The method of destroying undesirable target cells


(57) Abstract:

The invention relates to medicine and relates to a method of destruction of breast cancer cells or other carcinoma cells, preparation for destroying cells, and also set to perform. The invention consists in the destruction of undesirable target cells in a cell population, comprising containing the nucleus of the cells collected from the peripheral blood, or CD34+cells, or similar early precursor cells, while the cell population in vitro or in vivo exhibit with two or more immunotoxin that selectively destroy target cells. The advantage of the invention lies in the selectivity of action on malignant cells. 3 S. and 9 C.p. f-crystals, 7 PL.

The invention relates to selective purification of cell populations from target cells by treating the cell population with two immunotoxins.

The so-called autologous stem cells include cells isolated from the blood or bone marrow of cancer patients, containing, after pre-treatment of patients, a sufficient number of immature precursors of blood cells and immune cells iodine after re-introducing the cells into the blood of patients from which these cells were selected.

In the prior art purging autologous hematopoietic grafts with the use of antibodies in the case when the grafts are coming specially samples of bone marrow. This method of cleaning is published, for example, Myklebust, A. T., Godal, A., Juell, S. and Fodstad O. "Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow". Cancer Res. (USA) 1994, 54/1 (201-214) and Myklebust, A. T., Godal, A., Pharo, A., Juell, S. and Fodstad 0. "Eradication of small cell lung cancer cells from human bone marrow with immunotoxins". Cancer Res. (USA) 1993, 53 (16), 3784-3788. Both publications are used immunotoxins in which antibodies conjugated with toxin. The principle is to destroy the malignant cells collected from bone marrow cells before re-injection of cell suspension to the patient.

In recent years, methods that use actually the opposite principle. Applying the so-called stem-cell transplantation, trying to hold positive selection from the blood or bone marrow subpopulations of normal cells, which are able to restore normal bone marrow function after these cells are re-introduced to the patient. These "stem cells" are composed of a mixture. delanie such cells may be performed either by using the so-called afresa peripheral blood, procedures, occupying one or more days, or by immunoabsorption/selection of CD34+cells (immature precursor cells) from the blood or bone marrow using various known prior art techniques.

Stray K. M. et al., "Purging tumor cells from bone marrow or peripheral blood using avidin, biotin, iromunoadsorbtion" In: P. G. Adrian, Samuel G. and A. W-Diana W. (Eds), Advances in bone marrow purging and processing, pp.97-103, Orlando: Willelis Inc., 1994, describes the allocation of bone marrow cells or product afresa from peripheral blood. This cleaning procedure is performed in patients with lymphoma and in patients with breast cancer. The method includes a step of enrichment of CD34+cells before purification of b-cells or breast cancer cells using the so-called column Avidya. In this case, the treatment is carried out by an indirect method, so that the cell suspension is initially incubated with the primary antibodies, which interact with breast cancer cells, cell suspensions washed and again incubated with the antibody that interacts with the primary antibodies. This rat antibody biotinylation, that is connected to the column with beads, conjugated with Avidya, tumor cells are trapped by interaction between cells and complex primary antibody - biotinylated secondary antibody - avidin. The result of treatment using such a system is at most 3.2 log reduction of malignant cells. The application of this principle takes time and is cumbersome, as before application to the column with the cell suspension has to work on multiple stages, including incubation with the antibody and two washing. Thus, it is difficult to avoid damage to the stem cells or non-specific capture of stem cells in the column, resulting in undesirable loss of cells is essential to restore normal bone marrow function.

Tyer. C. L. et al., "Breast cancer cells are effectively purged from peripheral blood progenitor cells using an inimunomagnetic technique". Abstract to the first meeting of the International Society for Hematotherapy and Graft Engineering, Orlando, FL, 1993, describes immunomagnetic method similar to that used in the publication Miklebust et al., see above. This method, however, is used on peripheral blood cells. This principle is also different from the use of immunotoxins and cleaning efficiency varies from 3.3 to 4.8 log reduction is the use of indirect systems, including incubation with primary antibodies, followed by washing and a new incubation with beads associated with antibodies that interact with the primary antibody, but this assumption is legitimate. This procedure also includes additional and possibly traumatic treatment of normal cells, in addition, this procedure requires a lot of time. Its effectiveness is limited, and in the abstract does not mention anything about the selection of populations of CD34+cells, which is the main problem in this method, since the selection of CD34+cells per se takes a long time and is labor intensive. Thus, in most cases, selection of CD34+cells used immunomagnetic principle, which in this example is followed by one or two immunomagnetic stage to clean. Therefore, there is a significant risk of destruction of cells and their losses during the application of the method, requiring long lasting procedure and costly.

Lemoli, R. M. et al. (1994) Bone Marrow Transplant 13: 465-471, describe the purification of CD34+hematopoietic cells using the technique of immuno-absorption with avidin-Biotin. They increased the clearance of neoplastic cells by the x against infodelivery antigen CD30 and CD2. Tessa, R., et al. (1991) Int. J. Cancer, 49: 310-316, describe the allocation of autologous bone marrow prior to transplantation in patients with monocytic leukemia, using 2 immunotoxins specific to macrophage cell lines and United with caporino, and using 2 MoAbs (monoclonal antibodies with high specificity against circulating monocytes and 5b directed to cells of acute non-lymphoid leukemia (ANLL). In W091/09058 describes immunotoxins, including myelomonocytic b 195 used for treatment of ANLL cells in the bone marrow. Tonevitsky, A. G., et al., (1986) Int. J. Cancer, 37: 263-273, describe the allocation mouse erythroleukemia stem cells from bone marrow using immunotoxin comprising the conjugate of a-chain of ricin and b MAY 15, which interacts with the surface normal and neoplastic murine erythroid cells: a model for studies of therapy with bone marrow transplantation.

In the case of hematopoietic stem cells for transplantation is one of the main goals was that the cells that are supposed to re-enter the patients are allocated selectively with such a condition that the grafts should not contain any malignant clout malignant cells in a large number of test cases. Up to now there were a very limited number of attempts to remove or selectively destroy malignant cells in these grafts. This is partly due to the fact that experienced researchers in this field have not seen a need to, and because it was assumed that the existing known methods were not specific and, therefore, also had to destroy or remove vulnerable stem cells. Moreover, obtaining a bone marrow or mobilized peripheral blood from the patient is not simple and is not unlimited, and such a method for isolation of stem cells must be performed within a short period of time and should not have complications in order to avoid loss of or damage to normal cells. Thus, referring to the above, the argument in favor of the use of stem cells is, in part, that the transplant should be completely free of cancer cells, in part, that the restoration of bone marrow function is faster than after bone marrow transplantation without pre-selection. From this it follows that it is absolutely necessary to create a method, which is the procedure of obtaining stem cells.

The aim of the present invention is therefore to create a method of selection of stem cells, which does not include the above-mentioned inconveniences.

These objectives are achieved in the present invention are characterized in the accompanying claims.

The present invention relates to the purification of the collected populations of stem cells in the case of solid tumors using a method in which the cell population exhibited by composition of two or more antibodies associated with bacterial toxins. Used antibodies directed to antigens associated with target cells.

In the following summary of the invention is described in more detail using the example of the purification of stem cells isolated from peripheral blood, removal of breast cancer cells.

Known variants techniques highlight cells include immunoadsorption, the selection of stem cells in peripheral blood (PBSC) or CD-34+cells from blood or bone marrow. However, there is no known harmless and effective technique for treatment of these cell populations from tumor cells. It seems ocenie data must not contain CD34 receptors. It is essential that in the invention described below, unexpectedly also separated cancer cells with no toxicity against normal predecessors.

Before collecting the transplant of stem cells from the peripheral blood it is necessary to mobilize stem cells from the bone marrow by the use of chemotherapy or treatment by growth factors using methods known in the art. The collection of stem cells can be performed in accordance with one of several methods depending on what type of cells is desirable. In one method, the collected stem cells from peripheral blood. This can be done through the management of patients under the scheme, including leukapheresis on 10 and 11 days after administration of G-CSF (granulocyte-colony stimulating factor) (10 µg/kg/day) after receiving high doses of chemotherapy and total body irradiation. The speed of the flow of blood can be fixed, for example, at the level of 70 ml/min using a separator blood cells CS-300 Plus (Baxter Healthcare Corporation, Fenwal Division, Deerfield, IL, USA). The average volume of blood processed during this procedure, may be about 10 liters for 2.5 hours produ buffered saline (PBS), 1% serum albumin human (HSA), the CPU Sole 2991 to remove platelets. For use in the present invention, the concentration of cells (2-4l010/afares) can be adjusted to 1108/ml for negative selection (treatment) using immunotoxins.

If you want obtaining CD 34+cells, they can be isolated by positive selection using the ISOLEX 50or ISOLEX 300(Baxter). In this method, the product obtained by aparese or from the bone marrow, which can range from about 41010up to 61010cells can be together smashin and preincubation, for example, with anti-SV+monoclonal antibody IS at a concentration of 0.5 µg/1106cells at 4oC for 30 minutes with mild stirring. The treated cells are washed with PBS with 1% HSA processor b to remove unbound antibodies. DYNALpellets M-450 added to CD34+faction in the amount of 0.5 granules 1 having a core cell at 4oWith 30 minutes. Magnetic separation of the socket from the cells which were not targeted, can be performed using 2-or 3-fold flush unbound cells PBS, 1% HSA. CD34+cells can then be separated from Dynalthe ut at room temperature. Thus, CD34+cells can be collected by washing with PBS 5% sodium citrate. There are also other procedures for the selection of stem cells/early progenitor cells.

Binding profile of several antibodies to cell lines of breast cancer and tumor material was studied by other researchers and is partially confirmed by us. Antibodies that bind with a high percentage of breast cancer cells and is not associated with an important normal immature cells in the blood and bone marrow, konjugierte with exotoxin A of Pseudomonas (RE), and the ability to destroy breast cancer cells in culture tested, mainly by identifying the products of the colonies. Based on the binding profile of antibodies, the inventors of the present invention made all five different immunotoxins:

1. MOS-RE. This conjugate binds with a very high percentage of all breast cancer cells and has been very effective in model experiments and in active concentrations had very low toxicity against normal hematopoietic precursor cells.

2. NrLu10-PE. This conjugate binds to the same antigen, as described in ivalsa with the protein part of the antigen mucin, which is found mainly on breast cancer cells. The antigen is present in a large percentage of breast cancer cells, but not in all cells. Immunotoxin showed high specific activity against cancer cells, but was not as effective as the previous two immunotoxin.

4. VM-RE. This conjugate binds with charcterised epitope of the same antigen that VM-RE. Immunotoxin showed approximately the same efficiency that VM-D, in combination with very low toxicity against normal cells.

5. MLuCl-PE. This conjugate binds to a different antigen, namely LewisYthe antigen. The immunotoxin was slightly less active than the preceding, and also showed moderate toxicity against normal cells.

Immunotoxins in accordance with paragraphs 1,2,5 tested individually and in combination in model experiments for cleaning normal bone marrow samples from cancer cells (Myklebust et al., Cancer Research, 1994). As already noted, the great advantage of the use of transplants of stem cells is to reduce the interval to re-achieve normal function of the bone marrow (the EU is Adami and it was necessary to apply the toxins, able to destroy all the cancer cells in the graft in the absence of a significant effect on normal cells.

Started the search immunotoxins, more specific for breast cancer than MLuCl-PE, and more suitable for treatment in breast cancer.

During this search it has been unexpectedly discovered that the combination of MO-RE and VM-D was more effective than the sum of each previous toxins used separately. This is demonstrated below in Table 1. Further studies of the binding between the antibody and cell lines showed that the combination of immunotoxins leads to a stronger interaction than the interaction of the antibodies alone.

MOS associated with most breast cancer cells, including those that are less differentiated. VM recognizes the antigen mucin, which is expressed in a significant fraction of breast cancer cells, and cells that are more differentiated. NrLu10 and VM contact with the antigen recognizable MOS and VM, respectively, and, given this, it is not particularly likely that they would add something in the combination of immunotoxins MOS and VM. MLuCl-PE was theoretically interesting because it spasyuk, and model experiments did not show any obvious advantages included in the combination.

In the example below describes how to cleanse transplants, peripheral stem cell products afresa). In addition, the inventors have performed several experiments using a combination of MO-RE and VM-RE in the experiments, when the tumor cells were added to the collected peripheral stem cell or bone marrow before immunomagnetic positive selection CF-34+-cells. The results of one such experiment are shown in Table 2. Using two different cell lines it was shown that positive selection of CD 34+cells themselves (without any form of treatment) removes up to 3.8 log of tumor cells from the originally collected cell population. In other experiments the effect of "cleaning" in the selection of CD-34 cells varied from 2-3 log, which is also the case referring to the literature. When the processing of the immunotoxin was applied onto the positively selected population of CD-34 cells, the total effect of treatment was more than 4.7 log (table 2) for both cell lines. More than 4.7 log means in this case that all detectable tumor cells were removed. In other experiments we Imrali CD 34+cells after one-hour treatment with immunotoxin. In these experiments, we observed that tumor cells are destroyed or die within a short time after treatment, whereas tumor cells in the untreated control populations grew and formed colonies and/or proliferated, forming adhesive type cell cultures. Treatment had no effect on normal stem cells, so when testing three different systems viability of normal precursor cells was that they were only slightly reduced. Table 3 presents a similar experiment in which CD34+cells were incubated with immunotoxins for 2 hours at 37oWith, and it has been shown that stem cells are essentially survived handling immunotoxins.

A great surprise was that the use of two antibodies directed to antigens expressed by epithelial cells in accordance with the present invention, each of which was connected with the bacterial toxin is an exotoxin A of Pseudomonas, destroying cancer cells without any damaging effects on normal stem cells in the collected material from the peripheral blood and costner the Yan destruction increases when the binding of the toxin to the antibody, directed to epitopes expressed on target cells. However, it is also known that if the immature cells are exposed to one or more immunotoxins, there is a strong likelihood that this process will destroy the cell population of normal stem cells. Moreover, these normal stem cells sensitive to treatment ex vivo, accompanied by mechanical shocks and temperature changes. In the present invention the cell population, such as the transplant of stem cells collected from peripheral blood, exposed to the compositions of the two antibodies, each conjugated with PE. As one of immunotoxins was extremely active, unexpectedly it has been demonstrated that using a combination of two antibodies associated with bacterial toxin, the cleaning effect is greater than the sum of the effects, if immunotoxins are used separately. This sinergicheski effect is demonstrated in Table 4 of this opening using antibodies VM and MOS associated with exotoxin A of Pseudomonas, in respect of destruction RM of breast cancer cells human. Both immunotoxin are monoclonal the nom - the exotoxin A of Pseudomonas. One of the antibodies recognizes epithelial antigen encoded GA 733-2 gene, which is expressed by most cell carcinoma and, therefore, can be applied in all cases involving carcinomas (e.g., breast cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer and pancreatic cancer). Another antibody directed to the mucin, mucosal protein, which is slightly different from one type of cancer to another. In General, the antigen can be described as a complex of proteins encoded by genes MUC-1, MUC-2 and MUC-3. Examples of the above-mentioned antibodies are MOS and VM.

Conjugation of antibody and toxin can be done in different ways. The selection of one or more antibodies in the composition for binding to bacterial exotoxins was carried out in such a way that binding site of antibodies directed to epitopes expressed on the majority of target cells and neoexpressionist on normal cells. Previously, the problem in this area was that both cancer cells and normal blood cells Express the cell surface common antigens. In the example included in this opening, there are two mono is Auda in the peripheral blood, some are malignant. Antigen (whole protein) is encoded by gene GA 733-2. This antigen has, however, several epitopes, and it is important to choose the target of those epitopes that are most widely expressed.

Antibody VM represents one of the antibodies directed to an epitope of an antigen expressed by the MUCI gene. Several genes encode similar antigens, for example, (MUC2, MUC3).

Bacterial toxin Pseudomonas exotoxin a has a relatively moderate toxic effect on normal stem cells and cancer cells. However, when it binds with antibodies directed to antigens expressed on the target cells, toxic effects on them increases significantly. Table 5 demonstrates that the mixture of immunotoxins in accordance with the present invention, even after such a short time of incubation as 60 minutes, destroys T-47D cells, MCF7 cells and RM cells with a much higher degree of efficiency than other methods previously known. The combination of these two immunotoxins gives, therefore, unexpected results relative to what would be expected due to its electoral efficiency, simplicity, and only naznacite the use of multiple immunotoxins, consisting of exotoxin A of Pseudomonas conjugated with three different antibodies, see Myklebust et al., 1993 and 1994. One of the antibodies (MOS) was applied in a similar manner in the present invention, in order to clear the coming of bone marrow cells. However, the use of other antibodies does not seem optimal, among other things, because one of them is directed to the same antigen that MOS, and because other (MLuCl) has a cross-reaction with normal cells and therefore immunotoxin associated with this antibody can easily be toxic effect against the most immature stem cells. In the present invention on purified preparations of stem cells, we received another monoclonal antibody that enhances the effect MOS and found that the combination of these two antibodies used as immunotoxins, gives unexpected results.

Due to the high specific activity of open immunotoxins seems possible to introduce their mixture in the treatment in vivo of patients suffering from different types of carcinoma. If the cancer is limited, it will be possible to make an injection or infusion of each of them or their mixtures intravenously, for example, when the detection of the s one at a time or in combination to patients with the further spread of the disease, exciting abdominal fluid (ascites) or pleural fluid. A third option is to treat patients with the spread of cancer in the Central nervous system. In this case, immunotoxins can be injected directly into tumor tissue, cerebrospinal fluid or into the artery that supplies the brain with blood.

Do not know the use of these immunotoxins in vivo, except MO-RE, which was applied when leptomeningeal tumors as models for small cell lung cancer. VM-RE is not described in the literature in General.

An important problem when using immunotoxins in vivo is the fact that their half life is often very short, i.e. immunotoxin destroyed and removed from the blood before their concentration will be in tumors is high enough. US PS 5322678 Morgan et al. patented modified or antibody-based test part of immunotoxin, allowing to reduce the problem related to the short half-life in vivo. In the present invention serves a similar modification of the toxin part of the procedure, which was not previously running, and was not known.

Example 1

Effective relief from breast cancer cells is I dose chemoradiotherapy with supporting the introduction of autologous hematopoietic stem cells are used with increasing frequency for the treatment of patients with various forms of cancer (1, 2). In cases where this approach is unsuccessful, the most common cause is a relapse, not intoxication, infections and lack of engraftment of the graft (3). It is important that there is solid evidence that patients receiving treatment at high doses, reinfuse autotransplants containing clones of tumor cells, may contribute to relapse and to influence its outcome (4). Research cell autotransplants with the help of marker genes showed that the tumor cells remaining in reinfusion bone marrow (BM), contribute to the renewal of the disease (5). This conclusion is further supported by the results obtained in patients with follicular lymphoma, which show that efficient purification VM improves relapse-free period of life (6).

The use of sensitive immunocytochemical technique allows to detect contamination of tumor cells in histologically normal bone marrow autotransplants in patients with breast cancer were treated with high doses, the number 37-62% (7). The autotransplants stem cells perivertebral blood (PBSC) collected by afresa after preliminary licence these products will have a low probability of containing tumor cells. However, recently it was shown that, although the content of tumor cells in the PBSC autotransplants is less extensive compared with collections of VMS, the tumor cells are still often found in mobilized PBSC collections from patients with breast cancer (4, 7). In addition, recent data indicate that chemotherapy and/or growth factors can cause the tumor cells in the peripheral blood of patients, as with the original detected cancer cells in the bone marrow, and without their detection (7, 8), resulting in an additional increase in the risk of contamination PBSC transplants of tumor cells.

To avoid reinfused malignant cells in the PBSC autotransplants in carcinoma of the breast may need cleaning in vitro. Here we describe a practical and fast method of treatment, showing that the procedure incubation for 60 min with It (immunotoxins) directly added to the product of afresa selectively destroys more than 5 log of tumor cells.


Cell line. RM line of breast cancer cells was established in our laboratory from ascitic fluid taken from a patient with advanced illness who were cultured at 37oC in an atmosphere of 5% CO2in medium RPMI 1640 (RPMI) supplemented with 10% heated-inactivated serum fruits calves (FCS) and antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin). Medium and supplements were obtained from GIBCO (Paisley, UK).

Human bone marrow and precursor cells in the peripheral blood. The VM cells obtained from a healthy donor volunteers. The fraction of mononuclear cells (MNC) BM was obtained from Lymphoprep(Nycomed, Pharma, Oslo, Norway): and twice washed buffered saline (PBS) before use in experiments. PBSC were prepared, taking in patients with non-Hodgkin's lymphoma. To mobilize PBSC patients had previously received chemotherapy in combination with hematopoietic growth factors (G-CSF, Neupogen, Amgen/Hoffman-La Roche, Basel, Switzerland). After ten to twelve days after chemotherapy, when the number of CD34+-cells in the peripheral blood is high, the stem cells were collected from the CS-3000 plus separator blood cells (Baxter Healthcare Corp., Fenwal Division, Deerfield, IL).

Toxin antibodies and creating immunotoxins. Anti-MUCI (9) antibody VM (IgG1 was donated S. Kaul (Frauenklinik, University of Heidellberg, Germany), and anti-G2 (10) the antibody MOC-31 (IgG2a) was kindly provided by L. de Leij (University of Groningen, The Netherlands) and ACI Development is ostic, formed using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce, Rockford, IL) as described previously (11).

Treatment with immunotoxin. The effect of processing IT on the viability of forming clones of breast cancer cells was tested using incubation 2106exponential growing of tumor cells in RPMI with FCS with the specified concentrations IT at 37oWith mild agitation (100 rpm on an orbital incubator (Gallenkamp, Leicestershire, UK) for different periods of time, as indicated for each experiment. Cells are washed twice in PBS with 1% FCS prior to seeding to determine the formation of clones, in some experiments, 10% of tumor cells was mixed into it mononuclear cells BM or PBSC, incubated with IT, washed and determined the viability of tumor cells or forming clones of hematopoietic precursor cells.

Determination of the formation of colonies of tumor cells and hematopoietic cells predecessors. The applied definition of the ability of tumor cells to form clones in soft agar as described earlier (12). Three parallel samples of the culture were incubated for 14 days at 37oWith 5% CO25% OF O2and 90% Nto form clones treated and untreated normal precursor cells was evaluated using a CFU-GEMM method (13), in which 5104PBSC per ml were cultured individually in standard methylcellulose cultures (NCC-4433 Methocult, Terry Fox Labs, Vancouver, BC) in IMDM medium (GIBCO). 19 days of incubation BFU-E and CFU-GM colonies were counted with an inverted vasovasostomy microscope. Each determination was performed from three parallel cultures in 1 ml of 35 mm cups at 37oWith 5% CO2in the atmosphere of 100% humidity.


The growth of breast cancer cells human on soft agar. In several experiments we observed a linear relationship between the number of inoculated tumor cells and the number of formed colonies. Line RM cell efficiency education clones was in the range of from 20 to 30% (not shown). In experiments with T-47D and MCF7 cell lines were confirmed previously observed (14) linear relationship with D about 27% and 22%, respectively. These data were used to calculate the efficiency of reducing the number of breast cancer cells as a result of processing.

The effectiveness of individual immunotoxins and their mixtures in terms of destruction of breast cancer cells. In model experiments using three different concentrations of each of IT. As shown in TA is the time as 2.5 log of cell killing was achieved at a concentration of 1.0 µg/ml With MOC-31 at a concentration of 0.1 mg/ml was observed close to 3 log t cells at the highest concentration (1 µg/ml) efficiency was at least 5 log, the maximum effect that it is possible to determine if this method of analysis (14). Using the mixture of both IT, each at the indicated concentrations, all tumor cells are destroyed already at a concentration of 0.1 μg/ml (table 4). The results show that the mixture of two IT can destroy breast cancer cells very effectively, and the data also suggest that additivity can be obtained by the combination of two conjugates. Similar results were obtained when the efficiency of IT was tested when their negative effect on the other two cell lines breast cancer (not shown). Based on the expected heterogeneity in the expression of antigens on the target cells, it seems logical to use a combination of IT with the further development of a method suitable for clinical application.

PM1 cells were incubated with immunotoxins for 2 h at 37oWith, were sown on soft agar and identified the formed colonies, as described in "Materials and Methods".

The influence of the incubation time. In the above, experimento to be advantageous to use even shorter incubation time. To study whether to be reduced the exposure time with IT, without prejudice to destruction of tumor cells, a mixture of two conjugates used in a concentration of 1 µg/ml each, were tested in respect of actions against the three cell lines (breast cancer, using different time of incubation. In all cases, the 120 minute exposure time with IT led to the eradication of all tumor cells. It is essential that the treatment was just as effective when the incubation time was reduced to 90 minutes and even up to 60 min (table 5), and the data show that when applied concentrations IT the shortest incubation time sufficient to destroy all forming clones of tumor cells present in cultures of tumor cells.

Tumor cells alone (2x106/ml) or added (ratio 1:10) to the cells-the precursors of peripheral blood (total number 1x107/ml), incubated IT at 37oC for the indicated periods of time were sown on soft agao was defined as in table. 4.

To test whether the toxic effect on breast cancer cells to change in the presence of a large number of normal hematopoietic cells, was performed experiments in which outvie normal cells also destroy more than 5 log PM1 tumor cells already at the incubation time of only 60 minutes Since the results were similar for all three cell lines, the data show that the procedure of using IT can be effectively applied in the clinical setting.

The effect of incubation conditions and cell concentration. To test the effectiveness of treatments using IT in conditions similar to those that should be applied on clinical samples, RM tumor cells were entangled in the ratio of 1:10 to PBSC in experiments, when netmite cells taken directly from a bag of afresa, before incubation with IT resuspendable in normal saline with ACD (mother solution bag, 2220 R, Baxter Healthcare Corp., Fenwal Division). The results were comparable with that obtained in the initial experiments with cells that were washed and resuspendable in RPMI with 10% FCS. It was found (table 6) that in both cases, processing IT within 60 min destroys all RM cells, which indicates that the clinical application IT can be entered directly into the pouch afresa and that the low pH in these conditions does not affect the cytotoxicity of IT.

Cells from apartno bag was transferred into a three tubes containing 1x108cells each. Two tubes of cells (volumes 500-700 μl) rozvody the incubation with IT (group a). Cells in the third test tube was washed and resuspendable, bringing the volume up to 1 ml in RPMI with 10% FCS (Group B). In the groups exposed to the treatment, the cells were incubated at 37oC for 1 hour with 1 μg/ml each IT was cleaned and sown on soft agar. The definition and calculations as in table 1. The effectiveness of seeding in untreated control cultures was in the range of 20 - 30%.

In apparatnyh sacks total number of cells is high enough, and it was expected that at such high concentrations of cells of the effectiveness of the procedure may be reduced compared to the conditions used in the model experiments. However, in those experiments, when I tested this feature, no differences in effectiveness were not detected in the total cell concentration, increasing from source 1107up to 5107and then to 1108per ml (table 6).

The toxicity of IT with respect to the normal hematopoietic precursor cells. The effect of IT on the viability of CFU-GM and BFU-E were studied in the incubation conditions described above. It was found (table 1) that incubation with kernel PBSC with a mixture of IT even within 120 min did not reduce the viability of progenitor cells nor Kopecky, resuspendable in normal saline with ACD.

As for the clinical application of the treated cells will be frozen, followed by thawing before returning them to the patient, was studied also the effect of such procedures on precursor cells. It was found that freezing and thawing is only slightly reduce the number of CFU-GM and BFU-E (table 7). It should be noted that the processing of IT by itself did not reduce significantly the viability of progenitor cells, although there was a slight decrease in the average number of cell colonies in the group, where cells were treated at low pH. Data show that the concentration of IT, which effectively kills tumor cells after incubation for 60 min has only a minor effect on the survival of normal form clones of cells processed within two times longer time.


Autologous transplantation of circulating hematopoietic cells has recently attracted much attention due to their advantages compared with transplantation of BM (15, 16). In addition to the rapid recovery of bone marrow function assumes that the co was shown to the problem of contamination of tumor cells is reduced, but not eliminated (4). Also, it should be noted that the high dose chemotherapy, including the use of colony-stimulating factors, may cause the tumor cells in the peripheral blood (7, 8). Therefore, creating a fast and practical procedures for cleaning products afresa visokoobrazovna.

Describes several methods for removal of breast cancer cells from BM, including chemoimmunotherapy, immunomagnetic procedures and the use of immunotoxins(14, 17, 18, 19). In contrast, describes very little cleaning preparations PBSC (20, 21), although IT United with ribosome-inactivating protein, used for the destruction of lymphoid tumor cells, added to CD34-positive kletochnym collections obtained from BM (24). In a later study was obtained cleaning efficiency 2 log in addition to indirect clearing 3 log, achieved by using the procedure of selection of CD34-cells. The aim of this work was to develop reliable procedures for the allocation of breast cancer cells from PBSC. The results obtained in model experiments show that a 60-minute incubation with 1 μg/ml of each of the two conjugates without any toxic effect on normal precursor cells. It is essential that method allows you to add IT directly in the product afresa, and after incubation the cells are washed, centrifugeuse and ready for freezing. In particular, due to its simplicity and efficiency of the method should be attractive for use in the management of selected groups of patients with breast cancer in combination with high dose chemotherapy in combination with PBSC transplantation. Highly selective effectiveness of our procedures should be attributed to the following factors.

First, it is known that the antigen recognized MOS antibody is expressed on most cells in almost all the studied types of breast cancer (10). VM antibody, which recognizes cor-protein (apoprotein), expressed MUC-1 "gene" (9), is also associated with a large fraction of breast cancer cells (25). Together, these two monoclonal antibodies, apparently, largely overlapping the heterogeneity of expression of antigens detected in breast cancer. Secondly, we have previously shown that when designing IT is important to apply the toxin, which is combined with the antibody (11). We found that PE conjugates using a number of monoclonalantibody toxic, than equimolar concentrations of free D (11, 18), showing the specificity of such IT.

Require cleanup procedures were effective and reliable, it is also necessary that the method was practical and could be applied in the clinical scale. In addition to the advantage that IT can be added directly in aforizmy bag, our method involves only a 60-minute incubation to destroy all forming clones of tumor cells. Moreover, this treatment is not toxic to normal hematopoietic precursor cells, and in experiments on clean VM even much higher concentrations IT is well they are tolerated (14). We have also shown that freezing and thawing PBSC, processed IT, does not lead to any additional toxicity noteworthy also that the process of using IT does not lead to non-specific cell loss that the experience can be when you use methods involving the removal of tumor cells using immunogenum or immunoabsorption.

Previously, we estimate that the number of conjugate remaining in the treated BM IT after washing, is about 0.75% of the total added amount (26). In clinical at the Oia 2 µg 1T/ml (1108cells) should then lead, as expected, to the content in the final product as a 3 µg of IT. This is the amount of free toxin in 100 to 150 times smaller than theoretically calculated maximum dose tolerance (26). Thus, reinfuse purged PBSC should not be expected to show any systemic toxicity.

Unsuccessful therapy with high doses in combination with transplantation of autologous hematopoietic precursor cells may even more be determined by the efficacy of systemic treatment than cleaning efficiency grafts (1). Despite this, it is logical to remove malignant cells that must be present in the autograft, and recent evidence from studies of other types of tumors show the importance of such treatment (16). In breast cancer, we plan to apply simple, reliable and efficient procedure, which is the same as described here.

Example 2

Since breast cancer cells may be characterized by different sensitivity to immunotoxins so that the cleaning efficiency using VM and MOS may be different when using breast cancer cells from EP 1), was repeated with another cell line, MA. It was found that the effect of treatment immunotoxin was also good or even better in their effect on MA cells compared to the effect on RM cells (table 4). The results confirm the high specific activity treatment immunotoxins to clean.

In separate experiments, we studied the kinetics of activity immunotoxin in respect of destruction of cells in the experience, when RM of breast cancer cells was added to the cells-the precursors of peripheral blood (ratio 1:100). After incubation for 2 hours a mixed cell suspension was frozen and then thawed before cells were sown, and the viability of cancer cells and normal cells-precursors was determined in parallel experiments. It was found that the toxicity of breast cancer cells occurs rapidly, within about 72 hours, all tumor cells are killed. Compared to that found no differences in viability of normal precursor cells in cultures treated with immunotoxins and untreated cells in the same time frame.

Examples 3-4

Carcinoma cells, which also spread the urogenital tract, can be selectively destroyed by immunotoxins introduced into the tumor, fluid these body cavities or systemically, for example, in metastatic tumor cells in the target tissues such as blood, bone and bone marrow.

Example 3

MA-11 breast cancer cells human was injected into the left ventricle immunodeficient rats. Untreated control animals developed symptoms of compression of the spinal cord and they had to score through 34-37 days after injection of cells. In animals treated with intravenous single dose MOS-RE (20 μg/rat), revealed a long asymptomatic period of life, and some animals lived for over 50 days.

Another experiment using the same model confirmed these results, and in this case, some animals survived during the observation period the period of 110 days. In these experiments, one group of rats was treated with the immunotoxin comprising 425.3 antibody directed against the EGF receptor, conjugated with PE. All animals in this group survived.

In the third experiment with this model the control rats showed symptoms of compression of the spinal cord and it was necessary to score the kg 425.3-PE and one received 10 mg of each of the two immunotoxins. A significant increase in the duration of the asymptomatic period of life obtained with both immunotoxins used individually, giving 60% and 80% of the duration of survival with MO-RE and 425.3-PE, respectively. In experiments with a combination of all animals survived without disease symptoms.

In the fourth experiment with this model action MOS-D were compared with those of CIS-platinum and doxorubicin. In this experiment, all animals treated MOS-RE, survived for more than 70 days, while doxorubicin showed only a weak effect, and rats treated with CIS-platinum, lived no longer than control animals treated with saline. These data convincingly demonstrate that the applied immunotoxins are far ahead in respect of destruction of metastatic breast cancer compared with doxorubicin and CIS-platinum, the two drugs most commonly used in the clinic.

Example 4

MT-1 cell line breast cancer man was used in two different experiments. The first of these cells were injected into the left ventricle and control animals, it was necessary to score an average of 19 days for symptoms of compression of spintec, survived. In other experiments, MT-1 tumor cells were injected directly into the cavity of the tibia (tibial bones of rats containing bone marrow. All untreated animals had to score 20 days later due to the growth of the tibial tumors, whereas all rats treated by intravenous injection of 20 µg 425.3-PE through one day after injection of cells, lived for more than 100 days.

In addition, in this model, when MT-1 tumor cells were injected directly into the bone marrow of the tibial bone in rats, the effect VM-RE entered either on the first day or the seventh day, was compared with the effects 425.3-PE in groups of animals treated with it in those days, and that VM-RE. Moreover, we also analyzed the effect of doxorubicin (Adriamycin), administered intravenously on the 7th and 14th days. It was found that both immunotoxin was cured 80% of the rats was injected at the first or the seventh day. When we used the combination of the two immunotoxins with half concentration of each, all animals survived. Compared to the doxorubicin was clearly less effective, leaving alive only 35% of the animals after 90 days. Control animals had to score, as in previous experiments, che is the group of what VM-RE is as effective as 425.3-PE. Both agent efficiency actions have gone far ahead in comparison with doxorubicin, one of the drugs most frequently used in the treatment of breast cancer. Moreover, the combination of the two immunotoxins was cured from this disease all animals.

Example 5

Two types of experiments tested the effect obtained by recombinant method immunotoxin against product b2 gene, performed with recombinant method option RE. In the model described in Example 4, the highest concentration of recombinant immunotoxin significantly increased the life expectancy of animals, and 35% of rats survived. In the model, when MT-1 breast cancer cells were injected intrathecally (podvoloshino) immunodeficient rats, treatment with recombinant immunotoxin was also intrathecal (1st, 2nd and 3rd days), which led to a significant increase in life expectancy of animals. This effect was dose-dependent, and two different doses increased the lifespan of animals with 10.6 days (processed salt controls) to 23.4 days and 32.8 days after the use of two different doses of immunotoxin. At the highest doses is to function effectively at optimal doses may be even better.

1. The method of destruction of breast cancer cells or other carcinoma cells expressing the same antigens on the target cell population, comprising containing the nucleus of the cells collected from the peripheral blood, or CD34+cells selected from the above cells that have a nucleus or other immature/early precursor cells from the blood containing multipotent stem cells, wherein the cell population exhibiting a combination of the two immunotoxins, where each immunotoxin consists of a conjugate of antibody and cellular toxin fragments of an antibody and a toxin or recombinante obtained antibodies, toxins, immunotoxins or their fragments, and one of the antibodies, such as MOS, directed to the epitope of the antigen EGP2 expressed gene GA733, or one of the antibodies, such as 425.3, directed to the EGF receptor, and the other antibody, such as VM, directed to the epitope expressed genes MUC1, MUC2, or MUC3, or their combination, or another antibody, such as 425.3, directed to the EGF receptor, the toxin is an exotoxin A. Pseudomonas.

2. The method according to p. 1, characterized in that the antibodies used are MAC and VM aimed at antigenemia antibodies are MOS and VM or fragments thereof.

4. The method according to p. 1 or 2, characterized in that the antibodies used are MAC and 425.3 or fragments thereof.

5. The method according to p. 1 or 2, characterized in that the antibodies used are VM and 425.3 or fragments thereof.

6. The method according to p. 1, characterized in that the specific immunotoxins administered in vivo.

7. The method according to p. 6, characterized in that immunotoxins administered systemically, especially in the case of dissemination of malignant cells in tissues such as bone and bone marrow.

8. The method according to p. 6, characterized in that immunotoxins injected directly into the tumor or into the pleural or peritoneal cavity.

9. The preparation for the destruction of cells, characterized in that it contains a combination of two immunotoxins, where each immunotoxin consists of a conjugate of antibody and cellular toxin fragments of an antibody and a toxin or recombinante obtained antibodies, toxins of immunotoxins or their fragments, and one of the antibodies, such as MOS, directed to the epitope of the antigen EGP2 expressed gene GA733, or one of the antibodies, such as 425.3, directed to the EGF receptor, and the other antibody, such as VM, directed to the epitope expressed genes MUC1, MUC2, or MUC3 lactococci A. Pseudomonas.

10. The preparation for the destruction of cells by p. 9, wherein the antibody is selected from MO and VM and 425.3, or combinations thereof, or fragments, and the toxin is a native or recombinant exotoxin A. Pseudomonas or its fragments.

11. The drug under item 9 to obtain a therapeutic agent against cancer.

12. Set to perform the method according to p. 1, characterized in that it contains a combination of two immunotoxins, where each immunotoxin consists of a conjugate of antibody and cellular toxin fragments of an antibody and a toxin or recombinante obtained antibodies, toxins of immunotoxins or their fragments, and one of the antibodies, such as MOS, directed to the epitope of the antigen EGP2 expressed gene GA733, or one of the antibodies, such as 425.3, directed to the EGF receptor, and the other antibody, such as VM, directed to the epitope expressed genes MUC1, MUC2 or MUC3, or their combination, or another antibody, such as 425.3, directed to the EGF receptor, the toxin is an exotoxin A. Pseudomonas, in pharmaceutically acceptable form.


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