Method of producing immunotherapy vaccine

Abstract

A method of producing an immunotherapy vaccine is provided. The method includes performing a heat treatment to exosomes separated from cancer cells or body fluids including blood of a cancer patient to promote inactivation of proteolytic enzymes in the exosomes, and introducing or co-culturing the exosomes in dendritic cells derived from blood of the cancer patient or a healthy person to make antigen-presenting cells.

Claims

1. A method of producing an immunotherapy vaccine, the method comprising: performing a heat treatment under a condition of 56° C. for 30 minutes on exosomes separated from cancer cells or bodily fluids from a cancer patient to promote inactivation of proteolytic enzymes in the exosomes; and introducing or co-culturing the exosomes in the presence of dendritic cells derived from blood from the cancer patient or a healthy person to make antigen-presenting cells.

2. The method of producing an immunotherapy vaccine according to claim 1, wherein the exosomes are maintained as stable particles by inactivating the proteolytic enzymes by the heat treatment.

3. The method of producing an immunotherapy vaccine according to claim 1, wherein the dendritic cells are co-cultured with T cells to make cytotoxic T cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a microphotograph of exosomes with the heating conditions of 56° C. and 30 minutes.

(3) FIG. 2 is a microphotograph of exosomes with the heating conditions of 56° C. and 30 minutes.

(4) FIG. 3 is a microphotograph of exosomes with the heating conditions of 56° C. and 30 minutes.

(5) FIG. 4 is a graph showing the results of comparing the period after the heat treatment of heat-treated exosomes with the lysis activity (% of lysis).

(6) FIG. 5 is fluorescence (green fluorescence and red fluorescence) and bright-field microphotographs of proteins of dendritic cells (DCs) to which exosomes have been introduced, in which the upper row shows DCs to which non-heat treated exosomes have been introduced by electroporation, the middle row shows DCs to which exosomes heat-treated under the conditions of the present invention have been introduced by co-culturing, and the lower row shows DCs to which exosomes heat-treated under the conditions of the present invention have been introduced by electroporation.

DETAILED DESCRIPTION

(7) A preferred embodiment of the present invention will be described below. The scope of rights of the present invention is not limited by this embodiment.

(8) (A) Extraction of Exosomes from Cell Culture Supernatant Step 1: After culturing for 2 days in a medium supplemented with exosome-depleted fetal calf serum (FCS), exosomes were extracted from the culture supernatant. For the extraction, the miRCURY Exosome Cell/Urine/CSF Kit (Takara Bio Cat. 300102) is used as an isolation kit. Step 2: The number of exosome particles extracted in Step 1 was counted. Step 3: The exosomes were inactivated by performing a heat treatment (56° C. for 30 minutes).

(9) The cell membrane of an exosome easily breaks down when heated. Fusion with DCs is not possible when the cell membrane has collapsed. From this viewpoint, the upper temperature limit and the upper time limit of the heat treatment were determined by changing the heating conditions. The determination was made by observing the membrane structure of the exosomes from microphotographs taken at each condition.

(10) In all of FIGS. 1 to 3, heating conditions of 56° C. for 30 minutes were selected. In each figure, the circular dark part shows the membrane structures maintained by exosomes, and the circular whitish part which is larger than the dark part shows exosomes in which the membrane structure has collapsed. No nucleus is found in exosomes where the membrane structure is maintained, providing confirmation that the exosome is secreted from the cytoplasm. Among exosomes whose membrane structure has collapsed, there are also those in which the nucleus has disappeared.

(11) A temperature condition of less than 56° C., including 37° C. (body temperature), cannot be considered as heating, and so would be a normal exosome without denaturation of the active group of the enzyme. When the temperature exceeds 56° C., the number of exosomes whose membrane structure collapsed increased. The same applies to the treatment time. In the case of 56° C. for 30 minutes, as is clear from the microphotographs, exosomes in which the membrane structure has collapsed to some extent are also mixed therein. This proves that there are exosomes whose membrane structure has not collapsed and that the enzymatic denaturation in the exosomes is in progress. Therefore, it is desirable to perform the heat treatment under the conditions of 56° C. or lower and 30 minutes or less, and preferably close to 56° C. and 30 minutes as much as possible.

(12) (B) Lysis of tumor cells by activated cytotoxic T cells (CTLs)

(13) Step 1: After collecting blood from a healthy person, DCs were induced from peripheral blood mononuclear cells. Specifically, peripheral blood (50 ml) is collected. Then, centrifugation (c.f.g. 1800×g for 15 mins) is performed and the cells in the intermediate layer are collected. After this, the cells are washed twice with PBS (c.f.g. 1200 rpm for 5 mins), the number of cells is counted, and the cells are resuspended in a medium (CTS AIM V medium (Gibco, Thermo Fisher Scientific)) at 2×10.sup.6/ml.

(14) The cells are seeded at 1 ml/well in a 24-well plate and incubated at 37° C. in a 5% CO.sub.2 incubator for 2 hours. Then, the floating cells are removed, and a medium for the DCs (AIM V medium supplemented with cytokines (final 10 ng/ml GM-CSF and IL-4, Miltenyi Biotec) and 2% autologous plasma) is added at 1 ml/well, whereupon culturing is commenced.

(15) Subsequently, 500 ul of fresh dendritic cell medium is added to the well, and cytokines for maturation (final 10 ng/ml TNFα, Miltenyi Biotec) are added.

(16) Step 2: The following test sections (1) to (3) were performed as antigen stimulation of DCs. (1) Cellular fusion of DCs and tumor cells with PEG (polyethylene glycol) (2) Electroporation of DCs and exosomes (3) Co-culture of DCs and exosomes

(17) Step 3: Co-culture of DCs activated in the above test sections (1) to (3) and CTLs was performed.

(18) Step 4: Co-culture of the above co-cultured CTLs and tumor cells was performed.

(19) The lysis of the cytotoxic T cells (CTLs) was observed with an MTT assay. The results are shown in FIGS. 4 and 5.

(20) FIG. 4 shows the relationship between the target:effector ratio and the lysis of CTLs. The CTLs in which exosomes heat-treated by the method of the present invention are introduced to DCs by electroporation to make antigens exhibit the equal lysis to CTLs obtained by cellular fusion with current PEG (polyethylene glycol) and co-culture.

(21) In particular, when the effector is increased in the target:effector (horizontal axis), the lysis is higher than that of PEG cell fusion and co-culture.

(22) Further, it can be seen from FIG. 5 that the expression amount of protein by exosomes when heat-treated increases. Therefore, the vaccine obtained by the method of the present invention is effective for immunotherapy.

(23) The vaccine of the present invention, that is, a vaccine obtained by introducing heat-treated exosomes to DCs by electroporation or the like, functions as APCs. When these APCs are administered to the patient's body by subcutaneous injection or the like, the APCs migrate to the lymph node, educate the T cells in the lymph node, and thereby change the T cells to CTLs. As mentioned above, since the number of exosomes is extremely large compared to DCs, the number of DCs that take in exosomes is also large. As the number of such DCs increases, the number of APCs increases, and CTLs also increases, which enhances the immune effect.

(24) It is also possible to co-culture DCs to which exosomes have been introduced with T cells so as to make CTLs that can be used as a vaccine.