DE19735105A1 - New fusion protein - Google Patents

Abstract

A Fusion protein (I) comprises: (a) an interaction domain which can bind to a binding domain; and (b) a peptide chain which has to be transported into a target cell. Independent claims are also included for: (1) a polypeptide comprising a binding domain, which can specifically bind to the interaction domain of (I); (2) a protein transport system comprising (I); (3) a nucleic acid construct encoding (I) and the polypeptide; and (4) a medicinal agent comprising (I) and the polypeptide.

Description

With the most diverse molecular biological Areas of application is the transport of proteins through the Plasma membrane in tissue or culture cells and in the living Organism with very considerable difficulties.

Various methods are known from the prior art, those for the introduction of proteins into the cell interior are used, such as the permeabilization of Cells, microinjection of proteins, transfection of suitable vectors and so-called immunotoxins. Various bacterial toxins that act intracellularly are able to with the help of a complex transport system through the Plasma membrane to enter the cell. Some of these toxins are used to specifically kill certain cells.

The immunotoxins developed so far go from so-called Single chain toxins from a cell-binding domain, a Translocation domain and a cell toxic domain exist. Examples of this would be the diphtheria toxin or that  Pseudomonas Exotoxin A. It is a principle of immunotoxins, a selective binding through an altered cell binding domain the toxins to a specific population of target cells are enough to finally kill them through the toxin domain.

The previously known solutions of the transmembrane However, protein transport has significant disadvantages. So violate permeabilization and microinjection techniques Cell integrity and also these techniques are in the Usually only for individual cells or cell cultures, but not applicable to whole tissue.

In the case of transfections, suitable vectors are used that are common are made up of viral components into the cells introduced and the genes contained in the vectors should then be expressed in the target cells. Is problematic here often the lack of controllability of expression and the Fact that only some cells are transfected and that the transfection is usually not cell-specific.

The immunotoxins are usually relatively large Proteins that are often expressed with difficulty can be. They are also not suitable for certain Cell types. In addition, immunotoxins are generally toxic and therefore only to be handled with considerable difficulties.

Various bacterial exotoxins are known which attack eukaryotic cells through a covalent modification of intracellular target molecules. It is believed that several steps are usually required for these toxins to work:
First, the toxins bind to a receptor on the surface of the target cells via a binding region or a specific binding component. This is followed by endocytosis caused by the receptor. This is how the toxic protein gets into the cytosol, where the enzyme component changes a specific target molecule, which causes functional changes in the target cell. With the diphtheria toxin, which can be seen as a prototype of bacterial protein toxins, three functional domains could be identified which are responsible for receptor binding, membrane translocation and enzyme activity.

For most toxins, like the diphtheria toxin or the Pseudomonas exotoxin A are based on this functional area localized in a single toxin chain, with different Chains can be linked by disulfide bridges, or these functional domains can also not be covalently associated be like B. the cholera toxin or whooping cough toxin.

In contrast, the transport systems are not according to the invention of the single chain toxins mentioned above, but those of the so-called binary toxins used. These are the binary toxins Cell binding / translocation components and the toxic enzyme separate proteins that are on the surface of the target cell to meet.

The individual components of these binary toxins are used in the protein transport system according to the invention used. The protein transport system according to the invention has at least one Fusion protein and another polypeptide component on the interact, but are preferably separate. However, it is also possible that these components Fusion protein and polypeptide together in spatial terms available. Under the term "fusion protein" is a Polypeptide understood that at least two components has, which come from different proteins, the individual polypeptide chains covalently via peptide bonds are interconnected. Under a polypeptide in the sense of present invention is understood a compound that has at least 50, preferably at least 100 amino acids.

The present invention therefore relates to a fusion protein, which is characterized in that it

• a) an interaction domain that is specific to a binding Domain can bind, and
• b) a polypeptide chain that transports into a target cell shall be,

includes.

Fusion protein in the sense of the present application is a Polypeptide that has at least two components, which in nature not in the form of fused together Polypeptides occur. One part of the fusion protein (a) includes an interaction domain that acts as an adapter and Carrier serves for those proteins that are in the target cells should be transferred. This interaction domain binds specifically to a binding domain, which in turn to certain Binds components of the target cell. The interaction domain causes the associated polypeptide component (b) is bound to the target cell via the binding domain.

According to the invention, the interaction domain comes from one binary bacterial toxin, such as the anthrax toxin or the Clostridium perfringens iota toxin. The Clostridium perfringens iota toxin is a member of the binary actin ADP family ribosylating toxins.

The interaction domain particularly preferably comes from the C2 toxin from Clostridium botulinum, and more precisely it is in the interaction domain around the N-terminal C2I domain of C2 toxins from C. botulinum.

The C2 toxin from Clostridium botulinum consists of one Binding component C2II, which comprises a binding domain, the binds to the target cell and from a biologically active Toxin component C2I by the binding component in the Target cell is transported. The binding component has C2II a molecular weight of about 100 kDa and binds with a Receptor of the target cell, creating a binding site for the  enzymatic toxin component C2I is induced. After transportation through the cytoplasmic membrane and transfer into the cytosol ADP-ribolyzed C2I, more precisely the C-terminal part of C2I, monomeric G-actin on arginine 177 and thereby inhibits the Actin polymerization.

In the context of the present invention it was found that the C2I toxin consists of at least two domains. The most C-terminus localized domain C2I-C is for the catalytic Activity, namely ADP ribosyltransferase activity responsible. The other domain located at the N-terminus (C2I-N) is for detection and interaction with the Binding component (C2II) that binds to the target cells, essential. According to the invention, only the interaction domain (C2I-N) used in the fusion proteins according to the invention, namely as an "adapter" or "carrier" for such proteins that should be transferred to the target cells. A Fusion protein according to the invention does not have, for example toxic enzyme components (C2I-C) of the C2 toxin together with C2I-N on. According to the invention, the fusion protein associates via the Adapter part C2I-N with the binding component C2II, the in turn with a specific cell receptor on the Surface of the target cell is associated. The presence of the Interaction domain in the fusion protein enables that Translocation of the fusion protein into the target cell.

In contrast, the binding domain, that can bind specifically to the interaction domain, by the same bacterial toxin from which the Interaction domain comes from, because this is an optimal Interaction can be achieved. Because the bond between the Interaction domain and the binding domain usually highest is specific, the respective domains preferably originate from the same organism and usually also of the same toxin forth.  

Basically, within the scope of the present invention any polypeptide components with the help of the invention Protein transport system are introduced into the target cells. In a preferred embodiment, however, Polypeptide component around a toxin preferred by bacteria or plants, and is a polypeptide toxin. Examples of suitable polypeptide components (b) would be Diphtheria toxin or the Pseudomonas exotoxin A, which is this is only the toxin component, without those Components used for transport through the plasma membrane are responsible. However, plant toxins, such as ricin. To be favoured used in the context of the present invention neurotoxins that from Clostridium tetani or Clostridium botulinum and cytotoxins from C. difficle (e.g. toxins A and B), C. sordellii (lethal toxin and hemorrhagic toxin) and C. novyi (α-toxin). In a further preferred embodiment the toxin is the C3-like exoenzyme of Clostridium limosum. This exoenzyme has a molecular weight of about 23 kD and inactivates the small GTP-binding protein Rho by ADP ribosylation of aspartic acid in position 41.

The protein transport system according to the invention has another Component a binding separate from the fusion protein Domain (c) on the interaction domain (a) of the Can bind fusion proteins.

This binding domain (c) can either be separated from or in contact with the target cells together with the fusion protein to be brought. The polypeptide containing the binding domain binds to the target cells via one for each Target cell specific ligand domain. The ligand can is a molecule that is specific to certain Receptors of the target cells bind. In preferred In one embodiment, the polypeptide has a binding domain, those with a binding region from the variable range of a monoclonal antibody.  

The protein transport system according to the invention therefore also includes a polypeptide having a binding domain (c) which specifically to the interaction domain (a) one can bind fusion protein of the invention.

So that this polypeptide can bind to a target cell, it has continue to have a ligand domain for cell binding. This Ligand for cell binding specifically binds to one specific receptor on the surface of the target cell.

The ligand which is specific to a Cell receptor binds to the variable region of a monoclonal Antibodies represent. Such areas can by themselves known methods and cloned in a suitable manner with the binding domain (c).

The individual components of the invention Protein transport systems are preferred with genetically engineered means. For this, the for the single nucleic acid encoding components Introduced nucleic acid construct, which is then in a suitable Host cell is propagated, with an expression of the desired Gene is achieved. The fusion proteins expressed in this way are either excreted into the medium by the host cells or from the host cells isolated. With nucleic acid constructs are preferably vectors, wherein Plasmid vectors used for expression in bacteria or yeast are suitable, are particularly preferred.

The nucleic acid constructs according to the invention have at least the genetic information for an inventive Fusion protein and / or for a polypeptide according to the invention on as well as those nucleic acid fragments that are used for multiplication of the construct in the host cell and for expression of the Fusion protein and / or polypeptide in the host cell required are.  

Such is preferably one Nucleic acid construct around a vector, this vector can preferably be a viral vector or a plasmid.

The components of the invention are preferred as Medicines used. Such a drug can for example a polypeptide component that binds Domain (c) comprises, which polypeptide component can bind specifically to desired target cells. Both Target cells can be tumor cells, for example, that express certain tumor antigens on the cell surface. If the ligand portion of the polypeptide with the Tumor antigen binds, the target cells have on the surface the specific binding domain.

Now when the fusion protein contacts these cells the fusion protein binds via the interaction Domain (a) and the binding domain (c) to the target cell. Then the polypeptide (b) linked to the fusion protein, the is preferably a toxin, inside the cell transports and can develop its activity there.

The particular advantage of the solution according to the invention is in that the individual components are non-toxic and, that there are no handling problems. Only when Binding domain and interaction domain will come together the toxin is introduced into the cell and unfolds there intended activity. This makes it possible, for example, targeted killing of certain cells without the others To burden cells with the toxic components.

A medicament according to the invention can be used in each other separate units a fusion protein and a polypeptide include. This allows the two components to be in sequence or administered together. It is preferred is for parenteral administration suitable drug.  

The present invention is illustrated by the following reproduced examples explained in more detail.

example 1 Construction of C2IN and C2IN-C3

The C2I gene (1293 bp) was derived from a C. botulinum strain with the Designation KZZ 1577 using the polymerase chain reaction amplified. For this purpose, 300 ng of chromosomal DNA were combined in one Total volume of 100 µl with two units of Tag DNA polymerase in a conventional reaction mixture with the individual Nucleotides and 15 pmol of the primer C2IC (5'- AGATCTATGCCAATAATAAAAGAACCC-3 ', SEQ ID NO. 1) containing one BglII interface and C2IN (5'- GGATCCCTAAATCTCTTTATTTTGTATAAC-3 ', SEQ ID NO: 2) a BamHI interface. The amplification was done at usual Conditions carried out and the PCR product obtained was cloned into the vector pCR2.1.

The C2I gene was used for the expression experiments BgIII / BsaBI cut and digested with BamHI / SmaI Vector pGEX2T cloned, causing the plasmid pGEX2T-C2I originated. The pGEX2T-C2IN construct was obtained by BamHI digestion by pGEX2T-C2I followed by a religation of the 6000 base pairs long pGEX2T-C2IN fragment and this vector was transformed into competent E. coli cells. For the Construction of pGEX-C2IN-C3 was the C.limosum C3 gene from a C3-containing pCR2.1 vector cut out by digestion with BglII and BamH1, ligated with BamH1-digested pGEX-C2IN and transformed into competent E.coli cells. C2IN and C2IN-C3 were sequenced using the sequencing primers 5'pGEX2T-58 and 3'pGEX2T-43. To sequence the limits of C2IN-C3 became the primer C2IN-C3 '(5'GCTATTATAACTACTATAAAGGG-3', SEQ ID NO: 3) is used. The sequencing reaction was made after Instructions of the manufacturer carried out.  

Example 2 Expression and purification of the recombinant proteins

Recombinant GST fusion proteins were in E. coli, transformed with the corresponding DNA fragment in the pGEX2T vector and purified according to the manufacturer's instructions. The resulting fusion proteins with GST were digested with Thrombin was centrifuged and the supernatant was analyzed with SDS-PAGE and Western blot analysis with antiserum against C2I or C3 examined.

Example 3 Cloning, expression and characterization of the N-terminal Part of C2I (C2IN)

In order to analyze the N-terminal part of C2I (C2IN, amino acids 1-225) for ADP ribosyl transferase activity and for its ability to bind to C2II, C2IN was expressed as a GST-C2IN fusion protein in E. coli. pGEX2T-2CIN was carried out by BamHI digestion of pGEX2G-C2I and subsequent religation of the pGEX2T-C2IN DNA fragment and transformation of this vector into competent E. coli cells. The areas of interest here are shown schematically in FIG. 1. The identity of C2IN was confirmed by sequence analysis. The C2IN protein was purified and then analyzed with the aid of a Coomassie-stained gel ( FIG. 2A) and a Western blot analysis with antiserum against C2I. This is shown in Fig. 2B. The C2IN protein has a molecular weight of approximately 25 kDa and is recognized by specific anti-C2I antiserum. To demonstrate whether C21N has ADP ribosyltransferase activity, the ADP ribosylation test for G-actin was performed with rat brain lysate. It was found that no ADP ribosylation of Act in could be observed in the presence of C2IN. From this it could be concluded that the active center of the transferase is not on the N-terminal part of C2I. In addition, the C2IN polypeptide did not produce a cytotoxic effect in the presence of C2II in HeLa cells.

Example 4 The N-terminal part of C2I is responsible for the binding at C2II

In order to test whether the C2IN polypeptide affects the interaction of whole C2I and C2II, CHO cells (Chinese Hamster Ovary Cells) were grown with trypsin-activated C2II (200 ng / ml) and C2I (100 ng / ml) in the presence of Concentrations of C2IN (100, 300, 600, 1000 ng / ml) incubated at 37 ° C. After three hours, the cells were fixed on a slide and the number of rounded cells per field was determined. Fig. 3 shows that C2IN effectively inhibited cell rounding induced by C2I, from which it could be concluded that the N-terminal part of C2I has the toxin interaction site.

Example 5 Cloning and expression of the C2IN-C3 fusion toxin

A fusion toxin C2IN-C3 was produced using the N-terminal fragment C2IN and the C3 gene from Clostridium limosum. For this purpose, the C. limosum C3 gene was excised from the pCR2.1 vector by digestion with BglII / BamH1 and then the C3 DNA fragment was isolated from the agarose gel and ligated into the pGEX2T-C2IN vector digested with BamH1 . The construction is shown schematically in Fig. 4. After transforming this plasmid into competent E. coli cells, colonies were screened to determine whether they contained the pGEX2T-C2IN-C3 vector by Western blot analysis with anti-C2I antiserum. The C2IN-C3 fusion gene thus generated was isolated from a positive clone and sequenced using standard methods. The fusion protein expressed by this clone was identified and characterized biochemically. The DNA sequence determined confirmed the presence of the fusion protein.

The C2IN-C3 fusion protein was expressed in E. coli and on the one hand with anti-C2I antibodies and on the other hand with anti C3 antibodies as a fusion protein with a Molecular weight of about 50 kDa identified (C2IN ∼25 kDa plus C3 ∼23 kDa).

Figure 5 shows that the fusion protein actually contains the two components. In the left part of the figure, which is labeled Anti-C2I, antiserum against C2I was applied. No reactivity was found with the C.limosum transferase plotted in lane 4, but a reactivity could be demonstrated with C2I (lane 1), C2IN (lane 2) and the C2IN-C3 fusion protein (lane 3). The same fragments as in the left half were applied in the right half of FIG. 5, but reacted with an anti-C3 antibody. An immunological reaction with the C2IN-C3 fusion protein (lane 3) and the C.limosum transferase (lane 4 ) was found here. However, the anti-C3 antibody did not react with C2I (lane 1) or C2IN (lane 2).

Example 6 ADP ribosylation of Rho by the C2IN-C3 fusion toxin

To the catalytic activity and substrate specificity of the Investigating C2IN-C3 fusion toxin in vitro was a ADP ribosylation test performed with rat brain lysate. Here the gene products C2I, C2IN, C2IN-C3 and C3 were considered with regard to compared for their substrate specificities.

Fig. 6 shows the expected results. Actin is ADP-ribosylated by C2I (lane 1). No activity is obtained with C2IN (lane 2).

Rhc is ADP-ribosylated by the C3 toxin (lane 4) and that C2IN-C3 fusion toxin ADP-ribosylated exclusively Rho, however not actin, which suggests that the fusion protein is essential for C3 shows typical substrate specificity (lane 3).

Example 7 Cytotoxic effects of C2IN-C3 in cell cultures

It was examined whether C2II was able to transfer of the fusion protein C2IN-C3 in eukaryotic cells cause. For the cytotoxicity test, subconfluent, single-layer CHO cells with 200 ng / ml activated C2II with Incubate 100 ng / ml C2I or 200 ng / ml fusion protein C2IN-C3. After an incubation period of three hours, was through C2I / C2II and C2IN-C3 / C2II induced cell rounding and redistribution of the actin cytoskeleton was noted. It can be concluded from this that the destruction of the Actincytoskeleton by ADP ribosylation of G-actin in Case of C2I and by ADP ribosylation of Rho in the case of C2IN-C3 was effected.

The staining of F-actin with phalloidin-rhodamine showed that Difference between the destruction of the oytoskeleton by C2 or by the C2IN-C3 toxin. As a consequence of the total Disassembly of the act in filaments became only a very weak one Rhodamine fluorescence after treatment of the cells with the complete C2 toxin found. In contrast, the incubation resulted the cells with C2II and the fusion toxin C2IN-C3 an amorphous Coloring of the remaining F-actin, which is characteristic of the C3 toxin. The same cytotoxic effects of the different Toxin combinations were observed in HeLa cells. Around possible cytotoxic activities of the individual toxin proteins To test the cells were either 200 ng / ml C2II, with 200 ng / ml C2IN-C3 or with 1 µg / ml Olimosum C3 exoenzyme incubated. Incubation of the cells with the individual  Toxin components for three hours caused none Changes in cell morphology or in F-actin Arrangement of the cells.

The specificity of the C2II-induced absorption of the fusion toxin CHO-C2RK14 cells were used to test that most likely not a functional C2II receptor exhibit. There was no effect on C2II / C2IN-C3 treatment observed when these cells for three hours with the toxin were incubated. This result confirms the presumption that the transport of the C2IN-C3 fusion toxin into the cell a C2II-receptor-mediated route.

In order to analyze the time dependence of the effects induced by the fusion toxin, the polypeptides were added to cells that had grown on slides and a sample was fixed with PFA every 30 minutes. As shown in Figure 7A, no morphological changes were observed when the cells were treated with up to 30 µg / ml C.limosum C3 toxin. In the presence of the protein transport system according to the invention [C2IN-C3 / C2II (each 200 ng / ml)], the CHO cells began to round off after 60 minutes and more than 50% of the cells were round 120 minutes after the addition of the toxin. The HeLa cells were less sensitive, but more than 80% of the cells rounded off after three hours of incubation. These results are shown in Fig. 7A. As a control (represented by a triangle), CHO and HeLa cells were incubated with the C3 toxin only.

To check the C2IN-C3 catalyzed ADP ribosylation of Rho in intact cells, the cells were lysed and Rho was ADP ribosylated in lysates from treated cells in the presence of radiolabeled NAD and C3 toxin. As shown in Figures 7B and C, a time-dependent drop in radiolabeled Rho protein was observed: After 2.5 hours of incubation with C2IN-C3 / C2II, approximately 80% of the cellular Rho was ADP-ribosylated and after three hours ( FIG. 7C) no radiolabeling was found in the cell lysates, which indicated the modification of Rho by C2IN- C3 speaks in intact cells.

SEQUENCE LOG

Claims (23)

1. Fusion protein, characterized in that it

• a) an interaction domain that can bind specifically to a binding domain, and
• b) a polypeptide chain which is to be transported into a target cell,

includes. 2. Fusion protein according to claim 1, characterized in that the interaction domain of a binary bacterial toxin comes from. 3. Fusion protein according to claim 2, characterized in that the interaction domain comes from the anthrax toxin. 4. Fusion protein according to claim 2, characterized in that the interaction domain of the Clostridium perfringens iota Toxin originates. 5. Fusion protein according to claim 2, characterized in that the interaction domain of the C2 toxin from Clostridium botulinum. 6. Fusion protein according to claim 5, characterized in that the interaction domain the N-terminal C2I domain of the C2 toxin from C. botulinum. 7. Fusion protein according to any one of claims 2-6, characterized characterized in that the binding domain that is specific to the Interaction domain can bind from the same bacterial Toxin comes from which the interaction domain also comes.   8. Fusion protein according to one of the preceding claims, characterized in that the polypeptide component (b) Is toxin. 9. Fusion protein according to claim 8, characterized in that that the toxin is a vegetable toxin. 10. Fusion protein according to claim 8, characterized in that that the toxin is a bacterial toxin. 11. Fusion protein according to claim 10, characterized in that that the bacterial toxin is a neurotoxin produced by Clostridium tetani or Clostridium botulinum. 12. Fusion protein according to claim 10, characterized in that that the bacterial toxin is a cytotoxin from Clostridium difficile, C.sordellii or C.novyi. 13. Fusion protein according to claim 10, characterized in that that the bacterial toxin is the exoenzyme C3 from C.botulinum or is a C3-like toxin. 14. Polypeptide, characterized in that it is a (c) Has binding domain that is specific to the interaction Domain (a) of a fusion protein according to any one of claims 1-13 can bind. 15. A polypeptide according to claim 14, characterized in that it continues to be a ligand for binding to a target cell having. 16. Polypeptide according to claim 15, characterized in that the ligand specifically for binding to a target cell binds a specific receptor to the target cell.   17. A polypeptide according to any one of claims 14-16, characterized characterized in that the ligand that is specific to a Cell receptor binds to the variable region of a monoclonal Is antibody. 18. Protein transport system, characterized in that it at least one fusion protein according to any one of claims 1-13 and at least one polypeptide according to any one of claims 14-17 having. 19. Nucleic acid construct, characterized in that it is the genetic information for a fusion protein according to one of the Claims 1-13 and / or for a polypeptide according to one of the Claims 14-17. 20. Nucleic acid construct according to claim 19, characterized characterized that it is a vector. 21. A nucleic acid construct according to claim 20, characterized characterized in that the vector is a plasmid. 22. Medicinal product, characterized in that it is in units separated from one another by a fusion protein of claims 1-13 and a polypeptide according to any one of the claims 14-17 includes. 23. Medicament according to claim 21, characterized in that it is for parenteral administration suitable drug.