DE19545351C1 - Eukaryotic cells with hybrid receptor and thus controllable gene construct, process for their production and use of these cells in gene therapy - Google Patents

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

The present invention relates to eukaryotic cells that have been modified by genetic engineering methods, that these cells have at least one hybrid receptor and at least one controllable by this hybrid receptor Gene construct included.

Certain hybrid receptors are already in the prior art have been described. Brocker et al. (Eur. J. Immunol. (1993) 23, Pp. 1435-1439) describe a hybrid molecule comprising one Part of a single chain antibody Distance group with the transmembrane and cytoplasmic region of CD3ζ is connected. The CD3ζ chain is the Area of the T cell antigen receptor used for Signal transmission of the T cell antigen receptor in the cell responsible for.

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

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

Moritz et al. (Proc. Natl. Acad. Sci. 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 F ^ν antibody, more precisely from the connected 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.

The expression of genes is mostly in eukaryotic cells regulated by relatively complicated mechanisms. The Individual genes can be signaled from outside the cell can either be switched on or off. The path of the signal from the cell surface via a specific for the signal Receptor and optionally several intermediate molecules such as for example kinases or phosphatases in the cell nucleus and regulation of gene expression via DNA-binding Transcription factors are called signal transduction.

The object of the present invention is to use such cells Provide those through an outside cell existing signal the expression of a suitable gene in the Inside the cell can be effected.  

The present invention therefore relates to eukaryotic Cells with a hybrid receptor, one on the outside the cell localized for a particular signaling molecule specific receptor part and one from another receptor originating receptor part located inside the cell, which can trigger a signal inside the cell, comprises, the Cell contains at least one further gene construct, the one Control area that with that of the hybrid receptor emitted signal can interact and thereby the Expression of a related to this control area Can control transgene.

The part of the hybrid receptor is preferably which is located on the outside of the cell and for one specific signaling molecule is specific to a Antigen receptor, which can be selected from the group consisting of fragments of antibodies used for certain Antigens or haptens are specific, the on the Part of the hybrid receptor located outside the cell can also include a spacer.

The part of the hybrid receptor that is on the inside of the Cell is localized and trigger a signal inside the cell can, it is preferably a receptor part that can be selected from the group consisting of the intracellular and / or transmembrane areas of T cell anti gene receptors and B cell receptors.

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

The gene construct additionally introduced into the cell shows also at least any transgene. Is preferred the transgene selected from genes for gene products encode, such as cytokines, especially G-CSF, IL-2, IL-3, IL-4, IL-6, interferons, erythropoietin or insulin. In which 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 basically in all eukaryotic cells, such as animal cells Cells.

In a preferred embodiment, the cells used according to the invention around human cells, those cells which are selected are particularly preferred are from the group consisting of lymphocytes, hematopoietic stem cells, fibroblasts or tumor cells.

In the inventive method for producing the Cells, the target cell is transfected, causing the genetic information for at least one hybrid receptor and introduced at least one gene construct into the target cell becomes. The transfection is carried out by known Process in which electroporation is particularly preferred.

The genetic information for hybrid receptor and Gene construct can be isolated on one vector or on two Vectors be distributed.

The cells of the invention are preferred in the Gene therapy is used, taking the required Gene products can be expressed in the body, namely under control of the specific for the hybrid receptor Signal. A special area of application is Tumor therapy or diagnosis. If a tumor cell with a specific tumor antigen from a cell that has a variants of the antigen receptor against this tumor antigen  carries, is recognized, three reactions can be achieved a killing if a cytotoxic after tumor contact Cytokine like TNF is secreted or an immunostimulating by secretion of IL-2 or a purely diagnostic one Secretion of G-CSF. This latter, diagnostic Aspect is interesting because it is in the organism biologically detect isolated tumor cells, because just the antigen-receptor contact leads to the formation of G-CSF. G-CSF increases the number of leukocytes, you can see that still Tumor cells are present. This is especially for Residual tumors an elegant way of diagnosis.

The regulation of cellular genes is a process that takes many Intermediate stages from the cell surface (the ligand) extends to the cell nucleus (the regulated gene). It comes making mistakes in this way can lead to sustainable problems for the cell and the whole organism. On Activation signal that can no longer be switched off, could therefore lead to cell degeneration and thus to unregulated Leading growth, commonly referred to as cancer. The present invention provides cells that express a particular gene product, namely Excitation by a specific signaling molecule that is in the Area of the cell.

In the used in the context of the present invention Hybrid receptors are such constructs that the extracellular domain of a particular 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's internal signal is added of a ligand for (A). Under Ligand is here understood the specific molecule that is associated with the extracellular domain of the receptor (A) can interact. The consequence of this interaction is not that the signals are triggered in the cell that affects the receptor (A)  would be due, but it will be in the cell generates those signals that are under the control of the receptor (B) stand. Basically, within the scope of the present Invention used all receptors and their components will. Molecules or are used as ligands Connections used with the extracellular domain of the hybrid receptor can interact. This Ligands can be either in free form or bound to a carrier or on cells.

Receptors within the scope of the present invention the antigen receptors are preferably used. Also these receptors consist of different domains. Often these receptors are complex and exist from several associated protein chains that cross over Disulfide bridges are covalently linked. These chains will be generally called "light" and "heavy" chains because of their called different lengths. On these chains themselves individual sections are again defined as constant or variable regions. The interaction of the right ones variable and constant regions of heavier and lighter Chain enables highly specific recognition and binding an antigen or hapten. This of antibodies, in particular regions originating from monoclonal antibodies can use genetic engineering methods with others Areas of a receptor can be connected and thereby Antigen receptors are provided which are in the extracellular area with a special ligand (here Antigen or Hapten) bind and a signal inside the Hand over cell.

The cells of the invention now have in addition to the Hybrid receptor has at least one further gene construct, into the cell using genetic engineering methods was introduced. This gene construct has one tax area, i.e. a promoter or repressor who works with interact with the signal emitted by the hybrid receptor  can. The signal then switches that behind the promoter arranged transgene on or, if it is a Repressor acts, the transgene is through the signal switched off.

The one released into the cell interior by the hybrid receptor Signal usually also interacts with one in the cell existing control area. So if, for example, by the receptor the signal to activate an interleukin-2 gene is inserted into the cell, the receptor switches that Interleukin-2 gene on and Interleukin-2 is expressed. in the The scope of the present invention now also lies in the cell a gene construct before that - to close the previous example remain - has the promoter for interleukin-2 and on a transgene is connected to this promoter. If through the hybrid receptor the signal to activate the Interleukin-2 promoter is then introduced into the cell will not only be the transgene but also interleukin-2 expressed.

Other promoters, e.g. B. the CMV 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 such target cell lines in which the under Control of the promoter interacting with the signal standing gene is expressed only weakly or not at all. This can effectively regulate the transgene and the expression of possibly unwanted Genetic products are prevented.

Another type of receptor used in the context of present invention are used Cytokine receptors.  

The cytokines act as external messengers and enter Interaction with the cytokine receptors. In general cytokine receptors consist of at least three areas (Domains). The extracellular area is for the binding of the specific ligand responsible. With the ligands is the respective cytokine. To the extramellular area, the transmembrane area closes on, i.e. the area that extends through the cytoplasmic membrane extends and often for oligomerization of the Receptor molecule (homo- and heteropolymers) responsible is. The intracellular is inside the cell Area responsible for the transmission of the signal into the cell interior necessary is. This signal transmission can be different Way going on. An example of this would be one molecular inherent kinase activity or a forwarding of the Signals to associated cytoplasmic molecules.

When used in the present invention Gene construct has a repressor as the control area, the repressor can be activated by the hybrid receptor, which then inhibits its target sequence internally.

Within the scope of the present invention, it would also be conceivable in the cell to introduce a gene construct that consists of a control area (promoter), which corresponds to that of Hybrid receptor signal released into the cell interior interacts and has a repressor as a transgene. So if the hybrid receptor is by a signaling molecule is activated outside the cell, then a signal is sent to the Transfer cell interior and thereby expresses the repressor. This repressor can then use a naturally occurring one Gene interact in the cell and its expression repress.

A concrete example of the present invention is one Cell that is specifically stimulated to produce insulin can. This cell therefore has a hybrid receptor that  in the extracellular area, i.e. as antigen-specific Binding site, specifically with glucose as activator Ligands can interact. If there is enough in the blood high glucose concentration is achieved that binding the Glucose takes place at the hybrid receptor, this becomes Hybrid receptor its activation signal inside the Pass on the cell, with the help of another Receptor, for example T cell antigen receptor-derived Area. A signal is now triggered inside the cell for example with the promoter for interleukin-2 interact and turn on this promoter.

Because the cell contains a gene construct that acts as a transgene Contains gene for insulin and under the control of IL-2-Pro motors, production becomes this way switched on by insulin, depending on the im extracellular area existing glucose.

In the context of the present experiments, a hybrid receptor was used, which is shown schematically in FIG. 1. In the extracellular region of the hybrid receptor there is a region that is specific for a specific 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, thus consists of the light chain, bound to the variable part and a short section of the constant part of the heavy chain. In the variant antigen receptor, these areas were combined on a cDNA.

The antigen receptor is connected to the CD8 ^α region, the transmembrane part ζ and 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 became the Transfection of various lymphocytes used. In order to these lymphocytes express a variant Antigen receptor that specifically recognizes NIP, especially when this NIP is coupled to bovine serum albumin, and, which signals via the CD3ζ domain into the cell interior for example to the IL-2 promoter. Instead of one Plasmid vector can also use other suitable vectors, such as for example based on retroviral elements Vectors.

The cells provided according to the invention allow for advantageously other signals by arbitrarily selected signals, any genes can be switched on or off. The signals to which the hybrid receptors respond it can either be the body’s own signals, for example tumor antigen or the like, or around others Signals that are targeted, for example, in the context of gene therapy be brought into the body.

As a transgene, practically any desired gene can be attached to the Gene construct can be added.

In the context of the present invention, an example for Confirmation of the principle of the present invention carried out.

example 1

To test whether an endogenous antigen is a cell's own Promoter with a transgene behind it via a Variants of the antigen receptor was regulated on a System T cell + IL-2 promoter used.

The long-known Jurkat became the T cell line Line used (originated from a human acute T cell Leu kemia), which can be purchased in various ways, see e.g. B. American Tissue Culture Collection ATCC. This Line still has the option of adding the IL-2 promoter regulate, but generally forms very little IL-2 levels that are barely 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 FIG. 1.

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

For this purpose Jurkat cells (CR16s: without variants Antigen receptor; CR16s + NP8Z: with variant ζ chain re zeptor) electroporated, following the following steps were:

• - 5 × 10⁶ cells in 500 µl buffer in 0.4 cm electroporation give cuvette (biorad),
• - 10 µg pIL-2/1 + Luc (luciferase construct with murine Add 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, cells on 5 × 10⁵ / ml medium to adjust,
• - incubate in 24 wells overnight with and without induction, Induction with 0, 2, 10, 40 µg / ml NIP-BSA.

The next day the cells were made according to the protocol from Promega and the synthesized luciferase detected quantitatively using luciferin (Promega). All Experiments were carried out in quadruplicate and were reproducible.

Results

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

Table 1

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 (maximum increase of 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 was a Plasmid used that controls luciferase under human control CMV promoter expressed.

Cells used: CR16s + NP8Z

Results

The induction is about 2.3 times. Because the CMV promoter by itself from a very strong promoter was not a special one strong induction has been expected. This experiment only serves the confirmation of the first to general applicability to prove the system.

Example 3 Construction of a hybrid receptor

The construction of a chimeric single-chain antigen-Hy bridreceptor is described below. This Hybrid receptor has the internal name "pANP8Zn" receive.

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 β-actin promoter and the coding region of the following components of the single-chain receptor: CD8 ^α hinge region, TM and the cytoplasmic sequence of the TCR-ζ chain (T cell anti gene 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 above-mentioned precursor construct has now been replaced by an sc-Fv Fragment of the B1-8 antibody, which the hapten 3-hydroxy 4-Phenylacetyl (NP) binds, expanded. The amplification of the VH and VL sequences of a monoclonal antibody that 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. Sci. (1988) 85, pp. 5879-5883 described.  

The scFv fragment was cloned behind the leader sequence of the B1-8 antibody (Bothwell et al., Cell (1981) 24, pp. 625-637) and this fragment was fused in frame with the CD8 ^α- hinge 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 B1-8 antibody used in the present experiment also binds the hapten 4-hydroxy-5-iodo-3-nitro phenylacetate (NIP), whereby the affinity can be increased by a factor of about 10 by the iodine group present.

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

Fig. 4-A-4-C relate to the nucleotide sequence and the corresponding amino acid sequence of the construct.

Explanation of the figures

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

FIG. 2 shows the results obtained according to Example 2 in a graphical representation, the abbreviation CR16s meaning that these are cells which do not have a hybrid receptor but a gene construct with luciferase as a 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 that has the cytoplasmic sequence of the TCR-ζ chain and a sc-Fv fragment of an antibody that 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.

Claims (11)

1. Eukaryotic cell with a hybrid receptor that targets one located on the outside of the cell, for a particular one Signal molecule specific receptor part and one by one another receptor originating inside the cell localized receptor part that sends a signal inside the cell can trigger, comprises, wherein the cell at least one further Contains gene construct that has a control region that with the signal emitted by the hybrid receptor can interact and thereby express one with it Control area connected transgene can control. 2. Cell according to claim 1, characterized in that the Part of the hybrid receptor that is on the outside of the cell localized and specific for a particular signaling molecule is selected from the group consisting of Antigen receptors that work for certain antigens or haptens are specific, the one on the outside of the cell part of the hybrid receptor also located one Distance range can include. 3. Cell according to one of claims 1 or 2, characterized characterized in that the part of the hybrid receptor that is attached to the Inside the cell is localized and inside the cell Can trigger signal, is selected from the group consisting from the intracellular and / or transmembrane areas of T cell antigen receptors and B cell receptors. 4. Cell according to one of the preceding claims, characterized characterized that the control area further Gene construct that with that triggered by the hybrid receptor Signal can interact, is selected from the group consisting of promoters and repressors.   5. Cell according to one of the preceding claims, characterized characterized in that the transgene of the further gene construct is selected from genes selected for gene products the group consisting of cytokines, in particular G-CSF, TL-2, IL-3, IL-4, IL-6, interferons, erythropoietin and insulin or for Encode transcription factors. 6. Cells according to one of the preceding claims, characterized characterized that the cells are human cells, especially lymphocytes, hematopoietic stem cells, Fibroblasts or tumor cells. 7. A method for producing cells according to claims 1 to 6, characterized in that the target cell transfects which provides the genetic information for at least one Hybrid receptor and at least one gene construct in the Target cell is introduced. 8. The method according to claim 7, characterized in that the genetic information for hybrid receptor and gene construct is located in a vector. 9. The method according to claim 7, characterized in that the genetic information for hybrid receptor and gene construct two vectors is distributed. 10. Use of cells according to claims 1 to 6 in the Gene therapy. 11. Use of cells according to one of claims 1 to 6 in the treatment of tumors.