is delivered to the isolated immune cells by shear and squeeze induced loading.

3. The method of claim any one of claims 1 to 2, wherein the isolated immune cell is an antigen presenting cell.

4. The method of any one of claims 1 to 2 where the isolated immune cell is a monocyte or other cell derived from a monoctye that can be matured into a dendritic cell or antigen presenting cell.

5. The method of claim 3, wherein the antigen presenting cell is a dendritic cell.

6. The method of any one of claims 1 to 5, wherein the extracellular vesicle material comprises intact exosomes, intact microvesicles, an exosome lysate, a microvesicle lysate, or a combination thereof.

7. The method of claim 6, wherein extracellular vesicle material comprises one or more RNAs isolated from extracellular vesicles, one or more DNAs isolated from extracellular vesicles, one or more proteins isolated from the extracellular vesicles, or a combination thereof.

8. The method of claim 7, wherein the one or more RNA molecules and/or the one or more DNA molecules are amplified prior to delivery to the isolated immune cells.

9. The method of any one of claims 1 to 8, wherein the extracellular material is derived from diseased cells or tissue of a subject to which the vaccine composition is to be administered.

10. The method of any one of claims 1 to 8, wherein the extracellular material is derived from non-autologous diseased cells or tissues.

11. The method of claims 9 or 10, wherein the diseased cells or tissues are cancer cells or tissues, auto-immune disease cells or tissues, infected cells or tissues, or metabolic disease cells and tissues.

12. The method of claim 11, wherein the extracellular vesicle material is a tumor exosome or tumor exosome lysate.

13. The method of any one of claims 1 to 12, wherein the immune cells are isolated from a subject to which the vaccine composition is to be administered.

14. The method of any one of claims 1 to 12, wherein the immune cells are isolated from a non-autologous source.

15. The method of any one of claims 1 to 12, further comprising administering the composition comprising the primed immune cells to a subject in need thereof.