EP1234034A1

EP1234034A1 - Transfer compounds, the production and the use thereof

Info

Publication number: EP1234034A1
Application number: EP00985060A
Authority: EP
Inventors: Johannes Gerdes; Thomas Scholzen; Claudia Wohlenberg
Original assignee: Faustus Forschungs Cie Translational Cancer Research GmbH
Current assignee: Faustus Forschungs Cie Translational Cancer Research GmbH
Priority date: 1999-11-18
Filing date: 2000-11-17
Publication date: 2002-08-28
Also published as: CA2391881A1; AU2160401A; DE19955576B4; WO2001036629A1; US20030118600A1; US7189808B2; AU773085B2; JP2003514529A; CN1390257A; DE19955576A1

Abstract

Die vorliegende Erfindung betrifft die Verwendung eines carboxyterminalen Fragments des Ki-67 Proteins oder eines aktiven Teils, Fragments oder Homologs davon als eine Verbindung, die für den Transfer in Zellen und die Aufnahme und das Ausschleusen durch Zellen geeignet ist. Weiterhin umfasst die vorliegende Erfindung Transferverbindungen, die das o.g. carboxyterminale Ende des Ki-67 Proteins enthalten, sowie Vektoren kodierend dafür. Ausserdem fallen pharmazeutische Zusammensetzungen unter diese Anmeldung, sowie die Verwendung des Transferproteins als Träger oder als Wirkstoff bei der Behandlung von Krankheiten.

Description

Transfer connections., Their production and their use

DESCRIPTION

Technical field

The present invention relates to compounds which are capable of bringing associated compounds into a cell. In particular, the present invention relates to a transfer compound comprising the carboxy terminal fragment of the Ki-67 protein. Furthermore, this application comprises vectors which contain the sequence coding for the transfer compound, transfer compounds and pharmaceutical compositions comprising these transfer compounds and / or vectors. Processes for their production and the use of these transfer compounds are also claimed. Corresponding methods for the treatment or prevention of diseases by gene therapy with the aid of these transfer compounds are within the scope of the invention.

State of the art

Protein targeting is a fundamental biological process that is controlled by highly coordinated mechanisms.

For example, protein export or protein secretion takes place on specific reaction paths, for which characterized signal sequences are used to direct the proteins into the subcellular compartments involved, such as the endoplasmic reticulum, Golgi complex and vesicles. Signal sequences are also used for intracellular transfer. For example, nuclear localization sequences (NLS) are described for the transfer of proteins into the cell nucleus, which direct large proteins that cannot get into the nucleus through diffusion through the nuclear pores into the cell nucleus. The uptake of proteins into a cell is also complexly regulated. As an example, only the receptor-mediated endocytosis should be mentioned here, which serves to import specific proteins by binding to receptors on the cell membrane and then enclosing them in vesicles. This process serves on the one hand to supply cells with metabolites necessary for metabolism, and on the other hand to break down proteins. Receptor-mediated endocytosis also mediates cellular responses to many mediators such as peptide hormones or growth factors. After all, this process is used by viruses and toxins to get into cells.

For many areas of application in biomedical research, diagnostics and therapy, it is desirable to introduce substances, preferably proteins, nucleic acids, non-peptide molecules such as oligosaccharides, lipids or drugs or marker molecules, into cells. Since many of the substances mentioned cannot pass through the cell membrane, various methods are used for the introduction or intracellular production of these substances.

In addition to mechanical methods, such as, for example, microinjection, those skilled in the art are well known for this purpose, for example, molecular biological expression techniques. However, the latter methods are not very efficient; As a rule, expression is successful in only 2-20% of the cells, which makes, for example, in vivo application very problematic. This disadvantage has recently been overcome by using a structural protein (VP22) of the herpes simplex virus type 1 (HSV-1). After classic transfection with expression vectors, it was shown that, in contrast to another HSV-1 protein, which (as expected) could only be detected in 2-5% of the cells, the VP22 could be detected in 100% of the cells (PCT application no WO 97/05265). It has also been shown that this viral protein as a fusion protein introduces various polypeptides into target line populations can (WO 97/05265). However, it is well known to the person skilled in the art that viral proteins can trigger pleiotropic effects, preferably in mammalian cells, cell assemblies or the whole organism.

The EIA protein of the adenoviruses and the T antigen of Simian Virus 40 (SV40) trigger a multitude of processes in the cells. These include, for example, the initiation of DNA synthesis and the activation of various enzymes, such as dihydrofolate reductase, thymidine kinase and DNA polymerase (Nevins, JR Adenovirus E1A: Transcription regulation and alteration of cell growth control, in Doerfler, W and Böhm, P., The molecular repertoire of Adenovirus III: Biology and pathogenesis, Springer Verlag Berlin, Heidelberg, New York, 1995). Another example is the pleiotropic properties of the structural proteins of the reoviruses (Yue, Z. And Shatkin, AJ, Enzymatic and control functions of Reovirus structural proteins, in Tyler, KL and Oldstone, .B .A., Reoviruses I: Structure, Proteins, and Genetics, Springer Verlag Berlin, Heidelberg, New York, 1998).

The present invention is therefore based on the object of providing a transfer vehicle for connections in order to overcome these disadvantages. The transfer compounds can be used in gene therapy.

The transfer vehicle according to the invention is from a mammal, preferably of human origin.

Summary of the invention

The present object is achieved according to the invention by a carboxy-terminal fragment of the human Ki-67 protein.

Another aspect of the present invention relates to a vector which codes for this fragment. Furthermore, a transfer protein is described which has the carboxy-terminal fragment of the Ki-67 protein.

The transfer protein according to the invention can be the carboxy-terminal fragment of the Ki-67 protein of humans, mice, rats or other species.

In addition, the invention relates to methods for producing transfer connections and for producing vectors which code for these transfer connections.

Another aspect is a method for transferring compounds into a target group, selected from cell lines, cells in vitro, tumor cells, tissue, etc., with the aid of the above-mentioned transfer protein according to the invention or a vector which contains the sequence coding for a transfer protein according to the invention.

Furthermore, the present invention includes the use of the above. Compounds for the transfer of associated compounds, as well as methods for the therapy and prevention of diseases, in particular the use in gene therapy.

A pharmaceutical composition containing the transfer protein according to the invention alone or in association with another compound is also provided, as well as a method for its production.

Description of the pictures:

Figure 1: Representation of the nucleotide sequence of the Kon21 DNA insert. The numbering of the base pairs and the restriction sites used for cloning are indicated above the nucleotide sequence. The amino acid sequence of the derived Kon21 protein is shown below the nucleotide sequence. Bold Nucleotides are part of the restriction sites used. Underlined nucleotides have been introduced into the construct by the deoxyoligonucleotide primers used. For a better overview, only one of the two DNA strands was given in the 5 '-3' direction.

Figure 2: Map of the vector used, pCEP4-Kon21

Figure 3: Microscopic images of cells 6 hours (a-d), 10 hours (e-h) and 24 hours (i-1) after

Transfection, test conditions, see Example 1. Staining with MIB-21 (a, e, i, c, g, k) and counterstaining with

Propidium iodide (b, f, j, d, h, 1). The cells in the left

Half of the picture were taken with pCEP4-Kon21 (a, b, e, f, i, j) and the

Cells in the right half of the image transfected with pCEP4 (c, d, g, h, k, 1).

Figure 4: Microscopic images of cells 5 minutes (a-d) and 1 hour (e-h) after the addition of the high salt lysate, see Example 2. The cells in the left half of the image were stained with MIB-21 (a, c, e, g). The right half of the picture shows the same cells stained with propidium iodide (b, d, f, h). The upper half of the picture shows cells after 5 minutes (a-d), the lower half of the picture shows cells after 1 hour of incubation with the high salt lysate (e-h). Cells after addition of high salt lysate from pCEP4-Kon21 transfected cells (a, b, e, f) or high salt lysate from pCEP4 transfected cells (c, d, 9, h).

Detailed description of the invention

The fragment according to the invention, namely the carboxy-terminal region of the Ki-67 protein, encompasses the region of amino acids from 3037 to 3256 of the Ki-67 protein, as deposited in Swiss Prot under Accession No. P46013, or fragments of the region as described by the natural variation of the genome are present. The fragment can also only parts of the above Include fragments or homologs thereof as long as the function as transfer protein is retained.

Homolog here means that there is at least 80% homology in the amino acid residues which are essential for the function of the carboxy-terminal region as a transfer compound.

The human Ki-67 protein is expressed in all nuclei of proliferating cells in all active phases of the cell cycle, ie in Gl, S, G2 and mitosis, but not in resting phase GO cells (Gerdes et al. Cell cycle analysis of a cell proliferation -associated human nuclear antigen defined by the monoclonal antibody Ki -67 J. Immuno 1.13: 1710-15, 1984). The cDNA of the human Ki-67 and the murine equivalent are known and do not show any significant homologies with other proteins (Schlüter et al. The cell proliferation-associated antigen of antibody Ki -67: a very large, ubiqui tous nuclear protein wi th numerous repeated elements, representing a new kind of cell cycle -maintaining proteins J. Cell Biol. 123: 513 - 522, 1993, Starborg et al. The murine Ki -67 cell proliferation antigen accumulates in the nucleolar and heterochro atic regions of interphase cells ant at the periphery of the mi totic chromosomes in a process essential for cell cycle progression J. Cell Sei. 109: 143-153, 1996). The human Ki-67 protein has several NLS and is physiologically only detectable in the cell nucleus, except in mitosis. Only after the microinjection of antibodies could it be shown that the Ki-67 protein is formed in the cytoplasm and is transferred very quickly, presumably in supramolecular complexes, to the cell nucleus (Heyden et al. Cytoplasmic observation of the Ki -67 protein and immunofluorescence staining of i ts transport to the nucleus Eur. J. Cell Biol. Vol. 42: 33, 1996). A transfer from the nucleus or even from the cell has not been observed or described so far. The more surprising was our finding that we in the investigation of the function of partial structures of the Ki-67 protein.

A carboxy-terminal fragment of the human Ki-67 protein, called KON-21 (Figure 1), transiently expressed in CHO-Kl (Chinese Hamster Ovarian-Kl, ATTC No. CRL 9618) cell line cells showed a completely unexpected immunocytological distribution pattern of the produced polypeptide. As expected, the KON-21 was strongly cytoplasmic in 5-20% of the cells, as with control proteins. In addition, the KON-21 was also detectable in 100% of the cell nuclei. It could be shown that the Kon-21 peptide is first produced in 5-20% of the cells in the cytoplasm and, because it contains an NLS, is quickly transferred into the cell nucleus of these producer cells (Example 1 and Figure 3). In addition, the KON-21 is passed on to neighboring, non-transfected cells and localized in these recipient cells in the cell nucleus. This intercellular transfer of the KON-21 probably does not follow any of the conventional protein export or protein import routes described above, since the KON-21 lacks classic signal sequences for these processes. The intracellular transfer into the cell nuclei is presumably via the Ran-GTP-Importin-alpha system (Goerlich D. Transport into and out of the cell nucleus EMBO J. Vol. 17: 2721-27 1998) with the help of the NLS of the KON-21 accomplished.

Experiments with constructs that code for a fusion protein with the KON-21 protein showed that these fusion proteins are efficiently expressed and transferred intercellularly.

In experiments in which the KON-21 was added to the culture medium of target cells in cell extracts, the KON-21 alone or in conjunction with a fusion protein was transferred very efficiently and very quickly into the target cells (Example 2 and Figure 4). This aspect enables the further the transfer of non-peptidyl substances that cannot be expressed intracellularly.

These aspects mentioned above allow the use of these transfer compounds in the gene therapy of diseases such as cancer, allergy, autoimmune diseases, etc. the invention also includes methods for the treatment but also for the prevention of diseases.

The Kon21-DNA construct was produced using standard molecular biological techniques. For this, cDNA of the HeLa S3 cell line was amplified by means of PCR. The restriction sites for the subsequent cloning into a plasmid vector, as well as the sequence motifs necessary for the efficient translation of the mRNA, were introduced by using deoxyoligonucleotide primers which carried additional nucleotide sequences at their 5 'ends

(see Figure 1). The Kon21-DNA consuct was initially in the cloning vector pBluescript SK from Stratagene

(La Jolla, CA, USA) cloned. After sequencing the insert DNA, there were two deviations from the previously published DNA sequence of the Ki-67 cDNA (Schlueter et al. Supra). Sequence analysis of several independent clones confirmed the correctness of the sequences obtained. The insert DNA was expressed by means of the

Restriction enzymes HindiII and Notl were cut from the cloning vector and cloned into the eukaryotic expression vector pCEP4 from Invitrogen Corporation (Carlsbad, CA, USA). Figure 1 shows the complete nucleotide sequence of the DNA insert and the encoded amino acid sequence of the expression product. Figure 2 shows the structure of the Kon21 expression construct. Examples

example 1

After transfection, the Kon21 protein is passed on to all cells in a culture.

CHO cells were transiently transfected with the construct pCEP4-Kon21 and analyzed at different times. For this purpose, the slides covered with cells were rinsed in PBS / 10% FCS, air-dried for about 6 hours and then fixed in chloroform / acetone. This was followed by immunofluorescence staining with the monoclonal antibody MIB-21, which specifically recognizes the KON-21 protein. The binding of the antibody MIB-21 was then detected using an Alexa488 conjugated goat anti-mouse antibody (Molecular Probes Inc., Eugene, Oregon, USA). For better orientation, the DNA of the cells was additionally counterstained with propidium iodide. To control the staining, CHO cells were also transfected with the expression vector pCEP4. Microscopic images of cells 6 hours (ad), 10 hours (eh) and 24 hours (i-1) after transfection. Staining was done with MIB-21 (a, e, i, c, g, k) and counterstaining with propidium iodide (b, f, j, d, h, 1). The cells in the left half of the image were transfected with pCEP4-Kon21 (a, b, e, f, i, j) and the cells in the right half with pCEP4 (c, d, g, h, k, 1). While only a few nuclei of pCEP4-Kon21 transfected cells show staining with MIB-21 after 6 hours, the staining increases after 10 hours until after 24 hours all cell nuclei are stained with MIB-21. Cells which were transfected with pCEP4 as a control, however, show only a very weak non-specific background staining over the entire period. Example 2

After addition to the culture medium, the KON-21 protein is taken up by all cells of a culture.

Around 500,000 CHO cells were transiently transfected with the construct pCEP4-Kon21. As a control, 500,000 CHO cells were also transfected with the expression vector pCEP4. After 24 hours incubation in the incubator, the cells were harvested, sedimented and the cell sediment was frozen at -70 ° C. After thawing, the cell sediment was resuspended in 500 μl ice-cold high-salt buffer (10 mM HEPES, pH 7.9, 400 mM NaCl, 0.1 M EDTA, 0.5 mM DTT, 5% glycerol) and after 5 minutes incubation at 0 ° C sedimented again. The supernatant was added to CHO cells in 15 ml of culture medium and the cells were analyzed at different times. For this purpose, the slides covered with the cells were rinsed in PBS / 10% FCS, air-dried for about 6 hours and then fixed in chloroform / acetone. ' This was followed by immunofluorescence staining with the monoclonal antibody MIB-21, which specifically recognizes the KON-21 protein. The binding of the antibody MIB-21 was then detected using an Alexa488 conjugated Goat anti-mouse antibody (Molecular Probes Inc., Eugene, Oregon, USA). For better orientation, the DNA of the cells was additionally counterstained with propidium iodide. Microscopic images of cells 5 minutes (ad) and 1 hour (eh) after the addition of the high salt lysate. The cells in the left half of the image were stained with MIB-21 (a, c, e, g). The right half of the picture shows the same cells stained with propidium iodide (b, d, f, h). The upper half of the picture shows cells after 5 minutes (ad), the lower half of the picture shows cells after 1 hour incubation with the high salt lysate (eh). Cells after addition of high salt lysate from pCEP4-Kon21 transfected cells (a, b, e, f) or high salt lysate from pCEP4 transfected cells (c, d, g, h). After adding high salt lysate from pCEP4-Kon21 transfected cells, a weak staining of the cell nuclei with MIB-21 can be seen after only 5 minutes. Show after an hour all cell nuclei strongly stained with MIB-21. Cells that were incubated with high salt lysate from pCEP4 transfected cells, however, show only a very weak non-specific background staining.

Claims

Use of a carboxy terminal fragment of the Ki-67 protein or an active part, fragment or homologue thereof as a compound suitable for transfer into cells and uptake and delivery from cells.

Use according to claim 1, characterized in that the carboxy-terminal fragment contains amino acids 3037 to 3256 of the sequence of the Ki-67 protein as described in Swiss Prot under Acc. No. P46013 is shown, or contains homologous sequences thereof.

Use according to claim 1, wherein the carboxy-terminal fragment, the active part, a fragment or homologues thereof is associated with a second or more component (s) whose transfer into the target cell is desired.

Use according to claim 3, wherein the further component (s) are peptides or non-peptides.

Use according to one of the preceding claims 1 to 4, wherein the transfer compound, associated with one or more further compound (s), is added to a target population of cells and this is taken up by the target population.

Use according to claims 1 to 4, wherein the transfer compound, associated with one or more further compound (s), is transferred from a cell containing this compound and optionally produced to another cell and is taken up by the latter.

7. Transfer protein comprising the carboxy terminal end of the Ki-67 protein, the active part, a fragment or a homologue thereof, in connection with one or more further component (s) to be transferred.

8. Transfer protein according to claim 7, wherein the further component / further components comprises peptides and non-peptides.

9. Transfer protein according to claim 7 or 8, wherein the transfer protein is produced by a first cell and is taken up by another cell which does not itself produce this transfer protein.

10. Transfer protein according to claim 7 or 8, wherein the transfer protein is produced recombinantly.

11. Nucleic acid or homologs thereof which encodes the transfer protein mentioned in claims 7 to 9.

12. Vector containing a nucleic acid according to claim 11, which allows expression of a transfer protein according to one of claims 7 to 9.

13. Expression vector according to claim 12 with which a first cell is transfected or transformed and the product is then transferred to a second / further cell (s).

14. Eu or prokaryotic host cell containing a vector according to claim 12 or 13, a nucleic acid according to claim 11 and which produces the transfer protein according to claims 7 to 10.

15. Use of the carboxy-terminal end of the Ki-67 protein or the transfer protein according to claims 7 to 10 as a carrier for active ingredients in pharmaceutical compositions.

16. Pharmaceutical composition containing the carboxy-terminal end of the Ki-67 protein according to claims 7 to 10 as a carrier and transfer partner for other pharmaceutically active substances.

17. Use of the carboxy-terminal end of the Ki-67 protein according to claims 7 to 10 for the production of a pharmaceutical composition in gene therapy.

18. A method for transferring a compound to a cell population as a target population, comprising the steps:

a) introduction of the expression vector according to claim 12 or 13 into a host cell, b) expression of the transfer protein encoded by the vector according to one of claims 7 to 10 in the host cell and excretion of the same and c) transfer of the transfer protein to another cell population.

19. The method according to claim 18, wherein the expression vector is introduced into the host cell by transformation, transfection or microinjection.

20. A method for the transfer of compounds into cells in vitro, wherein the transfer protein is brought into the environment of the target cell.

21. A method for the transfer of compounds into cells comprising the steps of:

a) Recombinant expression of the transfer protein according to claims 7 to 10 with the aid of an expression vector according to claims 12 or 13 and b) combining the target cells and the transfer protein produced in step a.) in vitro so that the target cells absorb it.

22. A method for the treatment, prevention and therapy of diseases, associated compounds being introduced into cells by means of transfer protein according to claims 7 to 10 or with the aid of the expression vector according to claims 12 or 13.

23. The method according to claim 22, wherein the diseases are cancer, allergy, autoimmune diseases, inflammation, rheumatic diseases.

24. Use of the transfer protein according to claims 7 to 10 or the expression vector according to claims 12 or 13 in gene therapy.