CH716482A2 - Process for the preparation of an agent for suppressing metastases containing exosomes. - Google Patents

Abstract

Disclosed herein is a method of making a therapeutic agent containing exosomes, comprising the steps of: separating tumor cells from a biomaterial sample of a subject; Treating the separated tumor cells in a culture medium at 37 ° C. and 5% CO 2 for 24-72 h or treating the separated; Separating an exosome composition from the culture medium; Dissolving the separated exosome composition in a phosphate buffer to obtain therapeutic agents containing the exosome; characterized in that the culture medium comprises (i) either a 1: 1 mixture of DMEM / F12 (Dulbecco's Modified Eagle Medium: nutrient mixture F-12) and HEPES (4- (2-hydroxyethyl) -1-piperazine-ethanesulfonic acid), 2 mmol / ml or 3.65 mg / 10 ml L-glutamine, 100 units / ml penicillin, 100 µg / ml streptomycin, 10% fetal bovine serum; or (ii) 79 wt% RPMI, 20 wt% fetal bovine serum, and 1 wt% streptomycin.

Description

Area of revelation

The present invention relates to a method for producing an exosome-containing therapeutic agent for suppressing metastases and a method for treatment with an exosome-containing therapeutic agent.

Background, prior art

RO 2593003 C2 discloses a method for tumor immunotherapy based on the administration of an exosome-containing drug to the patient, cancer patients being treated with exosomes isolated from the cell culture K562 / i-S9, which does not allow specific Obtaining exosomes, which is why it is not possible to achieve a high efficiency of the immune response, in contrast to the exosomes, which are produced by the patient's own tumor cells - which contain specific antigens, similar to the antigens of tumor cells, which allow a highly effective immune response to achieve. As a result, the known method is not effective enough.

Summary of the disclosure

It is known that exosomes are involved in various stages of immune response reactions. In particular, exosomes that are secreted by B cells carry receptors for MHC II (the major histocompatibility complex II), co-stimulatory and adhesive molecules on their surface, which shows their ability to stimulate CD4-positive T cells directly. [B lymphocytes secrete antigen-presenting vesicles. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R., Harding CV, Melief CJ, Geuze HJ, J. Exp. Med., March 1, 1996; 183 (3): 1161-72.] In order to intensify this effect, exosomes are concentrated, for example, by magnetic beads and loaded with peptides. In addition to the ability to activate CD4-positive cells, exosomes that are obtained from dendritic cells and pooled with peptides from processed tumor antigens are able to activate tumor-specific cytotoxic lymphocytes and thereby inhibit tumor growth. [Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G, Amigorena S, Nat. Med., May 1998; 4 (5): 594-600.]

[0004] Exosomes secreted by tumor cells also act as activators of the immune system, but in the course of the tumor process, transformations occur that allow atypical cells to escape the effects of immunocompetent cells. One of these transformations is the inability of lymphocytes to recognize exosomes as carriers of genetic material from the tumor. When exosomes obtained from the patient are used per os, there is contact between the cells of the immune system and the exosomes synthesized by the tumor cells, which can lead to the activation of anti-tumor immunity.

It is a general object of the invention to provide an advanced therapeutic agent for suppressing metastases. In preferred embodiments, a therapeutic agent having increased effectiveness is provided.

In a first aspect of the invention, the general object is achieved by a method for producing a therapeutic agent containing exosomes, comprising the following steps: Separating tumor cells from a biomaterial sample of a subject; Treating the separated tumor cells in a culture medium at 37 ° C and 5% CO2 for 24-72 hours or treating the separated; Separating an exosome composition from the culture medium; Dissolving the separated exosome composition in a phosphate buffer to obtain the exosome-containing therapeutic agent; characterized in that the culture medium comprises: (i) either a 1: 1 mixture of DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, according to Dulbecco, R. & Freeman, G. (1959) Virology 8, 396, and Smith et al. (1960) Virology 12, 185) and HEPES (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), 2 mmol / ml or 3.65 mg / 10 ml L-glutamine, 100 units / ml penicillin, 100 µg / ml streptomycin, 10% fetal bovine serum; or (ii) 79 wt% RPMI, 20 wt% fetal bovine serum, and 1 wt% streptomycin.

As those skilled in the art will understand, RPMI, also known as RIMO 1640, is a growth medium used in cell culture (Atlas RM, Snyder JW (2006). Handbook of Media for Clinical Microbiology (2nd ed.). Boca Raton, Florida : CRC Press, p. 427).

It will be understood that the method for producing a therapeutic agent containing exosomes, and in particular the step of separating tumor cells, is carried out in vitro.

Typically, the separation of tumor cells from a biomaterial sample of a subject is accomplished by centrifugation.

In some embodiments, the dissolving of the separated exosome composition in a phosphate buffer is carried out such that a ratio of 1: 5 to 1:20 of separated exosome composition to phosphate buffer is obtained.

In further embodiments, the biomaterial sample consists of venous blood. Alternatively, it is also possible to use peripheral blood or tumor tissue of the subject obtained intraoperatively or an ascites / pleuritis fluid of metastatic origin.

In preferred embodiments, separating the tumor cells comprises: a. Exposure of the biomaterial to an EDTA anticoagulant; b. Dilute with Hanks' saline by a factor of 2; c. Applying the diluted mixture to a Ficoll-Urografin (F-U)) solution with a density of 1.077 g / cm 3 in a ratio of 1: 4, followed by centrifugation at 2000 rpm for 15 minutes; d. Collecting a generated interphase ring; e. Diluting the interphase ring with a phosphate buffer solution in a ratio of 1:15 to 1:20 and then centrifuging at 1500 rpm for 10 minutes to provide a sediment; f. collecting the sediment, adding lysing solution and keeping the collected sediment and lysing solution at 15-25 ° C for 10 minutes; G. Mix with a phosphate buffer with 0.9 M CaCl2 solution h. Collect the tumor cells.

The advantage of this method of isolating tumor cells is the ability to obtain the most quantitative pool of tumor cells possible while maintaining their viability and sterility. At the same time, this method allows, with minimal loss of tumor cells, to eliminate cells that do not fall into the pool of interest, such as red blood cells, leukocytes and, in some cases, platelets. The use of certain types of enzymes, such as Accutase, minimizes damage to cell surface receptors and antigenic determinants. The centrifugation conditions are chosen in such a way that a differentiation between cell debris (cell fragments and decomposition products) with exosomes of cells is made possible; the working conditions described allow a supernatant containing a precipitate with cell fragments, decomposition products and exosomes Obtain fraction with the highest content of exosomes from the total sediment.

In some embodiments, the step of separating the exosome composition from the culture medium further comprises differential centrifugation.

In further embodiments, differential centrifugation is carried out for 10 minutes at 3000 rpm, followed by collecting a generated first supernatant and centrifuging the first supernatant for 30 minutes at 10,000 rpm, followed by collecting a second supernatant generated and from ultracentrifuging the second supernatant for 2 hours at 100,000 rpm, with an exosome composition being separated after each centrifugation step.

In a second aspect, the invention is directed to a method of treating metastasis in a subject, comprising: Taking a biomaterial sample from the subject; Separating tumor cells from the biomaterial sample; Treating the separated tumor cells in a culture medium at 37 ° C and 5% CO2 for 24-72 hours or treating the separated; Separating an exosome composition from the culture medium; Dissolving the separated exosome composition in a phosphate buffer to obtain therapeutic agents containing the exosome; administering the therapeutic agent intravenously to the subject; characterized in that the culture medium comprises: (i) either a 1: 1 mixture of DMEM / F12 and HEPES, 2 mmol / ml or 3.65 mg / 10 ml L-glutamine, 100 units / ml penicillin, 100 µg / ml streptomycin, 10% fetal bovine serum; or (ii) 79 wt% RPMI, 20 wt% fetal bovine serum, and 1 wt% streptomycin.

In some embodiments, the dissolution of the separated exosome composition in a phosphate buffer is carried out so that a ratio of 1: 5 to 1:20 of separated exosome composition to phosphate buffer is obtained.

In further embodiments, the biomaterial sample consists of venous blood. Alternatively, it is also possible to use peripheral blood or tumor tissue of the subject obtained intraoperatively or an ascites / pleuritis fluid of metastatic origin.

In preferred embodiments, separating the tumor cells comprises: a. Exposure of the biomaterial to an EDTA anticoagulant; b. Dilute with Hanks' saline by a factor of 2; c. Applying the diluted mixture to a diatrizaoate) (urografin)) solution with a density of 1.077 g / cm 3 in a ratio of 1: 4, followed by centrifugation for 15 minutes at 2000 rpm; d. Collecting a generated interphase ring; e. Diluting the interphase ring with a phosphate buffer solution in a ratio of 1:15 to 1:20 and then centrifuging at 1500 rpm for 10 minutes to provide a sediment; f. collecting the sediment, adding lysing solution and keeping the collected sediment and lysing solution at 15-25 ° C for 10 minutes; G. Mix with a phosphate buffer with 0.9 M CaCl2 solution h. Collect the tumor cells.

In some embodiments, the step of separating the exosome composition from the culture medium further comprises differential centrifugation.

In further embodiments, differential centrifugation is carried out for 10 minutes at 3000 rpm, followed by collecting a generated first supernatant and centrifuging the first supernatant for 30 minutes at 10,000 rpm, followed by collecting a second supernatant generated and from ultracentrifuging the second supernatant for 2 hours at 100,000 rpm, with an exosome composition being separated after each centrifugation step.

Exemplary embodiments

Patient, 14 years old, schoolboy, medical history without abnormalities, acute B-lymphoblastic leukemia, cytogenetic variant t (1; 19) (q23; p13), E2A / PBX1. Blasts in the bone marrow aspirate (CD19 +, CD79 +, CD22cyt +, TdT +, HLADR +) 17.5%, the leukocyte component exceeds the upper limit, neutrophil metamyelocytes are the most elevated (up to 67%), myeloblasts are not defined. The hemogram shows: blast cells 9%, metamyelocytes 3%, erythropenia up to 2.9 * 10 ^ 12 / liter, leukopenia 2.3 * 10 ^ 9 / liter. At the maintenance therapy stage, exosomes obtained from autologous tumor cells were administered orally twice a week together with 6-mercaptopurine.

Two months after drug administration and on maintenance therapy, the patient showed a decrease in leukopenia from 2.3 * 10 ^ 9 / liter to 3.1 * 10 ^ 9 / liter, indicating a gradual increase in the proportion of mature leukocytes in the bloodstream indicates; the number of blasts in the bone marrow aspirate drops from 17.5% to 14%, blast cells are not found in the hemogram. The immunological parameters show an increase in the cytotoxic activity of NK (Natural Killer) cells from 65% before the drug administration to 78% after the drug administration (with a ratio of effector cells to target cells of 1: 5). These changes underpin the improvement in the patient's condition and the stabilization of the process.

The tumor cells were isolated from the patient's venous blood. The venous blood is drawn into a test tube with an anticoagulant EDTA, then the resulting blood is diluted 2-fold with Hanks' solution, then it is made into a Fikoll urographin solution with a density of 1.077 g / cm 2 at a Coated ratio of 1: 4, then centrifuged for 15 minutes at 4 ° C at 2000 rpm, then the interphase ring formed during centrifugation is collected and phosphate buffer-saline buffer is added at a ratio of 1:15, and then for 10 minutes centrifuged at 1500 rpm whereupon the precipitate is isolated and 2 ml of the lysis solution is added and the resulting mixture is kept at 15 ° C for 10 minutes, after which the phosphate buffer-saline buffer is added to the mixture at a ratio of 1:10 and stirred becomes. This is then centrifuged for 10 minutes at 1500 rpm, after which the supernatant is poured out and 4 ml of phosphate buffer saline solution buffer are added to the precipitate, further containing 0.9 M CaCl2, and stirred, then tumor cells from the suspension obtained are in the form of a precipitate isolated by passing it through a micro-tube. The resulting precipitate with tumor cells is then cultivated in a culture medium of the following composition: 79% RPMI, 20% FBS, 1% penicillin-streptomycin solution, under conditions of t = 37 ° C and 5% CO2, for 72 hours, The supernatant is isolated every 24 hours and culture medium of the following composition is added to the remaining precipitate: 79% RPMI, 20% FBS, 1% penicillin-streptomycin solution, in the volume of the isolated supernatant.

An isolation of exosome-containing sediment from the resulting culture medium is carried out by differential centrifugation, namely: The resulting culture medium is centrifuged for 10 minutes at 3000 rpm, then the resulting supernatant is collected and further centrifuged at 10,000 rpm for 30 minutes . Then the resulting supernatant is collected and ultracentrifugation is carried out at 100,000 rpm for 2 hours, after which the precipitate formed is isolated.

1.1. To create a tumor model for the purpose of studying the processes of metastasis and activation of the immune response, immunocompetent male mice of the C57BL / 6 line aged no more than 4 weeks were selected. n = 27.1 × 10 5 cells of the murine melanoma line F10 / B16 in phosphate-buffered saline solution (PBS, 100,000 cells / 0.1 ml PBS) were injected into the tail vein of all animals. The metastatic process took place for 8 days up to the time of exposure.

1.2. To obtain a preparation, the F10 / B16 line murine melanoma cells were thawed and cultured for 72 hours to reach the amount of 6,000,000. They were then centrifuged at 3000 rpm, and the supernatant was given to the customer to make the preparation.

1.3. The animals were divided into 3 groups: No. 1 (i.v. injection), No. 2 (oral administration), No. 3 (control).

1.4. The mice were housed under standard vivarium conditions with free access to briquette-shaped food and water. The preparation was administered on the 8th and 11th day after the inoculation date: • in group 1 100 µg of the preparation were injected into the tail vein, • in group 2 100 µg of the preparation was administered orally, • in group 3 100 µg of pure PBS ( without preparation) is injected into the tail vein.

The slaughter took place 16 days after the inoculation of the tumor cells using the broken neck method. To assess the process of metastasis, a visual assessment of the presence and number of metastases in the liver was carried out and their mass was measured. Statistical significance was assessed using a one-way analysis of variance in the SPSS 22 program. • Average number of liver metastases in group no. 1: 5.5 + -0.5. Average liver mass: 1.2 + -0.1. • Average number of liver metastases in group No. 2: 3.8 + -0.3. Average liver mass: 1.1 + -0.2 • Average number of liver metastases in group no. 3: 9.0 + -2.1. Average liver mass: 1.4 + -0.2

The differences in the number of metastases in the observation groups compared to the control are significant (p <0.05). The difference in the number of metastases between groups 1 and 2 is on the verge of significance (p <0.1). The liver mass in the observation and control groups differed insignificantly (p> 0.05).

To assess the activation of the immune system, splenocytes were isolated from the animal spleen, the percentage of cytotoxic CD8 <+> - T lymphocytes being determined by means of the flow cytometry method. For this purpose, the splenocytes were washed in PBS after isolation and incubated for one hour at 4 ° C. with anti-CD8 antibodies, with further analysis being carried out on the flow cytofluorometer from Beckman & Dickinson.

The average percentage of CD8 <+> - T lymphocytes in group No. 1: 27%

The average percentage of CD8 <+> - T lymphocytes in group No. 2: 36%

The average percentage of CD8 <+> - T lymphocytes in group No. 2: 17%

The “observation-control” values differ significantly (p <0.05). The difference between groups 1 and 2 is significant (p <0.05).

Oral administration of the preparation inhibits the metastasis process comparatively more strongly than intravenous injection. Similar observations were made during the analysis of the degree of activation of the immune system. The process may be related to the macrophage-mediated antigen presentation of the tumor to T-killers.

Claims (12)

A method of making a therapeutic agent containing exosomes, comprising the steps of: Separating tumor cells from a biomaterial sample of a subject; - treating the separated tumor cells in a culture medium at 37 ° C and 5% CO2 for 24-72 h or treating the separated; Separating an exosome composition from the culture medium; Dissolving the separated exosome composition in a phosphate buffer to obtain therapeutic agents containing the exosome; characterized in that the culture medium comprises: (i) either a 1: 1 mixture of DMEM / F12 and HEPES, 2 mmol / ml or 3.65 mg / 10 ml L-glutamine, 100 units / ml penicillin, 100 µg / ml streptomycin, 10% fetal bovine serum; or (ii) 79 wt% RPMI, 20 wt% fetal bovine serum, and 1 wt% streptomycin. 2. The method of claim 1, wherein the dissolving of the separated exosome composition in a phosphate buffer is carried out so that a ratio of 1: 5 to 1:20 of separated exosome composition to phosphate buffer is obtained. 3. The method according to any one of the preceding claims, wherein the biomaterial sample consists of venous blood. 4. The method according to any one of the preceding claims, wherein separating the tumor cells comprises: - exposure of the biomaterial to an EDTA anticoagulant; - dilute with Hanks saline by a factor of 2; - applying the diluted mixture to a Ficoll-urografin (F-U) solution with a density of 1.077 g / cm 3 in a ratio of 1: 4, followed by centrifugation for 15 minutes at 2000 rpm; - collecting a generated interphase ring; - Diluting the interphase ring with a phosphate buffer solution in a ratio of 1:15 to 1:20 and then centrifuging for 10 minutes at 1500 rpm to provide a sediment; - collecting the sediment, adding lysing solution and keeping the collected sediment and lysing solution at 15-25 ° C for 10 minutes; - Mix with a phosphate buffer containing 0.9 M CaCl2 solution - Collecting the tumor cells. 5. The method according to any one of the preceding claims, wherein the step of separating the exosome composition from the culture medium further comprises differential centrifugation. The method according to claim 5, wherein differential centrifugation is carried out for 10 minutes at 3,000 rpm, followed by collecting a generated first supernatant and centrifuging the first supernatant for 30 minutes at 10,000 rpm, followed by collecting a generated second Supernatant and from ultracentrifuging the second supernatant for 2 hours at 100,000 rpm, with an exosome composition being separated after each centrifugation step. 7. A method of treating metastasis in a subject comprising: - taking a biomaterial sample from the subject; - Separation of tumor cells from the biomaterial sample; - treating the separated tumor cells in a culture medium at 37 ° C and 5% CO2 for 24-72 h or treating the separated; Separating an exosome composition from the culture medium; Dissolving the separated exosome composition in a phosphate buffer to obtain therapeutic agents containing the exosome; - administering the therapeutic agent intravenously to the subject; characterized in that the culture medium comprises: (iii) either a 1: 1 mixture of DMEM / F12 and HEPES, 2 mmol / ml or 3.65 mg / 10 ml L-glutamine, 100 units / ml penicillin, 100 µg / ml streptomycin, 10% fetal bovine serum; or (iv) 79 wt% RPMI, 20 wt% fetal bovine serum, and 1 wt% streptomycin. 8. The method of claim 7, wherein the dissolving of the separated exosome composition in a phosphate buffer is carried out so that a ratio of 1: 5 to 1:20 of separated exosome composition to phosphate buffer is obtained. 9. The method according to any one of claims 7 or 8, wherein the biomaterial sample consists of venous blood. 10. The method according to any one of claims 7 to 9, wherein separating the tumor cells comprises: - exposure of the biomaterial to an EDTA anticoagulant; - dilute with Hanks saline by a factor of 2; - applying the diluted mixture to a Ficoll-urografin (F-U) solution with a density of 1.077 g / cm 3 in a ratio of 1: 4, followed by centrifugation for 15 minutes at 2000 rpm; - collecting a generated interphase ring; - Diluting the interphase ring with a phosphate buffer solution in a ratio of 1:15 to 1:20 and then centrifuging for 10 minutes at 1500 rpm to provide a sediment; - collecting the sediment, adding lysing solution and keeping the collected sediment and lysing solution at 15-25 ° C for 10 minutes; - Mixing with a phosphate buffer containing 0.9 M CaC12 solution; - Collecting the tumor cells. 11. The method according to any one of claims 7 to 10, wherein the step of separating the exosome composition from the culture medium further comprises differential centrifugation. 12. The method of claim 11, wherein differential centrifugation is carried out for 10 minutes at 3000 rpm, followed by collecting a generated first supernatant and centrifuging the first supernatant for 30 minutes at 10,000 rpm, followed by collecting a generated second Supernatant and from ultracentrifuging the second supernatant for 2 hours at 100,000 rpm, an exosome composition being separated after each centrifugation step.