Line of cells of human melanoma kg, secreting recombinant granulocytic macrofagal colony-stimulating factor

FIELD: biotechnologies.

SUBSTANCE: invention relates to field of biotechnology, namely, to obtaining genetically modified cell lines and can be applied in medicine for immunotherapy and immuno-prophylaxis in patients with malignant neoplasms. By means of recombinant method line of cells of human melanoma KG is obtained, which secretes recombinant granulocytic-macrofagal colony-stimulating human factor. Obtained line is deposited with Specialised cell culture collection of vertebrates of Russian cell culture collection under number RCCC ("П") 699"Д".

EFFECT: line of human melanoma cells KG possesses stable cultural, morphological and immunological characteristics and possesses ability to secrete recombinant human GM-CSF, remaining after cell inactivation with ionising irradiation.

 

The technical field of the present invention

The invention relates to the field of biology and medicine, in particular the production of genetically modified cell lines used for immunotherapy and immunization in patients with malignant tumors.

The prior art of the present invention

Activation of the immune system can stop the proliferation micrometastatic foci of tumor cells persist in the body of cancer patients after cytoreductive interventions [1]. Moreover, using immunotherapy is possible to achieve reduction and even disappearance of relatively large metastases and primary tumor sites in those cases when other treatments are not possible. The literature describes a case of regression of tumor size in 690 cubic centimeters in the adoptive transfer of antitumor T lymphocytes [2]. The observed cases of achieving a complete remission of cancer after antitumor vaccination with whole cell genetically engineered vaccines [3].

There are two different ways to generate protective immunity - active and passive. Active immunization or vaccination, call the stimulation of the formation of antigen-specific antibodies or T-cells using rst the value of the target antigen in combination with means, inducing the immune response. In contrast, passive immunization involves moving a formed in the other body effectors of the immune response - antibodies or lymphocytes. As an active immunotherapy, adoptive transfer can have a pronounced antitumor activity [2].

Among the most effective forms of activating private antitumor immunity of the patient allocate whole cell tumor vaccines secreting immunomodulatory cytokines. To receive these vaccines tumor cells transferout gene constructs encoding the appropriate cytokines, inactivating gamma-irradiation and injected into the body of patients. Secretion of tumor cells cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), IL-12 promotes the formation of a strong tumor-specific immune response. Recent advances in the field of active specific immunotherapy associated with the use of vaccines on the basis of cells secreting chimeric molecule consisting of the active sites of several cytokines (e.g., GIFT, connecting the activity of GM-CSF and IL-2) [4].

Vaccination with autologous tumor cells of patients is fraught with many technical difficulties, such as the availability of the tumor for hee is oricheskogo removal, establishing a short-term cultures of tumor cells, the number of received cells, the efficiency of delivery of a therapeutic gene and the level of its expression. In this regard, D.Pardoll proposed to use for vaccination allogeneic stable transfetsirovannyh cells with a high level of expression of GM-CSF [1]. The idea was supported by established fact cross-presentation of tumor antigens by dendritic cells of the patient, as well as the fact that many of immunological relevant tumor antigens were common to tumors of the same histological origin, and for tumors of various origins.

Despite the fact that the approach sharpens the question of the adequacy of vaccine antigens and the tumor, clinical trials have shown that the effectiveness of allogeneic vaccination is not inferior to the results obtained using autologous cells [5]. Moreover, the effect of allogeneic stimulation and the opportunity to enter a really large number of cells from patients with minor tumor size allowed us to obtain the most significant results in the entire history of active specific immunotherapy of tumors [3].

Some time after the introduction of the vaccine, which represents an inactivated genetically modified tumor cells, behold retiree GM-CSF, in the injection area creates a concentration gradient of the cytokine. GM-CSF induces positive chemotaxis and activation of monocytes and granulocytes in the blood and tissue macrophages. Immunohistochemical study of the introduction of the vaccine in 24-48 hours after injection reveals infiltration by granulocytes and cells of the monocytic/macrophage series [6]. Under the action of GM-CSF increases the production of IL-1 and tumor necrosis factor (TNF) by macrophages and develops local inflammatory response in which macrophages, granulocytes, and additionally engage in the field of injection of NK and NKT cells gradually destroy the entered cell vaccine.

A key factor contributing to the immunogenicity of GM-CSF-secreting vaccines is the ability of this cytokine to induce differentiation of early precursors in the direction of the most effective professional antigen presenting cells - dendritic cells [7]. It is shown that this process occurs in the injection area secreting GM-CSF tumor cells, if you enter cells produce a sufficient number of cytokine - not less than 36 ng to 106cells for 24 hours [3]. It is essential that the maximum concentration of GM-CSF is created in the immediate vicinity of the injection of genetically modified cells. In Mature dendritic the entrances (DC) differentiate their predecessors, localized near the cell vaccines, and, therefore, faguoqitirute the decay products of tumor cells. The study of sequential biopsy specimens designated for the vaccine shows that by 3-4 days among infiltrating the area of the cells there is a significant amount of DC expressing costimulation molecules, and to 5 days these cells are found in the regional lymph nodes [9].

Further stages of the immune response is the presentation of antigenic peptides from tumor antigens in the context of the molecules of the major histocompatibility complex (MHC) class II naive CD4+T-helper cells (Th0)that stimulates their differentiation into Mature T-helper type 1 and 2. As a result, the cross-presentation of tumor antigens by dendritic cells occurs in priming naive CD8+T-cells that stimulates their differentiation into functionally active cytotoxic T-lymphocytes. CD4+T-helpers of the first type contribute to the formation of clones of CD8+CTL. In addition, it was found that metastases vaccinated GM-CSF-secreting vaccines patients are abundantly infiltrated by lymphocytes, the majority (96%) which is a CD4+Th1cells, effectors hypersensitivity reactions slow (tuberculin) type[3, 10, 11].

Another subpop the isolation of T-lymphocytes - CD8+CTL - carry out the destruction of tumor cells and endothelial cells of tumor stroma in the case of the recognition of cross-prezentirani their tumor antigens. For the implementation of the antitumor effect the necessary secretion of interferon-gamma [12]. Under the action of interferon-gamma increases the expression of MHC-I on tumor cells and tumor stroma, which increases the efficiency of recognition and power activating signal and thereby allows the T-cell to carry out the induction of apoptosis in the target cell: secreted by cytotoxic cells (CTL) perforin forms pores in the cell membrane of the target, through which tumor cells penetrate Grasim In, which triggers a cascade of programmed cell death. Very likely, in addition to this mechanism, there are other ways of destroying tumor cells like T-lymphocytes and other cells. In particular, the ability to destroy cells of various tumors exhibit NK and NKT cells, and activated by various factors (including, and GM-CSF) granulocytes and macrophages.

In several clinical trials have shown that the use of GM-CSF-secreting vaccines affect the level of this cytokine in the serum of patients that stimulates the proliferation of early myeloid precursors and, in particular, the precursors of dendritic glue is OK, that may be essential for the antitumor effect of immunization [5].

Whole cell tumor vaccines based on GM-CSF may represent transtorno transfetsirovannyh autologous cells of the patient, obtained after surgical removal of the tumor, or allogeneic tumor cells cultured in vitro cell cultures secreting GM-CSF in the transient or stable transfection. Between these two approaches there are significant differences in immunological effects, and in the technology of preparation of the vaccine.

Autologous vaccination is always preceded by a surgical procedure is necessary in order to obtain tumor tissue. From distant tumor sites get monoclonal suspension, which is cultivated in vitro in the form of short-term cell culture. The cells transferout gene construct that encodes GM-CSF, inactivate by ionizing irradiation and injected patients; some cells preserved by freezing for subsequent vaccinations. The duration of the process of preparation of the vaccine varies from 20 days to three months [8].

A significant drawback of autologous vaccination is the need to do all these manipulations individually for each patient. This leads ktomu, that method is extremely difficult to standardize, and dramatically increases the cost of vaccination. It is also clear that the autologous vaccine therapy can be performed only for those patients, the tumor which are available for surgical resection.

A limitation of the approach is the number obtained from the patient's cells. As a rule, a significant number of cells could be obtained from patients with large and multiple tumor sites. Such patients are often in poor condition, have a marked secondary immunosuppression and, consequently, poorly respond to immunotherapy. In contrast, patients with a low degree of progression and neoplastic process that has a high chance of successful vaccination, in most cases, have tumors of smaller size, of which you cannot obtain a sufficient number of cells.

Clinical trials of autologous GM-CSF-secreting vaccines showed that the effectiveness of vaccine therapy depends not only on the number of input cells, but also on the amount of secreted their cytokine [5]. The productivity of the cells of the vaccine are usually estimated by the number of GM-CSF secreted per day one million cells in vitro. Accordingly, in terms of transient transfection this productivity depends both on the proportion transfiere the data cells, and the amount of cytokine produced by each cell. Although the currently used vectors based on adeno-associated viruses can achieve high productivity, maintaining a certain dependence of the vaccine therapy as to the efficiency of transfection and the ability of individual cells to produce sufficient amount of cytokine[5, 9, 13].

A significant factor affecting the effectiveness of autologous vaccine therapy, is the period of time required for preparation of the vaccine. It is known that immediately after removal of the primary tumor in the body of the patient changes, resulting in stimulation of growth of distant metastases [14]. In the same period disappears or weakens a number of factors, obuslavlivayuschih suppression of the antitumor immune response, and formed the conditions for the development of effective antitumor immunity. When immunization is carried out in the first days after the removal of the tumor, its efficiency becomes significantly higher. On the contrary, vaccine therapy initiated after 15 or more days after surgical rehabilitation, is less effective due to the fact that remaining in the body, tumor cells acquire the necessary time to adapt, to form the stroma and eff is active to resist the immune response [5].

At the same time, the vaccine therapy with autologous tumor cells has a significant advantage due to the maximum immunological match antigenic components of the vaccine and the original tumor. One of the most important components of the effector part of the immune response to the tumor is in the cytotoxic activity of CD8+T-lymphocytes that are specific for certain tumor antigens. A necessary condition for the implementation of the cytotoxic activity is the expression in cells of a tumor specific antigen and its presentation in the context of MHC molecules of class I. At the same time to generate CTL clones with antitumor activity requires the introduction of the same antigen in the composition of vaccinereports. It is established that tumor cells contain, as a rule, not one antigen, and some of their set of several dozen known antigens that are unique to each tumor [15]. In terms of experimental studies have shown that the closer the antigenic composition of vaccines and tumor, the greater the effect can be achieved using whole cell vaccine therapy. It is also important that the impact on the formation and the efficiency of the immune response renders not only the presence or absence of the specific antigen in the cells of the vaccine, but the ratio of the number of individual antigens. the I et, that antigenic composition of the surgically removed tumor and its remaining in the body metastases may vary, vaccines, created on the basis of autologous cells are maximally close antigenic composition to the tumor cells of the patient [16].

Unlike autologous allogeneic vaccines vaccines are modified cells previously obtained from other patients and some time in vitro. As a rule, it is a stable, homogeneous, well-proliferating cell lines with a high content of immunogenic tumor-associated antigens that are common to many types of tumors [17]. Selected for preparation of the vaccine line transferout gene constructs encoding GM-CSF, and then hold breeding and cloning, which gives a stable clone is a variant of the original cell line, all cells which secrete a certain amount of the cytokine. With the help of further modifications (re-transfection, sublimirovanny, amplification) may receive a stable variant cell lines with very high productivity. Thus obtained clones form the basis of allogeneic vaccines: the cells of one such clone, multiple modifications to the same source line, or even a combination of several different clones of lines grown in the need for the second number, inactivating gamma-irradiation and administered to patients.

In allogeneic vaccines are missing almost all manufacturing defects with autologous vaccination. All patients included in the program of biotherapy, enter the same preparation in advance is characterized by the level of productivity, the expression of tumor-associated antigens, immunophenotype etc. Allogeneic vaccines do not depend on the results of surgical treatment and can be applied in patients without surgical intervention. It is also important and something that started the vaccine therapy is possible in the first days after surgery, or even before it. When using allogeneic vaccination there is no technical limit to the number of input cells, which allows the use of large quantities of cells (500 million cells per vaccination), producing significant amounts of GM-CSF, and thereby to achieve the best results possible. An important factor is that allogeneic vaccine significantly cheaper autologous and that they receive after the development of the original clones is a much less labor-intensive process.

However, the introduction of alien patients for them allogeneic cells requires a careful selection of the varying composition of the vaccine for each patient. Irrelevant for autologous vaccine is AI, the question of compatibility antigenic components of the vaccine and tumor becomes crucial. In model systems have shown that it is sufficient to immunize experimental animals against one expressed in the tumor antigens to cause rejection of the entire tumor [18]. However, transformed by viral oncogenes murine cell line, as well as b-cell lymphomas are rare cases favorable match most critical for immunotherapy properties of tumor antigens, such as absolute specificity for the tumor and the need for antigen for the survival of tumor cells [19]. In the vast majority of other tumors can form variants of tumor cells escaping from the attack of T-lymphocytes and antibodies [20]. Losing the antigen or necessary for its presentation option MTL tumor subclones become immune to immune response, continue to grow and progress in the body and eventually lead to his death.

The formulation of tumor cells lose the expression of the tumor antigen is the result of random genetic events resulting in damage or disable the corresponding gene. Because these events are random, the probability of simultaneous loss of two or more antigens in the same cell is significantly lower than the probability of losing one antigen. Sootvetstvenno the simultaneous immunization with multiple antigens, expressed in tumors may be more effective than immunization against one. This expectation is confirmed by the results of clinical trials [21]. Therefore, it is highly likely that when allogeneic vaccination positive results can be expected only when the spectrum of tumor antigens expressed by cells of the vaccine, widely overlaps with the spectrum of tumor antigen specific patient's tumor. Because of the antigenic components of the vaccine are known in advance, it is advisable before starting immunotherapy to determine the antigenic profile of the patient's tumor. In addition, you must determine which of the variants of the MHC molecules are borne by the patient, because the presentation of a number of tumor-associated antigens (and, consequently, the possibility of an adaptive immune response to them) is unique for a limited number of options histocompatibility molecules.

A possible scenario when the tumor contains antigenic proteins, the products of which cannot be represented in the MTL of the patient, up to the present moment does not have a solution. Some researchers believe that it is advisable, however, immunisation of such antigens in the calculation that can be characterized variants of an antigen capable of pretentious in his MNF, or n is then, that, even if it is not presented in the context of histocompatibility molecules, the antigen may be targeted by the immune response [22]. At the same time some authors believe that the introduction of these antigens in a vaccine composition will adversely affect the final result due to the fact that at a critical time of vaccination, the immune system becomes too "loaded" with regard to irrelevant targets. This view is confirmed by the examples of infectious immunology: with most viral infections can potentially shape the immune response to hundreds of different epitopes of viral antigens, however, are practically immune response is only a few so-called immunodominant epitopes [23, 24]. Response to immunodominant epitopes may be ineffective and even harmful, because domination is not possible to generate an immune response to other antigens, more effective for protection against the pathogen, but less convenient for immune recognition [25].

Definition immunophenotype patients loci leukocyte antigens of human histocompatibility (HLA-A and HLA-B is also required in order to assess the degree of compatibility MTL patient cells and vaccines. As in the case of an antigenic composition, at the moment there is no consensus as to what the cue exactly options histocompatibility molecules in cells vaccine optimal for the introduction of certain patients. In General, there are three options allogeneic vaccines: 1) the maximum (or full) line input cells HLA phenotype of the patient, 2) the lack of conformity (low match) MTL patient and cell vaccine, or 3) the use of allogeneic tumor cell vaccines, not expressing the surface antigens of histocompatibility.

When used as a vaccine tumor cells with a high degree of compatibility allogeneic vaccination approaches for immunological effects to vaccination own tumor cells of the patient: the early introduction of the vaccine increases the residence time of cells in the body of vaccinated patients, as it does not transplant rejection, developing in the cells of the "other" HLA phenotype [26]. However, this effect remains only in the first few injections of the vaccine, and with continued immunization of input cells, in contrast, quickly destroyed because Express antigens in the context of "their" MNS, which can be recognized formed after the first vaccination cytotoxic T-lymphocytes. Another aspect of this vaccine is the fact that many variants of the molecules of the major histocompatibility class I serve as ligands inhibitory receptors of cytotoxic lymphocytes [20]. Pris is DTIE these ligands on tumor cells, as well as vaccines inhibits the cytotoxic activity of CTL, γδ-lymphocytes, NK and NKT cells, which may adversely affect the formation and implementation of the immune response [27]. The presence of lymphocytes of the patient corresponding receptors is an important argument in favour of using such a patient allogeneic vaccines, not bearing the appropriate HLA.

Vaccines, bearing molecules HLA non-native haplotype, can lead to the introduction of cells develop reactions transplant rejection, mediated CD8+T-lymphocytes, NK - and NKT-cells. With the development of alloimmunity activated cytotoxic cells that can recognize their own lack of histocompatibility molecules (NK-cells), or recognize the structure of foreign MHC molecules as a gross distortion of their own (T-lymphocytes and NKT cells). The result of the activation of effectors protivoinfektsionnogo immunity is accelerated rejection of the cells of the vaccine in the reactivation, resulting in 3-4 immunization of allogeneic cells completely eliminated within the first few days [28]. At the same time, allogeneic stimulation can have a positive effect on the antitumor immune response by stimulating a rare pool of cytotoxic limfotsitov is potentially automativcally activity. These lymphocytes are atypical for most T-lymphocytes T-cell receptor with differences in complementarity determining region (CDR) 2 and 1, which allows us to recognize as allogeneic MHC and modified molecules of the major histocompatibility tumor cells [29].

Another significant feature of alloimmunization is the property of allogeneic cells to induce polyclonal activation of T-lymphocytes, including inhibited by mechanisms of peripheral tolerance. Probably not physiologically high frequency of meetings of T-lymphocytes from allogeneic histocompatibility molecules is not dependent costimulation activate some part of T-cells without taking into account the specificity of their T-cell receptor (TCR). Among the respondents on alloantigen lymphocytes can be and CD4+and CD8+T cells specific for tumor antigens. Due to this stimulation of tumor-specific lymphocytes become activated phenotype and are able in certain circumstances to make several divisions, thereby increasing the total number of original rare clones. Thus, allogeneic polyclonal stimulation can produce antitumor T-lymphocytes to specific response to tumor antigens, which is practically impossible when using purely monoclone Inoi, specific stimulation.

One of the interesting aspects of allogeneic vaccine therapy is the ability to use tumor cells expressing molecules major histocompatibility complex class II. In a healthy organism the expression of HLA-DR, DQ and DP is limited to the population of professional antigen presenting cells (APC) (b-lymphocytes, macrophages and DC) and activated T-lymphocytes, endothelial and epithelial cells, fibroblasts and other parts of the tumor lines derived from patients with metastatic melanoma, also detected the expression of HLA-DR, the biological meaning of which is not clear. It is shown that the signals generated by the binding of HLA-DR with TCR, activate anti-apoptotic cascades that may promote the survival of tumor cells. In addition, the presence of MHC class II on tumor cell can have a value for the tolerance of the organism to tumor antigens, as presented epitopes (in the context of MHC class II) is not on ARS, and on tumor cells that do not have molecules that provide co-stimulating signals. According to modern concepts premirovanii such tumor cells naive T lymphocytes in the absence of signal 2 (costimulation) and cytokine necessary support can lead to apoptosis of T lymphocytes and those who amym to eliminate T-cells, specific to tumor antigens.

The application in allogeneic tumor cell vaccines expressing variants of MHC class II, coinciding with the haplotype of the patient, and not expressing the molecules of the family V7, can lead to the formation of tolerance to tumor antigens in response to the introduction of the vaccine [30]. In addition, significantly increases the risk of development of autoimmune complications and other immunopathy, because the conditions for stimulation of specific subpopulations of CD8+cells that can recognize MHC class II [31].

Another option vaccines based on allogeneic vaccine cells is the use of tumor lines, lost surface expression of molecules of the major histocompatibility complex as a result of various disorders. Many of these lines with the introduction into the organism of experimental animals relatively quickly destroyed by NK cells, which are able to implement cytotoxicity in the absence of an inhibiting signal from the molecules of MHC I on the target cell [32]. According to various estimates stay viable cells of the vaccine, not expressing HLA molecules in the body of the patient, is from 3 to 6 days, which is, in General, sufficient to make the necessary immunostimulation. In addition, the term of such vaccine kletok the patient's body almost does not change with repeated vaccinations. It is not clear whether there are any immunological peculiarities of the action of allogeneic vaccines on the basis of cells, not expressing antigens of histocompatibility. On the one hand, cells that were not exposed to the TCR-MHC-dependent cytotoxicity, represent a more stable source paracrine GM-CSF, suggesting greater efficacy of the vaccine when multiple immunizations. In addition, the lack of HLA molecules relieves the inhibitory effect of tumor cells to the cytotoxicity of NK-cells and creates conditions for the involvement of these cells in the inductive phase of the immune response. On the other hand, the mere receipt of such cell lines from metastatic aggressive tumors testifies to the fact that the MNF-negative lines are formed specific mechanisms of avoidance cytotoxic activity as NK-cells and T-lymphocytes. Many aspects of the use of vaccines on the basis of such cells require further investigation, especially considering the fact that the factors contributing to the stability of the MHC-negative tumor cells to the action of NK-cells, can have a negative effect on the formation of an immune response.

The disclosure of the present invention

The essence of the present invention is to provide a new unique cell line human melanoma KG, with the ability with createrobot immunoactivity cytokine - granulocyte-macrophage colony-stimulating factor. The uniqueness of this cell line is determined by using as material for genetic modification of a unique cell line human melanoma Mel Kor (RF patent No. 2287578). The ability of the cell line KG to secrete recombinant GM-CSF is achieved by genetic modification of the cells of melanoma Mel Kor gene construct containing the complementary DNA of GM-CSF human (SEQ ID NO:1)embedded in a vector designed for expression in mammalian cells.

The technical result is to obtain a cell line of human melanoma KG, stably transfected with the gene for GM-CSF and secreting this cytokine in the extracellular space under cultivation in vitro, as well as when using inactivated cells for administration to patients. The secretory cells KG GM-CSF leads to activation of the immune response against tumor antigens expressed by cells KG, with the introduction of inactivated cells of patients.

The aim of the present invention is an expanding collection of unique cell lines that can be used for immunotherapy of malignant tumors. This task is relevant to the modern practice of treating malignant tumors, because the range of antigens individual who's tumors is unique at that time, as the maximum effectiveness of immunotherapy is achieved through selection for patients of a vaccine with the most complete matching set of tumor antigens in the cells of the vaccine and tumor.

Technical result achieved when using the invention lies in the possibility of using inactivated cell line human melanoma KG for administration to patients suffering from cancer, to stimulate antitumor immunity, which gives the opportunity to increase the effectiveness of treatment and to increase life expectancy in the treatment of malignant tumors, and to immunoprophylaxis of malignant tumors.

Additional technical result achieved when using cells KG, is that the KG cells, cultured in vitro, can be a source of biologically active GM-CSF, which can be used for culturing cells, which require the presence of GM-CSF in the culture medium. Recombinant GM-CSF man, produced by cells of the KG, can be used as a stand-alone, high-purity protein and the composition of the culture medium or its derivatives.

The problem is solved in that the resulting new cell line human melanoma KG, secreting recombinant GM-CSF in which Alceste not less than 500 ng/10 6cells for 24 hours. The obtained cell line has a stable cultural, morphological and immunological properties and has the ability to secrete recombinant granulocyte-macrophage colony-stimulating factor as a result of genetic modification. All cell lines KG are characterized by stable secretion of GM-CSF, continued after inactivation of cells with ionizing radiation at a dose of 100 Gy, reliably preventing the proliferation of inactivating cells. The cell line KG deposited in the special collection of cell cultures vertebrate Russian cell culture collection under the number RCCC (P) D.

The implementation of the present invention

The procedure for obtaining genetically modified human melanoma cells KG, secreting GM-CSF

Obtaining clones

For transfection were purified using plasmid DNA gene construct, the coding sequence of the cDNA for GM-CSF, human, built-in vector designed for expression in mammalian cells, with the gene of resistance to selective antibiotic geneticin (G418) (SEQ ID NO:1).

Cell line Mel Kor were sown on Cup diameter 35 mm and were cultured for 24 hours to achieve a density of 16,000 cells/cm2.

After 24 hours of cultivation (after attaching and adaptation) to the cells is obavljale dissolved in binding buffer transfection complex, consisting of lipid and DNA associated with enhancers. For line Mel Kor used lipid and enhancer from a set Unifectin-M (Maxifectin), the amount of DNA used 1.5×106cells, was 5.6 ág. Preparation and submission transfection complex in the Cup with the cells was carried out in accordance with the manufacturer's instructions of the kit for transfection (Unifectin Group, Russia).

After 24 hours incubation with transfection mixture Wednesday in the cups were replaced with new cells and after a short adaptation perseval on cups with a diameter of 100 mm

After 24 hours incubation of the cells in culture medium were added selective antibiotic G418 (Sigma, USA) at a concentration of LD100(800 µg/ml).

After 1 week of incubation with the antibiotic (if necessary, the medium was replaced with the addition of a new portion of the antibiotic) resistant to the drug, the cells formed clones 75-150 cells in each.

Clones of cells was transferred into a 96-well plate using microdontia, to do this after removing the medium and washing the surface of the Cup on PBS clone under a microscope inflicted 10-15 ál of trypsin solution. After 30 seconds ukrepivshis cells captured by microdonation and transferred into prepared wells of 96-well plate with 200 μl of complete RPMI-1640 medium.

After 3-6 days successfully selected clones formed a monolayer, and PE is easily in a 24-well plate. After formation of the monolayer of cells was transferred into a 6-well plate.

From 6-hole tablet supernatant was collected for analysis of concentration of GM-CSF enzyme-linked immunosorbent assay, and the cells were perseval on 4-well plate, in which the clones were frozen at -135°C.

The concentration of GM-CSF enzyme-linked immunosorbent assay (ELISA)

After forming cells transfected clones density monolayer in 6-hole tablet (1,2×106of cells per well) culture medium was completely removed from the wells, the surface of the monolayer was washed once with 2 ml sterile PBS and the wells were added to 2 ml of complete RPMI-1640 medium.

After 24 hours, samples of the supernatant from each well was collected in cryoprobes and frozen at -20°C prior to the accumulation of optimum for the production of analysis number of samples.

After collecting 40 or more samples using a commercial kit ELISA GM-CSF human production Diaclone (USA) conducted the analysis of supernatants by ELISA in the following sequence.

The supernatant samples were thawed at room temperature, was stirred on a vortex for 10 seconds and centrifuged at 13400 rpm for 30 seconds. Each well pad production reaction were added to 50 ál analytical buffer. In the pre-marked holes were added to 50 ál of the article is ngarta GM-CSF in the wells for calibration 50 ál analytical buffer in the control wells and 50 ál samples to the remaining wells. The completed tablet incubated for 2 hours at room temperature on a rotator with a speed of 111 Rev/min After 2 hours from the wells was removed liquid, each of the wells washed four times with 400 μl of wash buffer. Later in the wells was added 100 μl of secondary antibody (conjugate antibodies against hGM-CSF and Biotin), and incubated for 1 hour at room temperature on a rotator at 111 rpm After 1 hour from the wells was removed liquid, each of the wells washed four times with 400 μl of wash buffer.

Later in the wells was added 100 μl conjugated with streptavidin enzyme (HRP-streptavidin) and incubated for 30 minutes at room temperature on a rotator at 111 rpm

After 30 minutes from the wells was removed liquid, washed four times each well with 400 μl of wash buffer.

Later in the wells was added 100 μl of color-forming substrate (stabilized Chromogen TMB) and incubated for 30 minutes at room temperature in the dark on the rotator at 111 rpm

After 30 minutes, the wells were added to 100 ál of stop solution and determined the optical density on the reader LabSystem (wavelength 450 nm).

Based on these results build the calibration curve and determine the concentration of GM-CSF in samples.

In the result of the method of transfection and subsequent selection was received line KG, producing at least 500 ng of GM-CSF is secreted 1 million cells per 24 hours).

The main characteristics of the cell line KG

Lineage cell lines:

The cell line obtained from a sample of tumor tissue of the patient CGA, and/b 96/10389, were treated at the research center of RAMS in 2001 on the occasion of disseminated melanoma. Material obtained from a sample of metastasis in the area of the femoral triangle. Stable transfection performed at the Institute of gene biology, Russian Academy of Sciences in 2004.

The number of passages:

At the time of preparing passports cell line was 40 passages.

Standard culturing conditions:

Culture medium RPMI (90%), fetal calf serum 10%, containing antibiotics (penicillin with streptomycin at a concentration of 100 u/ml and 100 µg/ml, respectively)

Method of removal: 0.25% solution of trypsin and 0.02% solution of versene in a 1:1 ratio.

Cultural properties:

For growing crops, you can use the culture flasks. Vials with an area of growth 25 cm2in 5 ml of medium seeded with 1×106cells. Passage 1 every 3-4 days. Cultivation at 37°C and 5% CO2. Cells have adhesive, monololy character growth.

Karyological characteristics of strain:

Culture is of uniform diameter cores, density, color and state of the chromatin. Analyzed 26 metaphases. The number of chromosomes is kolebletsja from 64 to 84. The modal number of chromosomes corresponds triploid set of (3n).

Well identifiable chromosome groups a, b, and D with no signs of translocations. The karyotype of this culture can be considered as stable. Chromosomal aberrations were not found.

they cell culture:

KG is characterized by the presence of multiple dense growth of tumor cells polymorphic melanoma cells elongated, rounded and irregular in shape. In cells detected normal - and hyperchromic nuclei rounded and oval in shape, containing a single nucleoli, and basophilic inhomogeneous, sometimes vacuolisation the cytoplasm. There are giant multinucleated cells, mitoses.

Marker features:

Surface antigen expression: CD63+; NMW+; MelanA+; Tyrosinase+; HMW+; HLA-A,B,C(-); HLA-DR(-). The cells KG there is no expression of the gene beta-2-microglobulin. Secrete GM-CSF person in an amount not less than 10 ng/106cells for 24 hours.

Contamination:

Bacteria and fungi in culture is not detected. Test for Mycoplasma negative.

Conditions of cryopreservation:

Cells cell line resuspending environment for freezing: nutrient medium RPMI 70%, fetal calf serum 20%, DMSO 10%. The mode of freezing: liquid nitrogen, the temperature reduction of 1°C per minute up to -25°C, then quick freezing to -70°C. Storage in liquid al is those at the temperature of -196°C. Defrost quickly, at 37°C. Cells diluted in 10 ml serum-free medium and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, and transferred into a culture flask with an area of growth 25 cm2. Cell viability is assessed according to the inclusion Trypanosoma blue. Cell viability after thawing is 90%.

Cell line human melanoma KG, secreting recombinant granulocyte-macrophage colony-stimulating factor human encoded by the DNA sequence SEQ ID NO:1, deposited in the special collection of cell cultures vertebrate Russian cell culture collection under the number RCCC (P) D.



 

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8 cl, 12 dwg

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to molecular biology, virology and genetic engineering. There are disclosed method of producing recombinant influenza virus with mutant gene NB of membrane protein, recombinant influenza virus produced by this method, and vector composition based on parts of recombinant influenza virus.

EFFECT: developed method of recombinant influenza virus with mutant gene NB of membrane protein.

24 cl, 8 dwg

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to biotechnology, specifically, to cell differentiation and can be used in medicine. Differentiated stromal cell is made of adipose tissue. This cell expresses one or more proteins, characteristic for endocrine pancreas cell, such as insulin, glucagon, somatostatin and pancreatic polypeptide.

EFFECT: production of differentiated and functionally relevant cell for researches, implantation, transplantation and development of tissue products for treating pancreas diseases and repairing pancreas tissues.

24 cl, 8 ex

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: substance of invention includes separated population of hematopoetic stem cells of negative lineage, (Lin- HSC), containing progenitor endotheliocytes (EPC), protecting retina blood vessels and neuronal eye networks, method of separation of hematopoetic stem cell population of negative lineage from bone marrow of mammal adult, herewith approximately 20% of cells in separated Lin- HSC express cell surface CD31.

EFFECT: possibility to apply for treatment of eye vascular diseases.

22 cl, 10 ex, 24 dwg

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of gene engineering and biotechnology and can be used for labeling biological objects. The nucleic acid molecule was extracted that encoded the fluorescing protein selected from fluorescing proteins represented by the Copepoda Crustacea biological kinds and fluorescing mutants of the aforesaid proteins. The aforesaid nucleic acid was functionally bonded to appropriate elements of expression regulation to be used in the method of producing aforementioned fluorescing protein. On the basis of the aforesaid extracted nucleic acid cloning and expressing vectors are produced as well as the expressing cassette. The cell and a stable cellular lineage, containing the aforesaid expressing cassette produce the fluorescing protein. Note that the said fluorescing protein, the nucleic acid encoding the protein above and expressive genetic structures containing the said nucleic acid, are used incorporated with the set designed to label a biological molecule. The fluorescing protein is also used in the methods of labeling a biological molecule, a cell or a cellular organella.

EFFECT: expanded biological objects labeling means.

12 cl, 9 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.

EFFECT: increased level of bioluminescence in comparison with natural protein.

11 cl, 6 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: human haemopoetic stem cells are separated either from bone marrow or mobilised peripheral blood, or blood separated from umbilical vein. The fractionation procedure is used to prepare in Ficoll gradient from total cell mass the suspension of mononuclear cells wherefrom enriched suspension of cells CD133+ and CD34+ is separated. The prepared suspension is cultivated in the environment Dulbecco M (IMDM) with insulin and transferrin, as well as the stem cell growth factor (SCF), tyrosine kinase ligand 3 of fetal liver (Flt3L), thrombopoietin (TPO) and ionomycin (Ca2+ ionophore). After 7-10 days of cultivation, the grown cell suspension contains 80-92% of CD34+ cells.

EFFECT: invention allows for short-term cultivation of higher number of low-differentiated haemopoetic stem cells to be used in therapeutic purposes.

8 dwg, 4 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: there proposed is the method for obtaining endotheliocytes from human embryonic stem cells, which involves differentiation of human embryonic stem cells on the substrate and in synthetic medium. Method allows obtaining endotheliocytes with high efficiency, choosing pure population of functional endotheliocytes that gained the same epigenetic status of key factors of transcription and homeostasis of genes sustaining as their analogues did, which are differentiated in embryo in vivo.

EFFECT: obtained endotheliocytes can be widely used in medical practice.

2 cl, 11 dwg, 4 tbl, 5 ex

FIELD: biology.

SUBSTANCE: present invention relates to biotechnology and molecular biology. Proposed is a medium for redifferentiation of dedifferentiated chondrocyte, as well as a method of redifferentiating dedifferentiated chondrocyte into the initial chondrocyte.

EFFECT: obtaining transplantation materials used in transplantology, plastic surgery, as well as cartilage regenerative therapy.

7 cl, 15 dwg

FIELD: medicine.

SUBSTANCE: claimed is human tumor cell and based on it vaccine.

EFFECT: invention can be used for therapeutic treatment of tumor diseases.

26 cl, 3 dwg, 3 tbl

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to biotechnology and can be used to reproduce stem cell cultures for various purposes. Precursor cell are recovered and additionally washed with buffer through two sequential centrifugations. It is followed with enzyme treatment of tissue with collagenase, several sequential centrifugation washings, plastic adherence of produced cell sample and following cultivation within 4 to 10 days in hypoxia environment at oxygen content not less than 5% to produce end cell culture. Cells of 1st to 2nd passages are used. Living, necrotic and apoptotic cells are quantified, while phenotype of mesenchymal stromal cells are analysed by identifying them with a kit of monoclones to hematopoietic and endothelial markers CD31, CD34, CD62L, CD62E, CD62P, CD117(c-kit), HLA-DR, as well as to markers being characteristic for mesenchymal stromal precursor cells CD9, CD54, CD71, CD90, CD105, HLA-ABC, vimentin.

EFFECT: reproducing multipotent mesenchymal stromal cells thus maintaining high yield, phenotype and high vitality.

2 dwg, 11 tbl, 3 ex

FIELD: medicine; biotechnology.

SUBSTANCE: present invention concerns biotechnology. The agent regulating a differentiation of cells-natural killers, containing in quality of an effective ingredient one or several genes is described. The genes used in the invention, are described in demand materials.

EFFECT: reception of the new agent regulating a differentiation of cells-natural killers.

6 cl, 4 dwg, 5 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to medicine, and particularly to immunotherapy. Proposal is given of a method of obtaining and/or multiplication of monocytic origin self-specific regulatory T-cells, co-expressing CD3 and CD14 antigens on their surface. The cells are suitable for preventing and/or curing diseases, caused by violation of self-tolerance, particularly, auto-immune diseases and allergies, as well as for production of pharmaceutical preparations. The given cells are self-specific for a patient, on which they are used.

EFFECT: design of a method of obtaining and/or multiplication of monocytic origin self-specific regulatory T-cells.

38 cl, 9 ex, 4 tbl, 12 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to molecular biology and bioengineering, and particularly to stem cells. Proposal is given of a composition for inhibiting proliferation of stem cells of a mammal, enclosed in a structure, containing agarose, as well as methods of inhibiting other cells, not entrapped, including stem cells and cancerous and/or hyper-proliferative cells.

EFFECT: obtaining a composition for treating cell proliferative diseases.

26 cl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to versions of compositions used for separating cells, particularly blood cells, containing dextran, an antibody against glycophorin A, anti-CD 15 antibody, anti-CD 9 antibody, anti-CD 94 antibody, anti-CD 161 antibody and if necessary, other anti-CD antibodies and other components, necessary for dividing cells, particularly heparin and bivalent cations. An assembly and a method of separating cells using the assembly are invented. The invented compositions and method allow for agglutination of cells through identification of their surface, and can be used for identifying even rare cells with high output.

EFFECT: agglutination of cells through identification of their surface.

50 cl, 4 dwg, 19 tbl, 14 ex

FIELD: gene engineering.

SUBSTANCE: invention can be used for production of recombinant polypeptide of human granulocyte colony-stimulating factor. Recombinant plasmid DNA is constructed in vitro. It includes synthetic gene of human granulocyte colony-stimulating factor, strong constitutive promoter A3 from the early stage of bacteriophage T7 and synthetic section - translation enhancer (TREN) of gene 10 in bacteriophage T7. This DNA in combination with high copy number of plasmid and optimisation of cultivation conditions ensures constitutive biosynthesis of target protein in transformed by this DNA race of Escherichia coli SGK25/pA3GF with high yield.

EFFECT: constitutive biosynthesis of target protein in cells; high yield.

2 cl, 4 dwg, 7 ex

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