EP1303183A1

EP1303183A1 - Somatic cloning gene transfer for the production of recombinant proteins, cells and organs

Info

Publication number: EP1303183A1
Application number: EP00953104A
Authority: EP
Inventors: Gottfried ApoGene GmbH & Co. KG BREM
Original assignee: Apogene GmbH and Co KG
Current assignee: Apogene GmbH and Co KG
Priority date: 2000-07-27
Filing date: 2000-07-27
Publication date: 2003-04-23
Also published as: WO2002009507A1; CA2416877A1; CA2416877C; AU2000265672A1; JP2004518405A; US8030537B1

Abstract

The invention relates to a method for recombinant production of substances, wherein cells are transformed with a nucleotide sequence coding said substance, the transformed cells are cloned and the cells thus obtained are inserted into a receiving organism. More particularly, the invention relates to the use of said method in the production of recombinant proteins, cells and tissues. The invention further relates to a method wherein cells of an individual are isolated, said cells are inserted into an immune-incompetent animal for further growth and the cells, tissue and/or organs which have grown in the animal are re-isolated and inserted into an individual.

Description

Somatic cloning gene transfer for the production of recombinant proteins, cells and organs

The present invention relates to a method for the recombinant production of substances, in which cells with a nucleotide sequence coding for the substance are transformed, the transformed cells are subjected to a cloning process, and the cells thus obtained are introduced into a recipient organism. The present invention relates in particular to the use of the method in the production of recombinant proteins, cells and tissues. According to a further aspect, the invention relates to a method in which cells of an individual are isolated, these cells are introduced into an immunocompetent animal for further growth and the cells, tissues and / or organs grown in the animal are isolated again and introduced into an individual ,

With the advent of recombinant genetic engineering, biological systems were increasingly incorporated into the production of useful substances. A large number of proteins are currently produced in in vitro cell culture systems. Based on the elucidation of the nucleotide sequence of the human genome, it is to be expected that the determination of new proteins and their role in the human body will be promoted and accelerated, so that in the near future a large number of new substances will be available for medical or other purposes, the Manufacturing is desired. However, the use of existing in vitro cell culture systems for this purpose has the disadvantage that it takes at least 1 to 2 years for the corresponding substances to be optimally produced. In addition, such systems are complicated to use, prone to failure and cost-intensive. For example, one of the greatest difficulties of large-scale cell culture systems is the risk of contamination of the culture solution.

h In recent years, animals have been increasingly included in the production of industrially and pharmaceutically interesting substances. In addition to the creation of so-called germline transgenic animals used somatic gene transfer, in which a DNA encoding a certain substance is introduced into somatic cells of the target individual. This can take place either in vivo, ie in the whole organism, or in vitro, ie in the laboratory in cells which have been obtained from the organism and are returned to these cells after the construct has been introduced into these cells. The construct is then expressed in the animal so that the substance of interest can develop its effect in the animal.

A disadvantage inherent in the transfer of gene constructs into somatic cells is, however, that the gene introduced is only expressed or acts in the treated individual and is limited to the life of the transformed cells. A transfer to subsequent generations is excluded.

Additional problems encountered with somatic gene transfer are, in particular, a sufficiently large number of specific target cells with stable transfection, which can often only be achieved with great difficulty, and the randomness of the integration of the introduced construct, which can negatively influence the properties and / or the behavior of the target cells. Furthermore, problems often arise with regard to inadequate expression of the polypeptide encoded by the introduced construct, which in addition to the difficulties lying at the DNA level can also be attributed to certain intra- and extracellular barriers and must be tested in each case. The problems described above therefore considerably restrict the use of the possible uses of somatic gene transfer in biotechnology in the broadest sense and in livestock production in particular.

It is therefore an object of the present invention to avoid the disadvantages of the prior art and to provide a new process for the production of certain interesting substances.

This object is achieved by a method for the recombinant production of substances, in which, in a first step, cells with a nucleotide sequence coding for the substance are transformed, the transformed cells are subjected to a cloning process, and the cells obtained in the cloning step are introduced into a recipient organism. The cells used in the first method step can be any cells isolated from an individual that can be subjected to a cloning process. Examples of these are, in particular, fetal or adult fibroblasts, primordial germ cells, granulosa cells, thymocytes, spleen cells, liver cells, macrophages, testes or ovary cells etc. The cells can be used as such, as isolated, or kept in culture, even before transformation may have been subjected to a cloning process.

According to a preferred embodiment, the cells used for the transformation are isolated from an existing cell clone, a cell culture or an organism. For the purposes of the invention, cell clones are to be understood as meaning in vitro cultured, cloned cells, fetuses from cloning or cloned animals.

The cells are then transformed with a nucleotide sequence that encodes the substance of interest. All substances that can be synthesized in the body are understood as substances, such as proteins, polysaccharides, lipids etc., but also the cells, tissues and organs that have the nucleotide sequence introduced. The nucleotide sequence coding for the substance is therefore not only a sequence which codes the substance directly, but also a sequence which codes the various polypeptides / enzymes which form the substance of interest in or outside the cells. For the purposes of the present invention, recombinant refers to genes which are either exogenous to the animal or also endogenous, but which have been modified in their expression pattern by means of recombinant genetic engineering, such as, for example, in their differentiation-specific expression pattern or the amount expressed.

All currently known techniques can be used for the transformation, such as physical, chemical or viral techniques. The use of homologous recombination is preferred with regard to the directional integration of the construct at specific locations in the genome. If large gene clusters are to be introduced into the recipient cell, it is also possible to use artificial chromosomes. Cell hybrids, such as those used for mapping, can also be created and selected so that they also contain specific chromosomal fragments from the target genome in addition to the farm animal genome. This is especially so advantageous if an entire system of the target genome, such as the immunoglobulin system, is to be transferred.

The cells are then subjected to a cloning process. Cloning methods are now known and are described, for example, in PCT / EP98 / 00230 (WO 99/36510) and PCT / EP / 00229 (WO), which is incorporated by reference here. The cloned cells are then grown to a certain stage of development in vivo and / or in vitro, for example to the fetus, and then introduced into the recipient organism (step c) of claim 1). Likewise, the cloned cells can be grown to such an extent that a certain differentiation is present, with certain, differentiated cells already being introduced into the recipient organism.

The cloned cells are then introduced into a recipient organism, which can be an animal, an animal fetus, an animal embryo or an animal cell aggregate, and also cloned animals, cloned animal fetuses, cloned animal embryos and cell aggregates. In a preferred embodiment, the parent cells that are transformed and subjected to a cloning process are of the same genotype as the recipient organism.

An advantage of such a procedure can be seen in the fact that an already existing, ie prepared recipient organism can be provided in a very short time with cells, preferably of the same genotype, which contain genes to be newly expressed, and produce recombinant substances. The time saved compared to conventional germline gene transfer can be up to 5 years until animals are ready for production. In addition, regardless of the time saved and the production system that can be obtained more quickly, with the associated cost savings, the present method is also more efficient since the producing organisms can be generated more economically. Thus it is possible to use in vitro existing cells of a clone, for which living animals are also present, as starting cells for the transformation and, after transformation and cloning, to introduce these into the recipient organism, which, since it belongs to the clone, inherently has the same genotype , Another advantage of, the process known as somatic cloning gene transfer described here can also be seen in the fact that can ^"respond quickly to new demands and needs in the expression of recombinant proteins. Normally, it takes a farm animal species with short generation interval, even when using , for example in a rabbit, at least 7 months until sufficient material is available for the first tests of the expressed genes after the constructs have been created. An additional year is required to produce the desired polypeptide the polypeptide of interest can be obtained by means of transgenic animals, at least 3 to 4. In contrast, the method according to the invention enables a considerable reduction in the time until an existing recipient organism is transformed with a new construct using cells and expression clones are created can be opened (only takes a few months).

The method according to the invention therefore makes it possible to arrive at the first protein amounts for the analysis in a time period comparable to the in vitro cell culture, whereby according to the invention a production system is already available with which the desired substance can be produced. The recipient organisms or animals can therefore be used quickly to produce the desired substances.

In certain cases, it is advantageous to prepare the recipient organism, which is preferably produced or to be produced in stock, specifically for the subsequent somatic cell gene transfer. This is particularly necessary if the applied transgenic or differently prepared cells are to be given an advantage over the cells or structures present in the recipient organism in order to achieve better expression or expression.

For this purpose, for example, certain cells, tissues or organs can be removed in the recipient organism, which can be achieved by physical, chemical, immunological, molecular genetic or surgical methods. In addition, animals or fetuses that are homozygous negative at crucial gene locations (screen-out concept) can be bred or kept in stock and used as required. One way of depleting certain cells, tissues or organs in the recipient organism is to use transgenic organisms which contain a construct which is under the control of a stage of development-specific or an inducible promoter. In the first case, when the promoter is activated during the specific development stage, a protein is formed automatically and tissue-specifically, which leads to the death / apoptosis of the corresponding cells, for example the diphtheria toxin. In the second case, a "suicide" gene construct, which has been preceded by a suitable tissue-specific promoter, is activated by application of a specific agent, such as, for example, tetracycline or ecdysone, which leads to the freely definable, specific depletion of the cells, tissues or organs in the Recipient organism leads.

A simple possibility for the provision and propagation of such organisms is their cloning, with which the construct once introduced into the organism can be passed on stably to offspring. It is clear that any cells, tissues or organs in the organism can be influenced in their proliferation or development as required.

The removal of cells / tissues from which the mammary gland normally arises can be cited as an example of a depletion of certain cells. When an animal is born, relatively few of these cells are present. These only develop in the course of the first pregnancy and shortly before birth there is a strong proliferation and the formation of the functional mammary gland. If these stem cells of the mammary gland, which are only present in a small number in the early development stage of the recipient organism, are removed in an animal organism and these removed cells are replaced by recombinant (transformed) cells of this type which carry an additional expression cassette for the synthesis of a specific recombinant substance of interest , it is achieved that in the animal organism thus obtained, the mammary gland that forms is formed from the applied transformed cells. As a result, the recombinant substance is also expressed in the introduced mammal system in the farm animal. The time saved with such a procedure is approximately 3 years.

Another example is the total or partial removal of the blood stem cells or the stem cells of the immunological system from the recipient organism by, for example, wise radiation or other methods and their replacement by transformed stem cells, which were transformed according to the invention and subjected to a cloning process and which, for example, enable the expression of humanized antibodies (AK) or AK fragments (for example bispecific AK) or other animal or human blood factors. If the animal's immunoglobulin genes are exchanged for those of humans in vitro with the cloned stem cells and if these cells are introduced into the recipient organism using the method according to the invention, these recipient organisms form polyclonal human-identical antibodies which are of great value for the therapy of human diseases.

In addition, with the aid of the method according to the invention, a particularly efficient and unproblematic production system can be created for factors which are present in the animals but are present in too low a concentration for economically interesting extraction. An example of this is the expression of several genes of heparin synthesis under very strong promoters. Since the heparin is naturally present in the organism, no immunological reactions would be elicited either against the cells, which preferably belong to the same genotype, or against the product.

The recloning of cells can be repeated at least three times. It has been found on this side that cells can complete up to 150 divisions, i.e. the cells from the cloning process survive much longer than normal cells and can therefore survive and express genes at least as long after several cloning processes in the animal and express the life span of the animal.

In an analogous manner and further development to the above fields of application, cells can be obtained from an existing animal, and embryos, fetuses and cell lines cultivated in vitro can be created. These cell lines are genetically modified using known methods. The cells are then differentiated directly in the in vitro culture or isolated after repeated cloning from fetal organs and introduced into the adult parent organism as "transgenic" but otherwise genotypically originating cells. Since these, with the exception of the transgene, do not differ immunologically from this, the cells are able to Establish animal and will produce, for example, recombinant substances according to the genetic transformation.

According to a further aspect, the invention also relates to a method for producing cells, tissues or organs in animal organisms, in which cells of an individual are isolated in a first step, the cells are introduced into an immune-incompetent animal organism, the animal organism is grown the cells of the individual grown in the organism are isolated, and the isolated cells are introduced into an individual.

This method is particularly suitable for culturing cells, tissues and organs, preferably of human origin, in animal organisms, since the cells in the recipient animal organism are replicated or also grow into tissues and organs.

According to a preferred embodiment, the cells of the starting individual, or the tissues or organs grown therefrom, are returned to the same individual from which they were removed, whereby tissues or organs can be made available which are used in a return (transplantation) the individual cannot be repelled.

Any animal or fetus or animal embryo / cell aggregate that does not reject the cells derived from the starting individual is understood as an immune-incompetent animal organism. In addition to animal organisms made immune deficient, organisms that are at such an early stage of development are suitable for this, during which the developing immune system has not yet learned to distinguish between its own and a foreign one. If cells of the starting individual are therefore introduced into such a recipient organism, then these cells are not rejected but essentially recognized as being their own and further developed accordingly.

Animal organisms obtained by cloning are therefore preferably suitable as the recipient animal organism. So it is possible to have multiple fetuses or embryos

To keep cell aggregates in stock and to treat them with the cells of the starting individual as required. The fetuses / embryos / cell aggregates are then up to one stage grown in which they have produced the desired effect, for example until the development of the mature organ or tissue or also until the development of a sufficient amount of cells of the starting individual. This tissue / organ or these cells are then obtained, for example by removing the organ from the animal, and transferring it to the starting individual. Depending on the distance of a particular organ, this will of course involve killing the animal.

The cells of the starting individual can be introduced into a conventional recipient organism. In this case, the animal is expected to chimerize the cells / tissue / organ with its own cells / tissue / organ. In order to ensure that only its "own" cells can be returned to the starting individual, the corresponding tissue / organ / the cells as a whole can be isolated and the animal cells separated, for example by means of labeling using antibodies directed against the animal cells and separation by means of FACS (Fluorescence Activated Cell Sorting) can be achieved.

According to a preferred embodiment, however, the cells / tissues / organs in question are removed from the recipient organism beforehand, which can be done as described above.

For example, in the case of a fetal recipient organism in which the organ system has been removed, the corresponding organ-specific stem cells of the starting individual, such as adult human stem cells which are obtained directly from the affected organ, are introduced into the fetus and will then replace the deputated cells in the fetus and form the appropriate organ. This creates a chimeric organism that contains a xenoorgan that is suitable for transplantation. The organ in question represents a xenoorgan in the animal organism, which, however, is not rejected due to the lack of immune competence of the fetus. After the transplantation to the original individual, for example a human patient, if an human stem cell has been used, there will be an xenogenous graft instead of an allogeneic one, or possibly even an autologous graft if the organ consists of adult stem cells from this individual / patient emerged, which is possible with certain organs and diseases despite the time requirement of about one year (e.g. kidney or liver).

The use of the present method thus allows the generation of cells, tissues or organs within a short period of time, so that even certain patients themselves can be quickly supplied with organs with an essentially or absolutely identical MHC type. The method according to the invention therefore solves the currently existing problems of the scarcity of available organs / tissues for the transplantation.

The following examples illustrate the invention without restricting it. The examples describe the production of bispecific antibodies from the serum of non-transgenic clone calves with transgenic blood stem cells. The following steps are carried out for the generation of calves with transgenic bone marrow cells, reference being made to PCT / EP98 / 00230 or PCT / EP98 / 0029 for the details, which are incorporated here by reference.

1. Extraction and genetic transformation of primary bovine fetal fibroblasts

2. Cloning by nucleus transfer and transfer of transgenic embryos to recipient animals

3. Obtaining the fetuses and isolating transgenic bone marrow stem cells from the fetal liver

4. Transfusion of these transgenic cells into existing clone calves of the same chromosomal genotype

5. Collection of serum by taking blood

6. Lysis of tumor cells using bispecific antibodies

example 1

1. Obtaining / genetic transformation of primary bovine fetal fibroblasts (BOFFs) a) Investigation of the sensitivity of bovine fetal fibroblasts (BOFFs) to various antibiotics (selection markers)

BOFFs were plated semi-confluently and the sensitivity to neomycin (G418), hygromycin and puromycin was examined in terms of concentration and effect / time. different Preparations from BOFFs showed clear differences (up to 2-100x) in relation to the necessary antibiotic concentrations in order to achieve the LDι ₀₀ . The time / effect windows between and within the preparations were sometimes greatly shifted. The best results were achieved with puromycin at a concentration of 1.5 μg / ml (3-5 times the concentration compared to the selection of stable human or murine fibroblast cell lines).

Different transfection methods that are commonly used to transfect established

Cell lines used were tested for their efficiency (antibiotic-resistant clones / transfection approach) in BOFFs. The following were tested:

DOSPER (Röche, Lipofectin, polycationic lipids);

LIPOFECTAMINETM (GBCOBRL Life Technologies, Lipofection, polycationic lipids);

CLONfectinTM (Clontech, Lipofection, cationic and amophilic lipids);

TransFastTM (Promega, Lipofection, cationic and amphophilic lipids); Ca ₃ (PO ₄ ) ₂ DNA precipitation.

The best results were achieved with Lipofectin using TransFastTM and transfection of Ca ₃ (PO ₄ ) ₂ DNA precipitates in the presence of 12% DMSO.

n In contrast to established fibroblast lines, which can be selected without problems in high dilution, so that antibiotic-resistant clones are formed, BOFFs cannot be plated below a certain critical density. When selecting for single cell clones or growing from extreme dilution to isolate single cell clones, BOFFs either die or go through one or more crises, which lead to changes in morphology and / or prophylactic behavior. The plating density of the BOFFs was therefore chosen in such a way that optimal growth was ensured. However, this means that the antibiotic-resistant cells represent a population and are not of clonal origin. Subsequent isolation and subcloning generate transgenic clonal cell lines b) Transfection with ScFv and pJWόpuro in BOFFs

Plasmids used:

MAR :: ScFv: the MAR sequences (chicken lysozyme gene matrix attachment regions; Castilla et al., Nat. Biotechnol. 16 (1998), 349) were cloned with ScFv in BlueScript (Stratagene). p77 (Brem et al., Theriogenology 43 (1995), 175) in ρUC18 (Norrander et al., Gene 26 (1983), 101).

pJWόpuro (Morgenstern & Land, Nucleic Acids Res. 18, 1068) p77 and MAR :: ScFv were linearized to guarantee a functional integration of the constructs. Due to the restriction enzyme interfaces available, the vector sequences could not be separated from the gene construct sequences. pJWόpuro was transfected circularly / supercoiled. The amounts of DNA used or the mixing ratio ScFv: selection marker was 8: 1 (2.5 μg p77 + 2.5 μgMAR :: ScFv + 0.6 μgpJW6puro = 5.6 μg total DNA) or 3: 1 (0.7 μg p77 + 0.9 μgMAR :: ScFv + 0.6 μg pJWόpuro = 2.2 μg total DNA).

BOFF # 32330201 pl cells were used, from which embryos had already been created more than a year before the transefection by cloning and after transfer also clone calves (10 calves born). After the trypsin treatment, the cell suspension was transferred to 10 mm culture dishes and with Dulbecco's modified Eagle's Medium (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum (Biochrom, Berlin), 2mM L-glutamine, 10-4mM 2-mercaptoethanol , 2 mM non-essential amino acids (Sigma, St. Louis, MO), 100 IU / ml penicillin and 100 μg / ml streptomycin. The cells are cultured at 37 ° C in 5% CO ₂ in air until the cell lawn is subconfluent (2 to 3 days) after which part of this "passage 0" is frozen (10% dimethyl sulfoxide, Sigma) and stored in liquid nitrogen ,

Transfection approaches: Before the transfection, BOFFs were sub-semi-confluent (approx. 5x10 - 10 cells) in 6-well culture dishes (0 35 mm) (MEM, 15% FCS, 1 x glutamine / penicillin / streptamycin). The transfection took place after the weaning and the expression of the fibroblast morphology of the cells. A transfection experiment was carried out for each 0 35 mm dish. Transfast® Lipofection: ratio DNA: liposome = 1: 2, Incubation time 1.5 hours

Ca ₃ (PO ₄ ) ₂ DNA precipitate transfection:

After an incubation period of 4 hours, the transfection efficiency was increased by adding 12% DMSO for 2 min (Müller et al., EMBO J. 12 (1993), 4221).

Selection, creation and analysis of transfectant pools:

The selection medium was added 24 hours after the transfection. After 2 days of selection in 6-well culture dishes, the cells of a 0 35 mm well were divided 1: 150 into a 24-well dish (concentration 2-8 x 10 ² cells / dish). The selection was continued until the shell was subconfluently covered with puro®-BOFFs (4-9 days). The pools were expanded, cryopreserved and tested for successful transfection with p77 and MAR :: ScFv using PCR.

Screening for ScFv transfectants: primer 377L2 / f 5'-CAGGTGTCCTCTCTGACATCG-3 ', and 377R2 S'CGCAGAGTCCACAGAGG-S'

(Annealing temperature, 66 ° C; 1 kb amplificate).

Screening for p77 transfectants: primers: 246 5'-GAAGACCCCATTTTGTCCCAAG-3 ', and

251 5'-GTCCCGAGGTAGATCTTCCC-3 '(annealing temperature, 62 ° C; 2.5 kb amplificate) or primer 248 5'-GATGCTTCTCTATTCCTCTG-3', and

251 5'-GTCCCGAGGTAGATCTTCCC-3 * (annealing temperature, 60 ° C; 1.2 kb amplificate)

PCR results:

Transfast® Lipofection: no p77 / MAR :: ScFv positive pools in both DNA ratios tested. Ca ₃ (PO4) ₂ DNA precipitate transfection: DNA-of-interest: selection marker = 3: 1: no p77 / MAR :: ScFv positive pools. Ca ₃ (PO ₄ ) ₂ DNA precipitate transfection: DNA of interest: selection marker = 8: 1 (pool 3): 6 of 24 pools positive for p77 and MAR :: ScFv Name of the positive clones / pools: A3, B2, B3, C2, C3, C6 (strongest signal)

Example 2 The animals were cloned according to the procedure described in PCT / EP98 / 00229 and the transgenic embryos were transferred to the recipient animals.

Example 3

Extraction of fetuses and isolation of transgenic bone marrow stem cells from the fetal liver

Transgenic cattle fetuses are obtained in the second trimester of pregnancy. During this period, the hepato-linear period of fetal development, the liver is the main site of blood formation and the

Seat of the stem cells of all blood cells, the hemocytoblasts. After immigration into the bone marrow cavity, the B lymphocytes also develop from these hemocytoblasts. The fetal liver is obtained aseptically and taken up in RPMI medium with 4 μg gentamycin sulfate and 200 IU / ml heparin as an anticoagulant. A

Separation into individual cells is carried out under strictly sterile conditions.

Example 4 Transfusion of the transgenic cells into clone calves of the same chromosomal genotype

The transplantation of the fetal blood cells takes place in a suspension in physiological saline or medium by intravenous infusion using a catheter (14 gauge, 1.7mm x 64mm, Teruno Co. Ltd).

Example 5

Collection and purification of serum by taking blood

Blood is obtained by puncturing the jugular vein at regular intervals after transfusion of the transgenic blood cells. The serum is separated from the blood cells by centrifugation of the blood (100 g, 30 min.). The serum is mixed with sodium azide (final concentration 0.02%) and filtered through cellulose acetate filters (0.22 μm). The supernatant is adjusted to pH 7.2 with 1 M NaOH. For bi-scFv molecular purification, the Culture supernatant was added to a protein L-agarose column (# 20520 Pierce, Rockford II, USA) equilibrated with 0.1M sodium phosphate buffer (protein L binds human IgG in particular also single chain variable elements (ScFv) but not bovine IgGl and IgG2). After the culture supernatant has been applied, the column bed is washed with 0.1 M phosphate buffer. The bound proteins are eluted step by step through a 0.1 M glycine buffer at pH values of 3.0 and 2.0. The eluate collected is dialyzed overnight against PBS, filtered 0.22 μm sterile and stored at 4 ° C.

Example 6 Lysis of Tumor Cells Using Bispecific Antibodies

The biological activity of the bispecific antibodies is tested in the cytotoxicity test with 5000 SK-Mel63 or M21 tumor cells. These two melanoma cell lines are very positive for the target antigen HMWG (high molecular weight glycoprotein), which is recognized by the monoclonal antibody (only scFv portion) 9.2.27. Peripheral blood lymphocytes (PBLs freshly obtained from healthy human donor and isolated by Ficoll gradient) are added in a ratio of 10: 1 to the target cells. Then 150 μg / ml of chemically conjugated bs-F (ab ') 2 with the specificity 9.2.27 x CD3 (melanoma x T-lymphocyte) is added to all cell culture dishes for panclonal stimulation of all T-lymphocytes, shows no mitogens at this concentration alone Property). The recombinant bi-scFv molecules purified from the serum are then added directly. The total volume of the test batch per cell culture dish in the microtiter plate is 150 μl. The plate is incubated for 5 days in an incubator at 37 ° C / 5% CO ₂ . The non-adherent blood lymphocytes are removed by washing them several times with PBS, so that only the remaining adherent tumor cells remain in the cell culture dishes (visual control), the cell culture dishes become 100 μl fresh medium and 10 μl of the Cell Proliferation Reagent dye WST (Boehringer Mannheim, Cat. No. 1644 807) was added. This is followed by another incubation (37 ° C / 5% CO ₂ ) in the incubator for 1 to 4 hours and then an evaluation using an ELISA reader (480 nm). The visual evaluation and the low optical density show a tumor cell killing of 100%. This shows that recombinant bispecific antibodies that are produced in transgenic blood cells are highly efficient.

Claims

claims

1. A process for the recombinant production of substances, in which a) cells with a nucleotide sequence coding for the substance are transformed, b) the transformed cells are subjected to a cloning process, and c) the cells obtained in step b) are introduced into a recipient organism ,

2. The method according to claim 1, wherein the cells to be transformed in step a) are subjected to a cloning process before the transformation.

3. The method according to claim 1 or 2, wherein the cells to be transformed in step a) are isolated from an existing cell clone or an existing organism.

4. The method according to any one of the preceding claims, wherein the recipient organism is an animal, a fetus, an embryo or cell aggregate.

5. The method according to any one of the preceding claims, in which certain cell types are removed in the recipient clone and these by recombinant cells of the same

Type to be replaced.

6. The method according to any one of the preceding claims, wherein the recipient organism has the same genotype as the cells to be transformed.

7. Use of transgenic animals, produced according to one of claims 1 to 6 for the production of recombinant substances.

8. A process for the production of cells, tissues or organs in animal organisms, in which a) cells are isolated from an individual, b) the cells are introduced into an immune-incompetent animal organism, c) the organism is cultivated, d) the cells of the individual grown in the organism are isolated, and e) the isolated cells are introduced into an individual.

9. The method of claim 8, wherein the cells grow into tissues and / or organs and the grown tissues / organs are introduced into the individual.

10. The method according to any one of claims 8 or 9, wherein the cells are derived from the individual to whom they are returned.

11. The method according to any one of claims 6 to 8, wherein the animal organism was obtained by cloning.

12. The method according to any one of claims 8 to 9, in which endogenous cells of a certain type have been removed in the animal organism.

13. Use of the method according to one of claims 8 to 12 for the production of grafts.

14. Use according to claim 13, wherein human grafts, in particular autologous human grafts are produced.