MXPA01001987A - Alginate capsules for use in the treatment of brain tumour - Google Patents

Abstract

Encapsulated producer cells which are capable of expressing a molecule which is an inhibitor of CNS tumour growth provide a novel approach to the treatment of tumours, such as braintumours which are localized within the central nervous system.

Description

Alginate capsules for use in the treatment of brain tumors This invention is in the field of the treatment of tumors which are located within the central nervous system (CNS) and cerebral-spinal, primary and secondary malignancies (metastatic), and new compositions and delivery systems useful in this therapy are provided. Brain tumors, primary (gliomas) have diverse biological characteristics, unique compared to other metastatic tumors. These are confined within the central nervous system and metastatic diffusion to other organs is virtually non-existent. Although these tumors show a high degree of invasion in the brain, they have a tendency to return after treatment in positions where they were originally found. The tumors are highly heterogeneous and consist of numerous cell types with different phenotypic properties. Nowadays, the selection treatment is surgery followed by radiotherapy and chemotherapy. Patients with most forms of malignancy of brain tumors (glioblastomas) have a severe prognosis with a survival of approximately 10 months REF: 127622 after the diagnosis. Therefore, there is an urgent need for new treatment strategies for this particular group of tumors. Since tumors have a tendency to return to their primary site, new local treatment strategies are necessary. In addition, since these tumors consist of numerous tumor cells with different phenotypic properties, the selection treatment must be capable of targeting different types of tumor cells. Other tumors which are located within the central nervous system and which are often difficult to treat successfully include tumors derived from astroglial and oliogodendroglial cells, for example: Astrocytomas Low grade astrocytomas (grade 1 and 2 astrocytomas) - Anaplastic astrocytoma (grade 3 astrocytoma) - Glioblastoma multiforme (astrocytoma grade 4) - including secondary glioblastoma, ie tumors that have differentiated from lower grade astrocytomas - primary glioblastoma, ie tumors that occur as de novo primary glioblastomas - giant cell glioblastoma - gliosarcomas - gliomatosis cerebri Oligodendrogliomas - including oligodendroglioma (WHO grade II) - anaplastic oligodendroglioma (WHO grade III) Mixed Gliomas - Oligoastrocytoma (WHO grade II) - Anaplastic oligoastrocytoma (WHO grade III) Tusao ^ s ep ^ nd.- 'arj.os - Ependymoma (WHO grade II) - Anaplastic ependymoma (WHO grade III] - Subependymoma (WHO grade I) Embryonal Tumors - Central Neuroblastoma - Ependymoblastoma - Medulloblastomas - PNETs supratentorial Neuroblastomas - Neuroblastoma olfactory - Neuroblastic tumors of the adrenal gland and sympathetic nervous system For most of these tumors, the first selection treatment is surgery followed with radiation therapy and / or chemotherapy. However, complete removal of the tumor is often difficult through surgical procedures, while radiotherapy and complementary chemotherapy are also sometimes not completely successful due to radio resistance and / or difficulties in the delivery of therapeutic doses of cytotoxic drugs. During recent years, much attention has been focused on gene therapy, where the reversion of the malignant phenotype has been treated by sub-regulation of the expression of oncogenes or the insertion of normal tumor suppressor genes. Immune system stimulating factors such as cytokines that are designed to increase the recognition and rejection of tumors by the immune system have also been introduced. In addition, the cells have been modified to allow the direct delivery of gene products to the tumor cells, increasing their susceptibility to the pharmacological agents. The documents which describe these developments include (i) Curr Opin Oncol, 1_, (1995), pages 94-100; (ii) Curr Opin Biotechnol, 5_ (1994), pages 611-616; (iii) Cancer Res, 5_3 (1993), pages 2330-7; (iv) Hum Gene Ther, 4_, (1993), pages 451-60; (v) Hum Gene Ther, 5_, (1994), pages 153-164; and (vi) Trends Pharmacol. Sci, 1, (1993), pages 202-208. Despite this extensive research in recent years, there are major obstacles which prevent the transition between experimental research and clinical treatment of brain tumors, malignancies. One problem is to prevent the immuno-rejection of genetically modified cells after the intracranial implant. This can be overcome by the encapsulation of producer cells. However, this results in other problems, to find materials specially adapted for use in the brain. Although, the brain is immunologically different from other areas of the body, for example in its lack of B lymphocytes, it is especially sensitive to the influence of biologically active compounds such as for example endotoxins.

It has been found, in accordance with the present invention, that the immuno-insulating alginate matrices are especially suitable for the encapsulation of producer cells proposed for the intercranial implant, in the treatment of CNS tumors. It is especially preferred that the immuno-insulating alginate matrices must be micro-accounts. Thus, in its broad aspect, the present invention provides an encapsulated producer cell capable of expressing a molecule which is a tumor growth inhibitor of the CNS, the producer cell that is encapsulated in an immune-insulating alginate matrix. It is preferred that this molecule must be a peptide, a protein or a polysaccharide and the most preferred molecule is a monoclonal antibody. The present invention also provides a method for the treatment of CNS tumors, which comprises implanting in the tumor site an encapsulated producer cell which is capable of expressing a molecule which is an inhibitor of tumor growth. Furthermore, the present invention provides a method for the preparation of a pharmacological product for the treatment of a CNS tumor, which comprises encapsulating within a matrix of immuno-insulating alginate a producer cell capable of expressing a molecule which is an inhibitor of the tumor growth. The present invention also provides the use of immuno-insulating alginate matrices for the encapsulation of producer cells proposed for the intercranial implant, in the treatment of CNS tumors. In one embodiment of the invention, producer cells contemplated for use herein include genetically engineered cells that produce molecules, e.g., proteins, peptides and polysaccharides, which will interact either directly with the tumor cells or indirectly with the communication pathways. of the tumor cells or hosts. Other useful producer cells, contemplated herein, are specialized cells which produce monoclonal antibodies such as for example hybridoma cells, or even naturally occurring cells which are capable of expressing tumor inhibitory molecules. It is well known that tumor growth is dependent on cellular interactions, specific with the host, mediated by the pathway of specific growth factors that regulate the growth of tumor cells in preferably complex forms. Tumors depend in this respect on the nutrients mediated via newly formed blood vessels supplied by the host. The various tumoral / host cell interaction pathways have been identified in recent years and have been described in the literature. Accordingly, a class of producer cells, useful herein, are those which can express proteins or peptides that will interact with the tumor / hopedane communication pathways. For example, useful producing cells include those that produce proteins and peptides which affect tumor neovascularization such as thrombospondin, endostatin, angiostatin and prolactin, proteins which interfere with the ratio of tumor cells to the extracellular matrix, for example protease inhibitors such as tissue inhibitors of metalloproteinases, and proteins and peptides which affect the immune system, including all the various classes of interleukins. Another preferred class of producer cells is constituted by those which express the proteins or peptides which interact directly with the tumor cells themselves. For example, producer cells useful in this category include: hybridoma cell lines that produce monoclonal antibodies which interact directly with a tumor receptor, eg, cell growth factor receptors which affect tumor cells such as the epidermal growth factor receptor (EGFr), the platelet-derived growth factor receptors AA and BB, the acidic or basic fibroblast growth factor receptors, the transforming growth factor receptor alpha and beta, the different classes of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2), tyrosine kinase receptors with immunoglobulin and EGF-like domains such as, for example, TIE-1 and TIE-2 / tek, the factor of hepatocyte growth (spreading factor); or monoclonal antibodies directed against the various classes of integrin receptors; monoclonal antibodies directed against CD-44; monoclonal antibodies directed against the CDK / cyclin complexes; monoclonal antibodies directed against FAS; monoclonal antibodies directed against glycolipids on the cell surface; monoclonal antibodies directed against glycoproteins; and monoclonal antibodies directed against proteins derived from the expression of specific oncogenes. Producer cells whose production of tumor growth inhibitory substances can be activated and deactivated by a pharmacological medium are of particular interest in some circumstances, for example producing cells with the expression of pharmacologically inducible genes such as, for example, the expression of genes activated with tetracycline. Any cell line which is transfectable can be used in accordance with this invention. The cell lines must be permanent, that is, capable of undergoing unlimited cell division, and are preferably non-human and non-tumorigenic. Examples of such cell lines which are freely and commercially available from the American Type Culture Collection, 10350 Linden Lake Plaza, Manassas, Virginia 20109, USA are: Cell line ATCC number Description H528 HB 8509 mouse B-cell myeloma 293 CRL 1573 primary embryonic kidney, human-transformed NIH / 3T3 CRL 1658 Swiss NIH mouse, COS-7 embryo CRL 1651 African green monkey, kidney, transformation SV40 BHK -21 CCL 10 Hamster kidney, normal CV-1 CCL 70 African green monkey, kidney, normal CHP-234 CRL-2272 Neuroblastoma, brain, human Rat2 CRL-1764 Embryo, thymidine kinase mutant, rat Namalwa CL-1432 Lymphoma Burkitt, human According to the present invention, the producer cells are encapsulated in alginate matrices of immuno-insulators which are capable of providing a stable, in-house delivery system of the expressed protein or another molecule which can interfere with growth and the progress of the tumor without immuno-rejection of the producer cells. Encapsulation of cells within alginate beads is a well-known technique for immobilizing cells and other substances, and has previously been used in the treatment of diabetes mellitus, in the production of monoclonal antibodies, and in other medical areas, as has described in the literature. From PCT / WO97 / 44065 this drug delivery technique for gene therapy in vivo has been proposed using encapsulated cells that release the gene transfer vectors at the site of a brain tumor. The capsules used for the encapsulation of the cells comprise two parts: a) a core comprising latent packaging cells and b) an outer shell that surrounds the core. The present invention provides a process and a product for much simpler encapsulation, wherein the producer cells are encapsulated directly in a one-step process using an immuno-isolation quality of the alginate. Alginate is a polysaccharide which is found mainly in brown seaweed. This consists of two types of monosaccharides; L-guluronic acid (G) and D-manuronic acid (M). These units of polysaccharides appear in blocks of alternating sequences of G and M (MG blocks) and the blocks consist mainly of units either G or M (blocks G / blocks M). The property of gel formation is achieved through a cross-linking of the G blocks with multivalent cations, especially Ca2 +. In order for an alginate not to be activated immunologically, the G content must be above 15%. However, it is more preferred, according to the present invention, to use a high G alginate, ie with a G content of 50% or higher in order to make the alginate immune-insulating. As is well known in the art, the G / M block relationships and the distribution of the different blocks are critical factors for the different properties of the resulting gene formed through crosslinking with a polyvalent cation. Another aspect, which is critical, is the purity of the alginate to be used. Thus, an advantage of the alginate matrices that can be used according to the present invention is that they can be produced in a high purity quality having a well defined constitution and a very low content of impurities such as endotoxins. A second advantage of the alginate matrices that can be used according to the present invention is that the alginate microbeads prepared by the dropwise addition of an alginate solution containing viable cells to a calcium solution have a concentration of Alginate rising from the center of the micro account to the outer surface. Because of that an optimal space is created in the center of the microbeads so that the cells live, proliferate and produce, by means of that enough nutrients and oxygen is available for the cells. The outer surface with its highest concentration of alginate gives rise to a barrier, so that the producer cells within the micro-accounts do not escape from the inside, or that the immune cells enter the accounts. In general, the use of alginate as an immobilization matrix for the cells involves mixing a suspension of the cells with a Na + alginate solution, then the mixture is poured dropwise into a solution containing multivalent cations (usually Ca2 +). The droplets form gel spheres that instantly trap the cells in a three dimensional grid of the ionically crosslinked alginate. This immobilization procedure can be carried out under very mild conditions and is therefore compatible with the majority of living cells. For a detailed description of both the theory and the practice of the technique, the reader addresses the document "Alginate as Immobilization Matrix for Cells" by Smidsr0d and Skjak-Braek in Trends in Biotechnology, March 1990, Vol. 8, No. 3, pages 71-78. A presently preferred method for forming calcium alginate beads to encapsulate producer cells according to this invention is as follows: Sodium alginate is dissolved in a concentration of 1-2% in water or isotonic saline. The alginate solution is sterilized with a membrane, and then the producer cells are added and the isotonicity is adjusted. Calcium alginate beads are formed by pouring the calcium alginate-producing cell solution drop-by-drop into a calcium chloride bath (0.05-0.25 M), either manually but preferentially using an electrostatic bead generator which establishes an electrostatic potential of 5 to 7 kV between the alginate feeding needle and the gelation bath. By adjusting the diameter of the needle (for example from 0.1 mm to 0.4 mm), the flow rate (for example from 5 ml / hr to 30 ml / hr) and the applied voltage, beads of comparatively uniform diameter can be generated. 100-400 μm. The homogeneity of the beads is controlled by adjusting the salt concentration in the gel bath, NaCl from 0 to 200 mM, with the highest salt concentration giving the highest homogeneity. The beads are allowed to harden in the gelling bath. It is contemplated that the encapsulated producer cells of this invention will be placed in the tumor cavity after the removal of the conventional bulky tumor by surgery. Shortly after surgery, the tumor weight is minimal and many patients have a symptom-free period before recurrence occurs. Since surgery is a traumatic event, the remaining tumor cells will try to establish new pathways of biochemical interaction with the host. This involves the formation of new blood vessels and new supplies of peptide growth factors to the remaining tumor cells. It is at this time, when the tumor weight is at a minimum, the treatment made possible by the present invention is more likely to be effective. In fact, it is a particular advantage of the present invention, according to one embodiment, that it readily allows the simultaneous implantation of several different types of producer cells to choose different phenotypic characteristics and microenvironmental factors that influence the progressive growth of tumors. of the brain or other tumors. For this purpose, a bank of producer cells containing the producer cells, stored, frozen at the temperature of liquid nitrogen could be established. The producer cells could then be removed from the bank to comply with the genotypic expression of the host tumor being treated. In order to establish that the producer cells are required for the treatment of a tumor, the following procedure could be used, by way of example. The characterization of the tumor that involves the determination of the state of the receptor and the phenotype is first performed on the material of the biopsy. Producer cells, appropriately selected which produce substances, for example monoclonal antibodies, directed against the host tumor receptor status are then implanted stereotactically up to 60 days after the surgical removal of the primary tumor. Alternatively, producing cells that produce anti-angiogenic substances can be implanted directly after surgical removal of the primary tumor.

The dosage of the producer cells to be implanted, of course, will depend on the precise circumstances of each patient, but typically the total number of implant cells would be in the range of 106 to 1012 per patient. The number of producing cells within each alginate encapsulation matrix or other will, of course, depend on the dimensions of the bead or other form of encapsulation. The encapsulated production cells will generally be surgically placed in the injured site after removal of the primary tumor. As the experiments to be described in detail later have shown, the encapsulated producer cells can survive, proliferate and maintain their specific expression periods in vi tro and in vivo. This discovery opens up the possibility of a new class of therapeutic treatment for patients with brain tumor conditions, whereby different production cells can be encapsulated, which are selected to target the selected characteristics of tumor growth and development. cerebral. In the experiments described herein, it has been shown that specific MAbs released from alginate beads can inhibit the migration of tumor cells as demonstrated by an interference with the epidermal growth factor receptor. It has also been shown that the specific products released from the encapsulated producer cells within the brain penetrate the parenchyma of the brain and can be distributed along the CSF pathways. The following experiments will help in the understanding of the invention and its advantages. Further reference will be made to the attached drawings in which: Figures 1A-1C Optical microscope images of NIH cells 3T3 encapsulated in alginate. All bars represent 250 μm.

Figure IA: The day of encapsulation.

Figure IB: Encapsulated cells after 3 weeks in culture.

Figure 1C Cells encapsulated after 9 weeks in the culture.

Figures 1D-1F: confocal laser scanning micrographs of NIH 3T3 cells encapsulated in alginate. Viable cells emit green fluorescence (in the present shown as lighter areas), while dead cells emit red fluorescence (not visible at present). All bars represent 250 μm.

Figure ID: On the day of encapsulation.

Figure 1E: Encapsulated cells after weeks in culture.

Figure 1F: Encapsulated cells after weeks in culture.

Figure 1G: D-galactosidase activity of BT4CnVIacZ cells encapsulated in alginate, after 9 weeks in the culture. The bar represents 500 μm.

Figures 2A-2D Flow cytometric histograms of NIH 3T3 cells encapsulated in alginate beads. The horizontal axis expresses the number of channels in the flow cytometer (relative DNA fluorescence), while the vertical axis expresses the relative number of cell nuclei in each channel.

Figure 2A: Control, monolayer culture.

Figure 2B: Cells encapsulated for 1 week.

Figure 2C: Encapsulated cells for 3 weeks.

Figure 2D Cells encapsulated for 9 weeks.

Figure 3 The release of antibodies from H528 hybridoma cells encapsulated in alginate (average value ± normal error). The horizontal axis represents the number of days in the culture, while the vertical axis shows the release of antibodies in the culture medium. The curve was estimated by a 3rd order regression analysis.

Figure 4 Migration of GaMg spheroidal cells after 4 days, untreated (control), stimulated with 10 ng / ml of EGF (EGF), or stimulated with 10 ng / ml of EGF in the presence of encapsulated hybridoma cells (EGF) / H528).

Figures 5A-5H Encapsulated H528 hybridoma cells implanted in the rat brain.

Figure 5A: Axial section of the rat brain. Staining with H &E, the bar represents 5 mm.

Figure 5B: The same section as Figure 5A, showing the H528 cells encapsulated within the implant site. Staining with H &E, the bar represents 500 um.

Figures 5C-5H: Confocal laser beam scanning micrographs of the release and dissemination of monoclonal antibodies within the brain. Figures 5C, E and D were taken with identical gain settings. Figures 5G and 5H were also taken with identical gain settings.

Figure 5C: A section of the parenchyma of the brain, with the H528 cells encapsulated on the left, opposite side. The bar represents 150 μm. An intense fluorescence in the parenchyma of the brain is observed on the left side, followed by a gradual decrease in intensity of at least 1000 μm within the brain.

The gradual change in the intensity of the fluorescence along the horizontal line is further shown in Figure 5D where the vertical axis represents the relative fluorescence intensity (0-255). An intense fluorescence is observed on the left side, with a gradual decrease in the parenchyma of the brain.

Figure 5E: The MAbs were found in the subarachnoid space and in the fundamental brain. The bar represents 75 μm.

Figure 5F: The weak fluorescence presented in the controls was probably caused by the non-specific binding. The bar represents 75 μm.

Figure 5G: The MAbs were further disseminated within the perivascular space. The bar represents 50 μm.

Figure 5H In comparison, the controls showed a weak immunoglobulin binding in the perivascular space. The bar represents 50 μm.

Figure 6: Radioimmunoassays showing the successful establishment of endostatin-producing cells. The figure shows the radioimmunoassays of endostatin release from the conditioned medium, in cell fractions and the medium of non-transfected cells in the second, third and fourth columns, respectively.

Figure 7 The effects of endostatin alginate therapy on tumor growth. Panel A shows an example of a control animal where the transfected, false, cells encapsulated in the alginate beads were implanted. The darker area of the brain shows the area of the tumor. Panel B shows the example of an animal treated with encapsulated endostatin-producing cells. The darker area shows the tumor, and a long necrotic area is visualized in the middle part of the tumor.

EXPERIMENTS MA ER ALES AND METHODS 1. Cell Lines In the experiments, four different cell lines were used Cell line Deposit details 1. NIH 3T3 ATCC CRL / 1658 2. BT4CnVIacZ Not deposited 3. H528 ATCC HB 8509 GaMg Not deposited The NIH 3T3 cells of mouse fibroblasts represent a line of producing cells, potentials that are capable of being genetically designed to express substances which show effects against the growth, progress and development of tumors. The NIH 3T3 cells were encapsulated in alginate, as described below and used to study the in vitro morphology, viability and kinetics of the cells. For studies of the viability of encapsulated cells in vivo, the alginate beads containing NIH 3T3 cells were also implanted in the rat brain. The BT4CnVlacZ cell line was originally developed from a rat glioma induced by ethylnitrosourea and was transfected stably with the bacterial cZ gene, cloned into a plasmid containing a terminal repeat cassette, long Moloney murine leukemia virus with a Resistance neomycin gene expressed from an internal Rous sarcoma virus promoter. See J. Nati Cancer Inst, 5_5 (1975), pages 1177-87 and Int. J. Cancer, 7_1 (1997), pages 874-80. The cells were encapsulated in alginate, and the synthesis in vi tro of the bacterial β-galactosidase was studied. Hybridoma cell line H528 was obtained from American Type Culture Collection (ATCC Rockville, MA). The cell line was generated by fusion of myeloma cells NS-l-Ag4-l with BALB / c mouse spleen cells and this produces a mouse monoclonal antibody (Mab) (IgG2a) that binds to and blocks the domain of EGF binding of human epidermal growth factor receptor (EGFR). The release in vivo of MAbs from cells encapsulated in alginate was studied using this cell line. The human glioma cell line GaMg has been described in Anticancer Res, (1988) pages 874-80, and it has previously been shown that it expresses the EGFR (Acta Neuropathol Berl, 84_ (1992), pages 190-197.) The specific inhibition of GaMg cell migration was studied in a co-culture system between the multicellular spheroids GaMg and the encapsulated H528 cells. 2. Cell culture NIH 3T3 and BT4CnVlacZ cell lines were cultured in 80 cm2 culture flasks (Nunc, Roskilde, Denmark) with a complete culture medium consisting of Dulbecco's modified Eagles (DMEM) medium supplemented with freshly inactivated calf serum. with 10% heat, four times the prescribed concentration of non-essential amino acids, L-Glutamine 2%, penicillin (100 IU / ml) and streptomycin (100 μg / ml) (all biochemical substances from BioWhittaker, Verviers, Belgium). Hybridoma cell lines H528 and GaMg were cultured in 80 cm2 culture flasks (Nunc) in an RPMI 1640 culture medium supplemented with 10% horse serum (BioWhittaker). The GaMg monolayers were treated with trypsin in confluence with 3 ml of 0.025% trypsin (BioWhittaker), and spheroids were started by seeding the 5 * 106 cells in 20 ml of the complete RPMI medium in 80 cm2 (Nunc) coated culture flasks. with bases with 0.5% noble agar (Difco, Detroit, MI) (30) in the complete RPMI medium. All cell lines were maintained in a normal tissue culture incubator at 37 ° C with 100% relative humidity, 95% air and 5% C023. Structure and properties of alginate In these experiments, sodium alginate from the brown seaweed Laminaria hyperborea (LF 10/60) (Protanal, Drammen, Norway) was used for the microencapsulation of the producer cells. This consists of two monosaccharides; a-L-guluronic acid (G) and ß-manuronic acid (M). The units G and M are joined together in three different types of blocks, GG, MM and MG, and the proportions and distributions of these blocks determine the chemical and physical properties of the alginate molecules. Some divalent cations similar to CA2 + bind strongly between the separated G blocks, which initiate the formation of an extended alginate network where the G blocks form firm bonds. The alginate which was used has a high content, above 60%, of G blocks, which results in a high mechanical stability and porosity, making it suitable for the encapsulation of the cells for the production of secondary metabolites (see Trends in Biotechnology, 8 ^ (1990), pages 71-78). Electron microscopy by scanning has shown pore sizes in alginate beads ranging from 5 to 200 nm (33,34). The mechanical strength, volume stability and porosity of the beads correlates with the guluronic acid content.

Encapsulation of the cells The encapsulation method used has been described in detail in "Alginate as Immobilization Matrix for Cells "by Smidsr0d and Skjak-Braek in Trends in Biotechology, March 1990, Vol. 8, No. 3, pages 71-78 In summary, the droplets of cells dispersed in 1.5% sodium alginate were released in a 0.1M Ca2 + solution After polymerization, the alginate beads were washed three times in Dulbecco's PBS (DPBS, Sigma, San Luis, MO) and once in the culture medium.The encapsulated cells were cultured in bottles of 175 cm2 culture (Nunc), containing 50 ml of the culture medium, the culture medium was changed every third day, and the bottles were replaced once a week.All the cells encapsulated in alginate were kept in a culture incubator. tissue culture, normal at 37 ° C, with 100% humidity, 95% air and 5% C02.For all experiments with the NIH 3T3 and BT4CnVlacZ cell lines, a cell density of 6 * 106 cells was used / ml of alginate and the sizes of the beads between 0.8 and 1.2 mm For the experiments in vi With the H528 cell line, a cell density of 3 * 10 5 cells / ml of alginate was used and the diameters of the beads between 2.3 and 2.5 mm. For in vivo experiments with the H528 cell line, a cell density of 3 * 10 5 cells / ml of alginate and diameters of the beads between 0.8 and 1.2 mm was used.

EXPERIMENTS IN VITRO 1. Morphology and viability of cells encapsulated in alginate The morphology of the NIH 3T3 cells encapsulated in alginate was investigated on the day of encapsulation, and then 3 and 9 weeks, in 6 beads transferred to a 6-well culture well (Nunc) with a 1.0 ml cover of DPBS. The beads were examined with a Nikon Diaphot optical microscope, and photographed with a Nikon F-301 camera. The morphology experiments were performed in duplicate. The viability of the cells within the alginate beads was investigated on the day of encapsulation, and after 3 and 9 weeks, by a two-color fluorescence viability assay (Live / DeadMR Viability / Cytotoxity Assay, Molecular Probes, Eugene, OR). A labeling solution was prepared with 2 μM of calcein-AM and 4 μM of ethidium homodimer in the complete culture medium. The alginate beads were individually placed in 16-mm multiple-well culture cuvettes (Nunc) with a 0.5 ml cover of 30-minute labeling solution at room temperature. Afterwards, these were transferred in DPBS and examined immediately. Fluorescence was measured in optical sections through the alginate using a confocal laser beam scanning microscope with an argon-krypton laser beam (Biorad MRC-1000, Hemel Hempstead, England), using Texas Red filter optics and FITC. Fluorescence was recorded in a 120 μm plane within the alginate beads. Feasibility experiments were performed in triplicate. The production of β-galactosidase was studied in BT4CnVIacZ cells encapsulated in alginate for 1, 3 and 9 weeks. The beads were washed for 1 minute in DPBS (pH = 8.4) and fixed for 10 minutes in glutaraldehyde 0.2% and 2% formaldehyde in DPBS. Then, these were washed 3 x 5 minutes in DPBS and stained for the β-galactosidase activity with 5-bromo-4-chloro-3-indole β-D-galacto-pyranoside (x-gal, Sigma). The substrate solution consisted of 1 mg / ml of x-gal dissolved in 100 μl of dimethylformamide, and mixed with 5 mM potassium ferricyanite, 5 mM potassium ferrocinate and 2 mM MgCl2 dissolved in DPBS (all biochemicals from Merck, Darmstadt, Germany). These were incubated at 4 ° C for a minimum of 24 hours and examined for the activity of ß-galactosidase, represented by a blue cell cytoplasm. 2. Cell kinetics of cells encapsulated in alginate Cell cycle distribution in encapsulated NIH 3T3 cells was determined by flow cytometric DNA analysis. The encapsulated cells were released from the alginate by dissolving the beads in the complete culture medium containing 1.5% tri-sodium citrate hydrate (E. Merck) for 15 minutes, followed by centrifugation at 140 g for 4 minutes, and the removal of the supernatant. The cells were resuspended twice in the complete culture medium, centrifuged at 140 g for 4 minutes, fixed in 96% ethanol cooled with ice and stored at 4 ° C. Before the flow cytometric analysis, the cells were incubated for 15 minutes with 0.5% pepsin (Sigma) in saline, physiological 0.9% (pH = 1.5) at 37 ° C before the isolated nuclei were washed in saline, physiological 0.9% and were treated for 1 minute with ribonuclease (Sigma) (1 mg / ml in saline, physiological 0.9%). DNA staining was obtained by adding propidium-iodine (Sigma) (50 μg / ml in saline, physiological 0.9%) to the nuclei. The content of the cellular DNA was measured using a Becton Dickinson FACSort flow cytometer (Becton Dickinson, Palo Alto, CA). Histograms of DNA were obtained by controlling the flow of a forward and lateral scatter histogram, from two parameters to a DNA histogram of a parameter. Each histogram was obtained by counting a total of 5000 controlled nuclei. The flow cytometric experiments were repeated three times, and the cell cycle distribution was determined as described in Radiat Environ Biophys, 12_ (1975), pages 31-39. 3. Release of antibodies from encapsulated hybridoma cells Alginate beads with diameters between 2.3 and 2.5 mm containing 1-5 * 103 H528 cells per count on the day of encapsulation were prepared as described above. After 0, 1, 5, 12, 19, 23, 30 and 33 days, respectively, 10 counts of the stem culture were removed and the release of Mabs in the RPMI medium was examined. The beads were transferred in 24 well culture wells (Nunc), in 0.5 ml of the complete RPMI medium (37 ° C). After 6 hours of incubation, four samples of 100 μl each were collected, placed in test tubes for 1.5 ml centrifuge (Treff AG, Degersheim, Switzerland) and frozen at -20 ° C). Flow cytometry was used to determine the concentration of MAbs in the samples. The GaMg monolayer cell cultures were treated with tipzin with 2 mM EDTA in DPBS. The cells were then centrifuged at 140 g for 4 minutes, the supernatant was removed, and the cells were fixed in a 2% paraformaldehyde solution in DPBS for 1 minute. Then, the cells were centrifuged at 140 g for 4 minutes and the supernatant was removed. Cells were then resuspended in DPBS containing 2 mM EDTA, 1% bovine serum albumin and 1 g / l glucose, and distributed in a 96-well conical plate (Nunc) with 1.7 * 195 cells / well. centrifuged at 340 g for 4 minutes, and the supernatant was removed. The cells were then placed in a vortex and incubated for 2 hours at 4 ° C with the RPMI medium with the Mab harvested (undiluted, and dilutions 1: 5, 1:20 1: 100 in DPBS). As a reference, an EGFR MAb antibody (528) (Santa Cruz biotechnology, Santa Cruz, CA) was used with a known MAb concentration (concentrations 20, 5, 1, 0.2, 0.1 and 0.05 μg / ml). The cells were washed twice in 2 mM EDTA, 1% BSA, 1 g / l of glucose in DPBS, and then incubated with goat anti-mouse immunoglobulins conjugated with FITC (Dako A / S, Glostrup, Denmark) (dilution 1:20) for 30 minutes at 4 ° C. Flow cytometry was performed on a Becton Dickinson FACSort flow cytometer. The individual cells were detected and visualized by a cytogram of forward and lateral spreading, of two parameters and the flow was controlled to a FITC histogram of a parameter, where the intensity of the fluorescence was determined. By using the various titers of EGFR MAbs with a known concentration in GaMg cells, a binding curve of reference antibodies to GaMg cells was obtained. By comparing the results obtained from the medium collected from the alginate beads containing the hybridoma, the MAb concentration curve was obtained. 4. Migration of the cells The GaMg spheroids were transferred individually to 16-mm multiple-well culture cuvettes (Nunc) in 1.0 ml of the complete RPMI medium containing 10 ng / ml of EGF (Sigma). Then, the tumor cells were exposed to the alginate beads containing H528 cells (three alginate beads in each well). As controls, the spheroids were exposed to the RPMI medium complete with or without 10 ng / ml of EGF. The orthogonal diameter of each colony was measured daily for four days, using an optical microscope with a calibrated reticle in the ocular device. The circular area converted by the cells that migrate out of the spheroids was then determined and used as an index of cell migration. The experiments were performed in duplicate, with six spheroids in each experiment.

. Establishment of endostatin-producing cells and testing: The endostatin release of the clients 5A. Establishment of endostatin-producing cells Methods: Cell line and culture conditions. 293 human fetal kidney cells (293-EBNA) expressing the Epstein-Barr virus (EBNA) -1 nuclear antigen were used as a producer cell line. The cells were transfected with the episomal expression vector pCEC-Pu containing the gene encoding human endostatin., by liposomal and were selected with 0.5 μg / ml puromycin. Transfected cells (293-endo) were cultured for confluence in 175 cm2 culture flasks (Nunc, Roskilde, Denmark) containing the culture medium consisting of Dulbecco's modified Eagles medium (DMEM) supplemented with fetal calf serum , inactivated with heat 10%, 4.5 g / l of D-glucose, penicillin (100 IU / ml) and streptomycin (100 μl / ml), 205 μg / ml of geneticin (G-418) and 0.5 μg / ml of puromycin . False transfectants were generated by transfecting 293 cells with the pCEP-Pu vector without the endostatin gene and cultured under the same conditions with the exception of puromycin (all biochemical products from Bio hitaker, Verviers, Belgium). The. Tumor cell line (BT4C) selected for these experiments was developed from a rat gliosarcoma induced by ethylnitrosourea (passage number 26) and is syngeneic in DB-IX. Cells were cultured for confluence in 80 cm2 culture flasks with the complete culture medium consisting of Dulbecco's modified Eagles medium (DMEM) supplemented with newborn calf serum, inactivated with 10% heat, 4 times the prescribed concentration of non-essential amino acids, L-Glutamine 2%, penicillin (10 IU / ml) and streptomycin (100 μl / ml). 5b. Estimating endostatin release from accounts Immunological treatments with blotting paper The conditioned medium of encapsulated endo-293 and 293-EBNA was collected and used for normal Western SDS / PAGE blotting to determine if endostatin was released from the beads. Briefly, the samples were separated on a 12% SDS gel and blotted on a PVDP nitrocellulose membrane. The blotting treatments were washed with 100% methanol for 5 minutes, dest. Water. 1 minute, blocking solution (Tris / HCL, I was born 0.45 M, Tween 2%, ph 10.2) 4 minutes and finally with wash buffer (Tris 0.05 M / HCL, I was born 0.15 M, Tween 20 0.05%, ph 10.2) for 15 minutes The blotting treatments were then incubated overnight with rabbit anti-human antisera (1: 1000 in wash buffer). After overnight incubation, the blotting treatments were washed in DPBS and incubated with pigmented anti-rabbit, alkaline, phosphatase-conjugated IgG (DAKO, Denmark). The visualization of the bands was done by incubation with the substrate staining solution (2-4 minutes).

EXPERIMENTS IN VIVO Intracranial implants BD-IX male inbred rats (36) weighing between 160 g and 250 g were kept on a normal pellet diet, given unlimited access to tap water and individually caged at a constant temperature and humidity in a program of light and darkness of 12 hours. The rats were anesthetized intraperitoneally with pentobarbitol at a concentration of 0.4 ml / 100 g of body weight. By means of an incision in the middle sagittal skin, a rough hole was made with a 3.5 mm drill, 4.2 mm posterior to the bregma point and 2.5 mm to the right of the sagittal suture. The cortical and white matter tissue was removed by suction to a depth of 2.0 mm and between 8 and 14 alginate beads (one-day manufacturing beads) were placed containing either NIH 3T cells or H528 cells in the tissue cavity. The rough hole was closed with bone wax and the skin was sutured with polyamide thread. Recovery was allowed under a heating lamp 1 hour. The care of the animal was in accordance with the institutional guidelines. Rats were observed once a day, and weighed every third day. All animals recovered quickly after the implants and showed no signs of disease or neurological deficiencies during the observation period. 2. Liberation and dissemination of immunoglobulins within rat brain After 3 and 9 weeks, the rats were sacrificed by inhalation of CO 2. The brains were removed, embedded in Tessue Tek (Miles Laboratories Inc., Naperville, IL) and frozen in 2-methylbutane (E. Merck) cooled with liquid nitrogen. The axial sections (14 μm) were cut in a cryocut 1800 cryotome Reichert-Jung (Leica, Wetzlar, Germany) and stored at -20 ° C. The cryosections obtained from rats were implanted with H528 encapsulated cells and sacrificed after 3 weeks, fixed in acetone for 5 minutes at room temperature, and then washed twice in DPBS for 5 minutes. The sections were then incubated with goat anti-mouse immunoglobulins conjugated with FITC (Dako A / S, 1:20 dilution) for 1 hour at room temperature, and then washed for 5 minutes with DPBS. The sections were treated for 30 seconds with ribonuclease (Sigma) (0.5 mg / ml in saline, physiological 0.9%), and staining of the nuclei was obtained by adding propidium-iodide (Sigma) (50 μg / ml in saline solution , physiological 0.9%) to the sections. In addition, the sections were washed with DPBS for 5 minutes, and then mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA). Fluorescence was measured using a Leica TCS NT confocal laser scanning microscope with an argon-krypton laser (Leica), using optical TRITC and FITC filter devices. The sections taken from the same depth inside the brains of the experimental animals were investigated, and the areas of maximum fluorescence intensity in both groups were studied. The cryosections obtained from rats implanted with NIN 3T3 cells and sacrificed after 9 weeks, were stained with Hematoxylin and Eosin for histological examination. 3. Immune responses to the producer cells encapsulated in alginate.

Methods The percentage of immuno-positive cells in the boundary zone between the brain for BD-IX rats and the alginate counts were evaluated 1, 3 and 9 weeks after implantation. The brains were mounted on supports or adapters, embedded in tessue-tek and frozen in liquid N2. Axial sections, serial 5-10 μm were cut in a Reichert Jung Cryiostat (Leica, Wetzlar, Germany) were mounted on slides and prepared for immunohistochemical analyzes. The sections were fixed in cold acetone for 5 minutes, incubated for 30 minutes at room temperature with 10% normal rabbit serum, diluted in PBS, and then incubated overnight at 4 ° C in a humidity chamber with monoclonal antibodies. of mice (mAbs) diluted in 10% rabbit serum. The following mAbs were used: mAbs of anti-rat macrophage 0X42, EDI, and ED2, positive T cells 0X19 against CD5, and 0X33 reactive with CD45RA positive B cells. The mAbs were obtained from Serotec, Oxford, UK. The biotinylated rabbit anti-mouse immunoglobulins, diluted 1: 300, were applied for 30 minutes. The avidin-biotin-peroxidase complex (ABCcomplex / HRP, Dakopatts, Glostrup, Denmark) was prepared as recommended by the manufacturer, and allowed to react with the sections for 30 minutes.

Finally, the sections were treated with a buffer containing 3-amino-9-ethyl-carbazole, for the development of a color reaction product. Washing in PBS followed all incubations. All preparations were counterstained with hematoxylin, mounted on Glycergel (Dakopatts), and analyzed by light microscopy. 4. Effects of alginate therapy with endostatin on tumor growth Adult BD-IX rats of both sexes (8 rats in total, plus 20 controls) were anesthetized by intraperitoneal injections of Equitesin in a dosage of 0.4 ml / 100 g of body weight. The rats were immobilized in a stereotactic structure (David Kopf Instruments, Tujunga, USA), an incision was made in the skin and a rough hole of 2 mm was made, 1 mm posterior and 3.0 mm to the right of the Bregma point, and inserted to a depth of 2.5 mm. Using an injection in the brain. After this, the 1 × 10 4 BT4c gliosarcoma cells were injected 1 mm lateral to the alginate beads at a depth of 2 mm. The alginate beads contained either 293 cells that produce endostatin or 293-mock transfectants as controls. Eight animals received implants from each cell line. In addition, as a control of normal tumor progress, 8 animals were infected with BT4C cells alone. Finally, as a control of the in vivo viability of the cells within the beads, the remaining 4 control animals received the alginate beads containing the 293-endo cells alone. The syringe was previously treated, slowly for 3 minutes (for all injections) and the closure was performed with bone wax and suture. The animals were allowed to recover from surgery under observation. During the experimental period the animals were housed in pairs at constant temperature and humidity, were fed a normal pellet diet and were given water from the tap to t libi tum.

RESULTS EXPERIMENTS IN VITRO 1 . Morphology and viability of cells encapsulated in alginate The alginate beads with diameters of 1.0 mm contained approximately 6.5 * 102 HIN 3T3 cells on the day of encapsulation (Figure IA). The cells were eventually distributed within the alginate beads, with a cell-free outer surface of 25-50 μm. During the cultivation, cell proliferation was observed within the alginate, which resulted in a cell density, increased after 3 weeks (Figure IB). After 9 weeks in the crop, multicellular spheroids were observed within the alginate beads (Figure 1C). Over 90% of the beads remained intact after 9 weeks in the culture, as assessed by the optical microscope. After about a week in the culture, a few individual cells migrated out of the alginate beads and into the culture medium, and this limited movement of the individual cells continued during the next 8 weeks of culture. The study with a scanning microscope with a confonal laser showed that about 90% of the encapsulated cells remained viable on the day of encapsulation (Figure ID). After 3 weeks in culture, about 50% of the cells originally encapsulated were viable (Figure 1E). Some of the surviving cells adapted to the alginate and formed viable multicellular spheroids, which could be clearly observed after 9 weeks (Figure 1F). At this point in time, it was difficult to assess the total number of viable cells within the beads due to the formation of multicellular spheroids. However, as shown in Figure 1F, most of the cells located in the spheroids were viable. The encapsulated BT4CnVlacZ cells expressed a constant β-galactosidase activity and eventually distributed during the total observation period of 9 weeks (Figure IG). 2. Cell kinetics of cells encapsulated in alginate Flow cytometric histograms of NIH 3T3 cells showed a change in cellular ploidy within the alginate beads 1 week after encapsulation (Figure 2B). This probably represents a polyploidization, compared to the diploid control (Figure 2A). However, after 3 and 9 weeks respectively (Figure 2C, 2D) a normalization was observed in the ploidy, with a diploid distribution, similar as for the controls. The fraction of proliferation of the cells in the S and G2M phases was 50% for the control, compared to 55% and 60% after 3 and 9 weeks in vi tro, respectively.

Release of antibodies from encapsulated hybridoma cells At the end of the first day of encapsulation, there was a release of 13 ng / (ml * hr) of MAbs in the culture medium (Figure 3). The diffusion of immunoglobulins out of the beads and in the medium increased steadily during the next days of culture, and reached a concentration of 457 ng / (ml * hr) after 12 days. MAbs production then stabilized around 400 ng / (ml * hr) during the last 3 weeks of the observation period. 4. Cell migration The migration of cells outside the GaMg spheroids stimulated with EGF was extensive, and the average excrescence area was doubled, compared with the controls (Figure 4). However, when the alginate beads containing the H528 cells were added in the presence of EGF, the cell migration was strongly inhibited, demonstrating that the encapsulated H528 producer cells effectively express an antibody directed against the EGF receptor.

. Estimating the endostatin release of the accounts.

As observed from western blotting of the conditioned medium, collected from the beads, a substantial amount of endostatin is released from the beads (Figure 6). Radioimmunoassays have shown that 10 alginate beads that produce endostatin (400 μm) with 25,000 encapsulated cells, secreted 2.5 μg / ml / 24 hours.

EXPERIMENTS IN VIVO 1. Intracranial Implants -10 The axial sections of the rat brains revealed little or no change in the parenchyma of the brain adjacent to the implant site containing the NIH 3T3 cells encapsulated in alginate (Figure 5A). HE observed little intracranial edema or swelling after 9 weeks. The alginate beads were free of any cell overgrowth and contained both individual, viable cells and multicellular spheroids (Figure 5b). The viable cells are distributed both in the center and in the periphery of the beads, with alginate cell free areas between the cells. A minimal aggregation of cells was observed around the border area between the implant and the parenchyma of the brain. 25 2. Liberation and dissemination of immunoglobulins within rat brain The beads implanted with encapsulated hybridoma cells were easily visualized after 3 weeks by intense green fluorescence (Figure 5C). Immunoglobulins were detectable in the brain tissue at a distance of at least 1 mm from the alginate beads (Figures 5C, 5D) with a gradual decrease in the fluorescence intensity of the boundary of the implant site and in the brain. For two of the experimental animals, the MAbs were detected in the entire cerebral hemisphere, where the implants were located (data not shown). The MAbs were found additionally in the leptomeninges in both hemispheres of the brain (Figure 5E), with the strongest fluorescence observed in the subarachnoidal area in the right hemisphere. Negative controls showed weak fluorescence in leptomeninges, probably caused by non-specific binding between immunoglobulins and epitopes in leptomenigeal cells (Figure 5F). However, the parenchyma of the brain was negative. The MAbs were also present in the perivascular space of the intracerebral blood vessels, with no apparent difference in fluorescence intensity between the two hemispheres (Figure 5G). The weak fluorescence present in the control was probably caused again by the non-specific binding (Figure 5H). 3. Immune responses to the producer cells encapsulated in the alginate.

Infiltration of mononuclear cells was observed in the brain adjacent to the alginate beads. The number of cells in the infiltrate decreased from week 1 to week 9. One week after implantation, a 0X42 positive microglia with a dendritic morphology in the parenchyma was observed and a reactive microglia and invading monocytes appeared in the borderline area towards the alginate accounts. EDI and ED2 stained the monocytes near the boundary zone while a few cells were stained by those mAbs elsewhere in the parenchyma of the brain. A limited number of T and B cells were also observed in the area of limit to the accounts (Table I). The number of 0X42 positive cells in the border area decreased from 62% at week one to 20% at week 9, while positive EDI cells decreased 34% at week one to 7% at week 9.

The amount of ED2 positive cells (5%), T cells (14%) and B cells (1%) changed only marginally during the observation period (Table I).

Table I Immune response of cells in rat brain tissue 9 weeks after implantation of NIH 3T3 cells in alginate Cells Immunoreactivity T cells (CD5) No reactivity B cells (CD45RA) without reactivity Microglia and macrophages (0X42) High reactivity Macrophages and monocytes (ED1 / ED2) Weak reactivity 4. Effects of alginate therapy with endostatin on the growth of thyroid Animals treated with endostatin-producing cells in alginate lived 20% +/- 4% longer than animals treated with transfected, false cells. Histological observations, > detailed revealed large areas. necrotic tumors that received the alginate therapy with endostatin (see Figure 7, panel B). Such necrotic areas were never observed in controls (transfected, false cells encapsulated in alginate; Figure 7, panel A).

DISCUSSION The results of the experiments described above clearly demonstrate that microencapsulated cells survive, proliferate and maintain their phenotypic expression for extended periods of time. It is also known that MAbs released from alginate beads have the ability to inhibit the migration of tumor cells in vi tro by interfering with EGFR, and that MAbs are released and disseminated within the rat brain. As observed by optical microscopy, NIH 3T3 cells adapted to the alginate in vi tro, and began to proliferate within a few days after encapsulation. The CLSM study revealed the viability of the cells around 90% on the day of encapsulation. During the first three weeks in culture, about 50% of the cells initially trapped died within the beads. However, after 9 weeks, the remaining cells showed the ability to form multicellular spheroids within the alginate. A cell death observed within the alginate has also been reported by others, and may be due to a reduced diffusion of oxygen, nutrients and waste products, which may eventually lead to a balance between the proliferation number and staining of the cells . A more favorable diffusion speed can be achieved by decreasing the size of the count, increasing the content of the G units, which would increase the dimensions of the pores, or change the concentration of the alginate. In addition, diffusion is dependent on the number of cells initially encapsulated within the accounts. The alginate itself is not toxic, and therefore can not be expected to contribute to observed cell death within the beads. The BT4CnVlacZ cells exhibited a strong β-galactosidase activity and eventually distributed during 9 weeks of culture. These results demonstrate that gene-specific products can also be produced for prolonged periods within the alginate accounts. The flow cytometric study showed that NIH 3T3 cells changed from a dipolide to a multiploid population after 1 week in the alginate. This indicates that the nuclei of the cells are divided, but due to the limited space within the rigid alginate network, the cells are not initially able to undergo cytokinesis. This will then result in individual cells with double and triple nuclei (Figure 2B). However, after 3 weeks, the distribution of the cell cycle was similar to the controls. This may indicate that the cells needed a certain period of adaptation within the alginate, where the individual cells with double and triple nuclei will either end their cytokinesis or die. Histograms after 9 weeks were similar to those after 3 weeks, but indicated an increase in cells in the proliferation phases. Analysis of the cell cycle distribution showed an increase in the number of proliferating cells, from 50% for control, to approximately 60% after 9 weeks. This may be due to a selection within the alginate accounts of the cells with a higher proliferative capacity during the prolonged culture of the NIH 3T3 cells. The release of the antibody from the hybridoma cells. H528 encapsulated was substantially constant around 400 ng / ml * hr from day 12 to day 33, which shows that a stable density of the hybridoma cells secreting MAbs has been established after 12 days in the culture. This finding is important for the clinical situation, since it shows that the stable production of monoclonal antibodies is achieved at a high level. The migration of cells away from the GaMg spheroids was stimulated in the presence of EGF. By adding encapsulated H528 cells to the spheroids stimulated with EGF, migration was inhibited, and the areas of growth were similar to the controls. This implies that the mechanisms of proliferation of paracrine cells were inhibited by these Mabs, probably by blocking the EGF binding domain of EGFR. Implantation of the producing cells, encapsulated in alginate in other organs outside the central nervous system (CNS) have shown an overgrowth of fibroblasts of the alginate beads, which leads to cell death and graft failure (Transplantation, 5_4 ( 1992), pages 769-774). Due to the unique localization, and the lack of fibroblasts in the CNS, the same cellular overgrowth was not observed in the present study (Figure 5A, B). Depending on the composition, alginates have been shown in some examples that initiate an immune response within the body by stimulating monocytes to produce high levels of cytokines. The cytokine stimulation part of the alginate is the M units. Therefore, an alginate with a high content of G units was selected for the experiments, in order to minimize the immune response within the brain. In the additional experiments, a low immune response has been found to cells encapsulated in alginate within the brain, with only a few microglial cells that are mounted in the brain tissue near the implanted beads. These observations further show that the producer cells, encapsulated in alginate, are an attractive treatment within the brain. A minimal aggregation of the cells around the border area between the implant site and the brain parenchyma was also observed. This may be due to NIH 373 cells escaping from the alginate beads, due to a mild immune response to the implants as discussed above, and / or due to a healing process of the tissue injury. However, the small number of producer cells which escape alginate is not considered to be a problem, since these cells would be taken into account by normal grafting against host rejection mechanisms. However, if desired, steps can be taken to prevent the escape of cells for example by covering the beads within a poly-L-lysine layer or by irradiating the cells before encapsulation., to inhibit its proliferative capacity due to that. Immunoglobulins were released from the alginate beads and disseminated in the brain parenchyma at a distance of at least 1 mm away from the boundary of the implant site. In two of the experimental animals, the MAbs were also detected in the entire cerebral hemisphere where the implants were located. This dissemination may be due to a passive diffusion process. The MAbs were also located in the subarachnoidal area and within the perivascular e of Virchov-Robin. This spreading is most likely mediated by the constant flow of cerebrospinal fluid within the CNS. Interestingly, the tumor cells follow the same dissemination pathways within the brain, which makes them accessible to the components produced by the cells encapsulated in alginate. In summary, the experiments which are described above show that the encapsulated producer cells survive and proliferate within the alginate for extended periods of time, as well as in vivo. Genetic products such as β-galactosidase are produced within the cellular cytoplasm of encapsulated BT4CnVlacZ cells for several weeks of culture. Encapsulated hybridoma cells also produce and release high amounts of MAbs in vi tro and in vivo. The migration of GaMg tumor cells is inhibited in the presence of the encapsulated H528 cells. Implants of encapsulated H528 cells also produce and release MAbs within the rat brain, and MAbs are disseminated within the brain parenchyma, as well as within the subarachnoid e and in the perivascular e. Therefore, the present invention represents a promising tool for the therapy of CNS tumors.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, property is claimed as contained in the following:

Claims (16)

1. An encapsulated producer cell capable of expressing a molecule which is an inhibitor of the growth of CNS tumors; characterized in that the encapsulation matrix is an immunoisolating alginate having a G content above 15% and the molecule is: a) a molecule that is capable of interacting with the tumor / host communication pathways; b) a molecule that is capable of affecting neovascularization of tumors; c) a molecule that is capable of interfering with the ratio of tumor cells to the extracellular matrix; or d) a monoclonal antibody capable of directly interacting with a tumor receptor, wherein the monoclonal antibody binds or interacts with the growth factor receptors derived from platelets AA and BB, the receptors of the basic fibroblast growth factor, the receptor of transforming growth factor alpha and beta, different classes of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2), tyrosine kinase receptors with inoglobulin-like and EGF-like domains such as, for example, example, TIE-1 and TIE-2 / tek, hepatocyte growth factor (spreading factor); CD-44; CDR / cyclin complexes; glycolipids on the surface of cells; glycoproteins; and proteins derived from the expression of specific oncogenes.

2. A producer cell, encapsulated according to claim 1, characterized in that the alginate has a G content above 50%.

3. A producer cell, encapsulated according to any of the preceding claims, characterized in that the alginate has a G content of 60% -80%.

4. A producer cell, encapsulated according to any of the preceding claims, characterized in that the alginate has a G content of 80% -100%.

5. A producer cell, encapsulated according to any of the preceding claims, characterized in that the expression of the molecule is capable of being activated and deactivated by an external pharmacological agent.

6. A producer cell, encapsulated according to any of the preceding claims, characterized in that the encapsulated producer cell is present in an account or a micro account.

7. A producer cell, encapsulated according to any of the preceding claims, characterized in that the CNS tumor is a brain tumor.

8. A producer cell, encapsulated according to any of the preceding claims, characterized in that the alginate is of a high purity quality, very low in endotoxin content or free of endotoxins.

9. A producer cell, encapsulated according to any of the preceding claims, characterized in that the encapsulated producer cell has a concentration of alginate which rises towards the outer surface of the capsule.

10. A producer cell, encapsulated according to any of claims 1 to 9, characterized in that the molecule is a molecule (eg, a protein, a peptide or a polysaccharide) that is capable of affecting neovascularization of tumors;

11. A producer cell, encapsulated according to any of claims 1 to 9, characterized in that the molecule is a monoclonal antibody capable of interacting directly with a tumor receptor, wherein the monoclonal antibody binds or interacts with the growth factor receptors platelet derivatives AA and BB, the receptors for the growth factor of basic fibroblasts, the transforming growth factor receptor alpha and beta, different classes of vascular endothelial growth factor receptors (VEGFR-1 and VEGFR-2), tyrosine kinase receptors with immunoglobulin-like and EGF-like domains such as, for example, TIE-1 and TIE-2 / tek, hepatocyte growth factor (spreading factor); CD-44; CDR / cyclin complexes; glycolipids on the surface of cells; glycoproteins; and proteins derived from the expression of specific oncogenes.

12. A method for producing a producer cell, encapsulated according to any of claims 1 to 11, characterized in that it comprises the encapsulation of producer cells in a one-step process.

13. A method for producing a producer cell, encapsulated according to any of claims 1 to 12, characterized in that it comprises the dropwise addition of an alginate solution containing viable cells to a calcium solution.

14. A producer cell, encapsulated according to any of claims 1 to 11, characterized in that it is for use in medicine.

15. The use of a producer cell, encapsulated according to any of claims 1 to 11 in the preparation of a medicament for treating a CNS tumor.

16. The use of an encapsulated producer cell according to any of claims 1 to 11 for treating a brain tumor.