WO2014007669A1 - Autologous cell vaccine for treating oncological diseases and method for producing same - Google Patents

Description

 Autologous cell vaccine for the treatment of cancer and method for its preparation

Technical field

 The invention relates to the field of medicine, oncology and can be used for the treatment of oncological diseases of various localizations.

 State of the art

 The problem of treatment of cancer, despite the huge number of developed and used therapeutic methods of treatment, is still practically unresolved. Of great interest is the question of the effectiveness of the immune response to a tumor in humans. One of the hypotheses of the onset of the malignant process is the failure of the immune system (Immunology of malignant growth. N.M. Berezhnaya, V.F. Chekhun. Kiev: Naukova Dumka, 2005, 791 p .; Kremetz E., M. Samuel, J. Wallace. " Clinical experience in the immunotherapy of cancer. Surg. Gynecol. Obstet. 1971, Vol. 133, P. 209-217; Baldueva IA "Antitumor vaccines. Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3 ), S. 66-70).

 Numerous studies have shown that an important role in protecting the body is assigned to the cellular link of the immune response, which is represented by a number of effector cells in the body (Immunology of malignant growth. Berezhnaya N.M., Chekhun V.F. Kiev: Naukova Dumka, 2005, 791 pp.). Initially, the role of natural killers (hereinafter referred to as NK), related to lymphoid cells, but differing in phenotype from T and B lymphocytes, was of particular interest.

The ability of these cells to exert a cytotoxic effect on tumor cells was discovered, which was confirmed in a number of in vitro experiments. In particular, a correlation was found between the frequency of the development of the tumor process and the quantitative level of NK in mice. The mechanism of action of NK is mediated by their ability to recognize antigens of the main class 1 histocompatibility complex (hereinafter HLA-I) on the cell surface and stop their lysis. Tumor cells may differ from normal cells in a reduced expression of HLA-I, and then such cells become targets for NK. In some cases, the expression level on tumor cells may be normal, such tumors escape from the control of NK (Immunology of malignant growth. Berezhnaya N.M. Chekhun. V.F. Kiev: Naukova Dumka, 2005, 791 s; Ivan Zanoni, Francesca Granucci, Maria Foti and et. al. "Self-tolerance, dendritic cell (DC) -mediated activation and tissue distribution of natural killer (NK) cells." Immunology letters, 2007, JVs 1 10, P. 6-17).

 The study of the activity of lymphoid cells under the influence of various cytokines showed that Interleukin-2 (hereinafter IL-2) plays an important role in the activity of T-lymphocytes and NK. In 1980, a report was published stating that lymphocytes cultured in the presence of IL-2 are capable of lysing sarcoma tumor cells. This phenomenon is called the LAK phenomenon. Studies have shown that lymphokine-activated killers (hereinafter LAK) come from a subpopulation of NK and have specific cytotoxicity with respect to tumor cells. The formation of LAK is the result of NK activation by a number of factors, the main of which is IL-2. This cytokine IL-2 helps to generate and enhance the cytotoxicity of lymphokine-activated killer cells (hereinafter LAK), participates in the regulation of the immune response and directs it along the T-helper 1 cell development pathway. In the body, IL-2 promotes the proliferation and differentiation of T-lymphocytes, stimulates the clonal proliferation of B-lymphocytes and antibody formation, increases the functional activity of phagocytes and NK cells (Immunology of malignant growth. Berezhnaya N.M. Chekhun V.F.Kiev: Naukova Dumka, 2005 , 791 pp., MV Kiselevsky, GV Casanova, SR. Varfolomeeva et al. “Experience in the use of interleukin-2 and lymphokine-activated killer cells in the treatment of hematologic diseases in children.” Immunology, 2002, T. 23, N ° l, S. 56-59; I.V. Kiseleva, S.F. Protasova, V.V. Zhunk , CM. Schekanova. "Cytokines in Cancer Treatment." Russian Journal of HIV / AIDS and related issues. 1999 T. 3, Jis 1 S.48-52).

In clinical studies of the LAK phenomenon, both allogeneic and autologous LAKs were cultured under various conditions. Moreover, LAK was administered both systemically and locally, in isolation and in combination with cytokines, including IL-2. However, these methods of therapy did not justify the hopes assigned to the LAK phenomenon: only in 50% of cases the clinical effectiveness of treatment was observed, the result was statistically unreliable, but in some cases the frequency of metastasis was significantly reduced. Moreover, the most effective methods for obtaining LAK and introducing them into the body were developed (Immunology of malignant growth. N.M. Berezhnaya, V.F. Chekhun. Kiev: Naukova Dumka, 2005, 791 c).

 In recent years, researchers of various specialties have paid great attention to the development and study of antitumor vaccines.

 Vaccination is a way to create active specific immunity using a vaccine containing an immunogenic antigen (Baldueva I. A. “Antineoplastic vaccines.” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66-70; Moiseenko V .M., Baldueva IA, Hanson KP. “Vaccine therapy of malignant tumors.” Oncology. 1999, T. 3, N ° 1, S. 327-332).

 It is known that antigens in tumors are components of tumor cells that are altered in structure relative to normal body cells.

 Recently, it has become clear that dendritic cells (hereinafter DC) play an important role in the process of tumor progression. The nature of this phenomenon is related to DC's ability to present tumor antigens to cytotoxic T-lymphocytes (CTLs, CTLs). It has been established that due to the absence of DC in the tumor or weak expression of MHC molecules of classes 1 and 2 (hereinafter HLA-I, HLA-II), costimulating CD80 and CD86 molecules, a stable antigen-specific T-cell response is not created on DCs that infiltrate the tumor ( Ptushkin VV “Dendritic cells and the role of cytokines in their differentiation and functioning. Clinical Oncohematology: A Guide for Physicians. Moscow, 2001. P. 6-72; Pashchenkov MV, Pinegin BV.“ The Main Properties of Dendritic cells. "Immunology. 2001, I ° 4, S. 7-16).

 This may be due to the production of factors by the malignant tumor: Interleukin-10 (hereinafter IL-10), transforming growth factor beta (hereinafter TGF-β), vascular endothelial growth factor (VEGF), endothelial cell growth factor (hereinafter beta-ECGF), and others that inhibit the differentiation, maturation, and functional activity of peripheral DCs (F. M. Marincola, E. M. Jaffee, DJ Hicklin and et. al. "Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance". Adv. Immunol., 2000, Vol. 74, P. 181-273).

It is known that the maturation of DC is a critical factor in triggering an immune response. Maturation can be initiated by various stimuli, including tumor antigens, microbial products, and inflammatory products: cytokines IL-1 (hereinafter IL-1), IL-6 (hereinafter IL-6), granulocyte macrophage colony stimulating factor (hereinafter GM-CSF), Tumor necrosis factor (hereinafter TNF-α), lipopolysaccharides (hereinafter LPS). During maturation, the antigen-capturing function of DC decreases, and the ability to activate T cells increases. Finally, differentiated DC is able to effectively activate T cells, after which the latter can carry out an immune response (Baldueva IA “Antitumor vaccines.” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66-70; Ptushkin VV "Dendritic cells and the role of cytokines in their differentiation and functioning. Clinical Oncohematology: A Guide for Physicians. Moscow, 2001, pp. 6-72; Pashchenkov MV, Pinegin BV." The main properties of dendritic cells. "Immunology. 2001, N ° 4, S. 7-16).

 In vivo studies have shown that DC are involved in the formation of an immune response in lymphoid organs. Moreover, they interact with all the main classes of T-cells and stimulate the development of a strong T-cell immune response (Baldueva IA “Antitumor vaccines.” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66 -70; Ptushkin VV “Dendritic cells and the role of cytokines in their differentiation and functioning. Clinical Oncohematology: A Guide for Physicians. Moscow, 2001, S. 6-72; Pashchenkov MV Pinegin B. V. .." The main properties of dendritic cells. "Immunology. 2001, N ° 4, S. 7-16).

 In vivo studies have also shown that DCs incubated with tumor antigens are able to generate a T-cell protective response against both tumor cells re-introduced into the body and existing tumors.

Recently, DC is considered as the main adjuvant in the creation of vaccines for the treatment of cancer (Baldueva IA “Antitumor vaccines” Practical oncology. Biotherapy of malignant tumors. 2003, N ° 4 (3), P. 66-70; Korostelev S. A. “Antineoplastic vaccines. Modern oncology. 2003, N ° 4 (3), pp. 9-15; Moskaleva E. Yu. Severin S. E.“ Prospects for the creation of antitumor vaccines using human dendritic cells. Immunology. 2002 . N ° 1. P. 8-15), since there is reason to believe that the use of antigen-loaded “professional GOVERNMENTAL "antigen-presenting dendritic cells will help create a strong specific immune response.

 The possibility of isolating tumor-specific antigens made it possible to implement an immunotherapy strategy based on the presentation of these antigens by HLA-I DC molecules with the generation of cytotoxic CD8 + T cells that can cause tumor rejection (Baldueva I. A. “Antitumor vaccines.” Practical oncology. Biotherapy of malignant tumors . 2003, N ° 4 (3), P. 66-70; Korostelev SA "Antitumor vaccines. Modern oncology. 2003, J e 4 (3), S.9-15; Moskaleva E. Yu. Severin S. Ye. “Prospects for the development of antitumor vaccines using human dendritic cells. "Immunology. 2002, N ° 1, S. 8-15).

 An important role is also played by the source of the tumor antigen.

 Stimulation of DC by individual peptides and proteins is known. However, the disadvantage of this approach is the heterogeneity and unstudied antigenic composition of tumor cells. The lysate obtained from the patient’s own tumor contains a wide range of antigens, including those not yet studied (A.E. Chag, BG Redman, JR Whitfield, et al. “A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced sapseg. "Clin. Cancer Res., 2002, Vol. 8, P.1021-1032).

 The method of administering DC is also important. Thus, it was shown that subcutaneously administered DCs have greater immunogenicity and the ability to migrate, in contrast to DCs administered intravenously (Korostelev SA “Antineoplastic vaccines.” Modern oncology. 2003, ° 4 (3), C. 9-15; Moskaleva E. Yu., Severin S.E. “Prospects for the development of antitumor vaccines using human dendritic cells. Immunology. 2002, N ° 1, S. 8-15).

Currently, the results of a series of trials of the use of DC-based antitumor vaccines are known. In particular, there is known data on the experience of using a vaccine containing autologous dendritic cells activated by tumor antigens in the presence of growth factors in case of follicular lymphoma, melanoma, myeloma, cancer of the prostate, breast, cervix, colon, thyroid gland (R. Soiffer, T. Lynch, M. Mihm, et al. "Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma." Proc Natl. Acad Sci USA. 1998, W ° 95, P.13141-13146; FJ Hsu, C. Benike, F. Fagnoni, et al. "Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells." Nat. Med. 1996, P. 52-58; Prokopovich S. K. Vinnitsky “Dendritic cells and the prospects for their use in immunotherapy of malignant neoplasms” Oncology. 2001, T. 3, N ° 2-3, S. 126-131).

 Known methods for immunotherapy of malignant tumors based on the formation of a T-cell protective response by phased administration of activated autologous cells into the body, and various methods for producing vaccines containing autologous lymphokinactivated killers (hereinafter LAK) and DC are known.

 Thus, a method of immunotherapy of malignant brain tumors (RU, 2262941, C1) is known, comprising locoregional administration in the tumor bed after its removal of LAK in combination with IL-2 and cytotoxic lymphocytes (CTL) stimulated by a tumor antigen in the presence of IL-2, in which sequentially:

 - conduct a course of cytokine therapy, including intramuscular injections of leukinferon interferon-alpha (IFN-α) with an interval of 48 hours,

 - conduct a course of adaptive immunotherapy with the introduction of LAK generated in the presence of recombinant IL-2, which are administered in combination with recombinant IL-2 with two generation procedures, and local-area administration of LAK with an interval of 24 hours,

 - then conduct a course of adaptive immunotherapy by introducing CTL in the tumor bed in combination with recombinant IL-2, while:

 using CTLs generated by culturing mononuclear cells of a patient’s peripheral blood with a DC loaded with a tumor antigen in the presence of recombinant IL-2, wherein:

 - to obtain DC from a patient before surgery, monocytes are isolated from peripheral blood, which are cultured with GM-CSF and IFN-a for 24 with maturation of DC in the presence of monocyte conditioned medium for 24 hours and incubation of DC in the presence of antigenic tumor material for 1 hours for their loading with a tumor antigen, while a tumor cell lysate can be used as antigenic material;

- then, after completion of the CTL immunotherapy course, a vaccine therapy course of DC loaded with tumor antigen is given as subcutaneous injections administered at 2-week intervals in combination with subcutaneous injections of recombinant IL-2.

 However, this method can only be used to treat malignant brain tumors. In addition, as a result of the separate production and administration of cell populations, therapy is more invasive and takes a long time.

 A known method of obtaining dendritic cells from increased populations of monocytes and T-cell activation (US, 6479286, B), in which:

 conducting leukapheresis of mononuclear cells of the peripheral blood of the patient, providing isolated peripheral blood, subsequent maturation of peripheral blood to obtain isolated monocytes,

 - then, to increase the population of monocytes from peripheral blood and differentiate the increased population in DC, carry out:

 - the first incubation of monocytes in vitro in the presence of IL-3 under conditions that do not lead to the differentiation of monocytes in DC; thereby increasing the population of monocytes; and

 - subsequent in vitro incubation of the increased population with GM-CSF and IL-4, thereby differentiating the increased population of monocytes in DC;

- then additionally activate DC by incubating them in vitro with TNF-a, temporarily activating DC to a pro-inflammatory status, while ensuring the presence of the peptide on the DC surface and increasing the population of antigen-presenting DC;

 - then, in vitro or in vivo, the T-cell population is activated with a population of antigen-presenting DCs to thereby provide an activated T-cell population and the effect of these antigen-presenting DCs on TNF-a.

In this case, the antigen is obtained from a protein or carbohydrate located on the surface of the transformed cell, using a peptide obtained from a protein selected from the group consisting of HIV Gag, HIV Env, C-EJAB- [beta] -2 HER2 / neu, PEM / MUC -1, Int-2, Hst, BRCA-1, BRCA-2, truncated EGFRvIII, MUC-1, p53, ras, RK, Myc, Myb, OB-1, OB-2, BCR / ABL, GIP, GSP, RET, ROS, FIS, SRC, TRC, WTI, DCC, NFl, FAP, MEN-1, ERB-Bl MART-1, gp-100, PSA, HBVc, HBVs, HPV E6, HPV E7, idiotypic immunoglobulin, tyrosinase, MAGE-1, trp-1, and mycobacterial antigen. Moreover, the differentially presented protein is selected from the group consisting of HIV Gag, HIV Env, HER-2, MART-1, gp-100, PSA, HBVc, HBVs, tyrosinases, MAGE-1, trp-1, mycobacterial antigens, and CEA.

 In addition, the method may further comprise contacting the DC with a protein differentially present on the cell, for example, a transformed cell selected from the group consisting of: a cancer cell, a bacterial cell, a parasitically infected cell, and a virus infected cell. In addition, the method may further include transducing said DC with a nucleic acid vector that differentially encodes a protein present on a cell selected from the group consisting of a cancer cell, a bacterial cell, a parasitically infected cell and a virus infected cell.

 When vaccinated, the patient is administered the above activated T cells and the above antigen presenting DC. As a result of vaccination, antigen-presenting DCs stimulate the patient's NK cell activity. In this case, the T cell may be a helper of T cells, and DC may be an antigenic protein comprising a peptide sequence derived from a peptide that appears on the surface of a cancer cell. However, the method described above has insufficient clinical efficacy due to the lack of LAK. In addition, the use of a peptide sequence as an antigen reduces the specificity of this vaccine.

 A known method of obtaining and using a combined cell transplant used for the prevention and treatment of cancer of various localization (RU, 2309753, C1), obtained on the basis of LAK and DC, containing lymphocytes activated by IL-2, and mature DC (mature DC, mDC) obtained by incubating immature DC (immature DC, iDC) with a tumor lysate, in which:

 - mononuclear cells (hereinafter MNCs) are isolated from the blood or bone marrow of a patient in need of treatment or subsequent prophylaxis of a malignant neoplasm, for example, by centrifugation in a single-stage ficol gradient;

 - MNCs are cultured by incubation in RPMI 1640 culture medium or in DMEM medium with 5% human patient serum or donor AB serum for 2-4 hours;

- divide MNCs into fractions of monocytes / macrophages adhered to the substrate (for example, to the wall of a culture bottle), and lymphocytes that do not adhere to the substrate;

- to obtain iDC CD83 ^low , CD86 ^low , CD80 ^low , GM-CSF and IL-4 are added to the attached monocyte / macrophage cells, proinflammatory factors IL-β, TNF-a, IL-6 are added to stimulate DC maturation, all in concentration 10 mg / ml;

- iDC is incubated with tumor lysate for 1.0 day and get mature DC (mDC) pulsed with antigens, CB83 ^{hi h} , CD86 ^{hi h} , CD80 ^{hi h} ,

 - and in order to obtain LAK, IL-2 at a concentration of 1000 IU / ml is added to lymphocytes that do not adhere to the substrate.

 Subsequently, mDC pulsed with antigens is administered subcutaneously, intravenously, interarterially or regionally (in the cavity), and LAK is administered intravenously.

 However, the vaccine obtained by the described method has insufficient clinical efficacy due to the absence of specifically activated in vitro T-lymphocytes, and specific cytotoxic cells are not obtained in the described method. According to the authors, a significant drawback of this method is the use of autologous serum of cancer patients or human heterologous serum, since autologous serum of cancer patients may contain dissolved factors that suppress the immune response, and heterologous serum may have an additional non-specific antigenic effect. In addition, the simultaneous introduction of antigen and acute phase cytokines into the medium, initiating the maturation of DC, can lead to early cell maturation with incomplete antigen presentation. The addition of mercaptoethanol to the medium increases survival and increases the yield of DC.

Closest to the present invention is a method of obtaining an autologous vaccine for the treatment of cancer (RU, 2392946, C1) consisting of two parts: a part containing T lymphocytes obtained from a non-adherent fraction of the patient’s peripheral blood MNCs, specifically activated by mature DC (mDC) and LAK and activated IL-2, and portions containing mature DC (mDC) pulsed by antigens obtained by incubating immature DC (iDC) of the attached fraction of peripheral MNCs blood of a patient with a tumor lysate. Moreover, the method of obtaining the specified autologous vaccines includes:

 - Isolation of MNCs from the patient’s peripheral blood on a gradient of ficolurourographin (p = 1.077 g / cm) with a blood to gradient ratio of 2: 1, respectively;

- cultivation of MNCs by in vitro incubation in vials for 1.0 hour at 37 ° C in DMEM supplemented with 10% FCS; HEPES, glutamine, 2-mercaptoethanol. gentamicin (calculated per 100 ml DMEM) in an atmosphere of 5% C0 ₂ ;

 - the separation of MNCs into monocytes that attach to the substrate, and lymphocytes that do not attach to the substrate;

 - cultivation of attached monocytes in vitro in a medium supplemented with a recombinant analogue of GM-CSF - a neupogen at a concentration of 50 ng / ml for 48 hours to obtain iDC pulsed by antigens that are capable of actively capturing antigen by phagocytosis and macropinocytosis;

 - stimulation of the maturation of iDC pulsed by antigens, in vitro autologous tumor lysate in a medium containing 2000 IU / ml rheopheron and 50 ng / ml betaleukin, followed by the addition of maturing factors, for 1, 0 days to obtain specifically activated mDC;

 washing the obtained specifically activated mDC by centrifugation in physiological saline for 10 minutes at 1500 rpm;

 - cultivation of non-attached fractions of MNCs in vitro for 72 h in a medium containing 100 IU / ml of roncoleukin, to obtain non-specifically activated lymphocytes - LAK;

 - washing the obtained LAK by centrifugation in physiological saline for 10 min at 1500 rpm;

 - co-cultivation of specifically activated mDC with LAK in vitro in fresh medium supplemented with 100 IU / ml Roncoleukin for 24 hours to obtain specifically activated killer lymphocytes,

 - separation and separate washing of the non-adherent fraction of cells containing T-lymphocytes specifically activated by mature DC (mDC) and LAK activated by IL-2, and the adherent fraction of cells containing mature DC (mDC).

At the same time, it was noted that the phased cultivation of monocytes, celled from the patient’s peripheral blood, it first produces immature dendritic cells with a pronounced ability to only absorb antigen, and then, after the addition of a tumor lysate and the aforementioned drugs that stimulate the dendritic cell maturation, it promotes the production of specifically activated mature DC (mDC) that effectively present antigens and activate T-helpers.

 However, the use of GM-CSF alone to generate immature DCs from monocytes of the adherent fraction of MNCs is not effective enough and yields a low percentage yield of immature DCs (iDC). In addition, the simultaneous addition of the tumor lysate and acute phase cytokines necessary for activation of DC maturation to the medium can lead to premature DC maturation with incomplete antigen presentation. Co-cultivation of mature DC and LAK does not exclude possible competition between the stages of specific and non-specific activation of cells, which ultimately reduces the number of specifically activated killers in the vaccine.

 Disclosure of invention

 The aim of the present invention was to develop an autologous cell vaccine for the effective treatment of oncological diseases of various localization by simultaneously activating specific and nonspecific units of the antitumor immune response in patients with tumors of various localizations, obtained on the basis of autologous mononuclear blood cells of a patient with cancer and containing mature vaccines in one part dendritic cells antigenactivated during maturation (hereinafter AA mDC ^ and in another part of the vaccine, T-lymphocytes specifically activated by the indicated mature dendritic cells, antigen-activated during maturation (AA mDC), and lymphokine-activated killers (LAK).

 When creating the present invention, the task was to create a method for producing an autologous cell vaccine containing mature dendritic cells antigenactivated during maturation (AA mDC) in one part of the vaccine and T-lymphocytes specifically activated with the indicated mature dendritic cells antigenactivated in one part of the vaccine maturation process (AA mDC), and lymphokinactivated killer cells (LAK cells).

The task was solved by creating an autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and nonspecific link of the antitumor immune response containing mature dendritic cells antigenactivated during maturation in one part of the vaccine (AA mDC), and in the other part of the vaccine containing T-lymphocytes specifically activated by these mature dendritic cells antigenactivated during maturation (AA mDC ^ and lymphokinactivated killers (LAK).

 The problem was also solved by creating a method for producing an autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and nonspecific link of the antitumor immune response, containing in one part mature dendritic cells antigenactivated during maturation (AA mDC) and containing in another parts of T-lymphocytes specifically activated by these mature dendritic cells antigen-activated during maturation (AA mDC), and lymphokinact Rowan killers (LAK), comprising the steps of:

 a) the allocation of mononuclear cells (MNCs) from the peripheral blood of a cancer patient;

 b) culturing the mononuclear cells obtained in step a) in vitro in a base medium with a high content of amino acids and vitamins, which provides cell viability, including lymphoid cells, supplemented with FCS, HEPES, Glutamine, Gentamicin, to obtain populations of monocytes and lymphocytes;

 c) separation of the mononuclear cells obtained in step b) into an adherent fraction of monocytes and an adherent fraction of lymphocytes;

 d) culturing the monocytes of the adherent fraction obtained in step c) in vitro in a fresh base medium, similar in composition to the medium used in step b), with the addition of 2-Mercaptoethanol, a recombinant analogue of granulocyte-macrophage colony stimulating factor (rhGM-CSF) and growth factor, to obtain immature dendritic cells (iDC), which are capable of actively capturing antigen by phagocytosis and macropinocytosis;

d) culturing the fraction obtained in step d) containing immature dendritic cells, in a fresh base medium, similar in composition to the medium used in step b), with the addition of 2-Mercaptoethanol, a tumor cell lysate obtained from a fragment of a patient’s autologous tumor, to produce antigen-activated immature dendritic cells (AA AL); f) culturing the fraction obtained in step e) containing antigen-activated immature dendritic cells in vitro in the medium used in step e) with the addition of recombinant human tumor necrosis factor alpha (rhTNF-a) to obtain mature dendritic cells antigenactivated in the process ripening (AA mDC);

 i) exposure of the lymphocytes of the non-adherent fraction obtained in step c) in a fresh medium, similar in composition to the medium used in step b), for a time sufficient to maintain the viability of the lymphocyte fraction;

 j) co-culturing the fraction obtained in step e) and part of the non-adherent fraction obtained in step i) in vitro in a ratio of 1: 10 in fresh medium, similar in composition to the medium used in step b), and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) and recombinant human Interleukin-12 (rhIL-12) or supplemented with recombinant human Interleukin-12 (rhIL-12) and recombinant human Interleukin-18 (rhIL-18), to produce T-lymphocytes specifically activated by mature dendritic cells antigenic during maturation (AA mDC);

 k) in vitro cultivation of the portion of lymphocytes of the non-adherent fraction not used in step k) obtained in step c) in a medium similar in composition to the medium used in step b) and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) obtaining lymphokinactivated killer cells (LAK cells);

 l) selection from the fraction obtained in step k) of adherent mature dendritic cells antigen-activated during maturation (AA mDC) and washing them to obtain one part of the vaccine;

m) selection from fractions of cells obtained in stage k) and in stage k) that did not adhere specifically activated T-lymphocytes and lymphokine-activated killer cells (LAK cells) and their washing with receiving another part of the vaccine.

 Moreover, according to the invention, it is advisable to isolate mononuclear cells from the patient’s peripheral blood on a ficol-urographin gradient with a density of 1.077 g / cm and a blood to gradient ratio of 2: 1.

 Moreover, according to the invention, it is advisable to cultivate in step b) for 1.0 hour at a temperature of 37 ° C in an atmosphere of 5% CO in a base medium supplemented with 100 ml of a base medium: FCS -1%, HEPES -5 mm, Glutamine - 2mM, Gentamicin -1000 μg / ml.

Moreover, according to the invention, it is advisable to cultivate in step d) for 48 hours in a basic medium supplemented with 100 ml of a basic medium: FCS -10%, HEPES -5 mM, Glutamine-2 mM, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, and supplemented with 1 ml of medium containing 1.0 million cells, recombinant human granulocyte-macrophage colony stimulating factor Neupogen (rhGM-CSF) - 50 ng / ml and recombinant human Interleukin -4 (rhIL- 4) - 100 ng / ml.

Moreover, according to the invention, it is advisable to cultivate in step e) for 20 to 24 hours in a base medium supplemented with 100 ml of base medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, with the addition of a tumor cell lysate at the rate of 100 μg per 1 ml of base medium.

Moreover, according to the invention, it is advisable to cultivate in step e) for 24 hours in the base medium supplemented with 100 ml of the base medium: FCS-10%, HEPES-5mM, Glutamine-2mM, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, with the addition of tumor cell lysate at the rate of 100 μg per 1 ml of the base medium and recombinant human tumor necrosis factor alpha (rhTNF-α) at the rate of 25 ng per 1 ml of the base medium.

 Moreover, according to the invention, it is advisable at step i) to carry out the exposure for 96 hours in the base medium supplemented with 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 μg / ml.

Moreover, according to the invention, it is advisable to cultivate in step k) for 48 hours in a basic medium additionally containing 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine-2 mm, Gentamicin-1000 μg / ml, supplemented based on 1 ml of medium containing 2.5 million cells, recombinant human Interleukin-2 (Roncoleukin, rhIL-2) -100 U / ml and recombinant human Interleukin-12 (rhIL-12) - 10 ng / ml or recombinant human Interleukin-12 (rhIL-12) - Jung / ml and recombinant human Interleukin-18 (rhIL-18) - 100ng / ml.

 Moreover, according to the invention, it is advisable to cultivate in step l) for 48 hours in a basic medium additionally containing 100 ml of medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 μg / ml and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) - 1000 IU / ml per 1 ml of medium containing 2.5 million cells.

 Moreover, according to the invention, it is advisable to use DMEM as the base medium.

 Moreover, according to the invention, it is advisable to wash the fractions of the cells in steps m) and n) twice in physiological saline for 10 minutes at 1500 rpm.

 Brief Description of the Drawings

 In the future, the method of obtaining an autologous cell vaccine according to the invention for the treatment of cancer is illustrated by examples of its implementation, and the properties obtained using it an autologous cell vaccine according to the invention are explained in examples of testing the activity of an autologous vaccine and are illustrated by the accompanying drawings, in which:

 Figure 1 - change in the expression of CD 14+ on cells in a monocytic pool at different stages of DC maturation, cultured from the adherent fraction of mononuclear cells of cancer patients with different localization at stages d), e). and e) a method for producing an autologous cell vaccine according to the invention: FIG. 1 A is for immature DC (iDC), FIG. IB - for mature DC (mDC);

 Fig. 2A, Fig. 2B shows the change in the expression of CD83 + H CD80 + on cells in a monocytic pool at different stages of DC maturation cultured from monocytes of the adherent fraction of blood MNCs of patients with cancer of different localization at stages d), e). and e) a method for producing an autologous cell vaccine according to the invention: FIG. 2A for immature DC (iDC), FIG. 2B for mature DC (mDC);

FIG. 3A, FIG. 3B - change in endocytosis ability in terms of average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation cultured from adherent fraction of blood MNCs cancer patients of different localization in incubation conditions at 37 ° C: Fig. ZA - for immature DC (iDC), Fig.ZV - for mature DC (mDC);

 Figure 4 - indicators of phenotypic markers at different stages of DC maturation cultivated by the method of obtaining the vaccine according to the invention from monocytes adhering fraction of blood MNCs of cancer patients;

 FIG. 5. - indicators of DC endocytosis activity, measured by the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation, cultivated by the method for producing the vaccine according to the invention from adherent fraction of blood MNCs from cancer patients;

 6 - indicators of the cytotoxicity of the culture of MNCs against tumor cells in the presence of mature DC antigen-activated during maturation (AA mDC) and cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2);

 Fig.7 - the results of tests to determine the content of IFN-γ in the culture of MNCs with the addition of cytokines (rhIL-12 and rhIL-18, rhIL-12 and rhIL-2) and after co-cultivation of MNCs with mature DC antigenactivated during maturation (AA mDC ), and the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhlL-2), with spontaneous and lysate-activated production;

 Fig. 8 shows the results of tests to determine the content of IL-4 in the conditioned medium of MNCs cultures when cytokines (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2) were added to the medium and tests to determine the content of IL-4 in the conditioned medium cultures of MNCs, co-incubated with mature DCs, antigen-activated during maturation (AA mDC) with the addition of cytokines (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2), with spontaneous and lysate-activated production.

 Best Mode for Carrying Out the Invention

 A method of obtaining an autologous cell vaccine for the treatment of cancer according to the invention was carried out using known means and known technologies. For cultivation, various well-known basic media can be used, traditionally used for cell cultivation and characterized by a high content of amino acids and vitamins and providing cell viability, including lymphoid cells, for example, DMEM or RPMI-1640 medium.

A method for producing an autologous vaccine according to the invention was implemented by the authors using DMEM as a base medium and known components:

 - DMEM medium - a medium with a high content of amino acids and vitamins, includes: transferrin, leucine, glucose, pyruvate and a number of other substances, and has high biological stability, provides normal vital activity of many types and cell lines, including lymphoid ones;

 - FCS - fetal calf serum inactivated for 30 minutes at a temperature of 60 ° C to reduce the likelihood of nonspecific antigenic stimulation, supports cell growth due to the growth factors, interleukins, hormones, albumin and other biologically active substances contained in it; it is used in the cultivation of various types of hematopoietic and lymphoid cells;

 - HEPES - a buffer that is not toxic to cells, widely used to maintain a pH of 6.8-8.2 in culture studies on the growth of lymphoid cells;

 - 2-Mercaptoethanol - an antioxidant that causes the reactivation of many enzymes and is used as an additive in the cultivation of lymphoid cells, increases the output of granulocyte cells - macrophage series;

 - Glutamine is an essential amino acid needed to nourish growing cells;

 - Gentamicin is an antibiotic that suppresses the growth of the bacterial flora, added in a concentration non-toxic to the growing cell culture.

 - Neupogen (rhGM-CSF) is a recombinant human analogue of GM-CSF, which is a glycoprotein, a hematopoietic growth factor that regulates the formation and functioning of immunocompetent cells: dendritic cells, macrophages and granulocytes. Since only about 1% DC is contained in human peripheral blood, the production of a sufficient amount of DC in vitro from peripheral blood monocytes can be carried out by culturing in the presence of growth factors, the main of which is GM-CSF.

- recombinant human Interleukin-4 (rhIL-4) is a differentiation factor for T and B lymphocytes. The most powerful effect of IL-4 has on the regulation of the formation of other cytokines through participation in numerous biological processes, such as the immune response and inflammatory reactions. In vitro monocytes under the influence of GM-CSF and IL-4 lose their ability to phagocytosis, acquire the morphology of dendritic cells.

 - autologous lysate of the patient’s tumor cells — a lysate obtained from a fragment of the patient’s autologous tumor by mechanical grinding, filtering, washing in three steps with a serum-free medium containing a double concentration of antibiotics and antimycotics, subsequent processing of the resulting tumor cell suspension with three cycles of freezing-thawing and subsequent filtration through a filter cartridge with a pore diameter of 0.45 μm (Yu JS. et all. "Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma." Cancer Res. 2004, Jul 15 ; 64 (14): 4973-9; Stift A.et all. “Dendritic cell vaccination in medullary thyroid carcinoma.” Clin. Cancer Res. 2004, May 1; 10 (9): 2944-53);

 - recombinant human tumor necrosis factor alpha (rhTNF-a) is a polypeptide. TNF-a is a pro-inflammatory cytokine that causes accelerated maturation of dendritic cells;

 - Roncoleukin (rhIL-2) - a preparation of the recombinant human cytokine IL-2. IL-2 causes polyclonal activation of T-lymphocytes and the generation of LAK in vitro. The formation of LAK is the result of the activation of natural killers by IL-2. This cytokine helps to generate and enhance the cytotoxicity of LAKs, which are more specific for tumor cells compared to NK;

 - recombinant human Interleukin-12 (rhIL-12) - a cytokine that activates the differentiation of T-lymphocytes, increases their cytotoxic activity, enhances the proliferation of DC and T-lymphocytes and the production of other cytokines. One of the most important effects of IL-12 is the ability to rotate the differentiation of T-helpers towards T-helpers of 1 way;

 - recombinant human Interleukin-18 (rhIL-18) - is involved in the activation of cytotoxic T-lymphocytes, ΝΚ cells, macrophages, dendritic cells and contributes to the formation of an effective anti-infection and anti-tumor immune response.

 A method of obtaining an autologous cell vaccine for the treatment of cancer according to the invention was carried out in stages as follows:

a) mononuclear cells (hereinafter MNCs) were isolated from autologous the peripheral blood of a cancer patient in a known manner on a gradient of ficol-urographin with a density of 1.077 g / cm ³ with a ratio of blood and gradient of 2: 1; b) to obtain populations of monocytes and lymphocytes, cultivation of 2 × 10 ⁶ in vitro obtained MNCs in vitro was carried out in culture bottles by incubation for 1.0 hour at 37 ° C in an atmosphere of 5% C0 ₂ in DMEM supplemented with 100 ml of DMEM: FCS -1%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin -1000 μg / ml;

 c) the MNCs obtained in step b) were separated into an adherent fraction of monocytes and an adherent fraction of lymphocytes by collecting the adherent fraction from the walls of the vials after culturing in step b);

d) to obtain iDCs that are capable of actively capturing antigen by phagocytosis and macropinocytosis, monocytes were cultured in the adherent MNCs fraction obtained in step c) in vitro for 48 hours in DMEM supplemented with 100 ml DMEM: FCS -10% , HEPES -5 mM, Glutamine - 2 mM, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, and supplemented with 1 ml of medium containing 1 million cells, Neupogen - 50 ng / ml and rhIL- 4 - 100 ng / ml The indicated cultivation time and concentration were chosen experimentally taking into account the maximum output iDC, while the phenotype is determined were added according to the main markers CD83 + and CD 86+, and it was found that adding to the adherent fraction in step d) Neupogen at a concentration of 50 ng / ml in combination with rhIL-4 leads to an increase in the percentage of iDC output;

e) to obtain AA iDC, the fraction obtained in step g) was iodinated for 20-24 hours in DMEM supplemented with 100 ml DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, 2 -Mercaptoethanol - 5x10 ^{" 5} M, Gentamicin - 1000 μg / ml, with the addition of 100 μg per 1 ml of DMEM autologous lysate of tumor cells obtained from a fragment of an autologous tumor of the patient;

f) in order to obtain AA mDC, the fraction obtained in step e) was cultured for 24 hours in vitro in DMEM supplemented with 100 ml DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, with the addition of an autologous tumor cell lysate at the rate of 100 μg per 1 ml of DMEM and rhTNF-a at the rate of 25 ng per 1 ml of DMEM; and) parallel lymphocytes of non-adherent fraction MNCs obtained in stage c) were kept in DMEM supplemented with 100 ml of DMEM: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 μg / ml for a time sufficient to maintain cell viability, which was 96 hours;

 j) to obtain T-lymphocytes specifically activated by AA mDC, the adherent fraction obtained in step e) and part of the non-adherent MNCs fraction obtained in step i) were co-cultured in vitro at a ratio of 1: 10 for 48 hours fresh DMEM medium, additionally containing per 100 ml of medium: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 μg / ml, supplemented with 1 ml of medium containing 2.5 million cells, Roncoleukin - 100 IU / ml and rhIL-12 - 10 ng / ml or rhIL-18 - 100 ng / ml and rhIL-12 - 10 ng / ml. It was found that the addition of Roncoleukin and rhIL-12, or rhIL-12 and rhIL-18 at the stage of co-cultivation with an increase in the period of co-cultivation up to 48 hours leads to a subsequent increase in the percentage yield of specifically activated T-lymphocytes;

 k) in order to obtain LAK, the part of the non-adherent fraction that was not used in step k) was cultured in vitro for 48 hours in DMEM medium additionally containing 100 ml of medium: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 μg / ml, and supplemented with Roncoleukin - 1000 IU / ml per 1 ml of medium containing 2.5 million cells. It was found that increasing the concentration of Roncoleukin to 1000 IU / ml at the stage of LAK production with a reduction in cultivation time to 48 hours allows a higher percentage LAK yield to be obtained in a shorter period;

 m) then the adherent AA mDC was selected from the cell fraction obtained in step k) and washed twice by centrifugation in physiological saline for 10 minutes at 1500 rpm to obtain one part of the vaccine;

 m) non-adherent T-lymphocytes, specifically activated by AA mDC, were selected from the cell fractions obtained in step k) and LAK obtained in step k), and they were washed twice by centrifugation in physiological saline for 10 minutes at 1500 rpm. / min to obtain another part of the vaccine.

In the process of research, the authors found that the phased cultivation of adherent fraction monocytes described above in the method according to the invention MNCs isolated from the peripheral blood of an oncological patient first produce immature dendritic cells (iDC) with a pronounced ability to only absorb antigen (step g), and then, after adding an autologous tumor lysate (step e) to obtain antigen-activated immature DCs (AA iDC) ) and the subsequent addition of components that stimulate the maturation of these iDCs (step e), helps to produce AA mDC (step e), which effectively present antigens and activate T-helpers (step k), which leads to the production of T-lymphocytes specifically activated AA mDC.

 It was found that the time diversity of the stages of adding a tumor lysate to the medium to immature DC and introducing the acute phase cytokine TNF-a necessary for their maturation, with an interval of 24 hours, eliminates the possibility of early maturation of DC with an incomplete process of antigen presentation, increases the efficiency of capture and presentation antigen, increases the maturation efficiency and the percentage yield of AA mDC.

According to the invention, co-cultivation in step k) of the fraction of MNCs obtained in step e) and a portion of the non-adherent fraction of MNCs obtained in step i), with the production of T-lymphocytes specifically activated by AA mDC, was carried out in the presence of Roncoleukin or rhIL-18 , while the process of differentiation of T-helpers is directed along the T-helper 1 way (YOU are the cellular version of the immune response), which, when both parts of the vaccine are administered to the patient, leads to the deployment of a specific antitumor immune response, activated cytotoxic Kie T-killer (CD8 ⁺⁾ T lymphocytes and delayed-type hypersensitivity (DTH T - CD4 ^+), which are the main effectors of the cellular immune response path. This starts the presentation of the antigen as part of autologous antigen-activated DC (AAA DC) naive helper T-lymphocytes. The addition of rhIL-12 to the medium at step k) of the method according to the invention activates the generation of cytotoxic T-lymphocytes, enhances their activity.

 The time necessary and sufficient for the effective phased cultivation of immature and mature DCs was determined experimentally by the authors and is optimal.

Moreover, LAK from a portion of the non-adherent fraction of MNCs was obtained in step l) parallel to the co-cultivation step in step k), but separately, which eliminates competition between the specific and non-specific activation of lymphocytes and leads to an increase in the output of LAK.

 The cultivation of MNCs in step b) in an environment with the addition of inactivated fetal calf serum (FCS) eliminates the possibility of suppressive effects on tumor cells of tumor growth products contained in the serum of a patient with cancer.

 The following are the results of tests performed in vitro to determine the effectiveness of an autologous cell vaccine obtained by the method according to the invention. Statistical processing of the results was performed using the nonparametric Mann-Whitney and Wilcoxon test, the differences were considered significant at p <0.05.

 1. Determination of the effectiveness of dendritic cells of the vaccine obtained by the method according to the invention.

 For testing, autologous peripheral blood and tumor samples of patients with cancer of various localization obtained during planned surgical intervention as described above were used.

 DC maturation was assessed by the expression of surface differentiation markers (CD 14+, CD80 +, CD83 +, CD205 +) and the change in DC endocytosis activity at different stages of DC maturity by flow cytofluorimetry using appropriate fluorochrome-labeled antibodies and FITC-labeled dextran (fluorescein isocyanothiocyanate ) The degree of capture of FITC-labeled dextran by the studied cells was determined as the relative value of the corresponding average fluorescence intensity (hereinafter MFI) of the labeled cells. The capture of dextran labeled with FITC fluorochrome was evaluated at 37 ° C and 4 ° C to assess the specific endocytotic activity of DC (at 37 ° C) and the non-specific activity of DC (at 4 ° C).

 FIGLA, 1B, FIGS. 2A, 2B and FIGS. 3A, 3B illustrate the induction processes of maturation of DC cultured according to steps d), e) and e) of a method for producing a vaccine according to the invention from monocytes of adherent fraction of blood MNCs of patients with cancer of different localization:

- FigLA for iDC and FigLV for mDC shows the change in the expression of CD 14+ on cells in a monocytic pool at different stages of DC maturation, while: “CD14-PE” is the luminescence intensity of the phycoerythrin labeled CD 14 marker (phycoerythrin, PE); "CD3-FITC" - the intensity of the glow of the marker CD 3, labeled fluorescein isothiocyanate (fluorescein isothiocyanate, FITC); “Cd 14 + mon” is the population of MNCs carrying the CD 14 monocyte marker, “cd 3 + mon” is the population of MNCs carrying the CD 3 marker. From FigLA and 1B it follows that for iDC the maximum number of CD14 + was 11.2% (FigLA ), and for mDC the maximum amount was 3.3% (FigLV);

 - Fig.2A for iDC and Fig.2B for mDC shows the change in the expression of CD83 + and CD80 + on cells at different stages of DC maturation, while; "CD80-PE" is the luminosity of the marker CD 80 labeled with phycoerythrin (phycoerythrin, PE); “CD83-FITC” is the luminosity of the CD 83 marker labeled with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC); "Cd80 + mon" - mononuclear cells carrying the marker CD 80; "Cd83 + mon" - mononuclear cells carrying the marker CD 83; “Cd83 + cd80 + mon” are mononuclear cells that simultaneously carry markers for CD83 and CD80. From Figures 2A and 2B, it follows that for iDC the maximum number of CD83 + / CD80 + was 34% (Figure 2A), and for mDC the maximum number of CD83 + / CD80 + was 78.1% (Figure 2B);

 - FIG. 3A, SV shows the change in endocytosis activity in terms of the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation cultivated from the adherent fraction of blood MNCs of patients with cancer of different localization, while “Dextran-FITC” is the luminescence intensity absorbed bacterial polysaccharide labeled with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC): in FIG. 3A for iDC under incubation conditions at 37 ° C, the endocytosis activity (according to MFI) was 1754, in FIG. 3B for mDC in incubation conditions at 37 ° C endocytotic activity l (according to MFI) was 120. According to the test results, it was found that after cultivation in steps d) and e) DC are characterized by an immature phenotype: low expression of CD 14+ (FigLA, region a), low expression of CD83 +, CD83 + / CD80 + (Fig. 2A, regions a, b), and high endocytotic activity (Fig. 3A). After adding rhTNF-α to AA iDC in step e) and incubating for 24 hours, DCs with a mature phenotype were obtained: low expression of C014 + (Fig. 1B, region a), high expression of CD83 + and CD83 + / CD80 + (Fig. 2B, regions a, b) “low endocytotic activity (Fig. 3B).

In FIG. 4. presents the indicators of phenotypic markers at different stages of DC maturation cultivated by the method of producing the vaccine according to the invention from monocytes of adherent fraction of blood MNCs of cancer patients in coordinates along the b axis percent (M ± m), moreover: for iDC obtained from MNCs monocytes in step d) of the method in the presence of Neupogen and rhIL-4, the expression of CD 14+ was 10.4 + 0.9 (left column in region a), the expression of CD83 + was 25.2 + 2.0 (the left column in region b is shown), the expression of CD83 + / CD80 + was 32.7 + 8.5 (the left column in region c was shown), the expression of CD205 + was 78.2 + 6.2 (the left column is shown in region d), and for AA mDC obtained in step e) of the method from AA iDC by subsequent cultivation in the presence of rhTNF-α, the expression of CD 14+ was 4 + 0.7 (the right column in region a is shown), express i CD83 + was 47.9 + 5.0 (the right column is shown in region b), the expression of CD83 + / CD80 + was 69.4 + 9.1 (the right column is shown in region c), the expression of CD205 + was 89.9 + 1.9 (the right column in area d is shown). Arrows indicate significant differences between groups (p <0.05);

 Figure 5 presents the indicators of DC endocytosis activity, measured by the average fluorescence intensity (MFI median fluorescence intensity) at different stages of DC maturation, cultivated by the method of obtaining the vaccine according to the invention from adherent fraction of blood MNCs of cancer patients, in axial coordinates units of MFI, (M + w), moreover: for iDC obtained from MNCs in step d) in the presence of Neupogen and rhIL-4, the MFI was 1878.0 + 227.3 (left column is shown); for AA mDC obtained in step e) of the method from AA iDC by culturing in the presence of rhTNF-α, the MFI was 116.8 + 1 1.0 (right column shown). Arrows indicate significant differences between groups (p <0.05);

 Table 1 shows the phenotypic markers and endocytotic activity of DC (according to the MFI indicator) at different stages of DC maturation cultivated by the method of obtaining the vaccine according to the invention from the adherent fraction of blood MNCs of cancer patients, (M ± t), wherein: iDC were obtained from MNCs on step g) of the method in the presence of Neupogen and rhIL-4, and AA mDC were obtained in step e) of the method from AA iDC followed by cultivation in the presence of rhTNF-a.

 Table 1

Indicators of phenotypic markers and endocytotic activity of DC (by MFI value) at different stages of DC maturation cultured from adherent mononuclear fraction

blood cells of patients with cancer, (M ± t) IDC mDC P Markers

CD 14+,% 10.4 + 0.9 4.0 + 0.7 p = 0.006

CD83 +,% 25.2 + 2.0 47.9 + 5.0 p = 0.01

CD83 / 80 +,% 32.7 + 8.5 69.4 + 9.1 p = 0.044

CD205 +,% 78.2 + 6.2 89.9 + 1.9 p = 0.2

MFI

 conditional 1878.0 + 227.3 1 16.8 + 1 1.0 p = 0.002 units

 Note: p is an indicator of the significance of differences between groups i DC and mDC.

 From the results presented in FIG. 4, FIG. 5 and Table 1, it follows that during the cultivation of mDC by the method of producing a vaccine according to the invention from monocytes of mononuclear blood cells of patients with cancer of different localization, there was a decrease in the expression of CD 14+ on the surface of mature DC and their endocytotic activity, which indicates the loss of nonspecific properties characteristic of monocyte cells - DC precursors. In addition, the increased expression of CD83 + and CD80 + on mature DCs reflects the directed differentiation of monocytes into mature DCs.

 2. Determination of the cytotoxic ability of the vaccine obtained by the method according to the invention.

 To evaluate the effectiveness of stimulation of the antitumor immune response of AA mDC, obtained by the method of obtaining the vaccine according to the invention, a direct cytotoxicity test was used.

 The cytotoxicity test of the non-adherent fraction of MNCs to the autologous tumor cells of the patient was carried out using a Promega kit according to the manufacturer's instructions.

 For testing, we used the AA mDC obtained in step e) of the method for preparing the vaccine according to the invention, which were then subjected to co-cultivation in step k) of the non-adherent fraction of MNCs obtained in step i) in a ratio of 1: 10, respectively, in an atmosphere of 5% С02 and 37 ° С in fresh DMEM medium, additionally containing: FCS, HEPES, Glutamine, Gentamicin, supplemented with Roncoleukin and rhIL-12 or supplemented with rhIL-18 and rhIL-12.

After 16-18 hours of specified incubation in the specified culture medium autologous tumor cells, previously obtained by mechanical homogenization of a tumor sample and stored at minus 70 ° C, were introduced.

 After incubation for 52 hours, tests were carried out for the cytotoxicity of the non-adherent fraction of MNCs to the patient's autologous tumor cells (group 1 of the tests),

 - after incubation in the complex MNCs + rhIL-12 + rhIL-18 (2nd group of tests);

- after incubation in the complex MNCs + rhIL-12 + rhIL-2 (3rd group of tests);

- after incubation in the complex MNCs + AA mDC (4th group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-18 (5th group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-2 (6th group of tests);

while the death of tumor cells was evaluated by the release of the intracellular enzyme lactate dehydrogenase. The results were expressed as a percentage relative to the absolute death of the cells when adding the lysis solution included in the kit.

 Figure 6 and table 2 presents the cytotoxicity indices of the MNCs culture against tumor cells in the presence of AA mDC and cytokines (rhIL-12 and rhlL-18 or rhIL-12 and rhIL-2), (M ± m), with confidence compared with cytotoxicity indices in the MNCs groups with the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2) in the absence of AA mDC, MNCs in the presence of AA mDC, without the addition of cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2).

 table 2

The cytotoxicity index of a mononuclear cell culture against tumor cells in the presence of AA mDC and cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2), (M ± m)

Experimental cytotoxic P group test group,%

 p2 = 0.0007

 p3 = 0.0009

 1 MNCs 7.4 ± 2.1 p4 = 0.00007

 p5 = 0.00002

p6 = 0.00001 2 pl = 0,0007

MNCs + rhIL-12 + rhIL-18 27.9 ± 5.6 p5 = 0.00002

 p6 = 0.00001

 pi = 0,0007

 3 MNCs + rhIL-12 + rhIL-2 24.9 ± 5.6 p5 = 0.00002

 p6 = 0.00001

 pl = 0,0008

 4 MNCs + AA mDC 39.6 ± 5.7 p5 = 0.0002

 p6 = 0.0001

 pi = 0.00002

 5 MNCs + AA mDC + rhIL-12 p2 = 0,0009

 62.9 ± 6.4

 + rhIL-18 p3 = 0.002

 p4 = 0.04

 pi = 0.00001

 6 MNCs + AA mDC + rhIL-12 p2 = 0,0007

 65.9 ± 6.4

 + rhIL-2 p3 = 0.001

 p4 = 0.03

 Note:

 PI - confidence indicator compared with group 1,

P2 is a confidence indicator compared with group 2,

p3 - confidence indicator compared with group 3,

p4 - confidence indicator compared with group 4,

p5 - confidence indicator compared with group 5,

RB is a confidence indicator in comparison with group 6.

 From the data presented in FIG. 6 and Table 2, it follows that the maximum cytotoxicity value was observed in the group of MNCs co-cultivated with AA mDC and cytokines. Thus, the most effective stimulation of the cytotoxic activity of MNCs is observed with the combined use of AA mDC and cytokines.

 3. Determination of antigen-specific stimulation of the immune response according to the T-helper type 1 using a vaccine obtained by the method according to the invention.

For analysis of antigen-specific stimulation of the immune response by T-helper Type 1 was determined by the content of IFN-γ in the conditioned medium of the culture of MNCs co-cultivated with AA mDC before and after additional stimulation with tumor antigens (lysate of autologous tumor cells at a concentration of 100 μg / ml) and cytokines. The IFNy content was determined using commercial ELISA kits (Vector-Best) according to the manufacturer's instructions.

 7 and table 3 presents the results of tests to determine the content of IFN-γ, (M ± t), in the culture of MNCs (7th group of tests) and with the addition of cytokines (rhlL-12 and rhIL-18 or rhIL- 12 and rhIL-2):

 - after incubation in the complex MNCs + rhlL-12 + rhIL-18 (8th group of tests);

- after incubation in the complex MNCs + rhIL-12 + rhIL-2 (9th group of tests);

and after co-cultivation of MNCs with AA mDC and the addition of cytokines (rhIL-12 and rhlL-18 or rbb-12 and rbb-2):

 - after incubation in the complex MNCs + AA mDC (10th group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-18 (1 1st group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-2 (12th group of tests),

during spontaneous production (“sp” - groups, left columns of the diagram) and during lysate-activated production when tumor cells are added to the lysate medium (“act” - groups, right columns of the diagram).

 Table 3

The content of IFN-γ in the culture of mononuclear cells co-cultivated with cytokines (rhIL-18 and rhIL-12 or rhIL-2 and rhIL-12)

 and / or with AA mDC in spontaneous

 and lysate-activated production, (M ± t)

 Experimental Content of IFN-γ, pkg / ml group of the Lysate group

Spontaneous activated

 producing

 niye (at

 (sp) adding to

lysate medium opuzole

 cells)

 (act)

 p5 = 0.051

7 MNCs 7.0 ± 0.5 11.9 ± 1, 3 p6 = 0.047

8 p5 = 0.049

MNCs + rhIL-12 + rhIL-18 12.8 ± 2.9 13.6 ± 2.2

 p6 = 0,049

9 p5 = 0.050

MNCs + rhIL-12 + rhIL-2 1 1.2 ± 1.2 14.4 ± 1.9

 p6 = 0.051

10 p5 = 0.052

MNCs + AA mDC 8.0 ± 0.8 11.2 ± 1.3

 p6 = 0.041 pl = 0.052

11 p2 = 0.057

MNCs + AA mDC + rhIL-12

 12.7 ± 4.6 22.2 ± 2.2 p3 = 0.059 + rhIL-18

 p4 = 0.084 pi = 0.042

12 MNCs + AA mDC + rhIL-12 p2 = 0.047

 12.0 ± 2.6 25.0 ± 2.0

 + rhIL-2 p3 = 0.057 p4 = 0.074

Note:

pi - confidence indicator in comparison with group 7,

P2 is a confidence indicator compared to group 8,

p3 - confidence indicator compared with group 9,

p4 - confidence indicator compared with group 10,

p5 - confidence indicator compared with group 1 1,

rb is a confidence indicator in comparison with group 12.

 The addition of rhIL-18 and rhIL-12 or rhIL-2 and rhIL-12 and rhlL-12 cytokines to the co-culture of MNCs and AA mDC significantly increased IFN-γ production in response to additional antigenic stimulation compared to the group of MNCs and AA mDC without adding cytokines. This indicates a stimulating effect of cytokines on the modulation of the immune response according to the T-helper type 1.

4. Determination of the content of IL-4 in the conditioned medium of cultures of MNCs with different modes of cultivation.

 The content of IL-4 in the conditioned culture medium of MNCs co-cultured with AA mDC before and after additional stimulation with tumor antigens (lysate of augologous tumor cells at a concentration of 100 μg / ml) and cytokines was determined. The content of IL-4 was determined using commercial ELISA kits (Vector-Best) according to the manufacturer's instructions.

 On Fig and table 4 presents the results of tests to determine the content of IL-4 in the conditioned medium of the culture of MNCs (13th group of tests), (M ± t), and the content of IL-4 in the conditioned medium of the culture of MNCs with the addition of cytokine medium (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2):

 - after incubation in the complex MNCs + rhIL-12 + rhIL-18 (14th group of tests);

- after incubation in the complex MNCs + rhIL-12 + rhIL-2 (15th group of tests);

and tests to determine the content of IL-4 in the conditioned medium of MNCs cultures co-incubated with AA mDC, prior to maturation with the treated tumor antigens, and with the addition of cytokines (rhIL-12 + rhIL-18 or rhIL-12 and rhIL-2):

 - after incubation in the complex MNCs + AA mDC (16th group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-18 (17th group of tests);

 - after incubation in the complex MNCs + AA mDC + rhIL-12 + rhIL-2 (18th group of tests),

during spontaneous production (“cn” - groups, left columns of the diagram) and during lysate-activated production when tumor cells are added to the lysate medium (“act” - groups, right columns of the diagram).

 Table.4

The content of IL-4 in the culture of mononuclear cells co-cultivated with cytokines (rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2), (M ± m) and / or AA mDC in spontaneous conditions

 and lysate-activated production, (M ± t)

 L Experimental Content of IL-4, pkg / ml

 groups of the group Spontaneous Lizat-R

producing activated production

 (sp) (when added to

 lysate medium

 tumor

 cells)

 (act)

 p2 = 0.22 p3 = 0.2

13 MNCs 9.3 ± 1.2 21.0 ± 1.3 p4 = 0.179 p5 = 0.56 p6 = 0.32 p3 = 0.56

14 p4 = 0.65

MNCs + rhIL-12 + rhIL-18 13, 1 ± 2.5 25.5 ± 1, 6

 p5 = 0.79 p6 = 0.42 p4 = 0.94

15 MNCs + rhIL-12 + rhIL-2 1 1.4 ± 3.2 27.2 ± 1.4 p5 = 0.44 p6 = 0.50 pl = 0.179 p2 = 0.65

16 MNCs + AA mDC 18.4 ± 1.1 25.5 ± 1.9 p3 = 0.94 p5 = 0.48 p6 = 0.38 pl = 0.56

17 MNCs + AA mDC + rhIL-2 p2 = 0.79

 1 1.2 ± 2.1 22.0 ± 2.3

 + rhIL- 18 p3 = 0.44 p4 = 0.48 pl = 0.52

18 MNCs + AA mDC + rhIL-12 p2 = 0.89

 1 1.0 ± 2.1 24.0 ± 2.3

 + rhIL-2 p3 = 0.34 p4 = 0.38

Note:

P1 is a confidence indicator in comparison with group 13, p2 - confidence indicator compared with group 14,

p3 - confidence indicator compared with group 15,

p4 - confidence indicator compared with group 16,

p5 - confidence indicator compared with group 17,

rb is a confidence indicator in comparison with group 18,

 As a result of the described testing, no significantly significant differences in the IL-4 content between the groups were revealed.

 Thus, according to the invention, a method has been developed for generating antitumor cytotoxic cells using antigen-activated autologous dendritic cells and rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2 in vitro to produce antigen-activated immature and mature dendritic cells antigenactivated during maturation, from mononuclear cells of patients with cancer of various localization, which are characterized by the corresponding functional activity and phenotype. Moreover, in comparison with the vaccine of the prototype, an increase in the expression of co-stimulatory molecules on the surface of antigen-activated dendritic cells was achieved, which indicates an increase in the effective process of antigen presentation.

 It was established that co-cultivation of mature DCs antigenactivated during maturation with lymphocytes of the non-adherent fraction of MNCs in combination with rhIL-12 and rhIL-18 or with rhIL-12 and rhIL-2 is an effective way to generate specific cytotoxic cells of mononuclear origin, which manifests itself in enhancing their cytotoxic antitumor response compared to the vaccine of the prototype. Using a combination of rhIL-12 and rhIL-18 or rhIL-12 and rhIL-2 to enhance the induction of the response of type 1 T helper cells allows a statistically significant increase in the cytotoxic potential of mononuclear cells and IFN-γ production in vitro.

Moreover, the method for producing an autologous vaccine according to the invention allows, at the method steps, to increase the yield of immature dendritic cells, increase the efficiency of capture and presentation of antigen, increase the yield of specifically activated killers, eliminates competition between specific and non-specific activation of cells, allows a higher percentage to be obtained in a shorter time. yield of non-specifically activated killers (LAC) and specifically activated killers (T-lymphocytes). Thus, the autologous cell vaccine according to the invention for the treatment of cancer obtained by the method according to the invention, in comparison with known vaccines, is more effective, does not require additional administration of cytokines directly into the patient’s body, and can be used to treat cancer patients with tumors of different localization .

 Industrial applicability

 The method of obtaining an autologous cell vaccine according to the invention for the treatment of cancer by simultaneously affecting a specific and non-specific link of the antitumor immune response can be implemented using known techniques and materials, and the vaccine obtained in this way is highly effective and can be used in the field of oncology for the treatment of patients with various oncopathology.

Claims

Claim 1. An autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and non-specific link of the antitumor immune response, containing mature dendritic cells antigenactivated during maturation (AA mDC) in one part of the vaccine, and T lymphocytes specifically containing in the other part of the vaccine activated by the indicated mature dendritic cells, antigen-activated during maturation (AA mDC), and lymphokinactivated killers (LAK).  2. A method of obtaining an autologous cell vaccine for the treatment of cancer by simultaneously acting on a specific and non-specific link of the antitumor immune response, containing in one part mature dendritic cells antigen-activated during maturation (AA mDC) and containing T-lymphocytes in its other part specifically activated by said mature dendritic cells antigen-activated during maturation (AA mDC) and lymphokinactivated killers (LAK), comprising the following step :  a) the allocation of mononuclear cells (MNCs) from the peripheral blood of a cancer patient;  b) culturing the mononuclear cells obtained in step a) in vitro in a base medium with a high content of amino acids and vitamins, which provides cell viability, including lymphoid cells, supplemented with FCS, HEPES, Glutamine, Gentamicin, to obtain populations of monocytes and lymphocytes;  c) separation of the mononuclear cells obtained in step b) into an adherent fraction of monocytes and an adherent fraction of lymphocytes; d) culturing the monocytes of the adherent fraction obtained in step c) in vitro in a fresh medium, similar in composition to that used in step b), with the addition of 2-Mercaptoethanol, a recombinant analog of granulocyte-macrophage colony stimulating factor (rhGM-CSF) and growth factor to produce immature dendritic cells (iDC) that are capable of actively capturing antigen by phagocytosis and macropinocytosis;  d) culturing the fraction obtained from step d) containing immature dendritic cells in a fresh medium, similar in composition to the medium used in step b), with the addition of 2-Mercaptoethanol, a tumor cell lysate obtained from a fragment of a patient’s autologous tumor, to obtain antigen-activated immature dendritic cells (AA iDC); f) culturing the fraction obtained in step e) containing antigen-activated immature dendritic cells (AA iDC) in vitro in the medium used in step e) with the addition of recombinant human tumor necrosis factor alpha (rhTNF-α) to obtain mature dendritic cells antigenactivated during maturation (AA mDC);  i) exposure of the lymphocytes of the non-adherent fraction obtained in step c) in a fresh medium similar to that used in step b) for a time sufficient to maintain the viability of the lymphocyte fraction;  j) co-culturing the fraction obtained in step e) and part of the non-adherent fraction obtained in step i) in vitro in a ratio of 1: 10 in fresh medium, similar in composition to the medium used in step b), and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) and recombinant human Interleukin-12 (rhIL-12) or supplemented with recombinant human Interleukin-12 (rhIL-12) and recombinant human Interleukin-18 (rhIL-18), to produce T-lymphocytes specifically activated by mature dendritic cells antigenactivated during ripening;  k) in vitro cultivation of the portion of lymphocytes of the non-adherent fraction not used in step k) obtained in step c) in a medium similar in composition to the medium used in step b) and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) obtaining lymphokinactivated killer cells (LAK cells);  l) selection from the fraction obtained in step k) of adherent mature dendritic cells antigen-activated during maturation (AA mDC) and washing them to obtain one part of the vaccine; m) selection from cell fractions obtained in step k) and in step k), not adhering specifically activated T-lymphocytes and lymphokinactivated killer cells (LAK cells) and washing them to obtain another part of the vaccine.  3. The method according to claim 2, in which the allocation of mononuclear cells from the peripheral blood of the patient is carried out on a gradient of ficol-urographin with a density of 1.077 g / cm3 with a ratio of blood and gradient of 2: 1.  4. The method according to claim 2, in which the cultivation in step b) is carried out for 1.0 hour at a temperature of 37 ° C in an atmosphere of 5% CO2. in the base medium supplemented with 100 ml of the base medium: FCS -1%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin -1000 μg / ml. 5. The method according to claim 2, in which the cultivation in step g) is carried out for 48 hours in a basic medium supplemented with 100 ml of a basic medium: FCS -10%, HEPES -5 mm, Glutamine-2 mm, 2-Mercaptoethanol-5x10 ^{" 5} M, Gentamicin - 1000 μg / ml, and supplemented with 1 ml of medium containing 1.0 million cells, a recombinant analog of granulocyte-macrophage colony stimulating factor Neupogen (rhGM-CSF) - 50 ng / ml and recombinant human Interleukin- 4 (rhIL-4) - 100 ng / ml. 6. The method according to claim 2, in which the cultivation in step d) is carried out for 20 to 24 hours in a base medium supplemented with 100 ml of a base medium: FCS-10%, HEPES - 5 mm, Glutamine - 2 mm, 2- Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, with the addition of a tumor cell lysate at the rate of 100 μg per 1 ml of base medium. 7. The method according to claim 2, in which the cultivation in step e) is carried out for 24 hours in a basic medium supplemented with 100 ml of a basic medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, 2-Mercaptoethanol - 5x10 ^"5 M, Gentamicin - 1000 μg / ml, with the addition of a tumor cell lysate at the rate of 100 μg per 1 ml of the base medium and recombinant human tumor necrosis factor alpha (rhTNF-α) at the rate of 25 ng per 1 ml of the base medium.  8. The method according to claim 2, in which at the stage i) the exposure is carried out for 96 hours in the base medium supplemented with 100 ml of the base medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 μg / ml 9. The method according to claim 2, in which the cultivation in step k) is carried out for 48 hours in a base medium additionally containing 100 ml of base medium: FCS -10%, HEPES -5 mM, Glutamine - 2 mM, Gentamicin - 1000 μg / ml, supplemented with 1 ml of medium containing 2.5 million cells, recombinant human Interleukin-2 (Roncoleukin, rhIL-2) -100 U / ml and recombinant human Interleukin-12 (rhIL-12) 10 ng / ml or recombinant human Interleukin-12 (rhIL-12) 10 ng / ml and recombinant human Interleukin-18 (rhIL-18) 100 ng / ml  10. The method according to claim 2, in which the cultivation in stage l) is carried out for 48 hours in a basic medium additionally containing 100 ml of the basic medium: FCS -10%, HEPES -5 mm, Glutamine - 2 mm, Gentamicin - 1000 μg / ml and supplemented with recombinant human Interleukin-2 (Roncoleukin, rhIL-2) - 1000 IU / ml per 1 ml of medium containing 2.5 million cells.  11. The method according to claim 2, in which DMEM is used as the base medium. 12. The method according to claim 2, in which the washing of the cell fractions in steps m) and n) is carried out twice in physiological saline for 10 minutes at 1500 rpm.