AU777526B2 - Pharmaceutical compositions comprising immortalised endothelial cells - Google Patents

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING IMMORTALIZED ENDOTHELIAL CELLS The present invention relates to pharmaceutical compositions for the detection and treatment of sources of angiogenesis and more particularly of tumours, based on the capability of endothelial cells injected into a patient to target primary or secondary angiogenesis sources.

Angiogenesis or the formation of new vessels is an essential step in tumour development In physiological situations it plays a fundamental part in reproductive functions (regeneration of the uterus, formation of corpus luteum and placenta), tissue repair following after injury and ischaemia, and in embryonic development. It is involved in certain diseases such as retinopathy, infantile haemangioma, rheumatoid polyarthritis, duodenal ulcers and evidently in tumour development.

Conventional cancer treatments have recourse to a multidisciplinary approach and combine surgery, radiotherapy, hormonal therapy and chemotherapy. Aside from their potential deleterious effects, their efficacy varies extensively depending upon the type of cancer. For example, patients suffering from acute lymphoblastic leukaemia (in children) or cancer of the testicle are very effectively treated by chemotherapy. Nevertheless, current chemotherapy is rarely effective against solid tumours, in particular after the onset of metastasis. In this context, other innovative gene therapy approaches have been put forward. They may be summarized as follows i) antisense nucleotide strategy using oligonucleotides, oligoribonucleotides and ribozymes; ii) gene therapy using tumour suppressor genes (anti-oncogenes); iii) gene therapy using cytokines or tumour vaccination; iv) viral therapy to induce enzyme activity on a prodrug; v) directed antibody therapy.

Approaches I) to iv) are characterized by their lack of selectivity, that is to say their impossible targeting of a tumour in the body. The directed antibody approach does allow targeting of a tumour. Numerous monoclonal antibodies have in vitro and in vivo anti-tumorous activity when coupled to cytotoxic agents such as doxorubicin, methotrexate, ricin A toxin, exotoxins of pseudomonas and radio-isotopes, or to an enzyme acting on a prodrug. The objective of all these approaches is to destroy the cancerous cells.

Another approach consists of endeavouring to target the formation of vessels associated with the cancerogenesis Numerous arguments support the fundamental role of non-controlled angiogenesis in the development of tumours, tumour development deprived of blood vessel supply being rapidly halted if a vascular network no longer provides nutrients. Under these conditions, the tumour does not exceed 1 mm 3 These data are confirmed both for primary tumours and for metastases. In addition, the density of tumour vascularization is associated with tumour aggressiveness as measured by the incidence of metastases and/or reduced patient survival.

This angiogenesis is dependent firstly upon angiogenic factors which include basic fibroblast growth factors (bFGF), and vascular endothelial growth factors (VEGF) and secondly upon their target, the endothelial cells. The influence of these factors on the endothelial cells is both proliferate and chemo-attractive. This attraction of the endothelial cells by angiogenic factors synthetized by tumour cells has, for example, been described in the PCT international patent application published under number WO93/13807.

For the treatment of angiogenesis sources, the prior art put forward various compounds for the inhibition of tumour angiogenesis. Some products are already at the human trial stage These compounds can be divided into several families: a) Compounds which inhibit the release of angiogenic factors such as interferons. They are used in efficient manner in tricholeucocyte leukaemia, chronic myeloid leukaemia and Kaposi's sarcoma.

b) Compounds which fix the free angiogenic factors such as tecogalan sodium (D-Gluco-D-galactan sulfate), pentosan polysulfate (analogue of heparin), suramin.

Trials in human therapy are very few for tecogalan and pentosan polysulfate. On the other hand wider testing has been possible with suramin in patients suffering from metastasis of cancer of the prostate, with results showing tumour resolution. It would appear, however, that suramin is highly toxic.

c) Compounds which block the effect of angiogenic factors such as platelet factor 4 TNP-470, thalidomide. Trials in human therapy are very limited for PF-4. In respect of TNP-470, a phase 1 trial has been conducted in patients with cancer of the cervix, or cancer of the prostate. Phase 1 trials are under way for thalidomide.

d) Compounds which inhibit interactions between the extra-cellular matrix and the endothelial cells such as Batimastat. A phase 1 trial is currently being conducted in patients suffering from cancerous ascites with intraperitoneal treatment, and another in patients suffering from cancerous pleural effusion.

e) Compounds which target the endothelial cells such as CM-101, an exotoxin (streptococcus B) or finally other compounds such as linomide, octreotide or the retinoids. A phase 1 trial has already been conducted with CM-101 in patients suffering from non-curable tumours, showing significant tumour reduction in some patients.

Linomide has already been used in a phase 1/II trial on renal carcinomas, melanomas and rectal cancers. No antitumorous activity has been identified. One trial currently being conducted is assessing the benefit of octreotide (Sandostatine) in patients suffering from breast cancer treated with tamoxifen. Finally, anti-tumorous activity has been observed with the use of retinoids in patients suffering from cervix or skin carcinoma when associated with alpha 2a interferon treatment.

Anti-angiogenesis treatments, although highly promising, have not yet shown their efficacy in Man.

As for the treatment and detection of angiogenesis sources, approaches based on the use of antibodies have been proposed, such as in US Patents 5 776 427 and 5 660 827 for example. These antibodies are directed against antigens present on the surface of vessels undergoing formation. Recently, Sipkins et al. [12] using antibodies directed against alphavbeta3 integrin labelled with gadolinium have been able to identify a tumorous angiogenic source in animals.

The present invention sets out to offer new pharmaceutical or diagnostic compositions for angiogenic sources, and more particularly for cancers, based on the selective attraction of endothelial cells by sources of angiogenesis.

However, it must be recalled that not all endothelial cells are identical. Indeed, in adults endothelial cells form a very heterogeneous cell population, not only between organs but also within one same organ between vessels of different sizes. Endothelial heterogeneity is characterized by morphological differences but also by the expression of molecular markers specific to one or more populations of endothelial cells. For example, in the central nervous system (CNS) the endothelial cells of cerebral microvessels, in association with the astrocyte cells of the brain parenchyma, form the blood-brain barrier (BBB).

In contact with the brain parenchyma, the cerebral endothelial cell develops morphologic and functional specialisations which are not encountered in other organs, such as the tight junctions. These specialisations of the cerebral vascular network (described under the term bloodbrain barrier) enable the cerebral endothelial cells to regulate the exchanges of circulating factors (circulating molecules, ions and cells) between the blood and tissue compartments in fine-tuned bi-directional manner.

The development of mammalian cell preparations for gene therapy therefore raises a problem regarding the homogeneity and characterization of said cells. An efficient solution to this problem consists of immortalizing the cells. In the prior art, the description has been made of brain endothelial cell lines, or of immortalized retinal endothelial and epithelial mammalian cell lines possibly carrying a transgene that can be used for the treatment of neurological diseases including tumours. Special mention may be made of the work conducted by the Applicant disclosed in international PCT patent applications published under numbers W096/11278 and W097/40139.

The immortalized lines of rat brain endothelial cells described in these patent applications, and of human brain or retinal cells may be used to treat brain tumours, in particular in patients suffering from glioblastoma.

Therefore, rat brain microvessel endothelial cells were immortalized by transfection of primary cultures with a plasmid containing the gene coding for the E1A antigen of Adenovirus and a neomycine-resistant neo gene. Several clones were obtained and one of them, called RBE4, is described in the above-mentioned PCT patent applications.

These immortalized cells have a non-transformed normal phenotype: in culture they form a monolayer, their proliferation is inhibited by contact and is dependent upon the addition of serum and bFGF growth factor, and finally they are not tumorigenic in vivo in Nuda mice.

Immunohistochemistry detected the expression by these cells of the endothelial differentiation markers i.e. the Factor VIII-bound antigen and the conversion enzyme of angiotensin. Like the primary cells from which they are derived, these immortalized cells do not express in structural manner some markers that are characteristic of endothelium with BBB: gamma-glutamyl transferase and alkaline phosphatase. This expression is however induced in the presence of astrocyte conditioned media, suggesting that these immortalized cells have maintained their differentiation potential in respect of a phenotype close to that of the in vivo brain endothelium These brain endothelial cell lines are capable, in vitro and after intra-cerebral transplantation, to show these specialisations. Implanted in the rat brain, these cells survive over the long term, integrate into the brain tissue and its vascularization. In particularly advantageous manner, electronic microscopy has shown the integration capacity of these cells in the vessel walls and parenchyma of the host These cells, genetically engineered to express the murine NGF growth factor, are also able to integrate into brain tissue and to secrete this factor in situ whose biological and neurotrophic activity vis-A-vis cholinergic neurones was able to be followed over several weeks after implantation The integration and proliferation capacity of these endothelial cells after co-implanting with 9L glioma cells into the brain of 344 Fischer rats has also been demonstrated In this same experimental model, it was shown that a brain endothelial cell line, genetically modified for the production of murine Interleukin-2 (IL-2) is able to considerably limit the tumour growth of coimplanted 9L cells and to significantly extend the survival of the grafted rats On the basis of these results, work has been conducted by the Applicant on an experimental glioblastoma model in the rat. Two rat brain endothelial lines were used, one producing the human immunostimulating cytokine Interleukin-2, and the other line expressing the HSV-I TK enzyme (suicide gene) The research work conducted by the Applicant on the immortalization of mammalian cells, and more particularly of brain endothelial cells, enabled the Applicant to obtain a substantial, homogeneous and fully characterized quantity of material to be grafted or injected, making possible the application of an efficient method for the gene treatment of disease in patients. Under the scope of this invention, it was found that after being injected into the blood compartment irrigating the CNS, the genetically modified immortalized endothelial brain cells, such as the RBE4 cells not expressing any transgene, the RBEZ and RBE4/GFP cells expressing a transgene, are able to survive and to integrate in the vascular wall of brain microvessels and in the brain parenchyma. The demonstration of the advantage of this approach required technical skills both in the development of the cell preparations and the compositions containing the same which were injected, and in the operating modes of injection.

The work conducted by the Applicant on the injection of cells into animals provided the opportunity of identifying the deleterious effect of the presence of cell aggregates in the injected compositions, such as vascular brain damage or pulmonary embolism. Yet, in surprising manner, the deleterious effect induced by the presence of these cell aggregates at the time of injection does not appear to have been given consideration to date. Yet it was proposed in the prior art to inject particles whether conjugate or not with an active agent to carry out diagnosis or implement therapy. For example, the studies may be cited which were conducted on synthetic microspheres of specifically determined size: the injection of 75 to 150-micron spheres into the heart vessels causes myocardial necrosis (Battler et al., 1993, J. Am. Coll. Cardiol., 22: 2001-2006), the injection of 7-micron spheres into swine arteries, to the proportion of 105 particles per gram of myocardium, does not cause any deleterious effect on myocardial tissue (Arras et al., 1998, Nature Biotechnology, 16: 159-162), the injection of microspheres 48 microns in diameter (900 microspheres) into the right internal carotid causes brain infarction in the parietal-temporal cortex, the corpus callosum, the hippocampus, the thalamus and the lenticular nucleus (Miyake et al., 1993, Stroke, 24: 415-420).

The injection into Man has also been described of radiolabelled albumin microspheres having a size of 15 to microns, into the common or internal carotid arteries to detect infarcted regions of the brain using brain tomoscintigraphy techniques (Verbas et al., 1976, J. Nucl.

Med., 17: 170-174) with no deleterious effect being reported.

In the area of extracorporeal circulation (ECC), in which the generated particles are likely to have a deleterious effect on the body, it was suggested to use 20-micron filters to reduce by 90 the number of potentially deleterious particles (Loop et al., 1976, Ann.

Thorac. Surg., 21: 412-420).

However, as indicated above, the rare work conducted in the prior art on the injection of cells, endothelial cells in particular, does not disclose any deleterious effects of injected cell compositions due to the presence of cell aggregates. Hence, international patent application PCT W093/13807 describes the intravenous injection of non-immortalized 2 x 106 endothelial cells into the tail vein of mice, and does not mention the observation of any deleterious effect related to the formation of cell aggregates Assuming that indeed no deleterious effect was observed, it is probable that the low number of cells injected in 30 g mice, in the order of 2 x 106, that is to say twice less than under the present invention in 300 g rats, does not induce a deleterious effect despite the formation of cell aggregates.

Likewise, the authors of the work concerning the intra-arterial injection (intra-femoral) of 1 to 2 x 106 non-immortalized endothelial cells into the lower limb of the rat, do not report the observation of any deleterious effect and do not suggest the problem of the formation of aggregates (Messina et al., 1992, proc., Natl. Acad. Sci., 89: 12018-12022) Even though this work does not consider the deleterious effects caused by the injection, it must be pointed out that the number of injected cells is low, of the order of 50 times less than the number used under the present invention. Moreover, the targeted organs in this work are the vessels of the lower limb, whose tolerance to ischaemia is greater than for other organs.

In addition, it is indicated that the experimenters clamped the femoral artery for one hour to reduce the blood flow rate and promote the adhesion of the cells to the vascular walls.

The purpose of the present invention is therefore to provide an efficient but simple solution with which to avoid the deleterious effects of injections of preparations of endothelial cells, and therefore to allow their application to the diagnosis or treatment of angiogenesis sources and more particularly of cancers.

This purpose is achieved in remarkable manner through the use of mammalian endothelial cells that are i) immortalized, ii) optionally contain an active substance for the diagnosis or treatment of angiogenic sources, iii) are able to fix themselves to the angiogenic sources, in order to prepare a pharmaceutical composition for the diagnosis and/or treatment of angiogenic sources and more particularly of cancers.

Advantageously, the above endothelial cells are immortalized and are non-tumorigenic.

In best preferred manner said composition does not contain any aggregate of said cells of a size likely to lead to transient or permanent dysfunction in patients.

The endothelial cells advantageously comprise an active substance for the diagnosis or treatment of angiogenic sources and more particularly of cancers. These may be cells: transfected with at least one gene encoding said active substance, such as for example a suicide gene, and/or loaded with said substance, such as for example a labelling compound of paramagnetic type, or an anticancer substance.

By active substance for diagnosis is meant any pharmacological agent, detectable either directly or indirectly after activation or not by another substance administered to the patient, for example by means of an imaging device or by assay of a sample of body fluid such as blood.

Also, by active substance for treatment, is meant any pharmacological agent having an activity against angiogenic sources, either directly or indirectly, after activation or not by another substance administered to the patient.

The present invention concerns the detection or treatment of angiogenic sources and in this respect particularly targets cancers, such as breast, colon, brain, liver, lung cancer etc., but also the treatment and more particularly the detection of all other active angiogenic sources, such as areas of inflammation, tissue lesions, grafts, retinopathies, etc..

The compositions of the invention are also remarkable in that they may be used to simultaneously conduct diagnosis and treatment of angiogenic sources and more particularly of cancers, evidently depending upon the active substance or substances contained by the endothelial cells.

Therefore, the compositions of the invention offer, among other advantages, the possibility of guiding a surgeon during diagnosis and/or therapy procedure by means of the use of endothelial cells labelled with an active substance that is directly or indirectly detectable.

In the prior art, international patent application PCT WO93/13807 is known, which describes the injection of non-immortalized endothelial cells via intravenous route for the treatment of tumours. But the endothelial cells described in this document are not immortalized and therefore are not able to deliver the same product to each patient. On the contrary, the immortalized cells which come within the scope of the present invention may be standardized and used in a pharmaceutical preparation both for diagnosis and for the treatment of cancers.

The invention more particularly concerns a pharmaceutical composition for use in the diagnosis or treatment of angiogenic sources through administration to a patient by systemic route, said composition containing mammalian cells optionally containing an active substance for the diagnosis and/or treatment of angiogenic sources and more particularly of cancers, characterized in that said cells are immortalized.

Preferably, said cells are immortalized and nontumorigenic The endothelial cells advantageously contain an active substance for the diagnosis and/or treatment of angiogenic sources and more particularly of cancers. As indicated above, they may in this case be cells transfected with at least one gene coding for said active substance or loaded with said substance.

The immortalization of the cells may be conducted using any method known to persons skilled in the art, such as those described in PCT patent application published under numbers WO96/11278 and WO97/40139. Under the invention particular preference is given to immortalized cells since the latter have the advantage of standardizing production and of producing cells in large quantities with high quality criteria. In addition, the non-tumorigenic character of the immortalized cells may be obtained using any method known to persons skilled in the art such as the one described in the PCT patent applications mentioned above.

According to one best preferred embodiment of the invention, said composition does not contain any aggregate of said cells of a size that is likely to lead to transient or permanent dysfunction in said patient.

The compositions of the invention may therefore contain a high number of cells, in the order of 1000 to 300 000 cells per microlitre of composition, which is much higher than the possible or allowed quantity in the prior art, with which it is possible to obtain a biological effect, for diagnosis or treatment, that is efficient without inducing a deleterious effect which could cause a transient or permanent decrease in the blood supply to the organ, of pulmonary embolism type, brain ischaemia, peripheral ischaemia, even death.

The compositions of the invention advantageously contain a pharmaceutically acceptable vehicle allowing the survival of said cells and not promoting their reaggregation.

The tests conducted under the invention made it possible to characterize the size of the aggregates likely to induce deleterious effects at the time of systemic injection of the composition containing the cells.

Therefore, in advantageous manner, a composition of the invention does not contain any cell aggregates of a size greater than approximately 200 microns, preferably greater than 50 microns and further preferably no greater than microns.

The invention concerns the endothelial cells derived from any type of mammalian organ or tissue, including of human origin, and more particularly brain or retinal cells.

The absence of any cell aggregate likely to lead to transient or permanent dysfunction in patients who have been administered a composition of the invention may be obtained using any biological, chemical or physical treatment of the cells preventing their aggregate formation or specifically eliminating the aggregates of said cells of a size greater than approximately 200 microns, preferably greater than 50 microns and further preferably greater than 30 microns. After this treatment the cells are advantageously suspended in a medium enabling their survival and not promoting their reaggregation. Such medium is for example any nutrient medium which does not promote aggregation such as PBS glucose not containing any calcium or magnesium.

A biological treatment of the cells according to the invention consists, for example, of genetically modifying said cells using a nucleic acid sequence expressing an agent preventing the formation of aggregates or inhibiting the expression of an agent promoting the formation of aggregates of said cells. Two approaches may therefore be used: deletion of sequences coding for adhesion molecules such as: Z01, Z02, E-selectine, V.E. Cadherine, ICAM-I, occludin, P-CAM, etc.., or insertion of sequences coding for molecules preventing the formation of aggregates, such as negative dominants of the adhesion molecules cited above, or coding for decoy proteins.

Physical treatment of cells according to the invention consists, for example, of filtering or screening. In addition to the removal of aggregate, this filtration or screening offers the advantage of making available a cell population of homogeneous size. This filtration or screening is conducted in the following manner: the cells are filtered using screening filters advantageously of 30-micron size, then diluted and gently detached for example by multiple pipetting, and the cell suspension is then aspirated into a syringe. The filter is previously soaked in sterile physiological serum then disinfected with 1000 alcohol, air dried, and re-soaked in sterile physiological serum. The filter is then positioned between the needle and the tip of the syringe containing the cells. The piston is advanced gently to achieve drop by drop outflow of the diluted cells.

But physical treatment may also be made by FACS type sorting (Fluorescent Analysis Cell Sorting) Chemical treatment of the cells according to the invention consists, for example, of trypsinizing the cells or subjecting them to the action of another protease.

The endothelial cells of the compositions of the invention may or may not be transfected with one or more genes encoding an active substance which can be used for the treatment or diagnosis of angiogenic sources. Under the present invention by transfection with one or more genes encoding an active substance for diagnosis or treatment of angiogenic sources is meant the transfection of cells with a fragment of nucleic acid, as expression vector, integrated in the genome or present in the cytoplasm of cells, and able to allow the expression of polypeptide(s), protein()s or a viral vector forming an active substance directly or indirectly. As examples which may be cited are immortalized non-tumorigenic endothelial brain cells transfected with a gene encoding an active substance that is the subject of the compositions described in international patent application PCT W096/11278 whose teaching is incorporated in this application by reference.

The size of the aggregates which are not likely, at the time of injection of the compositions of the invention into the patient, to lead to transient or permanent dysfunction depends upon the route of administration.

Therefore, organ selective arterial injections enter said organ directly without previously passing through a filtering organ such as a lung. Consequently for intraarterial injection, the tolerated aggregate size is smaller than for intra-venous injection. After injection into the vein of the fold of the elbow, the lung filter may come into action and limit the presence of aggregates in the other organs. However, the risk of a deleterious effect at the time of intra-venous injection does exist since the Applicant observed death in animals probably due to pulmonary embolism at the time of injection of endothelial cells which were not previously filtered.

In addition, interpretation of the data of the prior art and the experiments conducted by the Applicant, appear to indicate that spheres that are greater than 40 microns in size are likely to have a deleterious effect on target tissues via intra-arterial route. Therefore, if it is considered that a cluster of cells, endothelial cells for example, acts as a sphere it is recommended according to the invention to remove the aggregates that are larger than 30 microns. Nevertheless, the physical criteria of cell deformability in a microvessel differ from those for synthetic particles, and this parameter must be given consideration during cell treatment such as filtration, for which the use of a 30-micron filter permits the removal of aggregates over 30 microns in size and consequently the remaining cells, at least 90%, are isolated cells whose average diameter, for an endothelial cell for example, is 10 microns.

Consequently, the invention more particularly concerns: firstly a composition for intra-arterial administration to a patient, advantageously intra-carotid administration, characterized in that it does not contain any cell aggregate that is greater than 50 microns in size, and preferably no greater than 30 microns, and secondly, a composition for intra-venous administration to a patient, characterized in that it does not contain any cell aggregate greater than 200 microns in size, and preferably no greater than 100 microns.

These two routes of administration must be taken into consideration for the selection of cells injected into the target organ or tissue It is recommended to target an organ by injecting the compositions of the invention into the artery directly irrigating the target organ.

Conversely, the injection of said compositions via intra-venous route requires the prior selection or imparting to the cells of special properties enabling them to target the organ or tissue concerned. This may for example involve selecting endothelial cells having specific binding properties, or gene modification to impart the required properties to the target organ.

Injection by intra-arterial route, preferably by intra-carotid route, for treatment applications to the Central Nervous System, is a preferred embodiment for the use of the compositions of the invention. Even though injection by systemic route appears to be the most suitable, since it provides for the widest biodistribution, analysis of this parameter by the Applicant to optimise the gene therapy method using compositions of the invention led to giving preferential choice to the carotid vascular network. This network provides 80 of the cerebral blood flow required by Man for the proper functioning of the CNS, and is accessible in human clinics and for animal testing.

Therefore, under the present invention, the Applicant has shown that the injection of endothelial cells into the carotid is feasible while paying due heed to blood flow.

The choice of this route of administration minimizes as far as possible the changes in cerebral blood flow.

Indeed, the flow inside the internal carotid is never interrupted throughout this procedure. In addition, analyses made on control animals have shown no damage to the parenchyma. In the rat, the injection is made into the general carotid circulation and distributes itself to the entire area concerned. In man, it is possible, using neuroradiology treatment techniques, to inject smaller vessels using a catheter, such as the arteria cerebri media, the arteria cerebelli anterior or the arteria cerebelli posterior, or even branches of these arteries, and therefore to achieve potentially better targeting and reduced deleterious effect. Evidently, these techniques are invasive, but no more than an arteriography which requires the same procedure. On the other hand they are far less invasive than intraventricular or intracerebral injections for delivery of a gene therapy substance.

Under certain conditions, intra-carotid injection has led to death and parenchyma lesions. Death was in general immediate and most frequently associated with respiratory disorders. The most plausible explanation is that the injection of cells causes fatal pulmonary embolism.

Parenchyma lesions occurred when the quantities of endothelial cells were high and if the cell suspension was not filtered. This data confirms the concept of the present invention, according to which it is the cell aggregates which are responsible for brain parenchyma lesions and mortality since they are minimized after filtration. Brain parenchyma lesions most probably relate to brain infarction since they occur as a hypersignal at T2 and are located on the vascular territory of the internal carotid. Filtration made it possible to do away almost entirely with these deleterious effects, in some rare cases dilatation of the lateral ventricle was visible on the side of the injection.

The compositions of the invention can be used both in the area of diagnosis and for gene therapy of angiogenic sources, and more particularly of cancers. They can be used in remarkable manner to diagnose and/or treat at their earliest stage of development one or more tumour formations spread throughout the body.

As indicated above, the use of pharmaceutical compositions for the diagnosis or treatment of cancers is based on the affinity of the endothelial cells, injected into the blood compartment of a patient, for angiogenic sources.

This affinity has already been put forward [12] to detect angiogenic sources using antibodies labelled with gadolinium directed against a surface protein specific to endothelial cells (alphaVbeta3) and visualized under MRI.

The work conducted by the Applicant under the present invention shows that the endothelial cells injected by intra-carotid route or by intra-venous route are quickly identified in numerous organs, namely the kidney, lung, liver, brain, eye, heart, spleen. The injected endothelial cells then fix themselves and proliferate at the site of angiogenesis. The endothelial cells can then be used as angiogenesis tracers and allow the compositions of the invention to be used for the detection of angiogenic sources and more particularly for the diagnosis of cancers.

The invention therefore more particularly concerns a pharmaceutical composition as described previously for the detection of angiogenesis sources, and hence the diagnosis of cancers, by administration to patients via systemic route, characterized in that said cells express an active substance which has a tracer detectable by: direct visualization, assay of the signal produced by said tracer in a sample of the patient's body fluid, or a device able to use the signal produced by said tracer to produce images of at least part of the body of the patient possibly showing sources of angiogenesis.

More particularly, the invention concerns compositions in which said cells express a tracer that can be directly visualized or detected: by an imaging method such as scintiscanning, positron emission tomography, MRI, ultrasonography, laser, CT, or by a biological or biochemical assay method such as the ELISA, RIA, HPLC techniques, mass spectrometry, chemiluminescence, bioluminescence, western-blot or electrophoresis.

The invention concerns a method to detect or diagnose angiogenic sources in a patient, characterized in that it comprises the administration, by injection into the vascular network, of a pharmaceutical composition according to the invention, then taking a body fluid sample from the patient, and measurement of the quantity of active substance in said sample, the latter indicating the existence and extent of the angiogenic source.

Regarding the assay of a body fluid sample, after taking the sample from the patient the quantity of active substance is measured in said sample. Since the labelled endothelial cells fix themselves to the angiogenic source, the assay of the tracer will indicate the existence and extent of the angiogenic source.

The assay device is chosen from among the biological or biochemical assay devices such as ELISA, RIA, HPLC techniques, mass spectrometry, chemoluminescence, bioluminescence, western blot or electrophoresis.

As indicated above, the tracer expressed by the cells may be a substance: fixed to the cells during their preparation, such as a paramagnetic compound for example, gadolinium, MIONs or a radioactive compound, a fluorescent compound, etc..

encoded by the gene with which said cells are optionally transfected, such as a fluorescent compound for example, a secreted protein that can be assayed in a sample of body fluid.

Various tracer detection devices may be used under the present invention. Among these the invention considers the imaging techniques described below.

Consequently, the invention concerns a method for detecting and diagnosing angiogenic sources and more particularly cancers in patients, said method comprising the administration by injection into the vascular network of the above diagnostic composition, followed by exposure of all or part of the body of said patient to an imaging method that is able, using the signals emitted by the tracer, to produce images of the angiogenesis source possibly present in the patient.

Several imaging techniques for the detection of angiogenic sources are possible under the invention, among which preference is given to scintiscanning, positron emission tomography, and MRI, but mention may also be made of ultrasonography, CT or even laser.

Consequently, the compositions for the diagnosis or detection of angiogenic sources and the related methods of the invention more particularly concern the use as tracer or contrast medium carried by the endothelial cells of: technetium 99, to detect said cells at an angiogenic source using a scintiscanner a positron-emitting compound to detect said cells using positron emission tomography equipment, a paramagnetic compound to detect said cells at an angiogenic source using MRI equipment, an iodine compound to detect said cells at an angiogenic source using CT equipment.

MRI, ultrasonography, CT are generally used for a tumour that has already developed, causing a symptom which prompts the request for additional tests.

The compositions of the invention are therefore remarkable since they can be used to vector contrast medium used in imaging. Endothelial cells selectively fix themselves to angiogenic sources, and a substantial local concentration of contrast medium is observed which may be detected by MRI or any other technique indicated above.

Two approaches are then possible: If the risk zone is known in advance: for example the liver of a patient suffering from cirrhosis and likely to develop hepatocarcinoma, then localised MRI is conducted on this organ.

If the risk zone is not known beforehand: global investigation is carried out using entire body MRI imaging to detect angiogenic sources among the organs likely to contain a tumour formation.

The invention also concerns the treatment of angiogenic sources and possibly the prevention of their onset, more particularly of cancers. As indicated above, this use is based on the affinity of endothelial cells injected into a patient's blood compartment for angiogenic sources.

Some authors [13, 14] have put forward an ideal method for the distribution/release of active substance ("drug delivery system") which requires fulfilment of the following criteria: i) Selectivity: the method must allow conveying of the active substance with no leakage during transport, no degradation and no distribution in a non-affected tissue.

ii) The dose factor: the method must allow an active substance to be released at the proper dose onto the therapeutic target, and this dose may be released in controlled, predictable manner.

iii) Immunogenicity: the method must be able to be given repeated administration without causing an immune response and sensitisation.

iv) Field of application: it would be advantageous if several diseases could be treated using different active substances.

v) Pharmaceutical feasibility: the formulation should be convenient for mass production and easy to administer to patients.

The invention is characterized in that the active substance is a pharmacological agent having activity against an angiogenic source, either directly or indirectly, whether or not after activation by another substance or by a source of energy administered to the patient.

The compositions of the invention meet these criteria. Consequently, the invention relates to the previously described compositions for administration by systemic route, advantageously by intra-arterial route, as part of a gene therapy method to treat an angiogenic source and more particularly a cancer in patients, characterized in that the cells of said composition are transfected with at least one gene encoding an active substance for the treatment or prevention of said angiogenic source, and more particularly of a cancer.

The substance encoded by the gene with which the cells have been transfected may be directly active on the tumours or indirectly active, that is to say requiring: the administration to the patient of a second substance interfering with the first or with the gene encoding the latter, to achieve therapeutic effect on the tumour, exposure to a source of energy, transformation by a substance naturally present in the patient's body.

The gene with which the cells of the compositions of the invention are transfected is chosen for example from among the suicide genes such as thymidine kinase, immunomodulating cytokines such as 112 or 1112.

The compositions of the invention which can be used to treat a cancer are for example given at a dosage so as to allow administration of the order of 1 million de 200 million cells per kilogram body weight of the patient to be treated.

As an example embodiment of a method for treating cancers with the compositions of the invention, the method may be cited which consists of injecting a composition in which the immortalized endothelial cells contain a suicide gene. With this type of composition it is possible to meet all the criteria set forth above. Since: i) The endothelial cells will be attracted by the angiogenic foci, providing for good selectivity of the therapeutic product.

ii) Controlled release of the active substance is possible since the cells expressing the thymidine kinase enzyme will only be able to act when the prodrug (ganciclovir) has been ingested by the patient.

iii) The injected endothelial cells will cause immune reactions of xenogenic or allogenic type depending upon the type of cells chosen, but strategies, such as those described below, will make it possible to achieve maximum reduction of this risk. The transplantation of tissues, organs or cells between individuals (allografts) or between species(xenografts) generates a variety of immune responses.

Three types of rejection have been identified after xenograft: hyperacute rejection, acute vascular rejection and cell rejection Hyperacute rejection is due to pre-existing humoral immunity of the host vis-A-vis the donor. This reaction leads to the destruction of the graft within a few minutes or hours. This mechanism is dependent upon the activation of the complement. In this response, the endothelial cells are the first targets. Strategies have been developed to combat this hyperacute rejection.

They have recourse to the use of complement inhibitors whose leading molecules are: CD59, CD55 or DAF (decay accelerator factor), or CD46 or MCP (membrane cofactor protein) The other rejections occur several days after transplantation and therefore give time for possible MRI detection or even treatment.

If this approach is insufficient, the use of human cells may be made, corresponding in this case to conventional allografts. Immunosuppressive treatments are frequently associated therewith. It is also possible to consider transfecting these cells with constructs intended to reduce the immune response. The envisaged molecules are: TGF-betal, IL10 or the Fas ligand molecule iv) On account of the targeting of formations of angiogenic type and not of cellular type, the compositions of the invention are able to cover a wide field of application.

v) Each patient may benefit from one same product since the endothelial cell lines are perfectly stable and characterized.

The work already conducted has led to selection of a HSV-I TK (thymidine kinase) line. The injection of a prodrug such as ganciclovir can trigger the programmed death of endothelial cells solely at the angiogenic source with no toxic effect on the entire body.

004529?00 24A It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction.

o *o• oO o o* *o• o• BIBLIOGRAPHICAL REFERENCES 1) Folkman J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med. 1: 27- 31.

2) Gingras Bliveau R. (1997) L'angiog6nse tumorale. Med Sci 13: 1428-1435.

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Lal Indurti Couraud P.O, Goldstein G.W. Laterra J. (1994) Endothelial cell implantation and survival within experimental gliomas. Proc. Natl. Acad.

Sci. U.S.A. 91: 9695-9699.

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7) Ojeifo Forough Paik Maciag T., Zwiebel J.A. (1995) Angiogenesis-directed implantation of genetically modified endothelial cells in mice. Cancer Res. 55: 2240-2244.

8) Perico Remuzzi G. (1997) Prevention of transplant rejection. Drugs, 54: 533-570.

9) Quinon6ro Tch6ling6rian J-L, Vignais L, Foignant-Chaverot N, Catherine Colin, Philippe Horellou, Roland Liblau, Gilles Barbin, A. Donny Strosberg, Claude Jacque and Pierre-Olivier Couraud (1997) Gene Therapy 4, 111-119.

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Cell. Physiol. 159: 101-113.

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Claims (18)

1. Pharmaceutical composition when used in the diagnosis and/or treatment of angiogenic sources by administration to a patient via systemic route, said composition containing mammalian endothelial cells containing an active substance that is a pharmacological agent presenting an activity against the angiogenic sources, either directly, or indirectly after activation or not by another substance or a source of energy administered, for the diagnosis and/or the treatment of angiogenic sources, characterized in that said cells are immortalized, wherein said composition does not contain any aggregate of said cells of a size likely to lead in said patient to transient or permanent dysfunction.

2. Pharmaceutical composition according to claim 1, characterized in that said cells are not tumorigenic.

3. Pharmaceutical composition according to either claims 1 or 2, characterized in that it does not contain any aggregate of said cells of a size greater than 200 microns.

4. Pharmaceutical composition according to either claim 1 or 2 wherein it does not contain any aggregate of said cells of a size greater than 50 microns. Pharmaceutical composition according to either claim 1 or 2 wherein it does not contain any aggregate of said cells of a size greater than 30 microns. 20 6. Pharmaceutical composition according to any of the preceding claims, characterized in that it contains in the order of 1000 to 300,000 cells by microliter of composition.

7. Pharmaceutical composition according to any of the preceding claims, characterized in that the endothelial cells are associated in said composition with a pharmaceutically acceptable vehicle allowing survival of said cells and not promoting their re-aggregation. 004531465 28

8. Pharmaceutical composition according to any of the preceding claims for administration to a patient via intra-arterial route, characterized in that it does not contain any cell aggregate of a size greater than 50 microns.

9. Pharmaceutical composition according to any one of claims 1-7 for administration to a patient via intra-arterial route, characterised in that it does not contain any cell aggregate of a size greater than 30 microns. Pharmaceutical composition according to either claim 8 or 9, wherein the intra-arterial route is an intra-carotid route.

11. Pharmaceutical composition according to any of claims 1-7, for administration to a patient via intra-venous route, characterized in that it does not contain any cell aggregate of a size greater than 200 microns.

12. Pharmaceutical composition according to any one of claims 1-7 for administration to a patient via intra-venous route, characterised in that it does not ""contain any cell aggregates of a size greater than 100 microns.

13. Pharmaceutical composition according to any of the preceding claims, l characterized in that the endothelial cells are transfected with at least one gene i encoding an active substance and/or loaded with said substance.

14. Pharmaceutical composition of any one of the preceding claims, wherein g said active substance is a tracer detectable: 0 0 00 by direct visualization, or 000* by assay of a signal produced by said tracer in a sample of body fluid of the patient, or by a device able, from a signal produced by said tracer, to produce images of at least part of the body of the patient. 004531465 29 Pharmaceutical composition according to any one of the preceding claims, characterized in that said cells express a tracer that can be directly visualized or is detectable: by an imaging methods such as scintiscanning, positron emission tomography, MRI, ultrasonography, laser, CT, or by a biological or biochemical assay method, such as the ELISA, RIA, HPLC techniques, mass spectrometry, chemoluminescence, bioluminescence, western blot or electrophoresis.

16. Pharmaceutical composition according to any of the preceding claims, for administration to the patient via systemic route, under a gene therapy method to treat an angiogenesis source, characterized in that the cells of said composition are transfected with at least one gene encoding an active substance for the treatment or prevention of said angiogenesis source.

17. Pharmaceutical composition according to claim 16, wherein the systemic 15 route is an intra-arterial route.

18. Pharmaceutical composition according to either claim 16 or 17, characterized in that the cells of said composition are transfected with at least a suicide gene which will be activated by the subsequent administration to the patient of a prodrug. 20 19. Pharmaceutical composition according to either claim 17 or 18, characterized in that the cells of said composition are transfected with at least one immunomodulating cytokine gene. Pharmaceutical composition according to any one of claims 16 to 19, characterized in that its dosage is such that it allows administration in the order of 1 million to 200 million cells per kilogram of weight of the patient to be treated. 004531465

21. Pharmaceutical composition according to any of the preceding claims, characterized in that the angiogenesis sources are cancers, areas of inflammation, tissue lesions, grafts, retinopathies.

22. Method for detecting or diagnosing angiogenesis sources in a patient, characterized in that it comprises the administration by injection into the vascular network, of a pharmaceutical composition according to any of the preceding claims, followed by exposure of all or part of the body of said patient to an imaging device, from the signals emitted by the active substance, to produce images of angiogenesis sources possibly present in the patient.

23. Method according to claim 22, characterized in that the imaging device is chosen from among scintiscanning, positron emission tomography, MRI, ultrasonography, laser or CT.

24. Method for detecting or diagnosing angiogenesis sources in a patient, characterized in that it comprises the administration by injection into the vascular 15 network, of a pharmaceutical composition according to any of claims 1 to followed by taking a body fluid sample from the patient and measurement of the quantity of active substance in said sample, the latter indicating the existence and extent of the angiogenesis source. Method according to claim 24, characterized in that the assay device is 20 chosen from among biological or biochemical assay devices such as ELISA, RIA, HPLC techniques, mass spectrometry, chemoluminescence, bioluminescence, western-blot or electrophoresis. S. Neurotech S.A. By their Registered Patent Attorneys Freehills Carter Smith Beadle 23 August 2004