Compositions and methods of tumor vaccines based on gamma-delta T cell extracellular vesicles

Abstract

Gamma delta-T-extracellular vesicles (EV) have been found to have an immunologic adjuvant effect that promotes DC maturation, which effect is mediated by IFN-gamma associated with gamma delta-T-EV. Cell-free compositions comprising extracellular vesicles (EV) derived from gamma delta-T cells (gamma delta-T-EV) and methods of using the same are provided. The gamma delta-T-EV may bind to or encapsulate a tumor associated antigen (TAA) or a library of peptides to form a gamma delta-T-EV vaccine composition. The gamma delta-T-EV composition may be used as an adjuvant in cancer immunotherapy, or the gamma delta-T-EV vaccine composition may induce a tumor-specific T cell response and directly kill tumor cells in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 485,734, filed February 17, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The disclosed invention is generally in the field of cancer vaccines, and in particular in the field of personalized anti-cancer vaccines derived from gamma-delta T cells. Background Art

[0004] Tumor vaccines play a key role in cancer therapy because they can amplify antigen-specific anti-tumor responses. Dendritic cell (DC)-based tumor vaccines have been extensively studied; however, their clinical application is hampered by the high cost and difficulty in preparing personalized DCs. In addition, the immunosuppressive tumor microenvironment can weaken the anti-tumor efficacy of cell-based vaccines.

[0005] Vaccines based on cell-free nanoparticles (NPs) have also attracted attention for their potential in cancer therapy. NPs protect vaccine components from degradation, improving stability, prolonging bioavailability, and enhancing antigen uptake by antigen-presenting cells (APCs). NP size influences the uptake and specificity of NP-based vaccines. For example, smaller NPs (20-200 nm) can flow directly into lymph nodes and be readily taken up by resident DCs. Furthermore, smaller NPs are more capable of promoting DC maturation and inducing stronger antigen-specific immunity than larger NPs. Therefore, small NPs have great potential in vaccine development. Small extracellular vesicles (EVs; 20-200 nm), such as endosome-derived exosomes, shuttle lipids, proteins, and nucleic acids in intercellular communication, thereby influencing tumorigenesis and tumor treatment outcomes. EVs can be used to develop tumor vaccines. To date, tumor-derived EVs (TEVs) and DC-derived EVs (DC-EVs) have primarily been studied. TEVs are rich reservoirs of tumor-associated antigens (TAAs) and can function as tumor vaccines when loaded with immune adjuvants. Administration of TEV-based vaccines induced a broad anti-tumor immune response and showed higher efficacy than tumor lysates. However, TEVs were also found to promote tumor development and metastasis, which may limit the clinical application of TEV-based vaccines due to safety issues. Compared with TEVs, immune cell-derived EVs have a better safety profile due to their natural immune surveillance properties. DC-EVs carry effective antigen presentation and co-stimulatory molecules. After loading with immunogens, DC-EV-based vaccines can directly induce antigen-specific immune responses or be taken up by recipient APCs to indirectly promote anti-tumor immunity. However, the therapeutic efficacy of DC-EV-based tumor vaccines has been unsatisfactory in clinical trials. The heterogeneity of ex vivo expanded DCs may partly explain the poor treatment outcomes, as EVs derived from immature DCs (iDCs) have immune tolerance activity. In addition, the difficulty of large-scale ex vivo expansion of human DCs may also limit the preparation of DC-EVs for clinical use. Therefore, it is worth exploring other novel and more easily accessible EV-based vaccines.

[0006] Human γδ-T cells are a minor T cell population that acts as innate-like T cells and has direct antitumor activity against many different types of tumors. γδ-T cells also function as antigen-presenting cells (APCs) to induce antigen-specific T cell responses. More interestingly, γδ-T cells have an immune adjuvant effect to induce DC maturation and synergize CD4 T follicular helper cells to indirectly promote specific cellular and humoral immune responses. γδ-T cell-derived EVs (γδ-T-EVs) exhibit dual antitumor activity by carrying cytotoxic and immunostimulatory molecules, which can expand pre-existing tumor-specific T cells. However, whether γδ-T-EVs can be designed as tumor vaccines to further enhance antitumor immunity remains unknown.

[0007] Any discussion of documents, minutes, materials, apparatus, articles of manufacture or the like included in this specification should not be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0008] Throughout this specification, the word "comprise", or variations "comprises" or "comprising", will be understood to imply the inclusion of a stated element, elements or steps, or group of elements, elements or steps, but not the exclusion of any other element, element, step, or group of elements, elements or steps. SUMMARY OF THE INVENTION

[0010] It has been found that γδ-T-extracellular vesicles (EVs) have an immune adjuvant effect that promotes DC maturation, an effect mediated by IFN-γ associated with γδ-T-EVs. Cell-free compositions and methods of use thereof are provided, the cell-free compositions comprising, as "active agents," isolated extracellular vesicles (EVs) derived from γδ-T cells (γδ-T-EVs). In some forms, the EVs are small EVs.

[0011] γδ-T-EV can be part of a heterogeneous mixture factor (e.g., conditioned medium) or a fraction separated therefrom. In other embodiments, γδ-T-EV is separated from the conditioned medium or collected in other ways. Before administration to a subject, γδ-T-EV can be suspended in a pharmaceutically acceptable composition such as a carrier or matrix or reservoir. In some embodiments, γδ-T-EV can be combined with tumor-associated antigens (TAAs) or peptide libraries or encapsulate them to form a γδ-T-EV vaccine composition.

[0012] Also described are methods for using γδ-T-EV or γδ-T-EV vaccine compositions to induce tumor-specific T cell responses and directly kill tumor cells. The method comprises administering a composition of γδ-T-EV alone (as an adjuvant) or in combination with a tumor-associated antigen (TAA) or peptide library (as a vaccine) to a subject in need thereof. In one embodiment, the γδ-T-EV is allogeneic. In another embodiment, the γδ-T-EV is autologous. The γδ-T-EV or γδ-T-EV vaccine composition effectively increases the T cell response in the subject to which it is administered. The vaccine regimen effectively induces apoptosis of tumor cells. Vaccines based on allogeneic γδ-T-EVs show advantages over DC-EV-based vaccines through dual anti-tumor activity.

[0013] Additional advantages of the disclosed methods and compositions will be set forth in part in the description that follows and in part will be apparent from the description or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0015] Figures 1A-1G These results indicate that γδ-T-EV-based vaccines effectively induce tumor-specific T cell responses. Figure 1A is the size distribution plot; Figure 1B The expression of EV markers CD63 and CD81 is shown, and the gray histogram represents the isotype control; Figure 1C The expression of Alix, EBNA1, and LMP2a proteins in γδ-T-EV and γδ-T-EV(TAA) is shown; Figure 1D -1I is a graph showing equivalent amounts of EBNA1 peptide, TAA, γδ-T-EV, γδ-T-EV + free antigen, or γδ-T-EV-based vaccine were used to treat huPBMCs; seven days later, cells were restimulated with peptide pools and intracellular IFN-γ in T cells was measured: Figures 1D-1E is a graph showing the induction of EBNA1-specific CD4 and CD8 T cells by γδ-T-EV (EN1) and corresponding controls; Figures 1F-1G Figure 1 shows the induction of EBNA1- and LMP2a-specific CD4 and CD8 T cells by γδ-T-EVs (TAAs) and corresponding controls. Quantitative data are the mean ± SEM of four biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. EV: γδ-T-EV; EN1: EBNA1; EN1a: pooled EBNA1 peptides filtered in parallel during the preparation of the γδ-T-EVs-EN1 vaccine; TAAsa: ultracentrifugation pellet from equivalent unconditioned medium containing TAAs; HIV: cells treated with the γδ-T-EV-based vaccine but restimulated with the HIV p17 peptide pool.

[0016] Figures 2A-2N demonstrate that γδ-T-EV-based vaccines mobilize antigen-presenting cells to induce tumor-specific T cell responses. Figures 2A-2B are images of intact huPBMCs or HLA-DR+ cell-depleted huPBMCs treated with γδ-T-EV-based vaccines for 7 days, followed by detection of EBNA1 and LMP2a-specific T cells. Figures 2C-2D Purified CD3 T cells were treated with γδ-T-EVs or γδ-T-EVs (TAA) in the absence or presence of iDCs. TAA treatment was also used as a control (Ctr). After 7 days, LMP2a-specific T cells were detected. (Figures 2E-2F) iDCs were treated with γδ-T-EVs or γδ-T-EV-based vaccines for 48 h. TAA was used as a control (Ctr). Maturation markers (MHC-II, CD83, CD86, and CD40) were detected by flow cytometry. (Figures 2G-2H) TNF-α and IL-6 secretion from iDCs after 48 hours of treatment with γδ-T-EV or γδ-T-EV(TAA). (Figures 2I-2J) iDCs were treated with γδ-T-EV or γδ-T-EV(TAA) for 48 hours and then cocultured with CFSE-stained allogeneic CD3 T cells. After 7 days, proliferating T cells were measured by CFSE dilution using flow cytometry. (Figure 2J) IFN-γ secretion by pretreated iDCs in a coculture system with allogeneic CD3 T cells. (Figure 2K) IFN-γ expression on γδ-T-EV or γδ-T-EV(TAA). (Figures 2M-2N) iDCs were treated with γδ-T-EV or γδ-T-EV(TAA) in the presence of a neutralizing anti-IFN-γ antibody or an isotype control. After 48 hours, pretreated iDCs were cocultured with CFSE-stained allogeneic CD3 T cells. Seven days later, proliferating T cells were measured. Quantitative data are the mean ± SEM of four biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

[0017] Figures 3A-3M show that the γδ-T-EV-based vaccine retained direct antitumor activity both in vitro and in vivo. (Figure 3A) Apoptosis of EBV-LCL and (Figure 3B) SNU-719 cells was measured after 18-24 h of incubation with γδ-T-EV or the γδ-T-EV-based vaccine. Equivalent amounts of PBS, EBNA1, or TAA were used as controls. (Figure 3C) EBV-LCL cells were injected subcutaneously into Rag2 - / - γc - / -14 days later, mice bearing subcutaneous tumors were treated with γδ-T-EV or γδ-T-EV (TAA) at the indicated time points (n = 5). TAA served as a control (Ctr). Tumor volume (Figure 3D) and mouse survival (Figure 3E) were measured at the indicated times. (Figure 3F) Representative histochemical analysis of human Ki-67 in tumor tissue at endpoint, scale bar = 25 μm. (Figure 3G) Mice bearing subcutaneous SNU-719 tumors were treated with γδ-T-EV or γδ-T-EV (TAA) as indicated (n = 7). TAA served as a control (Ctr). Tumor volume (Figure 3H) and mouse survival (Figure 3I) were measured at the indicated time points. (Figure 3J) Representative histochemical analysis of human Ki-67 in tumor tissue at endpoint, scale bar = 25 μm. (Figure 3K) Apoptosis of C666-1 NPC cells after incubation with γδ-T-EV or a γδ-T-EV-based vaccine. (Fig. 3L-3M) Tumor volume of NPC tumors after treatment with the γδ-T-EV-based vaccine. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant.

[0018] Figures 4A-4H show that a γδ-T-EV-based vaccine inhibits tumor progression in humanized mice. (Figure 4A) A tumor model was established by subcutaneous injection of autologous EBV-LCL cells in humanized mice. γδ-T-EV or a γδ-T-EV-based vaccine was then injected into humanized mice (n = 7) as indicated. TAA was used as a control (Ctr). Tumor volume (Figure 4B) and mouse survival (Figure 4C) were measured at the indicated times. (Figure 4D) Representative histochemical analysis of human Ki-67 in tumor tissue at endpoint, scale bar = 25 μm. (Figures 4E-4F) γδ-T-EV or a γδ-T-EV-based vaccine was injected into humanized mice bearing EBV-induced B-cell lymphomas, with two doses administered every two weeks. Seven days after the booster injection, the percentage of LMP2a- and EBNA1-specific T cells in peripheral blood nucleated cells was measured. (Figure 4G) EBV-LCL cells died after 6 h of co-culture with γδ-T-EV or autologous T cells induced by a γδ-T-EV-based vaccine. EBV-LCL cells alone were used as a control (Ctr). Figure 4H EBV-LCL cells preloaded with EBV peptides died after 6 hours of coculture with autologous T cells induced by the γδ-T-EV-based vaccine. EBV-LCL cells preloaded with HIV p17 peptides served as a control. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant.

[0019] Figures 5A-5H show that allogeneic γδ-T-EV-based vaccines also promote antigen-presenting cells to induce tumor-specific T cell responses. Expression of MHC-II (Figure 5A) and CD83 (Figure 5B) on iDCs after 48 h of treatment with autologous, allogeneic γδ-T-EV or γδ-T-EV (TAA). TAA was used as a control (Ctr). (Figures 5C-5D) IL-6 and TNF-α secreted from iDCs after treatment. (Figure 5E) iDCs were pretreated with autologous, allogeneic γδ-T-EV or γδ-T-EV (TAA) for 48 h and then co-cultured with CFSE-stained allogeneic CD3 T cells. After 7 days, proliferating T cells were measured by CFSE dilution using flow cytometry. (Figures 5F-5G) Autologous, allogeneic γδ-T-EV or γδ-T-EV (TAA) were used to treat huPBMCs. After 7 days, cells were restimulated with EBNA1 (Figure 5F) or LMP2a (Figure 5G) peptide pools, and intracellular IFN-γ in T cells was detected. Figure 5H ) intact huPBMC or HLA-DR + Cell-depleted huPBMCs were treated with autologous or allogeneic γδ-T-EVs (TAA) for 7 days, and then LMP2a-specific T cells were detected. *p<0.05, **p<0.01, ***p<0.001. ns: not significant.

[0020] Figures 6A-6H show that allogeneic γδ-T-EV-based vaccines inhibit tumor progression in humanized mice. Figure 6A is a schematic diagram showing how a tumor model is established by subcutaneous injection of autologous EBV-LCL in humanized mice. Autologous or allogeneic γδ-T-EV (TAA) is injected intraperitoneally into humanized mice at the specified time. PBS, TAA or γδ-T-EV is used as a control (n=8). Tumor volume Figures 6B and 6C are graphs of mouse survival calculated as shown. Figure 6D. Representative histochemical analysis of human Ki-67 in tumor tissue at the end point, scale bar = 25 μm. Figure 6E is a schematic diagram showing how a tumor model is established by injecting autologous EBV-LCL into humanized mice reconstituted with complete huPBMCs or CD3 T cell-depleted huPBMCs. TAA or allogeneic γδ-T-EV (TAA) is injected intraperitoneally into humanized mice (n=6) at the specified time. Figures 6F and 6G show tumor volume and mouse survival calculated as indicated. Figure 6H shows representative histochemical analysis of human Ki-67 in tumor tissue at endpoint. Scale bar = 25 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant; Auto-: autologous; Allo-: allogeneic.

[0021] Figures 7A-7E show that vaccines based on allogeneic γδ-T-EVs prevent tumor progression in humanized mice. Figure 7A is a schematic diagram showing how humanized mice were primed and boosted with autologous or allogeneic γδ-T-EV-based vaccines as shown, and then subcutaneously injected with EGFP-expressing EBV-LCL cells (n=8) one week later. Equivalent amounts of PBS, TAA, or γδ-T-EVs were used as controls. Figure 7B is a graph of tumor incidence; Figure 7C is a whole-body fluorescent image of mice 42 days after tumor cell inoculation. Figure 7D is a graph of tumor volume, and Figure 7E is a graph showing mouse survival rates determined as indicated. ***p<0.001. ns: not significant; Auto-: autologous; Allo-: allogeneic.

[0022] Figures 8A-8J show that vaccines based on allogeneic γδ-T-EV have advantages over vaccines based on DC-EV by exhibiting dual anti-tumor activity. (Figures 8A-8B) are graphs of TNF-α and IL-6 secreted from iDC after 48 h of treatment with allogeneic γδ-T-EV, DC-EV, γδ-T-EV (TAA) or DC-EV (TAA). TAA is used as a control (Ctr). Figure 8C is a graph of iDC treated with allogeneic γδ-T-EV, DC-EV, γδ-T-EV (TAA) or DC-EV (TAA) for 48 h, with TAA used as a control (Ctr). Then, pretreated iDCs were co-cultured with CFSE-stained allogeneic CD3 T cells. After 7 days, proliferated T cells were measured by CFSE dilution using flow cytometry. Figure 8D is a graph showing the secretion of IFN-γ in a co-culture system of pretreated iDCs and allogeneic CD3 T cells. Figure 8E is a diagram of an allogeneic γδ-T-EV or DC-EV-based vaccine for treating huPBMC. After 7 days, the cells were restimulated with the LMP2a peptide library, and intracellular IFN-γ in T cells was detected. Figure 8F is a diagram of apoptosis of EBV-LCL cells after 18-24 h of culture with an allogeneic γδ-T-EV or DC-EV-based vaccine. Figure 8G is a schematic diagram showing how to establish a tumor model by subcutaneous injection of EBV-LCL cells in humanized mice. Then, allogeneic γδ-T-EV or DC-EV-based vaccines were intraperitoneally injected into humanized mice (n=6 or 7) at the specified time. TAA was used as a control (Ctr). Figures 8H-8I are diagrams showing tumor volume (Figure 8H) and mouse survival rate (Figure 8I) calculated as shown. Figure 8J Representative histochemical analysis of human Ki-67 in tumor tissue at endpoint. Scale bars: *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant.

[0023] Figure 9AFigure 2 shows the size distribution of DC-EV and γδ-T-EV-based vaccines. The size distribution of γδ-T-EV, DC-EV, γδ-T-EV (TAA), or DC-EV (TAA) was measured using a ZetaView particle tracking analyzer. Data are from one of three independent experiments. Figure 9B Figure 2. IFN-γ expression on DC-EV and γδ-T-EV-based vaccines. IFN-γ expression on γδ-T-EV, DC-EV, γδ-T-EV(TAA), or DC-EV(TAA) was determined by flow cytometry. Gray histograms represent isotype controls. Data are from one of three independent experiments.

[0024] Figures 10A-10D Apoptosis of various tumor cells after treatment with γδ-T-EV-based vaccines or PBS (Ctr) is shown.

[0025] Figures 11A-11C The results show that γδ-T-EV-based vaccines induced antigen-specific T cells from human PBMCs after 7 days of pulsing with TAAs (melanoma, cervical cancer) or recombinant tumor antigens (liver cancer). TAAs were obtained from cell lysates of the corresponding cancer cell lines after freeze-thawing (e.g., EBV-associated B-cell lymphoma, melanoma, cervical cancer, and liver cancer). Detailed Description of the Invention

[0027] The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and the examples included therein and the accompanying drawings and their preceding and following descriptions.

[0028] It should be understood that unless otherwise indicated, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques or specific reagents and can vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0029] Disclosed are materials, compositions, and components that can be used with, in conjunction with, or in preparing the disclosed methods and compositions, or as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, and the like of these materials are disclosed, while specific reference to each different individual and collective combination and arrangement of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if examples of classes A, B, and C molecules and classes D, E, and F molecules, as well as combination AD, are disclosed, each is contemplated individually and collectively, even though each is not recited individually. Thus, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the example combination AD. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the subgroups of AE, BF, and CE are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the example combination AD. Furthermore, each material, composition, component, etc. contemplated and disclosed above also specifically and independently includes or excludes any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically contemplated and should be considered disclosed.

[0030] I. Definition

[0031] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined.

[0032] As used herein, the term "preventing" refers to administering a composition prior to the onset of clinical symptoms of a disease or condition in order to prevent abnormal physical manifestations associated with the disease or condition.

[0033] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, nurse practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that a subject requires or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's expertise, but includes the knowledge that the subject is or will become ill with a condition treatable by the compositions of the present invention.

[0034] As used herein, "subject" includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and any other organisms or entities. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cattle, cat, guinea pig, or rodent), fish, bird, or reptile or amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insect and crustacean). The term does not represent a specific age or gender. Therefore, it is intended to encompass adult and neonatal subjects and fetuses, whether male or female. Patient refers to a subject suffering from a disease or condition. The term "patient" includes both human and veterinary subjects.

[0035] "Treatment" and "treating" refer to the medical management of a subject with the goal of curing, improving, stabilizing or preventing a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed at improving a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directly directed at eliminating the cause of the relevant disease, pathological condition or disorder. In addition, the term includes palliative treatment, i.e., treatment that is intended to relieve symptoms rather than cure the disease, pathological condition or disorder; preventive treatment, i.e., treatment that is intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition or disorder; and supportive treatment, i.e., treatment that is used to supplement another specific therapy that is intended to improve the relevant disease, pathological condition or disorder. It should be understood that although a treatment is intended to cure, improve, stabilize or prevent a disease, pathological condition or disorder, it does not actually need to produce a cure, improvement, stabilization or prevention result. The effect of the treatment can be measured or evaluated as described herein and known in the art as appropriate for the disease, pathological condition or disorder involved. These measurements and evaluations can be performed in qualitative and / or quantitative form. Thus, for example, characteristics or features of a disease, pathological condition or disorder and / or symptoms of a disease, pathological condition or disorder can be reduced to any effect or to any amount.

[0036] The term "effective amount" of the compositions provided herein refers to an amount of the composition that is non-toxic but sufficient to provide the desired result. As will be noted below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, the specific composition / agent used, its mode of administration, etc. Therefore, it is not possible to specify a precise "effective amount." However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

[0037] "Pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, e.g., a material that can be administered to a subject with a selected composition / agent without causing undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is contained.

[0038] As used herein, the term "pharmaceutically acceptable carrier" encompasses any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (eg, such as oil / water or water / oil emulsions), and various types of wetting agents.

[0039] As used herein, the terms "purified," "isolated," and similar terms refer to a molecule / agent that is isolated in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components with which the molecule / agent is normally associated in its natural environment.

[0040] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0041] The use of the term "about" is intended to describe values ​​that are within a range of about + / - 10% above or below the stated value; in other forms, these values ​​may be within a range of about + / - 5% above or below the stated value; in other forms, these values ​​may be within a range of about + / - 2% above or below the stated value; in other forms, these values ​​may be within a range of about + / - 1% above or below the stated value. The foregoing ranges are intended to be clear from the context and do not imply further limitations.

[0042] II. Composition

[0043] A. γδ-T-EV Composition

[0044] Provided are cell-free compositions comprising extracellular vesicles (EVs) derived from γδ-T cells (γδ-T-EVs), alone or in combination with tumor antigens.

[0045] γδ-T-EV can be a part of a heterogeneous mixture factor (e.g., conditioned medium) or a fraction separated therefrom. In other embodiments, γδ-T-EV, or one or more subtypes thereof, is separated from the conditioned medium or collected in other ways. Before being administered to a subject, γδ-T-EV, or one or more subtypes thereof, can be suspended in a pharmaceutically acceptable composition such as a carrier or matrix or reservoir. In some embodiments, γδ-T-EV is bound to or encapsulated in a tumor-associated antigen (TAA) or peptide library. In some forms, TAA is provided by a cell lysate obtained after freeze-thaw treatment of the corresponding cancer cell line. For example, TAA can be provided by obtaining a tumor lysate of EBV-LCL cells via a freeze-thaw method known in the art (Hatfield, et al., J Immunother. 2008, 31(7):620-32). TAA can be provided by obtaining tumor lysates of cancer cells via freeze-thaw, such as colorectal cancer, RCC (renal cell carcinoma), prostate cancer, cervical cancer, melanoma, lung cancer, breast cancer, B, T leukemia, lymphoma, pancreatic cancer, myeloma, nasopharyngeal carcinoma, gastric cancer cells, etc. In some forms, TAA is provided as a TAA source by a lysate separated from the patient's own tumor. However, TAA can be provided as a TAA source by a lysate separated from a source other than the patient's own tumor. Whole tumor lysate (WTL) constitutes an effective alternative source of cancer antigens, which avoids the need for antigen identification and verification.

[0046] The composition comprising γδ-T-EVs is provided in an amount effective to function as an adjuvant, for example, as measured by its ability to promote dendritic cell (DC) maturation, as revealed by enhanced expression of maturation markers, secretion of proinflammatory cytokines, and antigen presentation capacity of DCs following administration of γδ-T-EVs, for example, as measured by upregulation of CD83 and MHC-II expression by at least 30%, at least 40%, or at least 50% with TAA treatment compared to no γδ-T-EV treatment.

[0047] Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed structures. EVs, primarily composed of microvesicles and exosomes, are released from virtually all cells into the extracellular environment via shedding from the plasma membrane or originating from the endosomal system. Exosomes are small, 40-150 nm, endosomally derived EVs (sEVs) that are released by cells into the extracellular environment. In 2018, the International Society for Extracellular Vesicles (ISEV) recognized EV as a generic term for any cell-derived particle that lacks a nucleus and is bounded by a lipid bilayer. One type of characterization parameter includes: (1) EV size—small EVs (100-200 nm), in some forms 20-200 nm, large EVs (200-1000 nm); (2) EV deposition or density—low, medium, or high; (3) marker expression—e.g., EVs expressing CD63, CD81, or annexin A1; (4) cell type—e.g., EVs derived from γδ-T cells; and (5) biogenesis—e.g., plasma membrane or endosome. (Reviewed in Li, et al., Front in Immunol., vol. 11, article 1525 (2020). EVs contain a cargo consisting of proteins, mRNAs, noncoding (nc)RNAs such as microRNAs and long nc-RNAs, DNA, and lipids. This cargo can be transferred to recipient cells, constituting an important form of physiological intercellular communication. EVs disclosed express the markers CD63 and CD81.

[0048] γδ-T-EV can be bound to one or more tumor antigens or encapsulate one or more tumor antigens. Tumor antigens as used herein include tumor-associated antigens (TAA) (relatively restricted to tumor cells) and tumor-specific antigens (TSA) (specific to tumor cells). As demonstrated in the Examples section of this application, before EV separation, after pulsing, γδ-T-EV is directly loaded with the tumor antigen EBNA1 (γδ-T-EV (EN1)) or indirectly loaded with TAA (γδ-T-EV (TAA)). In some forms, before EV separation, after pulsing, γδ-T-EV is directly loaded with the tumor antigen EBNA1 (γδ-T-EV (EN1)) or indirectly loaded with TAA (γδ-T-EV (TAA)). As used herein, "pulsing" means: treating / exposing γδ-T cells with TAA at a specified time point before EV separation. As used herein, "direct loading" involves loading antigens onto EVs by physical or chemical methods after separating EVs from cell culture supernatants. As used herein, "indirect loading" requires feeding cells with antigens. These antigens will be engulfed by γδ-T cells and subsequently encapsulated in EVs. The antigen-containing EVs are then secreted into the cell culture supernatant, which can be used for EV isolation.

[0049] In some forms, the TAA is provided by cell lysates obtained following freeze-thaw treatment of the corresponding cancer cell line.

[0050] Tumor antigens are proteins expressed on the surface of tumor cells or secreted into the bloodstream. Tumor antigens for most human cancers have been identified. There are two major categories of tumor antigens. Tumor-specific antigens (TSAs) are present only on cancer cells and not on healthy cells, while tumor-associated antigens (TAAs) are highly expressed on tumor cells but also expressed at lower levels on healthy cells. Examples of tumor antigens include, but are not limited to, EBV antigens such as Epstein-Barr nuclear antigen 1 (EBNA1) and latent membrane protein 2a (LMP2a), as shown in Table 12-1 of Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003, available from: https: / / www.ncbi.nlm.nih.gov / books / NBK12961 / .

[0051] Table 1: General categories and examples of tumor antigens

[0052] category Example antigen Cancer histology Carcinoembryonic CEA (carcinoembryonic antigen) Colorectal cancer immature laminin receptor RCC TAG-72 Prostate cancer Tumor viruses HPV E6, E7 Cervical cancer Overexpression / accumulation BING-4 Melanoma calcium-activated chloride channel 2 lung cancer <![CDATA[Cyclin-B1]]> many 9D7 RCC Ep-CAM Breast cancer EphA3 many Her2 / neu many Telomerase many Mesothelin pancreatic ductal carcinoma SAP-1 Colorectal cancer Survivin many Cancer - Testicle BAGE family many CAGE family many GAGE family many MAGE family many SAGE family many XAGE family many CT9, CT10 many NY-ESO-1 / LAGE-1 many PRAME many SSX-2 Melanoma, multiple Lineage restrictions Melan-A / MART-1 Melanoma Gp100 / pmel17 Melanoma Tyrosinase Melanoma TRP-1 / -2 Melanoma P. polypeptide Melanoma MC1R Melanoma Prostate-specific antigen prostate mutation β-catenin Melanoma, prostate, HCC BRCA1 / 2 Breast and ovarian cancer CDK4 many CML66 CML Fibronectin many MART-2 Melanoma p53 many Ras many TGF-βRII Colorectal cancer Post-translational changes MUC1 (Mucin short variant S1) Ductal carcinoma, RCC Unique type Ig, TCR B, T leukemia, lymphoma, myeloma

[0053] BRCA = breast cancer antigen; CDK4 = cyclin-dependent kinase 4; CEA = carcinoembryonic antigen; CML66 = chronic myeloid leukemia (antigen) 66; CT = testicular cancer; HPV = human papillomavirus; Ep-CAM = epithelial cell adhesion molecule; Ig = immunoglobulin; MART-1 / -2 = melanoma antigen recognized by T cells-1 / 2; MC1R = melanocortin-1 receptor; SAP-1 = gastric cancer-associated protein tyrosine phosphatase-1; TAG-72 = tumor antigen-72; TCR = T-cell receptor; TGF-βRII = transforming growth factor-β receptor II; TRP = tyrosinase-related protein.

[0054] Other examples of tumor antigens include alpha-fetoprotein (AFP), epithelial tumor antigen (ETA), and mucin-16 (MUC-16), also known as ovarian cancer-associated tumor markers CA125 (cancer antigen 125); MAGE (melanoma-associated antigen); glypican-3 (GPC3); SOX2; WT1 (Wilms tumor 1); HMGA2 (high mobility group AT-Hook 2); HSP90 (heat shock protein 90); PSMA (prostate-specific membrane antigen); CA19-9 (carbohydrate antigen 19-9) and MUC16 (CA125). The identification and characterization of 9D7 are disclosed in Klade, et al., doi.org / 10.1002 / ijc.1582.

[0055] In some forms, γδ-T-EVs do not bind to or encapsulate microRNAs such as miR138.

[0056] B. Pharmaceutical Compositions

[0057] Also provided are pharmaceutical compositions comprising EVs. The pharmaceutical compositions can be administered parenterally (intramuscularly (IM), intraperitoneally (IP), intravenously (IV), subcutaneously (SubQ), subcutaneously), transdermally (passively or using iontophoresis or electroporation), or by any other suitable means, and can be formulated into dosage forms suitable for each route of administration.

[0058] The vesicles described herein can be conveniently formulated into pharmaceutical compositions together with a pharmaceutically acceptable carrier. See, for example, Remington's Pharmaceutical Sciences, latest edition, by EW Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods for preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the active agents described herein (i.e., γδ-T-extracellular vesicles). These are most typically standard carriers for administering compositions to humans. In one aspect, humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0059] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, which may also contain buffers, diluents and other suitable additives. Examples of non-aqueous solutions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose) etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents and inert gases etc.

[0060] For example, parenteral administration can include intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intravitreal, intratumoral, intramuscular, subcutaneous, subconjunctival, intracapsular, intrapericardial, intraumbilical, by injection, and by infusion into a patient.

[0061] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, such as solutions or suspensions; solid forms suitable for preparing solutions or suspensions after addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsions.

[0062] The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and / or by using a surfactant. In many cases, it is preferred to include an isotonic agent, for example, a sugar or sodium chloride.

[0063] Solutions and dispersions of the active agents disclosed herein (γδ-T-extracellular vesicles) as a free acid or base or a pharmacologically acceptable salt thereof can be prepared in water or another solvent or dispersion medium, suitably mixed with one or more pharmaceutically acceptable excipients, including but not limited to surfactants, dispersants, emulsifiers, pH adjusters, viscosity modifiers, and combinations thereof.

[0064] Suitable surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include but are not limited to those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long-chain alkyl sulfonates and alkylaryl sulfonates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as bis-(2-ethylthio)-sodium sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include but are not limited to quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethyl benzyl ammonium chloride, polyethylene oxide and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4 oleate, sorbitan esters, sucrose esters, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene laurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-lauryl-β-alanine, sodium N-lauryl-β-iminodipropionate, sodium myristoylamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0065] The formulation may contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent.

[0066] The formulation is typically buffered to a pH of 3-8 for parenteral administration after reconstitution. Suitable buffers include, but are not limited to, phosphate buffer, acetate buffer, and citrate buffer.

[0067] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinyl pyrrolidone, dextran, carboxymethyl cellulose, and polyethylene glycol.

[0068] Sterile injectable solutions can be prepared by mixing the desired amount of active ingredient with one or more of the excipients listed above (as needed) into a suitable solvent or dispersion medium, and then filter sterilizing. Typically, dispersions are prepared by mixing a variety of sterilized active ingredients into a sterile vehicle containing an alkaline dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile-filtered solution. Powders can be prepared in a porous manner in nature, which can increase the dissolution of the particles. Methods for preparing porous particles are known in the art. Parenteral formulations as described herein can be formulated for controlled release, including immediate release, delayed release, extended release, pulsed release, and combinations thereof.

[0069] For parenteral administration, the active agent and optionally one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the active agent and / or one or more additional active agents. In embodiments where the formulation contains two or more drugs, the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsed), or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and pulsed, etc.).

[0070] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0071] Compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, pouches or tablets. Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups and lozenges. Tablets can be prepared using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can be prepared as hard or soft capsule shells that can encapsulate liquid, solid and semi-solid filler materials using techniques well known in the art. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or adhesives can be desired.

[0072] The formulations can be prepared using pharmaceutically acceptable carriers. As generally used herein, "carriers" include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0073] The carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizers, and glidants.

[0074] Examples of suitable coating materials include, but are not limited to, cellulosic polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; vinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic acid resins sold under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0075] Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can be formulated for controlled release. For example, the active agent (γδ-T-extracellular vesicles) and optionally one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in soft or hard gelatin or non-gelatin capsules or dispersed in a dispersion medium to form an oral suspension or syrup. The particles can be formed from the drug and a controlled-release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled-release coatings before being incorporated into the finished dosage form.

[0076] Preparation can be in the form of suspension or emulsion. Generally, there is provided a pharmaceutical composition, including one or more activating agents of an effective dose, the activating agent optionally including a pharmaceutically acceptable diluent, preservative, solubilizing agent, emulsifying agent, adjuvant and / or carrier. These compositions can include diluent, sterile water, a plurality of buffer contents (for example, Tris-HCl, acetate, phosphate) under multiple pH and ionic strengths; and optionally, additives such as detergents and solubilizing agents (for example, TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), antioxidants (for example, ascorbic acid, sodium metabisulfite) and preservatives (for example, thimerosal, benzyl alcohol) and filler materials (for example, lactose, mannitol). The example of a non-aqueous solvent or vehicle is propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate. Preparation can be lyophilized and redissolved / resuspended immediately before use. The formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0077] "Diluents," also called "fillers," are often required to increase the bulk of a solid dosage form to provide a practical size for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, and powdered sugar.

[0078] "Binders" are used to impart cohesiveness to solid dosage forms, thereby ensuring that the tablets, beads, or granules remain intact after the dosage form is formed. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as gum arabic, tragacanth, sodium alginate, celluloses (including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, and veegum), and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinyl pyrrolidone.

[0079] "Lubricants" are used to facilitate tablet preparation. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0080] "Disintegrants" are used to facilitate the disintegration or "breakup" of the dosage form after administration and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginic acid, gums, or cross-linked polymers such as cross-linked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0081] "Stabilizers" are used to inhibit or delay drug decomposition reactions, including, for example, oxidation reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; vitamin E, tocopherol and its salts; sulfites such as sodium metabisulfite; cysteine ​​and its derivatives; citric acid; propyl gallate and butylated hydroxyanisole (BHA).

[0082] Immune adjuvants stimulate the immune system's response to the target antigen, but do not provide immunity themselves. Adjuvants can present antigens to the immune system in various ways. Adjuvants can act as a reservoir for antigens, presenting antigens over a longer period of time, thereby maximizing the immune response before the body clears the antigen. An example of a reservoir adjuvant is an oil emulsion. Adjuvants can also act as stimulants, which participate in and enhance the body's immune response.

[0083] The adjuvant can be, but is not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of Q. saponaria, such as Quil A (a mixture of over 25 different saponin molecules), or subcombinations or individual molecules thereof, such as QS21 (a glycolipid that elutes at peak 21 by HPLC fractionation; Antigenics, Inc., Worcester, MA); poly[bis(carboxyphenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulatory complexes containing mixed saponins, lipids, and forming virus-sized particles with pores that can accommodate antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham Adjuvant System #4, which contains alum and MPL; SBB, Belgium), micelle-forming nonionic block copolymers such as CRL 1005 (which contains linear chains of hydrophobic polyoxypropylene pendant with polyoxyethylene chains, Vaxcel, Inc., Norcross, Ga) and Montanide IMS (e.g., IMS 1312, water-based nanoparticles combined with a soluble immunostimulant, Seppic).

[0084] Adjuvant can be TLR part, such as those discussed above. The adjuvant worked by TLR3 includes but is not limited to double-stranded RNA. The adjuvant worked by TLR4 includes but is not limited to derivatives of lipopolysaccharide, such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide associated with lipid A; OM Pharma SA, Meyrin, Switzerland). The adjuvant worked by TLR5 includes but is not limited to flagellin. The adjuvant worked by TLR7 and / or TLR8 includes single-stranded RNA, oligoribonucleotides (ORN), synthetic low molecular weight compounds such as imidazoquinoline amine (for example, imiquimod (R-837), resiquimod (R-848)). The adjuvant worked by TLR9 includes DNA from viral or bacterial origin, or synthetic oligodeoxynucleotides (ODN), such as CpG ODN. Another class of adjuvants are phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids containing phosphorothioate backbone linkages.

[0085] The adjuvant can also be an oil emulsion (e.g., Freund's adjuvant); a saponin preparation; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0086] Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

[0087] The immunostimulatory complexes known as ISCOMs are particulate antigen delivery systems comprising an antigen, cholesterol, a phospholipid, and a saponin (Quil A or other saponin) with strong immunostimulatory activity. ISCOMATRIX® is a particulate adjuvant containing cholesterol, a phospholipid, and a saponin (Quil A) but without an antigen. See, for example, U.S. Patent No. 9,149,520. This adjuvant has a structure substantially identical to that of ISCOM, consisting of perforated cage-like particles with a diameter of approximately 40 nm. Antigens can be formulated with ISCOMATRIX® to produce vaccines with antigen presentation and immunostimulatory capabilities, which are similar to ISCOM-type formulations but have broader applicability because their use is not limited to hydrophobic membrane proteins. Modifications of ISCOM formulations and ISCOMATRIX® have also been developed to achieve better binding of some antigens, as described in WO 98 / 36772.

[0088] ISCOM and ISCOMATRIX® combine the advantages of a particulate delivery system with the in situ presence of an adjuvant (Quil A) and have therefore been found to be more immunogenic than other colloidal systems such as liposomes and protein micelles. ISCOM and ISCOMATRIX® formulations retain the adjuvant activity of Quil A while increasing its stability, reducing its hemolytic activity, and producing less toxicity. They also generate an immune response similar to that obtained by immunization with a simple mixture of antigen and saponin, but allow the use of significantly smaller amounts of antigen. Several ISCOM-type vaccine formulations or vaccine formulations containing ISCOMATRIX® have been approved for veterinary use, for example against equine influenza virus.

[0089] Other liposome systems composed primarily of saponins from Q. saponaria and sterols (primarily cholesterol) have been described, one of which is called ASO1B. See, for example, WO 96 / 33739, which is also formulated as an emulsion as described in US 2005 / 0220814. See also US Published Application No. 2011 / 0206758.

[0090] Iscomatrix-like adjuvants, such as ISCOMATRIX®, are thought to act via classical inflammasome activation and subsequent release of proinflammatory cytokines such as IL-18 and IL-1β (Wilson, et al., Journal of Immunology. 2014;192(7):3259-68. doi: 10.4049 / jimmunol.1302011. PubMed PMID: 24610009). This mechanism is thought to be mediated, at least in part, by endosomal degradation and NRLP3 activation, which releases cathepsins into the cytosol.

[0091] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may include penetration enhancers. Chemical enhancers and physical methods including electroporation and microneedles can be used in conjunction with this method. Typically, penetration enhancers are selected so that they do not disrupt and / or eliminate the biological activity of EVs.

[0092] C. Methods for Preparing γδ-T-EVs

[0093] Methods for isolating EVs derived from γδ-T cells (γδ-T-EVs) are described in Wang, X., et al., Sci Transl Med, 2020. 12: p. eaaz3426; Wang, X., et al., J Immunother Cancer, 2022. 10(2): e003832 (2022) and CN 108103026. Compared with the method in CN 108103026, the improved method herein increases the yield, and its protocol includes amplifying γδ-T cells using 5 μM zoledronate and 1000 IU / ml IL-2, which only produces γδ-T cells with a purity of approximately 60% after amplification. Before preparing γδ-T-EVs, γδ-T cells need to be sorted to obtain high purity (CN 108103026, Figure 1B). However, the cell sorting procedure is time-consuming and reduces cell viability, potentially affecting the production and function of γδ-T-EVs. In contrast, the improved protocol disclosed herein uses pamidronate (about 9 μg / ml) IL-2 (about 200 IU / ml IL-2) and includes restimulating γδ-T cells three days after the initial activation; this results in a high purity of γδ-T cells (>95%). This expansion protocol allows the expanded γδ-T cells to be directly subjected to the preparation of γδ-T-EVs without the need for prior cell sorting. This approach is quite convenient in practice and better preserves the viability of γδ-T cells and the function of γδ-T-EVs.

[0094] γδ-T-EVs are bound to or encapsulated with tumor-associated antigens (TAAs) (here γδ-T-EVs (TAAs) or peptide libraries) to form γδ-T-EV vaccine compositions. CN 108103026 discloses γδ-T-EVs carrying miR-138, whereby γδ-T cells are transfected with lentiviral (miR-138) vectors. However, due to the innate-like nature of γδ-T cells with intrinsic antiviral capabilities, transfection with exogenous viruses can be challenging and may reduce the cell viability of γδ-T cells. In addition, the use of vectors derived from retroviruses in the preparation of γδ-T-EVs may introduce additional biological activity hazards. In addition, only a single anti-tumor agent (miR-138) is loaded into its γδ-T-EV product using a lentiviral vector.

[0095] The disclosed method prepares γδ-T-EVs (TAAs) by directly exposing γδ-T cells to TAAs. Leveraging their natural properties as antigen-presenting cells, γδ-T cells can phagocytose TAAs and can be restimulated for further activation and expansion. γδ-T cells then secrete TAAs into γδ-T-EVs (TAAs), which contain multiple tumor antigens and can induce a broad anti-tumor immune response.

[0096] In some embodiments, the vesicles are isolated from primary cells that were isolated from the subject to be treated. However, the cells can also be isolated from untreated subjects.

[0097] Extracellular vesicles can be isolated using differential centrifugation, flotation density gradient centrifugation, filtration, high performance liquid chromatography, and immunoaffinity capture. Ultrafiltration and high performance liquid chromatography (HPLC) are additional methods for separating EVs based on their size differences. EVs prepared by HPLC are highly purified.

[0098] I. How to use

[0099] Also provided are methods of using the disclosed compositions. In some embodiments, the method comprises contacting a cell or administering to a subject in need thereof an effective amount of a composition comprising an extracellular vesicle.

[0100] The frequency of application of the method of treatment can be, for example, every day, every week, every two weeks or monthly once, twice, three times, four times or more times. In some embodiments, the composition is applied once to the subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. In some embodiments, the frequency of application is once a week, twice or three times, or once, twice or three times every two weeks, or once, twice or three times every four weeks. In some embodiments, the composition is applied 1-3 times, preferably 2 times, to the subject weekly.

[0101] In one aspect, the compositions described herein can be administered to a subject, including humans or animals, including but not limited to mice, dogs, cats, horses, cows, sheep, etc., who is in need of alleviation or improvement of a recognized medical condition.

[0102] Vesicles and pharmaceutical compositions described herein can be applied to subjects in a variety of ways, depending on whether local or systemic treatment is needed, and the area to be treated. Thus, for example, pharmaceutical compositions described herein can be applied to the surface of the eye as ophthalmic solutions and / or ointments. In addition, pharmaceutical compositions can be administered to subjects vaginally, rectally, intranasally, orally, by inhalation or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal, and intratracheal routes. Parenteral administration (if used) is typically characterized by injection. Injections can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms or emulsions suitable for dissolving or suspending in liquids before injection. Recently revised methods for parenteral administration involve the use of slow-release or sustained-release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795.

[0103] In some embodiments, the composition is administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective to deliver the composition to target cells.

[0104] In a preferred embodiment, the composition is administered locally, for example, by direct injection into or adjacent to the site to be treated. Typically, local injection results in an increased local concentration of the composition that is greater than that achieved by systemic administration.

[0105] The dose or amount of the active agent described herein is large enough to produce the desired effect in the method in which delivery occurs. The dose should not be so large as to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Generally, the dose will vary with the age, condition, sex, and degree of disease of the subject and can be determined by one skilled in the art. The dose can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, dosing regimen, and route of administration can vary.

[0106] Exemplary dosages for in vivo methods are discussed in the experiments below. As further research proceeds, information on appropriate dosage levels for treating various conditions in various patients will emerge, and taking into account the recipient's treatment background, age, and general health, a person of ordinary skill will be able to determine the appropriate dosage. The selected dosage depends on the desired therapeutic effect, route of administration, and desired duration of treatment.

[0107] The disclosed methods of use include co-administering one or more chemotherapeutic agents. Examples include, but are not limited to, alkylating agents such as altretamine; bendamustine; busulfan; carboplatin; chlorambucil; cisplatin; cyclophosphamide; dacarbazine; ifosfamide; mechlorethamine; melphalan; oxaliplatin; temozolomide; thiotepa; trabectedin; carmustine; lomustine; streptozocin. Antimetabolites include 5-fluorouracil; 6-mercaptopurine; azacitidine; capecitabine; cladribine; clofarabine; cytarabine; decitabine; floxuridine; fludarabine; gemcitabine; hydroxyurea; methotrexate; nelarabine; pemetrexed; pentostatin; pralatrexate; thioguanine; and trifluridine / tipiracil combination. Topoisomerase inhibitors include etoposide, irinotecan, irinotecan liposomal, mitoxantrone, teniposide, and topotecan. Mitotic inhibitors include cabazitaxel, docetaxel, albumin-bound paclitaxel, paclitaxel, vinblastine, vincristine, and vinorelbine. Antitumor antibiotics include daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, bleomycin, actinomycin D, and mitomycin.

[0108] The disclosed compositions and methods can be understood by reference to the following non-limiting examples. Example

[0109] Example 1: Development of a γδ-T cell extracellular vesicle-based tumor vaccine with dual anti-tumor activity

[0110] method

[0111] Cell culture

[0112] EBV-LCL cell lines were established using huPBMCs isolated from healthy donors provided by the Hong Kong Red Cross as previously described (Xiang, et al., Cancer Cell, 26(4): p.565-76 (2014)). Briefly, huPBMCs were treated with supernatant from the B95.8EbfaV-GFP or B95-8 cell lines containing EBV (Speck and Longnecker, ArchVirol, 144(6): p.1123-37 (1999)). (Generously provided by Diane Hayward, Johns Hopkins University, Baltimore) and cultured in 15% fetal bovine serum (FBS)-RPMI-1640 medium supplemented with cyclosporine A. The EBV-positive gastric cancer SNU-719 cell line (Korean Cell Line Bank) was cultured in 10% FBS-RPMI-1640 medium. In practice, γδ-T cells / DCs were cultured for EV production, and EBV-LCL cell lines were induced immediately after huPBMC isolation. The remaining huPBMCs and autologous transformed EBV-LCL cells were frozen and used to establish tumor-bearing humanized mouse models. All research protocols and animal work in this study were approved by the Institutional Review Board of the University of Hong Kong / Hospital Authority Hong Kong West Cluster and the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.

[0113] Preparation of γδ-T-EV and γδ-T-EV-based vaccines

[0114] γδ-T cells were expanded for EV isolation using a modified procedure from previous studies (Wang, et al., Sci Transl Med, 12: p. eaaz3426 (2020); Wang, et al., J Immunother Cancer, 10(2) (2022); Qin, et al., J Virol,. 85(19): p.10109-16 (2011)). Briefly, huPBMCs were isolated from buffy coats of healthy donors from the Hong Kong Red Cross. huPBMCs were activated with 9 μg / ml pamidronate on days 0 and 3 and cultured in 10% FBS-RPMI-1640 medium. Human recombinant interleukin-2 (IL-2; Invitrogen) was added at a final concentration of 200 IU / ml from day 3. After 14-20 days, γδ-T-EVs were prepared using a protocol modified from a previous study. γδ-T cells were cultured in EV-free medium supplemented with 200 IU / ml IL-2, from which FBS-derived EVs were previously removed by ultracentrifugation at 100,000 g for 18 h (SW32Ti rotor, Beckman). After 48 h, the conditioned medium was harvested for EV isolation.

[0115] To prepare γδ-T-EVs loaded with tumor-associated antigens (TAAs), γδ-T cells were pulsed with 500 μg / ml TAA (a tumor lysate of EBV-LCL cells obtained by freeze-thawing). After 24 h, the γδ-T cells were washed thoroughly with PBS and cultured in EV-free medium supplemented with 200 IU / ml IL-2. After 48 h, the conditioned medium was harvested and EVs were isolated. In the functional assay, an equivalent amount of TAA to that in the γδ-T-EV-based vaccine was used as a control after quantification and normalization of EBV antigens by Western blotting.

[0116] To prepare γδ-T-EVs (EN1), the EBNA1 peptide library was loaded directly onto EVsomes using the method described previously (Mu, et al., Cell Mol Immunol, (8): p. 944-956 (2022)). Briefly, γδ-T-EVs were mixed with a peptide library (Miltenyi) and an equal volume of acetate buffer (pH 5.2) at 4°C for 30 min. After acidification, the preparation was neutralized to pH 7.0 using 2 mol / L Tris buffer (pH 11) on ice for 45 min. Unbound peptides were removed after filtration through a 100-kDa cutoff filter (Pierce). The same amount of γδ-T-EVs or peptide library used for peptide loading was filtered in parallel, and the fraction above the filter was collected and used as a control.

[0117] Preparation of DC-EVs and DC-EV-based vaccines

[0118] To generate iDCs, human CD14+ cells were isolated from huPBMCs using magnetic beads (Miltenyi Biotec) and incubated for 6 days in 10% FBS-RPMI medium supplemented with 10 ng / ml IL-4 and 50 ng / ml GM-CSF (Peprotech) (Wu et al., BMC Infect Dis, 11: p. 201 (2011)). iDCs were pulsed with or without 500 μg / ml TAA for 10 hours. Then, 0.5 μg / ml lipopolysaccharide was added. After 14 hours, cells were washed extensively with PBS and cultured in 10% FBS-RPMI medium without EVs. After 48 hours, the conditioned medium was harvested for EV isolation.

[0119] EV isolation and characterization

[0120] EVs were isolated by differential centrifugation at 4°C as previously described (Wang et al., Sci Transl Med, 12: p. eaaz3426 (2020); Wang et al., J Immunother Cancer, 10(2) (2022)). Conditioned medium was centrifuged at 300 x g for 10 min, 2,000 x g for 10 min, and 10,000 x g for 30 min. The supernatant was then filtered using a 0.22-μm syringe filter and ultracentrifuged twice at 100,000 x g for 70 min (SW32Ti rotor, Beckman). EVs were solubilized in PBS. EV protein concentration was determined using a BCA protein assay kit (Pierce, Bonn). EV size distribution was determined by nanoparticle tracking analysis. For flow cytometric analysis, EVs were conjugated to 4-μM aldehyde / sulfate latex beads overnight (Thery, et al., Curr Protoc Cell Biol,. Chapter 3: p. Unit 322 (2006)) and incubated with glycine to block remaining binding sites. EV-loaded beads were stained with antibodies and corresponding isotype controls. For western blot analysis, proteins from cell lysates or EVs were obtained by lysis in radioimmunoprecipitation assay buffer in the presence of a protease inhibitor cocktail (Thermo Fisher Scientific) and separated by SDS-electrophoresis on 8% to 12% gels. Subsequently, the proteins were transferred to a membrane and blocked with 5% skim milk. The membrane was incubated overnight with anti-Alix (Abcam), anti-EBNA1 (Santa Cruz), or anti-LMP2a antibodies (Abcam). After incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody, the chemiluminescent signal was detected with Immobilon Classico Western HRP substrate (Millipore).

[0121] Induction of tumor antigen-specific T cells in vitro

[0122] Intact huPBMCs from EBV-seropositive donors or huPBMCs depleted of HLA-DR+ cells using anti-HLA-DR microbeads (Miltenyi) were incubated with 20 μg / ml of EV preparations and cultured in RPMI-1640 medium supplemented with 10% human AB serum. Seven days later, cells were restimulated with 1 μg / ml of the corresponding or irrelevant peptide pool for 6 h, followed by the addition of 10 μg / ml brefeldin A (BFA, Sigma-Aldrich) 2 h later. Antigen-specific cells were detected by flow cytometry by staining for T cell markers and IFN-γ (Biolegend).

[0123] Determination of DC maturation and function

[0124] To determine the effects of EV-based vaccines on DC maturation and function, iDCs were cultured with 20 μg / ml EVs or EV-based vaccines. In some experiments, neutralizing anti-IFN-γ antibodies or isotype antibodies were added. After 48 h, cells were harvested to detect maturation markers (e.g., CD40, CD83, CD86, and MHC-II). Cell culture supernatants were also harvested to detect IL-6 and TNF-α secretion. Mixed lymphocyte reactions (MLRs) were performed to determine the functional activity of DCs as previously described (Leung, et al., Blood, 2013. 121(9): p. 1584-94). Briefly, allogeneic CD3 T cells were isolated from huPBMCs using a pan T cell isolation kit (Miltenyi Biotec) and stained with carboxyfluorescein succinimidyl ester (CFSE). iDCs pretreated with EV preparations were then co-cultured with allogeneic CD3 T cells at a ratio of 1:10. Five days later, T cell proliferation was measured by flow cytometry. Culture supernatants were also collected for MLR assays and assayed for IFN-γ secretion.

[0125] Apoptosis and proliferation assays

[0126] EBV-LCL or SNU-719 cells were treated with 30 μg / ml EVs or EV-based vaccines. After 18–24 h, apoptosis was assessed by Annexin V expression, and cell proliferation was assessed by Ki67 expression (Biolegend).

[0127] Cytotoxicity assay

[0128] As effector cells, T cells were negatively selected from huPBMCs pretreated with γδ-T-EVs or γδ-T-EV-based vaccines using a pan-T cell isolation kit (Miltenyi). T cell cytotoxic activity was measured against the following targets: (i) autologous EBV-LCL, and (ii) autologous EBV-LCL pulsed with 1 μg / ml EBNA1, LMP2a, or HIV p17 peptide pools. Effector and target cells were co-cultured at a ratio of 10:1. To distinguish the two cell populations in the assay, target cells were stained with CFSE before co-culture. Six hours later, cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Target cell death was measured as the percentage of PI+ cells within the CFSE-positive population.

[0129] Establishment and treatment of EBV-associated tumors in Rag2− / −γc− / − and humanized mice

[0130] Rag2- / -γc- / - mice were maintained in the laboratory animal facility of the University of Hong Kong. Four to six-week-old Rag2- / -γc- / - mice were used to construct humanized mice by reconstitution with EBV seropositive CD3 T cell-depleted huPBMCs or complete huPBMCs using previously described methods. After injection of huPBMCs, symptoms of graft-versus-host disease (e.g., weight loss, diarrhea) were monitored daily. Typically, weight loss occurs between days 1 and 10 and returns to normal 15-20 days after huPBMC injection. Four weeks after huPBMC reconstitution, mice are referred to as "humanized" mice because they have a functional human peripheral immune system (Tu, et al., J Exp Med, 2011. 208(7): p. 1511-22; Xiang, et al., Cancer Cell, 2014. 26(4): p. 565-76). EBV-associated tumor models were established by subcutaneously implanting SNU-719 or EBV-LCL cells in Rag2- / -γc- / - mice or humanized mice. For the humanized mouse model, EBV-LCL cells and huPBMCs were autologous. Without treatment, almost all mice injected with tumor cells developed tumors within two weeks. Mice with palpable tumors were randomized for treatment experiments. Mice were age- and sex-matched, and no significant differences in tumor volume were found between groups before treatment. Mice were intraperitoneally injected with autologous, allogeneic EVs, or EV-based vaccines, as indicated. Tumor volume, tumor incidence, and animal survival were monitored and calculated at the indicated times. Tumor volume was measured by digital caliper and calculated as length x [width] 2 x 0.52. Mice bearing subcutaneous tumors reaching a diameter of 17 mm were euthanized according to the University of Hong Kong protocol. Tumor tissue was harvested for immunohistochemical analysis.

[0131] Preventive effect of γδ-T-EV-based vaccines against EBV-associated tumors in humanized mice

[0132] Humanized mice were established as described above. Mice were vaccinated with γδ-T-EV or γδ-T-EV(TAA). Two weeks later, animals were boosted with the same formulation. Seven days after the booster vaccination, EBV-LCL cells (1x10 per mouse) were added. 6 The mice were inoculated with the 100 mg / kg sera (100 mg / kg) of WT mice. Tumor incidence, tumor volume, and mouse survival were calculated at the designated time points. Tumor progression was also verified using an IVIS Spectrum in vivo imaging system (Caliper Life Sciences).

[0133] In vitro detection of tumor-specific T cells

[0134] Humanized mice bearing EBV-induced B-cell lymphomas were injected (ip) twice every two weeks with 50 μg / mouse of γδ-T-EV or γδ-T-EV (TAA). An equivalent amount of TAA was also used as a control. Seven days after the booster injection, peripheral blood nucleated cells were isolated and restimulated with peptides to detect EBNA1- or LMP2a-specific T cells.

[0135] Histological and immunohistochemical analysis

[0136] Tumor tissues were fixed with 10% formalin and embedded in paraffin for sectioning. Sections were subjected to immunohistochemical staining. Ki67 was detected by immunohistochemistry using an anti-human Ki67 antibody (Abcam, UK) and visualized using a diaminobenzidine detection kit (Maixin, China).

[0137] Flow cytometric analysis

[0138] The following antibodies were used for surface staining of cells: anti-CD63 (H5C6), anti-CD3 (HIT3a), anti-CD4 (RPA-T4), anti-CD8 (SK1), anti-MHC-II (Tü39), anti-CD83 (HB15e), anti-CD86 (GL-1), and anti-CD40 (5C3). For intracellular staining, cells were fixed, permeabilized, and then stained with anti-IFN-γ (B27) or isotype control as previously described (Ni, et al., Am J Respir Cell Mol Biol,. 58(6): p. 684-695 (2018)). All samples were detected using a FACSL SSR II flow cytometer (BD, USA) and analyzed using FlowJo software (Tree Star, USA).

[0139] Cytokine secretion assay

[0140] The cytokine concentrations in the culture supernatant were detected and analyzed using the LEGENDplex Human Cytokine Detection Kit (BioLegend).

[0141] statistics

[0142] Quantitative data are expressed as mean ± SEM. Comparisons between groups were performed by Mann-Whitney U test. One-way analysis of variance (ANOVA) with Bonferroni correction was used to compare multiple groups. Two-way ANOVA analysis was used to determine the effects of multiple variables and corresponding interactions. Animal survival and tumor incidence were analyzed via the Kaplan-Meier log-rank test. Statistical significance was calculated using a two-tailed test, and P < 0.05 was considered significant. The specific sample size, P value, and other details of each test are described in the text or figure legends.

[0143] result

[0144] γδ-T-EV-based vaccines effectively induce tumor-specific T cells

[0145] EVs were isolated from γδ-T cells derived from different donors by differential centrifugation. Typically, 4.1±0.3 μg of γδ-T-EV protein was obtained from 106 cells (N=4). To prepare the vaccine regimen, γδ-T-EVs were directly loaded with the tumor antigen EBNA1 (γδ-T-EV(EN1)) or indirectly loaded with TAA (γδ-T-EV(TAA)) after pulsing γδ-T cells before EV isolation (Damo, et al., Sci Rep, 2015. 5: p. 17622; Bu, et al., J Mol Neurosci, 2015. 56(3): 631-43).

[0146] Characterization analysis showed that the antigen loading procedure did not alter the size distribution and expression of EV markers on the γδ-T-EV-based vaccine ( Figures 1A-1B In addition, γδ-T-EV (TAA) contains complete tumor antigens, including EBV antigens such as Epstein-Barr nuclear antigen 1 (EBNA1) and latent membrane protein 2a (LMP2a) ( Figure 1C ), which is a key therapeutic target for EBV-associated tumors (Cui, and Snapper, Front Immunol, 2021. 12: p. 734471). These data suggest that TAAs can be loaded onto γδ-T-EVs using an indirect antigen loading strategy.

[0147] The results showed that both γδ-T-EV (EN1) induced more antigen-specific CD4 and CD8 T cells compared with the other groups. Removal of free peptide did not enhance the antigen-specific T cell response, indicating that free peptide can be effectively removed by the filtration procedure after loading peptide on γδ-T-EV ( Figures 1D-1E ). In addition, preloading of γδ-T-EV with tumor peptides significantly enhanced the efficacy of inducing tumor-specific T cell responses compared with free peptides without preloading. In addition, γδ-T-EV (TAA) induced more EBNA1- and LMP2a-specific CD4 and CD8 T cells compared with γδ-T-EV, TAA, or γδ-T-EV + free TAA ( Figures 1F-1G Cells treated with the γδ-T-EV-based vaccine did not respond to an irrelevant HIV p17 peptide, confirming the antigenic specificity of these T cell responses ( Figures 1D-1G Together, these data suggest that γδ-T-EVs can be engineered to be tumorigenic.

[0148] γδ-T-EV vaccine enhances tumor-specific T cell immunity

[0149] γδ-T-EVs exhibit immune adjuvant effects on antigen-presenting cells

[0150] To determine whether recipient APCs would affect the efficacy of γδ-T-EV-based vaccines, anti-HLA-DR microbeads were used to deplete APCs in huPBMCs as previously described (Walker, et al., J Immunol, 2009. 182(3): p. 1548-59). As shown in Figures 2A-2B, HLA-DR+ cell-depleted huPBMCs had significantly fewer antigen-specific T cells induced by the γδ-T-EV-based vaccine than intact huPBMCs, indicating that the efficacy of the γδ-T-EV-based vaccine is dependent on recipient APCs. Therefore, hypothetically, the γδ-T-EV-based vaccine can mobilize APCs to induce antigen-specific T cell responses.

[0151] To confirm this hypothesis, purified CD3+ T cells were treated with the preparation in the absence or presence of immature DCs (iDCs). γδ-T-EV(TAA) co-cultured with iDCs increased the efficacy of γδ-T-EV(TAA) in inducing tumor-specific T cell responses (Figure 2C). Although γδ-T-EV+free TAA also showed a trend of enhanced tumor-specific T cells in the presence of iDCs, its efficacy was significantly lower than that of γδ-T-EVs pre-loaded with TAAs (γδ-T-EV(TAA)) (Figure 2C). Therefore, co-delivery of antigens and γδ-T-EVs to recipient iDCs is important for promoting tumor-specific T cell responses. In summary, these findings demonstrate that vaccines based on γδ-T-EVs can induce tumor-specific T cells through recipient APCs.

[0152] To elucidate the effects of γδ-T-EV on APCs, iDCs were cultured with TAA, γδ-T-EV, or γδ-T-EV (TAA) for 48 h. Compared with TAA, both γδ-T-EV and γδ-T-EV (TAA) promoted the expression of DC maturation markers, including MHC-II, CD83, CD86, and CD40 (Figures 2D-2G). In addition, after treatment with γδ-T-EV or γδ-T-EV (TAA), the secretion of proinflammatory cytokines IL-6 and TNF-α from iDCs was significantly increased (Figures 2H-2I). In addition, iDCs pretreated with γδ-T-EV or γδ-T-EV (TAA) had higher antigen presenting capacity, as revealed by promoting greater proliferation of allogeneic CD3+ T cells (Figure 2J) and IFN-γ secretion (Figure 2K) in mixed lymphocyte reaction (MLR) assays. These data suggest that γδ-T-EVs promote iDC maturation and enhance their antigen-presenting capacity, as demonstrated by dendrite induction from iDCs by γδ-T-EVs or γδ-T-EV-based vaccines. Furthermore, the effects of γδ-T-EVs (TAAs) on iDCs were comparable to those of γδ-T-EVs (Figure 2L), indicating that the antigen-loading procedure does not hinder the adjuvant effect of γδ-T-EVs (TAAs).

[0153] The potential mechanism of the adjuvant effect of γδ-T-EV on DCs was further determined. Similar to their parental cells, γδ-T-EVs express potent IFN-γ, a well-known cytokine that promotes DC maturation

[29] . Blocking IFN-γ with neutralizing antibodies inhibited the antigen presentation ability of γδ-T-EV-enhanced iDCs (Figure 2L), indicating that the carried IFN-γ mediates the immune adjuvant effect of γδ-T-EVs. In summary, these results suggest that vaccines based on γδ-T-EVs can promote tumor-specific T cell responses by delivering antigens and acting as immune adjuvants on recipient APCs.

[0154] γδ-T-EV-based vaccines retain direct antitumor activity both in vitro and in vivo

[0155] Subsequent studies determined whether vaccines based on γδ-T-EVs inherited this direct anti-tumor property. As shown in Figures 3A and 3B, both γδ-T-EVs and γδ-T-EVs (TAAs) induced more tumor cell apoptosis compared to TAAs. There was no significant difference between γδ-T-EVs or γδ-T-EVs (TAAs) (Figures 3A-3B).

[0156] To determine the direct anti-tumor effect of the γδ-T-EV-based vaccine in vivo, Rag2- / -γc- / - mice bearing EBV-induced B-cell lymphoma were treated with γδ-T-EV or γδ-T-EV(TAA) (Figure 3C). When compared with TAA, mice treated with γδ-T-EV or γδ-T-EV(TAA) effectively inhibited tumor growth and prolonged mouse survival (Figures 3D-3E). No significant difference was observed in the anti-tumor effect between γδ-T-EV and γδ-T-EV(TAA) (Figures 3D-3E). It was found that tumor cells in mice treated with γδ-T-EV or γδ-T-EV(TAA) had lower proliferation capacity, as immunophenotyping analysis found fewer Ki-67-positive cells in the residual tumors.

[0157] Other tumor models based on EBV-associated gastric cancer or nasopharyngeal carcinoma were also used to verify the direct anti-tumor activity of the γδ-T-EV-based vaccine in vivo (Figure 3F). Similarly, after treatment with γδ-T-EV or γδ-T-EV(TAA), tumor-bearing mice were found to have smaller tumor volume and longer survival (Figures 3G-3H). Consistently, no significant differences were found between γδ-T-EV or γδ-T-EV(TAA) (Figures 3G-3H). It was found that tumor cells in mice treated with γδ-T-EV or γδ-T-EV(TAA) had lower proliferation capacity, as fewer Ki-67-positive cells were found in the residual tumors by histological analysis. The γδ-T-EV-based vaccine also identified consistent anti-tumor effects on NPC tumors (Figures 3I-3K). In summary, the data indicate that the γδ-T-EV-based vaccine retains its direct anti-tumor activity both in vitro and in vivo.

[0158] γδ-T-EV-based vaccine inhibits tumor progression in humanized mice

[0159] Humanized mice carrying functional human immune cells (e.g., B cells, T cells, and antigen-presenting cells) are particularly suitable for preclinical modeling of vaccine therapies. To determine the therapeutic efficacy of γδ-T-EV-based vaccines in humanized mice, a tumor model was constructed by subcutaneous injection of autologous EBV-LCL in humanized mice as described above. As shown, mice with developing subcutaneous tumors were treated with γδ-T-EV or γδ-T-EV-based vaccines (Figure 4A). Compared with the control group, γδ-T-EV or γδ-T-EV-based vaccines inhibited tumor growth and prolonged mouse survival (Figures 4B-4C). It was also found that tumor cells in mice treated with γδ-T-EV or γδ-T-EV-based vaccines had lower proliferation capacity, as fewer Ki-67-positive cells were identified in the corresponding residual tumors by histological analysis. In addition, unlike the similar therapeutic effects in immunodeficient mice (Figures 3C-3H), the γδ-T-EV-based vaccine had higher therapeutic efficacy than γδ-T-EV in humanized mice (Figures 4B-4C). Interestingly, γδ-T-EV(TAA) had higher therapeutic efficacy than γδ-T-EV(EN1) (Figures 4B-4C), suggesting that the tumor-specific T cell responses induced by γδ-T-EV(TAA) may be more effective than those induced by γδ-T-EV(EN1), thereby clarifying the role of tumor cells in tumorigenesis.

[0160] Consistent with the results of in vitro studies, both γδ-T-EV(EN1) and γδ-T-EV(TAA) induced tumor-specific T cell responses in humanized mice. However, γδ-T-EV(TAA) induced antigen-specific T cell responses against multiple tumor proteins (e.g., EBNA1, LMP2a), while γδ-T-EV(EN1) only targeted the EBNA1 antigen (Figures 4D-4E). In addition, the data showed that tumor-specific T cells induced by γδ-T-EV(TAA) had higher cytotoxic effects against autologous EBV-LCL than tumor-specific T cells induced by γδ-T-EV(EN1). In addition, preloading EBNA1 or LMP2a on autologous EBV-LCL, rather than HIV p17 peptide, resulted in higher cytotoxic activity (Figures 4F-4G). These data indicate that γδ-T-EV-based vaccines carrying tumor lysates can induce antigen-specific T cells against multiple tumor targets and exhibit higher therapeutic efficacy against EBV-associated tumors. Therefore, γδ-T-EV (TAA) was further investigated in the following studies.

[0161] Allogeneic γδ-T-EV-based vaccines mobilize recipient APCs to induce tumor-specific T cell responses

[0162] Since many cancer patients are immunocompromised due to cancer or anti-cancer therapy, it is difficult to obtain enough patient γδ-T cells and expand them ex vivo to prepare autologous γδ-T-EVs in clinical practice. In addition, it is much easier to collect γδ-T cells and prepare γδ-T-EVs from allogeneic healthy donors, and has advantages in standardization and centralization. Therefore, additional studies further determined the anti-tumor potential of vaccines based on allogeneic γδ-T-EVs. As shown in Figures 5A-5B, autologous and allogeneic γδ-T-EVs or γδ-T-EVs (TAA) promoted iDC maturation, as revealed by the upregulation of CD83 and MHC-II compared with TAA treatment. The secretion of proinflammatory cytokines (Figures 5C-5D) and the antigen presentation ability of iDCs were also comparable after treatment with autologous and allogeneic γδ-T-EVs or γδ-T-EV-based vaccines. These data indicate that allogeneic

[0163] Vaccines based on γδ-T-EVs have similar immune adjuvant effects as their autologous counterparts.

[0164] We further compared the efficacy of autologous and allogeneic γδ-T-EV-based vaccines in inducing tumor-specific T cells. Compared with γδ-T-EV, both autologous and allogeneic γδ-T-EV (TAA) induced more tumor-specific T cells, with comparable levels of tumor-specific T cells (Figures 5F-5G). Similarly, depletion of HLA-DR+ APCs from huPBMCs prevented the induction of tumor-specific T cells by both autologous and allogeneic γδ-T-EV (TAA) ( Figure 5H ), indicating that recipient APCs are essential for the function of both autologous and allogeneic γδ-T-EV-based vaccines. Taken together, these results indicate that allogeneic γδ-T-EV-based vaccines are comparable to their autologous counterparts in mobilizing recipient APCs and inducing tumor-specific T cell responses.

[0165] Allogeneic γδ-T-EV-based vaccine inhibits tumor progression in humanized mice

[0166] The anti-tumor efficacy of vaccines based on allogeneic and autologous γδ-T-EV in humanized mice was further compared. As shown in Figure 6A, humanized mice carrying EBV-induced B cell lymphoma were treated with allogeneic or autologous γδ-T-EV (TAA). PBS, TAA or γδ-T-EV were used as controls. Mice treated with γδ-T-EV had smaller tumor growth and longer mouse survival than mice treated with PBS or TAA. After loading tumor antigens, autologous or allogeneic γδ-T-EV (TAA) showed better anti-tumor efficacy than γδ-T-EV (Figures 6B-6C). In addition, allogeneic γδ-T-EV (TAA) was not inferior to autologous γδ-T-EV (TAA) in tumor control (Figures 6B-6C). Tumor cells in mice treated with autologous or allogeneic γδ-T-EV (TAA) were also found to have lower proliferation capacity, as fewer Ki-67 positive cells were found in the corresponding residual tumors. Considering the advantages of allogeneic EVs in clinical applications, these data suggest the strong translational potential of allogeneic γδ-T-EV-based vaccines in cancer therapy.

[0167] To evaluate whether human T cells are involved in the anti-tumor activity of allogeneic γδ-T-EV-based vaccines, humanized mice reconstituted with intact huPBMCs or CD3 T cell-depleted huPBMCs were used for tumor establishment and treatment (Figure 6D). Consistently, treatment of intact huPBMC-reconstituted humanized mice with allogeneic γδ-T-EVs (TAA) significantly inhibited tumor growth and prolonged mouse survival compared to TAA (Figures 6E-6F). However, in humanized mice reconstituted with CD3 T cell-depleted huPBMCs, therapeutic efficacy was reduced. In addition, after treatment with allogeneic γδ-T-EVs (TAA), tumor cells in mice reconstituted with CD3 T cell-depleted huPBMCs had increased proliferative capacity than tumor cells in mice reconstituted with intact huPBMCs, as more Ki-67-positive cells were found in the corresponding residual tumors. Therefore, these results indicate that allogeneic γδ-T-EV-based vaccines control tumor progression in humanized mice.

[0168] Allogeneic γδ-T-EV-based vaccine prevents tumor progression in humanized mice

[0169] To determine whether vaccines based on allogeneic γδ-T-EV can prevent tumor development, humanized mice were intraperitoneally injected with PBS, TAA, allogeneic γδ-T-EV, or γδ-T-EV (TAA) on days 21 and 7 before tumor cell inoculation. Autologous γδ-T-EV (TAA) was also used as a control (Figure 7A). One week after the booster injection, mice were challenged with autologous EBV-LCL cells (Figure 7A). Rapid tumor development was observed in all mice immunized with PBS, TAA, or γδ-T-EV. However, only 25% and 12.5% ​​of mice developed tumors after immunization with allogeneic and autologous γδ-T-EV (TAA), respectively (Figure 7B). In vivo imaging analysis was also used to verify tumor development in each group 6 weeks after tumor cell inoculation. In addition, immunization with allogeneic or autologous γδ-T-EV (TAA) effectively inhibited tumor growth (Figure 7C) and prolonged mouse survival (Figure 7D). There was no significant difference in the prophylactic efficacy of allogeneic and autologous γδ-T-EVs (TAAs) (Figures 7B-7D). Therefore, our data suggest that allogeneic γδ-T-EV-based vaccines have a prophylactic effect against tumor development.

[0170] Allogeneic γδ-T-EV-based vaccines demonstrate superiority over DC-EV-based vaccines through dual antitumor activity

[0171] Because allogeneic DC-EVs also have adjuvant effects and can be used as nanocarriers in tumor vaccines, we compared the efficacy of allogeneic γδ-T-EVs with DC-EV-based vaccines. Both vaccines were prepared by pulsing parental cells with TAAs before EV isolation. Figure 9A Figure 2 is a graph of the size distribution of DC-EV and γδ-T-EV-based vaccines. Size distribution of γδ-T-EV, DC-EV, γδ-T-EV(TAA), or DC-EV(TAA) measured by ZetaView particle tracking analyzer. Data shown are from one of three independent experiments. Vaccines based on γδ-T-EV and DC-EV have similar size distribution characteristics, with a peak at ~160 nm ( Figure 9B). In addition, γδ-T-EV and γδ-T-EV(TAA) carried robust IFN-γ, while DC-EV and DC-EV(TAA) did not. Both allogeneic DC-EV- and γδ-T-EV-based vaccines induced dendrites from iDC. However, allogeneic γδ-T-EVs and γδ-T-EV(TAA) induced more proinflammatory cytokines from iDC than DC-EV or DC-EV(TAA) (Figures 8A-8B). In MLR assays, iDCs pretreated with allogeneic γδ-T-EV and γδ-T-EV(TAA) also promoted more T cell proliferation and IFN-γ secretion than their DC-EV counterparts (Figures 8C-8D). These data indicate that allogeneic γδ-T-EV-based vaccines have a higher immune adjuvant effect than DC-EV-based vaccines.

[0172] The vaccine efficacy of allogeneic DC-EV and γδ-T-EV-based vaccines was further compared. TAA, allogeneic γδ-T-EV, γδ-T-EV (TAA), DC-EV, or DC-EV (TAA) was used to treat huPBMC. Compared with TAA or allogeneic EVs, allogeneic γδ-T-EV (TAA) and DC-EV (TAA) both induced more tumor-specific T cells (Figure 8E). However, allogeneic γδ-T-EV (TAA) induced tumor-specific T cells more effectively than allogeneic DC-EV (TAA) (Figure 8E). More importantly, γδ-T-EV and γδ-T-EV (TAA) can directly induce tumor cell apoptosis, while DC-EV or DC-EV (TAA) cannot (Figure 8F). These data suggest that γδ-T-EV-based vaccines have advantages over DC-EV-based vaccines by exhibiting dual anti-tumor activity.

[0173] The therapeutic efficacy of allogeneic γδ-T-EV-based and DC-EV-based vaccines in humanized mice was further compared. As shown in Figure 8G, humanized mice carrying EBV-induced B cell lymphoma were treated with allogeneic γδ-T-EV (TAA) or DC-EV (TAA), respectively. TAA or allogeneic EV was used as a control. Mice treated with allogeneic γδ-T-EV (TAA) or DC-EV (TAA) had smaller tumor size and longer mouse survival than TAA. However, allogeneic γδ-T-EV (TAA) exerted a higher therapeutic effect than allogeneic DC-EV (TAA) (Figures 8H-8I). The proliferation of tumor cells in residual tumors also supported these findings. Therefore, our results show that allogeneic γδ-T-EV-based vaccines show advantages over allogeneic DC-EV-based vaccines in inhibiting tumor progression in humanized mice. In addition to EBV-associated tumors, Figures 10A-10Dand 11A-11C show more examples of γδ-T-EVs loaded with other tumor antigens, such as lung cancer, liver cancer, melanoma, cervical cancer, and ovarian cancer, and indicate that γδ-T-EV-based vaccines can kill a variety of tumor cells and further induce tumor antigen-specific T cells against multiple tumors.

[0174] Summarize

[0175] This study demonstrates that γδ-T-EVs can serve as nanocarriers to deliver TAAs and function as immune adjuvants to induce antigen-specific T cell responses. Furthermore, γδ-T-EV-based vaccines retain direct antitumor activity. Administration of γδ-T-EV-based vaccines effectively controlled different tumors in vivo. Importantly, allogeneic γδ-T-EV-based vaccines also exhibited potent therapeutic effects and demonstrated superiority over allogeneic DC-EV-based vaccines by exerting dual antitumor activity. Therefore, the application of γδ-T-EV-based vaccines has excellent potential in cancer therapy.

[0176] As a bridge between the innate and adaptive immune systems, γδ-T cells play a key role in regulating APC function (Ismaili, et al., Clin Immunol, 2002.103(3 Pt 1): p. 296-302). Here, we demonstrate for the first time that γδ-T cells can promote APC maturation in a cell-free manner via EVs, which is mediated by IFN-γ carried by γδ-T-EVs. The adjuvant effect of γδ-T-EVs is sufficiently potent that no additional adjuvant is required for γδ-T-EV-based vaccines, the preparation methods of which are generally different from TEV-based or DC-EV-based vaccines (Naseri, et al., Oncoimmunology, 2020. 9(1): p. 1779991; Xia, et al., Biomed Pharmacother, 2022. 152: p. 113250). Therefore, the preparation of γδ-T-EV-based vaccines may be more convenient because no pre-loading of immune adjuvants is required. Furthermore, γδ-T-EV-based vaccines may have a better safety profile compared to TEV-based vaccines due to their properties in tumor surveillance, whereas TEVs can drive tumorigenesis (Whiteside, Adv Clin Chem, 2016. 74: p. 103-41).

[0177] In clinical practice, preparing personalized vaccines for cancer patients is very difficult because the process is time-consuming and labor-intensive. In addition, due to immune dysregulation caused by cancer or anti-cancer therapies (such as radiotherapy and chemotherapy), it is difficult to expand γδ-T cells of cancer patients in vitro to produce EVs. In contrast, it is much easier to prepare γδ-T cells from healthy donors on a large scale using protocols modified by us or other groups (Alexander, et al., Clin Cancer Res, 2008. 14(13): p. 4232-40; Bonneville and Scotet, Curr Opin Immunol, 2006. 18(5): p. 539-46; Kouakanou, et al., Cell Mol Immunol, 2020. 17(5): p. 462-473; Qin, et al., J Infect Dis, 2012. 205(11): p. 1646-53; Qin, et al., J Infect Dis, 2009. 200(6): p. 858-65; Xiang, et al., Cancer Cell, 2014. 26(4): p. 565-576; Peters, et al. al., Cell Mol Immunol, 2022. 19(10): p. 1198-1200), which ensures the production of γδ-T-EVs for clinical applications. The data showed that allogeneic γδ-T-EVs also have a strong immune adjuvant effect by inducing bystander activation of recipient APCs. When used as nanocarriers for tumor antigens, allogeneic γδ-T-EVs are comparable to their autologous counterparts in inducing tumor-specific immune responses and tumor control. More importantly, the allogeneic γδ-T-EV-based vaccine not only showed potent therapeutic anti-tumor effects but also effectively prevented tumor development. After two doses of immunization with the γδ-T-EV-based vaccine, most mice were protected from tumor development and survived longer. Therefore, the γδ-T-EV-based vaccine has a wide range of application scenarios in clinical practice for the prevention and treatment of tumors. Some previous studies have shown that vaccines based on allogeneic and syngeneic DCs have similar anti-tumor efficacy, which supports our findings. Considering the advantages of preparing γδ-T-EVs from healthy donors in terms of quality control, standardization, and centralization, allogeneic γδ-T-EV-based vaccines could be used as a “ready-to-use” approach in cancer therapy.

[0178] DC-EV-based vaccines have been extensively studied for cancer immunotherapy and have demonstrated promising therapeutic potential in preclinical models. However, their therapeutic efficacy has been limited in clinical trials (Besse, et al., Oncoimmunology, 2016. 5(4): p. e1071008; Escudier, et al., J Transl Med, 2005. 3(1): p. 10). The heterogeneity of ex vivo expanded DCs may partly explain the poor therapeutic outcomes of DC-EV-based vaccines, as EVs derived from immature DCs exert immunosuppressive activity. In contrast, homogeneous and immunostimulatory human γδ-T cells can be expanded on a large scale using phosphoantigens, which ensures the preparation of γδ-T-EV-based vaccines for clinical use. Similar to our findings, allogeneic DC-EVs were shown to have a significant adjuvant effect. Natural γδ-T-EVs have a higher adjuvant effect than DC-EVs and are more effective in inducing tumor-specific T cell responses after antigen loading. Furthermore, the γδ-T-EV-based vaccine retained direct antitumor activity, whereas the DC-EV-based vaccine was unable to directly kill tumor cells. Therefore, the γδ-T-EV-based vaccine has an advantage over the DC-EV-based vaccine through its dual antitumor activity.

[0179] In summary, this study provides strong preclinical proof-of-concept for the use of γδ-T-EV-based vaccines in cancer therapy. As a cell-free approach, the γδ-T-EV-based vaccine exhibits dual antitumor activity. Importantly, the allogeneic γδ-T-EV-based vaccine also exhibits robust antitumor effects. Therefore, this approach has promising translational potential.

[0180] It should be understood that the disclosed methods and compositions are not limited to the specific methods, protocols and reagents described, as these may vary. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0181] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0182] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", mean "including but not limited to", and are not intended to exclude, for example, other additives, components, elements or steps.

[0183] "Optional" or "optionally" means that the subsequently described event, circumstance, or material can or cannot occur or exist, and that the description includes instances where the event, circumstance, or material occurs or exists and instances where it does not.

[0184] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, ranges from one particular value and / or to another particular value are also specifically contemplated and considered disclosed, unless the context specifically indicates otherwise. Similarly, when values ​​are expressed as approximate values ​​by using the antecedent "about," it should be understood that the particular value forms another specifically contemplated embodiment that should be considered disclosed, unless the context specifically indicates otherwise. It should also be understood that the endpoints of each range are significant relative to the other endpoint and independent of the other endpoint, unless the context specifically indicates otherwise. It should be understood that all individual values ​​and subranges of values ​​contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed, unless the context specifically indicates otherwise. Finally, it should be understood that all ranges refer to both the recited range as a range and the range as a collection of individual numbers from a first endpoint to a second endpoint and including the first and second endpoints. In the latter case, it should be understood that any individual number can be selected as a form of the amount, value, or characteristic referred to by the range. In this manner, ranges describe a set of numbers or values ​​from a first endpoint to a second endpoint, including the first endpoint and the second endpoint, from which a single member of the set (i.e., a single number) can be selected as the amount, value, or characteristic to which the range refers. The foregoing applies regardless of whether some or all of the embodiments are explicitly disclosed in a particular instance.

[0185] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the disclosed methods and compositions belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the present disclosure, particularly useful methods, devices and materials are described below. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. It is not admitted that any reference constitutes prior art. The discussion of the references states the assertions of their authors, and the applicant reserves the right to question the accuracy and relevance of the cited documents. It should be clearly understood that although many publications are cited herein, such citation does not constitute an admission that any of these documents constitute a part of the common general knowledge in the art.

[0186] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. An immunogenic composition comprising extracellular vesicles (EVs) derived from γδ-T cells (γδ-T-EVs) in a pharmaceutically acceptable carrier.

2. The composition of claim 1, further comprising one or more tumor antigens (TA).

3. The composition of claim 1 or 2, wherein the one or more tumor antigens are selected from the group consisting of Epstein-Barr nuclear antigen 1 (EBNA1) and latent membrane protein 2a (LMP2a).

4. The composition of claim 1 or 2, wherein the tumor antigen is selected from the group consisting of: immature laminin receptor, BING-4, calcium-activated chloride channel 2, cyclin-B1, carcinoembryonic antigen; breast cancer antigen; cyclin-dependent kinase-4; chronic myeloid leukemia (antigen) 66; cancer-testis antigen; human papillomavirus E6 / E7 protein; epithelial cell adhesion molecule; immunoglobulin; T cell recognition melanoma antigen-1 / -2; melanocortin-1 receptor; gastric cancer-associated protein tyrosine phosphatase-1; tumor antigen-72; T cell receptor; transforming growth factor-β receptor II; tyrosinase-related protein, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, P. polypeptide (P. polypeptide), prostate-specific antigen, β-catenin, BRCA1 / 2, fibronectin; p53; Ras and mucin short variant S1.

5. The composition of claim 1 or 2, wherein the tumor antigen is selected from the group consisting of: alpha-fetoprotein (AFP); MAGE (melanoma-associated antigen); Glypican-3 (GPC3); SOX2; WT1 (Wilms tumor 1); HMGA2 (high mobility group AT-Hook 2); HSP90 (heat shock protein 90); PSMA (prostate-specific membrane antigen); CA125 (cancer antigen 125); and CA19-9 (carbohydrate antigen 19-9).

6. The composition of any one of claims 2-5, wherein the tumor antigen is obtained from a cancer cell lysate.

7. The composition of any one of claims 2-5, comprising a cancer cell lysate.

8. The composition of claim 7, wherein the cancer cells are selected from the group consisting of colorectal cancer, RCC (renal cell carcinoma), prostate cancer, cervical cancer, melanoma, lung cancer, breast cancer, and B, T leukemia, lymphoma, pancreatic cancer, myeloma, nasopharyngeal carcinoma, and gastric cancer cells.

9. The composition of any one of claims 1-8, further comprising an adjuvant.

10. The composition of any one of claims 1-9, prepared by contacting the γδ-T cells with a composition comprising TAAs prior to EV isolation, thereby indirectly loading the γδ-T cells with the one or more tumor antigens.

11. The composition of any one of claims 1-10, wherein the EV is a small EV.

12. The composition of claim 11, comprising Ev having a size of about 20-200 nm.

13. The composition of claim 11, comprising Ev having a size of about 100-200 nm.

14. The composition of any one of claims 1-13, in an effective amount to promote dendritic cell maturation as measured by upregulation of CD83 and MHC-II expression by at least 30%.

15. The composition of any one of claims 1-13, in an effective amount to promote dendritic cell maturation as measured by upregulation of CD83 and MHC-II expression by at least 50%.

16. A method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 1-15.

17. The method of claim 16, wherein the subject has been diagnosed with at least one type of cancer selected from the group consisting of colorectal cancer, RCC (renal cell carcinoma), prostate cancer, cervical cancer, melanoma, lung cancer, breast cancer, and B, T leukemia, lymphoma, pancreatic cancer, myeloma, nasopharyngeal carcinoma, and gastric cancer.

18. The method of claim 16 or 17, wherein the composition is administered intranasally.

19. The method of claim 16 or 17, wherein the composition is administered intravenously.

20. The method of any one of claims 16-19, comprising obtaining a tumor antigen from a cancer cell lysate.

21. The method of claim 20, wherein the cancer cells are selected from the group consisting of colorectal cancer, RCC (renal cell carcinoma), prostate cancer, cervical cancer, melanoma, lung cancer, breast cancer, and B, T leukemia, lymphoma, pancreatic cancer, myeloma, nasopharyngeal carcinoma, and gastric cancer cells.

22. The method of any one of claims 16-21, further comprising administering one or more chemotherapeutic agents to the subject.

23. The method of claim 22, wherein the chemotherapeutic agent is selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a mitotic inhibitor, and an antitumor antibiotic.

24. The method of claim 23, wherein the alkylating agent is selected from the group consisting of altretamine, bendamustine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, mechlorethamine, melphalan, oxaliplatin, procarbazine, temozolomide, thiotepa, trabectedin, carmustine, lomustine, and streptozocin.

25. The method of claim 23, wherein the antimetabolite is selected from the group consisting of 5-fluorouracil; 6-mercaptopurine; azacitidine; capecitabine; cladribine; clofarabine; cytarabine; decitabine; floxuridine; fludarabine; gemcitabine; hydroxyurea; methotrexate; nelarabine; pemetrexed; pentostatin; pralatrexate; thioguanine; and trifluridine / tipiracil combination.

26. The method of claim 23, wherein the topoisomerase inhibitor is selected from the group consisting of: etoposide; irinotecan; irinotecan liposomal; mitoxantrone, teniposide, and topotecan.

27. The method of claim 23, wherein the mitotic inhibitor is selected from the group consisting of: cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, vinorelbine.

28. The method of claim 23, wherein the antitumor antibiotic is selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, bleomycin, actinomycin D, and mitomycin C.

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