RU2309753C1 - Combined cell transplant based on lymphokine-activated killers and dendrite cells, method for production thereof and method for treatment and prophylaxis of malignant, infective diseases and immunodeficient conditions - Google Patents

Abstract

FIELD: biopharmacology and medicine.

SUBSTANCE: disclosed is method for production of mononuclear blood cells, namely activated lymphocytes (ACL) and dendrite cells (DC). Claimed method includes a) isolation of mononuclear leucocytes (MNL) from blood and marrow of patient suffering from malignant neoplasm, or infective disease, or immunodeficient condition; d) incubation of isolated mononuclear lymphocytes in cultural medium; c) separation of MNL cultural bottles adhering to plastic and non-adhering thereto.; d) treatment of adhering MNL with growth factors to produce dendrite cells therefrom; e) treatment of DC with lysates from patient tumor; f) cultivation of non-adhered lymphocytes with interleukine-2 to produce ACL-cells. Also disclosed is method for treatment and prophylaxis malignant and infective diseases and immunodeficient conditions in patients by combined application of ACL-cells and dendrite cells.

EFFECT: new method for treatment and prophylaxis of malignant, infective diseases and immunodeficient conditions.

3 cl

Description

The present invention relates to medicine, relates to the production of lymphokine-activated killer cells and dendritic cells and can be used to prevent and treat cancer, especially in “risk groups” and in “family forms” of cancer, immunodeficiency states, both congenital and acquired , in particular with viral and bacterial infections.

The history of studies of the role of the immune system in the antitumor defense of an organism is about 40 years old. Numerous studies conducted during this period have shown that an important role in this defense is given to the cellular component of immunity, which is represented in humans and animals by a number of effector cells. Among this series, NK cells belonging to lymphoid cells, but differing phenotypically from T and B lymphocytes [Davydov M.I., Normantovich V.A., Kiselevsky M.V., Volkov S.M. and other Adaptive immunotherapy for tumor pleurisy: a clinical and laboratory study. - Russian Oncology Journal. 2000. - No. 6 - p. 14-17].

The special role of NK in antitumor immunity is confirmed both by the ability of these cells to lyse tumor cells in vitro, and by the confirmed correlation between the quantitative level of these cells in the body and its ability to develop antitumor defense. In particular, observations of inbred line mice showed that different lines of these animals are characterized by different quantitative levels of NK. In mice with a high level of NK, spontaneous or induced tumors are rare; in mice with a low level, the frequency of spontaneous tumors, on the contrary, is high. Cell transplantation from mouse lines with a high level of NK to lines with a low level of these cells in the experiment increases the resistance of the recipients to some forms of tumors [Davydov MI, Normantovich VA, Kiselevsky MV, Volkov SM and other Adaptive immunotherapy for tumor pleurisy: a clinical and laboratory study. - Russian Oncology Journal. 2000. - No. 6 - p. 14-17].

It should be noted that one of the features of NK cells is the presence of a killing inhibitory receptor (KIR) on their surface. This receptor recognizes antigens of the main histocompatibility complex of class I (MHC-I), present on almost all normal cells of the body, and stops the lysis of the target cell. Tumor cells may differ from normal ones by the absence or decrease in the expression of antigens of the main histocompatibility complex, and then such cells become targets for NK. If MHC-I is expressed on the tumor cells and the level of this expression is normal, lysis by the NK cell of such a tumor cell will probably be impossible.

A study of the activity of lymphoid cells under the influence of various cytokines has shown that interleukin-2 (IL-2), the active component of T-cell growth factor (FRTK), plays an important role in the activity of both T-lymphocytes and NK cells. In 1980, Yron et al published a report that in a normal mouse splenocyte culture containing PRTC, cells are generated that can lyse freshly isolated or cultured syngeneic sarcoma cells. These results were further confirmed in experiments on mice and human lymphocytes, and the described phenomenon was called the LAC phenomenon, defined as “lysis of freshly isolated or cultured tumors in vitro by lymphoid cells activated in IL-2 culture”. LAC cells differ from NK cells present in samples of freshly isolated lymphoid tissue, both phenotypically and in their ability to lyse freshly isolated NK-resistant tumor target cells [Pashenkov MV, Pinegin BV The main properties of dendritic cells. - Immunology. 2001. - p. 7-16].

The study of the LAC phenomenon quickly moved from laboratories to clinics: the attempt to use LAC cells as a therapeutic agent in the treatment of cancer began to be undertaken by the US National Cancer Institute (NCI) from 1980-1981 [A. Yarilin The basics of immunology. - M .: Medicine, 1999. - p. 247-278]. For twenty years of studying the phenomenon, both autologous and allogeneic LAC cells, LAC cells with different phenotypes incubated over different periods were used, attempts were made to use various cytokines to induce the activity, the features of tumors lysed by LAC cells were studied, and various doses and modes of administration of LAC cells in various tumors.

Clinical studies showed that the LAC phenomenon, which demonstrated high antitumor activity in preclinical studies in vitro and in vivo in animals, subsequently did not justify the expectations placed on it in the treatment of human oncological diseases. Numerous clinical studies conducted over more than 20 years have shown that the effectiveness of systemic use of LAC cells against such tumors as melanoma and kidney cancer does not exceed the effectiveness of standard methods of treatment [Fernandez N.C., Flament C., Crepineau F. , Angevin E., Vivier E., Zitovgel L. Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC / NK cross talk. - Eur. Cytokine Netw. 2002. - vol. 13. - p.17-27].

With the use of IL-2 / LAC-therapy, individual successes were noted with the use of LAC cells in the local-regional mode, in particular with metastatic lesions of the liver and lungs [Freedman R.S., Platsoncas C.D. // Immunotherapy for peritonel ovarian carcinoma metastasis using ex vivo expanded tumorinfiltrating lymphocytes. // 1996. - Vol. 82. - p. 115-116]. The effectiveness of this therapy in the treatment of tumor pleurisy with the introduction of IL-2 / LAK into the pleural cavity was also noted. Traditionally, this relatively higher efficiency is associated with the fact that the locoregional use of LAK cells allows for a higher concentration of LAK cells in the tumor area. Nevertheless, the results of the application of locoregional therapy, as well as systemic, cannot be considered satisfactory, since there are no sufficiently convincing, statistically reliable data indicating the constancy of the antitumor effect of this therapy achieved in some studies. There are also reports that indicate unsatisfactory results of this type of therapy [Ferlazzo, G., Wesa, A., Wei, WZ, Guli, A. Dendritic cells generated from CD34 + progenitor cells or from monocytes differ in their ability to activate antigen-specific CD8 + T cells. - J. Immunol. 1999 .-- vol. 163. - p. 3597]. However, the presence of certain successes in the use of IL-2 / LAC-therapy stimulated interest in studying the patterns of distribution of LAC-cells in vivo in order to find out the reasons for the relative effectiveness of locoregional therapy. These works show that with systemic administration, only a very small fraction of the cells reach the target tumor.

In 1991, Basse P. et all examined NK cells labeled with a fluorescent compound and found that some time after administration, they are able to accumulate 10-fold concentrations of lung and liver metastases in the tissue [Ardavin C., Martinez del Hoyo G., Martin P., Anjuere P., Arias S.P., Marin AR, Ruiz S., Parrillas V., Hemandes H. Origin and differentiation of dendritic cells. - Trends in Immunology. 2001 .-- vol.22. - p.691-700].

In 1999, J. Kjaergaard et all published a study “Biodistribution and tumor localization of lymphokine-activated T-killers depending on the route of administration to tumor-bearing animals” [Banchereau J., Steinman R.M. Dendritic cells and the control of immunity. - Nature. 1998 .-- vol. 392. - p.245-252].

This study demonstrated that the accumulation of labeled T-LAC cells in organ metastases directly depends on the route of introduction of cells into the body. With intravenous administration, the intensity of accumulation of LAK cells in metastases located in the lungs was 5 times more intense than in the surrounding lung tissue. In metastases and healthy tissue of other organs, the accumulation of LAC cells with this route of administration was practically not observed. On the contrary, with the intraportal route of administration, the highest accumulation of LAC cells was observed precisely in the liver, and in the lungs and other organs the cells were not detected. With intracardiac (left ventricular) administration of LAC cells, their accumulation in adrenal gland tissues was insignificant, and with intraperitoneal administration, it was found that LAC cells accumulate in those metastases that are not separated from the introduced LAC cells by peritoneal tissue. Researchers conclude that there are practically insurmountable obstacles in the path of LAC cells in the microvasculature of organs such as the liver and lungs.

It should be noted that despite the fact that LAC cells accumulate in the liver and lung metastases with appropriate routes of administration, their concentration for the lysis of the tumor is apparently insufficient. Thus, it can be assumed that the effectiveness of IL-2 / LAC therapy depends not only on the route of administration, but also on the number of LAC cells introduced and the size of the tumor mass. In addition, with an excess of tumor cells, the opposite is possible.

The data and conclusions of these studies are consistent with the results of individual clinical studies that have demonstrated the efficacy of locoregional IL-2 / LAK therapy in adjuvant and therapeutic regimens for metastatic lung and liver lesions. Feuerstein B. et all in 2000 publish the results of a small clinical trial on the adjuvant locoregional therapy of pancreatic cancer metastases in the liver [Feuerstein B., Berger T.G., Maczek C., Roder C., Schreiner D., Hirsch U., Haendle I., Leisgang W., Glaser A., Kuss O., Diepgen TL, Schuler G., Schuler-Thurner B. A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use. - J. Immunol. Methods 2000, vol. 245, p.15-29]. 29 patients with pancreatic cancer III and IV Art. (according to the Japan Pancreas Society) were divided into two groups, in both groups a radical resection of pancreatic cancer and intraoperative radiotherapy were performed, in the study group, intraportal IL-2 / LAC-therapy was additionally performed. Both groups were subsequently compared in terms of survival and the incidence of liver metastases. After 3 years in the experimental group (n = 12) of survivors - 36% (n = 4), in the control (n = 17) - 0%, the result is statistically unreliable (p = 0.082). The frequency of liver metastases was significantly lower in the experimental group than in the control group: 2 out of 12 versus 10 out of 15 (p <0.05).

Researchers who have received the effect of this therapy also note the need to use high doses of injected LAC cells, a long treatment regimen and maximum preliminary cytoreduction of the tumor mass in order to achieve the optimal ratio of LAC cells / tumor cells.

The next intractable question related to the effectiveness of LAC-therapy is the mechanism of recognition of a tumor cell by a killer cell and its lysis. Recognition of a target cell by a killer cell may or may not be related to the presentation of foreign antigens in complex with the MHC class I protein molecules on the surface of the target cell. According to classical concepts in immunology, cytotoxic lymphocytes lyse cells that represent such complexes on their surface, and the lysis performed by NK cells is not associated with MHC-I, because NK cells express a killing inhibitory receptor blocking the lysis of the target cell in the presence of an autologous MHC on its surface [MV Kiselevsky, AG Blumenberg Adaptive immunotherapy of ovarian cancer. - A collection of articles dedicated to the European School of Oncology, 2001, p.164-176].

It was shown that with tumor pleurisy, the efficiency of introducing LAK cells into the pleural cavity is about 90%. The expression of MHC-I on tumor cells was not determined in this study, however, it can be assumed that it was unlikely to be close to 0% in all patients [Bindon J., P. Ruell et al. //. J. Immunol. 1984. - vol. 3. - p. 475-478].

These facts hypothetically may indicate that the LAC phenotype is not similar to the NK cell phenotype and that the cytotoxicity of LAC cells may not be limited to MHC-I ³² either in terms of antigen presentation or inhibition of killing via the killing inhibitory receptor (KIR). It is also possible that the cytotoxicity of LAK cells depends on some other factors that are currently unknown.

In any case, in connection with the foregoing, it seems necessary to phenotypize the used LAC cells, as well as to determine the correlation between their cytotoxicity and expression on the already phenotyped tumor cell lines of certain antigens.

In addition, other properties of the tumor undoubtedly play an important role in the implementation of the antitumor effect: the severity of expression of signaling molecules, adhesion molecules, a number of enzymes that may not be expressed at all or not expressed enough to make contact between the killer and the target. It is also possible that factors secreted by the tumor play a role - immunosuppressive and others.

In recent years, researchers of various specialties have paid great attention to the study of the antitumor effect of vaccines based on dendritic cells (DC). The nature of this phenomenon is associated with the ability of DCs to present tumor antigens to cytotoxic T-lymphocytes. To date, certain types of biological activity of DC have been studied. At the same time, many methods of preparation and antitumor properties of DC-based vaccines remain unclear.

Mature DCs for antitumor vaccines are most often obtained from CD14 + monocytes by a 2-stage procedure. In the first stage, monocytes develop into immature DCs in the presence of granulocyte / macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs have a high ability to capture antigens by macrophinocytosis and phagocytosis (Banchereau J., Steinman RM Dendritic cells and the control of immunity. - Nature. 1998. - vol.392. - p.245-252; Keller R. Dendritic cells: their significance in health and disease. - Immunol. Letters. 2001. - vol. 78. - p. 113-122). In morphological and phenotypic terms, these are large, irregularly shaped cells that weakly or do not adhere to plastic, significantly expressing the co-stimulator CD86 (B7-2), weakly expressing the CD14 monocyte / macrophage marker and the mature CD83 marker CD83 (Hasebe, H., Nagayama, H. , Sato, K., Enomoto, M., Takeda, Y., Takahashi T.A., Hasumi K., Eriguchi M. Dysfunctional regulation of the development of monocyte-derived dendritic cells in cancer patients. - Biomed. & Pharmacother. 2000. - vol. 54. - p. 291-298; Morse MA, Vredenburgh JJ, Lyerly HK A comparative study of the generation of dendritic cells from mobilized peripheral blood progenitor cells of patients undergoing high dose chemotherapy. - J. Hematother. Stem Cell Res. 1999. - vol. 8 - p. 577-584). In the vast majority of experiments, another co-stimulator CD80 (B7-1) is expressed much weaker than CD86, and its expression level, according to various authors, varies significantly - 2-87%.

In the second stage, the maturation of DC is initiated. Maturation of DC can occur under the influence of a number of factors: bacteria (living or dead), bacterial products (LPS), viruses, double-stranded RNA or its analog poly-I: C, pro-inflammatory factors and their combinations (IL-1β, TNF-α, IL -6, prostaglandin-E2 (PGE-2)) and ligand CD40 (CD40L) (Dhodapkar M.V., Steinman RM, Sapp M., Desai H., Fossella C., Krasovsky J., Donahoe SM, Dunbar PR, Cerundolo V., Nixon DF, Bhardwaj N. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. - J. Clin. Invest. 1999. - vol. 104. - p. 171-180; Keller R. Dendritic cells: their significance in health and disease. - Immunol. Letters. 2001. - vol. 78. - p. 113-122). As they mature, they significantly lose their ability to endocytize and process antigens. However, mature DCs retain the ability to present immunogenic peptides that do not require prior processing inside the cell (Sallusto F., Nicolo C., De Maria R., Corinti S., Testi R. Ceramide inhibits antigen uptake and presentation by dendritic cells. - J. Exp Med. 1996. - vol. 184. - N 6. - p.2411-2416). Mature DCs stably express on their surface complexes of molecules of the main histocompatibility complex (MHC) with peptides (Cella M., Engering A., Pinet V., Pietras T., Lanzavecchia A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. - Nature. 1997. - vol. 388. - p. 782-787). They also differ from immature DCs by the expression of characteristic markers of mature DCs - CD83 and DC-LAMP - a member of the family of membrane glycoproteins associated with lysosomes; in addition, the number of co-stimulating molecules increases significantly. On the surface of mature DCs, the number of adhesive molecules CD54 and CD58 that promote interaction with T-lymphocytes increases (Keller R. Dendritic cells: their significance in health and disease. - Immunol. Letters. 2001. - vol. 78. - p. 113-122 ) Maturation under various conditions can significantly affect the essential functional characteristics of DCs, such as secretion of IL-12 (Ebner S., Ratzinger G., Krosbacher B., Schmuth M., Weiss A., Reider D., Kroczek RA, Herold M. , Heufler C., Fritsch P., Romani N. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation and is further enhanced by IL-4. - J. Immunol. 2001. - vol. 166 - p.633-641) and the ability to migrate (Luft T., Jefford M., Luetjens P., Toy T., Hochrein H., Masterman KA, Maliszewski C., Shortman K., Cebon J ., Maraskovsky E. Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E2 regulates the migratory capacity of specific DC subsets. - Blood. 2002 - vol. 100. - p.1362-1372). Both CD40L and killed bacteria significantly stimulate the formation of IL-12. Moreover, the maximum amount of cytokine is produced at the initial moments of maturation. At the same time, the medium conditioned by monocytes leads to the maturation of DCs that do not synthesize functional IL-12 (Ebner S., Ratzinger G., Krosbacher B., Schmuth M., Weiss A., Reider D., Kroczek RA, Herold M ., Heufler C., Fritsch P., Romani N. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation and is further enhanced by IL-4. - J. Immunol . 2001. - vol. 166 - p. 633-641).

DCs treated with a tumor antigen in the presence of factors that increase its immunogenicity (TNF-α; IL-1β; CD40L; LPS; monocyte-conditioned medium; viral RNA analogue - polyninosin-cytidylic acid) are able to effectively present the antigen and cause the development of T-cell the immune response to it, as shown in a number of in vitro studies, in animal experiments and even in experiments on healthy volunteers (Chen Z., Dehm S., Bonham K., Kamencic H., Juurlink B., Zhang X., Gordon JR, Xiang JS DNA array and biological characterization of the impact of the maturation status of mouse dendritic cells on their phenotyp e and antitumor vaccination efficacy. - Cell Immunol. 2001. - vol. 214. - p.60-71; Dhodapkar, MV, Bhardwaj, N. Active immunization of humans with dendritic cells. - J. Clin. Immunol. 2000. - vol.20. - p. 167-173; Nouri-Shirazi M., Banchereau J., Fay J., Palucka K., Dendritic cell based tumor vaccines. - Immunol. Letters. 2000. - vol. 74. - p. 5-10; Sallusto F., Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte / macrophage colony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis factor alpha. - J. Exp. Med. 1994 .-- vol. 179. - p. 1109-1118). An experiment on volunteers showed that the immune response to DC developed rapidly, within 7 days, but the peak of both CD4 + and CD8 + T-cell responses to the presented antigen was observed only after 1-3 months. The development of T-cell memory was observed. However, with a natural viral infection, a much stronger and stronger immune response occurs (Dhodapkar M.V., Steinman RM, Sapp M., Desai N., Fossella C., Krasovsky J., Donahoe SM, Dunbar PR, Cerundolo V., Nixon DF, Bhardwaj N. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. - J. Clin. Invest. 1999. - vol. 104. - p. 171-180).

In addition to the type of DC used, obviously, an important role is also played by the source of tumor antigen and the method of administration of DC. In clinical practice, mainly proteins and peptides, as well as tumor lysates (Dhodapkar MV, Bhardwaj N. Active immunization of humans with dendritic cells. - J. Clin. Immunol. 2000. - vol. 20. - p.167-173; Keller R. Dendritic cells: their significance in health and disease. - Immunol. Letters. 2001. - vol. 78. - p.113-122). The disadvantage of vaccines based on specific peptides or proteins is, firstly, that not all OAAs have already been identified. In addition, the tumor cell population is heterogeneous and some cells may not express a separate antigen (Dhodapkar MV, Bhardwaj N. Active immunization of humans with dendritic cells. - J. Clin. Immunol. 2000. - vol.20. - p.167-173 ) The tumor lysate, obviously, contains a wide range of different antigens, including unknown OAAs. Recently, there has been a preliminary report on the use of DCs transfected with total tumor RNA for the treatment of common forms of colon and lung cancer (Nair SK, Morse M., Boczkowski D., Cumming RI, Vasovic L., Gilboa E., Lyerly HK Induction of tumor -specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells. - Ann. Surg. 2002. - vol. 235. - p.540-549). DCs induced the development of CTLs that can specifically lyse a tumor.

The method of administering DCs can significantly affect their migration into lymphoid tissue and their immunogenicity. As shown, DCs successfully reached the lymph nodes when administered intradermally, but not intravenously (Morse MA, Coleman RE, Akabani G., Niehaus N., Coleman D., Lyerly HK Migration of human dendritic cells after injection in patients with metastatic malignancies .- Cancer Res., 1999, vol. 59, p. 56-58). However, information on the immunogenicity of human DC, introduced in various ways, is absent. A comparative study in mice suggests that DCs administered intravenously are less immunogenic than those administered subcutaneously (Lappin M.V., Weiss JM, Delattre V., Mat B., Dittmar H., Maier C., Manke K., Grabbe S., Martin S., Simon JC Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection. - Immunology. 1999. - vol. 98. - p. 181-188).

At this point in time, a series of clinical trials of DC-based antitumor vaccines have been conducted. Hsu et al. (Hsu FJ, Benike C., Fagnoni F., Liles TM, Czerwinski D., Taidi B., Englemann EG, Levy R. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. - Nature. 1996. - vol.392. - p.245-252) was administered to 4 patients with follicular lymphoma of blood DC, loaded with tumor-specific idiotypic protein. Lymphoma regression was observed in 3 patients. Nestle et al. used to treat patients with a common form of mODK melanoma (Nestle F.O., Alijagic S., Gilliet M., Sun Y., Grabbe S., Dummer R., Burg G., Schadendorf D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. - Natur. Med. 1996. - vol. 2 - p. 328-332). Either specific melanoma peptides or a tumor lysate were used as a tumor antigen. DCs were injected into 16 healthy lymph nodes in 16 patients. In 11 patients, a delayed-type hypersensitivity reaction (HRT) was observed in response to DCs loaded with peptides, and in 5 patients, objective tumor regression was observed. Another study involved 11 patients with advanced stage IV melanoma (Thurner B., Haendle I., Roder C., Dieckmann D., Keikavoussi P., Jonuleit H., Bender A., Maszek C., Schreiner D., von den Driesh P., Brocker EB, Steinman RM, Enk A., Kampgen E., Schuler G., Vaccination with mage-3A1 peptide-pulsed mature, monocyte derived dendritic cell expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. - J. Exp. Med. 1999. - vol. 190. - p. 1669-1678). Mature DCs obtained from monocytes loaded with the melanoma peptide MAGE-3A1 were used. DC was administered in 5 doses: 3 - subcutaneously and 2 - intravenously. The development of immune responses to the antigen and expansion of cytotoxic lymphocytes (CTLs) specific for MAGE-3A1 were observed. 6 out of 11 patients regressed individual metastases. Interestingly, non-regressive metastases did not express MAGE-3A1 mRNA. There are also similar data on the promising use of DC for the treatment of common forms of cancer of the prostate, kidney, thyroid, breast, colon, cervix, myeloma (Dhodapkar MV, Bhardwaj N. Active immunization of humans with dendritic cells. - J. Clin. Immunol. 2000. - vol.20. - p.167-173; Jefford M., Maraskovsky E., Cebon J., Davis ID The use of dendritic cells in cancer therapy. - Lancet Oncol. 2001. - vol.2. - p. 343-353; Keller R. Dendritic cells: their significance in health and disease. - Immunol. Letters. 2001. - vol. 78. - p. 113-122; Nouri-Shirazi M., Banchereau J., Fay J., Palucka K., Dendritic cell based tumor vaccines. - Immunol. Letters. 2000. - vol. 74. - p.5-10).

As a prototype, a DC-based vaccine obtained as a result of pulsation of mature DCs by a tumor lysate, activated IL-2 human lymphocytes (LAC) can be considered. At the same time, each of these methods has its drawbacks, since it affects only one of the links (innate or acquired) of the immune system. In addition, with the joint introduction of DCs and LACs, they can increase the duration of each other's existence in an active state.

The present invention is to obtain a transplant for the implementation of combination immunotherapy, characterized in that it combines the simultaneous effect on innate and adaptive immunity and can be used for adaptive immunotherapy of cancer and infectious diseases and immunodeficiency states.

To solve this problem, a group of inventions is proposed, united by a common inventive concept.

A method is proposed for producing mononuclear blood cells: activated lymphocytes (LAC) and DC, which includes the following stages:

a) the isolation of mononuclear leukocytes (MNCs) from the blood or bone marrow of a person in need of treatment or prevention of a malignant neoplasm, or an infectious disease, or an immunodeficiency state;

b) incubation of isolated mononuclear lymphocytes in a culture medium;

c) the separation of the culture vial of MNCs adhering to and not adhering to plastic;

e) treatment with growth factors of adherent MNCs to obtain dendritic cells from them;

f) treatment of DC with a lysate of a tumor of a patient;

g) the cultivation of non-adherent lymphocytes with interleukin-2 to generate LAC.

A method for the treatment and prevention of malignant and infectious diseases and immunodeficiency states in patients in need of this, consisting in the combined use of an antitumor vaccine and LAC.

A method for producing a cell transplant is proposed, characterized in that the selection of MNCs from peripheral blood or bone marrow is carried out by centrifugation in a single-stage ficoll gradient.

The proposed method allows to obtain a sufficient amount of MNCs from 100-400 ml of peripheral blood.

The cultivation of MNCs takes place in RPMI 1640 or DMEM medium with 5% human serum of the patient (or AB serum of the donor) in disposable plastic bottles, 250 ml in volume from Costar or similar bottles from other companies. To separate cells into monocytes / macrophages (cells that attach to the substrate) and lymphocytes (cells that do not attach to the substrate), MNCs are placed in culture bottles with nutrient medium for 2 hours to a day and then the non-adherent lymphocytes are poured into another bottle. To obtain immature DCs, granulocyte / macrophage colony stimulating factor (GM-CSF) and interleukin-4 (10 ng / ml) are added to the remaining adhering cells (monocytes / macrophages) remaining in the vial. To stimulate the maturation of immature DCs in mature DCs, the pro-inflammatory factors IL-1β, TNF-α, IL-6 (10 ng / ml) can be used. Preferred in accordance with the present invention is the maturation under the action of TNF-α (20 ng / ml). DCs obtained from monocytes / macrophages of peripheral blood during incubation with growth factors have characteristic features of the morphological structure and ultrastructure. For these purposes, immature DC dendritic cells obtained under extracorporeal conditions (CD83 ^low , CD86 ^low , CD80 ^low ) are incubated with tumor lysate with viral and bacterial antigens and DC maturation induction factors for 1 day and mature pulsed DC antigens are obtained (CD83 ^high , CD86 ^high , CD80 ^high ). In a bottle with lymphocytes (non-adherent cells), IL-2 is added at a concentration of 1000 IU / ml.

A method for the treatment and prevention of malignant and infectious diseases and immunodeficiency states in patients in need thereof, which consists in the combined use of dendritic cells and LAC cells, characterized in that the vaccine obtained is administered subcutaneously, intravenously, intraarterially or regionally (intraperitoneally, intrapleurally and other cavities ) The present invention is effective for the prevention and treatment of cancer, especially in “risk groups” and in “family forms” of cancer, immunodeficiency states, both congenital and acquired, in particular with viral and bacterial infections.

Experiments in mice indicate that the effectiveness of the vaccine protection is weakened after 3 months. and revaccination is required. Given the difference in the life expectancy of mice and humans, re-vaccinations with an interval of 5-7 years can be recommended for the latter.

For use in the clinic, the method is effective and safe.

Clinical example

Patient B., 36 years old.

Diagnosis: metastases of colon cancer in the liver, retroperitoneal lymph. nodes. Condition in the course of complex treatment. Blood type: 0 (1) positive.

11.22.02 right-sided hemicolectomy for cancer of the ascending colon (histology - moderately differentiated adenocarcinoma, metastases to the lymph nodes). During the operation, multiple metastases were detected in both lobes of the liver from 1 to 3.5 cm, for which reason chemoembolization (doxorubicin - 70 mg + 10 ml lipiodol) was performed on the right hepatic artery.

01/22/03 left-sided hemihepatectomy; high-frequency thermalablation of the remaining available metastatic nodes. Histol. research 1028/03 - in the liver metastases of low-differentiated adenogenic cancer. Tumor tissue is negative for thymidine phosphorylase and thymidylate synthetase.

From February 14 to February 17, 2003, a 96-hour infusion into the 5-FU hepatic artery was performed - 1400 mg / day and leucovorin 50 mg / day. In March 2003, metastases in retroperitoneal lymph were detected. nodes. From March to August 2003, 12 courses of chemotherapy were carried out according to the scheme: oxaliplatin - 170 mg - 1st day; 5 - fluorouracil - 800 mg - the 1st day in / in the stream and 5200 mg in / drip for 46 hours. Given the continuing positive dynamics from the remaining focal changes in the liver and the absence of new lesions, the patient was transferred to a long-term administration of celecoxite at 200 mg 2 times a day.

Taking into account the urgent request of the patient and the existing expediency of continuing the antitumor treatment on October 14-16, 2003, three bolus injections of LAC in diabetes mellitus were made - 920 million cells in the hepatic artery. During treatment, there was a slight weakness, discomfort in the right hypochondrium, nausea.

With the control MRI from 11/17/03, positive dynamics - a lesion of 1.4 × 0.7 cm is determined in the right lobe of the liver (it was 1.8 × 1.2 cm from 03/03/03). Other foci are not visualized. Retroperitoneal lymph. nodes are not enlarged. 15-17.12.03 intrahepatic LAC-therapy in diabetes - 632 million cells. During the control study in the results of clinical and biochemical blood tests, deviations from the norm were not detected. Upon admission in February according to MRI with contrast (05.02) in the right lobe of the liver, a section with clear contours of up to 1.5 cm is the ablation zone. Convincing data for metastatic lesion was not received. 16-18.02.04 in / art., In / 780 million LAC was introduced in the liver. Upon receipt in April, a puncture biopsy of the remaining lesion in the liver was performed - no signs of tumor damage were obtained. REA - 0.74, CA 19.9-2.0.

19-21.04.04 in / art, in / hepatic injected 740 million LAC. Upon receipt in June, signs of disease progression were not detected. An attempt to catheterize the hepatic artery failed (obliteration and spasm). 07/07/06/04 620 million LAK introduced in / in. 16-18.08.04 iv LAC - therapy in DM-850 million cells. 09-11.11.04 I / O 605 million LAC. 24-26.01.05 - in / in 710 million LAC. April 18-20, 2005 in / in 700 million LAC. 06/29/01/01.05 in / in cap 675 million LAC. September 19-21, I / O 485 million LAC. 15-17.11.05 505 million LAC. 17-19.01.06 - 605 million LAC. 13-15.03.06 - 500 million LAC. There are no reactions. There are no signs of disease progression.

Claims (3)

1. A cell transplant for the treatment and prevention of cancer, infectious diseases and immunodeficiencies, characterized in that it contains lymphocytes activated by interleukin 2 and dendritic cells (DC) obtained by incubating immature DCs with tumor lysate, or viral or bacterial antigens and factors induction of maturation of DC (TNF-α; IL-1β; IL-6). 2. A method of obtaining a cell transplant for the treatment and prevention of cancer, infectious diseases and immunodeficiencies, characterized in that the blood or suspension of bone marrow is centrifuged in a single-stage ficoll gradient, the cultivation of MNCs is carried out in RPMI 1640 or DMEM medium with 5% of the patient’s human serum or AV donor serum, then the cells are divided into monocytes / macrophages that attach to the substrate and lymphocytes that do not attach to the substrate, MNCs are placed in the culture medium for 2-4 hours and then drained non-adherent lymphocytes, to obtain immature DCs, granulocyte / macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) are added to the remaining adherent cells of monocytes / macrophages, then pro-inflammatory factors IL-1β, TNF-a are used to stimulate DC maturation α, IL-6 (10 ng / ml), while immature dendritic cells (CD83 ^low , CD86 ^low , CD80 ^low ) obtained under extracorporeal conditions are incubated with tumor lysate, or viral or bacterial antigens, and DC maturation induction factors for 1 day and get mature n lsirovannye antigens DK (SV83 ^{high, CD86 high, CD80 high)} , to produce LAK cells to lymphocytes IL-2 added in a concentration of 1000 IU / ml. 3. A method for the treatment and prevention of cancer, infectious diseases and immunodeficiency conditions, characterized in that the cell transplant according to claim 1, and obtained according to claim 2, is administered subcutaneously, intravenously, intraarterially or regionally at a dose of 50 million - 2 billion cells.