Hemopoietic cell cd34+ and use thereof

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically obtaining hemopoietic cells from blood, and may be used in medicine. The homopoietic cell CD34+ is extracted from peripheral blood of cancer patients undergone a growth factor treatment course. The obtained cell is transduced by a ligand which induces apoptosis with participation of the tumour necrosis factor, and is used to treat tumours.

EFFECT: invention enables to obtain a hemopoietic cell CD34+ which has anti-tumuor activity.

6 cl, 3 dwg, 8 tbl, 3 ex

 

The technical field to which the invention relates

This invention relates to migrate to tumor cells expressing the ligand (TRAIL)induces apoptosis with the involvement of tumor necrosis factor and their use in cancer therapy.

The level of technology

Dysregulation of the mechanisms of apoptosis plays a key role in the pathogenesis and progression lymphoproliferative disorders, which allows neoplastic cells to survive longer than their normal life expectancy and may be purchased chemo - and radioresistance [1]. Thus, the path of apoptosis represent an attractive therapeutic target in restoring the sensitivity of malignant cells to apoptosis or activation of apoptosis agonists [2]. For modulation of genes and proteins of apoptosis may be considered several strategies that focus on either the inner (Bcl-2, Bcl-XL)or external (DR4, DR5, FLIP)or convergent (cIAP2) pathways of apoptosis [3].

The ligand (TRAIL)induces apoptosis involving tumor necrosis factor, belongs to the family of factors tumor necrosis (TNF) - receptor ligands death [cells] [4].

The selected DNA sequence encoding TRAIL, expression vectors comprising said DNA sequence, and recombinant polypeptides TRAIL described patentei application WO 97/01633.

TRAIL binds to the receptor cell death 4 (DR4) and death receptor cells 5 (DR5), which is expressed on the cell surface of many cancer cells [5]. Binding of soluble TRAIL to DR4 or DR5 leads to activation of caspase and apoptosis [6].

Liu Zhongyu et al., J Clin Invest. Vol 112, no.9, 9.11.2003, 1332-1341 describe dendritic cells, using recombinant adenovirus transmoldovan TRIAL, and potential medical use in the treatment of arthritis. No mention of the possible use of the above-mentioned cells in the treatment of tumors has not been done.

Studies in vitro and in vivo suggest that due to its high specificity against cancer cells, as well as high antitumor activity, soluble recombinant TRAIL should play a key role in anti-cancer therapy [7]. Indeed, TRAIL selectively induces apoptosis in many transformed cells without affecting normal cells [4]. In addition, the TRAIL making mice demonstrated a significant efficacy against tumor xenografts carcinoma of the intestine [8, 9], breast cancer [10], multiple myeloma (a disease of Rusticola-Calera) [11] or gliomas [12, 13].

Study of toxicity in mice and nonhuman primates have shown that normal hepatocytes and keratinocytes resistant to trimeresurus TRAIL version [14, 15], then to the to they showed significant sensitivity to induction of apoptosis unoptimized drugs TRAIL or cross-linked to the antibody variants of the ligand [16]. Whether liver toxicity can be a big problem when using trimeresurus version TRAIL in high doses, remains unclear.

Additional restrictions on the use of soluble TRAIL associated with a weak apoptotic response observed in a sufficient number of cases the study of various lines of tumor cells, which require either prolonged incubation or very high doses of soluble TRAIL in order to undergo apoptosis [17, 18]. Obtained after intravenous pharmacokinetic profile TRAIL (half-life in plasma is 32 minutes, and the half-time of elimination of 4.8 hours), conditions of prolonged exposure and high concentrations are not applicable in treatment strategies in vivo [10]. To overcome the limitations associated with soluble recombinant TRAIL and especially with tumor target cells, present in different animal models used several approaches that use the TRAIL gene transfer mediated by adenovirus [19, 20].

However, the effectiveness of approaches using gene transfer largely depends on the efficiency of infection of the tumor and how you can avoid effective immune clearance [21]. In addition, in terms of systemic injection of adenoviral vectors is still essentially the t a few questions, associated with their security [22]. For example, adenoviral gene transfer overcomes TRAIL attenuated apoptotic response of hepatoma cells, but causes aggressive apoptosis in primary human hepatocytes [19]. Currently, gene therapeutic approaches using adenoviruses are mainly based on intratumoral release of adenovirus encoding TRAIL [23]. Despite local anti-tumor activity, intratumoral release of TRAIL has no systemic antitumor activity, loosing thus, any clinical value.

It is known that different types of cells migrate preferentially in tumors [24], and can be loaded structures necessary for the production of anti-cancer molecules. Thanks migration characteristics of CD34+ cells [25] and natural killer cells (NK) [26] can be used as carriers for delivery of anticancer molecules.

After infusion of CD34+ cells demonstrate specific properties of migration, including the ability to permanently colonize the bone marrow and temporary colonization of the liver and spleen [27-30]. These migratory properties are strictly related with the expression of adhesion receptors (e.g., CXCR-4, VLA-4, VLA-5, CD44, and so on)that interact with specific ligands (in the example, SDF-1, VCAM-1, and so on), expressed on stromal cells, inherent in the microenvironment of the bone marrow, as well as the microenvironment

tumors [31-34].

NK cells are a subpopulation of lymphocytes that play an essential role in the cellular immune defense against tumor cells through mechanisms that are not limited to, major histocompatibility complex [35]. After intravenous infusion of NK-cells migrate to the bone marrow and lymphoid organs and penetrate into the tumor sites due to activity of signaling cytokines. So many groups [researchers] have tried to use NK-cell migration in tumors with therapeutic purposes [36-38].

Although the ability of NK cells to migrate into tumors known, genetically modified cells undergo stress, which depends on the type of the transferred gene. The stress cells may lose the original ability to migrate into the tumor after gene transfer TRIAL. So there is no special reason to expect success, and in each case the required experimental verification to confirm viable whether this particular approach.

Now it was found that CD34+ cells and NK-cells, engineered to Express TRAIL, can be successfully used for obtaining mediated cell antitumor act is vnesti in vivo.

Description of the invention

In accordance with this invention, mediated by adenovirus gene transfer in migratory tumor cells are designed for producing ligand (TRAIL)induces apoptosis with the involvement of tumor necrosis factor. Cells according to the invention can be introduced systematically to highlight TRAIL in place of [finding] the tumor without causing the above problems.

Accordingly, this invention relates to cells that migrate into the tumor and expressing TRAIL and the containing cell preparations. The invention also relates to the use of the above-mentioned cells in preparations of cell compositions for cancer therapy, in particular for the treatment of lymphoma in humans.

Cells according to the invention can be obtained by transduction of migrating to the tumor cells defective replicated adenovirus encoding gene TRAIL man (Ad-TRAIL) under the control of a suitable promoter, such as CMV. Transduction can be performed in accordance with methods known molecular biologists, preferably following the procedure described in the examples.

The term "migratory in the tumor cells used in accordance with this invention for labeling cells, capable of (1) after intravenous injection to migrate into tumor tissue is ü and (2) to Express an adequate amount of membrane-bound TRAIL (mTRAIL) for at least several days.

Examples of migrating tumor cells are able to migrate from the blood into the tumor metastases include hematopoietic cells, namely hematopoietic CD34+ cells from the peripheral blood of patients with cancer treated with growth factor; NK cells from peripheral blood; cytokine-induced killer cells (CIK), obtained from the culture ex vivo mononuclear peripheral blood cells; endothelial cells and endothelial precursor cells; the tumor infiltrates of lymphocytes (TIL); lymphokine-activated killer cells (LAK); macrophages; mesenchymal stem cells (MSC) (newly allocated from the peripheral blood or human tissues or cultured ex vivo) and even cell line human, preserving the ability to detect the tumor and constructed for the expression of mTRAIL. In order to get an adequate expression of mTRAIL, can be addressed several ways with the use of plasmids and viral vectors with the appropriate regulatory genetic elements, such as tissue-specific promoters and/or enhancers.

CD34+ cells and NK is particularly preferred.

Optimal transduction efficiency of NK-cells can be achieved by inactivated NK cells within 18 hours with N-acetylcysteine (10 mm). After inactivated NK-cells were subjected to gradually (50-500) multiple Tr is Specchi (MOI) of Ad-TRAIL (50-500) in serum-free conditions at 37°C. Then made environment with the addition of serum (RPMI-1640/FBS (fetal bovine serum) 20%), a few hours later, the culture was added Gene Booster (1:200)and then further incubated for another 18 hours.

Similar conditions can be applied to cells CD34+, with the exception of inactivated with N-acetylcysteine.

Compositions according to the invention can be obtained by the use of carriers suitable for parenteral, in particular intravenous, such as indicated in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

For example, the fillers that may be used include any pharmaceutical agents that are not harmful for the patient who receives the composition, such as water, saline, glycerol and ethanol, if necessary, mixed with additional substances, such as moisturizing or emulsifying agents, buffers, etc. Appropriate dose depends, among other factors, gender, age and condition of the patient treated, the severity of the disease. An appropriate effective amount can be easily determined by any specialist practising in this area, and can be in any way determined by clinical studies. A therapeutically effective dose is usually from about 103up to 1015transduced cells. The other is their dosage, undoubtedly, can be installed by conventional experiments, i.e. on the basis of curves "dose-response, determine the maximum tolerated dose (MTD) or holding on a limited number of patients 1-phase clinical studies. Dosage for treatment can be a single dose or mnogorazovyj mode. Further, the patient can enter as many doses as is required. The person skilled in the art will easily determine the most effective dosage regimen.

Hereinafter the invention will be further illustrated by the following examples, presenting clear and convincing evidence that

(i) stem cells CD34+, as well as NK-cells, can be efficiently transpulmonaryin vitroadenovectors expressing TRAIL;

(ii) after transduction with these cell subtypes temporarily Express TRAIL on its cell surface;

(iii) a brief duration of expression of TRAIL associated with a strong apoptotic effect found in TRAIL-sensitive and TRAIL-resistant tumor cells, cultured together with translotsirovannoi cells;

(iv) injection of transduced cells [mice] NOD/SCIDin vivoin an early or advanced stage of tumor development is associated with a significant prolongation of survival of mice, suggesting that TRANS lirovannye adenovirus CD34+ cells and NK-cells can effectively serve as carriers for systemic delivery TRAIL for therapeutic purposes.

The effectiveness of intravenous is a distinct advantage of the present invention in comparison with protocols based on intratumoral administration.

While the mechanism of the antitumor effect of mTRAIL-expressing human cellsin vitromost likely, apoptosis is triggered by a direct reaction between mTRAIL and recognizes its receptor on the cell tumors, the mechanism of its antitumor activityin vivounknown to us. Direct cytolysis of tumor cells may be associated with any abscopal indirect growth effects arising from apoptosis located in the tumor non-neoplastic cells expressing receptors for TRAIL (vascular, perivascular, interstitial, and other types of cells that play a critical role in tumor development and survival of tumor cells). The following experiments aimed at better understanding of the mechanism observedin vivoantitumor activity, but are in no way intended to limit the invention to any of the hypotheses of the actual mechanism of the observed activity.

Example 1 - apoptosisin vitrocell lines lymphoma, cultivated together with CD34+ cell or NK-cells expressing TRAIL after mediated by adenovirus transfer g is on

Evaluated the effects of co-culture cell lines lymphoma cells CD34+ or NK cells, are designed so that they are expressed TRAIL after mediated by adenovirus gene transfer.

In initial experimentsin vitroan adenoviral vector expressing TRAIL (Ad-TRAIL)was used to establish the optimal conditions adenoviral infection of CD34+ cell and NK-cells. The period of validity of the expression of TRAIL on the cell surface CD34+ cell and NK-cells are then controlled using conjugated with phycoerythrin (PE) anti-TRAIL monoclonal antibody (clone Rik-2). Finally, the ability of membrane-bound TRAIL to induce apoptosis in cell lines lymphoma was evaluated by co-cultivation of cells Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells and cell lines lymphoma.

Methods

Adenovirus encoding gene TRAIL man. In these experiments used a defective replicating adenovirus encoding gene TRAIL man (Ad-TRAIL), expressed under the CMV promoter [39]. Ad-TRAIL contains the complete coding sequence of the TRAIL of the person cloned into the websites ofXhoI andNoiI pAd5CMVK-NpA. The obtained plasmid and adenovirus major sequences (Ad5) with a deletion of the E1 gene was transfusional in embryonic kidney cells [line] 293 people, were isolated viral part of the s and amplified for analysis of expression of TRAIL. Recombinant adenoviruses were tested for the presence of replication competent virus with the help of test belascoaran A549, and the titer of virus was determined by blagoobrazov on cells [line] 293. Purified viruses were placed in a phosphate buffer (PBS) with 3% sucrose and stored at -80°C until use. The TRAIL gene product expressively on the cell surface of transduced cells and could be detected using flow cytometry.

Adenoviral transduction of CD34+cell. CD34+ cells for transduction of Ad-TRAIL were obtained from peripheral blood gave consent for cancer patients receiving chemotherapy and treated with hematopoietic growth factors. CD34+ cells from samples leukopheresis was enriched with immunomagnetic techniques and positive selection (Miltenyi Biotech). For adenoviral transduction of CD34+ cells was applied [density] 2 × 106/ml in 35 mm Petri dishes in 1 ml serum-free medium (IMDM)containing the appropriate dilution of the original Ad-TRAIL, allowing to obtain the final level of infection in the range from 100 to 1000. After incubation (37°C, 5% CO2within 2 hours the cultures were added to 1 ml IMDM/FBS (fetal calf serum) 20%, and 4 hours later was added GeneBooster (1:200). Then cells were incubated for 18 hours, then thoroughly washed (3×) in a medium containing serum, and, finally, OTS is aligned transduction efficiency by direct immunofluorescence assay using PE-conjugated anti-TRAIL antibodies.

Adenoviral transduction of NK-cells. NK-cells for transduction was obtained from peripheral blood gave consent for that of healthy donors. NK cells were enriched using immunomagnetic techniques and positive selection (Miltenyi Biotech). Before transduction of NK-cells were incubated (18 hours, 37°C) RPMI-1640 with the addition of 10% FBS and N-acetylcysteine (10 mm). For adenoviral transduction of NK-cells were placed [density] 2 × 106/ml in 35 mm Petri dishes in 1 ml serum-free medium (RPMI-1640)containing an appropriate dilution of the original Ad-TRAIL, with a final multiplicity of infection (or intrusion) (MOI) ranging from 50 to 500. After incubation (37°C, 5% CO2within 2 hours the cultures were added to 1 ml of RPMI-1640/FBS20%, and 4 hours later was added GeneBooster (1:200). Cells additionally incubated for 18 hours, then thoroughly washed (3×) in a medium containing serum, and, finally, the transduction efficiency was assessed by direct immunofluorescence assay using PE-conjugated anti-TRAIL antibodies.

Joint culture. The activity of CD34+ cell or NK-cells expressing TRAIL on its outer membrane, apoptosis testedin vitroin experiments on co-cultivation. Briefly, cells Ad-TRAIL/CD34+ or Ad-TRAIL/NK cells (effector cells) were cultured together with lymphoma cells (target cells). Induction is popdose was evaluated after 24 and 48 hours after the start of culturing. In these experiments, the ratio of effector cells: target cells was calculated based on the transduction efficiency of effector cells.

Results

Adenoviral transduction of CD34+cell.Preliminary experiments showed that the optimal transduction efficiency of CD34+ cell was consistently achieved effect on CD34+ cells (2 × 106/ml) graduating MOI of Ad-TRAIL (range 100 - 1000) in serum-free conditions for 2 hours (37°C). Then added an equal volume of medium with addition of serum (IMDM/FBS 20%), and 4 hours later the cultures were added Gene Booster (1:200)and further incubated for 18 hours. In the end, the cells were thoroughly washed (3×) in a medium containing serum, and evaluate transgene expression by direct immunofluorescence assay using PE-conjugated anti-TRAIL antibodies.

Whereas in control cells background signal of the TRAIL was not detected in cells that are affected in Ad, revealed [defined] the percentage of TRAIL-positive cells CD34+, which increased, depending on the MOI. In a representative experiment, the percentage of TRAIL-positive CD34+ cell was 25% and 84% at MOI 100 and 1000, respectively.

The above Protocol infection, respectively resulted in maximum efficiency gene transfer into CD34+cells expressing TRAIL, when MOI equal to 1000, with an average mn of the rising (± standard deviation (SD)) 83 ± 8% (range 70 - 95%, n = 5). Cell viability, as measured by the test for the release of dye Trypanosoma blue, MOI 1000 had no effect (>85% viable cells).

To assess the temporal nature of transgene expression, CD34+ cells were transducible MOI 1000 and analyzed for expression of TRAIL up to 7 days after transduction. In these experiments, in order to ensure the survival of CD34+cell in culture was added a small dose (3 ng/ml), granulocyte colony-stimulating factor (G-CSF). As shown in table 1, a significant percentage of the CD34+ cell (25%) continued to Express the TRAIL up to 120 hours after transduction.

Table 1
Additional time of transgene expression by CD34+cell, translotsirovannoi Ad-TRAIL
Hours after infection244872120168
% of CD34 cells*expressing TRAIL9570422513

Adenoviral transduction of NK-cells. Preliminary experiments showed that the optimal efficiency transdu the tion of NK-cells, respectively, is achieved by inactivated NK cells within 18 hours with N-acetylcysteine (10 mm). After inactivated NK-cells (2 × 106/ml) were subjected to gradually MOI of Ad-TRAIL (range 50-500) in serum-free conditions for 2 hours (37°C). Then added an equal volume of medium with addition of serum (RPMI-1640/FBS 20%), and 4 hours later was added Gene Booster (1:200), and further incubated for 18 hours. Finally, the cells were thoroughly washed (3×) in a medium containing serum, and evaluate transgene expression by direct immunofluorescence assay using PE-conjugated anti-TRAIL antibodies.

Cells that are affected in Ad, showed the percentage of TRAIL-positive NK cells, which was increased depending on the MOI. In a representative experiment, the percent of TRAIL-positive NK cells was 30%, 45% and 46% at MOI of 50, 100 and 500, respectively.

The above Protocol infection bravely gave maximum efficiency of gene transfer at MOI of 100, with a mean (±SD) 61 ± 18% (range 45 - 84%, n = 4) NK-cells expressing TRAIL. Cell viability, as measured by the test for the release of dye Trypanosoma blue, MY level 100 had no effect (>85% viable cells).

To estimate the period of transgene expression, NK-cells were transducible MOI 100 and analyzed for the expression of TRAIL up to 7 days after transduction. As shown in Table 2, transduced NK-cells continued to Express the TRAIL up to 168 hours after TPA is succee.

Table 2
Additional time of transgene expression of NK-cells, transduced with Ad-TRAIL
Hours after infection244872120168
% of CD34 cells*expressing TRAIL4155606355

Joint culture. In the end evaluated the ability of membrane-bound TRAIL run apoptosis in lymphoid cell lines by culturing cells of Ad-TRAIL/CD34+ or Ad-TRAIL/NK cells (effector cells) and tumor cell lines (target cells). For these experiments were selected two cell lines according to their sensitivity to soluble TRAIL. In particular, we used sensitive to TRAIL cell line KMS-11 and TRAIL-resistant cell line JVM-2.

CD34+ cells or NK-cells transduced with the optimal conditions in accordance with the protocols of infection, described above, were collected 24 hours after the primary exposure to adenovirus, thoroughly washed and the cult who were verovali together with cells KMS-11 or JVM-2.

When cells Ad-TRAIL/CD34+ were cultured together in the ratio of effector cells: target cells" to 0.8:1, a substantial portion (81%) of apoptotic and necrotic cells detected among cells KMS-11 after 24 hours of culturing. The number of apoptotic cells then increases after 48 hours of co-culture (93% of apoptotic cells).

When Ad-TRAIL/NK-cells were co-cultured at a ratio of effector cells: target cells" of 0.6:1, a substantial portion (83%) of apoptotic and necrotic cells detected among cells KMS-11 after 24 hours of culturing. The number of apoptotic cells then increases after 48 hours of co-culture (85% of apoptotic cells).

For TRAIL-sensitive cell lines KMS-11 power apoptotic effect caused by membrane-bound TRAIL, was similar to that caused by a 24 - to 48-hour exposure to large doses (100 ng/ml) of soluble TRAIL (table 3). However, this pharmacokinetic profile TRAIL after intravenous injection (half-life in plasma is 32 minutes, and the half-life of excretion is 4.8 hours) continuous 48 - or even 24-hour exposure at 100 ng/ml could not be achievedin vivo.

Table 3
The effect of the s soluble TRAIL on cell KMS-11
The amount of soluble TRAIL% viable cells after 24 hours% viable cells after 48 hours
0 ng/ml7875
100 ng/ml272

Were also performed experiments on joint incubation of cells with Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells resistant to soluble TRAIL cell line JVM-2.

When cells Ad-TRAIL/CD34+ was cultivated together with the ratio of effector cells: target cells" to 0.8:1, after 48 hours of co-culture showed a significant part (51%) of apoptotic and necrotic cells in TRAIL-resistant [line] JVM-2.

When Ad-TRAIL/NK-cells were cultured together with the ratio of effector cells: target cells" of 0.6:1, after 48 hours of co-culture showed a significant part (53%) of apoptotic and necrotic cells in TRAIL-resistant [line] JVM-2.

On the TRAIL-resistant cell line JVM-2 power apoptotic effect induced membrane-bound TRAIL, was significantly higher than that caused by a 48-hour exposure to large doses (100 ng/ml) of soluble TRAIL (table 4).

Table 4
The influence of soluble TRAIL on cell JVM-2
The amount of soluble TRAIL% viable cells after 24 hours% viable cells after 48 hours
0 ng/ml8784
100 ng/ml8680

For further study power apoptotic activity mediated cell allocation TRAIL effector cells (cells of Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells) were cultured together with target cells (cell line KMS-11) within 24 hours at various ratios effector: target".

As shown in Table 5, the cells of the Ad-TRAIL/CD34+ trigger apoptosis or necrosis in a significant proportion (66%) of cells KMS-11 by co-cultivation with a ratio of effector: target of 0.4:1.

Table 5
Induction of apoptosis by co-cultivation of cells Ad-TRAIL/CD34+ cells and KMS-11 for 24 hours with increasing ratios effector: target (E:T)
The ratio of CD34+/KMS-110:1008:1 0,4:10,8:1
% residual viable cells71673422

As shown in Table 6, Ad-TRAIL/NK-cells trigger apoptosis or necrosis in a significant proportion (64%) of cells KMS-11 by co-cultivation with a ratio of effector: target 0,3:1.

Table 6
Induction of apoptosis by co-cultivation of Ad-TRAIL/NK-cells and cells of KMS-11 for 24 hours with increasing ratios E:T
The ratio NK/KMS0:10,06:10,3:10,6:1
% viable cells66683619

Example 2 - Evaluation of anticancer activity of CD34+ cell or NK-cells, engineered to Express TRAIL after mediated by adenovirus gene transfer, mice NOD/SCIDin vivo

To explore therapeutic potential of cell Ad-TRAIL/CD34+ or Ad-TRAIL/NK cells, mice NOD/SCID was administered to the TRAIL-sensitive cell line multiple myeloma KMS-11. Then the mice were injected with cells of Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells, and the survival of the mice showed the antitumor efficiency of allocation TRAIL cells.

Ways

Evaluation of antitumor activity of cells Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells. Female mice NOD/SCID at the age of six to eight weeks with a body weight of 20-25 g were purchased at the firm Charles River (Milan, Italy). Mice were placed in a standard laboratory conditions in accordance with established rules. Experimental procedures on animals were approved by the ethics Committee of animal experiments of the National Cancer Institute (Istituto Nazionale Tumori) and were performed in accordance with the rules of the coordinating Committee for Cancer Research UK.

Mice intravenously (I/V) was injected cells KMS-11 (0,5 × 106on the mouse). Treatment of cells with Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells (1 × 106mouse) was weekly/in-injections within 4 weeks, starting from the 7th or 17th days after injection of tumor cells. Average transduction efficiency re-entered cells Ad-TRAIL/CD34+ or Ad-TRAIL/NK-cells was 83 ± 8% and 61 ± 18%, respectively. Thus, each mouse received an average of 3.32 × 106expressing TRAIL CD34+ cell and is 2.44 × 106expressing TRAIL NK cells for four injections. Mice twice a week checking on the appearance of swollen is whether, body weight and toxicity. Registered term survival in each group and evaluated the differences between groups using statistical analysis. Appropriate control group consisted of (i) mice injected only with tumor cells, (iii) mice injected with tumor cells plus retranslations cells CD34+ or NK-cells.

Results

Cells Ad-TRAIL/CD34+.Mice transplanted cells KMS-11 (0,5 × 106on the mouse), were treated in four-injections of cells Ad-TRAIL/CD34+ on a weekly basis in accordance with two different modes, i.e. the treatment of early stage tumors, which began on the 7th day, and the treatment of advanced stage tumors, which began on the 17th day after the injection of cells KMS-11. Since the average transduction efficiency re-entered cells Ad-TRAIL/CD34+ was 83 ± 8% (mean ± SD, n = 9), each mouse received an average of 3.32 × 106expressing TRAIL CD34+ cell for four injections.

As shown in figure 1, the median survival of mice NOD/SCID injected only cells KMS-11, was 56 days. Injection of CD34+ cell wild type had no effect on median survival (56 days), whereas mouse, NOD/SCID, which was treated cells Ad-TRAIL/CD34+ early-stage tumors had a median survival in 111 days (P <0,0001).

The effect of treatment with Ad-TRAIL/CD34+ at an advanced stage is the tumor shown in figure 2. The median survival of mice NOD/SCID injected only cells KMS-11, was 56 days. Injection of CD34+ cell wild type had no effect on median survival (56 days), whereas treatment of cells with Ad-TRAIL/CD34+ at an advanced stage of the tumor was associated with a significant increase in average survival rate (98 days, P <0,007).

Ad-TRAIL/NK-cells. Mice transplanted cells KMS-11 (0,5 × 106on the mouse), were treated in four-injections of Ad-TRAIL/NK-cells on a weekly basis. Re-infusion of NK-cells was started on day 7 after injection of cells KMS-11 at an early stage of tumor growth. In these experiments, the average efficiency of transduction of the input Ad-TRAIL/NK-cells was 61 ± 18%. Thus, each mouse received an average of 2.44 × 106expressing TRAIL NK cells for four injections.

As shown in Fig. 3, the median survival of mice NOD/SCID injected only cells KMS-11, was 56 days. Injection of NK-cells wild type had no effect on median survival (50 days), whereas the treatment of Ad-TRAIL/NK-cells at an early stage of the tumor was associated with a significant increase in the average survival rate (74 days, P <0.03 in).

Example 3 - Antitumor activity of CD34 cells/Ad-TRAILin vitroagainst cell lines of breast cancer

A significant part of the cell lines breast cancer resistant to induced TRAI apoptosis as a result of multiple mechanisms, includes scenery TRAIL nonspecific receptors TRAIL, loss of expression of the receptors R1 and R2, overproduction FLIP and so on [40]. Based on previous results showing that resistant to soluble TRAIL (sTRAIL) cell lines lymphomas really become sensitive to TRAIL when exposed to membrane-bound TRAIL (mTRAIL), the authors investigated the sensitivity of the two cell lines breast cancer to sTRAIL and mTRAIL.

The sensitivity of cell lines breast cancer to unichtozhayuschego effect sTRAIL was assessed by comparison with evaluation of apoptosisin vitroin sTRAIL-sensitive and sTRAIL-resistant cell lines breast cancer, cultivated together with CD34 cells/Ad-TRAIL.

Used two cell lines breast cancer, i.e. MCF-7 and MDA-MB-361. To assess the sensitivity of individual cell lines to unichtozhayuschego effect sTRAIL, tumor cells (5-10 × 104/ml) for 72 hours were subjected to low (10 ng/ml) and high (100 ng/ml) doses of sTRAIL. Then apoptosis was assessed by double staining with annexin-V/propidium iodide.

Ways

Cell line. Cell lines breast cancer MCF-7 and MDA-MB-361 were acquired firms DSMZ (Braunschweig, Germany, EU) and ATCC (Manassas, VA, USA), respectively. Cells periodically overali polymerase chain reaction for contamination by Mycoplasma. All experimentsin vitrowere performed on exponentially growing cells.

The expression of annexin-V Analysis using annexin V-FITC (fluoresceinisothiocyanate) (PharMingen) was used to quantify the percentage of cells undergoing early or late apoptosis and necrosis. Briefly, the analyzed cells were washed twice with cold PBS, and then resuspendable in binding buffer (10 nm HEPES, 140 nm NaCl, 5 nm CaC12, pH 7,4). After incubation, 0.1 ml of cell suspension was transferred into a 5 ml culture tube and add 5 µl of annexin V-FITC and 10 μl of propidium iodide. After intensive mixing, the samples were incubated for 15 min at room temperature in the dark. At the end of incubation was added 0.4 ml of binding buffer and cells were immediately analyzed using flow cytometry.

Results

To explore whether incubation with sTRAIL with apoptosis, cell lines MCF-7 and MDA-MB-361 were subjected to sTRAIL (10 - 100 ng/ml, 72 hours), and then the percentage of apoptotic and necrotic cells was determined by analysis of cell sorting device with excitation of fluorescence (FACS). As shown in Table 7, the impact of sTRAIL was not able to induce an apoptotic response in MCF-7 cells, whereas it showed up in the authentic response in the form of cell death of MDA-MB-361 when exposed to 100 ng/ml sTRAIL within 2 hours. In accordance with these results MCF-7 is sTRAIL-resistant cell line, whereas MDA-MB-361 is sTRAIL-sensitive cell line.

Table 7
The sensitivity of cell lines breast cancer to sTRAIL
Cell linesTRAIL (ng/ml)
Phenotype010100
Apoptotic + necrotic cells (%)
MCF-7BC10913
MDA-MB-361BC6946

After that evaluated the apoptosisin vitrocell lines breast cancer, cultivated together with CD34+cell expressing TRAIL after mediated by adenovirus gene transfer (CD34/Ad-TRAIL).

Ways

Adenovirus encoding gene TRAIL man. For these experiments we used the defective replicating adenovirus, the encoding gene TRAIL man (Ad-TRAIL), expressed under the CMV promoter [39]. Ad-TRAIL contains the complete coding sequence of the TRAIL of the person cloned into the websites ofXhoI andNotI pAd5CMVK-NpA. The resulting plasmid, and the main adenoviral sequences (Ad5) with a deletion of the E1 gene was transfusional in embryonic kidney cells [line] 293, was isolated viral particles and amplified for analysis of expression of TRAIL. Recombinant adenoviruses were tested for the presence of replication competent virus with the help of test belascoaran A549, and the titer of virus was determined by blagoobrazov on cells [line] 293. Purified viruses were placed in PBS with 3% sucrose and stored at -80°C until use. The TRAIL gene product expressively on the cell surface of transduced cells and could be detected using flow cytometry.

Adenoviral transduction of CD34+cell. CD34+cells, transducible Ad-TRAIL, were obtained from the peripheral blood gave consent for cancer patients receiving chemotherapy and treated with hematopoietic growth factors. CD34+ cells from samples leukopheresis was enriched with immunomagnetic techniques and positive selection (Miltenyi Biotech). For adenoviral transduction of CD34+ cells was applied [density] 2 × 106/ml in 35 mm Petri dishes in 1 ml serum-free medium (IMDM)containing sootvetstvuyshee breeding source Ad-TRAIL, which gave the opportunity for a multiplicity of infection (MOI) of 500. After incubation (37°C, 5% CO2within 2 hours the cultures were added to 1 ml IMDM/FBS 20%, and 4 hours later was added GeneBooster (1:200). Next, cells were incubated for 18 hours, then thoroughly washed (3×) in a medium containing serum, and finally, evaluated the transduction efficiency by direct immunofluorescence assay using PE-conjugated anti-TRAIL antibodies.

A collaborative culture. Activity mTRAIL, triggering apoptosis was tested in vitro using experiments on co-cultivation. Briefly, cells CD34/Ad-TRAIL (effector cells) were cultured together with breast cancer cells (target cells). Induction of apoptosis was assessed 48 hours after the start of culturing. In these experiments we used the ratio of effective cells: target cells"that make up 1:1 and 4:1.

Results

Adenoviral transduction of CD34+cell. Optimal transduction efficiency of CD34+ cell was achieved consistent effect on CD34+ cells (2 × 106/ml) Ad-TRAIL at MOI of 500 in serum-free conditions for 2 hours (37°C). Whereas in control cells the background signal TRAIL were not found, cells expressing TRAIL, showed the percentage of TRAIL-positive cells CD34+ 93 ± 8% (mean ± SD). Cell viability, caniveau in the test for release of dye Trypanosoma blue, MOI -1,000 had no effect (≥ 85% viable cells).

Co-culture. The authors assessed the ability of mTRAIL run apoptosis in cell lines of breast cancer by co-culture of CD34 cells/Ad-TRAIL (effector cells) and tumor cells (target cells). Cells CD34/Ad-TRAIL was collected 24 hours after the initial exposure to adenovirus, thoroughly washed and cultured together with cells MCF-7 or MDA-MB-361. In these experiments we used two ratios of effector cells: target cells", i.e. the 1:1 and 4:1. The results are summarized in Table 8.

50
Table 8
Destroying tumor cells activity mTRAIL against cell lines of breast cancer
Cell lineConditions of cultureApoptosis + necrosis
MDA-MB-361
Control6
CD34/Ad-TRAIL ratio (E:T =1:1)28
CD34/Ad-TRAIL ratio (E:T = 4:1)
CD34/Ad-Mock ratio (E:T = 1:1)12
CD34/Ad-Mock ratio (E:T = 5:1)19
Ad-TRAIL 106plaque-forming units (pfu)6
MCF-7
Control5
CD34/Ad-TRAIL ratio (E:T =1:1)17
CD34/Ad-TRAIL ratio (E:T = 4:1)64
CD34/Ad-Mock ratio (E:T = 1:1)21
CD34/Ad-Mock ratio (E:T = 4:1)22
Ad-TRAIL 106pfu9

Significant percentage of cell death was detected by co-cultivation within 48 hours of CD34 cells/Ad-TRAIL with sTRAIL-sensitive cells MDA-MB-361 with a ratio of E:T :1 (cell death = 28%) and the ratio E:T 4:1 (cell death = 50%).

In respect of sTRAIL-sensitive cell line MDA-MB-361 power apoptotic effect caused by exposure to mTRAIL was similar to that caused by a 72-hour exposure to high doses (100 ng/ml) sTRAIL (table 7).

To verify the fact that the cytotoxic effect of CD34/Ad-TRAIL really occurs because of mTRAIL, cells MDA-MB-361 were cultured together with falsely translotsirovannoi a CD34+cell. As shown in Table 2, the joint cultivation of cells MDA-MB-361 with false translotsirovannoi cells CD34+ was associated with a moderate effect cell death detected only at the highest ratio of E:T. a mild induction of cell death, probably associated with excessive density of the culture.

To exclude the possibility that the activity of CD34/Ad-TRAIL, [leading] to cell death, associated with the free adenoviral particles that have not laundered at the end of the infection of CD34 Ad-TRAIL, cells MDA-MB-361 were subjected to 106plaque-forming units (pfu). No evidence of cell toxicity was not able to detect when exposed to 106viral particles to cells MDA-MB-361 (table 8).

The amount of pfu was calculated as follows.

To infect 5 × 105CD34+ cell with the value of the MOI, part 500, used 250 × 106pfu, which was added to 1 ml of suspension cleto is CD34+.

At the end of the infection of CD34+ cells 3 times washed in culture medium. For each washing procedure used a dilution of 1:20, i.e. the culture suspension was diluted at least 8000 times.

Assuming that at the end of the washing procedure in suspension culture remains 250 × 106pfu, it was diluted 8000 times, i.e. expected to co-culture was added <remaining 50,000 pfu.

Thus, in order to exclude that activity, causing cell death detected in co-culture, connected with free adenoviral particles that have not been washed, cell lines breast cancer was affected by the number of pfu equal to theoretical value of the residual pfu (see #3), multiplied by 20 (i.e. 50000 × 20 = 106pfu).

Then experiments on co-cultivation was performed by incubation of CD34 cells/Ad-TRAIL with sTRAIL-resistant cell line MCF-7. In these experiments showed significant proportion of cell death by co-cultivation within 48 hours of CD34 cells/Ad-TRAIL with sTRAIL-resistant MCF-7 cells at a ratio of E:T equal to 4:1 (cell death = 64%), while the ratio E:T of 1:1, cell death could be located.

In respect of sTRAIL-resistant cell line MCF-7 power apoptotic effect caused by the 48-hour who is esteem mTRAIL, was significantly higher than that caused by a 72-hour exposure to large doses (100 ng/ml) sTRAIL (Tables 7 and 8).

To ensure that the cytotoxic effect of CD34/Ad-TRAIL really is associated with mTRAIL, cells MCF-7 were cultured together with falsely translotsirovannoi a CD34+cell. As shown in Table 8, the joint cultivation of MCF-7 cells with false translotsirovannoi cells CD34+ was associated with a moderate effect cell death, which is probably caused by the excess density of the culture.

To exclude the possibility that the activity of CD34/Ad-TRAIL, causing cell death, associated with the free adenoviral particles which have not laundered at the end of the infection of CD34 Ad-TRAIL, cells MCF-7 were subjected to 106pfu. Evidence that cellular toxicity was observed when exposed to 106pfu cells MCF-7 were not detected (table 8).

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1. Hematopoietic cell CD34+ peripheral blood of cancer patients treated with growth factor, translationa ligand, inducing apoptosis with the involvement of tumor necrosis factor, and with antitumor activity.

2. The cell according to claim 1, which can be obtained by transduction of CD34+ cell adenoviral vectors encoding the ligand, inducing apoptosis with the involvement of tumor necrosis factor.

3. The use of cells according to claim 1 or 2 for the preparation of drugs for the treatment of tumors.

4. The use according to claim 3, where the tumor is a lymphoma.

5. The use according to claim 3, where the fuck is al is a carcinoma of the breast.

6. Use PP, 4 or 5, where the medicinal product is intended for intravenous administration.



 

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