Cancer vaccine comprising exosomes obtained from or derived from activated and mature human b lymphocytes

Abstract

The present invention relates to compositions comprising one or more population of activated exosomes suitable for use as cancer vaccines. The activated exosomes in the first population each exhibit CD19 and one or more additional surface molecules that are characteristic of mature or activated B lymphocytes, and each comprise or exhibit one or more tumor antigens selected from the group consisting of MAGEA4, GAGE2D, and 5T4. Also provided are methods of using such compositions to prevent or treat cancer, and methods of producing such compositions.

Description

Technical Field

[0001] The present invention relates to a composition comprising one or more activated exosome populations, which is suitable for use as a cancer vaccine. The activated exosomes in the first population each display CD19 and one or more additional surface molecules that are characteristic of activated or mature human B lymphocytes, and each contain or display one or more tumor antigens selected from MAGEA4, GAGE2D and 5T4. Methods for preventing or treating cancer using such compositions are also provided, as well as methods for producing such compositions. Background Art

[0002] Exosomes are a subpopulation of extracellular vesicles (EVs). They are lipid bilayer membrane structures of small size of 30nm-150nm produced by almost every cell type (Théry, Zitvogel and Amigorena, 2002). The unique process of their biogenesis enables exosomes to contain molecules from the original parent cells and regulate the function and gene expression in the recipient cells (Valadi et al., 2007)(Zhou et al., 2016). They have been found to exist in biological fluids such as saliva, urine, breast milk and blood (Raposo and Stoorvogel, 2013), (Jakobsen et al., 2015) and they play an important role in physiological and pathological conditions by regulating biological processes between cells (Zaborowski et al., 2015).

[0003] Due to their natural ability to package and transport active biomolecules (such as proteins, lipids, and nucleic acids) from short to long distances in the human body, they are considered promising drug delivery systems (Doyle and Wang, 2019). One of the biggest advantages of exosomes is their ability to cross physiological barriers, including blood-lymphatic, blood-air, blood-brain, blood-cerebrospinal, blood-retinal, and blood-placental barriers; this overcomes the key obstacles found when using artificial nanoparticles as another type of delivery platform (Elliott and He, 2021).

[0004] To date, there are several therapeutic cancer vaccines under development based on different platforms such as peptides, DNA / mRNA, proteins, viruses, autologous patient-derived whole tumor vaccines, allogeneic whole cell vaccines, and dendritic cell-based vaccines (DeMaria and Bilusic, 2019)(Thomas and Prendergast, 2016). Therapeutic cancer vaccines are designed to target specific signaling pathways, growth factors, or specific antigens that are highly expressed in cancer cells but not expressed or expressed only in limited amounts in normal tissues (Tagliamonte et al., 2014; Loria et al., 2019). However, the development of cancer vaccines is very challenging due to poor immunogenicity (Mandelboim et al., 1995), transplant rejection (Aguirre et al., 2019), targeting of appropriate specific tumor antigens, and the high cost of the production process; all of which limit their application. For example, in the case of autologous cell vaccines, the biggest obstacle is related to the preparation of the appropriate amount of tumor sample required for treatment from the patient (Morton et al., 1992). Dendritic cell-based vaccines have aroused great expectations in the field of immunotherapy, but the results of clinical trials have not been convincing. In addition, the process of vaccine development is expensive and difficult to standardize; this leads to problems with the stability of the vaccine, that is, it loses its effectiveness during long-term storage (Muenst et al. 2016).

[0005] Currently, the FDA has approved only three non-exosome therapeutic cancer vaccines for clinical use in patients. These vaccines are a vaccine for metastatic castration-resistant prostate cancer (PROVENGE), a vaccine for metastatic melanoma (IMLYGIC), and a vaccine for patients with early-stage bladder cancer (TheraCys). Clinical trials have shown a slight improvement in overall survival only in patients with early-stage cancer (Gatti-Mays et al., 2017); however, there was no benefit for patients with advanced or metastatic disease (Dillman, 2017).

[0006] The new concept of cell-free, exosome-based immunotherapy has received much attention in cancer vaccine development. Compared with peptide-based or nucleic acid-based vaccine therapies, exosomes are stable in vivo, well tolerated by the human body, have few side effects, and have a long circulation half-life; this enables them to reach cells in distant tissues (Lai et al., 2013). Due to their phospholipid bilayer membrane, exosomes are able to fuse with cell membranes and deliver their cargo to recipient cells. Due to their biocompatibility, stability in the blood circulation system, small size, and specificity for targeted delivery, exosomes are very good candidates for immunological purposes and cancer vaccine applications (Temizoz, Kuroda, and Ishii, 2016)(Harari et al., 2020). In addition, it has been found that exosomes derived from certain immune cells may act as antigen presenting structures and stimulate anti-tumor immune responses (Raposo et al., 1996). Recently, the development of dendritic cell-derived exosome (DEX) vaccines as antigen delivery systems has attracted much attention. Compared to dendritic cells, DEX contain more MHC-I and MHC-II complexes, are more resistant to immunosuppressive mechanisms within tumors (Fu et al., 2020), and due to their unique lipid composition, they maintain higher stability for longer periods of time (Viaud et al., 2010). Preclinical in vitro and in vivo studies in mice have shown that DEX can activate CD4+ and CD8+ T cells, which can become memory T cells (Tkach et al., 2017).

[0007] However, a recent clinical trial using immature dendritic cell-derived exosomes loaded with HLA-restricted melanoma-associated antigen (MAGE3A) peptides in HLA A2+ non-small cell lung cancer patients showed only a mild specific T cell immune response during the phase I clinical trial, and no specific CD4+ and CD8+ T cell responses during the phase II clinical trial (Morse et al., 2005). In addition, another study in phase II clinical trials using a vaccine based on exosomes derived from mature dendritic cells and loaded with IFN-γ did not show any effect on cancer-specific T cell immune responses in patients with non-small cell lung cancer (Besse et al., 2016).

[0008] A nonrandomized phase I / II clinical trial using exosomes derived from nonactivated dendritic cells pulsed with the SIRT1 biomarker initially showed very promising results. The vaccine induced a specific immune response in cytotoxic T lymphocytes and achieved stability for up to 20 months in one patient (out of a total of seven patients who developed progressive metastatic disease and died within 10 months); however, this responding patient later developed lung metastases (Narita et al., 2015).

[0009] Additionally, a vaccine used in a phase I clinical trial for colorectal cancer, consisting of ascites-derived exosomes (AEX) mixed with granulocyte-macrophage colony-stimulating factor (GM-CSF), has shown a strong anticancer cytotoxic T-lymphocyte response (Dai et al., 2008); however, no follow-up results were obtained from this study.

[0010] The main reason why the success of exosome-based vaccine immunotherapy from the above clinical trials is so low is that it is difficult to identify and target specific tumor antigens associated with certain types of cancer. The above clinical studies did not show any convincing antigen-specific responses.

[0011] Therefore, more effective immunotherapies for the prevention and / or treatment of cancer are needed. Summary of the invention

[0012] The present invention aims to overcome one or more of the above problems / limitations by providing an exosome-based vaccine that contains a specific combination of tumor antigens to stimulate appropriate anti-cancer properties in effector T cells and also targets the adaptive immune system response.

[0013] The present invention combines two technologies: using in vitro activated exosomes derived from mature B lymphocytes as a delivery system; and bioengineering these exosomes with three selected broad-spectrum tumor antigens. These in vitro activated exosomes will have the same or similar capabilities as B lymphocytes, so they will be able to present tumor antigens in an unprocessed form on their exosome surface, and they will directly act as antigen presenting structures to elicit immune responses. In addition, bioengineering their exosome surfaces with three broad-spectrum tumor antigens will enhance their anti-cancer immune response properties, thereby being able to target various tumor cells. In addition, the fusion of bioengineered exosomes with dendritic cells will induce the processing of these tumor antigens so that they are further displayed on the surface of dendritic cells and expose these antigens to certain immune cells.

[0014] Vaccines based on the exosomes of the present invention will not only be able to stimulate and enhance appropriate direct anti-cancer immune responses in pre-formed tumors, but will also be able to train the adaptive immune system (which will recognize these tumor antigens and eliminate cancer cells expressing them immediately after these antigens appear). The use of exosomes from in vitro activated B lymphocytes will bypass several steps in the immune system activation process (e.g., processing antigens, activating B cells after contact with antigens, producing antigen presenting cells, etc.), and the selected tumor antigens will more effectively activate and train immune cells, which in turn will fight against a wide spectrum of tumor cells generated by various cancer tissues. The use of the anti-cancer vaccine of the present invention will target cells from solid tumors and liquid tumors, as well as cells expressing the above-mentioned tumor antigens from their metastases.

[0015] In some embodiments, tumor antigens are all full-length proteins. Most tumor antigens used in vaccines are peptides with short amino acid sequences. Using full-length proteins makes the vaccines of the present invention highly immunogenic because immune cells can recognize them with higher efficiency.

[0016] The present invention relates to the development of novel cell-free cancer vaccines based on in vitro activated exosomes, which are derived from activated and mature human B lymphocytes or activated and mature human peripheral blood mononuclear cells (PBMCs) (which also contain a large number of B lymphocytes). Both types of mature and in vitro activated exosomes (i.e., from B lymphocytes and PBMCs) can be bioengineered with a combination of tumor antigens (i.e., GAGE2D, MAGEA4, and the extracellular domain of 5T4 ex5T4), which will also increase the immunogenicity of the vaccine. The present invention also relates to in vitro activated exosomes from genetically engineered B lymphocytes or PBMCs expressing one or more tumor antigens (e.g., the extracellular domain of GAGE2D, MAGEA4, and 5T4). Since these tumor antigens are widely expressed by cancer cells in different types of tumor tissues, new cancer vaccines based on these antigens will be able to stimulate a wide range of anti-tumor responses in immune cells.

[0017] The exosome surface of in vitro activated B cells is bioengineered to display three selected tumor antigens, so that they can serve as antigen presenting structures for B lymphocytes, which can even recognize unprocessed antigens in natural form and produce adaptive memory. In addition, the additional in vitro activation of bioengineered exosomes will allow them to be easily taken up by other immune cells (such as dendritic cells or macrophages), which can process antigens and present antigens on their surface to naive T lymphocytes. The vaccine of the present invention can trigger the immune system through different mechanisms while inducing a high level of immune response. This can be achieved through the immunogenic properties of bioengineered activated exosomes, through the direct display of tumor antigens on their surface, and through the unique ability of exosomes to cross and internalize with the plasma membrane of receptor cells, where tumor antigens can be processed and displayed to the immune system again. The interaction of these activated exosomes with T lymphocytes and B lymphocytes and other immune cells leads to the activation and differentiation of T cells and B cells into cytotoxic memory cells, and also activates other types of immune cells involved in the immune response. Although cytotoxic T cells are designated to kill pathogens or diseased cells (including cancer), the presence of memory cells in the human body is part of adaptive immunity. These cells are able to generate very rapid immune responses when exposed to the same antigen, even many years later. In this context, the cell-free activated and tumor antigen-expressing exosome-based vaccines of the present invention can be used not only as immunotherapeutic treatments for cancer patients, but also as preventive vaccines to stimulate adaptive immunity in healthy people.

[0018] In one embodiment, the present invention provides a composition comprising one or more exosome populations, wherein the exosomes in the first exosome population: (a) each display CD19 and one or more additional surface molecules that are characteristic of mature activated B lymphocytes, and; (b) each contain (preferably display) one or more tumor antigens selected from MAGEA4, GAGE2D and 5T4, wherein the exosomes in the first exosome population contain (preferably display) all 3 tumor antigens in total.

[0019] In another embodiment, there is provided a pharmaceutical composition comprising a composition of the invention, optionally together with one or more pharmaceutically acceptable carriers, diluents, adjuvants or excipients.

[0020] In another embodiment, a pharmaceutical composition is provided, comprising:

[0021] (A) a first exosome population, wherein the exosomes:

[0022] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0023] (b) each comprises (preferably displays) a first tumor antigen, wherein the first tumor antigen is MAGEA4; and

[0024] (B) a second population of exosomes, wherein the exosomes:

[0025] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0026] (b) each comprises (preferably displays) a second tumor antigen, wherein the second tumor antigen is GAGE2D;

[0027] and optionally

[0028] (C) a third exosome population, wherein the exosomes:

[0029] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0030] (b) each comprises (preferably displays) a third tumor antigen, wherein the third tumor antigen is 5T4,

[0031] Preferably, wherein the surface molecule, exosome population and / or tumor antigen is as defined herein,

[0032] As a combined preparation, it is used for treating or preventing cancer, or for inducing an immune (eg, T cell or B cell) response against the tumor antigen in a subject in a form suitable for simultaneous, separate or sequential use.

[0033] In another embodiment, there is provided a composition of the invention for use in therapy or as a medicament.

[0034] In another embodiment, there is provided a use of the composition of the present invention in the preparation of a medicament, wherein:

[0035] (i) for preventing or treating cancer in a subject; or

[0036] (ii) for use in a method of inducing a T cell or B cell response against a cancer antigen in a subject; or

[0037] (iii) for inducing an adaptive or innate immune cell response in a subject.

[0038] In another embodiment, a method of preventing or treating a subject susceptible to or suffering from cancer is provided, the method comprising administering to a subject in need thereof an effective amount of a composition of the present invention.

[0039] In another embodiment, a method for inducing a T cell or B cell response or other immune cell against a cancer antigen in a subject, or a method for inducing an adaptive or innate immune cell response in a subject is provided, the method comprising administering an effective amount of a composition of the invention to a subject in need thereof.

[0040] In another embodiment, a method for generating a population of activated CD19+ exosomes is provided, wherein the exosomes contain (preferably display) one or more tumor antigens, the method comprising the following steps:

[0041] (a) maturing immature B lymphocytes to generate mature B lymphocytes displaying CD19;

[0042] (b) producing exosomes from the mature B lymphocytes; and

[0043] (c) incorporating one or more tumor antigens into each exosome (preferably into the (cell surface) membrane of the exosome), wherein the tumor antigens are MAGEA4, GAGE2D and 5T4;

[0044] To generate an activated CD19+ exosome population, each of which contains (preferably displays) one or more of the tumor antigens, and wherein the exosome population contains (preferably displays) all three tumor antigens in total.

[0045] The composition of the present invention comprises one or more exosome populations, for example 1, 2, 3, 4, 5 or more exosome populations. As used herein, the term "exosome" refers to a cell membrane-derived extracellular vesicle having a lipid bilayer outer membrane and a size of 30 nm to 150 nm.

[0046] As used herein, the term "exosome population" refers to a group of exosomes that have some common characteristics (eg, they all display surface molecules that are characteristic of mature activated B lymphocytes).

[0047] The exosomes of the present invention can be described as "activated" because they are derived from mature B lymphocytes that are then activated by exposure to one or more tumor antigens; or they are derived from mature activated B lymphocytes (which are activated by exposure to one or more tumor antigens). Exosomes can be described as "in vitro activated" because they have been activated in vitro.

[0048] Exosomes can be obtained (or can be obtained), for example, from human B lymphocytes from blood or from conditioned B lymphocyte culture medium (from which the cells have been removed) by continuous ultracentrifugation at 2000×g for 40 minutes, at 10,000×g for 60 minutes, and at 100,000×g for 1.5 hours, or by size exclusion chromatography.

[0049] The first exosome population displays surface molecules that are characteristic of mature activated B lymphocytes. These surface molecules are displayed on the outer membrane of the exosomes. Preferably, the exosomes have been obtained or derived from mature activated B lymphocytes.

[0050] B lymphocytes (also called B cells) are a type of white blood cell in the lymphocyte subtype. B lymphocytes participate in humoral or antibody-mediated adaptive immune responses. B lymphocytes are defined by the B cell receptor (BCR) on their cell surface. Therefore, the first population of exosomes will at least display the B cell receptor on its surface.

[0051] Mature activated B lymphocytes are CD19 and CD138 positive. Therefore, the exosomes of the first group will also display CD19 and CD138 proteins as some of their surface molecules. Mature activated B lymphocytes also express CD86 or CD80 and MHC I and MHC II proteins on their cell surface, so the activated exosomes of the present invention may also display one or more of these proteins on the external or internal exosome membrane. Therefore, preferably, the surface molecules that are characteristic of mature activated B lymphocytes are BCR, CD19, CD138, CD86 or CD80, MHC I and MHC II. The presence of at least one of these surface molecules together with tumor antigens on the surface of the exosomes of the first group makes the exosomes act as antigen presenting structures not only for naive T lymphocytes but also for B lymphocytes, and participate in the activation of immune responses of other immune cells. (The surface molecules that are characteristic of normal, mature activated B lymphocytes are not tumor antigens.)

[0052] The display of such surface molecules on cells or exosomes can be readily determined by flow cytometry or by Western blotting using appropriate antibodies against the surface molecules.

[0053] Immature B lymphocytes (eg, isolated from donor blood or from a cell line) can be matured in vitro by contacting them with CD40L. Preferably, the proliferation of the mature B lymphocytes is then induced by IL-2.

[0054] Preferably, the first population of exosomes accounts for at least 5%, more preferably at least 10% of the total exosomes in the composition.

[0055] The composition may additionally comprise one or more other exosome populations that do not display surface molecules characteristic of mature activated B lymphocytes.

[0056] For example, the composition may additionally include exosomes, which display surface molecules as PBMC features other than B lymphocytes. In particular, the composition may additionally include exosomes obtained from or derived from PBMCs other than B lymphocytes. For example, the composition may additionally include exosomes, which display surface molecules as bone marrow cells (such as monocytes and dendritic cells) and / or other lymphoid cells (such as NK cells and T lymphocytes). In particular, the composition may additionally include exosomes obtained from or derived from bone marrow cells (such as monocytes and dendritic cells) and other lymphoid cells (such as NK cells and T lymphocytes). Although these other cells (and exosomes obtained from or derived from them) may not display surface molecules as mature activated B lymphocyte features, these other cells (and exosomes) can display one or more of these tumor antigens.

[0057] The first exosome population also comprises (e.g., displays) one or more tumor antigens. In some embodiments, these tumor antigens are displayed on or in the outer cell membrane of the exosomes. Therefore, these tumor antigens are membrane-associated antigens.

[0058] In other embodiments, the one or more tumor antigens are contained within exosomes. In other embodiments, some of the one or more tumor antigens are displayed on or in the outer cell membrane of the exosomes, and some are contained within the exosomes.

[0059] In some embodiments, the one or more tumor antigens are independently selected from:

[0060] (i) products of mutated oncogenes and tumor suppressor genes;

[0061] (ii) products of other mutant genes;

[0062] (iii) overexpressed or aberrantly expressed cellular proteins;

[0063] (iv) Tumor antigens produced by oncogenic viruses

[0064] (v) carcinoembryonic antigen;

[0065] (vi) altered cell surface glycolipids and glycoproteins; and

[0066] (vii) Cell type specific differentiation antigens.

[0067] In some embodiments, the one or more tumor antigens are 1, 2, 3, 4, or 5 or more (e.g., 5-10) tumor antigens. Each exosome may, for example, contain (e.g., display) 1, 2, 3, 4, or 5 or more (e.g., 5-10) of the one or more tumor antigens on its outer surface.

[0068] In embodiments where the number of tumor antigens is two or more, the exosomes in the first population contain (e.g., display) these two or more tumor antigens in total, i.e., each of these two or more tumor antigens is present (e.g., displayed) individually or in combination by one or more exosomes in the first population. Thus, some exosomes in the first population may contain (e.g., display) one tumor antigen; some exosomes may contain (e.g., display) two tumor antigens; some exosomes may contain (e.g., display) three tumor antigens; and so on. However, in general, when the first exosome population is considered as a whole, at least some of the exosomes in the first population will contain (e.g., display) each of the different tumor antigens.

[0069] In some embodiments, one or more of these tumor antigens are tumor-associated antigens. In other embodiments, one or more of these tumor antigens are tumor-specific antigens.

[0070] Examples of tumor-associated antigens include MAGEA4, GAGE2D, and 5T4. Additional examples of tumor-associated antigens include NYESO-1, HER-2 / NEW, MAGE family, GAGE ​​family, XAGE family, RAGE family, BAGE family, SSX, Mammaglobulin A, tyrosinase, WT1, MUC-1, hTERT, CA-125, gp-100, HPV, HBV, EBV, HTLV, SV40, GA7330 / EpCam, SART1, SART3, survivin, mesothelin, and AFP.

[0071] Examples of tumor-specific antigens include PSA, PSMA, CEA, PAP, MART1, and melanA.

[0072] The one or more tumor antigens are selected from MAGEA4, GAGE2D and 5T4.

[0073] In some preferred embodiments, the exosomes in the first population collectively comprise (e.g., display) all three tumor antigens MAGEA4 and GAGE2D and 5T4 (i.e., wherein each of these three tumor antigens is present (e.g., displayed) by one or more exosomes in the first population, either alone or in combination).

[0074] Preferably, tumor antigens are full-length proteins. Most tumor antigens used in vaccines are peptides with short amino acid sequences. Using full-length proteins makes the vaccine of the present invention highly immunogenic because immune cells can recognize them with higher efficiency.

[0075] The MAGEA4 gene encodes melanoma-associated antigen 4; it is also referred to as MAGEA4. The human MAGEA4 gene has the UniProtKB database accession number P43358 (MAGA4_HUMAN). The amino acid sequence of the human MAGEA4 polypeptide is given herein as SEQ ID NO: 1. As used herein, the term "MAGEA4" or "MAGEA4 polypeptide" preferably refers to a polypeptide whose amino acid sequence comprises or consists of the amino acid sequence given in SEQ ID NO: 1, or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith. Preferably, the variant is a variant that binds to a histone deacetylase.

[0076] The GAGE2D gene encodes G antigen 2D. The human GAGE2D gene has the UniProtKB database accession number Q9UEU5 (GGE2D_HUMAN). The amino acid sequence of the human GAGE2D polypeptide is given herein as SEQ ID NO: 2. As used herein, the term "GAGE2D" or "GAGE2D polypeptide" preferably refers to a polypeptide whose amino acid sequence comprises or consists of the following: the amino acid sequence given in SEQ ID NO: 2, or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity therewith. Preferably, the variant is a variant that binds to FLT3 (CD135 antigen).

[0077] The 5T4 (TPBG) gene encodes an inhibitor of Wnt / β-catenin signaling. The human 5T4 gene has the UniProtKB database accession number Q13641. The amino acid sequence of the human 5T4 polypeptide is given herein as SEQ ID NO: 3. As used herein, the term "5T4" or "5T4 polypeptide" preferably refers to a polypeptide whose amino acid sequence comprises or consists of the following: an amino acid sequence given as SEQ ID NO: 3, or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto. Preferably, the variant is an inhibitor of Wnt / β-catenin signaling.

[0078] In some embodiments, the term "5T4" or "5T4 polypeptide" as used herein also encompasses only the extracellular domain of the 5T4 polypeptide. As used herein, the term "extracellular domain of a 5T4 polypeptide" preferably refers to a polypeptide whose amino acid sequence comprises or consists of the amino acid sequence given in SEQ ID NO: 4, or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto. Preferably, the variant is an inhibitor of Wnt / β-catenin signaling.

[0079] Preferably, the one or more tumor antigens are selected from MAGEA4, GAGE2D and 5T4. In some embodiments, the exosomes in the first population comprise (e.g., display):

[0080] (i) MAGEA4;

[0081] (ii) GAGE2D

[0082] (iii) 5T4;

[0083] (iv) MAGEA4 and GAGE2D;

[0084] (v) MAGEA4 and 5T4;

[0085] (vi) GAGE2D and 5T4; or

[0086] (vii) MAGEA4, GAGE2D, and 5T4;

[0087] wherein at least some of the exosomes in the first population comprise (e.g., display) MAGEA4

[0088] At least some of the exosomes in the composition comprise (e.g., display) GAGE2D; and optionally

[0089] At least some of the exosomes in the composition comprise (e.g., display) 5T4. In some preferred embodiments, each exosome comprises (e.g., displays):

[0090] (i) MAGEA4 and GAGE2D; or

[0091] (ii) MAGEA4, GAGE2D and 5T4.

[0092] The presence of such tumor antigens can be readily determined by flow cytometry or by Western blot using appropriate antibodies against the antigen.

[0093] There are many established algorithms that can be used to compare two amino acid sequences. Usually, a sequence is used as a reference sequence, and a test sequence can be compared with it. The sequence comparison algorithm calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The comparison of the amino acid sequence for comparison can be carried out by a computer-implemented algorithm (for example, GAP, BESTFIT, FASTA or TFASTA) or BLAST and BLAST 2.0 algorithm.

[0094] Percent amino acid sequence identity and nucleotide sequence identity can be obtained using the BLAST alignment method (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http: / / www.ncbi.nlm.nih.gov / BLAST). Preferably, standard or default alignment parameters are used.

[0095] Standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When the sequence similarity spans the entire sequence, blastp will also report the overall comparison, which is a preferred result for protein identification purposes. Preferably, standard or default comparison parameters are used. In some cases, the "low complexity filter" can be removed.

[0096] BLAST protein searches can also be performed with the BLASTX program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distance relationships between molecules. (See Altschul et al. (1997) supra). When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs can be used.

[0097] About nucleotide sequence comparison, MEGABLAST, discontinuous megablast and blastn can be used to achieve this goal.Preferably, standard or default comparison parameters are used.MEGABLAST is specially designed to effectively find long comparisons between closely similar sequences.Discontinuous MEGABLAST can be used to find nucleotide sequences similar but not identical to nucleic acid of the present invention.

[0098] The BLAST nucleotide algorithm searches for similar sequences by dividing the query into short subsequences called words. The program first identifies words that fully match the query word (word hits). The BLAST program then expands these word hits in multiple steps to generate the final gap comparison. In some embodiments, BLAST nucleotide searches can be performed with the BLASTN program, score = 100, word length = 12.

[0099] One of the important parameters that controls the sensitivity of a BLAST search is the word length. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word length (11). Therefore, blastn is better than MEGABLAST at finding alignments with related nucleotide sequences from other organisms. The word length is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.

[0100] More sensitive searches can be achieved by using the newly introduced discontinuous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html). This page uses an algorithm similar to that reported by Ma et al. (Bioinformatics. 2002 March; 18(3):440-5). Discontinuous megablast does not require exact word matches as seeds for alignment extensions, but instead uses discontinuous words within a longer template window. In encoded mode, third base wobbles are taken into account by focusing on matches at the first and second codon positions and ignoring mismatches at the third position. Searches in discontinuous MEGABLAST using the same word length are more sensitive and efficient than standard blastn using the same word length. The unique parameters for discontinuous megablast are: word length: 11 or 12; template: 16, 18, or 21; template type: encoding (0), non-coding (1), or both (2).

[0101] In some embodiments, the BLASTP 2.5.0+ algorithm (as available from NCBI) can be used with default parameters.

[0102] In other embodiments, the BLAST global alignment program (eg, available from NCBI) can be used, which uses a Needleman-Wunsch alignment of two protein sequences with a gap penalty of 11 for presence and 1 for extension.

[0103] As used herein, the term "sequence identity" in the context of amino acid sequences may alternatively be replaced by "sequence similarity". The term "similarity" allows conservative substitutions of amino acid residues with similar physicochemical properties within a defined length of a given comparison. The percentage of similarity can be determined using any reasonable similarity scoring matrix.

[0104] In one embodiment, the composition is an immunogenic composition or a vaccine composition. As used herein, the term "immunogenicity" refers to the ability to elicit a specific immune response against a tumor antigen. For example, such a response may occur when the composition of the invention is administered at an appropriate dose and in an appropriate formulation, which may include / require an appropriate adjuvant. A booster containing a dose similar to or less than the initial dose may be required to obtain the desired immunogenic response.

[0105] In particular, the immunogenic composition of the present invention is capable of inducing antibodies (preferably neutralizing antibodies against one or more tumor antigens, preferably against MAGEA4, GAGE2D and / or 5T4 in a subject, or capable of inducing the activity of T lymphocytes, B lymphocytes, NK cells, dendritic cells or macrophages).

[0106] The ability of a composition of the present invention to induce neutralizing antibodies in a subject (eg, a human subject) can be tested by purifying serum from the blood of a subject to which the composition has been administered.

[0107] Antibodies can be measured using ELISA or pseudotyped microneutralization (pMN) assays. ELISA is the most sensitive of the two assays; it quantifies all antibodies. In contrast, pMN is less sensitive, but it quantifies neutralizing antibodies.

[0108] The detection of T lymphocyte activity can be performed by detecting (e.g., by Western blot or ELISA, or flow cytometry) the production of specific cytokines or chemokines expressed by different activated T cell populations. For example, Th1 T helper cells: IL-2, IFN-γ, TNF-γ; Th-2 T helper cells: IL-4, IL-10, IL-13; Th-17 T helper cells: activation of Th17.

[0109] The immunogenic composition may additionally include one or more pharmaceutically acceptable carriers. Materials suitable for use as pharmaceutically acceptable carriers are known in the art. Non-limiting examples of pharmaceutically acceptable carriers include water, saline and phosphate buffered saline. However, in some embodiments, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative (such as thimerosal or sodium azide) for convenient long-term storage. Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate buffered saline (PBS; pH 7.4). In addition to a pharmaceutically acceptable carrier, the composition of the present invention may also be combined with one or more of salt, excipient, diluent, adjuvant, immunomodulator and / or antimicrobial compound.

[0110] In one embodiment, the composition of the invention may contain 5% to 95% active ingredient (i.e., exosomes), such as at least 10% or 25% active ingredient, or at least 40% active ingredient, or at least 50%, 55%, 60%, 70% or 75% active ingredient.

[0111] The products of the present invention may be administered in a manner compatible with the dosage form, and in an amount that is prophylactically and / or therapeutically effective.

[0112] The compositions of the present invention are usually administered by conventional routes, such as intravenous, subcutaneous, intraperitoneal, oral or mucosal routes. Administration can be by parenteral administration; for example, subcutaneous or intramuscular injection.

[0113] Therefore, the composition of the present invention can be prepared as a vascular injection, which is a liquid solution or suspension. Alternatively, a solid form suitable for dissolving or suspending in a liquid before injection can be prepared. The preparation can also be emulsified, or the peptide is encapsulated in a liposome or microcapsule. The active ingredient is usually mixed with a pharmaceutically acceptable and compatible excipient with the active ingredient. Suitable excipients are, for example, water, saline, glucose, glycerol, ethanol, etc., and combinations thereof. In addition, if necessary, the product of the present invention can also contain a small amount of auxiliary substances, such as a wetting agent or an emulsifier, and / or a pH buffer.

[0114] Preferably, the composition of the present invention is a vaccine composition, e.g., suitable for parenteral administration, optionally with one or more adjuvants. As used herein, a vaccine is a formulation that stimulates a protective immune response when administered to a subject. The immune response can be a humoral and / or cell-mediated immune response. Thus, a vaccine can stimulate B cells and / or T cells, as well as other immune cell types.

[0115] The composition may also comprise a surfactant. Examples of suitable surfactants include Tween (such as Tween 20), Brij and polyethylene glycol.

[0116] Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al., eds., University Park Press, Baltimore, MD, USA, 1978. Encapsulation within liposomes is described, for example, by Fullerton in US Pat. No. 4,235,877.

[0117] The amount of exosomes of the invention present in each vaccine dose is selected to be an amount that induces an immunoprotective response in a typical vaccine without significant adverse side effects. This amount will vary depending on which specific immunogen is used and whether the vaccine contains an adjuvant.

[0118] Typically, it is expected that each dose will contain 1 μg / ml-1000 μg / ml of protein, for example 1 μg / ml-200 μg / ml, such as 10 μg / ml-100 μg / ml, and more specifically 10 μg / ml-40 μg / ml. The optimal amount of a particular vaccine can be determined by standard studies, including observation of antibody titers and other responses in the subject. After the initial vaccination, the subject preferably receives a booster immunization within about 4 weeks, followed by repeated booster immunizations every 6 months as long as there is a risk of infection. The immune response to the composition of the present invention is enhanced by using adjuvants and / or immunostimulants.

[0119] The compositions of the invention do not contain cells, i.e. they are cell-free. Preferably, these compositions contain no DNA / RNA or substantially no DNA / RNA (except for exosomes).

[0120] Also provided is an immunogenic composition comprising two or more populations of exosomes as defined herein, as a combined preparation, for use in treating or preventing cancer, or for inducing a T cell or B cell response against a tumor antigen (preferably against MAGEA4, GAGE2D or 5T4) in a subject in a manner suitable for simultaneous, separate or sequential use.

[0121] In particular, the immunogenic composition may comprise:

[0122] (A) a first exosome population, wherein the exosomes:

[0123] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0124] (b) each comprises (e.g., displays) a first tumor antigen, wherein the first tumor antigen is MAGEA4; and

[0125] (B) a second population of exosomes, wherein the exosomes:

[0126] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0127] (b) each comprises (e.g., displays) a second tumor antigen, wherein the second tumor antigen is GAGE2D;

[0128] and optionally

[0129] (C) a third exosome population, wherein the exosomes:

[0130] (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and

[0131] (b) each comprises (e.g., displays) a third tumor antigen, wherein the third tumor antigen is 5T4;

[0132] Preferably, wherein the surface molecule, exosome population and / or tumor antigen is as defined herein,

[0133] As a combined preparation, it is used for treating or preventing cancer, or for inducing T cell or B cell response against tumor antigen in a subject in a form suitable for simultaneous, separate or sequential use.

[0134] The present invention also provides a kit consisting of the first, second and third exosome populations defined above.

[0135] In yet further embodiments, the present invention provides a composition of the present invention for use in treatment or as a medicament. In another aspect, the present invention provides a method for the use of a composition of the present invention in preventing or treating cancer in a subject. In a further embodiment, the present invention provides a method for the use of a composition of the present invention in inducing a T cell, B cell or other immune cell response to a cancer antigen in a subject. In a further embodiment, the present invention provides the use of a composition of the present invention in the preparation of a medicament for preventing or treating cancer in a subject. In a further embodiment, the present invention provides the use of a composition of the present invention in the preparation of a medicament for inducing a T cell, B cell or other immune cell response to a cancer antigen in a subject. The present invention also provides a method for preventing or treating a subject susceptible to or suffering from cancer, the method comprising administering an effective amount of the composition of the present invention to a subject in need thereof. The present invention also provides a method for inducing a T cell, B cell or other immune cell response to a cancer antigen in a subject, the method comprising administering an effective amount of the composition of the present invention to a subject in need thereof. The present invention also provides the use of a composition of the present invention in treating cancer; and providing a composition of the present invention when used to treat cancer. The cancer antigen is preferably one of the tumor antigens as defined herein.

[0136] The compositions of the present invention can also be used in similar uses and methods for producing neutralizing antibodies, or methods for activating T lymphocytes against tumor antigens in vivo.

[0137] The efficacy of the uses and methods for treating / preventing cancer can be tested (eg, by ELISA) by determining the presence of neutralizing antibodies against tumor antigens in the subject's blood or the activity of T lymphocytes in the subject's blood.

[0138] As used herein, the term "prevention" includes preventing the occurrence of cancer and / or reducing the severity of cancer. Thus, "prevention" encompasses vaccination.

[0139] As used herein, the term "treatment" includes treatment and preventive / preventative measures (including post-exposure prophylaxis), and includes post-infection treatment and improvement of cancer. Each of the above methods and uses may include the step of administering to a subject an effective amount (such as a therapeutically effective amount) of a composition of the present invention.

[0140] As used herein, an effective amount is a dose or amount sufficient to achieve a desired biological result. As used herein, a therapeutically effective amount is an amount effective for treating, preventing, curing, delaying a disorder or recurring disorder (e.g., cancer), reducing its severity, improving at least one symptom, or prolonging the survival of a subject beyond the expected survival period in the absence of such treatment, after single or multiple doses are administered to a subject (such as a mammalian subject, particularly a human subject).

[0141] Therefore, the amount of active ingredient to be administered depends on the subject to be treated, the ability of the subject's immune system to produce a protective immune response, and the degree of protection required. The exact amount of active ingredient to be administered may depend on the physician's judgment and may vary from subject to subject. Administration to a subject may include administering to a subject an exosome group or composition of the present invention (i.e., a product of the present invention), wherein the product of the present invention is administered multiple times in sequence (e.g., wherein the composition is administered twice, three times, or four times). Therefore, in one embodiment, an exosome group or composition of the present invention is administered to a subject, and then the same product of the present invention (or substantially similar product) is administered again at a different time.

[0142] In one embodiment, administering to a subject comprises administering to a subject a population of exosomes or a composition of the invention, wherein the product of the invention is administered substantially before, simultaneously with, or after another immunogenic composition.

[0143] The present invention also extends to a primary immunization-boosting immunization regimen. For example, one or more products of the present invention can be used to achieve primary immunization and / or boosting immunization. These products can be administered to a subject sequentially, simultaneously or separately.

[0144] The compositions of the invention may be administered in a single dose regimen (ie, substantially the entire dose is administered at once). Alternatively, the compositions of the invention may be administered in a multiple dose regimen.

[0145] A multiple-dose regimen is one in which the initial course of treatment (e.g., vaccination) may employ 1-6 individual doses, followed by additional doses given at subsequent intervals as required to maintain and / or enhance the immune response, for example (for human subjects), a second dose is given at 1-4 months, and subsequent doses are given after an additional 1-4 months if necessary.

[0146] The dosage regimen will be determined, at least in part, by the individual need and will be subject to the judgment of the practitioner (eg, a physician or veterinarian).

[0147] Simultaneous administration means administration at (substantially) the same time.

[0148] The sequential administration of two or more compositions of the present invention refers to the administration of the compositions at (substantially) different times, one after the other. For example, sequential administration may encompass the administration of two or more compositions of the present invention at different times, with different time intervals of several days (e.g., 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150 or 200 days).

[0149] For example, in one embodiment, the compositions of the invention may be administered as a vaccine as part of a "prime-boost" vaccination regimen.

[0150] In one embodiment, the compositions of the present invention can be administered to a subject such as a mammal in combination (simultaneously or sequentially) with one or more immunomodulators selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-17) and / or cytokines (e.g., IFN-γ).

[0151] The subject is preferably a mammalian subject. The subject can be a human or non-human. For example, the subject can be a farm mammal (e.g., sheep, horse, pig, cow or goat), a companion mammal (e.g., cat, dog or rabbit) or a laboratory test mammal (e.g., mouse, rat or monkey).

[0152] Preferably, the subject is a person. The subject can be male or female. People can be, for example, 0-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 or more than 100 years old. People can be people suffering from a specific disease or condition (e.g., lung cancer) or people suffering from a specific disease or condition (e.g., lung cancer). In some preferred embodiments, the subject is a subject suffering from cancer or having previously suffered from cancer. Control subjects can be defined as disease-free subjects, cancer-free subjects, normally developed subjects or healthy aging subjects. As used herein, the term "control subject" refers to an individual or individual group belonging to the same species as the subject being tested. For example, if the subject is a person, the control will be a person.

[0153] The cancer may be a malignant or benign cancer. Preferably, the cancer is selected from colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, blood cancer (e.g., leukemia, lymphoma and myeloma).

[0154] In yet a further embodiment, a method of preparing the exosome population of the present invention is provided.

[0155] In particular, a method for generating a population of CD19+ exosomes is provided, wherein the exosomes contain (e.g., display) one or more tumor antigens, the method comprising the following steps:

[0156] (a) Maturation of immature B lymphocytes to generate mature B lymphocytes displaying CD19

[0157] (b) producing exosomes from the mature B lymphocytes; and

[0158] (c) incorporating one or more tumor antigens into each exosome (preferably into the membrane of the exosome), wherein the tumor antigens are MAGEA4, GAGE2D and 5T4;

[0159] to produce a population of activated CD19+ exosomes, each of which comprises (preferably displays) one or more tumor antigens, and

[0160] The exosome population comprises (preferably displays) all three tumor antigens.

[0161] In yet another embodiment, a method for generating a population of activated CD19+ exosomes is provided, wherein the exosomes contain (e.g., display) one or more tumor antigens, the method comprising the following steps:

[0162] (a) expressing one or more tumor antigens (preferably cell surface tumor antigens) in each cell in a cell population comprising immature or mature B lymphocytes, wherein the tumor antigens are MAGEA4, GAGE2D and 5T4, thereby maturing and activating the immature or mature B lymphocytes to produce mature activated B lymphocytes; and

[0163] (b) producing activated CD19+ exosomes from the mature activated B lymphocytes;

[0164] to produce a population of activated CD19+ exosomes, each of which comprises (preferably displays) one or more tumor antigens, and

[0165] The exosome population comprises (preferably displays) all three tumor antigens.

[0166] In yet another embodiment, a method for generating a population of activated CD19+ exosomes is provided, wherein the exosomes contain (e.g., display) one or more tumor antigens, the method comprising the following steps:

[0167] (a) maturing immature B lymphocytes in a cell population comprising immature B lymphocytes to generate mature activated B lymphocytes displaying CD19+;

[0168] (b) expressing one or more cellular tumor antigens (preferably cell surface tumor antigens) in each mature B lymphocyte, thereby activating the B lymphocyte, wherein the tumor antigens are MAGEA4, GAGE2D and 5T4; and

[0169] (c) Production of activated CD19+ exosomes from mature activated B lymphocytes,

[0170] To generate an activated CD19+ exosome population, each of which contains (e.g., displays) one or more of the tumor antigens, and wherein the exosome population contains (preferably displays) all three tumor antigens in total.

[0171] Immature B lymphocytes can be obtained from any suitable source, including from a blood sample obtained from a subject. Blood contains peripheral blood mononuclear cells (PBMCs), i.e., a mixed population of bone marrow cells and lymphoid cells. Therefore, PBMCs include some mature and immature B lymphocytes. Therefore, in the context of the claimed method, immature B lymphocytes can be provided in the form of PBMCs (e.g., mixed with other PBMCs).

[0172] B lymphocytes and PBMCs can be obtained from blood samples by standard methods (e.g., by density gradient centrifugation). Individual cell types can be isolated from PBMCs by standard immunomagnetic separation or FACS (fluorescence activated cell sorting) techniques.

[0173] Immature and mature activated B lymphocytes (and PBMCs) can be engineered to express tumor antigens (rather than being pulsed with tumor antigens). For example, these cells can be transfected (stably or transiently) with a suitable expression construct encoding a tumor antigen, for example under the control of an inducible or constitutive promoter. Methods for expressing such antigens in cells are known in the art (e.g., "Molecular Cloning: A Laboratory Manual" (4th edition), Green, MR and Sambrook, J. (2014 update)).

[0174] Immature B lymphocytes or PBMCs containing immature B lymphocytes can be activated by any suitable method. For example, immature B lymphocytes can be activated (e.g., in vitro) by contact with CD40L. Preferably, the proliferation of mature activated B lymphocytes is subsequently induced by IL-2.

[0175] As described above, mature activated B lymphocytes and exosomes derived therefrom are positive for CD19 and CD138; they may also naturally express BCR, CD86 or CD80, and MHC I and MHC II proteins on their cell (or exosome) surfaces.

[0176] Thus, activation can be verified by assaying for the presence of such molecules by flow cytometry or Western blotting using appropriate antibodies against these molecules.

[0177] Exosomes can be produced from mature activated B lymphocytes by any suitable method. PBMCs containing mature B lymphocytes can also be used instead of mature B lymphocytes. Such methods include centrifugation or ultracentrifugation; this can be combined with size exclusion chromatography (SEC) or not. Size exclusion chromatography columns can be used, for example, using porous gel columns. In such columns, the pore size is preferably 30nm-150nm to allow exosomes to pass but not larger vesicles to pass. Other methods include immunoprecipitation using exosome markers (e.g., CD9, CD81, CD63, Alix, TSG-101, etc.). This can be achieved, for example, by direct immunoprecipitation using a commercially available kit.

[0178] One or more tumor antigens can be incorporated into the exosome surface membrane of the exosomes to produce exosomes displaying the desired tumor antigen. This incorporation can be achieved by any suitable means. For example, the exosomes can be electroporated, incubated or pulsed with tumor antigens in an oscillator. Cationic polymers (e.g., polybrene) can be added to improve the incorporation efficiency. Preferably, under oscillating conditions, optionally in the presence of polybrene, the exosomes are pulsed (e.g., incubated) with tumor antigens at about 37° C. for about 90 minutes.

[0179] The present invention also encompasses a population of exosomes comprising (e.g., displaying) one or more tumor antigens obtained or obtainable by the methods of the present invention.

[0180] The present invention also provides a method for producing a pharmaceutical composition, the method comprising a method for producing a population of exosomes, the exosomes comprising (e.g., displaying) one or more tumor antigens of the present invention, and wherein the method further comprises the following steps: (d) mixing the population of exosomes with one or more pharmaceutically acceptable diluents, excipients, adjuvants or carriers. The present invention also encompasses a composition obtained or obtainable by the method for producing a pharmaceutical composition of the present invention.

[0181] Preferably, the method steps are performed in the order specified (one after the other).

[0182] In other embodiments, the present invention provides one or more of the following embodiments:

[0183] Embodiment 1. A composition comprising one or more exosome populations, wherein the exosomes in the first exosome population: (a) each display a surface molecule that is characteristic of mature activated B lymphocytes; and (b) each contain (preferably display) one or more tumor antigens.

[0184] Embodiment 2. The composition according to claim 1, wherein the surface molecules characteristic of mature activated B lymphocytes are BCR, CD19, CD138, CD86 or CD80, MHC I and MHC II.

[0185] Embodiment 3. The composition according to embodiment 1 or embodiment 2, wherein the first exosome population has been obtained or derived from: (i) mature activated B lymphocytes; or (ii) a PBMC population comprising mature activated B lymphocytes.

[0186] Embodiment 4. A composition according to embodiment 3, wherein: (i) the mature activated B lymphocytes; or (ii) the PBMC population comprising mature activated B lymphocytes has been modified to contain (preferably display) one or more of the tumor antigens after the exosomes are produced.

[0187] Embodiment 5. A composition according to Embodiment 3, wherein: (i) the mature activated B lymphocytes; or (ii) the PBMC population comprising mature activated B lymphocytes has been modified to comprise (preferably display) the one or more tumor antigens before the exosomes are produced.

[0188] Embodiment 6. A composition according to any of the preceding embodiments, wherein the composition further comprises one or more additional exosome populations that have been obtained or derived from monocytes, dendritic cells, NK cells and / or T lymphocytes.

[0189] Embodiment 7. A composition according to embodiment 6, wherein one or more of the additional exosome populations comprises (preferably displays) one or more of the tumor antigens.

[0190] Embodiment 8. A composition according to any one of the preceding embodiments, wherein the exosomes in the first exosome population contain (preferably display) 1, 2, 3, 4 or 5 (preferably 3) of the tumor antigens.

[0191] Embodiment 9. A composition according to any one of the preceding embodiments, wherein the exosomes in the first exosome population all contain (preferably display) 1, 2, 3, 4 or 5 (preferably 3) of the tumor antigens.

[0192] Embodiment 10. A composition according to any of the preceding embodiments, wherein one or more of the tumor antigens is a tumor-associated antigen or a tumor-specific antigen.

[0193] Embodiment 11. A composition according to any of the preceding embodiments, wherein the one or more tumor antigens are selected from MAGEA4, GAGE2D and 5T4.

[0194] Embodiment 12. The composition of embodiment 11, wherein 5T4 is the extracellular domain of a 5T4 polypeptide.

[0195] Embodiment 13. A pharmaceutical composition comprising the composition of any of the preceding embodiments, optionally together with one or more pharmaceutically acceptable carriers, diluents, adjuvants or excipients.

[0196] Embodiment 14. A pharmaceutical composition comprising: (A) a first population of exosomes, wherein the exosomes: (a) each display a surface molecule characteristic of mature activated B lymphocytes; and (b) each contain (preferably display) a first tumor antigen; and (B) a second population of exosomes, wherein the exosomes: (a) each display a surface molecule characteristic of mature activated B lymphocytes; and (b) each contain (preferably display) a second tumor antigen; and optionally (C) a third population of exosomes, wherein the exosomes: (a) each display a surface molecule characteristic of mature activated B lymphocytes; and (b) each contain (preferably display) a third tumor antigen, preferably wherein the surface molecules, exosome populations and / or tumor antigens are as defined in any one of Embodiments 2-12, as a combined preparation, in a form suitable for simultaneous, separate or sequential use for treating or preventing cancer, or for inducing T cell or B cell responses against tumor antigens in a subject.

[0197] Embodiment 15. A composition according to any one of embodiments 1 to 14 for use in therapy or as a medicament.

[0198] Embodiment 16. A composition according to any one of claims 1 to 14: (i) for use in a method of preventing or treating cancer in a subject; or (ii) for use in a method of inducing a T cell or B cell response to a cancer antigen in a subject.

[0199] Embodiment 17. Use of the composition of any one of Embodiments 1 to 14 in the preparation of a medicament for: (i) preventing or treating cancer in a subject; or (ii) inducing a T cell or B cell response against a cancer antigen in a subject.

[0200] Embodiment 18. A method of preventing or treating a subject susceptible to or suffering from cancer, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of Embodiments 1 to 14.

[0201] Embodiment 19. A method of inducing a T cell or B cell response against a cancer antigen in a subject, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of Embodiments 1 to 14.

[0202] Embodiment 20. The composition, use or method of any one of Embodiments 16 to 19, wherein the cancer is selected from colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, and blood cancer (e.g., leukemia, lymphoma and myeloma).

[0203] Embodiment 21. A method for producing a population of exosomes, wherein the exosomes contain (preferably display) one or more tumor antigens, the method comprising the following steps: (a) activating immature B lymphocytes to produce mature activated B lymphocytes; (b) producing exosomes from the mature activated B lymphocytes; and (c) incorporating one or more tumor antigens into the exosomes (preferably into the cell surface membrane of the exosomes); to produce a population of exosomes containing (preferably displaying) one or more tumor antigens.

[0204] Embodiment 22. A method for producing a population of exosomes, wherein the exosomes contain (preferably display) one or more tumor antigens, the method comprising the following steps: (a) expressing one or more tumor antigens (preferably cell surface tumor antigens) in a cell population comprising immature B lymphocytes; (b) activating the immature B lymphocytes to produce mature activated B lymphocytes; and (c) producing exosomes from the mature activated B lymphocytes; to produce a population of exosomes containing (preferably displaying) one or more tumor antigens.

[0205] Embodiment 23. A method for producing a population of exosomes, wherein the exosomes contain (preferably display) one or more tumor antigens, the method comprising the following steps: (a) activating immature B lymphocytes in a cell population containing immature B lymphocytes to produce mature activated B lymphocytes; (b) expressing one or more tumor antigens (preferably cell surface tumor antigens) in the mature activated B lymphocytes; and (c) producing exosomes from the mature activated B lymphocytes to produce a population of exosomes containing (preferably displaying) one or more tumor antigens.

[0206] Embodiment 24. A method according to any one of Embodiments 21 to 23, wherein the immature B lymphocytes are present in a PBMC population, or wherein the cell population comprising immature B lymphocytes is a PBMC population.

[0207] Embodiment 25. A method for producing a pharmaceutical composition, the method comprising the method for producing a population of exosomes according to any one of embodiments 21 to 24, wherein the exosomes contain (preferably display) one or more tumor antigens, and wherein the method further comprises the following steps: (d) mixing the population of exosomes with one or more pharmaceutically acceptable diluents, excipients, adjuvants or carriers.

[0208] Embodiment 26. A population of exosomes or a pharmaceutical composition obtained or obtainable by the method of any one of Embodiments 21 to 25.

[0209] The disclosure of each reference listed herein is specifically incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] Figure 1 : Western blot analysis of HEK293T cells stably transfected with tumor-associated antigens.

[0211] a. GAGE2D protein (GAGE2D-His-tag or empty vector) was expressed in cell lysate (left) or conditioned medium (right).

[0212] b. MAGEA4 protein (MAGEA4-His-tag or empty vector) was expressed in cell lysate (left) or conditioned medium (right).

[0213] c. The extracellular domain of 5T4 protein (5T4-His-tag or empty vector) was expressed in cell lysate (left) or conditioned medium (right).

[0214] Figure 2 : Western blot analysis of tumor-associated antigens isolated from conditioned media after purification by fast protein liquid chromatography (FPLC) and size exclusion chromatography (SEC).

[0215] a. Expression of GAGE2D protein in the collected fractions obtained after SEC.

[0216] b. Expression of MAGEA4 protein in the collected fractions obtained after SEC.

[0217] c. Expression of the extracellular domain of 5T4 protein in the pooled fractions obtained after SEC.

[0218] Figure 3 a and Figure 3b: Nanoparticle Tracking Analysis (NTA). Size distribution of exosomes isolated from conditioned medium from a. activated B lymphocytes or from b. activated PBMCs.

[0219] Figure 3 c and Figure 3 d: Western blot analysis of engineered activated exosomes pulsed with TAA.

[0220] c. Expression profile of activated exosomes derived from mature B lymphocytes and engineered for GAGE2D, MAGEA4 and the extracellular domain 5T4.

[0221] d. Expression profiles of exosomes derived from activated PBMCs and engineered for GAGE2D, MAGEA4 and the extracellular domain 5T4. CD9 was used as a general marker for exosomes.

[0222] Figure 4 : 3D spheroid T cell cytotoxicity assay (tumor killing assay).

[0223] a. GFP fluorescence expression profiles of H1299 tumor spheroids co-cultured with freshly isolated PBMCs and treated or not with activated exosomes derived from mature B lymphocytes and engineered against the extracellular domains of GAGE2D, MAGEA4, and 5T4.

[0224] b. GFP fluorescence expression profiles of H1299 tumor spheroids co-cultured with freshly isolated PBMCs and treated or not with activated exosomes derived from mature B lymphocytes and engineered against GAGE2D and MAGEA4.

[0225] c. GFP fluorescence expression profiles of H1299 tumor spheroids co-cultured with freshly isolated PBMCs and treated or not with activated exosomes derived from mature PBMCs and engineered against the extracellular domains of GAGE2D, MAGEA4, and 5T4.

[0226] Figure 5 : Cytokine response was determined by ELISA method.

[0227] a. IL-2 production in freshly isolated PBMCs or in PBMCs co-cultured with empty activated exosomes derived from mature B lymphocytes or with activated exosomes derived from mature B lymphocytes and engineered for GAGE2D, MAGE4A and the extracellular domain 5T4.

[0228] b. IFN-γ production in freshly isolated PBMCs or in PBMCs co-cultured with empty activated exosomes derived from mature B lymphocytes or with activated exosomes derived from mature B lymphocytes and engineered for GAGE2D, MAGEA4 and the extracellular domain 5T4.

[0229] c. IL-17 production in freshly isolated PBMCs or in PBMCs co-cultured with empty activated exosomes derived from mature B lymphocytes or with activated exosomes derived from mature B lymphocytes and engineered for GAGE2D, MAGEA4 and the extracellular domain 5T4.

[0230] Figure 6 : Localization of tumor antigens in B lymphocytes and PBMC-derived and bioengineered exosomes.

[0231] a. Western blot analysis of tumor antigens MAGEA4, the extracellular domain of 5T4, and GAGE2D after exosomes were treated with trypsin. CD9 was used as a broad exosome marker. DETAILED DESCRIPTION

[0232] Example

[0233] The present invention is further illustrated by the following examples, wherein parts and percentages are by weight, and degrees are degrees Celsius, unless otherwise stated. It should be understood that these examples, although representing preferred embodiments of the present invention, are given by way of illustration only. Through the above discussion and these examples, those skilled in the art can determine the essential features of the present invention, and without departing from its essence and scope, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, according to the foregoing description, in addition to those shown and described herein, various modifications to the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0234] The following materials and methods were used in one or more embodiments.

[0235] Materials and methods:

[0236] Generation of stable cell lines producing tumor-associated antigens (TAA)

[0237] Stable HEK293T cell lines producing the target proteins were prepared according to Elegheert et al., 2018 (Elegheert et al., 2018). Briefly, the full-length and extracellular domain coding sequences of the extracellular domain genes of human GAGE2D, MAGEA4, and 5T4 (TPBG) were cloned into the lentiviral plasmid pHR-CMV-TetO2-3C-Avi-His6 (Addgene #113887) and then transfected into virus-producing HEK293 Lenti-X cells using PEI transfection reagent together with the packaging plasmid psPAX2 (Addgene #12260) and the envelope plasmid pMD2.G (Addgene #12259). Three days after transfection, the culture medium containing the viral particles was collected and added to the HEK293T target cells. Three days after infection, polyclonal stable cell lines were established and protein production was verified from the culture medium and cell lysates by Western blotting.

[0238] Transfection of B lymphocytes or PBMCs with TAA vectors

[0239] HEK293T cells were transfected with a lentiviral plasmid pHR-CMV-TetO2-3C-Avi-His6 (Addgene #113887) containing the full-length coding sequence of the human GAGE2D gene, the MAGEA4 gene, or the extracellular domain sequence encoding the extracellular domain of the 5T4 (TPBG) gene, together with a packaging plasmid and an envelope plasmid pMD2.G that allow viral particle production. The culture medium containing the viral particles was collected and added to human isolated B lymphocytes. The stable transfection of TAAs producing genetically modified B lymphocytes was verified by Western blotting. Genetically engineered TAA-B lymphocytes are activated by CD40L and induced to proliferate and grow by IL-2. This allows the production of activated exosomes derived from TAA-B cells in the culture medium. The blood used to obtain mature human B lymphocytes and peripheral blood mononuclear cells was obtained in the Czech Republic with the consent of the donor.

[0240] Preparation of H1299 lung cancer cells stably expressing GFP

[0241] HEK293 cells were transfected with pHR-CMV-TetO2-EmGFP plasmid (Addgene #113892) together with packaging plasmid psPAX2 (Addgene #12260) and envelope plasmid pMD2.G (Addgene #12259) for the production of viral particles. Three days later, H1299 cells were transduced with medium containing viral particles. Another three days after infection, H1299 cells were FACS sorted and a polyclonal GFP-expressing cell line was established.

[0242] Protein purification from the culture medium of the stable HEK293T protein-expressing cell line

[0243] Stable HEK293T cells are inoculated into 2xT175 cell culture bottles with about 70% confluence and cultured for 5-7 days. The culture medium is collected, centrifuged briefly (5 minutes, 2000xg, 4°C, universal 320R centrifuge), and then the supernatant is ultracentrifuged at 4°C for 30 minutes at 50,000xg (Optima XPN-90, Beckman Coulter, rotor 70TI), and then dialyzed overnight to PBS. The next day, the dialyzed culture medium is used to use AKTAgo FPLC instrument (Cytiva) to carry out protein purification by His affinity chromatography, anion exchange chromatography and size exclusion chromatography (SEC). The quality of the purified protein is assessed by Western blotting and Coomassie staining using antibodies for His-tags and specific antibodies for the target protein.

[0244] Isolation of activated exosomes from mature B lymphocytes or PBMCs

[0245] According to Wu et al., 2010 (Wu et al., 2010), in culture medium (1×10 7 PBMCs or B lymphocytes were cultured and activated with 4% paraformaldehyde (100 cells / condition) for up to 5 days.

[0246] Conditioned media were collected and analyzed by Optima XPN-80 (Beckman, MA) using UltraClear Thinwall tubes. ) ultracentrifuge at 2000xg for 40 minutes, 10,000xg for 60 minutes, and 100,000xg for 1.5 hours to separate EVs. Exosomes were washed once in 1ml PBS and purified by centrifugation at 100,000xg for 80 minutes in an Optima MAX-XP ultracentrifuge (Beckman Coulter). The exosome precipitate was collected and passed through a SEC column (IZON, 70nm) to obtain exosomes with a size of 70nm-150nm. Exosome protein concentration was measured using MicroBCA assay (Thermo Scientific).

[0247] Nanoparticle Tracking Analysis (NTA)

[0248] Before starting the analysis, the total exosome pellet was resuspended in 1 mL PBS (Gibco) by vigorous pipetting and kept on ice.

[0249] Before starting any measurement, NanoSight NS300 (Malvern Panalytical) was washed three times by loading distilled water onto the syringe pump using a 1mL syringe and pressing the liquid into the flow cell top plate of NanoSight. PBS is used to perfuse the instrument and control the purity of the diluent (i.e., there are no particles in the solution or there are particles with a concentration below the detectable level). After perfusion, 1mL of sample was carefully loaded onto the syringe pump. Each measurement was automatically performed with the help of the syringe pump, and a data acquisition script was generated on the NTA 3.2 software. Once each sample was loaded into the sample chamber, it was automatically recorded three times, each for 60 minutes, and the focus of the particles in the solution was manually adjusted.

[0250] Bioengineering exosomes with TAA

[0251] 1 μg of exosomes were pulsed with 100 μg of MAGEA4, 100 μg of GAGE2D and 7.5 μg-10 μg of 5T4 protein in the presence of polybrene in a thermostatic shaker by mixing at 500 rpm for 90 minutes at 37°C. After pulsing, the activated exosomes were washed in PBS and centrifuged at 100000 g for 75 minutes at 4°C (Optima Max-XP Beckman Rotor MLA150), and the precipitated exosomes were then reconstituted in PBS and used for treatment.

[0252] Isolation of PBMCs

[0253] Use of generic Ficoll-Paque TM Protocol Peripheral blood mononuclear cells (PBMCs) were isolated from freshly collected EDTA-treated human female donor blood. Briefly, the collected blood was diluted with fetal bovine serum in saline and filtered through Ficoll-Paque TM The resulting solution was carefully centrifuged and the PBMC-containing layer was separated from the Ficoll-Paque TM The medium / plasma interface was separated. The PBMC layer was then washed continuously and centrifuged to remove contaminants such as platelets and plasma. Finally, the separated PBMCs were resuspended and cultured in RPMI medium containing 10% human serum AB and penicillin / streptomycin in a T25 flask.

[0254] SDS-PAGE and western blotting

[0255] Briefly, cells or exosomes were lysed and resuspended in 2x sample buffer containing 1 M DTT. The samples were then boiled at 100 °C for 10 min, 20 μg of protein was loaded onto the gel, and Precast gels (10% or 4%-12%) (Thermo Scientific) were separated by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked and incubated with primary antibodies in 5% skim milk diluted in PBS-Tween 20. Secondary antibodies were always incubated in 5% skim milk. Before exposure to film (Fujifilm), the membranes were covered in ECL solution from Thermo Scientific, Millipore or GE Healthcare and developed in XoGraph developer.

[0256] Cleavage of surface TAAs for western blot analysis.

[0257] Exosomes derived from B lymphocytes or PBMCs and exosomes bioengineered with TAAs were resuspended in PBS and treated with trypsin at 37°C for 30 minutes. After treatment, the exosomes were ultracentrifuged at 100,000 x g for 80 minutes to separate the exosomes from the cleaved surface proteins. The supernatant after trypsin digestion (which contains cleaved surface exosomal TAA proteins) and exosomes were collected and analyzed by Western blot with specific antibodies against certain tumor antigens.

[0258] Tumor Killing Assay—Cytotoxic T Cell Response

[0259] H1299-GFP cells were generated by U-shaped plates and co-cultured with or without PBMCs and with or without exosomes derived from activated B lymphocytes and / or PBMCs engineered (pulsed) with the extracellular domains of GAGE2D, MAGEA4, and 5T4 at a concentration of 4 μg / ml. GFP fluorescence was measured by TECAN instrument for 24 hours to 120 hours according to the manufacturer's protocol.

[0260] Enzyme-linked immunosorbent assay (ELISA)

[0261] Freshly isolated PBMCs were co-cultured with activated exosomes derived from mature B lymphocytes and / or PBMCs engineered (pulsed) with the extracellular domains of GAGE2D, MAGEA4, and 5T4 at a concentration of 4 μg / ml. The culture medium was collected every 24 hours and used for ELISA according to the manufacturer's user guide (IL-17 Human Elisa Kit, Catalog number: RAB0262; IFN-γ Human Elisa Kit, Catalog number: RAB0222 and IL-2 Human Elisa Kit, Catalog number: BMS221) were used for ELISA assay to measure the concentration of produced cytokines.

[0262] Example 1: Generation of TAA-engineered exosomes

[0263] First, we prepared HEK-293T cell lines that were stably transfected with GAGE2D, MAGEA4, and ex5T4 extracellular domains. The expression and production of these proteins in the culture medium were verified by Western blot analysis ( Figure 1 a, b, c). The synthesized protein from the conditioned medium was collected, purified by fast protein liquid chromatography (FPLC), and after a size exclusion chromatography (SEC) step, the purity of the obtained protein-containing fractions was confirmed by specific antibodies using the Western blotting technique ( Figure 2 a, b, c). In order to accept exosomes derived from "active" mature immune cells as delivery cargo for the vaccine platform, we activated human B lymphocytes or peripheral blood mononuclear cells from healthy donors by CD40L. Their expansion and proliferation were provided by the additional presence of IL-2 to enhance the production of exosomes. Both types of activated isolated exosomes were validated by NTA analysis to confirm their size ( Figure 3 a, b) and then bioengineered them with all three tumor-associated antigens GAGE2D, MAGEA4, and ex5T4. The efficiency of the loading process was tested by immunoblotting ( Figure 3 c, d).

[0264] Example 2: Stimulation of immune cells

[0265] To investigate whether the designed vaccine could stimulate immune cells to fight cancer, we performed an in vitro "tumor killing assay" on three-dimensional H1299-GFP cancer spheroids co-cultured with immature peripheral blood mononuclear cells (PBMCs), which mimic tumors in the human body. Figure 4 a, b) compared to exposure to our mature B cell (B lymphocyte) derived activated exosomes (which were pulsed with GAGE2D, MAGEA4 and ex5T4 tumor-associated antigens ( Figure 4 a), or pulsed with GAGE2D and MAGEA4 ( Figure 4 b)) The 3D spheroids show not only a reduced size but also a “shrinking” pattern over a longer period of time ( Figure 4 a). A similar pattern was also observed when cancer spheroids were treated with activated PBMC-derived exosomes pulsed with all three tumor-associated antigens, indicating that a fraction of the activated PBMC-exosomes used for the vaccine contained a population of mature B cell exosomes ( Figure 4c) These results suggest that not only are exosomes derived from mature immune cells required, but also that the presence of our selected tumor-associated antigens is critical for the activation of specific immune cell subtypes from PBMCs that actively participate in the elimination of cancer cells within the tumor.

[0266] Example 3: Stimulation of cytokine production

[0267] To further characterize the effects of activated exosome-based TAA vaccines on stimulating immune responses, we investigated the effects of treatment on the production of cytokines of T cell activation. We selected IL-2 and IFN-γ cytokines, which represent type 1 T helper cell (Th-1) responses and are therefore involved in cell-cell mediated immunity and phagocyte-dependent protection mechanisms. Using an ELISA approach, we found that PBMCs produced higher levels of both types of cytokines in the presence of designed mature B cell-activated exosome-derived TAA vaccines compared to control treatments, in which mature B cell exosomes were either “empty,” not pulsed with the selected TAA, or not treated at all ( Figure 5 a, b). In addition, we also observed an increase in IL-17 cytokine production by PBMCs after treatment of PBMCs with the designed exosome-based vaccine ( Figure 5 c). IL-17 cytokine expression characterizes the presence of the Th-17 T helper cell subset, i.e. cells involved in mediating the host's defense mechanisms against various infections, including cancer cells.

[0268] Example 4: Study on the localization of TAA in bioengineered immune exosomes.

[0269] We further investigated whether immune exosomes bioengineered with tumor-associated antigens could directly stimulate and interact with surface molecules on immune cells responsible for anticancer responses. To answer this question, we examined the localization of TAAs in our engineered activated B lymphocyte- and PBMC-derived exosomes. Surprisingly, the extracellular domain of 5T4 and the intact MAGEA4 protein were localized or inserted into the exosome membrane, and in B lymphocyte- and PBMC-TAA-derived immune exosomes, only the GAGE2D tumor antigen was retained as cargo within the exosomes ( Figure 6 a). Our results suggest that our proposed therapy should be able to induce immune responses with higher efficiency through direct binding of TAAs localized on the surface of exosomes to specific molecules on immune cells.

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[0302] sequence

[0303] The Sequence Listing filed with this patent application is incorporated herein in its entirety as a part of the specification.

[0304] SEQ ID NO:1

[0305] Gene name: MAGEA4_txt; Melanoma-associated antigen 4 · Homo sapiens (human) · Gene: MAGEA4 (MAGE4); Uniprot: P43358 · MAGA4_HUMAN

[0306] MSSEQKSQHCKPEEGVEAQEEALGLVGAQAPTTEEQEAAVSSSSPLVPGTLEEVPAAESAGPPQSPQGASALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAESLFREALSNKVDELAHFLLRKYRAKELVTKAEMLERVIKNYKRCFPVIFGKASES LKMIFGIDVKEVDPASNTYTLVTCLGLSYDGLLGNNQIFPKTGLLIIVLGTIAMEGDSASEEEIWEELGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGSNPARYEFLWGPRALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGV

[0307] SEQ ID NO:2

[0308] Gene Name: GAGE2D_txt; G antigen 2D·Homo sapiens (human)·Gene: GAGE2D; GAGE8; Uniprot: Q9UEU5·GGE2D_HUMAN

[0309] MSWRGRSTYRPRPRRYVEPPEMIGPMRPEQFSDEVEPATPEEGEPATQRQDPAAAQEGEDEGASAGQGPKPEADSQEQGHPQTGCECEDGPDGQEMDPPNPEEVKTPEEGEKQSQC

[0310] SEQ ID NO:3

[0311] Full amino acid sequence of 5T4:

[0312] MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLGIVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYHYRYEINADPRLTNLSSNSDV

[0313] SEQ ID NO:4

[0314] Extracellular domain of 5T4_txt; sp|Q13641|TPBG_HUMAN|32 - 355 OS=Homo sapiens OX=9606 GN=TPBG

[0315] SSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTS

Claims

1. A composition comprising one or more exosome populations, wherein the exosomes in the first exosome population: (a) each display CD19 and one or more additional surface molecules characteristic of mature activated B lymphocytes, and; (b) each comprises (preferably displays) one or more tumor antigens selected from the group consisting of MAGEA4, GAGE2D and 5T4, The exosomes in the first exosome population contain (preferably display) all three tumor antigens.

2. The composition according to claim 1, wherein the tumor antigens are all full-length proteins.

3. A composition according to claim 1 or claim 2, wherein the one or more additional surface molecules are selected from BCR, CD138, CD86, CD80, MHC I and MHC II.

4. The composition according to any one of the preceding claims, wherein the first population of exosomes is obtained or derived from: (i) mature or activated B lymphocytes; or (ii) A PBMC population comprising mature or activated B lymphocytes.

5. The composition according to claim 4, wherein the exosomes are obtained or derived from: (i) the mature B lymphocyte; or (ii) said PBMC population comprises mature B lymphocytes, It is subsequently modified to contain (preferably display) one or more of said tumor antigens.

6. The composition according to claim 4, wherein: (i) the activated B lymphocytes; or (ii) said PBMC population comprises activated B lymphocytes, The exosomes are modified before production to contain (preferably display) the tumor antigen.

7. A composition according to any one of the preceding claims, wherein the mature or activated B lymphocytes are matured in vitro, preferably by using CD40L and IL-2.

8. A composition according to any one of the preceding claims, wherein: (i) the mature B lymphocyte; or (ii) said PBMC population comprises mature B lymphocytes, Obtained from healthy donors.

9. The composition according to any one of the preceding claims, wherein the composition additionally comprises one or more additional exosome populations which have been obtained or derived from immune cells, preferably selected from monocytes, dendritic cells, NK cells and T lymphocytes.

10. The composition of claim 9, wherein one or more of the additional exosome populations comprises (preferably displays) one or more of the tumor antigens.

11. The composition according to any one of the preceding claims, wherein the exosomes in the first exosome population all comprise (preferably display) all three of the tumor antigens.

12. The composition of any one of claims 1 or 3 to 11, wherein 5T4 is the extracellular domain of a 5T4 polypeptide.

13. A pharmaceutical composition comprising the composition of any one of the preceding claims, optionally together with one or more pharmaceutically acceptable carriers, diluents, adjuvants or excipients.

14. A tumor vaccine, comprising the pharmaceutical composition of claim 13.

15. A pharmaceutical composition, comprising: (A) a first exosome population, wherein the exosomes: (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and (b) each comprises (preferably displays) a first tumor antigen, wherein the first tumor antigen is MAGEA4; and (B) a second population of exosomes, wherein the exosomes: (a) each display CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and (b) each comprises (preferably displays) a second tumor antigen, wherein the second tumor antigen is GAGE2D; and (C) a third exosome population, wherein the exosomes: (a) each displays CD19 and one or more additional surface molecules characteristic of mature, activated B lymphocytes; and (b) each comprises (preferably displays) a third tumor antigen, wherein the third tumor antigen is 5T4; Preferably, the surface molecule, exosome population and / or tumor antigen is as defined in any one of claims 2 to 12, As a combined preparation, it is used for treating or preventing cancer, or for inducing immune cell response against the tumor antigen in a subject in a form suitable for simultaneous, separate or sequential use.

16. A composition according to any one of claims 1 to 13 for use in therapy or as a medicament.

17. A composition according to any one of claims 1 to 13: (i) for use in a method of preventing or treating cancer in a subject; or (ii) for use in a method of inducing a T cell or B cell response against a cancer antigen in a subject; or (iii) for inducing an adaptive or innate immune cell response in a subject.

18. Use of the composition according to any one of claims 1 to 12 in the preparation of a medicament, wherein: (i) for preventing or treating cancer in a subject; or (ii) for inducing a T cell or B cell response against a cancer antigen in a subject; or (iii) for inducing an adaptive or innate immune cell response in a subject.

19. A method for preventing or treating a subject susceptible to or suffering from cancer, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 1 to 13.

20. A method for inducing a T cell or B cell response against a cancer antigen in a subject or for inducing an adaptive or innate immune cell response in a subject, the method comprising administering an effective amount of the composition of any one of claims 1 to 13 to a subject in need thereof.

21. The composition, use or method of any one of claims 17 to 20, wherein the cancer is selected from colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, and blood cancer (e.g., leukemia, lymphoma and myeloma).

22. The composition for use, the use or the method of claim 21, wherein the cancer is a solid tumor, a liquid cancer or a metastasis.

23. A method for producing a population of activated CD19+ exosomes, said exosomes comprising (preferably displaying) one or more tumor antigens, said method comprising the following steps: (a) maturing immature B lymphocytes to generate mature B lymphocytes displaying CD19; (b) producing exosomes from the mature B lymphocytes; as well as (c) incorporating one or more tumor antigens into each of the exosomes (preferably into the membrane of the exosomes), wherein the tumor antigens are MAGEA4, GAGE2D and 5T4; To generate an activated CD19+ exosome population, each of which contains (preferably displays) one or more of the tumor antigens, and wherein the exosome population contains (preferably displays) all three of the tumor antigens.

24. A method for generating a population of activated CD19+ exosomes, said exosomes comprising (preferably displaying) one or more tumor antigens, said method comprising the following steps: (a) expressing one or more tumor antigens (preferably cell surface tumor antigens) in each cell in a cell population comprising immature or mature B lymphocytes, wherein the tumor antigens are MAGEA4, GAGE2D and 5T4, thereby maturing and activating the immature or mature B lymphocytes to generate mature activated B lymphocytes expressing CD19; as well as (b) producing activated CD19+ exosomes from the mature activated B lymphocytes; To generate an activated CD19+ exosome population, each of which contains (preferably displays) one or more of the tumor antigens, and wherein the exosome population contains (preferably displays) all three of the tumor antigens.

25. A method for producing a population of activated CD19+ exosomes, said exosomes comprising (preferably displaying) one or more tumor antigens, said method comprising the following steps: (a) maturing immature B lymphocytes in a cell population comprising immature B lymphocytes to generate mature activated B lymphocytes that display CD19; (b) expressing one or more tumor antigens (preferably cell surface tumor antigens) in each of the mature B lymphocytes, thereby activating the B lymphocytes, wherein the tumor antigens are MAGEA4, GAGE2D and 5T4; and (c) producing activated CD19+ exosomes from the mature activated B lymphocytes, To generate an activated CD19+ exosome population, each of which contains (preferably displays) one or more of the tumor antigens, and wherein the exosome population contains (preferably displays) all three of the tumor antigens.

26. The method according to any one of claims 23 to 25, wherein the immature B lymphocytes are present in a PBMC population, or wherein the cell population comprising immature B lymphocytes is a PBMC population.

27. The method according to any one of claims 23 to 26, wherein the immature B lymphocytes are obtained from a healthy subject.

28. The method according to any one of claims 23 to 27, wherein the immature B lymphocytes are matured in vitro using CD40L and IL-2.

29. A method for producing a pharmaceutical composition, the method comprising the method for producing a population of exosomes according to any one of claims 23 to 28, the exosomes comprising (preferably displaying) one or more tumor antigens, and wherein the method further comprises the following steps: (d) mixing the exosome population with one or more pharmaceutically acceptable diluents, excipients, adjuvants or carriers.

30. A population of exosomes or a pharmaceutical composition obtained or obtainable by the method of any one of claims 23 to 29.

Images