WO1997020938A2

WO1997020938A2 - Cells with hybrid receptor and gene construct which can be controlled by said hybrid receptor, and use of said cells in gene therapy

Info

Publication number: WO1997020938A2
Application number: PCT/DE1996/002334
Authority: WO
Inventors: Roland Mertelsmann; Peter Kulmburg; Felicia Rosenthal
Original assignee: KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
Priority date: 1995-12-05
Filing date: 1996-12-03
Publication date: 1997-06-12
Also published as: WO1997020938A3; AU1868697A; DE19545351C1

Abstract

The present invention relates to cells with a hybrid receptor comprising a receptor part localised on the outside of the cell and specific to a particular signal molecule and a receptor part originating from another receptor, localised on the inside of the cell and capable of setting off a signal inside the cell. The cell contains at least one other gene construct with a control region which can interact with the signal sent out by the hybrid receptor and thereby control the expression of a transgene bound to this control region. Also disclosed are methods of producing the claimed cells and their use in gene therapy.

Description

CELLS WITH HYBRID RECEPTORS AND CONTROLLABLE GENE CONSTRUCTS, AND USE OF THESE CELLS IN GENE THERAPY

The present invention relates to cells which have been modified by genetic engineering methods such that these cells contain at least one hybrid receptor and at least one gene construct which can be controlled by this hybrid receptor.

Certain hybrid receptors have already been described in the prior art. Brocker et al. (Eur.J. Immunol. (1993) 23, pp. 1435-1439) describe a hybrid molecule comprising part of a single-chain antibody which is linked via a short spacer group to the transmembrane and cytoplasmic region of CD3ζ. The CD3ζ chain is the area of the T cell antigen receptor that is responsible for the signal transmission of the T cell antigen receptor in the cell.

The ζ chain is traditionally assigned to the CD3 complex, which forms the actual T cell receptor with the α and ß chains of the classic TCR (T cell receptor). It is citizens it is becoming more and more common to call the ζ chain part of the TCR. A strict nomenclature is not adhered to in the literature.

Eshhar et al. (Proc.Natl. Acad.Sei. USA, Vol. 90 (January 1993), pp. 720-724) describe hybrid receptors in which the variable regions (Fv) of an antibody have been linked to the constant regions of the T cell receptor chains, whereby T lymphocytes with a specificity dependent on the antibody type were created and the hybrid receptor transmitted a signal for the IL-2 expression. This controlled the lysis of the target cells depending on the antigen.

Moritz et al. (Proc. Natl. Acad. Se. USA, Vol. 91 (May 1994), pp. 4318-4322) describe cytotoxic T lymphocytes which have a hybrid gene. The hybrid gene was constructed from a single-chain Fv antibody, more precisely from the linked heavy and light chains of the variable regions of a monoclonal antibody against the extracellular region of the ERBB2 receptor, a connecting region as a spacer group and the ζ chain of the T cell antigen receptor (TCR) . This gene was introduced into the cytotoxic T lymphocytes using retroviral gene transfer.

In eukaryotic cells, the expression of the genes is usually regulated via relatively complicated mechanisms. The individual genes can either be switched on or off by signals from outside the cell. The path of the signal from the cell surface via a receptor specific for the signal and possibly several intermediate molecules such as kinases or phosphatases into the cell nucleus and the regulation of gene expression via DNA-binding transcription factors is known as signal transduction. The object of the present invention is to provide those cells in which the expression of a suitable gene in the cell interior can be brought about by a signal present outside the cell.

The present invention therefore relates to cells with a hybrid receptor which comprises a receptor part which is located on the outside of the cell and is specific to a specific signal molecule and a receptor part which is derived from another receptor and is located in the interior of the cell and which can trigger a signal inside the cell , wherein the cell contains at least one further gene construct which has a control region which can interact with the signal emitted by the hybrid receptor and can thereby control the expression of a transgene connected to this control region.

The part of the hybrid receptor which is located on the outside of the cell and which is specific for a specific signal molecule is preferably an antigen receptor which can be selected from the group consisting of fragments of antibodies which are specific for specific antigens or haptens , wherein the part of the hybrid receptor located on the outside of the cell can also comprise a spacer.

The part of the hybrid receptor that is located on the inside of the cell and can trigger a signal inside the cell is preferably a receptor part that can be selected from the group consisting of the intracellular and / or transmembrane regions of T cell Antigen receptors and B cell receptors.

The cells of the invention also have a gene construct that was introduced into the target cells using genetic engineering methods. In a preferred embodiment, the control area of the further gene construct is which can interact with the signal triggered by the hybrid receptor, to cell-specific or non-cell promoters or repressors.

The gene construct which is additionally introduced into the cell also has at least one transgene. The transgene is preferably selected from genes which code for gene products, such as cytokines, in particular G-CSF, IL-2, IL-3, IL-4, IL-6, interferons, erythropoietin or insulin. The transgene can also be transcription factors that can play a role in the repression of genes.

The hybrid receptors used according to the invention can in principle be used in all eukaryotic cells, such as animal cells.

In a preferred embodiment, the cells used according to the invention are human cells, with particular preference being given to cells which are selected from the group consisting of lymphocytes, hematopoietic stem cells, fibroblasts or tumor cells.

In the method according to the invention for producing the cells, the genetic information for at least one hybrid receptor and at least one gene construct is introduced into the target cell in a manner known per se. Transfection is preferred, with electroporation being particularly preferred.

The genetic information for the hybrid receptor and gene construct can be localized on one vector or distributed over two vectors. For the vectors, retroviral vectors or preferably plasmid vectors are possible. However, it is also possible for the genetic information to be on naked nucleic acid. The present invention also relates to nucleic acid constructs for the genetic modification of a cell, which contain the genetic information for

a) a hybrid receptor which comprises a receptor part located on the outside of the cell, specific for a specific signal, and a receptor part which originates from another receptor and is located in the interior of the cell and which can trigger a signal inside the cell, and

b) a further gene construct which has a control region which can interact with the signal emitted by the hybrid receptor and can thereby control the expression of a transgene connected to this control region.

The nucleic acid constructs according to the invention preferably contain the genetic information for constituents a) and b) on a single unit. The nucleic acid constructs are usually present in Foππ of DNA.

The cells according to the invention are preferably used in gene therapy, it being possible for the gene products required in each case to be expressed in the body, specifically under the control of the signal specific for the hybrid receptor. A special area of application is tumor therapy or diagnosis. If a tumor cell with a specific tumor antigen is recognized by a cell that carries a variant antigen receptor against the same tumor antigen, three reactions can be achieved, a killing if a cytotoxic cytokine such as TNF (tumor necrosis factor) is secreted after tumor contact or an immunostimulatory by secretion of IL-2 or a purely diagnostic by secretion of G-CSF. This last-mentioned, diagnostic aspect is interesting because it can be used to biologically detect isolated tumor cells in the organism, since the antigen-receptor contact to form G-CSF leads. G-CSF increases the number of leukocytes, which shows that tumor cells are still present. This is an elegant way of diagnosis, especially for residual tumors.

The regulation of cellular genes is a process that spans many intermediate stages from the cell surface (the ligand) to the cell nucleus (the regulated gene). If errors occur in this way, this can lead to sustainable problems for the cell and the whole organism. An activation signal that can no longer be switched off could therefore lead to cell degeneration and thus to uncontrolled growth, this is commonly referred to as cancer. The present invention provides cells that express a particular gene product, upon excitation by a particular signaling molecule that is in the vicinity of the cell.

The hybrid receptors used in the context of the present invention are constructs which have the extracellular domain of a specific receptor (A) coupled to the transmembrane and intracellular domain of another receptor (B). The transmembrane domain could of course also originate from receptor A. In this case, the cell-internal signal is triggered by the addition of a ligand for (A). Ligand here means that specific molecule which can interact with the extracellular domain of the receptor (A). The consequence of this interaction, however, is not that the signals are triggered in the cell which would be attributable to the receptor (A), but rather those signals are generated in the cell which are under the control of the receptor (B). In principle, all receptors and their components can be used in the context of the present invention. Molecules or compounds that can interact with the extracellular domain of the hybrid receptor are used as ligands. These ligands can be either in free form or bound to a support or to cells. Receptors which are preferably used in the context of the present invention are the antigen receptors. These receptors also consist of different domains. These receptors are often complexly structured and consist of several associated protein chains that are covalently linked via disulfide bridges. These chains are commonly referred to as "light" and "heavy" chains because of their different lengths. On these chains, individual sections are again defined as constant or variable regions. The interaction of the right variable and constant regions of heavy and light chains enables the highly specific recognition and binding of an antigen or hapten. These regions derived from antibodies, in particular monoclonal antibodies, can be connected to other areas of a receptor by means of genetic engineering methods and thereby antigen receptors can be provided which bind in the extracellular area with a special ligand (here antigen or hapten) and send a signal to the interior of the Hand over cell.

In addition to the hybrid receptor, the cells according to the invention now have at least one further gene construct which was introduced into the cell using genetic engineering methods. This gene construct has a control region, that is to say a promoter or repressor, which can interact with the signal emitted by the hybrid receptor. The signal then turns on the transgene located behind the promoter or, if it is a repressor, the transgene is turned off by the signal.

The signal delivered into the cell interior by the hybrid receptor normally also interacts with a control area present in the cell. If, for example, the signal for activating an interleukin-2 gene is introduced into the cell by the receptor, the receptor switches on the interleukin-2 gene and interleukin-2 is expressed. In the context of the present invention, the cell now also lies a gene construct that - to stay with the previous example - has the promoter for interleukin-2 and a transgene is connected to this promoter. If the signal for activating the interleukin-2 promoter is introduced into the cell by the hybrid receptor, then not only the transgene but also interleukin-2 is expressed.

Other promoters, e.g. the CMV promoter or the CD 69 promoter can be used as long as a specific activation of the promoter leads to transgene expression.

It is therefore preferred in the context of the present invention to use target cell lines in which the gene under the control of the promoter interacting with the signal is expressed only weakly or not at all. This can effect an effective regulation of the transgene and the expression of possibly undesired gene products is prevented.

Another type of receptors used in the present invention are cytokine receptors.

The cytokines act as external messengers and interact with the cytokine receptors. In general, cytokine receptors consist of at least three domains. The extracellular region is responsible for the binding of the specific ligand. The ligands are the respective cytokines. The extracellular area is followed by the transmembrane area, that is to say the area which extends through the cytoplasmic membrane and which is often responsible for oligomerization of the receptor molecule (homo- and heteropolymers). Inside the cell is the intracellular area, which is necessary for the transmission of the signal into the cell interior. This signal transmission can take place in various ways go. Examples of this would be a molecule-inherent kinase activity or a forwarding of the signal to associated cytoplasmic molecules.

If the gene construct used in the context of the present invention has a repressor as the control region, the repressor can be activated by the hybrid receptor, which then inhibits its target sequence inside the cell.

In the context of the present invention, it would also be conceivable to introduce a gene construct into the cell which consists of a control region (promoter) which interacts with the signal emitted by the hybrid receptor into the cell interior and which has a repressor as a transgene. If the hybrid receptor is activated by a signaling molecule outside the cell, then a signal is transmitted into the cell interior and the repressor is thereby expressed. This repressor can then interact with a naturally occurring gene in the cell and repress its expression.

A specific example of the present invention is a cell that can be specifically stimulated to produce insulin. This cell therefore has a hybrid receptor that can specifically interact with glucose as an activating ligand in the extracellular region, ie as an antigen-specific binding site. When a sufficiently high concentration of glucose is reached in the blood that the glucose binds to the hybrid receptor, this hybrid receptor will pass on its activation signal to the interior of the cell, using the region derived from another receptor, for example a T cell antigen receptor . A signal is now triggered inside the cell, which, for example, can interact with the promoter for interleukin-2 and switch this promoter on. Since the cell contains a gene construct that contains the gene for insulin as a transgene and is under the control of the IL-2 promoter, the production of insulin is switched on in this way, depending on the glucose present in the extracellular region .

In the context of the present experiments, a hybrid receptor was used, which is shown schematically in FIG. 1. In the extracellular area of the hybrid receptor there is a region that is specific for a particular hapten, namely 4-hydroxy-5-iodo-3-nitrophenylacetate (hereinafter NIP). This hapten is recognized by a specific B cell receptor and the antigen receptor itself consists of two chains, namely a heavy (long) and a light (short), which is encoded by two different cDNA's in the cell nucleus. The two chains themselves consist of different domains, namely the variable and constant areas. The receptor is designed so that the heavy chain is chemically linked to the light chain via disulfide bridges and is then exposed on the cell surface, anchoring in the cell membrane via the heavy chain. The extracellular part of the receptor, which can then specifically recognize the antigen, consists of the variable part of the light and heavy chain of an antibody. In the variant antigen receptor, these areas were combined on a cDNA.

The antigen receptor is connected to the CDβα region, the transmembrane and signal-conducting intracellular part of the CD3ζ molecule. In the present case, instead of the naturally occurring transmembrane / intracellular part, a new part was fused as cDNA to the cDNA encoding the antigen-binding part. This new part comes from the CD3 region of the T cell receptor complex and normally has the function of ensuring the signal transmission from the T cell receptor into the cell interior. This cDNA construct was cloned into a plasmid (expression vector) and this recombinant plasmid was used to transfect various lymphocytes. These lymphocytes thus express a variant antigen receptor which specifically recognizes NIP, in particular if this NIP is coupled to bovine serum albumin, and which transmits signals into the cell interior via the CD3 Domäne domain, for example to the IL-2 promoter. Instead of a plasmid vector, other suitable vectors, such as vectors based on retroviral elements, can also be used.

By means of the cells provided according to the invention, other, also arbitrarily selected, genes can be switched on or off in an advantageous manner by signals of any chosen type. The signals to which the hybrid receptors react can either be the body's own signals, for example tumor antigen or the like, or other signals that are introduced into the body, for example, as part of gene therapy.

Virtually any desired gene can be added to the gene construct as a transgene.

In the context of the present invention, the following examples were carried out to confirm the principle of the present invention.

example 1

A system T cell + IL-2 promoter was used to test whether an endogenous antigen can regulate a cell's own promoter with a transgene behind it via a variant antigen receptor.

The long-known Jurkat line (originated from a human acute T-cell leukemia) was used as the T-cell line, which is commercially available, see for example American Tissue Culture CoUection ATCC. This Line still has the possibility to regulate the IL-2 promoter, but generally only forms very small amounts of IL-2, which are hardly detectable.

This line was transfected with the variant antigen receptor plasmid by electroporation, then clones with stably integrated DNA were grown by selection with 2000 μg / ml neomycin. This results in the cell line CR16s + NP8Z. The variant antigen receptor itself has the structure shown in Figure 1.

The experiment was intended to show that cells carrying the antigen receptor variant can transmit a signal into the cell interior by stimulation with NIP and that this signal can activate a promoter inside the cell. This promoter drives a foreign gene, here as a marker gene, luciferase, and the genesis of this product can be demonstrated. In addition, it should be shown that the increasing NIP concentrations lead to an increased transgene expression when stimulated.

For this purpose, Jurkat cells (CR16s: without variant antigen receptor; CR16s + NP8Z: with variant ζ chain receptor) were electroporated, the following steps being carried out:

- Put 5 x IO ⁶ cells in 500 μl buffer in 0.4 cm electroporation cuvette (biorad),

Add 10 μg pIL-2/1 + Luc (luciferase construct with murine IL-2 promoter,

- put on ice for 10 min,

- electroporation with GenePulser (Biorad) at 280 V, 950 μF (20-30 ms pulse),

- put on ice for 10 min,

- wash once with buffer, adjust cells to 5 x 10 / ml medium,

- Incubate in a 24 well hole overnight with and without induction, induction with 0, 2, 10, 40 μg / ml NIP-BSA. The next day, the cells were lysed according to the Promega protocol and the synthesized luciferase was detected quantitatively using luciferin (Promega). All experiments were carried out in quadruplicate and were reproducible.

Results:

The following table shows the amounts of luciferase that were measured in the individual experiments (mean value from 4 measurements).

Table 1

Plasmid = pIL-2 / l + Luc cells = CR16s cells = CR16s + NP8Z

NIP = 0 μg / ml 212.7 LU 270.1 LU

NIP = 2 µg / ml 239.8 LU 307.9 LU

NIP = 10 µg / ml 239.3 LU 374.9 LU

NIP = 40 µg / ml 282.7 LU 827.2 LU

LU: Luciferase light units (relative size)

As can be seen from Table 1 above and the graph in FIG. 2, NIP-BSA has only a marginal effect on the luciferase expression (increase at most 30%), while the influence of the stimulated ζ chain after NIP contact is pronounced (increase maximum 300%). In addition, a good dependence of the expression on the amount of added NIP-BSA can be seen.

Example 2

Experimental setup as in Example 1, but a plasmid was used which expresses luciferase under the control of the human CMV promoter.

Cells used: CR16s + NP8Z Results:

(Double values)

Stimulation of luciferase units

Plasmid: none none 26 28

NIP 20 μg / ml 23 22

CMV luciferase no 27101 26546

NIP 20μg / ml 63682 62463

The induction is about 2, 3 times. Since the CMV promoter is already a very strong promoter, no particularly strong induction was expected. This experiment only serves to confirm the first to demonstrate the general applicability of the system.

Example 3

Construction of a hybrid receptor

The construction of a chimeric single chain antigen

Hybrid receptor is described below. This

Hybrid receptor has received the internal name "pANP8Zn".

The vector was compiled from a precursor construct with the internal name "pFvCD8-5-OM". This precursor construct contains the resistance genes for ampicillin and neomycin, the human ß-actin promoter and the coding region of the following components of the single-chain receptor: CDδαhinge region, TM and the cytoplasmic sequence of the TCR-ζ chain (T cell antigen receptor). The primers necessary for the amplification of these sequences and the origin of the templates are described in Brocker et al. (Eur.J. Immunol. (1993) 23, pp. 1435-1439).

The abovementioned precursor construct has now been expanded to include a sc-Fv fragment of the Bl-8 antibody which binds the hapten 3-hydroxy-4-phenylacetyl (NP). The amplification of the VH and VL sequences of a monoclonal antibody, the necessary primers and the Gly / Ser linker are in Hoogenboom et al., Nucleic Acids Research (1991) 19, pp. 4133-4137); Clackson et al. , Nature (1991) 352, pp. 624-628 and Huston et al., Proc.Acad. (1988) 85, pp. 5879-5883.

The scFv fragment was cloned behind the leader sequence of the Bl-8 antibody (Bothwell et al., Cell (1981) 24, pp. 625-637) and this fragment was fused in frame with the CD8αhinge region. The restriction sites relevant for the cloning of these fragments were introduced by means of suitable synthetic linker fragments. The Sc-Fv fragment of the Bl-8 antibody used in the present experiment also binds the hapten 4-hydroxy-5-iodo-3-nitrophenylacetate (NIP), whereby the affinity can be increased by a factor of about 10 by the iodine group present.

FIG. 3 shows a schematic representation of the individual fragments of the construct.

Figures 4-A-4-C relate to the nucleotide sequence and the associated amino acid sequence of the construct.

Example 4

Human T cells can be activated using a variety of stimuli (e.g. phorbol esters such as PMA, also called TPA). EA 1, now better known as CD69, is one of the first genes to be induced (Hara et al., (1986), J.Exp.Med., 164, 1988-2005). In their publication, the authors show that CD69 can be found more on the cell surface of cells that have been induced with PMA, and more so than on cells that did not receive any activation. The CD69 was detected on the cell surface by an antibody reaction. The differences in CD69-showing cells from non-activated cells to activated cells are extremely large at 1% to 85.9%. According to the invention, the CD69 gene was therefore selected in order to monitor the activation of T cells. In the experiment, a human T cell line (Jurkat) was used, which was provided by Prof. Serfling from Würzburg. FACS analysis (cell sorting) using the Becton Dickinson antibody CD69PE (No. 347827) showed (FIG. 5) that these Jurkat cells from 3.3% to 98.8% CD69 expression by means of PMA / ionomycin activation (10 ng / ml or 0.25 μg / ml overnight) can be induced. The cells used therefore reacted in a manner which is also described in the literature.

The plasmid pAN98ZN was then introduced into these Jurkat cells by electroporation. This plasmid contains the genes for the expression of the variant antigen receptor (NIP-BSA as antigen) and the neomycin resistance. The process of electroporation has been described above. Jurkat cells containing this plasmid can be isolated on the basis of the neomycin resistance mediated with it. These cells then also express the variant antigen receptor (now VAR for short) on the surface, but depending on the clone in different amounts (5-68%).

These Jurkat cells (once untransfected without VAR or on the other hand pAN98ZN transfected with VAR) were used for the further experiments.

The aim of the experiments was to show that the cells with VAR can transmit an antigen-dependent signal into the cell nucleus, whereas the cells cannot do so without the VAR. The schematic structure of the cell is shown in Figure 6:

The VAR, which is encoded by the plasmid pAN98ZN (not shown in FIG. 6, to avoid excessive complexity) is integrated into the cell membrane (circle). After loading with the antigen NIP-BSA, this VAR can transmit a signal into the cell interior, which inter alia acts on the CD69 promoter (CD69 Prom). This signal to the CD69 promoters causes two sequelae: first, it will natural CD69 gene located in the chromosome (the "chromosomes" are shown schematically and have the gene for a protein [FACS]) activated. This increase in the CD69 protein can - as already mentioned above - be measured by FACS. Second, the CD69 promoter encoded on a plasmid in the cell is activated. Under his control is now not the natural CD69 gene, but the luciferase gene as a transgene. The expression of the luciferase is then measured as a "transgene" as in Example 1 in the luminometer.

Because of the complexity of the processes, the steps are now discussed in detail, with only the parts that are actually present in the cell being shown in the associated illustrations.

a) Non-specific activation of the chromosomal copy of the CD69 gene (FIG. 5)

The Jurkat cell (completely unchanged, i.e. without any gene construct) is stimulated with PMA + ionomycin overnight. The expression of CD69 on untreated cells (3.3%) and on stimulated cells (98.8%) is measured. It can be concluded that the chromosomal copy of the CD69 gene can be activated and is functional.

b) Specific activation of the chromosomal copy of the CD69 gene (FIG. 7)

The Jurkat cells already carry the VAR after transfection using plasmid pAN98ZN. Again, activation of the cells with PMA + ionomycin shows an enormous increase in CD69 expression (from 1.8% to 99.3%). The VAR does not appear to affect the ability of the cell to be activated. By adding NIP-BSA to these cells, activation of CD69 expression to 53.7% can be achieved. This means that the VAR can transmit a specific signal to an inducible promoter after antigen contact. This example primarily demonstrates the functionality of the VAR in the cells according to the invention; in the second place, a possible cross-interference activation:: VAR will also be excluded.

In contrast to Examples 1 and 2, stimulation with NIP-BSA was not effected here (and in all the experiments described below) by simply adding the NIP-BSA to the medium. Rather, 24-well holes were coated with 200 μl of a solution containing 50 μg / ml of NIP-BSA for 2 h at 37 ° C., the excess NIP-BSA was then washed away and only then were the cells introduced into these 24-well holes . The incubation then takes place overnight as usual. This type of activation is stronger and gives a better signal inside the cell.

c) Construction of the CD69 luciferase reporter plasmid:

In the vector pXP2 (Nordeen, (1988), Biotechniques, £, pp. 454-457), a 1.4 kBP fragment with the CD69 promoter (Pvull-Sacl; see Lopez-Cabrera et al ., (1995), J.Biol.Chem., HO, 21545-21551). The finished plasmid is already the reporter plasmid pCD69 luciferase.

d) activation of the chromosomal CD69 gene after transient transfection of the cells with the luciferase reporter plasmid (FIG. 8)

The reporter plasmid pCD69-luciferase is introduced into the Jurkat cells (with and without VAR) by electroporation (see Example 1). It is measured whether NIP-BSA via VAR has an influence on the expression of the chromosomal copy of the CD69 gene. In the cells without VAR there is no change in CD69 expression (23.5% against 19.9%), in the cells with VAR, however, an increase from 29.9% without NIP-BSA to 39% with the antigen can be achieved. The basic activatability of the chromosomal copy of the CD69 gene is therefore retained, even if (at least) another copy of the CD69 promoter is introduced into the cell on a plasmid. The regulation of the promoters appears to be largely independent.

It is striking in this experiment that the basic values of CD69 expression are very high here. While non-electroporated, unstimulated cells only express about 1-5% of CD69, this percentage is much higher for the electroporated cells (23-30%). This effect is clearly due to the electroporation as such and not to the additionally introduced plasmid. The values of corresponding electroporation experiments without the addition of plasmid are not given, but have confirmed the above statement. It is also easy to see that a current surge, which must be held in such a way that at least 50% of the cells die from it, also influences the remaining 50% more or less, that is, activates (membrane repairs, etc.). However, a clear residual activatability remains as shown.

e) Specific activation of the transgene (luciferase) by the VAR (FIG. 9)

The reporter plasmid pCD69-luciferase is introduced into the Jurkat cells (with and without VAR) by electroporation. It is measured whether NIP-BSA via VAR has an influence on the expression of the luciferase gene under the control of the CD69 promoter. In the cells without VAR, the addition of NIP-BSA leads to a slight non-specific increase in luciferase expression from 29.2 to 45.1 units. In the cells with VAR, NIP-BSA causes a significant increase in luciferase activity from 129.9 to 298.8 units.

All luciferase values here are mean values from 4 independent measurements. The specific increase in luciferase activity in the cells with VAR is significantly larger than that non-specific in the cells without VAR. The statistical significance for a specific activation of the cells with VAR was calculated with a p-value of 0.037. This shows that the VAR can transmit a specific signal to activate a transgene introduced into the same cell.

It does not need to be particularly emphasized that the gene coding for luciferase can easily be replaced by a suitable other transgene. In practical use, the transgene could encode a cytokine or other therapeutically usable gene product. Likewise, the nucleic acid constructs used according to the invention can have the information for the hybrid receptor and the further gene construct on one unit.

Explanation of the figures

Figure 1 is a schematic representation of the hybrid receptor with the CD3-ζ chain.

FIG. 2 shows a graphical representation of the results obtained according to Example 2, the abbreviation CR16s meaning that these are cells which do not have a hybrid receptor, but a gene construct with luciferase as the transgene.

The abbreviation "CR16s + NP8Z" means that these are cells which have a gene construct with luciferase as a transgene and additionally a hybrid receptor, which is shown schematically in FIG. 1.

FIG. 3 is a schematic representation of a construct which has the cytoplasmic sequence of the TCR-ζ chain and an sc-Fv fragment of an antibody which binds the hapten NP or NIP. FIG. 4 shows the nucleotide sequence and the amino acid sequence of the construct explained in more detail in Example 3.

Figure 5 schematically depicts a cell that does not contain a nucleic acid construct. Within the cell nucleus, a region from the chromosome is shown schematically, which has the CD69 promoter (CD69 Prom) and behind it a gene for a protein is shown, namely the surface marker CD69. The arrow indicates that the surface marker can be found on the cell surface and can be determined quantitatively using a cell sorter analysis (FACS). Furthermore, FIG. 5 shows that the expression of CD69 can be induced by PMA / ionomycin activation (PMA / Iono).

FIG. 6 schematically represents a cell according to the invention. The gene construct which codes for the variant antigen receptor (VAR) is not shown. In the cell nucleus there is a chromosome with the promoter for CD69 and the surface marker CD69, which can be detected by cell sorting (FACS). In addition, a nucleic acid construct containing the CD69 promoter (CD69 Prom) and a transgene connected behind it is located in the cell nucleus of the cell according to the invention. In FIG. 6, the luciferase was mentioned as an example as a transgene, since this gene product can easily be measured experimentally. In practical use, the luciferase can be replaced by a desired other transgene.

FIG. 7 schematically represents a cell which contains a gene construct for the variant antigen receptor. Since the variant antigen receptor can be induced by the antigen NIP, the expression of the surface marker CD69 increases with this induction. Since the cells can be stimulated with PMA / ionomycin, the chromosomal copy of the CD69 gene must still be inducible and the gene expression of CD69 is not influenced by the variant antigen receptor.

FIG. 8 schematically shows a cell according to the invention in the lower part; the cell shown in the upper part contains no variant antigen receptor.

The graphic in the right part of FIG. 8 shows that the addition of the antigen NIP-BSA has no effect on cells without variants of the antigen receptor. If, on the other hand, the variant antigen receptor is present, a considerable increase in CD69 expression can be achieved.

FIG. 9 shows a cell according to the invention in the lower left part. In the upper left part, a cell is shown that has no variant antigen receptor. The right part of FIG. 9 shows the inducibility of the luciferase via the variant antigen receptor. 0 ELPO means that the cell has no variant antigen receptor. NIP ELPO means that the cell has a variant antigen receptor.

Claims

claims

1st cell with one

a) Hybrid receptor, which comprises a receptor part located on the outside of the cell, specific for a certain signal molecule and a receptor part originating from another receptor, located inside the cell and which can trigger a signal inside the cell, the cell comprising at least one

b) contains a further gene construct which has a control region which can interact with the signal emitted by the hybrid receptor and can thereby control the expression of a transgene connected to this control region.

2. Cell according to claim 1, characterized in that the part of the hybrid receptor, which is located on the outside of the cell and is specific for a certain signal molecule, is selected from the group consisting of antigen receptors which are specific for certain antigens or haptens, wherein that part of the hybrid receptor located on the outside of the cell can also comprise a spacer.

3. Cell according to one of claims 1 or 2, characterized in that the part of the hybrid receptor which is located on the inside of the cell and can trigger a signal inside the cell is selected from the group consisting of the intracellular and / or transmembrane regions of T cell antigen receptors and B cell receptors.

4. Cell according to one of the preceding claims, characterized in that the control region of the further gene construct, which can interact with the signal triggered by the hybrid receptor, is selected from the group consisting of promoters and repressors.

5. Cell according to one of the preceding claims, characterized in that the transgene of the further gene construct is selected from genes which are selected for gene products from the group consisting of cytokines, such as G-CSF, IL-2, IL-3, IL- 4, IL-6, interferons, erythropoietin and insulin or code for transcription factors.

6. Cell according to one of the preceding claims, characterized in that it is eukaryotic cells.

7. Cell according to one of the preceding claims, characterized in that the cells are human cells, in particular lymphocytes, hematopoietic stem cells, fibroblasts or tumor cells.

8. A method for the production of cells according to claims 1 to 7, characterized in that the genetic information for at least one a) hybrid receptor and at least one b) further gene construct is introduced into the target cell in the target cell.

9. The method according to claim 8, characterized in that the genetic information for hybrid receptor and gene construct is located on eir ^Q m vector.

10. The method according to claim 8, characterized in that the genetic information for the hybrid receptor and gene construct is distributed over two vectors.

11. Use of cells according to claims 1 to 7 in gene therapy.

12. Use of cells according to one of claims 1 to 7 in the therapy of tumors.

13. Nucleic acid construct for the genetic modification of a cell according to one of claims 1 to 7, characterized in that it contains the genetic information for

a) a hybrid receptor which comprises a receptor part located on the outside of the cell, specific for a specific signal, and a receptor part originating from another receptor, located in the interior of the cell, which can trigger a signal inside the cell and

14. Nucleic acid construct according to claim 13, characterized in that it has the genetic information for the components a) and b) in a single unit.