ES2209627B1 - CELLULAR VEHICLE ABLE TO LOCATE OVER OR IN THE NEIGHBORHOOD OF CANCER INJURY. - Google Patents

Abstract

Cellular vehicle capable of being located on or in the vicinity of cancerous lesions. It consists of autologous tumor cells manipulated ex vivo by the introduction inside of a variety of antitumor agents; suicide genes, antiangiogenic genes, oncolytic viruses, antitumor drugs, antitumor antibodies, or any substance that will exert a therapeutic action on metastases and the primary tumor. Due to its capacity for colocalization in cancerous lesions, said cell vehicle will act on the neoplastic lesions locally, achieving a localized therapeutic effect, without systemic toxicities. Said cellular vehicle may be used in patients with cancer, with the intention of carrying out a therapy comprising the distal administration to said patient of an effective amount of the cellular vehicle described herein.

Description

Cellular vehicle capable of being located on or in the vicinity of cancerous lesions.

Object and field of application

The present invention relates to a cellular vehicle capable of being located on or in the vicinity of the primary tumor and its cancerous metastases. This cell vehicle consists of autologous tumor cells manipulated ex vivo so that they are capable of transporting a variety of different antitumor products inside. Said cellular vehicle may be used in patients with cancer, with the intention of carrying out a therapy comprising the distal administration to said patient of an effective amount of the cellular vehicle described herein. Due to its capacity for colocalization in cancerous lesions, said cell vehicle will act on the neoplastic lesions locally, achieving a localized therapeutic effect without systemic toxicities. It will act synergistically with current antitumor treatments (radiotherapy, chemotherapy, surgery).

Background of the invention

Solid malignant tumors are characterized by the appearance of an initial lesion (primary tumor) from the which tumor cells spread through the bloodstream and / or lymphatic These cells that leave the primary tumor reach distant organs, where they settle, grow and originate the metastasis, representing metastases a relatively step advanced in neoplastic disease. They occur when one or more clones of primary tumor cancer cells acquire damage additional molecules that give them the ability to abandon the primary tumor and distributed throughout the body via blood and / or lymphatic Cancer cells that leave the primary tumor and they enter the bloodstream they end up settling in organs determined, which depends on the type of tumor and the blood flow and lymphatic differences between the different organs There are three theories to explain the fact that Selectively metastatic tumors (Liota, 2001). First, the tumor cells reach all organs but only develop metastasis in those where they find growth factors adequate (Fidler, 2000). Second, the endothelial cells of the blood vessels express adhesion molecules that act as anchor points for circulating tumor cells (Al-Medhi, 2000). And third, there are molecules chemoattractants (chemokines) specifically secreted by different tissues, which stimulate cell migration Tumors from the circulation to the parenchyma of the organs Diana (Muller, 2001).

Metastases are the main problem for the cure of solid tumors. The forecast of patients who are diagnosed with cancer in metastatic stages is significantly worse than those with localized disease, and unfortunately, between 60-70% of patients they have metastases at the time of diagnosis, macro and microscopic (Welch, 1997). The presence of size metastases greater than 2-3 millimeters (macroscopic metastases) It is diagnosed with current methods (CAT and / or MRI), however micrometastases cannot be visualized with the methods of use Daily Clinic The problem with these microscopic metastases is particularly illustrative of the situation of many patients since patients receiving treatment for tumors seemingly resectable and curable end up presenting the disease in organs distant from the tumor, where the presence of metastatic lesions at the time of diagnosis. This situation occurs in almost all types of tumors, since They are typically childish as well as adults.

Current treatments have limitations to eradicate metastases. The surgery cannot be used to microscopic lesions, and chemotherapy and radiotherapy not they can reach all tumor cells at sufficient doses, without causing lethal toxicities for the patient. Therefore it they need new treatments that provide greater efficiency and specificity in addition to minor side effects.

A treatment against mestastatic disease should be able to exercise its action specifically on cancerous lesions, to avoid systemic toxicities that They give today. To do this we would have to have a way of achieve micrometastases and act locally on them. Others groups of researchers have tried different approaches experimental in this regard, which we could classify in two large groups:

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Immunological strategies: It aims to use the ability of the immune system to recognize and destroy cancer cells. Many of these strategies pretend that they are the cells of the patient's immune system those that destroy the tumor cells, for which they are manipulated the tumor cells of the patients (see patents ES2080341; US2137172; EP0733373A2; EP0904786A1; US006039941A), lymphocytes tumor infiltrants (Freedman, 1996), or macrophages (Killion, 1998). On other occasions monoclonal antibodies are used, capable of recognizing specific antigens on the surface of tumors (Carter, 2001).

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Nonimmunological strategies: It consists of the administration to the patients of capable vehicles of locating in metastases, and bearers of principles anti-tumor Such vehicles can be cellular, as per example endothelial cells (see WO9313807 patent) or even bacteria (see patent WO0125397), or non-cellular, such as microspheres located in delimited capillary beds (see US4345588).

We have developed a different approach to the already known. Our hypothesis is that whatever the main mechanism that explains the development of metastases (the three theories noted above are not exclusive), the same factors that act on the cells that leave the tumor Primary should act on tumor cells that are injected into blood circulation Therefore, tumor cells that are Infused intravenously (IV) will settle in the organs target where metastases develop. There is, therefore, the possibility that cancer cells infused via IV end located in the places where it has previously settled and developed a metastatic lesion. We have verified that This hypothesis is correct. We have also developed strategies Therapeutics that have proven effective in controlling metastatic disease

It is an objective of the present invention to provide of a cell vehicle capable of being located on or in the vicinity of the primary tumor and cancerous metastases.

It is another objective of the present invention provide a method to administer the cell vehicle distally to metastatic lesions and the primary tumor.

It is another objective of the present invention provide such a method so that it is unnecessary to inject directly the cell vehicle to metastatic lesions or to primary tumor

Description of the invention

The cellular vehicle object of the present invention consists of autologous tumor cells manipulated ex vivo so that they are capable of transporting a variety of products, including antitumor agents: suicide genes, antiangiogenic genes, oncolytic viruses, antitumor drugs, antitumor antibodies. , or any substance that will exert a therapeutic action on metastases and the primary tumor. Due to its capacity for colocalization in cancerous lesions, said cellular vehicle will act on the neoplastic lesions locally, achieving a localized therapeutic effect without systemic toxicities.

An additional advantage to the decrease in systemic toxicity is to camouflage possible antigens that stimulate an immune response from the patient, and It would make repeated use of modified tumor cells difficult. This fact is more important in the case of suicide gene therapy and in the case of monoclonal antibodies. Gene therapy suicide with the TK gene has been hindered in the clinic by the emergence of an anti-TK immune response (Tiberghien, 1997). In our strategy, tumor cells do not would present the antigen (CD protein, oncolytic adenovirus, monoclonal antibody) as continuously and effectively as in the case of suicide gene therapy against graft disease against host

The use of tumor cells that are given Insert foreign genetic material for later administration a cancer patient has already been contemplated in other patents before ours (among them, those already mentioned ES2080341; US2137172; EP0733373A2; EP0904786A1; US006039941A). exist notable differences between these inventions and ours, some Of which are the following:

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All those patents are based in the introduction to the tumor cells of at least one gene immunomodulator, with the intention of enhancing the response immune against said tumor, since what is intended is Stimulate an action of the patient's lymphocytes. The effect is not local but systemic, it is not toxic but immune.

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Those in which the introduction of a toxic or lethal gene (ES2080341, US006039941A) is contemplated, is always done together with the immunomodulatory gene. That is, none of these patents contemplates the possibility of generating a tumor cell that only carries the toxic or lethal gene. Moreover, the destruction of the tumor cell would be carried out according to these patents "when justified""for example, approximately 14 days after the administration of transfected tumor cells" (extracts from the patent of Philip Frost et al , ES2080341). This is so since the beneficial effect of such inventions derives from the presence of the immunomodulatory gene (or genes), and the lethal gene never has a therapeutic effect.

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The administration of cells to the patient is not specified to have to be via systemic (intravenous). When you comment directly you contemplates that it is "an immunization of a local nature" (ES2080341), which is in full agreement with the administration of vaccines, which is done subcutaneously or intradermally usually.

The use of vehicles has also been patented cell phones, different from the one we propose, for Cancer treatment by non-immunological methods (patents WO9313807 and WO0125397). There is a fundamental difference between these inventions and ours: none of them contemplate the use of autologous tumor cells as a vehicle to transport anticancer substances but it is about other types Cellular: autologous endothelial cells (WO9313807) or bacteria (WO0125397).

The therapeutic possibilities of colocalization of ex vivo modified tumor cells can be amplified by transferring genes from adhesion molecules or genes from chemokine receptors to the tumor cells to be injected, in addition to the antitumor principles. These molecules are involved in the settlement of metastases in different organs depending on the type of tumor.

The above and other related points they will be more apparent in view of the description given to then, when they are read together with the drawings.

Brief description of the drawings

Figure 1. Expression of human cytokeratin 19 (CK-19) in organs of mice that are given implanted subcutaneous breast cancer cells. Seventy days after subcutaneous cell implantation MDA-MB-231, the RNA was purified from the following organs: primary tumor (T), lung (P), liver (H), contralateral ganglion (GC), kidney (R), brain (C), femur contralateral (FC), spleen (B) and ovary (0), (R street corresponds to the reaction reagents). Using RT-PCR, amplified the sequence corresponding to the cytokeratin 19 gene  human, expressed only by tumor cells.

Figure 2. Graphic description of the model of colocalization of intravenously injected tumor cells, about the metastatic tumor.

TO)

Tumor Subcutaneous primary followed by hematogenous dissemination: It was generated a metastatic tumor by subcutaneous implantation of cells human neoplastic (MDA-MB-231).

B)

Intravenous injection of modified tumor cells according to the invention: At 70 days, tumor cells bearing the lac-Z gene were injected intravenously.

C)

Colocalization of the cells injected over pre-existing lesions: Mice are they sacrificed at 24 and 48 hours after infusion and analyzed where they had settled the infused cells, by different methods

Figure 3. Death due to contiguity with the system Cytosine deaminase / 5- Fluorocytosine (CD / 5-FC). In the ordinate axis reflects the viability of the mixtures cell after culture with 5FC. The viability was determined with Trypan Blue

Figure 4. Death due to contiguity with the adeno-oncolytic Staining with violet crystal cell mixtures (cells MDA-MB-231 on the top plate and PC3 cells in the lower plate) to show the viability of the culture. Viable cells stain with the dye while that damaged cells don't.

Figure 5. Results of in vivo treatment with AdCD. The amount of residual primary tumor was calculated using the formula (volume = rr / 6 * a * b), "a" being the major axis and "b" the minor axis. The amount of metastasis was quantified by "dot blot" on lung and kidney DNA samples, with a specific probe for human DNA.

Figure 6. Results of in vivo treatment with oncolytic adenovirus. Lung weight (indirect measurement of metastatic tumor burden) of treated mice and controls.

Description of a preferred embodiment

In accordance with the present invention, we have verified that the intravenous injection of our cellular vehicle ( ex vivo modified tumor cells) to a mammal, is followed by the colocalization of said cellular vehicle on metastatic lesions in different organs, and in the primary lesion . Therefore, ex vivo modified tumor cells are able to migrate through the bloodstream to places where they settle and develop metastases, as well as to the primary tumor. Once the ex vivo modified tumor cells reach the metastatic lesions and the primary tumor, they can induce several local therapeutic effects: death by contiguity, release of oncolytic vectors, segregating antiangiogenic molecules, etc. Any metastatic tumor, including sarcomas, neuroblastomas, melanomas, epithelial tumors such as breast, lung, pancreas, prostate, ovarian, uterine, cervical, gastrointestinal, can be targets for ex vivo , modified tumor cells of in accordance with this invention.

The mechanism of action of our vehicle cell phone follows two steps: first it sits in the vicinity of the metastasis and in the primary tumor, and second exerts its effect Negative about them. The effect of the cellular vehicle is therefore local. Our invention complements the therapeutic strategy Current metastatic tumors. Since its rationality is different, act synergistically with treatments current antitumor agents (radiotherapy, chemotherapy, surgery) and may be administered repeatedly in patients.

The cellular vehicle of the present invention It can be used in several therapeutic methods:

1.- Death by contiguity: It is transferred to the tumor cells that are going to inject a gene that codes for a enzyme capable of transforming a harmless substance into a drug antitumor, such as the CD gene that transforms the 5-FC in 5-Fluorouracil (5-FU). The cells transduced with the CD gene are capable of generating 5-FU in the presence of 5-FC, and die, freeing the microenvironment that they surrounds enough amounts of 5-FU to lead to the death of surrounding cells that do not possess the CD transgene (Figure 3, and Aboody, 2000). Cell death by contiguity ("bystander effect") is a known phenomenon and exploited in antitumor gene therapy using suicide genes such as thymidine kinase (TK) from the herpes simplex virus (Mesnil, 2000) or the CD, but an approach like the one we've never used described us.

Another strategy is to transfer to cells tumors that are going to inject a defective oncolytic virus, able to replicate in tumor cells and destroy them (Alemany, 2000), respecting non-tumor cells. One time destroyed the cells, the defective virus is released and returns to infect and destroy the tumor cells that are in the neighborhood (Figure 4). Our invention provides a solution to problem of the administration of oncolytic viruses in tumors and metastases not accessible, so currently the virus injection directly into the bloodstream (with accumulation of it in the liver tissue) or to the tumor, if this is accessible from the outside (eg, head and neck cancer).

Another different strategy is to incubate the tumor cells in the presence of an antitumor drug and then inject them, similar to the liposome preparations that They are currently used (Alberts, 1997). In this way the tumor cells will transport antineoplastic substances to the neighborhood of metastases and the primary tumor, and there they will spread and will exert a local effect.

You can also manipulate the cells so that transport monoclonal antitumor antibodies. In this situation, our invention solves a limitation of use Current monoclonal antibody, which is the lower case proportion of molecules that are located on the lesions metastatic tumors when an intravenous treatment is performed direct (0.001% -0.01% of the injected dose, according to the review of Sump).

2.- Decrease in local blood supply: tumor cells that will be injected are transduced with genes that encode antiangiogenic molecules (angiostatin, endostatin, binding domains for FIk1, Flt1 and neuropilin receptors) (Kuo, 2001), in addition to what has already been pointed out in the first point above. In this way, when they are located in metastatic lesions produce substances that will decrease the development of the vessels blood that need metastases for growth.

In general, the ex vivo modified tumor cells of the present invention can be transduced using any conventional method of gene transfer (using viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, alphaviruses, or non-viral vectors such as liposomes, electroporation ) of gene expression cassettes: regulatory elements + promoter + transgene.

Tumor cells used and manipulated ex vivo are obtained from the same patient, either from the primary tumor, peripheral blood, bone marrow or from a metastatic nodule that is removed. Its infusion to the patient is done intravenously, at doses that can vary, in the form of a slow infusion.

Another issue related to the strategy therapeutic that we describe is the fact of infusing cells live tumor, which in theory could generate metastasis if the cells were not destroyed. The use of adenoviral vectors achieves a transduction efficiency of up to 100%. In addition, the manipulated cells can be inactivated or attenuated by irradiation with gamma or X-rays and / or treatment with mitomycin C by well known procedures.

We now describe the materials and methods Experimental for the sole purpose of illustrating the procedure and never with the intention of being limiting.

Material and methods 1.- Cell lines

Cells were used MDA-MB-231, line derived from a pleural effusion in a patient with breast cancer, and PC3 cells, human prostate cancer cells. The cells remained in DMEM medium with 10% fetal calf serum.

2.- Animals

NOD / SCID immunodeficient mice were used 6-8 weeks. They were obtained from Jackson Laboratory (Bar Harbor, Maine, USA) and were housed in the animal shelter of the Unit of Molecular and Cellular Biology and Gene Therapy of CIEMAT

3.- Generation of a metastatic human cancer model in immunodeficient mice

Cells MDA-MB-231 were implanted in the area mammary of NOD / SCID mice, and PC3 cells in the right flank. The animals were followed for 3 months before being slaughtered by CO2. At the end of the experiments collected different organs from the mice for study histological (in paraffin and OCT) and PCR (frozen at -70 degrees). The presence of metastases can only be explained by the hematogenous and / or lymphatic spread of tumor cells from the primary injury The presence of cells could be evidenced tumor in many organs of the mice and in some of them (lung, kidney) the growth of metastatic nodules. Some of these animals that developed metastatic tumor received transduced cells (see point 4) intravenously and were slaughtered 24/48 hours later. Figure 1 shows how to detects the presence of metastatic cells in different organs of the mice. Figure 2 explains how the experiment was performed of colocalization.

4.- Transduction of tumor cells with adenovectors and oncolytic adenovirus

Cell suspensions of MDA-MB-231 or PC3 cells were infected with adenoviral vectors or oncolytic viruses, depending on the type of experiment in which they were to be used. An adenoviral vector carrying the lac-Z gene under the control of the cytomegalovirus (CMV) promoter (AdLacZ) was used in the colocalization experiments. In contiguous death experiments a) an adenoviral vector with the CD gene under the CMV promoter (AdCD), or b) an oncolytic adenovirus (AdOncoEGFP) that replicates exclusively in dividing human cells was used. In the animal model used, this adenovirus behaves the same as ONYX-type selective replication oncolytic adenoviruses. It also expresses the EGFP gene under the MLP promoter (Major Late Promoter), which will result in an expression of this protein only when the adenovirus is replicating, since this promoter is only activated in the final phase of virus replication. Viral replication can be evaluated by fluorescence analysis (infected cells in which the adenovirus is replicating appear green in a fluorescence microscope, or in a flow cytometer).

The transduction of tumor cells is performed in DMEM medium without serum, at a m.o.i (multiplicity of infection) optimized according to the type of vector. The effectiveness of transduction was 100%, so that all cells injected carried the corresponding vector or virus.

5.- Detection of tumor cells by RT-PCR

Total RNA from the extracted organs was purified using TRIzol. To detect the presence of tumor cells RT-PCR was performed for a sequence of the human gene of cytokeratin 19, according to a protocol already published by We (Lillo, 2000). Figure 1 is an example of this technique.

6.- Immunohistological study

The organs were fixed in formalin and embedded in paraffin. Sections at different levels were stained with hematoxylin-eosin and analyzed at microscope. Tumor cells were identified with the CAM 5.2 antibody, which recognizes several human keratins and Transduced tumor cells were identified with the antibody Ab1 that recognizes the enzyme? -Galactosidase. Freezing cuts of organs preserved in OCT are labeled with the CAM 5.2 antibody and the transduced cells were identified by staining with X-gal. The transduction efficiency was 100% (all infused cells they carried the transgene). The accumulations of neoplastic cells (recognized by CAM 5.2 antibody) that did not express the transgene (not blue in staining with X-gal) they could only represent metastatic lesions and not cells tumor injected through IV. Cell identification β-gal + in different cuts of the renal or lymphatic metastases (not shown) showed that the phenomenon colocalization does not happen only in the lung, an organ that receives the cells injected at the first moment but also It occurs in metastases of other organs.

7.- Evaluation of death due to contiguity in vitro

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Transduced tumor cells with the AdCD adenovector were mixed with non-tumor cells transduced, to different proportions. The cell mixtures are they were grown in the presence of 5-FC, and the viability was determined at 4 days by staining with violet crystal or by exclusion of positive cells with Trypan Blue (figure 3).

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Tumor cells infected with the oncolytic adenovirus were mixed with non-tumor cells transduced, to different proportions. The cell mixtures are cultivated for 4 days and the viability was determined (figure 4) and The proportion of green cells.

8.- Evaluation of death due to contiguity in vivo

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Cells transduced with AdCD : Groups of mice that had developed a tumor (as explained in point 3) underwent surgical removal of the tumor mass. Next, a group received 5 intravenous doses of transduced cells, 3x105 cells per dose, 1 dose every 4 days. This group of mice was implanted with an osmotic pump for the continuous diffusion of 5-FC. One week after the last injection of transduced cells, all mice were sacrificed and the major diameters of the remains of the primary tumor were measured and the target organs of the metastases were collected, for histological and molecular study. The amount of metastasis was studied by "dot blot" on DNA of these organs, using a specific probe for human DNA (Figure 5).

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Cells infected with AdOncoEGFP : Groups of mice that had developed a tumor (as explained in point 3) underwent surgical removal of the tumor mass. Next, a group received 5 intravenous doses of transduced cells, 3x105 cells per dose, 1 dose every 4 days. One week after the last injection of transduced cells, all mice were sacrificed, the major diameters of the remains of the primary tumor were measured, the lungs were weighed (Figure 6) and the target organs of the metastases were collected, for histological study. and molecular, and quantification of metastases.

The results of these experiments demonstrate that our invention is able to decrease both the primary tumor such as metastases without causing significant systemic toxicity.

In accordance with the present invention, any mammal could be treated: cats, dogs, horses, cows, Pigs, sheep, goats. The most indicated is to use this invention in humans.

The parameters described in the present invention (number of cells, transduction conditions, cell dose injected) can be used in a general way. By Of course, the duration of treatment will vary depending on the particular conditions of each treatment.

Following the description made of this invention is clear to anyone familiar with this field of knowledge that can be done many modifications and changes to what is described respecting the spirit and essence of the invention. It is the intention of the applicants in the claims included in this document cover all these modifications and equivalent variations that fall within the true spirit and scope of the invention.

Bibliographic references

The bibliographic references included in Then they can facilitate understanding or setting practice of certain aspects of the present invention. The inclution of a bibliographic reference in this list is not intended to be nor constitutes an admission that such reference represents a antecedent with respect to the present invention.

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Claims (8)

1. Cellular vehicle capable of being located on or in the vicinity of cancerous lesions of a mammal, characterized by comprising autologous tumor cells that have been manipulated ex vivo by the introduction of one or more antitumor agents or principles of systemic non-immunological action, and because the cancerous lesions on which it is located refer to a primary tumor and its metastases. 2. Cellular vehicle according to claim 1, characterized in that said autologous tumor cells are modified to contain one or more genes capable of expressing and secreting therapeutic products. 3. Cellular vehicle according to claim 2, characterized in that said modified autologous tumor cells contain one or more of the genes of the following families: suicide genes (eg, herpes virus tyrosine kinase), antiangiogenic genes (eg ., angiostatin, endostatin, binding domains for the FIk1, FIt1 and neuropilin receptors). 4. Cellular vehicle according to claim 1, characterized in that said autologous tumor cells are modified to contain one or more oncolytic viruses, capable of replicating. 5. Cellular vehicle according to claim 1, characterized in that said autologous tumor cells are modified to contain at least one antitumor drug. 6. Cellular vehicle according to claim 1, characterized in that said autologous tumor cells are modified to contain one or more monoclonal anti-tumor antibodies. 7. Cellular vehicle according to claim 1, characterized in that said cancer can be sarcoma, melanoma, carcinoma (breast, lung, pancreas, prostate, ovarian, uterine, cervical, gastrointestinal), neuroblastoma. 8. Cellular vehicle according to claim 1, characterized in that said mammal is a human.