Patent Application
20130078219
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file “5402P453-US_ST25.txt”, file size 41 KiloBytes (KB), created on 19 Oct. 2012. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5). The substitute sequence listing in the ASCII text file entitled “5402P453-US.txt” is hereby incorporated by reference in its entirety. The ASCII text file entitled “5402P453-US.txt” was created on 4 Dec. 2012 and the size is 45 KB.
The present invention relates to a genetically modified Paramyxovirus, a pharmaceutical composition comprising this Paramyxovirus, the use of a genetically modified Paramyxovirus for the therapeutic and/or prophylactic treatment of a tumor disease, and a method for the production of a pharmaceutical composition for the therapeutic or prophylactic treatment of a tumor disease.
Statistically, every third European develops cancer in his lifetime. In Germany every year about 395,000 human beings develop cancer, about 195,000 thereof are women and 200,000 are men. Most of these cases develop at the age of over 60 years.
Solid tumors are still a big challenge of the clinical oncology. A significant improvement of the prognosis of individual tumor diseases can almost exclusively be reached by establishing new principles of therapy, integrated into multimodal concepts.
One of these new principles of therapy relates to the application of replicating viruses for the treatment of tumors. This approach is referred to as virotherapy or oncolysis. Numerous viruses have oncolytic properties with a preferred replication in different tumor cells in comparison to a reduced replication in healthy parenchyma cells. Currently, multiple virotherapeutic agents are subject of several clinical trials.
Viruses of the family of Paramyxoviridae are of particular interest. Important members of this family of enveloped viruses are the Newcastle disease virus which belongs to the genus of Avulavirus, the Measles virus which belongs to the genus of Morbilliviruses, and the Sendai virus belonging to the genus of the Respiroviruses.
The genome of the Paramyxoviruses comprises a negative single-stranded RNA, i.e. an RNA molecule encoding genes or open reading frames (ORFs) in the anti-sense mode. In a Sendai virus the 3′-head region of the RNA genome is followed by the viral genes N (Nucleocapsid), P (Phospho), M (Matrix), F (Fusion), HN (Hemagglutinin-Neuraminidase) and L (Large), followed by the 5′-tail region.
The N, P, and L proteins are required for the expression of the genes encoded by the genomic RNA and for the autonomous replication of the RNA. The HN protein supports the infection of specific cell types. The so-called Matrix protein (M) is a structure protein in the virus particle which is associated with the membrane.
The F protein has a central function in the infection by inducing the cell membran fusion which is necessary for the initial infection and the virus expansion to the neighboring cells. It is synthesized in virus-infected cells as an inactive precursor F0 and anchored in the lipid envelope of the virus which originates from the plasma membrane of the host cell. F0 is cleaved into the active subunits F1 and F2 by the tryptase “Clara” which can be found in the respiratory tract of rats and mice and is secreted from the bronchial epithelium cells. F1 and F2 have the capability to fuse cell membranes, thereby initiating the infection of the host by the virus. Therefore, the cleavage of F0 is a decisive determinate for the infectiousness and pathogenity of the Sendai virus. The protease restriction is an important determinant by which the infection with the Sendai virus in mice is restricted to the respiratory tract and cannot result in a systemic infection.
Kinoh et al. (2004), Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinses, Gene Ther. 11, p. 1137-1145, propose the use of a genetically modified Sendai virus for the treatment of tumor diseases. The principle of the genetic modification is the introduction of an artificial cleavage site into the viral F protein, which is recognized and can be cleaved by tumor-specific matrix metalloproteinases and, thereby, should enable a tumor-specific replication of the modified viruses. Furthermore, the known genetically modified Sendai virus comprises a deletion in the viral M protein resulting in an inhibition of the release of offspring viruses in such a way that an expansion of the virus is only possible by cell-to-cell contacts via fusion. This modified Sendai virus is also disclosed in EP 1 505 154.
Kinoh et al. (2009), Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies, Gene Ther. 16, p. 392-403, reports a genetically modified Sendai virus having a truncation of amino acids in the viral F protein which should result in an increase of the fusion activity. Furthermore, the viral F protein comprises a so-called “Urokinase Type Plaminogen Activator (uPA) Sensitive Sequence” (SGRS) by which a cleavage and activiation of F0 through tumor-specific proteases should extend the replication capacity of the viruses to a multitude of tumors.
Elankumaran et al. (2010), Type I Interferone sensitive recombinant Newcastle-Disease-Virus for oncolytic virotherapy, Journal of Virology, online publication, propose the use of recombinant Newcastle disease viruses (rNDV) as an anti-tumor agent which either comprise a mutation in the V protein and is referred to as “rBC-Edit”, or a mutation in the F protein and is referred to as “rLaSota V.F.”.
US 2009/0175826 reports using a recombinant Newcastle disease virus (rNDV) as an oncolytic agent, which comprises a transgene which should induce apoptosis in tumor cell lines.
The oncolytic viruses described in the art so far have not proven of value. A clinical application with a defined proof of effectiveness is still to be demonstrated. In particular, the oncolytic viruses so far used in the art have the disadvantage of also infecting and destroying non-tumor cells to an extended degree. On these grounds the oncolytic viruses used so far are unusable in a clinical application. In addition, it is not clear for which tumor diseases a good effect of individual virus systems can be reached. For this reason the oncolytic viruses used so far are not usable in clinical applications.
Against this background an object underlying the present invention is to provide an improved oncolytic virus by means of which the disadvantages of the oncolytic viruses used so far could be largely avoided. In particular, such oncolytic viruses should be provided which can replicate in tumor cells and destroy the latter, however which replicate in non-tumor cells only in a strongly restricted fashion and, thereby, fulfill a sufficient epidemiological safety aspect.
This object is achieved by the provision of a genetically modified Paramyxovirus, preferably a genetically modified Sendai virus (SeV) which, in reference to the wild type (wt), comprises in its F gene at least a first genetic modification and in its P gene at least a second genetic modification.
As used herein, “Paramyxovirus” refers to such a virus which belongs to the family of Paramyxoviridae and a virus resulting therefrom by the way of propagation. Paramyxoviruses encompass the subfamily of Paramyxovirinae with the genera of Respiroviruses, Rubulaviruses, Avulaviruses, and Morbilliviruses as well as the subfamily of Pneumovirinae with the genera of Pneumoviruses, and Metapneumoviruses. Details on the taxonomy of the Paramyxoviruses can be found, for example in Kneipe et al. (2007), Fields Virology, 5th Edition, Lippincot Williams & Wilkins.
As the inventors were able to find out, basically every strain of the Sendai virus is qualified for the modification according to the invention. Especially suitable strains of the Sendai virus are “Fushimi”, “Harris”, “Z-Strain”, “Ohita”, “Hamamatsu”, “Cantell”, and “52”.
The inventors have realized for the first time that a genetic modification of the F gene or the F protein, respectively, and the abolishment of the protease restriction provides for an efficient expansion of the virus in the tumor tissue so that a broad spectrum of different tumors can be infected.
The nucleotide sequence of the F gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 1 and has the GeneID 1489775. The amino acid sequence of the F protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 2 and has the database access nos. BAA 24390.1 or NP—056877.
The inventors have also realized that the second genetic modification in the P gene or P protein, respectively, results in an attenuation of the virus. Because of the genetic modification or mutation in the P gene, respectively, the reading frames for the genes or proteins C′, C, Y1, Y2 are shifted. However, the P protein remains completely intact.
The nucleotide sequence of the P gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 3. The amino acid sequence of the P protein is depicted in the enclosed sequence listing under SEQ ID NO: 4 and has the database access nos. BAA 24386.1 and AAB 06279.
The P gene encodes for several accessory non-structural proteins referred to as C′, C, Y1, Y2, V and W. These proteins are generated by overlapping reading frames and “RNA editing”. The exact functions of the accessory non-structural proteins are not fully illucidated in detail, whereas it is assumed that they can interact with the unspecific defense system of the infected cells, such as the interferon system.
The Paramyxoviruses as modified according to the invention are, in contrast to the wild type viruses, in the position to independently replicate in the tumor tissue and to destroy the latter, whereas no restriction to a specific tumor cell type can be observed. Surprisingly, the virus according to the invention can only replicate to a very limited extend in non-tumor cells, such as primary human hepatocytes or fibroblasts. As a result, the virus according to the invention infects tumor cells with a very high preference in reference to nontumor cells. Damages of normal tissue are significantly reduced whereas the therapeutic scope of the virus is increased.
As used herein “genetic modification” refers to a preferably targeted alteration of the genome, the genetic information or the encoded proteins of the Paramyxovirus of the invention in reference to the wild type, which can e.g. be introduced by targeted mutagenesis.
In one embodiment the first genetic modification is designed in such a manner that it results in a tropism extension.
This measure has the advantage that the virus according to the invention becomes independent from specific proteases which e.g. are only expressed by a small number of tissues or tumor cells. In this context, for example the wild type Sendai virus depends on a tryptase which is only expressed by bronchial epithelium cells and, for this reason, it can only infect these cells. The virus described by Kinoh et al. (2009; I.c.) depends on the matrix metalloproteinase (MMP) and, therefore, can only infect such tumor cells which express MMP. In contrast, the further developed virus according to the invention is in the position to infect and lyse all tumor cells by reason of the first genetic modification in the F gene.
In another embodiment the second genetic modification is configured to result in a tropism restriction.
This measure has the advantage that the virus safety is increased and side effects are significantly reduced. The virus according to the invention selectively lyses tumor cells, however not healthy cells or only to a very limited extend. The inventors have realized that through the targeted genetic modification of a P gene, i.e. a non-structure protein, the mode of response of the cell to be lysed can be influenced in such a way that the virus replicates in tumor cells in a targeted manner. This approach differs from those described by Kinoh et al. (2009; I.c.) where a deletion of the M gene which encodes a structure protein, results in the synthesis of an incomplete virus particle. That also results in the reduction of the lysis activity. Surprisingly, the lysis activity of the virus according to the invention is, restricted to tumor cells, very high.
It is preferred if through the first genetic modification in the F gene that the nucleotide sequence encoding for the SeV WT protease cleavage site, which comprises the amino acid sequence VPQSR (SEQ ID NO: 5), is replaced by a nucleotide sequence encoding for a ubiquitous protease cleavage site, preferably from the F gene of the Newcastle disease virus (NDV), which comprises the amino acid sequence RRQKR (SEQ ID NO: 6).
The modification of the proteolytic SeV-WT protease cleavage site in the F protein according to the invention abolishes the protease restriction of the wild type. The F protein can then be cleaved by ubiquitous proteases by which an independency from specific proteases which might only be present in the respiratory tract of mice or also from tumor-reducing proteases can be reached. As a result, the genetically modified virus can be used for a broader spectrum of tumors, whereas the risk of the development of a resistance is significantly reduced.
In another embodiment the second genetic modification in the P gene results in a modification of an accessory non-structural protein encoded by the P gene, preferably in such a way that at least one of the latter is not transcribable, further preferred not transcribable because of a functional destruction of the start codon.
These measures have the advantage that the virus as modified according to the invention is attenuated in such a way that the replication in non-malign cells and the infection of healthy tissue is significantly restricted. Consequently the viruses according to the invention infect tumor cells in a more targeted manner and are capable to lyse them.
It is preferred if the accessory non-structural proteins are selected from the group consisting of: C′, C, Y1, Y2, V and W, wherein it is preferred if through the second genetic modification in the P gene at least the C′/C and Y1/Y2 proteins, further preferred at least the C′/C, V, and W proteins, further preferred at least the C′/C, V, W and Y1/Y2 proteins are modified or functionally destructed, respectively.
As the inventors have been able to find out in a model system viruses which are modified in such a manner comprise a particularly high selectivity for tumor cells and a particularly poor replication capacity in non-tumor cells.
The nucleotide sequence of the C′ gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 7 and comprises the GeneID AB005796.1. The amino acid sequence of the C′ protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 8 and comprises the database access number BAA 24394.
The nucleotide sequence of the C gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 9. The amino acid sequence of the C protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 10 and has the database access number BAA 24396.
The nucleotide sequence of the V gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 11. The amino acid sequence of the V protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 12 and comprises the database access number BAA 20021.
The nucleotide sequence of the W gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 13. The amino acid sequence of the W protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO:14 and comprises the database access number AAX07444.
The nucleotide sequence of the Y1 gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 15. The amino acid sequence of the Y1 protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 16 and has the database access number BAA 24388.
The nucleotide sequence of the Y2 gene of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 17. The amino acid sequence of the Y1 protein of the Sendai virus is depicted in the enclosed sequence listing under SEQ ID NO: 18 and has the data base access number AAX07449.
In another embodiment the virus according to the invention comprises, in reference to the wild type (wt), at least one transgene, preferably a suicide gene or other cell death inducing or immunostimulating genes.
This measure has the advantage that the cytotoxicity of the virus according to the invention is again increased as the product of the transgene additionally contributes to an intensified destruction of the infected tumor cells. Transgenes having an anti-tumor effect are also encompassed.
Against this background a further embodiment of the present invention is a pharmaceutical composition comprising the genetically modified Paramyxovirus according to the invention and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are comprehensively described in the state of the art, for example in Bauer et al. (1999), Lehrbuch der Pharmazeutischen Technologie, Wissenschaftliche Verlagsgesellschaft mbH Stuttgart, Kibbe et al. (2000), Handbook of Pharmaceutical Excipients, American Pharmaceutical Association and Pharmaceutical Press. The content of the before-mentioned publications is subject of the present disclosure. It goes without saying that the pharmaceutical composition may comprise further accessory and active agents, such as cytostatics.
Another embodiment of the present invention is the use of the genetically modified paramyxovirus according to the invention for the therapeutic and/or prophylactic treatment of a tumor disease, preferably the development of a solid tumor, further preferred the development of a hepatocellular carcinoma.
The hepatocellular carcinoma has an incidence of one million each year and thus is worldwide one of the most frequent malignomas. Only in few cases a curative therapy is possible by means of resection and liver transplantation. So far convincing alternative concepts of therapy are lacking, since a distinct resistance against all chemotherapeutics tested so far can be observed. These needs are effectively met by the invention.
Another embodiment of the present invention is a method for the production of a pharmaceutical composition for the therapeutic and/or prophylactic treatment of a tumor disease, preferably the development of a solid tumor, further preferred the development of a hepatocellular carcinoma, comprising the following steps: (1) Providing the genetically modified Paramyxovirus according to the invention, and (2) formulating the genetically modified Paramyxovirus into a pharmaceutically acceptable carrier.
It is to be understood that the features mentioned before and those to be explained in the following cannot only be used in the combination as specifically indicated but also in other combinations or in isolated manner without departing from the scope of the invention.
The present invention will now be explained by means of embodiments resulting in further features, advantages and characteristics of the invention. Reference is made to the enclosed figures.
Sendai viruses are negative strand RNA viruses with a genome of approximately 15 kb. The genomic organization is shown in
The basis of the invention is provided in form of the so-called pSVV10 plasmid, more precisely the pSVV10IdGFPMFHN plasmid with a size of 19.774 by which encodes the cDNA of the Sendai virus of the strain Fushimi, ATCC VR-105. The plasmid is depicted in
The accessory proteins C′, C, Y1, Y2, V and W of the Sendai virus are encoded by the P gene. The ORFs of the C and the Y proteins are shifted by +1 base. The V and W proteins are generated according to the number of the inserted G proteins at the so-called editing site (ES). The P-ORF sequence region with the accessory genes is shown in
To insert point mutations into the genes of the accessory proteins a mutagenesis PCR was performed. However, for this method only plasmids having a maximum size of 8 kb can be used. The P/V/C region of the genome of the Sendai virus was subcloned from the Sendai virus vector pSVV10 (19774 bp) via the restriction enzymes EcoRI and SphI into the cloning vector pSL1180 (3422 bp). For this, both of the vectors of pSVV10 (
By means of targeted mutation the transcription of the accessory genes C′, C, Y1 and Y2 should be prevented. A mutation in the editing site has also the effect that the editing can no longer happen. The following overview shows the relevant cloning area of pSL1180 with all gene starts and the editing site (SEQ ID NO: 19):
reading frame of P/W/V + 1
reading frame 1
reading frame of P/W/V + 1
reading frame of P/W/V + 1
reading frame of P/W/V + 1
Three specific primers were developed which, on one side, functionally destruct the start sequences of the genes for the accessory proteins, on the other side which do not result in a change of an amino acid in the P reading frame.
With the primers in the mutagenesis PCR the following mutations have been generated: Desired mutations:
The “QuickChange Multi Site directed Kit” of the company Stratagene® enables a targeted insert of point mutations for plasmids of a size of up to 8 kb in three succeeding steps. In the first step the mismatched primers comprising individual point mutations aneal to the denaturated template single strand when given to the reaction. It has to be taken care that all primers bind to the same template strand. The PfuTurbo polymerase, beginning at the primers, extends the complementary sequence without displacing the primers. The newly generated DNA strand now incorporates the mutation and single overhanging ends, so-called “nicks”, which are adequately displaced by components of the enzyme Blends.
In the second step a digestion with Dpnl is made resulting in a digestion of specifically methylized and hemimethylized DNA. Since plasmids which have been amplified in Escherichia coli are dam methylized only the parental template is digested however not the copies generated in the PCR, which contain the mutations.
In the last step the ssDNA is transformed in XL10 gold ultra-competent cells and there converted into dsDNA in vivo. Now the plasmids can be isolated from the bacteria and analyzed for the inserted mutation by means of sequencing. The principle of the mutagenesis PCR is depicted in
The mutagenesis assay is made up as follows (Tab. 1):
The mutagenesis PCR resembles a conventional PCR, only the extension time is very long since the complete vector has to be complemented and, therefore, it varies for each vector: Two minutes per kb; here: pSL1180+Eco-SphI fragment from pSVV10˜5.5 kb corresponds to 11 minutes extension; cf. Tab. 2:
Directly after the amplification 1 μl of Dpnl restriction enzyme (10 U/μl) is given to the PCR assay, resuspended with the pipette, shortly centrifuged and digested for one hour at 37° C. The resulting ssDNA was transformed into XL10 gold ultra-competent cells and isolated from the bacterial as dsDNA-mutated plasmid by means of Miniprep; cf. Tab. 3:
$Gotoh et al. FEBS letters, 1999
The resulting mutated plasmids were verified for the correct insert of the mutations. The primer procedure with the inclusion of the entire mutation region is shown in
The mutated sequence region had to be recloned back into a vector with the complete cDNA sequence of the Sendai virus. Since viruses are to be produced which should have a ubiquitous F cleavage site instead of a cleavage site only activatable by the tryptase “Clara” in the respiratory tract of rodents the eco-eco region of the vector pRS Id-EGFP Fmut (19958 bp, Sascha Bossow, MPI Munich) has been used. Instead of the cleavage site in the F protein of the Sendai virus with the nucleotide sequence (GTTCCACAGTCGAGA; SEQ ID NO: 33) it incorporates a ubiquitous cleavage site of the F protein from the Newcastle disease virus with the sequence (CGTCGTCAGAAGAGA; SEQ ID NO: 34).
At first the different mutated SphI-EcoRI fragments were excised from the pSL1180 Sph1-Eco pSVV10 cloning vector and cloned into a Sendai virus vector pSVV13 which is similar to the vector pSVV10, however lack of the regions for the F gene and the HN gene. Then into such vector the lacking regions for the F gene and the HN gene were cloned with the modified cleavage site in the F gene from pRS Id-EGFP Fut via EcoR I. The resulting vectors are referred to as pSeVmut. Functional Sendai viruses were produced by the “rescue” method via the transfection of BSR-T7 cells. Step 1 of the recloning is shown in
By the “rescue” method for Paramyxoviridae it is possible to produce genetically modified viruses. For this BSR-T7 cells which constitutively express the T7 RNA polymerase were co-transfected by lipofection with helper plasmids and plasmids encoding the cDNA of the Sendai virus. The plasmids controlled by a T7 promoter are transcribed in the cell resulting, in several steps, in viral proteins and viral negative strand RNA genomes and in the generation of functional viruses.
BSR-T7 cells (3×105 per cavity of a plate with six cavities) are seeded and cultivated overnight at 37° C. For the transfection 200 μl of DMEM with FuGENE 6 are put into a vial (the amount of Fugene 6 corresponded to a ratio of 2 μl per μg of DNA) and were incubated for five minutes. After the addition of the DNA components which are listed in the Tab. 4 another incubation step is performed for 25 minutes at room temperature.
The BSR-T7 cells were washed for two times with DMEM; in the following 1.8 ml of DMEM medium+2% FCS is provided. The incubated transfection mixture was added dropwise under agitation and the cells were incubated for three days at 33° C. Thereafter the transfected BSR-T7 cells were washed for three times each with 1 ml DMEM to remove plasmid remainders. 1 ml of fresh DMEM medium+2% FCS was added to each assay and the newly generated viruses were harvested after one day.
Before the transfer to Vero cells for the amplification the virus containing supernatant was centrifuged at 300 rpm for four minutes at room temperature. Vero cells which were prepared four days before (plated: 2×105 cells per 3.5 cm dish; set: approx. 106 cells per 3.5 cm dish at the day of the infection) were two times washed with DMEM and infected with 100 to 500 μl of BSR-T7 supernatant (ad 500 μl adsorption volume with culture medium) for one hour at 33° C. under agitation (every 15 minutes). The inoculum was removed, the cells were washed for two times with DMEM and incubated in 1 ml culture medium for two to five days under daily exchange of medium at 33° C. As soon as eGPF was detectable as viral encoded marker protein in the fluorescence microscope the culture supernatant was removed. With this initial virus passage further passages (passages two and three) were produced at a larger scale. The titer of the virus offspring was quantified by the TCID50 method.
The following genetically modified or recombinant Sendai viruses according to the invention were produced:
The generated recombinant Sendai viruses were extensively characterized on several levels. The virus replication was analyzed on non-transformed Vero producer cells and on tumor cells, in particular on hepatoma cells (Hep3B, HuH7, PLC/PRF/5), and on several non-tumor cells such as the human fibroblast cell line MRC 5 and primary human hepatocytes (PHH) of various donors.
Growth curves
The titration of the virus particles was repeated in three independent assays, the mean value and the standard deviation are indicated (IO=inoculus).
In the three human standard cell lines for the hepatocellular carcinoma HuH7, PLC/PRF/5 and Hep3B it has been able to demonstrate that all recombinant viruses show a very good replication in tumor cells. Therefore, one decisive precondition for a relevant oncolytic activity of the recombinant viruses according to the invention is fulfilled.
The human fibroblast cell line MRC-5 and primary human hepatocytes (PHH) of several different donors were exemplarily analyzed as non-tumor cells. The result is shown in
The titration of the virus particles was repeated in three independent assays (for PHH with three different donors). Indicated are the mean value and the standard deviation (IO—inokulum).
In a further experiment the virus expansion in vivo was analyzed.
The result is shown in
The inventors were able to demonstrate by means of different recombinant Sendai viruses which have been genetically modified in respect to the wild type in their F gene and their P gene that they can be used as oncolytic viruses in the anti-tumor therapy.
The following sequences are listed:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2010 018 961.8 | Apr 2010 | DE | national |
This application is a continuation of copending international patent application PCT/EP2011/056290 filed on 20 Apr. 2011 and designating the U.S., which has been published in German, and claims priority from German patent application DE 10 2010 018 961.8 filed on 23 Apr. 2010. The entire contents of these prior applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2011/056290 | Apr 2011 | US |
| Child | 13656913 | US |
