LT6263B - Obtaining method of dendritic cells (ld) from peripheral blood, bone marrow or apherasis product and use thereof - Google Patents

Abstract

The invention is related to a particular field of medicine - obtainment of dendritic cells (DL) from human peripheral blood, blood apheresis or bone marrow aspirates, and their use in immunotherapy in treating such pathology as various oncological diseases regardless of the stage of the disease and the type of auto-immune diseases, allergic diseases, other immune system diseases. Peripheral blood or blood apheresis or bone marrow aspirate is separated and distinguishing only mononuclear cell fractions, this fraction is seeded on the cells culture medium in culture flasks and incubated for two hours. After incubation, flasks are washed with brine, loose cells are removed, adherent cells with existing flasks are further cultured in a special medium. After three days, the media inside the flask is changed from half to 85 % with new DL maturation medium with antigens and adjuvants. The cells are cultivated for six hours and their removal is carried out with specially designed standard solutions. The cells are washed and filtered through a 100 µm filter and are frozen at a dose of 5 million cells in a cooling medium with a autologous serum of patient.

Description

1

LT 6263 B

FIELD OF THE INVENTION The invention relates to the field of medicine, in particular, to the extraction of dendritic cells from peripheral blood or bone marrow or tissue monocytes for their maturation, antigen loading and use in immunotherapy to induce a specific immune response according to the antigen used.

TECHNICAL LEVEL

More than two decades ago, having demonstrated that the immune system can recognize and eliminate cancer cells, great attention has been paid to research on immune cells in an attempt to identify the effects they have on the genesis of cancer. It is now known that both congenital and acquired immune systems fight cancer [Teng MWL, Swann JB, Koebel CM, Schreiber RD, Smyth MJ. Immune-mediated dormancy: An equilibrium with cancer. Journal of Leukocyte Biology 2008; 84: 988-93.]. Despite the great variety of combat cells, cancer, through its defense mechanisms, avoids destruction [Dunn GP, Old LJ, and Schreiber RD. The three Es of cancer immunoediting. Annual Revievv of Immunology 2004a; 22: 329-60]. Because of the pressure of the immune system, the cancer, to survive, acquires properties that reduce its immunogenicity, and / or inhibits the components of the immune system [Chen DS and Mellman I. Oncology meets immunology: The cancer-immunity cycle. Immunity 2013; 39: 1-10.]. If you notice that patients with an immune system triggered by the infection have a better prognosis, immunotherapy has been started [Cann SAH, van Netten JP, van Netten C. Dr VViIliam Coley and Tumor Regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80.], Which aims at activating or targeting immune cells to the destruction of cancer cells.

Immunotherapy has a great advantage over traditional methods of treatment, because it has more specificity and does not cause healthy cell death [Eggermont LJ, Paulis LE, Tel J, Figdor CG. Efficient cancer immunotherapy: Advances in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65]. One of the methods of immunotherapy treatment is vaccination with autologous vaccines that can be prepared from an autologous tumor lysate or produced on the basis of dendritic cells whose function is to deliver antigens to naive T cells. Already done 2

Many studies have been conducted with various anticancer vaccines and, although a better immune response to the tumor is achieved, the expected treatment effect has not yet been attained [Kim J and Mooney DJ. In vivo modulation of dendritic cells by engineered materials: Towards new cancer vaccines. Nano Today 2011; 6: 466-77].

It is investigated which method of presenting cancer-related antigens to use, which adjuvant to choose for vaccines to be even more effective: to enhance the immune response, overcome the immunosuppressive tumor environment. Methods of maturation of dendritic cells are being improved, as this determines the function of these cells. It is known that immature dendritic cells induce immune tolerance while mature activate cytotoxic T cells, which are the most important components of specific immune response against malignant cells [Palucka K and Banchereau J. Dendritic-cell-based therapeutic cancer vaccines. Immunity 2013; 39: 38-48].

Dendritic Cells (DL) are professional antigen-presenting cells that combine innate and acquired specific immunity and play an important role in killing cancer cells [Kim SK, Yun CH, Han SH. Enhanced anti-cancer activity of human dendritic cells with gamma-irradiation-induced apoptotic colon cancer cells. Cancer Letters 2013; 335: 278-88]. These cells are located in the tissues and receive antigens by endocytosis and pinocytosis. Subsequent caught antigens DL, together with MHC Class I and MHC Class II molecules, are provided by lymph nodes in naive T helper, which in turn activate cytotoxic T lymphocytes. The environment in which the "captured" antigens were found determines whether DL will cause anti-tumor immunity or immune tolerance [Chen DS and Mellman I. Oncology meets immunology: The cancer-immunity cycle. Immunity 2013; 39: 1-10; Harris TJ and Drake CH. Primer is tumor immunology and cancer immunotherapy. Journal for Immunotherapy of Cancer 2013; 1: 12]. Immature DL, which has received the antigen in peripheral tissues, usually causes immunotolerance, since there are no costimulating molecules in their membranes. DL maturation is associated with certain changes, such as reduced efficiency of antigen capture, increased expression of MHC class II and costimulatory molecules on cell surface, acquires receptors that direct their migration, acquire cytokine secretion function. One of the DL secreted cytokines IL-12 promotes T cell differentiation into effector T cells [Palucka K and Banchereau J. Dendritic-cell-based therapeutic cancer vaccines. Immunity 2013; 39: 38-48]. In addition, because dendritic cells work 3

LT 6263 B as APL, they can directly eliminate cancer cells through death signals such as TRAIL (a TNF-associated apoptosis-inducing ligand) or Fas ligand [Kim SK, Yun CH, Han SH. Enhanced anti-canceractivity of human dendritic cells with gamma-irradiation-induced apoptotic colon cancer cells. Cancer Letters 2013; 335: 278-88].

Despite significant advances in science, the development of new drugs and the knowledge of tumor biology, cancer remains one of the main causes of death not only among developing countries but also in developed countries. Currently, the most important cancer treatment methods are three: surgery (tumor removal), chemotherapy (drug use) and radiotherapy (radiotherapy). Each method has limitations or side effects, for example, surgical treatment can only be used to remove a solid, tangible tumor, and problems arise due to untreated primary tumor residues or metastases. Chemotherapy is based on the destruction of rapidly dividing cells by various cytostatics, but not only the cancer cells, but also the hair follicles, intestinal epithelium, bone marrow cells, etc., rapidly proliferate. [Aly HAA. Cancer therapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23]. Immune system cells, such as macrophages, can inhibit the effectiveness of chemotherapy depending on the type of tumor. One of the first studies to investigate the effect of macrophages on chemotherapy has shown that doxorubicin, a medicine used to treat cancer in mice, enhances the anti-cancer effect of macrophages. Another example is B cells that indirectly promote the progression of orthotopic epithelial tissue carcinoma. Chemotherapy without inhibiting B cells is ineffective. Only in combination with anti-CD20 antibodies, paclitaxel is effective against this type of cancer [Coffelt SB and de Visser KE. Immune-mediated mechanisms influencing the efficacy of anticancer therapies. Trends in Immunology 2015; 36 (4): 198-216].

Combined multi-method treatment is used to achieve better results. Treatment with conventional chemotherapy methods is harmful because of the damage to healthy cells, and hence, great achievements are expected after extensive application of immunotherapy specifically directed against cancer cells. Immunotherapy is a method of treating cancer, when the components of the immune system are actively or passively activated and directed to the destruction of malignant cells. Increased specificity prevents side effects caused by conventional methods of treatment [Eggermont LJ, Paulis LE, Tel J, Figdor CG. Tovvards efficient cancer immunotherapy: Advances 4

LT 6263 B in developing artificial antigen-presenting cells. Trends in Biotechnology 2014; 32 (9): 456-65].

The beginnings of first immunotherapy date back to the 19th century. when Coley noticed that a patient with complete tumor remission had previously had an acute streptococcal infection. Later, after developing a vaccine against dead Streptococcus pyogenes and Serratia marcescens, people with non-operative cancer (usually sarcoma) were treated. The purpose of the vaccine was to induce the immune system [Cann SAH, van Netten JP, van Netten C. Dr VViIliam Coley and Tumor Regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80; Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13]. Approximately at the same time, Pearl reported that tuberculosis had anti-cancer effects, and therefore invented a vaccine for the treatment of cancer, used for the treatment of tuberculosis - BCG (baccile Calmette-Guérin). The BCG vaccine is currently the only bacterial vaccine used in immunotherapy for the treatment of non-invasive bladder cancer [Mayer G. Immunology -chapter one: innate (non-specific) immunity. Microbiology and Immunology On-Line Textbook. University of South Carolina School of Medicine. http://pathmicro.med.sc.edu/qhaffar/innate.htm [citing 01/05/2015]. Cann SAH, van Netten JP, van Netten C. Dr VViIliam Coley and Tumor Regression: A place in history or in the future. Postgraduate Medical Journal 2003; 79: 672-80], BCG for lung cancer immunotherapy is only used as an adjuvant for stimulation of the immune system [Raez LE, Fein S, Podack ER. Lung Cancer Immunotherapy. Clinical Medicine & Research 2005; 3 (4): 221-8].

The discovery of antigens associated with tumor (TAA) has led to the development of specific immunotherapy [Parish CR. Cancer immunotherapy: the past, the present and the future. Immunology and Cell Biology 2003; 81: 106-13; Vayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. Personalized view on cancer immunotherapy. Cancer Letters 2014; 352: 113-25], which aims to induce an immune response to the tumor [Bluestone JA, Small EJ. VVill it include immunotherapy? Cancer Cell 2012; 22: 7-8], knowing that an effective immune response is not due to reduced recognition of immune cells, weak tumor immunogenicity, and immunosuppressive tumor environment [Kumar S and Mason M. Principles of cancer treatment by immunotherapy. Surgery 2012; 30 (4): 5

LT 6263 B 198-202]

By function, tumor immunotherapy is divided into passive and active. Passive immunotherapy involves all immunologically active substances, immunomodulating cells and independent of the patient's own immune system, as it is provided with cells with anti-cancer effects [Aly HAA. Cancertherapy and vaccination. Journal of Immunological Methods 2012; 382: 1-23] Further passive immunotherapy may be further subdivided into monoclonal antibody stimulating cytokines and adoptive T cells [Wayteck L, Breckpot K, Demeester J, De Smedt SC, Raemdonck K. Personalized view on cancer immunotherapy. Cancer Letters 2014; 352: 113-25], Bone marrow transplantation is still attributed to another source for passive immunotherapy [Zavala VA and Kalergis AM. New clinical advances in the treatment of solid tumors. Immunology 2015]

Active immunotherapy requires the proper functioning of the patient's immune system, as the recipient's immune mechanisms are triggered during the treatment of cancer cells. Various vaccines are included in active immunotherapy: BCG, peptide, DNA, dendritic cells, etc., which should be as toxic to the body as possible, could lead to an antitumor immune response not only against the primary tumor, but also against metastases that produce immune memory. Preventing Disease Recovery [Moingeon P. Cancer Vaccines. Vaccine 2001; 19: 1305-26; Yannelli JR and Wroblewski JM. 20 years of cellular immunotherapy. Vaccine 2004; 23: 97-113].

Dendritic Cell Vaccines In 1973, Steinman, together with Z. Cohn, presented a work on newly discovered cells that have been named dendritic cells (DL) by cytoplasmic growths [Rovvley DA and Fitch FW. The road to the discovery of dendritic cells, a tribute to Ralph Steinman. Cellular Immunology 2012; 273: 95-8]. Due to the ability of DL to capture and deliver antigens to T cells, they become an important weapon for the development of anti-cancer vaccines [Vasaturo A, Verdoes M, de Vries J, Torensma R, Figdor CG. Restoring immunosurveillance by dendritic cell vaccines and manipulation of the tumor microenvironment. Immunobiology 2015; 220: 243-8]. Also, cancer, especially in the advanced stage, is associated with a decrease in the number of DL and does not have an immunostimulatory but immunosuppressive function, so patients with 6

For LT 6263 B cancer, it is important to restore the amount and function of DL. There are two types of DL vaccines prepared ex vivo and in vivo [Strioga MM, Darinsk A, Pasukoniene V, Mlynska A, Ostapenko V, Schijns V. Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: ? Vaccine 2014; 32: 4015-24.].

Autologous ex vivo DL vaccines are usually prepared from functionally intact monocytes that are loaded with tumor antigens, matured ex vivo and injected back into the patient [Strioga MM, Darinsk A, Pasukoniene V, Mlynska A, Ostapenko V, Schijns V. Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: Tooth or not? Vaccine 2014; 32: 4015-24.]. Maturation of DL is an important step since immature cells induce immunotolerance, which is manifested by the removal of cytotoxic T cells and the expansion of regulatory T cells [Palucka K and Banchereau J. Dendritic-cell-based therapeutic cancer vaccines. Immunity 2013; 39: 38-48; Radford KJ, Tullett KM, Lahoud MH. Dendritic cells and cancer immunotherapy. Current Opinion in Immunology 2014; 27: 26-32], The mature, charged DL travels to lymph nodes, where it activates CTL that can eliminate cancer cells.

Studies of therapeutic vaccines for more than 10 years have shown that DL-based vaccines are safe, increasing the amount of circulating T-cell helper and CTL-specific tumor antigens [Palucka K and Banchereau J. Cancer immunotherapy via dendritic cells. Nature Revievvs Cancer 2012; 12: 265-77] and are suitable for treating many tumors [Radford KJ, Tullett KM, Lahoud MH. Dendritic cells and cancer immunotherapy. Current Opinion in Immunology 2014; 27: 26-32]. However, although the efficacy of such vaccines has been demonstrated in preclinical and clinical studies, treatment results could be better. Therefore, special attention is currently given to improving the maturation methods of the DL vaccines themselves and to the search for additional adjuvants.

In vivo DL vaccines, in addition to not requiring manipulation of cells outside the body, have another advantage. In the case of ex vivo, only one population of DL, usually derived from monocytes, is commonly used for the production of DL vaccine, and various subtypes of myeloid and plasmacitoid DL that are differentiated by localization, migration pathways, functions and effects on specific immune response are used in vivo [Strioga MM, Darinskas A, Pancreatic V, Mlynska A, Ostapenko V, Schijns V. Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: Is it a tick or not? Vaccine 2014; 32: 4015-24]. During the last vaccination 7

LT 6263 B antigens are provided directly in DL in vivo. Antigen sources may include irradiated cancer cells, tumor lysate, DNA plasmids with tumor antigens and others [Strioga MM, Schijns V, Powell DJ, Pasukoniene V, Dobrovolskiene NT, Michalek J. Dendritic cells and their role in tumor immunosurveillance. Innate Immunity 2013; 19 (1): 98-111]. Such specific antigen delivery to dendritic cells results in a strong response from CD4 + and CD8 + cells. It is also important that without DL maturation signals, such vaccination induces tolerance [Palucka K and Banchereau J. Cancer immunotherapy via dendritic cells. Nature Revievvs Cancer 2012; 12: 265-77], since the primary function of DL is to maintain tolerance, which is necessary because not all autoreactive T cells are destroyed during selection. And if the tissue or inflammatory response breaks, DL simultaneously delivers both its own and foreign antigens, so tolerance to its own proteins allows T cells to focus on fighting against aliens, while avoiding autoimmune diseases [Havviger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, Ravetch JV, Steinman RM, Nussenzvveig MC. Dendritic cells induce peripheral T cell unresponsiveness under steady static conditions in vivo. Journal of Experimental Medicine 2001; 194: 769-80].

Certain components that additionally activate the immune response are very important to increase the effectiveness of anticancer vaccines. The most common bacterial frigments, proteins, etc. are used. products, because precisely the bacterial antigens develop the fastest and most active response. Adjuvants are substances that enhance the antigen-specific immune response. They are important components of therapeutic vaccines because they increase the immunogenicity of weak antigens and also help overcome the immunosuppressive environment of the tumor and trigger the CTL response [Dubensky TW and Reed SG. Adjuvants for cancer vaccines. Seminars in Immunology 2010; 22: 155-61]. Adjuvants can be used for different purposes [Petrovsky N and Aguilar JC. Vaccine adjuvants: Current statė and future trends. Immunology and Cell Biology 2004; 82: 488-96]: Increase the immunogenicity of antigens, since "dead" vaccine antigens are poorly immunogenic; reducing the amount of antigens or the number of immunizations required to produce an adequate immune response; improve the effectiveness of vaccines in patients with weakened immune systems; deliver antigens.

Substances isolated from bacteria due to their immunostimulatory properties can be used as adjuvants. Lipopolysaccharide of Gram-negative bacteria, co-administered with antigens, induces an immune response despite the absence of FPS itself 8

LT 6263 B is highly immunogenic. Such an adjuvant activates the immune system by activating TLR receptors that support alarms. The FTA is not widely used in human treatment due to its pyrogenic effect [Petrovsky N and Aguilar JC. Vaccine adjuvants: Current statė and future trends. Immunology and Cell Biology 2004; 82: 488-96]. Also adjuvants may include other substances / antigens that, according to their origin, can activate different TLR receptors: TLR-1: bacterial lipoproteins and peptidoglycans TLR-2: bacterial peptidoglycans TLR-3: double-stranded RNA (viruses) TLR-4: lipopolysaccharides TLR -5: bacterial scabs TLR-6: bacterial lipoproteins

TLR-7: Single RNR TLR-8: Uniform RNA Rich G Sequence

TLR-9: CpG and DNA TLR-10: Unknown

BRIEF DESCRIPTION OF THE INVENTION

Unlike analogues that refer to the classic methods of dendritic cell extraction and maturation, our invention is characterized by the rapid (three-day) monocyte transit to immature dendritic cells and rapid (six hours) maturation, using a special maturing mixture and conditions.

Peripheral blood or blood apherate or bone marrow aspirate is separated by separating only the mononuclear cell fraction, this fraction is inoculated in cell culture medium on cell culture vials and incubated for two hours. After incubation, the vials are washed with saline (PBS or physiological), the stuck cells are removed, the adherent cells with the existing vials are further cultured in a special medium. Three days later, the vial is replaced by half to 85% of the medium with new antigen and antigenic maturation medium for DL. The cells are cultivated for six hours and removed for a specific purpose 9

EN 6263 B with standard solutions. The cells are washed, filtered through a 100 µm filter and frozen at 5 million cells in the cooling medium with autologous patient serum. Performing a sterility test and other mandatory tests.

DETAILED DESCRIPTION OF THE INVENTION I. Isolation of mononuclear cells from blood, apherate. bone marrow aspirate

The blood is diluted with a solution of hydroethyl starch for infusion as a plasma substitute, diluted 1: 1. The mixed solution is left to stand at room temperature for 20-40 minutes, usually two layers are released for a period of time: the lower, rich in red blood cells, and the upper with red blood cells, plasma and nuclear blood cells. The top layer is collected in centrifuge tubes and centrifuged at 1000-1500G for 5-15 min. at room temperature, the supernatant is centrifuged after centrifugation and the precipitate is suspended in saline or in the remaining upper mixture of blood / hydroxyethyl starch solution. Typically, at 150-300 ml of blood, the centrifuge is diluted to 40 ml of finite volume. If there is more blood, the centrifuge is diluted proportionally accordingly. The vials of 15 or 50 ml are then filled with a solution of ficol or percol at a density of 1.0 to 1.12 g / ml. The solution is filled with up to 1/3 of the volume of the tube. Pipette slowly from this solution by tilting the tube as carefully as possible by gently pouring the diluted cell centrifuge to the top of the tube. Centrifuge tubes at 1100-1500 G for 10-20 min. at room temperature, the brake is not used. Three centrifuges are seen in the tubes after centrifugation: the bottom is a fraction of red blood cells, the monoclonal layer above the erythrocytes floating above the clear layer of the ficol, and the platelets together with the diluted hydroxyethylramramol plasma above the mononuclear layer. The pipette carefully collects the mononuclear layer and is poured into new tubes containing brine or culture medium. The stage of cell washing from the gradient material (ficol or percol) is underway. The cells are well mixed and centrifuged at 500-850 G for 10-20 min. at room temperature. The brake is not used. The procedure can be repeated several times. After washing, the cells are counted and dispensed into cell culture vials 10

LT 6263 B in a ratio of 175 cm2 / 100-120 million cells and 10 ml medium for T lymphocytes or dendritic cells. The vials are incubated in cell cultures at 37 ° C and 5-15% CO2 for 2 hours. After incubation, the flasks are washed three times with PBS or saline, and the adherent cells are removed. They are sown separately so that the MLR test can be performed later (it is not part of this patent). Cells remaining in the vials are filled with 20 ml of a special monocyte differentiation medium consisting of Interferon alpha (60,000 IU) (5μΙ of working solution from 6 million interferon alpha diluted in 0.5 ml medium) and 200 µl of GM- CSF 1000 U / ml (working solution 100,000 U / ml) and 1% autologous patient serum. II. Monocyte differentiation into dendritic cells After addition of differentiation media, the vials are cultured for three days. An additional day function is possible, but then 30% of the new differential medium should be added on the second day of cultivation. The cells in the vials in such cultivation conditions are 50-75% dendritic cells. III. Maturation of dendritic cells, presentation of antigen

The third or fourth day of cultivation involves maturation of DL and antigen loading. the vial is filled with 10-18 pg / ml of antigen at the concentration of the protein, together with a differentiation cocktail and an antigen for finite loading. The antigen at 10-18 pg / ml is mixed with the adjuvant (100-200 U LPS and 50-100 U CpG or Poly l: C, or ampligen, or Poly ApU). The mixture is incubated for one hour at 37 ° C with stirring. Prepare antigen loading and maturation medium. Composition and contents per vial: 10 ml medium, 200 μΙ GM-CSF (2000 U / ml), 5 μΙ IFN-alpha (6000 U / ml), 1000 U / ml IFN-gamma, 1% autologous plasma or serum. After an hour of incubation, the antigen with the adjuvant is mixed with the antigen loading and maturation medium, the medium in the vials is collected, the cells are centrifuged, and the cells are suspended in antigen loading and maturation (already mixed with antigen and adjuvants) after leaving up to 10% of the medium. . Everything is poured back into growing bottles, the cells are still 6 11

LT 6263 B hours are stored in the incubator. After incubation time, a significant change in cell morphology is observed, becoming oblong, elongated. This indicates successful antigen opsonization and activation of DL into immunogenic cells. IV. DL removal and cooling

After 6 hours of incubation with antigen and antigen loading and maturation, the cells are removed and frozen.

The medium is collected in tubes, the vials are filled with PBS / EDTA solution (PBS standard EDTA concentration 0.02-4%). Keep warm for 15 minutes. or cold at +4 ° C to 2 or. The cells are harvested, the flasks are rinsed twice with PBS or saline, collected in tubes and centrifuged at 450-800 G for 10 min. At + 4 ° C. The centrifuged cells are passed through a 100 µm nylon filter and counted. The cells are frozen at a concentration of 5-10 min. cells / ml. The cooling medium consists of 10% DMSO (dimethylsulfoxide) in serum or plasma of an autologous patient. DMSO is never added as a pure solution to the cell suspension, it is only mixed with diluted serum. Mixing with DMSO heat. The cells mixed with the refrigerant medium are packed into refrigerated tubes which are placed in a freezer and cooled in a standard protocol by placing them at -80 ° C for a couple of hours. V. DL thawing and injection Frozen tube is withdrawn from the refrigeration room and immediately placed in a container with warm water at + 37 ° C. Defrost until the ice is completely dissolved and the same volume of physiological fluid or autologous serum is added. The use of a 27-29G needle to inject the vaccine into the subcutaneous vaccine should be used to contract the vaccine into a syringe, preferably 21G, preferably 21G. Indications for use of DL and examples of avdvmo

For the indication procedure: 12

EN 6263 B a) Oncological diseases of all types and stages; b) Autoimmune diseases (multiple sclerosis, rheumatoid arthritis, lupus erythematosus, psoriasis).

Examples of dendritic cell therapy for patients and their results

Patient R.N. Stage IV metastatic melanoma, no chemotherapy treatment, patient survival prognosis - 2-3 months. Autologous DL treatment applied since 2013. Three DL courses were conducted in seven vaccines, and the patient received a total of 21 DL of vaccine over two years. The patient is in a stable state, the disease does not progress.

Ligonė E.R., 2009 diagnosed breast cancer, surgical treatment, chemotherapy, tumor metastasized to lungs and liver, prognosis poor, autologous DL vaccination started in 2012 (three courses of 5 vaccines), patient's disease stabilized, metastasis stopped, metastasis stopped (inactivation), metastasis stopped . The patient remains stable.

Patient T.K., diagnosed ovarian cancer in 2009, stage IV with metastases in the peritoneum. Operational treatment was applied to remove metastases of peritoneal tissue, chemotherapy. In 2011, an autologous DL vaccine was used, one vaccination course, and a total of 8 vaccines. The disease does not progress, the patient's condition remains stable.

Patient J.S., in 2010 kidney carcinoma was diagnosed, IVstasis, lung and liver metastases. Applied chemotherapy treatment, after two of the six chemotherapy courses due to the patient's intolerance, was treated with an autologous DL vaccine, four vaccination courses per six vaccines have been conducted since 2011, with a standard schedule. The patient's primary tumor and metastasis have disappeared, the patient is considered to be fully recovered.

Claims (7)

6263 B DEFINITION OF DEFINITION 1. A method of obtaining dendritic cells (DL) from peripheral blood, apheresis or bone marrow aspirate, comprising the extraction and maturation of dendritic cells, characterized in that: a) secretes only a mononuclear cell fraction by separating peripheral blood; blood apheresis or bone marrow aspirate, b) this fraction is passed to cell culture flasks in cell culture medium and incubated for two hours; after three days in the vial replaces half to 85% of the medium with new DL maturation medium with antigens and adjuvants, the cells are cultured for six hours and removed by dedicated standard solutions, e) wash the cells, filter through a 100 µm filter and freeze-ready for use in a refrigerated medium with an autologous patient serum, f) prior to use, place the frozen tube in a vessel with warm water at + 37 ° C, thaw until the ice is completely dissolved and add the same volume of physiological fluid or autologous serum; The syringe uses a 28G needle, preferably a 21G needle, to inject the vaccine into a subcutaneous injection - 27-29G needle. 2. The method of step a) of claim 1 wherein the dilution is diluted with 1: 1 hydroethyl starch solution and left to stand at room temperature for 20-40 minutes until two layers are released: lower, rich in red blood cells, and upper containing red blood cells. , plasma and nuclear blood cells; - Collecting the top layer in centrifuge tubes and centrifuging at 1000-1500 G for 5-15 min. at room temperature, the supernatant 14 LT 6263 B formed after centrifugation is shaken and the precipitate is suspended in saline or in the remaining upper blood / hydroxyethyl starch mixture, diluting to 150 ml with 40 ml of blood; - Add up to 1/3 of the volume of the vial to a 15 ml or 50 ml tube with a density of 1.0-1.12 g / ml in a solution of ficol or percol. - slowly pipetting on this solution by tilting the tube to lie down the top of the diluted cell centrifuge to the top of the tube; - Centrifuge tubes at 1100-1500 G for 10-20 min. at room temperature without brake; - gently pipette a mononuclear layer, formed after centrifugation between the lower layer of erythrocytes and the upper platelet layer, and transfer to new tubes containing brine or culture medium; - Rinse cells from gradient material, mix well and centrifuge at 500-850 G for 10-20 min. at room temperature without brake; - The procedure is repeated several times if necessary. A method according to claim 1, step b), characterized in that - the flushed cells are counted and passed to the cell culture vials in a ratio of 175 cm2 / 100-120 min cells and 10 ml medium for T lymphocytes or dendritic cells; - Incubate vials at 37 ° C and 5 to 15% CO2 in incubators for 2 hours. 4. The method of step c) of claim 1, wherein, after incubation, the vials are washed three times with PBS or saline, uncoated cells, and the cells remaining in the vials are infused with 20 mL of a special monocyte differentiation medium consisting of Interferon alpha 60,000. IU and 200 μΙ GM-CSF 1000 U / ml and 1% autologous patient serum, and vials filled with differentiation medium, where 50-75% of cells are dentritic cells, cultured for three days. 15 LT 6263 B 5. The method of step d) of claim 1 wherein the vial is filled with 10-18 pg / ml of antigen at a concentration of protein or RNA, the antigen is mixed with an adjuvant at 100-200 U LPS and 50-100 U CpG or Poly 1: C, or Ampigene, or Poly ApU, incubate the resulting mixture for one hour at 37 ° C with stirring; - prepare the antigen loading and maturation medium in the following composition and amounts per vial: 10 ml medium, 200 μΙ GM-CSF (2000 U / ml), 5 μΙ IFN-alpha (6000 U / ml), 1000 U / ml IFN-gamma, 1% autologous plasma or serum; - after an hour of incubation, the antigen with adjuvant is mixed with the antigen loading and maturation medium, the medium in the vials is collected, the cells are centrifuged, and the cells are suspended in antigen loading and maturation medium already mixed with antigen and adjuvants, leaving up to 10% of the old medium; - everything goes back to growing vials, the cells are kept in the incubator for another 6 hours until a significant change in cell morphology is observed, they become oblong, flared, indicating the successful antigen opsonization and activation of dendritic cells into immunogenic cells. 6. The method of claim 1, step e), wherein: - collecting the medium into the tubes, the PBS / EDTA solution is infused into the vials (PBS standard EDTA concentration 0.02 to 4%) and kept warm for 15 min. or cold at + 4 ° C to 2 or; - Collecting the cells, flushing the vials twice with PBS or saline, collecting everything into tubes and centrifuging at 450-800 G for 10 min. At + 4 ° C; - centrifuged cells are passed through a 100 μητι nylon filter, counting and freezing at the ready-to-use doses of 5 million cells / ml of refrigerated medium consisting of 10% DMSO (dimethylsulfoxide) autologous patient serum or plasma; - The cells mixed with the refrigerant were blown into the freezer tubes which were placed in a freezer and cooled to a temperature of -80 ° C for a couple of hours. 16 LT 6263 B 7. Dendritic cells obtained by the method of claims 1-6 for use in immunotherapy for the treatment of all types and stages of oncological diseases and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, psoriasis.