MXPA98007252A - Method to destroy cells objective does not wish - Google Patents

Abstract

A method for destroying unwanted target cells in a cell population comprising nucleated cells harvested from peripheral blood or CD-34 + cells or similar early progenitor cells from anterior nucleated cells or from bone marrow aspirates is described, method in which the population of cells is exposed in vitro or in vivo to 2 or more immunotoxins that selectively destroy the malignant cells. In addition, the invention relates to the mixture of immunotoxins, the use of the mixture and an equipment for performing the method

Description

METHOD TO DESTROY UNDESIRED OBJECTIVE CELLS DESCRIPTION OF THE INVENTION The present invention relates to the selective purging of a population of cells for target cells by exposing the population of cells to a combination of two immunotoxins. The so-called autologous pluripotent stem cell transplantation comprises cells isolated from blood or bone marrow of cancer patients which, after pretreatment. patients contain adequate amounts of immature blood and immune progenitor cells to restore in a short time the function of a bone marrow that does not work, or over a longer period of time after reinstillation of cells in the blood of patients who have been harvested the cells . Purging of autologous hematopoietic transplants using antibodies is known in the art when transplants represent samples of unselected bone marrow. The purge was published, for example, by Myklebust, A.T., Godal, A., Juell, S. and Fodstad, 0. "Comparison of two antibody-based methods for elimination of breast cancer cells from human bone marrow". Cancer Res. (USA) 1994, 54/1 (209-214) and Myklebust, AT, 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), 3784-88. Both publications use immunotoxins in which the antibody is conjugated to a toxin. The principle is to destroy malignant cells from harvested bone marrow cells before reinjection of the cell suspension in the patient. In recent years methods have been developed in which the principle is actually the opposite. Using what is called pluripotent stem cell transplantation, it is attempted to select positively from a subset of blood or bone marrow from normal cells which are capable of restoring normal bone marrow function after the cells are reinstalled in the patient. These "pluripotent cells" consist of a mixture of most of the immature precursors for blood and immune cells, and also more differentiated cells. The harvest of such cells can be performed either by what is termed afaresis of peripheral blood, a procedure that requires one or more days, or by immunoabsorption / selection of CD34 + cells (immature progenitor cells) of blood or bone marrow using different techniques known in the art. Stray, K.M. et al., "Purging tumor cells from bone marrow or blood using avidin, biotin, immuno adsorption" In: P.G. Adrián, G. Samuel and A. -. Diana (Eds), Advances in bone marrow purging and processing, pp. 97-103, Orlando: Willelis Inc., 1994 describes the purging of bone marrow cells in a peripheral blood afares product. This procedure is performed for purging 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, with what is called the avidin column. In this case, the purging is performed indirectly so that the cell suspension is initially incubated with primary antibodies which bind to the breast cancer cells, the cell suspension is washed and incubated once more with an antibody to which binds to the primary antibodies. This rat antibody is biotinylated, i.e., connected to a molecule which binds strongly to avidin. When this cell suspension is finally loaded onto a column with spheres conjugated with avidin, the tumor cells are trapped by the binding between the cells with primary antibody-biotinylated secondary antibody-avidin. The results of purging using such a system is at most a 3.2 log abatement of malignant cells. The principle is time consuming and problematic since the cell suspension must be managed in several stages including incubation with antibody and two washes before it is loaded onto the column. Therefore, it is difficult to avoid damage to pluripotent cells or there is an unspecific trapping of the pluripotent cells in the spine, which results in an unfortunate loss of cells crucial for the recovery of normal function of the bone marrow.

Tyer, C.L. et al., "Breast cancer cells are effectively purged from peripheral blood progenitor cells using an immunomagnetic technique". Excerpt from the first meeting of the International Society for Hematology and Graft Engineering, Orlando FL, 1993 describes an immunomagnetic method similar to one used in the publication of yklebust et al. before However, this method is used in peripheral blood cells. This principle is also completely different from the use of immunotoxins and the effectiveness of purging varies from a depression of 3.3 to 4.8 log for malignant cells in experimental models. The extract makes no mention of any additional use of an indirect system with incubation of primary antibodies followed by washing and re-incubation with spheres connected to antibodies which bind to the primary antibody, but this is a reasonable mption. In addition, this method comprises an additional and possibly traumatic treatment of normal cells and the procedure requires time. The effectiveness is limited and the extract does not mention anything about purging the population of CD34 + cells, which would be a major problem with this method, since the selection of CD34 + cells per se is time consuming and laborious. Therefore, in most cases an immunomagnetic principle is used for the selection of CD34 + cells, which in this example, is followed by one or two immunomagnetic steps for purging purposes. Therefore, there is a considerable risk of cell destruction and cell loss, with a method that requires a long-term procedure and involves high costs. Lemoli, R.M. et al. (1994) Bone Marrow Transplant 13: 465-471 describes the purification of human CD34 + hematopoietic cells using the avidin-biotin immunoabsorption technique. They increased the purging of neoplastic cells by using various immunotoxins that contain the inactivating protein ribosome saporin and directed towards lymphoid ciated antigens CD30 and CD2. Tecce, R. et al (1991) Int. J. Cancer, 49: 310-316 describes the purging of autologous bone marrow before transplantation in patients suffering from onocitic leukemia with two immunotoxins specific for the line of monocytic cells constructed with saporin and two highly specified monoclonal antibodies (MoAb) for circulating monocytes and acute non-lymphoid leukemia (ANLL) M5b. WO91 / 09058 discloses immunotoxins comprising MoAb 195 specific myelomonocytic useful for removing ANLL from bone marrow. Tonevitsky, A.G. et al (1986) Int. J. Cancer, 37: 263-273, describes the elimination of mouse erythroleukemic pluripotent cells from bone marrow using an immunotoxin comprising a ricin-chain A conjugate and MoAb MAE15 which binds to the Neoplastic normal mouse erythroid cell surface: a model for studies of bone marrow transplant therapy.

When isolating pluripotent cells for transplantation, one of the main objectives is that the cells which are re-inserted in patients are selectively isolated in such a way that the transplants do not contain any malignant cells. In the prior art it has been recently demonstrated that such preparations of pluripotent cells comprise, surprisingly, malignant cells in a significant number of the cases examined. Hitherto, very limited efforts have been made to eliminate or destroy selected malignant cells in such transplants. This is partly because a person familiar with the technique does not seem to have the need, and also because it has been expected that currently known methods are not specific and therefore would also destroy or remove vulnerable pluripotent cells. In addition, a supply of bone marrow or peripheral blood mobilized from patients is not simple and unlimited, and such method of elimination of pluripotent stem transplants should be performed within a short period of time and should not be complicated in order to avoid losses or damage to normal cells. Therefore, with reference to the above, the reason for using pluripotent stem cell transplants is in part that the transplant must be completely free of cancer cells, partially this reconstitution of the bone marrow function is faster than after the transplant with bone marrow not selected. It follows that it is absolutely necessary to invent a method which leaves normal and fragile pluripotent cells intact, and which is practical to perform in combination with the procedures for the isolation of pluripotent cells. The object of the present invention therefore is to provide a method for purging in pluripotent stem transplants, which does not involve the above disadvantages. Such objects are obtained by the present invention characterized by the appended claims. The present invention relates to the purging of populations of pluripotent cells collected in cases of solid tumors, a method in which the population of cells is exposed to a composition of two or more antibodies connected to bacterial toxins. The antibodies used are directed to antigens associated with the target cell. In the following, the invention will be described in greater detail by using an example of removing transplants from pluripotent cells harvested from peripheral blood to remove breast cancer cells. Known techniques for collecting cells comprise the immunoadsorption / selection of peripheral blood pluripotential cells (PBSC) or CD-34 + cells from blood or bone marrow. However, currently there are no sufficiently innocuous and effective enough techniques to eliminate the tumor cells of these tumor populations. It is considered evident that even among these immature cells there are malignant cells which, according to previous knowledge, do not possess CD34 receptors. Importantly, the invention described below also surprisingly eliminates cancer cells without toxicity to normal progenitors. Before harvesting a pluripotent peripheral blood cell transplant, it is necessary to mobilize the stem cells from the bone marrow using chemotherapy or treatment with growth factors by methods known in the art. The harvesting of pluripotent cells can be carried out according to one or several methods, based on the type of cells desired. In a In this method, peripheral blood pluripotential cells are collected. This can be done by establishing protocols in patients to experience leukapheresis on days 10 and 11 of the administration of G-CSF (10 ug / kg / day) after receiving high doses of chemotherapy and irradiation of full body. The blood flow rate can be set, for example at 70 ml / min using a CS-300 Plus blood cell separator (Baxter Healthcare Corporation, Fenwal Division, Deerfield, IL, USA). The average volume of blood treated during such procedure can be approximately 10 liters for 2.5 hours to a central venous catheter in the double lumen. 15 ml of PBSC can be collected and washed with phosphate buffered saline (PBS), 1% human serum albumin (HSA) in a 2991 Cobe Processor for purging platelets. For use in the present invention, the cell concentration (2-4 x 10 10 / afaresis) can be regulated to a 1 x 108 / ml space for negative selection (purged) with immunotoxins. If CD-34 + cells are desired, it can be carried out by positive selection with ISOLEX 50MR or ISOLEX 300MR (Baxter). In this method, the product of bone marrow afaresis, which can be from about 4 x 10 10 to 6 x 10 10 cells can be mixed together and incubated with, for example, monoclonal antibody against CD34 + 9C5 at 0.5 μg / 1 x 10 6 cells , at 4 ° C for 30 minutes in a low speed centrifuge. The treated cells are washed with PBS with 1% HSA in Cobe Processor to remove unbound antibody. DYNALME M-450 spheres are added to the CD34 + fractions, at 0.5 spheres per 1 nucleated cell at 4 ° C for 30 minutes. Magnetic separation of non-target cell rosettes can be performed by washing out unbound cells two or three times with PBS, 1% HSA. Then, CD34 + cells can be released from the Dynal ™ spheres by adding, for example ChymoCell-R (Chymopapain) to a final concentration of 200 pKat / ml in 15 minutes at room temperature. Therefore, CD34 + cells can be harvested by washing with PBS in 5% sodium citrate. In addition, other methods for selecting pluripotenial cells / early progenitor cells are known. The binding profile of the various antibodies in the breast cancer cell lines and the tumor materials has been examined by other investigators and partially confirmed by us. Antibodies which bind to a large percentage of breast cancer cells and not to immature normal cells important in blood and bone marrow, were conjugated to Pseudomonas exotoxin A (PE) and examined the ability to kill cancer cells of breast in culture, mainly in trials producing colonies. Based on the binding profile of the antibodies, the present inventors produced five different immunotoxins: 1. MOC31-PE: This conjugate binds in a large percentage to all breast cancer cells and is very effective in experimental models and in the actual concentrations with marginal toxicity to normal hematopoietic progenitor cells. 2. NrLulO-PE: This binds to the same antigen as the previous one, but to another epitope. It is slightly less active than M0C31-PE. 3. BM7-PE: This binds to part of the protein of a mucin antigen which is found mainly in breast cancer cells. The antigen is present in a large percentage of breast cancer cells, but not in all.

The immunotoxin shows a highly specific activity by cancer cells, but it is not as effective as the two previous immunotoxins. 4. BM2-PE: This binds to an epitope that contains sugar in the same antigen as BM7-PE. The immunotoxin shows approximately the same effectiveness as BM7-PE, concomitant with a very low toxicity for normal cells. 5. MLuCl-PE: This binds to a totally different antigen, the Lewisy antigen. The immunotoxin is slightly less active than the previous ones and also shows a moderate toxicity to normal cells. The immunotoxins according to 1, 2 and 5 were tested individually and in combination in experimental models of elimination of regular bone marrow samples for cancer cells (Myklebust et al., Cancer Research, 1994). As already mentioned, it is a great advantage to use pluripotent stem cell transplants because of the shorter interval to regain normal bone marrow function (ie, a safer procedure). However, despite what was expected, such transplants are contaminated with tumor cells and it was necessary to apply immunotoxins capable of destroying all the cancer cells in the transplant without significantly affecting the normal cells.

The search was initiated for more specific immunotoxins for breast cancer compared to MLuCl-PE and more suitable for eliminating breast cancer. During this investigation, it was surprisingly discovered that the combination of M0C31-PE and BM7-PE is more effective than the sum of each of the previous immunotoxins used alone. This is demonstrated in Table 1 below. Additional studies of binding between the antibody and the cell lines have shown that the combination results in a stronger binding than the antibodies alone. MOC31 binds to most of the breast cancer cells, in addition to those which are less differentiated.

BM7 recognizes a mucin antigen which is expressed in a considerable fraction of breast cancer cells, also on the cells which are more differentiated. NrLulO and BM2 bind to the antigen recognized by M0C31 and BM7, respectively, and with this in mind, it was not very likely that they would add anything to a combination of immunotoxins M0C31 and BM7. MLuCl-PE was theoretically interesting in that it binds to > an antigen different from the immunotoxins mentioned above. However, MLuCl-PE is toxic to normal cells and the experimental models showed no clear advantage when included in the combination. In the following example, the purging of transplants of peripheral pluripotent cells (afaresis products) is described. In addition, the inventors have developed several experiments using the combination of M0C31-PE and BM7-PE in experiments where tumor cells are added to peripheral pluripotent cells harvested or to bone marrow before a positive selection of CD-34 + immunomagnetic cells. The results of each such experiment are shown in Table 2. With two different cell lines, it is shown that the positive selection of CD34 + cells in itself (without any other form of purging) removes up to 3.8 log tumor cells from the population of original harvested cells. In other experiments, the "purge" effect of the CD34 selection varies from 2-3 log to which is also referenced in the literature. When the immunotoxin treatment was used in a positively selected CD34 population, the total purge effect was greater than 4.7 log (Table 2) for both cell lines. More than 4.7 log means in this case that all the detectable tumor cells were removed. In other experiments we have grown in separate assays, tumor cells and normal progenitor cells taken from a CD34 + population after one hour of immunotoxin treatment. In these experiments we have observed that the tumor cells are destroyed or dried shortly after treatment, while the tumor cells in the non-eliminated control populations grow and create colonies and / or proliferate in cell type adhesion cultures. Normal pluripotent cells are not influenced by the treatment so that in the three different test systems, the survival of normal progenitor cells is only significantly reduced. Table 3 shows a similar experiment in which CD34 + cells are incubated with the immunotoxins for 2 hours at 37 ° C, and it is shown that pluripotent cells essentially survive treatment with immunotoxins.

Table 1 Effect of immunotoxins involving PE in the description of breast cancer cells PM1 Table 2 Purging effects of positive immunomagnetic selection of CD34 + alone and combined with immunotoxin M0C31 and BM7 against breast carcinoma, in experimental models with breast cancer cells PM1 and T-47D mixed (1%) to peripheral blood pluripotential cells. s > to. Calculated from the observed number of colonies, taking into consideration the efficiency of plating the number of cells destroyed by the treatment. b. Average of the results obtained in independent experiments, each carried out in triplicate. c. The immunotoxins M0C31-PE and BM7-PE were used at a concentration of 1 μg / ml of each.

Table 3 Effect of IT on the survival of colonies in CD34 + cells selected from mobilized peripheral blood. 1 x 10B CD34 + cells were incubated with the immunotoxins for 2 h at 37 ° C, seeded in CFU-GM and CFU-GEMM cultures (5 x 103 / well) and assayed as described in "Materials and methods" in Example . oo It was very surprising that the use of two antibodies, directed to antigens expressed by epithelial cells according to the invention, each combined with the bacterial toxin Pseudomonas exotoxin A, destroyed malignant cells without causing any damage to normal pluripotent cells in the harvested peripheral blood and bone marrow. It is known in the art that cells can be destroyed by bacterial exotoxins and that the destructive effect is increased by connecting the toxin to antibodies directed to epitopes expressed by the target cells. However, it is also known that immature cells are exposed to one or more immunotoxins and that there is a great possibility that this treatment will destroy normal pluripotent cells in the cell population. In addition, these normal pluripotent cells are sensitive to ex vivo treatment that accompanies mechanical traumas and temperature changes. In the present invention, the cell population, for example a transplantation of pluripotent cells harvested from peripheral blood, is exposed to a composition of two antibodies, each conjugated to PE. Since one of the immunotoxins is excessively active, it is surprisingly demonstrated that, by using two antibodies connected to a bacterial toxin, the elimination effect appears to be greater than the sum of the effects when the immunotoxins are used separately. This synergistic effect is demonstrated in Table 4 of the present disclosure using antibodies BM7 and MOC31 connected to Pseudomonas exotoxin A by killing human breast cancer cells PM1. Immunotoxins are monoclonal antibodies directed against tumor-associated antigens connected to the bacterial toxin exotoxin A of Pseudomonas. One of the antibodies recognizes an epithelial antigen encoded by the gene GA 733-2, which expresses most of the carcinoma cells and therefore can be used in all cases involving carcinomas (for example breast cancer, cancer rectal colon, prostate cancer, ovarian cancer, lung cancer and pancreatic cancer). The other antibody is targeted at mucin, a mucosal protein which is slightly different from one type of carcinoma to the other. Commonly, the antigen can be described as proteins encoded by the MUC-1, MUC-2 and MUC-3 genes. Examples of monoclonal antibodies mentioned above are MOC31 and BM7. The conjugation of antibody and toxin can be performed in different known ways. The selection of two or more antibodies in the composition to bind them to bacterial exotoxins is performed in such a way that the antibody binding is directed to epitopes expressed in most of the target cells and not on normal cells. The problem in the prior art is that both malignant cells and normal blood cells express common antigens on the cell surface. In the example included in the present description, the two monoclonal antibodies M0C31 and BM7 are used. The first of these antibodies is directed to epithelial cells which are found in the peripheral blood and which are malignant. The antigen (the complete protein) is encoded by the GA 5 733-2 gene. However, this antigen has several epitopes and it is important to direct the epitopes that are expressed more abundantly. The BM7 antibody is one of the antibodies directed to an epitope of the antigen expressed by the MUC1 gene. Several genes 0 code for similar antigens, for example (MUC2, MUC3). The bacterial toxin exotoxin A of Pseudomonas has a relatively moderate toxic effect on normal pluripotent cells and malignant cells. Nevertheless, when they are connected to antibodies directed to antigens expressed in 5 target cells, the toxic effect of these is very pronounced. Table 5 shows that the immunotoxin mixture according to the invention, even after an incubation time as small as 60 minutes, destroys T-47 D cells, MCF7 cells and PM1 cells at a much higher efficiency level. than that known in the prior art with other methods. Therefore, the combination of these two immunotoxins provides surprising results in relation to what would be anticipated due to selective efficacy, simplicity, and only marginal toxicity to normal progenitor cells.

It has been stated that the use of several immunotoxins consisting of Pseudomonas exotoxin A conjugated to three different antibodies is known, see Myklebust et al. 1993 and 1994. One of the antibodies (M0C31) was used in a manner similar to the present invention in order to eliminate unselected bone marrow cells. However, the other antibodies used do not seem to be optimal, among other things because one of them is directed to the same antigen as M0C31 and because the other (MLuCl) cross-reacts with normal cells and therefore the bound immunotoxin This antibody can easily be toxic to most pluripotent and mature cells. In the present invention, in the preparations of pluripotent cells subjected to purging, we have prepared another monoclonal antibody which adds to the effect of MOC31 and it is observed that the combination of these two antibodies used as immunotoxins provides profoundly surprising results. Due to the high specific activity of the immunotoxins described, it is possible to administer the mixture for in vivo treatment of patients suffering from different types of carcinoma. If the cancer disease is limited, it will be possible to inject or infuse each or the mixture intravenously, for example, when the spread of the disease to the bone marrow is demonstrated. It is also possible to inject the immunotoxins alone or in combination in patients with additional dissemination of the disease with abdominal fluid (ascites) or with pleural effusion. A third possibility is to treat patients with cancer spread to the central nervous system. In this case, the immunotoxins can be injected directly into the tumor tissue, into the spinal fluid or into the artery that supplies blood to the brain. The use of these immunotoxins in vivo is not known, except that M0C31-PE has been used in a leptomeningeal tumor model for small cell lung cancer. BM7-PE has not been described in the literature in any way. A major problem in using immunotoxins in vivo is that their half-lives are often very short, ie the immunotoxins are broken down and removed from the blood before their concentration is adequately elevated in the tumor. In US Pat. No. 5,322,678, Morgan et al. they have patented a modification of the antibody part of an immunotoxin in order to reduce the problem of a short average duration in vivo. The present inventor suggests a similar modification of the toxin part, a product not produced or previously known.

Use 1 Effective purging of breast cancer cells harvested from peripheral blood pluripotent cells with immunotoxins.

Introduction High-dose chemoradiation therapy with autologous hematopoietic stem cell support has been used increasingly frequently to treat patients with various malignant cancers (1,2). In cases in which this approach is unsuccessful, the most common reason is the relapse of the disease, instead of toxicity, infections and lack of grafts (3). Importantly, there is strong evidence that in patients receiving high-dose treatment, reinfusion of autografts containing clonogenic tumor cells may contribute to relapse and may influence the outcome (4). Gene tagging studies of autografted cells have indicated that tumor cells remaining in bone marrow (BM) administered again contribute to the recurrence of the disease (5). This conclusion is further supported by the results in patients with follicular lymphocytes, which indicate that efficient elimination of BM improves disease-free survival (6).

By using sensitive immunophitochemical techniques, the contamination of tumor cells in histologically normal bone marrow autografts can be observed, in the 37-62% range of breast cancer that undergo treatment with high doses (7). Autografts of peripheral blood pluripotent cells (PBSC) collected by afares after pretreatment with hematopoietic growth factors and chemotherapy have increasingly been used in the belief that these products will have a low probability of containing tumor cells. However, it has recently been found that although the tumor cell ratio is less extensive in PBSC autografts compared to BM harvested, malignant cells are still frequently found in PBSC collections mobilized from breast cancer patients (4, 7) . In addition, recent findings show that chemotherapy and / or growth factors can mobilize tumor cells in peripheral blood in patients with and without detectable cancer cells in bone marrow (7, 8), results that further increase the risk of contamination by tumor cells in PBSC grafts. To prevent reinfusion of malignant cells, in vitro purging of PBSC autografts from breast carcinoma cells may be necessary. Here we report a practical and rapid purging method, which shows that a 60-min incubation procedure with IT added directly to the afaresis product selectively destroys more than 5 log of tumor cells.

MATERIALS AND METHODS Cellphone line. The breast cancer cell line PM1 was established in our laboratory from ascitic fluid extracted from a patient with advanced disease. The MCF7 and T-47D cell lines were obtained from the American Type Culture Collection (Rockville, MD) (ATCC HTB 22 and ATCC HTB 133 respectively). Cells were cultured at 37 ° C in a 5% C02 atmosphere in RPMI 1640 medium (RPMI) supplemented with 10% heat inactivated fetal bovine serum (FCS) and antibiotics (100 U / ml penicillin, 100 μg streptomycin). The medium and supplements were purchased from GIBCO (Paisley, United Kingdom).

Human bone marrow and peripheral blood progenitor cells. The BM cells were obtained from healthy voluntary donors. The fraction of BM mononuclear cells (MNC) is obtained by Lynfoprep ™ (Nycomed Pharma, Oslo, Norway) and washed twice in phosphate-buffered saline (PBS) before being used in the experiments. PBSC was prepared from patients without Hodgkin lymphoma. To mobilize PBSC, patients were treated with chemotherapy plus hematopoietic growth factors (G-CSF, Neupogen, Amgen / Hoffman-La Roche, Basel, Switzerland). At 11 to 12 days after chemotherapy, when the number of CD34 + cells in the peripheral blood is high, pluripotential cells were collected by using a CS-3000 Plus blood cell separator (Baxter Healthcare Corp., Fenwal Division, Deerfield, IL).

Toxin, antibodies and immunotoxin construction. The antibody BM7 (IgGl) against MUC1 (9) was a gift from S. Kaul (Frauenklinik, University of Heidelberg, Germany). And the antibody MOC-31 (IgG2a) against EGP2 (10) was kindly provided by L. de Leij (University of Groningen, Netherlands), and by MCA Development (Groningen). PE was obtained from Swiss Serum and Vaccine Institute (Bern, Switzerland). Each antibody was conjugated to PE by means of a thioether bond formed with sulfo-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce, Rockford, IL) as previously described (11).

Treatment with immunotoxin. The effect of IT treatment on clonogenic breast cancer survival was tested by incubating 2 x 10s tumor cells that grow exponentially in RPMI with FCS, with the indicated concentrations of IT at 37 ° C, with gentle shaking (100 rpm in a Orbital incubator (Gallenkamp, Leicestershire, United Kingdom)) for varying periods of time, as indicated for each experiment. The cells were washed twice in PBS with 1% FCS before being seeded in the clonogenic assay. In some experiments, 10% of cells were mixed tumors to BM or PBSC mononuclear cells, incubated with IT, washed and the effect was determined for tumor cells or for the survival of hematopoietic progenitor clonogenic cells.

Colony assays for hematopoietic tumor and progenitor cells. The soft clonogenic agar assay for tumor cells used has been previously described (12) Cultures were incubated in triplicate for 14 days at 37CC in 5% C02, 5% 02 and 90% N2, and colonies over 50 cells were counted in a Zeiss stereomicroscope. The clonogenic capacity of treated and normal untreated progenitor cells was determined in CFU-GEMM assays (13) in which 5 x 104 PBSC per ml were individually cultured in standard methyl cellulose cultures (HCC-4433 Methocult, Terry Fox Labs, Vancouver, BC) in IMDM medium (GIBCO). After 19 days of incubation, the BFU-E and CFU-GM colonies were counted in a reverse phase contrast microscope. Each assay was performed with cultures in triplicate in 1 ml, the container is 35 mm at 37 ° C in a 100% humidified atmosphere, with 5% C02.

RESULTS Growth of human breast cancer cells on soft agar. In several experiments, a linear relationship was observed between the amount of thin tumor cells and the number of tumor colonies formed. With the PM1 cell line, the cloning efficiency was in the range of 20 to 30% (not shown). In experiments with T-47D and MCF7 cell lines, the previously reported linear relationships (14) with PE of 27% and 22%, respectively, were confirmed. These data were used to efficiently calculate the suppression of breast cancer cells obtained with the treatment.

Efficacy of the individual immunotoxins and in a mixture, for the destruction of breast cancer cells. In experimental models, three different concentrations of each IT were used. As shown in Table 4, only marginal effects were obtained with the BM7 conjugate at the two lower concentrations, while 2.5 log of cell destruction was reached at 1.0 μg / ml close to 3 log cell destruction observed, and at the concentration Higher (1 μg / ml) efficacy was at least 5 log, the maximum possible effect to be determined in this trial (14). With a mixture of both ITs, each at the indicated concentrations, all the tumor cells were destroyed at 0.1 μg / ml (Table 4). The results show that the mixture of two ITs can destroy breast cancer cells very efficiently, and the data also suggest that an additive condition can be obtained by combining the two conjugates. Similar results are obtained when testing the effectiveness of IT against the other two breast cancer cell lines (not shown). Due to the expected heterogeneity in the expression of antigens on the target cells, it is considered logical to use the combination of IT in the further development of a method suitable for clinical use.

Table 4 Efficacy of immunotoxins involving Pseudomonas exotoxin A in the destruction of human breast cancer cells PM1. PM1 cells were incubated with immunotoxins for 2 h at 37 ° C, plated on soft agar, and colony formation was determined as described in "Materials and Methods". co Calculated from the observed number of colonies, taking into account the efficiency of plating, and determined as the logarithm of the number of cells inactivated by the treatment. b Average of the results obtained in independent experiments of soft agar, each carried out in triplicate. c Each immunotoxin is used at the indicated concentration.

Influence of incubation time. In the previous experiments, an incubation time of 120 min was used with IT. In a clinical setting it would be advantageous, for practical reasons, to use an even shorter incubation time. To study whether the time of exposure to IT can be reduced without affecting the specific destruction of tumor cells, the mixture of the two conjugates, used at a concentration of 1 μg / ml each, was tested with different incubation time against three breast cancer cell lines. In all cases, a 120 min exposure to IT resulted in the eradication of all tumor cells. Importantly, the treatment was equally effective when the incubation time was reduced to 90 min and even to 60 min (Table 5) and the data show that at the IT concentrations used, the shorter incubation time is sufficient to destroy the totality of clonogenic tumor cells present in the cultures of tumor cells.

Table 5 Influence of incubation time on the efficacy of a mixture of immunotoxins3 MOC-31 and BM7 in the destruction of breast cancer cells. Tumor cells alone (2 x 10s / ml) or mixed (1:10 ratio) to peripheral blood progenitor cells (total, 1 x 10 7 cells / ml) were incubated with IT at 37 ° C for the indicated periods of time, they were seeded on soft agar and tested as in Table 4. ○ Each IT used at a concentration of 1 μg / ml b Result obtained in two independent experiments with T-47D and PM1 cells and in an experiment with MCF7 cells.

To examine whether the toxicity to breast cancer cells can be altered in the presence of a large number of normal hematopoietic cells, experiments were performed in which tumor cells were mixed in a 1:10 ratio with PBPC collected by afaresis. As demonstrated in the Table 5, ITs destroy more than 5 logs of PM1 tumor cells also in the presence of normal cells, already after an incubation time after 60 min. Since the results were the same for all three cell lines, the data indicate that the IT procedure can be effectively used in a clinical system.

Influence of incubation conditions and cell concentration. To examine the efficacy of the IT procedure under conditions similar to those used in clinical samples, PM1 tumor cells were mixed in a ratio of 1:10 with PBPC with experiments in which unwashed cells taken directly from the afaresis bag were resuspended before of incubation with IT, in a normal saline solution with ACD (concentrated solution bag, R 2220, Baxter Healthcare Corp., Fenwal Division). The results were compared with those obtained in the initial experiments with cells that were washed and resuspended in RPMI with 10% FCS. It was found (Table 6) that in both cases, treatment with IT for 60 min destroys all of the PMl cells, which indicates that for clinical use, it can be injected directly into the afaresis bag, and that the pH decrease under such conditions does not affect the cytotoxicity of IT.

Table 6 Effect of incubation conditions on the efficacy of a mixture of immunotoxins MOC-31 and BM7 in the destruction of mixed breast cancer cells PMl (ratio, 1:10) with peripheral blood progenitor cells isolated by separation afaresis of blood cells.

The cells of the afaresis pouch were placed in three tubes, each containing 1 x 108 cells. In two of the tubes, the cells were diluted (in volumes of 500-700 μl) in PBS with 20% ACD and 1% human albumin to a final volume of 1 ml. One of the "tubes was used as control and for IT incubation (Group A). Cells in the third tube were washed and resuspended to 1 ml in RPMI with 10% FCS (Group B). In the treatment groups, the cells were incubated at 37 ° C for 1 h with 1 μg / ml of each IT, washed and plated on soft agar - the assay and calculations are as in Table 1. Efficiency of planting in untreated control cultures it is in the range of 20% -30%.

J In the afaresis pocket, the total number of cells will be too high and it is conceivable that at such high cell concentrations, the efficiency of the procedure may be reduced compared to the conditions used in the model studies. However, in experiments where this possibility was tested, no difference in efficacy was observed when the total cell concentration was first increased from 1 × 10 7 to 5 × 10 7, and then to 1 × 10 8 per ml (Table 6).

Toxicity of IT to normal hematopoietic progenitor cells. The effect of IT on the survival of CFU-GM and BFU-E under incubation conditions was studied as described above. It was found (Table 7) that even at 120 minutes of incubation of PBPC nucleated with the IT mixture the survival of the progenitor cells was not reduced, either tested after washing and resuspension of the cells in RPMI with 10% FCS, or when the unwashed cells were resuspended in normal saline with ACD. Since in a clinical system the treated cells will be frozen and reheated before being administered to the patient again, the effect of such procedures on the progenitor cells was also studied. It was found that freezing and rewarming reduced only slightly the number of CFU-GM and BFU-E (Table 7). Notably, treatment with IT in itself does not significantly reduce progenitor cell survival, although a slight reduction in the average number of cell colonies is observed in the group in which the cells have been treated under low pH conditions. The data demonstrate that the concentration of IT that effectively eradicates tumor cells after 60 minutes of incubation has only a negligible effect on the survival of normal clonogenic cells treated twice in this manner.

Table 7 Toxicity of a mixture of immunotoxins MOC-31 and BM7 to CFU-GM and BFU-E in fresh human PBPC harvested after mobilization with G-CSF. Effect of incubation conditions and freezing procedure. The control and treatment groups are as in Table 6. Nucleated PBPCs were incubated with 1 μg / ml of each of the IT for 2 h at 37 ° C before seeding (5 x 10 4 cells / vessel) in the assay as described in "Materials and Methods". or The mean ± SD of the results is obtained from cultures in triplicate in two individual experiments DISCUSSION Autologous transplantation of circulating hematopoietic stem cells has recently attracted considerable interest due to its advantages compared to BM transplantation (15, 16). In addition to the rapid reconstitution of bone marrow function, it has been assumed that the use of PBSCs can eliminate the risk of reinfusing tumor cells that contaminate the transplant. However, it has been shown that the problem of contamination of tumor cells is reduced, but not eliminated (4). It should also be noted that high-dose chemotherapy involving the use of colony-stimulating factors can recruit tumor cells in the peripheral blood (7, 8). Therefore, a quick and practical procedure to purge afares products is very sought after. Several methods have been reported to remove BM breast cancer cells, including chemoimmunosuppression, immunomagnetic procedures and the use of immunotoxins (14, 17, 18, 19). In contrast, only very few studies have been described regarding the purging of PBSC preparations (20, 21), but the IT prepared with an inactivating ribosome protein. (22, 23) have been used to destroy lymphoid tumor cells added to collections of CD34-positive cells prepared from BM (24). In this last study, a purge efficiency of 2 log was obtained in addition to an indirect purification of 3 log obtained by the CD34 selection procedure. The objective of the present study was to develop a safe IT procedure to purge breast cancer cells from PBSCs. The results obtained in experimental models demonstrate that 60 min of incubation with 1 μg / ml of each of the two conjugates that involves antibodies against carcinoma and PE, efficiently destroys all the tumor cells mixed with the PBSC without toxicity to the normal progenitor cells. Importantly, the method allows IT to be added directly to the afares product and after incubation, the cells are washed, centrifuged and ready for freezing. Particularly, due to its simplicity and effectiveness, the method is attractive for use in the management of selected groups of breast cancer patients together with high-dose chemotherapy combined with PBSC transplantation. The high selective efficiency of our procedure can be attributed to the following factors: Firstly, it is known that the antigen recognized by the M0C31 antibody is expressed on most of the cells in almost all breast cancer samples examined (10). ). In addition, the BM7 antibody, which recognizes the core protein expressed by the MUC-1 gene (9), binds to a high fraction of breast cancer cells (25). Together, these two monoclonal antibodies seem to cover, to a reasonable extent, the heterogeneity in antigen expression found in breast cancer. Second, we have previously shown that, when constructing IT, it is important to use a toxin that matches the antibodies used (11). We have found that PE conjugates that involve several of the monoclonal antibodies that include those that were used here are very effective (14). In addition, IT with PE are always more toxic than equimolar concentrations of free PE (11, 18), which demonstrates the specificity of such IT. Purging procedures need to be efficient and safe, and it is also necessary that the method is practical and can be used on a clinical scale. In addition to the advantage that IT can be added directly to the afaresis pocket, our method includes an incubation time of only 60 min to destroy all of the clonogenic tumor cells. In addition, this treatment is not toxic to normal hematopoietic progenitor cells, and in BM purging experiments, even much higher concentrations of IT are well tolerated (14). We have also found that freezing and rewarming of PBSCs treated with IT does not cause additional toxicity, and it is notable that the IT procedure does not involve the loss of nonspecific cells that can be experienced with methods involving the removal of tumor cells with immunospheres or immunosorption . We have previously calculated the amount of conjugate that remains in BM treated with IT after washing, which is about 0.75% of the total amount added (26). In a clinical system, the treatment of PBSC containing approximately 2 x 1010 mononuclear cells, with the recommended concentration of 2 ug IT / ml (1 x 108 cells), can then be expected to result in a maximum of 3 μg of IT. in the final product. This represents 100-150 times less free toxin than the theoretically calculated maximum of the tolerable dose (26). Therefore, reinfusion of purged PBSCs should not be expected to provide any systemic toxicity. The success or failure of high-dose therapy combined with the transplantation of autologous hematopoietic progenitor cells may depend even more on the efficacy of the systemic treatment than on the purging efficiency of the grafts (1). However, it is logical to remove cancer cells that may be present in the autograft, and recent evidence from studies of other types of tumor demonstrates the importance of purging (16). In breast cancer, we suggest using a simple, safe and effective procedure like the one described here.

Example 2 Since the breast cancer cells may have different sensitivity to immunotoxins, so that the purging activity of BM7 and M0C31 may be different in breast cancer cells from different patients, the same experiments were repeated as previously performed with cells of breast cancer PMl (Example 1) with another cell line, MAll. It was found that the effect of the immunotoxin treatment is good, or even better, against MAll cells compared to PMl cells (Table 4). The results confirm the high specific activity of the purging treatment with immunotoxin. In separate experiments, the kinetics of the killing activity of immunotoxin cells was studied in an experiment where PMl breast cancer cells were added to peripheral blood progenitor cells (ratio, 1: 100). After incubation for 2 hours, the suspension of mixed cells was frozen and subsequently reheated before the cells were seeded, and the viability of normal cancer cells and progenitor cells was determined in parallel experiments. It was found that intoxication of breast cancer cells occurs rapidly, within approximately 72 hours all the cells were dead. In comparison, no difference in the viability of normal blood progenitors was found in cultures of cells treated or not treated with immunotoxin within the same time interval.

Examples 3-4 5 Carcinoma cells that disseminate to bone or bone marrow, to pleural and abdominal cavity, to brain and spinal cord tissue, and to the urogenital tract can be selectively destroyed by immunotoxins administered in the tumor, in the bodily or systemically fluid, for example, to target metastatic tumor cells in tissues such as blood, bone and bone marrow.

E p e 3 15 Human breast cancer MA-11 cells were injected into the left cardiac ventricle of immunodeficient rats. The untreated control animals developed symptoms of spinal cord complexation and had to be sacrificed 34- 20 37 days after the injection of cells. Animals treated intravenously with a single dose of MOC31-PE (20 μg / rat) showed prolonged, symptom-free survival, and some of these animals lived for more than 50 days. Another experiment in the same model confirms the results, and in this case some of the animals survived during an observation period of 110 days. In these experiments, a group of rats was treated with an immunotoxin consisting of antibody 425.3 directed against EGF receptor conjugated to PE. All the animals in this group survived. In a third experiment of this model, the control rats showed compression symptoms of the spinal cord and had to be sacrificed between day 40 and 60 after injection of the cells. In this experiment three treatment groups were included, one with 20 μg of 425.3-PE, and one that received 10 mg of each of the two immunotoxins. Significant prolongation of disease-free survival was obtained with both immunotoxins used individually, which provides 60% and 80% long-term survival with MOC31-PE and 425.3-PE respectively. In the combination experiments, all the animals survived free of disease. In a fourth experiment in this model, the effect of M0C31-PE was compared with that of cis-platin and doxorubicin. In this experiment, all of the animals treated with M0C31-PE survived for more than 70 days, while those treated with doxorubicin only showed a marginal effect, and the rats treated with cis-platin did not live longer than the control animals treated with Saline solution. These data demonstrate convincingly that the immunotoxins used are highly superior in the destruction of breast cancer metastases compared to doxorubicin and cis-platina, two of the most commonly used drugs in the clinical setting.

Example 4 The human breast cancer cell line MT-1 was used in two different experiments. In the first of these, the cells were injected into the left cardiac ventricle and the control animals had to be sacrificed due to symptoms of spinal cord compression after a median time of 19 days. All animals treated with 425.3 -PE intravenously one day after cell injection survived. In the other experiments, the MT-1 tumor cells were injected directly into the bone marrow cavity in the rat tibia. All untreated animals had to be sacrificed 20 days later due to the growth of tumors in the tibia, while rats treated with 20 μg of 425.3 -PE intravenously on the day after the injection of the cells survived for more than 100 days . In addition, in the model where the MT-1 tumor cells were injected directly into the bone marrow of the rat tibia, the effect of BM7-PE administered either on day 1 or on day 7 was compared, compared to the effects of 425.3-PE on groups of animals treated on the same day as for BM7-PE. In addition, the effect of doxorubicin (Adriamycin) administered intravenously on day 7 and day 14 was also studied. Both immunotoxins were found to cure 80% of the rats, whether administered on day 1 or on day 7 When half of the concentrations of each immunotoxin are combined, all the animals survived. In comparison, doxorubicin was clearly less effective, leaving only 35% of the animals alive after 90 days. The control animals had to be sacrificed 20 days after the cells, as in the previous experiments. The data confirm the effect of 425.3-PE shown previously. Importantly, it was found that BM7-PE is equally effective as 425.3-PE. Both agents are clearly superior in efficacy compared to doxorubicin, one of the most commonly used clinical drugs against breast cancer. In addition, the combination of the two immunotoxins cured all the animals of their disease.

Example 5 In two groups of experiments, the effect of immunotoxin produced recombinantly against the product of the erbB2 gene and with a recombinantly manufactured variant of PE was tested. In the model described in Example 4, the highest concentration of the recombinant immunotoxin significantly prolongs the life span of the animals and 35% of the rats survived. In a model where MT-1 breast cancer cells were injected intrathecally into immunodeficient rats, treatment with recombinant immunotoxin was also delivered intrathecally (on days 1, 2 and 3) resulting in a significant prolongation in duration of life of the animals. This effect depends on the dose, and the two different doses increase the duration of life of the animals of 10.6 days (controls treated with saline solution) to 23.4 days and 32.8 days with the two different doses of the immunotoxin. At the highest dose, 20% of the rats survived. Since no toxicity was observed even with the highest immunotoxin dose, it is expected that the effect at optimal doses may be even better.

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Claims (12)

1. A method for destroying breast cancer cells or other carcinoma cells expressing the same target antigens, in a population of cells, comprising nucleated cells harvested from peripheral blood, or CD-34 + cells selected from the above nucleated cells, or Other immature / early blood progenitor cells containing multipotent pluripotent cells, the method is characterized in that the population of cells is exposed to a combination of two immunotoxins, wherein each immunotoxin is constituted by a conjugate between an antibody and a cellular toxin, fragments of antibodies and toxin, or recombinantly produced antibodies, toxins, immunotoxins or fragments thereof, wherein the antibodies are directed to epitopes on the EGP2 antigen expressed by the GA733 gene and to epitopes on the antigen expressed by the MUC1, MUC2 or MUC3 genes , respectively, or a combination of these, and the toxin is exo Pseudomonas A toxin.

2. The method according to claim 1, characterized in that the antibodies used are MOC31 and an antibody directed to the antigens encoded by the MUC1, MUC2, MUC3 genes or a combination of these.

3. The method according to claims 1 and 2, characterized in that the antibodies used are M0C31 and BM7, or fragments thereof.

4. The method according to the claims 1 and 2, characterized in that the antibodies used with M0C31 and BM2 or 12H12, or fragments thereof.

5. The method according to claims 1 and 2, characterized in that the antibodies used are M0C31 and 595A6 or fragments thereof.

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

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

8. The method according to claim 6, characterized in that the immunotoxins are administered directly in the tumor or in the pleural and abdominal cavities.

9. A preparation of two immunotoxins to destroy cells, according to the method according to claim 1, characterized in that it contains two immunotoxins directed to antigens present in malignant cells.

10. The preparation according to claim 9, characterized in that the antibodies are selected from among MOC31, and BM7, 595, BM2, 12H12 or combinations thereof, or fragments thereof, and the toxin is Pseudomonas exotoxin A, native or recombinant, or fragments thereof.

11. The use of a preparation of two immunotoxins according to claim 9, characterized in that it is used to produce a therapeutic agent against cancer.

12. A kit (kit) for carrying out the method according to claim 1, characterized in that it contains a preparation of two immunotoxins in a pharmaceutically acceptable formulation.