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EP0637967A1 - Procede ameliore - Google Patents

Procede ameliore

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Publication number
EP0637967A1
EP0637967A1 EP92923698A EP92923698A EP0637967A1 EP 0637967 A1 EP0637967 A1 EP 0637967A1 EP 92923698 A EP92923698 A EP 92923698A EP 92923698 A EP92923698 A EP 92923698A EP 0637967 A1 EP0637967 A1 EP 0637967A1
Authority
EP
European Patent Office
Prior art keywords
cells
vertebrate
human
recipient
donor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92923698A
Other languages
German (de)
English (en)
Inventor
Nils BRÜNNER
Mogens Spang-Thompsen
Erik C/O Lombardi Cancer Center Thompson
James C/O Lombardi Cancer Center Zwiebel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kraeftens Bekaempelse
Original Assignee
SPANG THOMSEN MOGENS
Kraeftens Bekaempelse
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SPANG THOMSEN MOGENS, Kraeftens Bekaempelse filed Critical SPANG THOMSEN MOGENS
Priority claimed from PCT/DK1992/000306 external-priority patent/WO1993008301A1/fr
Publication of EP0637967A1 publication Critical patent/EP0637967A1/fr
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to improvements in the fields of expe ⁇ rimental animals, drug screening and drug development, and to related developments, including new means and a new method for treating can- cer.
  • Dissemination of cancer involves escape of cells from the primary tumour, degradation of the normal tissue and migration, intravasa- tion, homing, extravasation, and colonization in an environment potentially different from the original location.
  • cancer cells are known to produce proteolytic enzymes, adhesion molecules and integrins which may all contribute to the invasive and metastatic phenotype, the mechanisms involved in the metastatic process are not fully understood.
  • fibroblasts endothelial cells, parenchymal cells and other host cells capable of non-immune host reactions
  • Hostomycin et al. 1989; Basset et al., 1990; Gr ⁇ ndahl-Hansen et al. , 1991.
  • the cancer cells exist in a competitive microenvironment with surrounding normal cells as well as non-malignant tumour-infiltrating cells, and the ability of the cancer cells to dominate and evade host non-immune and immune responses determines their invasive/metastatic capacity.
  • the present invention materials and techniques are provided which make it possible to perform detections, determinations and investigations which were previously either not possible or had only been performed in less realistic environments, both in connec- tion with cancer drug and cancer treatment development, and in con ⁇ nection with the development and testing of drugs and treatment principles in relation to other diseases.
  • the invention leads the way to new drugs and treatments and new drug and treatment principles, and as an example, one novel cancer therapy according to the invention is also disclosed herein.
  • the gist of this aspect of the present invention is to provide testing and screening materials and methods which are much closer to the natural human environment.
  • the invention makes it possible to establish or mimic a broad range of the factors which influence or are influenced by the growing cancer cells in the tissues in which the cancer cells operate, in that the invention makes it possible to directly expose human cancer cells to realistic environments with respect to the factors influencing their growth, and at same time monitor them extremely sensitively and accurately.
  • these advantages are critical and decisive for a number of important new developments.
  • the model has been found to be useful not only for examining interactions between cancer cells and host tissue in rela ⁇ tion to the invasive and malignant phenotype, but also for quantita- tively evaluating the effect of drugs which may interfere with biolo ⁇ gical events involved in the invasive and metastatic process.
  • the gene product of this gene is not directly visual ⁇ ly distinguishable, but rather latently distinguishable in the sense of the use of the term "latently” herein, which means that the gene product must be subjected to a conversion treatment in order to develop the visual distinction.
  • the conversion of the lacZ gene product into a coloured product is per ⁇ formed by staining with the chromogenic substrate 5-bromo-4-chloro-3- indolyl- -D-galactopyranoside (X-gal) resulting in a blue staining of the labelled cell.
  • Alternative staining procedures like red gal or FDG may be used.
  • suitable genes are selected from the group consisting of a Drosophila alcohol-dehydrogenase gene, and a placental alkaline phosphatase gene, both of which give rise to latently distinguishable gene products, and a gene encoding melanine, which is directly distinguishable or a gene encoding luciferase or peroxidase.
  • the donor cells comprises a genetic marker and, furthermore, a second transgene which is different from the genetic marker, such as a gene encoding a polypeptide, such as a growth factor (e.g. TGF-3 or mammostatin) or a growth factor receptor (e.g. EGF receptor, PDGF receptor or IGF-1 receptor), a cytokine (e.g. IL-1, IL-2 or TNF- ⁇ ) , or an oncogene product.
  • a growth factor e.g. TGF-3 or mammostatin
  • a growth factor receptor e.g. EGF receptor, PDGF receptor or IGF-1 receptor
  • a cytokine e.g. IL-1, IL-2 or TNF- ⁇
  • an oncogene product e.g. IL-1, IL-2 or TNF- ⁇
  • the most important aspects of the present invention are the ones in which the donor cell genotype is human.
  • the donor cell genotype is human.
  • the invention also relates to a method for detecting, in a non-human recipient vertebrate, a cell whicTi has the genotype of a donor vertebrate, and which is identical to or derived from a cell transferred from a donor of the vertebrate genotype into the non-human recipient vertebrate, the donor being a vertebrate of the donor vertebrate genotype or a cell line comprising cells from such vertebrate, the method comprising using, as the recipient vertebrate, a vertebrate cells of which are marked with a genetic marker which, either directly or latently, permits distinc- tion between the donor cell genotype and cells of the recipient and, when the marker is one which latently permits distinction, developing the latent distinction.
  • the present invention relates to an ex ⁇ pression system or transcription unit comprising a genetic marker, the system or unit comprising a 5'-flanking sequence capable of mediating expression of said genetic marker.
  • a transgenic animal contains one or more transgenes within its geno ⁇ me.
  • a transgene is a DNA sequence which has been introduced into the genome of the animal and is generally integrated at a locus of a genome, wherein the' transgenic DNA sequence is not otherwise normally found at that locus in that genome.
  • Transgenes may comprise heterolo- gous DNA sequences (sequences normally not found in the genome of species in question) or homologous DNA sequences (sequences derived from the genome of the species in question) .
  • Homologous recombina ⁇ tion i.e. the exchange of an endogenous gene with a variant of the gene, is also within the definition of the transgene as used herein.
  • the 5' regulatory sequence includes the transcribed por- tion of the endogenous gene upstream from the translation initiation sequence (the 5' untranslated region or 5' UTR) and those flanking sequences upstream therefrom which comprise a functional promoter.
  • a "functional promoter” includes those necessary un- transcribed DNA sequences which direct the binding of RNA polymerase to the endogenous gene to promote transcription.
  • Such sequences typically comprise a TATA sequence or box located generally about 25 to 30 nucleotides from the transcription initiation site.
  • the TATA box is also sometimes referred to as the proximal signal.
  • distal 5' expression regulation sequences In addition to such proximal 5' expression regulation sequences, it is preferred that additional 5' flanking sequences (referred to herein as “distal 5' expression regulation sequences") also be inclu- ded in the transgene.
  • Such distal 5' expression regulation sequences are believed to contain one or more enhancer and/or other sequences which facilitate expression of the endogenous gene and as a conse ⁇ quence facilitate the expression of the genetic marker when operably linked to the distal and proximal 5' expression regulation sequences.
  • These 5' expression regulation sequences regulate the spatial and temporal distribution of gene expression. The amount of distal 5' expression regulation sequences depends upon the endogenous gene from which the expression regulation sequences are derived.
  • the use of both 5' and 3' expression regulation sequences are preferred, in some embodiments of the invention, endogenous 3' regulation sequences are not used. In such cases, the 3' proximal expression regulation sequences normally associated with the genetic marker are used to direct polyadenylation. As with the 5' expression regulation sequences, the optimal amount of 3' expression regulation sequence may be readily determined by varying the amount of 3' flank- ing sequence to obtain maximal expression of the genetic marker poly ⁇ peptide. In general, the distal 3' regulation sequence, be it from an endogenous gene or a heterologous gene, will not extend into the adjacent gene from which it is derived and will exclude any sequences which adversely effect the level of transgene expression.
  • the term “genetic marker” when used, it should be understood that this term relates to a gene encoding a polypeptide, glycoprotein or RNA the presence of which can be detected as well as to any modifica ⁇ tions or analogues of said gene which do not have any significant adverse effect on the expression or activity of the polypeptide or RNA to be detected. Such modifications or analogues may be obtained by e.g. substitution, addition, insertion or deletion of the DNA sequence encoding the genetic marker.
  • substitution When “substitution” is performed, one or more nucleotides in the full nucleotide sequence are replaced with one or more different nucleo ⁇ tides* when “addition” is performed, one or more nucleotides are added at either end of the full nucleotide sequence; when “insertion” is performed one or more nucleotides within the full nucleotide sequence is inserted; and when “deletion” is performed one or more nucleotides are deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it.
  • the donor vertebrate cells further comprises a second, third or further transgenes which is different from the genetic marker.
  • this method is defined as encompassing both the embodiment where the donor cell is genetically labelled and the recipient cells are not r and the reverse embodiment, in accor ⁇ dance with the explanation of these two principles above. (Evidently, it is also possible - and within the scope of the present invention and the above definitions - to have both the donor cells and the recipient cells labelled with different genetic markers allowing, and even enhancing, distinction therebetween.)
  • the donor cells or cells of the recipient are modified to either directly or latently permit distinction bet ⁇ ween on the one hand cells which are identical to the donor or cells derived therefrom, and on the other hand cells of the other non-human recipient vertebrate, the determination of any effect of the drug or the treatment being performed on the basis of detection or investiga ⁇ tion, in the other recipient vertebrate, of cells identical to or derived from the donor cells, utilizing, in the detection or inves- tigation, the distinction obtained through modification of the donor cells or of cells of the recipient, whereby, when the modification is one which latently permits distinction, the latent distinction is developed prior to or in connection with the detection or investiga ⁇ tion.
  • This method may be performed by injecting a transcription unit com ⁇ prising said genetic marker into a fertilized egg or a cell of an embryo of a vertebrate so as to incorporate the transcription unit into the germline of the vertebrate and developing the resulting injected fertilized egg or embryo into an adult vertebrate.
  • a preferred embodiment of the method described above is a method, wherein the modification of the donor cells or the cells of the recipient comprises labelling the cells with a genetic marker which, either directly or latently, permits or facilitates distinction between the donor cell genotype and cells of the recipient vertebra- te.
  • the modified donor or recipient cells further comprises a second, third, fourth or further transgene which is different from the genetic marker.
  • the genetic marker is a gene encoding a product which in itself is visually distinguishable from non-marked cells, or which is capable of being made visually distinguishable from the non-marked cells, the gene product can thus be a coloured or fluorescent product or a product which can be converted into a coloured or fluorescent pro ⁇ duct.
  • the donor cells are human cells such as human cancer cells.
  • a further, important, aspect of the invention is a mammal in which at least one of the cell lines MDA-MB-231, MDA-MB-435, or OVCAR-3 is capable of metastasizing, and which mammal has been modified by labelling cells of the mammal, preferably " substantially all cells of the mammal, with a genetic marker as defined above.
  • a very advan ⁇ tageous mammal of this kind is a mammal, in particular a thymus- deficient or nude mouse, in which the cell lines MDA-MB-231, MDA-MB- 435, and OVCAR-3 are capable of metastasizing.
  • mice are the nu/nu META/Bom mouse available from Bomholtgaard, Gammel Ry, Denmark, the SCID mouse available from Bomholtgaard, Gammel Ry, Denmark, and the BAR mouse available from the Bartholin Institute, Karlunehospitalet, Denmark, whenever they have been modified with a genetic marker as defined above.
  • Another, also very interesting aspect of the invention is a cancer cell which is labelled with a genetic marker in the form of a gene encoding a product which in itself is visually distinguishable from non-marked cells, or which is capable of being made visually distin ⁇ guishable from the non-marked cells, in particular a gene the product of which is a coloured or fluorescent product or a product which can be converted into a coloured or fluorescent product, such as explain ⁇ ed above.
  • Example 1 herein describes the construction of such a human cancer cell wherein the genetic marker is a lacZ gene, the conversion of the gene product of which into a coloured product can be performed by staining with the chromogenic substrate 5-bromo-4-chloro-3-indoyl-3- D-galactopyranoside (X-gal) resulting in a blue staining of the labelled cell.
  • the genetic marker is a lacZ gene
  • labelled cancer cells are labelled cancer cells of one of the cell lines MDA-MB-231, MDA-MB- 435, MDA-MB-435, and OVCAR-3.
  • fibroblasts which are capable of finding and infiltrating a malignant tumour of cancer cells of a human genotype in an immunodeficient non-human recipient vertebrate into which cancer cells of the said mammal genotype have been intro ⁇ quizzed, are modified so that they contain, and are capable of expres ⁇ sing, a gene which is a candidate for the gene therapy.
  • non-labelled human cancer cells are introduced into a non-human recipient, such as a nude mouse e.g. a nu/nu META/Bom mouse, the cells of which are labelled with, e.g. lacZ.
  • a non-human recipient such as a nude mouse e.g. a nu/nu META/Bom mouse
  • the cells of which are labelled with, e.g. lacZ.
  • the tumours with the infiltrating fibro ⁇ blasts are stained, which will reveal presence of host cells in the tumour.
  • a next step could be to introduce the candidate gene in the fibroblasts in such a way that it is expressed when the fibroblasts have been transferred to a recipient.
  • particularly preferred fibroblasts for use in a patient are fibroblasts of a tissue type which is compatible with the tissue type of the patient, most prefe ⁇ rably fibroblasts from the patient.
  • One aspect of the gene therapy according to the invention are fibro ⁇ blasts which are capable of finding and infiltrating a malignant tumour in a mammal and which contain, and are capable of expressing, a gene producing a gene product which is capable of controlling the progression of cancer cells in the mammal.
  • One strategy for exploiting the gene therapy method would be to keep the gene-expressing fibroblasts in cell cultures, ready for use in gene therapy, which therapy is preferably instituted briefly after removal of a malignant tumour from a patient.
  • a collection of cell cultures should be maintained, comprising the modified fibroblasts of a multitude of human tissue types.
  • Another strategy is to isolate, immediately after removal of a tumour from a patient, fibroblasts from the tumour, modifying the fibroblasts with a gene which has been found to be able to control the growth of the cancer cells in question, and reintroducing the so modified fibro ⁇ blasts into the patient.
  • Within the scope of the present invention is thus a method of treating a patient in need thereof with the above- mentioned fibroblasts.
  • Test systems for carcinogenesis and mutagenesis are known and descri- bed e.g. in W091/15579 which relates to mutagenesis testing using transgenic non-human mammals having a genome characterized by the presence of an excisably-integrated lambda phage containing a target gene system comprising LacI and LacZ operatively linked to prokaryo- tic expression signals.
  • the carcinogenesis testing system according to the present invention uses a different approach as described below:
  • the invention also relates to a method for determining the carcino ⁇ genicity of a substance or a treatment on cells of a vertebrate donor genotype, comprising either
  • the donor cells being modified by labelling with a genetic marker to either directly or latently permit distinction between on the one hand cells which are identical to the donor or cells derived there ⁇ from, and on the other hand cells of the non-human recipient verte- brate, and determining any capability of the substance or the treat ⁇ ment to convert the donor cells into cancer cells by examining any invasive and metastatic activity of the donor cells, either in vitro, in the recipient, or after transfer of the donor cells to another recipient, on the basis of detection of any metastasis utilizing the distinction obtained through the modification of the donor cells.
  • the donor cells of particular interest are human cells, in particular leukocytes, bone marrow cells or keratinocytes.
  • the other parameters of this method such as the choice of the recipient and the kind of genetic marker, are preferably the same as described above.
  • Example 14 An example of such a carcinogenicity test according to the invention is given in Example 14.
  • the invention also relates to the use of a drug, the effect of which with respect to preventing, diminishing, controlling or inhibiting a disease has been established using the methods according to the invention for the preparation of a pharmaceutical composition for preventing, diminishing, controlling or inhibiting the disease, and to the use of a drug, the effect of which with respect to preventing, diminishing, controlling or inhibiting the progression of metastases has been established using the methods according to any the inven ⁇ tion, for the preparation of a pharmaceutical composition for preven- ting, diminishing, controlling or inhibiting the progression of metastases.
  • human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal.
  • human cancer cells is transduced or transfected with a genetic marker allowing subsequent identification of the human cancer cells following inoculation into immunedeficient mice. The following example describes the labelling of the human cancer cells.
  • the MCF-7 and MDA-MB-231 human breast cancer cell lines were origi- nally obtained from ATCC, Maryland, USA, and the MDA-MB-435 human breast cancer cell line was kindly provided by Dr. Janet Price, MD Anderson Hospital, Houston, Texas, USA.
  • the cells were routinely propagated in DMEM (Flow Laboratories, Scotland) with 10% Fetal Calf Serum (D10) or IMEM without phenol red and supplemented with 5% charcoal stripped fetal calf serum.
  • Viral stocks of the BAG vector (Fig. 1) (Price et al. , 1987) packaged in PA317 cells (Miller et al., 1986) were used to transduce the breast cancer cell lines.
  • the human breast cancer cells were plated one day before infection at a 1:10 split ratio.
  • the culture medium was replaced with 5 ml of viral supernatant con ⁇ taining 4 ⁇ g/ml of polyprene and incubated for 2 hours at 37°C in a 5% CO2 incubator. An additional 5 ml of D10 was then added and the cells were returned to the incubator. The medium was replaced with fresh D10 the next day.
  • the resulting cell lines were named MDA-MB- 231 BAG and MDA-MB-435 BAG, respectively.
  • Resistant colonies were isolated by means of cloning cylinders and tested for lacZ expression according to the method described below. Positive cell clones were expanded and used for further studies. The resulting cell lines were named MCF-7 pRSVIacZ, MDA-MB-231 pRSVIacZ and MDA- MB-435 pRSVIacZ, respectively.
  • the cells can be genetically labelled by transfection methods such as calcium phosphate precipitation, DEAE- dextran, popyprene, protoplast fusion, electroporation, lipofection, lipid-mediated transfection, laser-mediated transfection and scrape- loading or by transduction methods by use of retrovirus, SV40, BPV or vaccinia virus, and similarly, other antibiotics, e.g. hygromycin, can be used for the selection of transfectants.
  • transfection methods such as calcium phosphate precipitation, DEAE- dextran, popyprene, protoplast fusion, electroporation, lipofection, lipid-mediated transfection, laser-mediated transfection and scrape- loading or by transduction methods by use of retrovirus, SV40, BPV or vaccinia virus, and similarly, other antibiotics, e.g. hygromycin, can be used for the selection of transfectants.
  • the trans- prised or transfected cell populations were grown in medium containing G418.
  • the selected cells did not all express ?-galactosi- dase as determined by X-gal staining. Therefore, cells were subjected to fluorescein-di-9-D-galactopyranoside (FDG)-FACS selection, as described by Nolan, et al. (1988). In this procedure, the release of fluorescein from a non-fluorescent substrate within cells expressing the lacZ gene product, 3-galactosidase, allows cell separation in a flow cytometer.
  • FDG fluorescein-di-9-D-galactopyranoside
  • a confluent 100 mm dish was trypsinized and the single cell suspension was adjusted to 1-5 x 10' cells/ml in D10.
  • 100 ⁇ l of the cell suspension was incubated at 37 ⁇ C in a Falcon 2058 tube. Uptake of the substrate FDG by the cells was accomplished by hypotonic shock with the addition of 100 ⁇ l of 2mM FDG in dtt ⁇ O. Following a 1 minute incubation at 37°C, the substrate was trapped within the cells by the addition of 1.8 ml ice cold D10. The cells were incubated on ice for 1 hour and sorted on a Becton Dickinson dual laser FACStar Plus flow cytometer set to a 488 nm wavelength.
  • malignant and non-malignant human cells may be labelled.
  • MDA-MB-231 and MDA-MB-435 cells were infected with the BAG vector (Fig. 1). These cells do not stain with X-gal unless they have been infected with the BAG vector as it is shown in Fig. 3 for MDA-MB-435 cells. Because of a low viral titre (5 x 10 ⁇ " cfu/ml) , only about 1% of either cell line was initially found to stain positive with X-gal. Following G418 selection, approximately 60 - 70% of the G418- resistant cells expressed the lacZ gene as based upon X-gal staining. In order to enrich for lacZ-expressing cells, both breast cancer cell lines were subjected to FDG-FACS selection.
  • mice 6-8 weeks-old intact female nu/nu-META/Bom (Bomholtgaard, Denmark) mice were used. The mice were kept in sterile laminar flow clean benches at 25 ⁇ C and 50% humidity. At the end of the experiment, the mice were killed by cervical dislocation.
  • the purpose of transducing the human tumour cells with the lacZ gene was to be able to find the cells after their dissemination in the nude mouse.
  • Primary subcutaneous tumours of each transduced cell line demonstrated highly specific X-gal staining, whereas tumours from non-transduced cells did not stain blue. This is shown for MDA-MB-231 and MDA-MB-231 BAG tumours in Figure 4A and 4B, respectively.
  • Identi ⁇ cal results were seen following staining of MDA-MB-435 and MDA-MB-435 BAG primary xenotransplants. Both BAG lines were serially passaged in nude mice, and they retained lacZ expression after at least four pas- sages.
  • X-gal staining was also confined to the BAG-transduced human cancer cells (Fig. 5). Both untransduced and transduced tumours was locally invasive, pene ⁇ trating the peritoneal wall of the animals.
  • X-gal staining of mouse liver, spleen, pancreas, intestine and lungs from mice with trans ⁇ quizd sc tumours of either tumour line demonstrated blue staining of secondary tumour formation within organs in the peritoneal cavity (Fig. 5) .
  • Untransduced and lacZ transduced secondary tumours were locally invasive in various intraperitoneal organs: MDA-MB-231 BAG preferentially spread to pancreas and the hepatic portal tract, whereas secondary MDA-MB-435 BAG tumours most often were on the omentum of the intestine, and in the spleen, pancreas, and hepatic portal tract.
  • Lung metastases were found constantly in mice with either tumour (Fig. 5). Histologic examination of the blue stained areas in lungs confirmed the presence of micrometastases (Fig. 5) .
  • lacZ expressing malignant or non-malignant human cells may be introduced into immunedeficient animals intra ⁇ venously, intraperitoneally, intracranially or into various organs. Comparative studies between non- ransduced and transduced tumour cells
  • the aim of the study is to obtain a model for studying the invasive and metastatic phenotype of human cancer cells. Determination of possible effect of a retroviral transduction on the transformed phenotype of the tumour cells was to be performed. First, it was observed that the transduction altered neither the tumourigenicity nor the lag period or growth rate of the tumours. Second, comparison betwen in vitro and in vivo invasion of the cells before and after transduction was performed. Since the invasion process of tumour cells includes infiltration of the basement membrane, we studied the cells' ability to cross an artificial basement membrane (Matrigel) (Albini et al.,1987). In this assay, no significant activity diffe ⁇ rence was found between non-transduced and transduced cells.
  • lacZ expressing cells were shown to be capable of crossing the Matrigel.
  • transduction did not interfere with the cells' ability to locally invade the peritoneal wall or to invade into the peritoneal cavity and into internal organs.
  • a reliable in vivo comparison between the invasive and metastatic activity of wild-type cells ver ⁇ sus transduced cells were not possible, since micrometastases from wild-type tumours could not be readily identified.
  • transduced and non-transduced cells seem to behave similarly in both in vitro and in vivo invasion assays, indicating that the retro ⁇ viral integration of the lacZ gene did not significantly change the invasive behavior of the cells.
  • Intravenous injection of lacZ transduced human cancer cells (accord ⁇ ing to Example 1) into the tail vein of immunedeficient mice provides a means of investigating in vivo adhesion capability as well as capa ⁇ bility of those cells to form experimental metastases.
  • This model might be used 1) for studying the specificity for various organs of different tumour cells and 2) as a quick in vivo screening for the effect of experimental anti-metastatic adjuvant therapy (ct. Example 5).
  • the cells were harvested non-enzymatically by scraping using a rubber policeman. After centrifugation and resuspension the cells were counted for viability using Trypan-Blue, and kept on ice until in- jected. Each mouse was injected in the tail vein with 50.000 cells per 0,1 ml medium. Surplus of cell suspension was incubated as a viability control for the cells.
  • Fig. 6 shows the results from an experiment where MDA- MB-231 BAG cells were injected into the tail vein of nu/nu META/Bom mice. Lungs were removed at 5 min, 24 hours and 2 weeks following cell injection. The lungs were stained with X-gal according to the method described in Example 2. Few minutes after cell injection, a large number of the lacZ expressing human cancer cells were trapped in the capillaries of the lungs (Fig. 6A) , but most of these cells were cleared from the lung tissue following another 24 hours ( Figure 6B) . After two weeks a small number of tumour nodules (experimental metastasis) consisting of several tumour cells had been established in the lungs ( Figure 6C) .
  • MDA-MB-231 BAG having the best in vivo adhesion properties followed by MCF-7 BAG.
  • MDA-MB-435 BAG was found to be almost non-adhering.
  • u-PA urokinase type plasmi- nogen activator
  • the human breast cancer cell line MDA-MB-231 was routinely propagated in DMEM with 5% Fetal Calf Serum (D5). Cells for nude mouse experi ⁇ ments were harvested using a cell scraper instead of using enzymes. The cell lines were tested and found free from Mycoplasma contamina ⁇ tion.
  • mice 6-8 weeks-old nu/nu META/Bom (Bomholtgaard, Denmark) were used. The mice were kept in laminar flow clean benches and all equipment used was autoclaved.
  • a total number of 2 x 10 6 MDA-MD-231-BAG tumour cells were inoculated subcutaneously into each flank of the animals.
  • the tumour cells were always placed inferior to the thoracic wall.
  • 250 ⁇ g of antibodies were injected into the mice. Based on pharmacokinetic studies of the antibody used, a second injection of 250 ⁇ g antibody was given on day 21 after cell inoculation. The mice were sacrificed 6 weeks after cell inoculation.
  • u-PA-antibodies clone 5 (Nielsen et al. , 1986) were used to inhibit the enzymatic activity of uPA, and an IgGl mouse monoclonal antibody directed against a barley protein was used as an irrelevant control antibody.
  • the transduced cell line was examined for the presence of mRNA coding for both u-PA and u-PAR by Nothem blotting.
  • the cDNA for u-PA comprising pHUK8 (Verde et al., 1984) and the cDNA for the u-PAR com ⁇ prising p-u-PAR-1 (Roldan et al., 1990) were used. Following hybridization, filters were washed with three changes of 0.1 x SSC for a total of 1 hour at 65°C. Autoradiography was performed at -80 C C using two Chronex Quanta III intensifying screens. X-gal staining of cells in culture
  • Tumours were measured twice weekly in two dimensions and tumour growth curves were constructed on the basis of a transformed Gompertz function.
  • liver, diaphragm, spleen, pancreas, intestine and lungs were excised from each animal.
  • the organs were processed with X-gal according to the method described in Example 2.
  • blue areas from various organs were fixed in 4% formalin and processed for routine histology. If an organ presented one or more blue areas it was registered as positive for metastatic lesion.
  • an ELISA for 3-D-galac osidase as described by Fugiwara et al. could be used as an objective measurement of metastatic spread.
  • MDA-MB-231 BAG cells The capacity of MDA-MB-231 BAG cells to activate plasminogen was determined by a modification of the method previously described for use with suspension-growing cells (Ellis et al. , 1990) . Briefly, the MDA-MB-231 BAG cells were grown to confluency in 24 well Costar trays maintained in serum-free DMEM (Flow Laboratories, Scotland) in the presence of the plasmin inhibitor aprotinin (10 ⁇ g/ml) (Bayer, Ger- many).
  • the cells Prior to assay the cells were washed 3 times i Hepes-buffered DMEM, followed by incubation for 15 minutes at room temperature in the presence or absence of 10 ⁇ g/ml of a monoclonal antibody to u-PA (clone 5). After 2 subsequent washes the cells were incubated with plasminogen (20 ⁇ g/ml) and the specific fluorogenic plasmin specific substrate H-D-Val-Leu-Lys-AMC (0.2 mM) in PBS containing 0.2% BSA (200 ⁇ l in each of 12 wells per incubation) at 37°C was added.
  • plasminogen 20 ⁇ g/ml
  • H-D-Val-Leu-Lys-AMC 0.2 mM
  • BSA 200 ⁇ l in each of 12 wells per incubation
  • Nunc Immunoplates (Flat bottom Maxisorb, Nunc A/S, Denmark) were coated over-night at 4°C with 1 ⁇ g/ml of u-PA (UKIDAN Leo) in 0.1 M Na 2 C0 3 , pH 9.8, Excess u- PA was washed away and additional protein binding sites were blocked by a one hour incubation with 1% skimmed milk powder in PBS. After washing, the plates were incubated for 1 hour at 37°C with standard dilutions of anti-u-PA antibody clone 5 in mouse serum or sample to be tested. The plates were washed again and then incubated for an ⁇ other hour with biotinylated rabbit anti-mouse IgG (Dako) .
  • u-PA UKIDAN Leo
  • MDA-MB-231 BAG tumours in mice not injected with anti-u-PA antibodies were growing invasively into the peritoneal muscle layer of the mice.
  • the cancer cells expanded into the muscle fibers, and often tumour cells were located at the peritoneal side of the muscles.
  • the irrelevant antibody had no effect on local tumour invasion.
  • the anti-u-PA antibodies inhibited to some extent local spread of tumour cells.
  • Metastatic spread of the human cancer cells was confirmed by the presence of cancer cells in the blue spots, as determined by conven ⁇ tional histologic examinations.
  • other potential anti-invasive, anti-metastatic and/or anti-angiogenic compounds can be tested in the described model (see Example 5) .
  • mice were divided into 7 groups of 5 mice each.
  • mice After 2 weeks the subcutaneous tumours were extirpated from one group of mice, following which the animals lived until 9 weeks after the cancer cell introduction. At the same time a similar group of 5 mice were sacrificed and examined for lung metastases according to the method described in Example 2.
  • tumours were measured in two dimensions prior to other procedures in order to calculate the average tumour burden for statistical comparison between the two groups.
  • Anti-proliferative therapy such as conventional chemotherapy (Cyclophosphamid, Methotrexat, 5-Fluoro-Uracil and Adriamycin) and endocrine therapy (Tamoxifen) and combinations hereof.
  • the processes involved in invasion and metastasis can be divided into 3 main parts: A) dissolution of the extracellular matrix and basement membranes; B) cancer cell migration; and C) attachment either by cell-to-cell or cell-to-substrate interaction.
  • the adjuvant model (Example 5) will be used for experiments of neo- adjuvant therapy by administering therapy prior to tumourectomy.
  • the frequency of lung metastases as visualised by staining of the product encoded by the genetic marker in treated animals compared to untreated animals will be used.
  • lacZ expressing transgenic nu/nu META/Bom mouse As a tool in the study of tumour/stroma cell interactions a lacZ expressing transgenic nu/nu META/Bom mouse is established.
  • the lacZ gene is attempted expressed ubiquitously, but also tissue/cell specific expression is pursued.
  • the stromal component of the xenotransplanted human tumours is -D-galactosidase expressing whereas the human cancer cells will not express the lacZ gene.
  • the different cell types can be distinguished from each other, either by X-gal staining or by FACS as described in Example 1.
  • a plasmid is prepared in which the lacZ gene is brought into such a context that it is efficiently transcribed and translated in mammalian cells.
  • the enhancer/promoter combination is chosen to give the desired specificity of expression, e.g. in vir ⁇ tually all cells of transgenic animals.
  • Examples of enhancer/promo ⁇ ters with very general expression are derived from the eEF-1 alpha gene, the HMG gene, the UBI gene, and the early genes of CMV. Each of these includes enhancer, promoter, an intron, the lacZ gene with a nuclear targeting sequence, a polyadenylation site, and a MAR ele ⁇ ment. Introns increase transgene expression (Palmiter et al.
  • the MAR element matrix attachment region
  • the MAR element is optional but has been reported to increase consistent expression of transgenes (McKnight et al., 1992; Phi-Van et al., 1990; Stief et al. , 1989).
  • the EF enhancer/promoter is derived from the eukaryotic translation elongation factor 1 alpha (eEF-1 alpha) , which normally directs the synthesis of one of the most abundant proteins in eukaryotic cells (Mizushima and Nagata, 1990). It is active in all cells that synthe- size protein. The derivative is active in transfection experiments with many cell types.
  • eEF-1 alpha eukaryotic translation elongation factor 1 alpha
  • the HMG enhancer/promoter belongs to the housekeeping gene for murine hydroxy-methylglutaryl-coenzyme A reductase, and it has also been used for ubiquitous expression (Mehtali et al., 1990).
  • the UBI enhancer/promoter normally drives the synthesis of ubiquitin, a protein present in all cells (Wiborg et al. , 1985).
  • the CMV enhancer/promoter belongs to the early transcription unit of cytomegalovirus. Like the parent virus, it is active in many cell types, and can be used for ubiquitous expression (Schmidt et al., 1990).
  • the sequence contains a nuclear localization signal so that the ⁇ -galactosidase is targeted to the nucleus.
  • Cells containing this transgene and developed with the chromogenic substrate X-gal have the blue stain concentrated to the nucleus and are therefore more easily detected than if the stain were dispersed over the whole cell volume.
  • the one-cell zygotes used for microinjection are derived from crosses between male nu/nu META/Bom mouse and female +/+ META/Bom mice.
  • Progeny is tested for lacZ expression by X-gal staining in important cell types in tail biopsies. After breeding to preserve transgene and nu character, the tissue specificity is further characterized in descendants of the transgenic founders.
  • fibroblast and endothelial cells from nude off-springs of the transgenic animals is stained with X-gal as described in Examples 1 and 2.
  • Animals with a positive X-gal staining reaction is further tested for lacZ expression in other cell types using X-gal staining as described in Examples 1 and 2.
  • Animals with high expression of ⁇ - D-galactosidase is used for establishment of a lacZ transgenic strain of nu/nu META/Bom mice.
  • the capability of the mouse cells from the transgenic immunedeficient mouse having been in contact with the human donor cells to induce or transfer a disease to other mouse cells may be examined by transfer- ring cells from the immunedeficient transgenic mouse to another immunedeficient non- ransgenic mouse.
  • the experimental scheme may be used to identify substan ⁇ ces which can be used to inhibit or counteract the progress of the disease or which can be used for prophylaxis and thereby as drugs to prevent, control and/or combatting the autoimmune disease.
  • the scheme opens widespread possibilities for examining human cells in vivo and for examining the effect of various substances on the human cells in vivo .
  • the effect of a substance on the development of the human donor cell, the interaction between the human donor cells and the cells from the lacZ transgenic nu/nu META/Bom mouse, the pro ⁇ gress of the disease and the potential as a prophylactic treat ⁇ ment is examined by treating the mouse with the substance in question using various treatment schemes, various treatment ways and various doses of the substance In question.
  • the treat ⁇ ment may be locally or generally applied and may be combined with e.g. surgical treatment in some instances. Due to the possibility of visualizing the human donor cells, the direct effect of the treatment on the human donor cells and the pos- sible Interaction between the human donor cells and the cells from the lacZ transgenic nu/nu META/Bom mouse can be determined.
  • the above outlined experiment for determination of the effect of a substance on the human donor cells may also be performed on the additional transfer of cells from an lacZ transgenic nu/nu META/Bom mouse to a recipient mouse as described above under 3) .
  • Autoimmune diseases which may be examined using the above outlined experimental scheme comprises various types of diabetes such as diabetes mellitus in which pathological processes In the islets of pancreas takes place before clinical manifestation of diabetes mel ⁇ litus (type 1)). Furthermore, conditions related to the functions of the thyroid gland, In particular hyper- or hypofunctioning of the gland, e.g.
  • the substance which may be. used in the treatment may be a chemical compound, antibodies or antigens, amino acids, proteins such as a growth factors, lymphokines, interferons, interleukines, insulin, hormones, leukotrienes, anti-sense RNA etc.
  • human cells may be genetically engineered with a gene the product of which allows visuable detection.
  • the gene could be the lacZ gene or other genes the product of which can be visualized by specific staining procedures.
  • lacZ transgenic animals may be crossed into other transgenic animals resulting in animals expressing both genes.
  • Brain tissue is excised from new-borne or embryos of the lacZ trans ⁇ genic mouse described in Example 7.
  • each of the human breast cancer xenografts has a characteristic and constant fraction of diploid cells during tumour growth, suggesting that the stromal compartment of the tumours proliferates at a rate proportional with the epithe- lial human breast cancer cells.
  • tumour infiltrating fibroblast from xenotransplanted human breast cancer cells growing in lacZ transgenic nu/nu META/Bom mice will be described.
  • human breast cancer cells (MDA-MB-231 or MCF-7) are inoculated subcutaneously into nu/nu META/Bom mice.
  • the primary subcutaneous tumour is removed and transferred to a sterile petri dish containing tissue culture medium.
  • the tumour is disaggregated mechanically and/or enzymatically (Engelholm et al. , 1985) into a single cell suspension.
  • the fibroblasts can be isolated from the single cell suspension using FACS.
  • tumour infiltrating fibroblasts may be separated from the human tumour cells by differences in growth requirements in vi tro .
  • Fig. 9A the first passage in vitro of an MCF-7 human breast cancer xenograft resulted in the outgrowth of epithelial tumour cells and fibroblasts.
  • Fig. 9B already in the second in vitro passage, the tumour infiltrating fibroblast overgrew the tumour cells, resulting in a pure fibroblast culture.
  • the fibroblasts have now been serially passaged for more than 10 passages without any sign of epithelial tumour cells.
  • tumour Infiltrating fibroblasts are able to repopulate a human xenograft, either by being inoculated simultaneously with the human cancer cells or by being in ⁇ jected Intravenously into a mouse carrying a human tumour xenograft.
  • tumour infiltrating fibroblasts from lacZ transgenic mice or fibroblasts which in vitro are transfected or transduced with lacZ are mixed with 10° human tumour cells and inoculated subcutaneously into a nu/nu META/Bom mouse. 6-8 weeks later, the tumour is removed. In order to evaluate the fate of the tumour infiltrating fibroblast, the tumour is stained for ?-galactosidase production according to the procedure described in Example 2.
  • tumour infiltrating fibroblast from lacZ trans ⁇ genic nu/nu META/Bom mice might be introduced by intravenous injec- tion into nu/nu META/Bom mice carrying human breast cancer xenografts with subsequent extirpation and X-gal staining of the primary human breast cancer.
  • tumour infiltrating lymphocytes may be candidate cells for such a therapy strategy.
  • Another possibility would be to genetically engineer tumour infil ⁇ trating fibroblasts.
  • tumour growth and invasion/metastasis will be recorded and compared to the same parameters in a control experiment where the tumour infiltrating fibroblasts are untransfected. Furthermore, the primary tumours are stained with X-gal in order to test for the presence of lacZ expres ⁇ sing fibroblasts.
  • Precllnical drug screening programs most often include sensitivity testing of human cancer cells in vitro and murlne ascites tumours grown in vivo in syngenic mice.
  • a more appropriate assay system would be the in vivo testing of human cancer cells.
  • human tumours grown as solid tumours in nude mice often have a slow growth rate resulting in time-consuming and expensive assay conditions.
  • a more rational assay would be one similar to the murine ascites tumour systems, where the human cancer cells are inoculated intra- perltoneally into nude mice with subsequent application of therapy.
  • the end-point should be survival time of the mice in relation to that of a control group consisting of nude mice injected in a similar manner with the human ascites tumour cells but without treatment.
  • Smears of ascites fluid were produced on glass slides from the intra ⁇ peritoneal cavity. The slides were dried and subsequently fixed and stained as described for cells In culture (see Example 1) .
  • Example 2 The method described in Example 2 was used.
  • the drug in question is administered intraperitoneally at the same time as the tumour cells. Different treatment schedules and routes of administration is used.
  • Measurements such as increase in life span, body weight, or formation of metastasis is used as end-points in the evaluation of treatment effect.
  • the in vitro sensitivity patterns of MDA-MB-231-derived ascites- tumour forming cells and MDA-MB-435-derived ascites-tumour forming cells are summarized In Table 5 below.
  • the cell lines showed a diffe- rential sensitivity to the six drugs tested.
  • the MDA-MB-231-derived cells were more sensitive than the MDA-MB-435-derived cells to ADR, BCNU and VP-16, whereas the cell lines were similar in sensitivity to the other drugs.
  • the LD50 values of the individual drugs are within the ranges found for other human cancer cells (Jensen et al. , 1992). TABLE 5
  • test for determination of the carcinogenicity of various substances is outlined.
  • the test may be performed in vivo on human cells whereby a much more biologically correct and informative test on the actual carcinogenicity of a substance on human cells being placed in a biologically active environment is provided as compared to known in vitro tests.
  • the test is performed as follows:
  • Example 1 Human cells labelled with the lacZ-gene as described in Example 1 are transferred to nu/nu META/Bom mice prior to exposure to the substance to be tested or after exposure which may take place in vitro by exposing the cell culture for the substance in question.
  • the human cells are Identified using visualization of the lacZ-gene with X-gal staining (Examples 1 and 2) and the human cells are examined for the occurrence of characte- ristics known to be present in cancer cells such as cyto- logical and morphological characteristics for malignant cells. Also various DNA and RNA analysis using techniques like, Southern Blot and Northern Blot may be used to exa ⁇ mine the cells for malignancy criterias, e.g., gene ampli- fIcations, gene deletions etc.
  • test for identification of substances which may be used in the treatment of human pathogenic organisms such as bacteria, vira and unicellular parasites is described.
  • the test is performed as follows below:
  • the infected lacZ transformed human cells are transferred to an immunedeficient mouse, such as a nu/nu META/Bom mouse using a method suitable for the cell in question.
  • an immunedeficient mouse such as a nu/nu META/Bom mouse
  • the pathogens for which substance capable of combatting the pathogens are sought may be bacteria such as Mycobacterium tuberculosis , Myco- bacterium lepra, Pneumocystis carinae , vira such as retrovira, in particular HIV, herpes vira, cancer-inducing vira etc., unicellular parasites such as Plasmodium falciparum, Toxoplasma gondii, flagel ⁇ lates such as Giardia sp., Trypanosoma sp., Leishmania sp., Trichomo - nas sp. and amoebae such as Entamoeba sp. and fungi.
  • bacteria such as Mycobacterium tuberculosis , Myco- bacterium lepra, Pneumocystis carinae , vira such as retrovira, in particular HIV, herpes vira, cancer-inducing vir
  • FIG. 1 Schematic illustration of the BAG vector (Price et al. , 1987).
  • the BAG vector consists of the lacZ gene under the transcrip ⁇ tional control of the Moloney murine leukemia Long Terminal Repeat (LTR) promotor and the SV40 promoted neo® gene.
  • LTR Moloney murine leukemia Long Terminal Repeat
  • Fig. 3 X-gal staining of tumour cells grown in vitro .
  • Cells were fixed and processed for X-gal staining (see Example 1) .
  • E Macroscopic appearance of lung metastases
  • F Single lung metastasis (arrow)
  • G Histological section of the lung metastasis (arrow) seen in 3F.
  • Fig. 6 MDA-MB-231 BAG A few minutes after intravenous injection, a large number of tumour cells are trapped in the capillaries of the lungs ( Figure 6A) , but most of these cells are cleared from the lung tissue following another 24 hours ( Figure 6B). After 2 weeks a small number of tumour nodules (experimental metastases) consisting of several tumour cells is been established in the lungs ( Figure 6C) .
  • Fig. 7 Macroscopic growth of untreated control tumours, tumours in mice treated with an irrelevant antibody and tumours exposed to anti- u-PA antibodies.
  • Fig. 8 Serum concentration of anti-u-PA antibodies in nude mice following injection of 100 ⁇ g anti-u-PA antibodies from clone 5.
  • FIG. 9 Schematic Illustration of the EF-lacZ-MAR vector (11515 bp)
  • FIG. 10 Schematic illustration of the HMG-IacZ-MAR vector (14.50 Kb)
  • FIG. 11 Schematic illustration of the UBI -lacZ-MAR vector (11664 bp)
  • FIG. 12 Schematic illustration of the CMV-lacZ-MAR vector (10094 bp)
  • Fig. 13 MCF-7 human breast cancer xenografts established in tissue culture. A: After 1 passage in culture both tumour cells and fibroblasts are present B: After second in vitro passage only fibroblasts are seen. ⁇ 7
  • Lin W-C, Pretlow TP, Pretlow II TG, Culp LA "Bacterial lacZ Gene as a Highly Sensitive Marker to Detect Micrometastasis Formation during Tumor Progression", Cancer Res . 50, 2808-2817, 1990
  • Lin WC, Pretlow TP, Pretlow II TG, Culp LA "Development of Micrometastases: Earliest Events Detected With Bacterial lacZ Gene-Tagged Tumor Cells", J " . Natl . Cancer Inst. 82, 1497-1503, 1990
  • Price J, Turner D, Cepko, C "Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer", Proc . Natl . Acad. Sci . USA 84, 156-160, 1987
  • Roderick TH and Guidi JN "Strain distribution of polymorphic variants", In Lyon MF and Searle, AG: Genetic Variants and Strains of the Laboratory Mouse. Gustav Fischer Verlag, Stuttgart, N.Y. , 2nd ed. 76-77, 1989
  • Rygaard K "A Rapid Method for Identification of Murine Cells in Human Malignant Tumours Grown in Nude Mice.” In: Rygaard et al. (eds), Immune-deficient Animals in Biomedical Research , Basel, Karger, 268-272, 1987
  • Shreffler DC "Linkage of the mouse transferrin locus", J. Jfered. 54, 127-129, 1963
  • Wilson EL, Gartner M, Champbell JAH, Dowdle EB "Metastasis of a human melanoma cell line in the nude mouse", Int . J. Cancer 41, 83-86, 1988

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Abstract

Procédé de détermination de l'effet d'un médicament ou d'un traitement et qui consiste à introduire, dans un receveur vertébré non humain qui peut être immuno-déficitaire, notamment une souris nue, des cellules provenant d'un vertébré donneur tel qu'un homme, et assurant la médiation de la maladie, les cellules du donneur ou du receveur étant modifiées, par exemple par marquage au moyen d'un marqueur génétique tel qu'un gène lacZ; à administrer le médicament au vertébré receveur non humain, ou à soumettre ce dernier au traitement; puis à déterminer l'effet du médicament ou du traitement. Lorsque le marqueur génétique est un gène lacZ, la détection est réalisée par coloration avec le substrat chromogène X-gal, ce qui entraîne une coloration en bleu de la cellule marquée. En outre, on a prévu un procédé de limitation de la progression de cellules cancéreuses chez un malade homme ou animal. Ce procédé consiste à administrer au malade des fibroblastes que l'on a transformé à l'aide d'un produit génétique exprimant un produit génique inhibiteur de la progression des cellules cancéreuses.
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