RU2593003C2 - Exosomes-onkosom preparation and method for immunotherapy of solid tumours using thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to medicine, particularly to a method for immunotherapy of solid tumours. Method for immunotherapy of solid tumours involves the following steps: vaccinating a patient with a combination of an exosomes preparation and P-type oligodesoxynukleotide, wherein the preparation contains exosomes recovered by K562/i-S9 cell culture, and contains protein, CEA and 5T4 antigens, ligands FasL and TRAIL, and the P-type oligodesoxynukleotide represents a sequence of nucleotides GGGGCGCCGTGATGGCGAGCGCGCC, the said combination is introduced subcutaneously for at least 2 months and at least 1 time a week in an effective amount, preferably in doses of 0.01-0.04 mg/kg each. Preparation of exosomes recovered by K562/i-S9 cell culture containing as per the immune-enzyme analysis data protein, CEA and 5T4 antigens, ligands FasL and TRAIL in certain proportions.

EFFECT: use of the above described preparation increases the level of immune response and reduces the titre of tumor markers in volunteers.

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Description

FIELD OF THE INVENTION

The invention relates to medicine, in particular to oncology, and can be used to obtain a highly effective agent for the treatment of solid tumors, precancerous conditions and the prevention of malignant neoplasms.

State of the art

For the treatment of certain types of malignant neoplasms and the prevention of relapses, the embryonic antitumor modulator “EPOM” was proposed (RU 2240810 C2, publ. November 27, 2004). The preparation contains a set of normal fetal proteoglycans in which cancer embryonic antigens or tumor associated antigens (TAAs) are detected, of which a significant amount is cancer embryonic antigen - CEA, trophoblastic β-glycoprotein (TBG) and α-fetoprotein (AFP) .

CEA - the English name for CEA, as well as CEACAM5 or CD66e, is a 180-kDa glycoprotein that is overactively formed by colorectal cancer cells, as well as in epithelial tumors (Hammarstrom S. The carcinoembryonic antigen (CEA) family: structures, suggested function sand expression nin normal and malignant tissues // Semin Cancer Biol 1999, v. 9, pp. 67-81). This circumstance makes CEA attractive for immunotherapy (S. Dai, Tao W., Baomei W., Xiangyang Z., Fangming X., Taoyong S., Yanfeng W., Xuetao C. More Efficien tInduction of HLA-A * 0201-Restricted and Carcinoembryonic Antigen (CEA) - Specific CTL Response by Immunization with Exosomes Prepared from Heat-Stressed CEA-Positive Tumo rCells // Clin Cancer Res 2005; 11 (20): 7554-7563).

TWG - the English designation “5T4” - is a glycoprotein formed by epithelial tumors (Myers K.A. Isolation of a cDNA Encoding 5T4 Oncofetal Trophoblast Glycoprotein // The Journal of Biological Chemistry, vol. 269, No. 12, Mar. 25, 1994, p. 9319-9324). The antigen is a member of the 72-kDa transmembrane glycoprotein family, the extracellular part of the polypeptide chain of which includes two leucine-rich repeating domains separated by a “gap” of hydrophilic amino acids with 7 N-glycosylation sites and an endoplasmic tail of 44 amino acid residues (Starzynska, T ., Wiechowska Kozlowska, A., Marlicz, K., Bromley, M., Roberts, SA, Lawniczak, M., Kolodziej, B., Zyluk, A., Stern, PL 5T4 oncofetal antigen in gastric carcinoma and its clinical significance // Eur. J. Gastroenterol. Hepatol. 1998, v. 10 (1), pp. 479-484). The 5T4 antigen is concentrated on the outer surface of the cytoplasmic membrane of the microvilli, and its excessive expression in epithelial cells is associated with changes in morphology and increased mobility. Like CEA, 5T4 antigen is found in exosomes that secrete tumor cells (US Patent Application 20140141986) by budding from the cytoplasmic membrane, similar to the process of maturation of human immunodeficiency virus (HIV). Thus, we can consider replacing the source of the two most important tumor antigens (usually solid), which is currently EPOM, with a human tumor cell line producing exosomes containing CEA and 5T4.

However, among the proteins contained in exosomes, often possessing a powerful immunosuppressive property. Such proapoptotic molecules are FasL and TRAIL (Valenti R., V. Huber, P. Filipazzi, L. Pilla, G. Sovena, A. Villa, A. Corbelli, S. Fais, G. Parmiani, L. Rivoltini. Human Tumor -Released Microvesicles Promote the Differentiation of Myeloid Cells with Transforming Growth Factor-B-Mediated Suppressive Activity on T-Lymphocytes // Cancer Res 2006; 66 (18): 9290-9298). To overcome the immunosuppressive effect of exosomes in vivo (G. Andreola, L. Rivoltini, C. Castelli, V. Huber, P. Perego, P. Deho, P. Squarcina, P. Accornero, F. Lozupone, L. Lugini, A. Stringaro, A. Molinari, G. Arancia, M. Gentile, G. Parmiani, S. Fais. Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles // J. Exp. Med. May 20 2002, Volume 195, No. 10, 1303-1316) currently use two methodological approaches: the use of in vivo exosome inhibitors using reverse transcriptase inhibitors (RT) and the use of adjuvants such as unmethylated oligodeoxynucleotides (ODN). The use of RT inhibitors known to those working with HIV-infected individuals can significantly reduce exosome formation (US Patent Application 2007269878).

Another way to increase the effectiveness of tumor immunotherapy is the use of adjuvants, among which NK adjuvants, which are unmethylated ODNs, are distinguished by their activity (US Patents 8129351 and 8158592). The unique ability of unmethylated ODN to activate tumor immunotherapy (Hartmann, G., GJ Weiner, AM Krieg. 1999. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc. Natl. Acad. Sci. USA 96: 9305), is associated with the ability of ODN to penetrate human cells and bind to TLR receptors of innate immunity (Giordani L., M. Sanchez, I. Libri, M. G. Quaranta, B. Mattioli, M. Viora. IFN-α amplifies human naive In cell TLR-9-mediated activation and Ig production // J. Leukoc. Biol. 86: 261-271; 2009). In addition, unmethylated ODNs alone are capable of suppressing the growth of tumor cells (Krieg A.M. Development of TLR9 agonists for cancert herapy // J. Clin. Invest. (2007) 117 (5): 1184-1194).

An adjuvant based on viral particles has been described previously (Kirillova I.V., Sivov I.G., Samokhvalov E.I., Kulish D.M. The drug Isvak, based on recombinant virus-like particles of the hepatitis C virus, induces interferon alpha and stimulates B-cells // Technology for the production of biological products 2009, v. 1 (2), pp. 25-36). However, further studies showed that the active principle of Isvak was ONE with the original structure, but P-type (Bode C., G. Zhao, F. Steinhagen, T. Kinjo, DM Klinman. CpG DNA as a vaccine adjuvant // Expert Rev Vaccines 2011 April; 10 (4): 499-511).

The method of obtaining the EPOM preparation (RU 2240810 C2) consists in the fact that the embryonic native tissue is removed, washed, kept in alcohol, crushed, frozen in liquid nitrogen, homogenized to a powder state, diluted with water and disintegrated in a cooled ball mill until revealing rectified cell nuclei (more than 80%), the smallest cytoplasmic breaks and fragments of fibrous structures, process the resulting homogenate with ultraviolet light, centrifuge and treat the resulting supernatant with alcohol to isolate After sedimentation of glycoproteins, they process the precipitate of glycoproteins in a ball mill and separate them into a precipitate by centrifugation, and then sterilize the filtration and lyophilization of the target product.

A method for the prevention and treatment of precancerous pathological conditions with the use of EPOM (RU 2240810 C2) is characterized in that it includes vaccination of persons belonging to high cancer risk groups according to the test results, by subcutaneous administration of EPOM once a year in an effective amount, preferably at a dose of 0 , 01-0.04 mg / kg.

SUMMARY OF THE INVENTION

Thus, the object of the present invention is to expand the arsenal of agents for the effective treatment and prevention of malignant neoplasms using a new exosome preparation, ONCOSOM, in combination with P-type ONE (ONE).

The problem is solved by the preparation of exosomes isolated by the K562 / i-S9 cell culture, and containing, according to the enzyme immunoassay, protein in the amount of 0.9-1.5 μg / 10 ⁶ cells, CEA antigens in the amount of 0.045-0.075 μg / 10 ⁶ cells and 5T4 0.03-0.05 μg / 10 ⁶ cells, and FasL ligands less than 0.005 μg / 10 ⁶ cells and TRAIL less than 0.004 μg / 10 ⁶ cells.

The problem is also solved by the method of immunotherapy of solid tumors, which consists in vaccinating the patient with a combination of ONCOSOM exosome preparation and P-type oligodeoxynucleotide ODNP, which is a sequence of GGGGCGCCGTGATGGCGAGCGCGCC, which are administered subcutaneously for at least 2 months and at least 1 time, preferably at least once a week, preferably doses of 0.01-0.04 mg / kg each and preferably 3-8 times for 2 months.

To increase the effectiveness of treatment during the vaccination period, a revertase inhibitor drug can be orally administered. Revertase inhibitors inhibit the formation of the patient’s own exosomes of tumor cells that mask the effects of antibodies to tumor antigens. You can use any known drugs - revertase inhibitors (Stavudine, Phosphazide, Lamivudine and others) and apply them according to well-known specialist schemes.

The technical result of the invention is to increase the level of the immune response (humoral and cellular) and lower titers of tumor markers in volunteers.

As a producer of ONCOSOM, the K562 / i-S9 cell line, known to specialists as a powerful producer of CEA and 5T4 antigens, with reduced ability to produce FasL and TRAIL, is proposed. Within the framework of this aspect, it is proposed to co-administer ONCOSOM and ODNP subcutaneously at doses of 0.01-0.04 mg / kg each, preferably at a dose of 0.03 mg / kg and 0.02 mg, respectively. Combination therapy using the introduction of RT inhibitors according to schemes known to those skilled in the art gives even better results.

One aspect of this task is the development and use of P-type oligodeoxynucleotide (ODN) (ODN), the hallmark of which are two palindromes. They allow the formation of a duplex that activates B- and plasmacytoid dendritic cells, and also cause a significantly higher amount of IFN-α compared to C-type ODNs (Samulowitz U., Weber M., Weeratna R. A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies high potency in type Interferon induction with preferred structural properties // Oligonucleotides 2010; 20 (2): 93-101).

To achieve the solution of the problem allows the use in the proposed method ONKOSOM, ODNP and preferably RT inhibitors. Proposed in the invention ODNP (ONE R-type) has a powerful antimutagenic, anticlastogenic effect, interferonogenic and immunomodulatory effects, as well as antiviral activity.

Without claiming to be an exhaustive and final scientific justification of the positive effect of the proposed composition on the body, it is believed (based on existing knowledge in this field) that the specific activation of the immune response plays a key role in the mechanism of realization of the antitumor effect of ODNP.

The reason for this conclusion is the data obtained by the authors on the stimulation of the production of α- and γ-interferons, IL-6.

A positive immunomodulatory effect was also detected in ONCOSOM-vaccinated volunteers suffering from tumor pathology, with parallel treatment with RT inhibitors.

The invention is further illustrated by non-exhaustive examples of embodiments and drawings, which are intended only to demonstrate the feasibility of the invention, but not to limit.

Brief Description of the Drawings

In FIG. 1 presents electronic photos of ONCOSOM and EPOM preparations, in which particles of the bacteriophage MS2 are present.

In FIG. 2 presents the results of an enzyme immunoassay (ELISA). On the axes: abscissa - antigens (name indicated), ordinate - optical density at 435 nm (OD ₄₃₅ ). The first twelve columns are EPOM, the second twelve columns are ONCOS.

In FIG. 3 presents the results of ELISA ONCOSOM using anti-EPOM IgG.

In FIG. 4 presents the results of the induction of interferon-α of a series of ODNP.

Detailed Disclosure of Invention

The method of obtaining ONCOSOM

In order to obtain exosomes from K562 / i-S9 cells by heat shock, the cells were first cultured at 37 ° C for 43 h, then transferred to 43 ° C (water bath) for 1 h, and then regenerated at 37 ° C for 4 h. Then the cells were frozen with liquid nitrogen and homogenized to a powder state. The homogenate was irradiated with UV light for 25 minutes, and then cell fragments were precipitated for 15 minutes at 6000 rpm. 96 ° ethyl alcohol was added to the clear supernatant to a concentration of 84 °. The precipitate obtained by precipitation at 60,000 g for 20 minutes was dissolved in distilled water and dried by freeze drying after ultrafiltration. The purity of the obtained ONCOSOM preparation was checked by electron microscopy. Particle concentration was determined in μg per 10 ⁶ cells. The presence of CEA and 5T4 antigens, as well as FasL and TRAIL, was determined using ELISA with commercial kits.

The toxicity of the drug was determined in the Lal test.

The method of obtaining one.

ODNP is synthesized in the direction from the 3′- to 5′-end. During the synthesis, the 3'-terminal oligonucleotide is covalently linked to a solid-phase carrier - CPG (controlled poreglass). The synthesis monomers are nucleoside phosphoramidites, and the synthesis begins with the release of the nucleoside located at the 5′-end of the synthesized ODN, while the dimethoxytrityl (DMTr) protective group is removed from the 5′-OH group (stage 1).

The next stage is condensation, in which the activated nucleoside phosphoramidite covalently joins the free 5′-hydroxyl group of the terminal detritylated nucleoside (stage 2). The efficiency of such an attachment is about 99%. In order to prevent erroneous oligonucleotides from accumulating due to the remaining 1% of unreacted 5′-terminal hydroxyl groups, they are blocked (“capped”) by the acetylation reaction. The minimum number of molecules that turned out to be unblocked during the “capping” reaction continues to participate in the synthesis, and as a result, after the last synthesis cycle, shorter products (oligonucleotides with missing bases) are formed.

After the condensation and “capping” reaction, the two terminal bases of the synthesized ODN are bound by an unstable phosphite tri-ether bond. Its translation into a stable phosphotriether bond is carried out due to the oxidation reaction (stage 3), which completes the cycle of addition of one base. Then the detritylation step follows again (step 4) and the cycle repeats. After the addition of the last base, the oligonucleotide is removed from the solid phase and the remaining protections are removed from it, which leads to the desired ODNP.

The yield of the target product in the synthesis process is equal to the efficiency of the condensation reaction to the degree of (n-1), where n is the length of the oligodeoxynucleotide. The condensation efficiency during the reaction is at the level of 99-99.5%. Thus, the yield, for example, of an oligodeoxynucleotide of 25 nucleotides in length is 80-90%.

Storage of ONP. For long-term storage or long-term transportation, ODNP is recommended to be lyophilized. In this state, at a temperature of -20 ° C, single-dose formulations can be stored for several years, at room temperature - from several months to several years. Dissolving ODNP is recommended in sterile deionized water or in sterile TE buffer (10 mM Tris pH 8.0, 1 mM EDTA).

Determination of the concentration of ODNP.

The concentration of ODNP (in pmol / μl) is determined by the formula:

[C] = OD ₂₆₀ · 100 / 1.54 · A + 0.75 · C + 1.17 · G + 0.92 · T

where A, C, G, T is the number of corresponding nucleotides in the ODNP sequence. When using the indicated amounts of reagents, the optical density is usually 18-20. The calculation of the concentration of ODNP shows that this value ranges from 65 to 75 pmol / μl. Based on the fact that the final concentration of oligodeoxynucleotides in the kit is 2.5 pmol / μl, and the flow rate of each oligodeoxynucleotide per reaction is 2 μl, the obtained amount of ODNP is sufficient to complete 70-80 sets, depending on the yield of synthesized ODNPs.

The resulting preparation is stored in the refrigerator at a temperature of minus (20 + 5) ° С. Under these conditions, the drug remains active for up to 1 year.

Biological activity

A study of the interferon-inducing activity of ODNP was carried out by direct induction in donated blood. Heparinized blood was obtained from 10 anonymous healthy donors of various ages, sex and complexion. For each experiment, 250 μl of blood serum was mixed with 250 μl of RPMI-1640 medium (Sigma, USA), as well as with 50 μl of PBS containing various amounts of an ONP drug. The mixture was incubated for 20 hours at 37 ° C, and then used to determine interferon. Determination of interferon-α was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using an ELISA kit (LLC "Cytokine", Russia) according to the manufacturer's recommendations. The cell interferon test was performed at the N.F. Research Institute of Epidemiology and Microbiology. Gamalei RAMS on the method of processing VSV culture of human fibroblasts. Statistical analysis of the experiments showed satisfactory reliability, which does not require a further increase in the number of experiments.

In FIG. 4 is a graph illustrating the induction of interferon with the administration of ODNP.

The study of stimulation of B-cells with the drug ODNP was carried out in the SE “SSC Institute of Immunology FMBA of Russia” (Moscow) by a standard method. We studied the effect of ODNP on the humoral thymus-dependent immune response, estimated by the number of antibody-forming cells in the spleen of mice immunized with sheep erythrocytes. For the experiment, 4 groups of mice were used, consisting of 10 animals each. Each group was injected, respectively representing a negative control (PBS), a positive control (polyacrylate 80 kDa) and two concentrations of ODNP dissolved in PBS. Three hours after the injection, 5 × 10 ⁶ sheep erythrocytes were immunized. The number of antibody-forming cells (AOKs) per mouse spleen was determined by local hemolysis in agar by Jern and Nordin. In parallel, the number of mononuclear cells per spleen was determined. Statistical analysis of the experiments showed satisfactory reliability, which does not require a further increase in the number of experiments.

Example 1. Obtaining ODNP.

Automatic synthesis of oligonucleotide blocks is based on the solid-phase method, which allows the synthesis of polynomial oligonucleotides on a programmable synthesizer. For this, solutions of phosphoramidite derivatives of each nucleotide and tetrazole are prepared. The procedure consists of the steps of preparing a sample of each preparation in penicillin vials (previously very well washed and sterilized with dry heat), drying each phosphoramidite derivative and tetrazole under vacuum for 2 hours at a pressure in the desiccator of not more than 0.2-0.3 atm. For greater dewatering efficiency, a pre-calcined sorbent (e.g., CaCl ₂ ) is placed on the bottom of the desiccator.

Preparation of solutions of tetrazole and phosphoramidites A, G, C, T (for one pair of oligonucleotides). To 280 mg of tetrazole in a vial, 20 ml of acetonitrile was added with a 20 ml measuring pipette. To 150 mg of dried phosphoramidite in vials, add with the help of measuring pipettes 5 and 10 ml acetonitrile in the following amounts:

to obtain a solution of phosphoramidite A - 4.6 ml;

to obtain a solution of phosphoramidite G - 4.7 ml;

to obtain a solution of phosphoramidite C - 4.8 ml;

to obtain a solution of phosphoramidite T - 5.3 ml.

Preparation of the DNA synthesizer for operation, filling the containers with the initial solutions included in the instrument set, as well as solutions of tetrazole, phosphoramidites A, G, C, T and acetonitrile, connecting two cassettes with a polymer carrier containing the first nucleotides of the synthesized ODNP oligonucleotides (in each cassette - one of a pair of oligonucleotides) and the synthesis is performed in accordance with the Instructions for use of the synthesizer.

The cassettes containing the synthesized oligonucleotides are transferred from the DNA synthesizer each into a separate 1.5 ml Eppendorf tube with a cap. The tubes are balanced on a technical balance, placed in a centrifuge rotor and centrifuged at 2000 rpm for 1 min. After the rotor stops, each of the cassettes is transferred to a similar clean tube. The supernatant is discarded.

In tubes with drained cartridges, 1 ml of 35% ammonia is added, balanced on a technical balance, placed in a centrifuge rotor and centrifuged at 2000 rpm for 1 min. After the rotor stops, each of the tubes with the cartridge is transferred to a separate bottle, tightly closed with a lid and placed in a thermostat for 16 hours at 4 ° C.

Vials containing tubes with cassettes are removed from the thermostat and left in the room for 1 hour to cool to room temperature. Then, each of the cassettes is transferred to a similar clean tube. The liquid remaining in each tube containing one of the synthesized oligonucleotides is retained. Tubes with cassettes are balanced on a technical balance, placed in a centrifuge rotor and centrifuged at 2000 rpm for 2 minutes. After stopping the rotor, the cassettes are removed, and the remaining liquid, also containing the synthesized oligonucleotides, is combined with the previous portions obtained after incubation of the cassettes.

The process of purification of oligonucleotides consists of the release of oligonucleotides from the polymer base on which the synthesis of oligonucleotides takes place. Oligonucleotides are transferred to a solution of ammonia, freed from the latter and low molecular weight impurities by chromatography on a column with Sephadex G-25. Using these amounts of reagents, 0.7 ml of a solution of each oligonucleotide in deionized water is obtained. Eight chromatographic columns of 5 ml each, filled with Sephadex G-25, are equilibrated with sterile deionized water. On the surface of Sephadex of each column, a liquid collected during the operation is applied using a syringe (each oligonucleotide is placed on a separate column). After complete penetration of the applied liquid into Sephadex, 0.7 ml of sterile deionized water is again layered onto the surface using a syringe. After passage of the free volume (5 ml), 0.7 ml of eluate was collected from each column.

10 μl of a solution of each oligonucleotide obtained during execution is taken. Each sample of the Sephadex G-25 eluate was transferred to a separate 1.5 ml Eppendorf tube and the volume was adjusted to 1 ml with sterile deionized water. Each sample is transferred to a quartz cuvette, placed in a spectrophotometer and the optical density (OD) of the samples is determined at 260 nm.

The obtained ODNPs have a generalized structure:

GGGGCGCCGXGXXGGCGXGCGCGCC

Example 2. Determination of protein concentration in the ONCOSOM preparation

To determine the protein concentration, the ONCOSOM preparation obtained from 10 ⁶ cells was suspended in 20 mM Tris-HCl buffer with 1% Triton-X100. Protein content was measured spectrophotometrically (at 280 nm) using a solution of bovine serum albumin (BSA) in a similar buffer as standard control.

Example 3. Determination of the concentration of antigens in the drug ONCOSOM

To determine the concentration of antigens in the ONCOSOM preparation obtained from 10 ⁶ CEA, 5T4, FasL and TRIAL cells, commercial kits known to those skilled in the art were used.

In FIG. 2 shows the results of ELISA. On the axes: abscissa - antigens (name indicated), ordinate - optical density at 435 nm (OD ₄₃₅ ). The first 12 columns are EPOM (three series of drugs), the second 12 columns are ONCOSOM (three series of drugs).

In FIG. 3 presents the results of inhibition of enzyme-linked immunosorbent assay (ELISA) of ONCOSOM using anti-EPOM IgG depleted in antigens of TBG; CEA; FasL and TRIAL.

Rows B-E (horizontal) present the results of ELISA inhibition of four determinations of the same ONCOSOM preparation using four different anti-EPOM IgG preparations. The control was the results of the determination of CEA in the preparation of EPOM using anti-EPOM IgG (F row).

The vertical rows (labeled TWG; CEA; FasL and TRIAL) are formed by wells containing the standard ONCOSOM preparation, with which IgG interacting, depleted of the corresponding antigens, is present. Results with unpainted or slightly colored wells indicate the presence of only the corresponding antigen in the anti-EPOM IgG preparation or a very low concentration of the other three antigens. Thus, the presented result shows that only one anti-EPOM IgG preparation contains high concentrations of antibodies against all of the above antigens. The ONCOSOM preparation in the same experiments showed the presence of all these types of antigens (tumor markers) in high concentration.

Example 5. The determination of the number of exosomes in the drug ONCOSOM

Drops of a concentrated ONCOSOM preparation (5 μl) diluted in PBS1: 10 and 10 ¹⁰ particles of phage MS2 were placed on Famvar-coated microscopic nets (200 or 300 cells). The mixture was stained with 1% uranyl acetate and the grid was observed in JEM-100C (accelerating voltage of 120 kV). The size and number of exosomes was calculated from the ratio of particle sizes of phage MS2 and exosomes.

From the electronic photos in FIG. One of the ONCOSOM preparations (left) and EPOM (right) shows that the structures identified as exosomes are present in both preparations (typical structures are circled by the dashed line), however, the sizes of exosomes in the ONCOSOM preparation are more regular (for comparison, a baseline length 100 μm and a higher electron density (darker), which indicates a higher concentration of protein included in the structure.

Table 1 summarizes the results of the use of the combination of ONCOSOM and ODNP with revertase inhibitors for treatment.

Evaluation of the amount of human DNA retrotransposons in the blood of patients (retroDNA titer) was carried out by quantitative PCR with primers, the structure of which is known to specialists (carried out by Invitro LLC).

Assessment of QL100 (indicator of quality of life) was carried out by a survey conducted by the attending physician, according to the questionnaire OCRC - 100 (Questionnaire of the World Health Organization).

Table 1 shows a decrease in all patients with the FasL tumor marker (TRIAL) and an increase in the quality of life.

Table 2 shows the results of using the combination of ONCOSOM and ODNP for prevention. You can also see a decrease in the patient's FasL tumor marker (TRIAL) and an increase in interferon content.

The following are additional examples of treatment using the invention.

Example 6

Patient G., 57 years old. Does not smoke, nutrition is correct, teacher, regular sports, until recently, a large number of sexual partners (women), uses Viagra (100 mg), suffers from prostate adenoma for 15 years (after diagnosis, the amount of PSA (bound) in the blood - 47.8 ng / ml, free - 140 ng / mg).

A multiple (once every two weeks) vaccination with ONCOSOM (dose - 0.3 mg) was carried out against the background of the introduction of an OND adjuvant (dose - 2 mg) for 3 months. A positive skin reaction appeared after the third vaccination (5 weeks after the start of vaccination; mild infiltration). After 7 weeks, the skin reaction intensified and lasted 10 weeks. The lifestyle has not changed, the indicators are close to normal (14 weeks after vaccination, the amount of PSA (bound) in the blood is 4.2 ng / ml, free - 14 ng / mg).

Example 7

Patient Ms, 59 years old, businessman, choleric type, poor and irregular nutrition, blood count is normal. Identified gastric cancer (metastatic).

Weekly vaccination with ONCOSOM (dose - 0.3 mg) was carried out against the background of the introduction of the OND adjuvant (dose - 2 mg). Vaccination was performed multiple for 3 months. At the same time, Stavudine was taken at 0.03 g 2 times a day for the first 1.5 months. The lifestyle has not changed. After 1.5 years of observation, positive changes were revealed (see table 1). 2 years after the start of vaccination, the patient went abroad for permanent residence.

Table 1

A patient Age diagnosis (stage) Treatment, period Exosome quality / μl; before / after treatment Title (pg) FasL (TRIAL); before / after treatment Title retroDNA (genome equivalents); before / after treatment QL100-quality of life; before / after treatment Mr. A. 57; prostate cancer (3) Introduction ONCOSOM and ONP; 4 months 1 r / wk 10 ³ / <10 7 (12) / 2 (4) 10 ⁵ / <10 ² 73/91 M-in L. 59; stomach cancer (4) After surgery, the introduction of ONCOS and ONP; 3 months 1 r / week; Stavudin 10 ⁴ / <10 14 (23) / 4 (5) 10 ⁷ / <10 ³ 63/79 (went abroad for permanent residence after 1.5 years of observation) Mrs B. 58; pancreatic cancer (3) Introduction ONCOSOM and ONP; 4 months 1 r / wk 10 ³ / <10 9 (11) / 2 (5) 10 ⁶ / <10 ² 70/92 (after discharge from the ONC, the patient lived another 2.5 years)

table 2

A patient Age diagnosis (stage) Treatment, period Exosome quality / μl; before / after treatment Title (pg) FasL (TRIAL); before / after treatment Title retroDNA (genome equivalents); before / after treatment QL100-quality of life; before / after treatment St. I. 65; prostate adenoma (3) Introduction ONCOSOM and ONP; once a year 10 / <10 5 (8) / 3 (2) 10 ² / <10 ¹ 83/92

Claims (3)

1. The method of immunotherapy of solid tumors, which consists in vaccinating a patient with a combination of an exosome preparation and a P-type oligodeoxynucleotide, the preparation comprising exosomes isolated by a K562 / i-S9 cell culture, and contains, according to the enzyme immunoassay, an amount of 0.9- 1.5 μg / 10 ⁶ cells, CEA antigens in the amount of 0.045-0.075 μg / 10 ⁶ cells and 5T4 - 0.03-0.05 μg / 10 ⁶ cells, and FasL ligands - less than 0.005 μg / 10 ⁶ cells and TRAIL is less than 0.004 μg / 10 ⁶ cells, and the P-type oligodeoxynucleotide is a sequence of nucleotides GGGGCGCCGTGATGGCGAGCGCGCC, The combination indicated is administered subcutaneously for at least 2 months and at least 1 time per week in an effective amount, preferably in doses of 0.01-0.04 mg / kg each. 2. The method according to p. 1, characterized in that during the vaccination period, a drug is administered orally, a revertase inhibitor in effective doses. 3. The exosome preparation for use in the method according to claim 1 or 2, comprising exosomes isolated by K562 / i-S9 cell culture and containing, according to enzyme immunoassay, a protein in the amount of 0.9-1.5 μg / 10 ⁶ cells , CEA antigens in the amount of 0.045-0.075 μg / 10 ⁶ cells and 5T4 - 0.03-0.05 μg / 10 ⁶ cells, as well as FasL ligands - less than 0.005 μg / 10 ⁶ cells and TRAIL - less than 0.004 μg / 10 ⁶ cells.

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