Patent Application
20140171620
The invention relates to ciliary neurotrophic factor (CNTF) variants which, owing to the loss of affinity for the interleukin 6 receptor (IL-6R), can be used as a therapeutic with particularly low side effects for disorders or diseases which are associated with CNTF.
During the development of new medicaments which not only reduce weight safely and effectively, but at the same time positively influence metabolic disarrangements which are often associated with adiposity, such as insulin resistance and glucose intolerance, a class of molecules naturally produced by the body has become the subject of current investigation. These molecules are the so-called glycoprotein 130 (gp130) ligands from the group of interleukin (IL)-6-like cytokines, wherein the ciliary neurotrophic factor (CNTF) and its analogs are particularly investigated as potentially active therapeutics against adiposity (Febbraio M A, gp130 receptor ligands as potential therapeutic targets for obesity. J. Clin Invest 2007; 117(4):841-9).
CNTF was originally identified as a trophic factor that supports the survival of neurons in the ciliary ganglion of chick embryos (Adler R, Landa K B, Manthorpe M. Varon S. Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons. Science 1979; 204(4400):1434-6). Additional investigations revealed that this effect was not only found with parasympathetic but also with other periphery neurons, such as sympathetic, sensory and motor neurons (Barbin G, Manthorpe M, Varon S. Purification of the chick eye ciliary neuronotrophic factor. J Neurochem 1984; 43(5): 1468-78; Sendtner M, Kreutzberg G W, Thoenen H. Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature 1990; 345(6274):440-1) and also with different central neurons (Ip N Y, Li Y P, van d S, I, Panayotatos N, Alderson R F, Lindsay R M. Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures. J Neurosci 1991; 11(10):3124-34; Hagg T, Quon D, Higaki J, Varon S. Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS. Neuron 1992; 8(1): 145-58; Ip N Y, McClain J, Barrezueta N X, Aldrich T H, Pan L, Li Y, Wiegand S J, Friedman B, Davis S, Yancopoulos G D. The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron 1993; 10(1):89-102; Clatterbuck R E, Price D L, Koliatsos V E. Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system. Proc Natl Acad Sci USA 1993; 90(6):2222-6). CNTF is a molecule that has been strongly conserved during evolution and which functions are mainly restricted to the neuromuscular axis (Perret D, Guillet C, Elson G, Froger J, Plun-Favreau H, Rousseau F, Chabbert M, Gauchert J M, Gascan H. Two different contact sites are recruited by cardiotrophinlike cytokine (CLC) to generate the CLC/CLF and CLC/sCNTFRalpha composite cytokines. J Biol Chem 2004; 279(42):43961-70). CNTF shows a number of structural and functional characteristics that distinguish this factor from most other cytokines of the gp130 group. Firstly, it does not possess a signal peptide and consequently cannot be secreted. This led to the early assumption that it is a protein which is stored in the cytosol and released only upon damage of the nerve fibers (Masiakowski P, Liu H X, Radziejewski C, Lottspeich F, Oberthuer W, Wong V, Lindsay R M, Furth M E, Panayotatos N. Recombinant human and rat ciliary neurotrophic factors. J Neurochem 1991; 57(3):1003-12; Stöckli K A, Lottspeich F, Sendtner M, Masiakowski P, Carroll P, Gotz R, Lindholm D, Thoenen H. Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor. Nature 1989; 342(6252):920-3). Further, CNTF in high concentration is able not only to bind to its original alpha receptor (CNTFR), but also to the IL-6 receptor (IL-6R) (Schuster B, Kovaleva M, Sun Y, Regenhard P, Matthews V, Grotzinger J, Rose-John S, Kallen K J. Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF. J Biol Chem 2003; 278(11):9528-35).
Due to its neurotrophic potential, CNTF was tested as a potential therapeutic for different neurodegenerative diseases. Protective effects were shown in animal models (Anderson K D, Panayotatos N, Corcoran T L, Lindsay R M, Wiegand S J. Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease. Proc Natl Acad Sci USA 1996; 93(14):7346-51; Emerich D F, Winn S R, Hantraye P M, Peschanski M, Chen E Y, Chu Y, McDermott P, Baetke E E, Kordower J H. Protective effect of encapsulated cells producing neurotrophic factor CNTF in a monkey model of Huntington's disease. Nature 1997; 386(6623):395-9), and a phase I study showed the safety of intracerebrally administered CNTF for Huntington's disease (Bloch J, Bachoud-Levi A C, Deglon N, Lefaucheur J P, Winkel L, Palfi S, Nguyen J P, Bourdet C, Gaura V, Remy P, Brugieres P, Boisse M F, Baudic S, Cesaro P, Hantraye P, Aebischer P, Peschanski M. Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study. Hum Gene Ther 2004; 15(10):968-75). In contrast, in a clinical phase III study subcutaneously injected CNTF showed no influence on disease progression in patients with amyotrophic lateral sclerosis (Miller R G, Petajan J H, Bryan W W, Armon C, Barohn R J, Goodpasture J C, Hoagland R J, Parry G J, Ross M A, Stromatt S C. A placebo-controlled trial of recombinant human ciliary neurotrophic (rhCNTF) factor in amyotrophic lateral sclerosis. rhCNTF ALS Study Group. Ann Neurol 1996; 39(2):256-60). Some patients in this study developed a dose-dependent weight loss which confirmed the previously observed cachexia after systemic administration of CNTF to mice (Henderson J T, Seniuk N A, Richardson P M, Gauldie J, Roder J C. Systemic administration of ciliary neurotrophic factor induces cachexia in rodents. J Clin Invest 1994; 93(6):2632-8). Accordingly, work was focused on the elucidation of the mechanisms which lead to such a reduction of the body weight to allow the targeted modulation of these mechanisms. It is however to be concluded that CNTF is not a physiological regulator of the weight, and its cachectic effects were observed exclusively after exogenous administration in animal experiments and clinical studies.
Early on it was already known that CNTF from rats has a fourfold higher activity than human CNTF (Masiakowski P, Liu H X, Radziejewski C, Lottspeich F, Oberthuer W, Wong V, Lindsay R M, Furth M E, Panayotatos N. Recombinant human and rat ciliary neurotrophic factors. J Neurochem 1991; 57(3):1003-12). This is due to an amino acid in position 63 of the protein which is arginine (R) in rats and glutamine (Q) in humans. By the substitution Q63R, it was possible to generate a human CNTF variant whose activity corresponds to that of rat CNTF (Panayotatos N, Radziejewski E, Acheson A, Pearsall D, Thadani A, Wong V. Exchange of a single amino acid interconverts the specific activity and gel mobility of human and rat ciliary neurotrophic factors. J Biol Chem 1993; 268(25):19000-3). Further studies showed that also N-terminal or C-terminal deletions to some extent resulted in an increase of activity or at least did not negatively influence CNTF activity (Negro A, Corsa V, Corona G, Grandi C, Skaper S D, Callegaro L. Structure-function studies of human ciliary neurotrophic factor. Neurochem Res 1994; 19(2):223-7; Krüttgen A, Grotzinger J, Kurapkat G, Weis J, Simon R, Thier M, Schroder M, Heinrich P, Wollmer A, Comeau M. Human ciliary neurotrophic factor: a structure function analysis. Biochem J 1995; 309 (Pt 1):215-20). These findings ultimately led to the generation of Axokine®, a CNTF analog which differs from naturally occurring human CNTF by targeted modifications (deletion of the 15 C-terminal amino acids, replacement of cysteine in position 17 by alanine, and replacement of glutamine in position 63 by arginine) (Peterson W M, Wang Q, Tzekova R, Wiegand S J. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci 2000; 20(11):4081-90) and which is tested as a potential therapeutic for adiposity (Preti A. Axokine (Regeneron). IDrugs 2003; 6(7):696-701). Moreover, evidence has been obtained recently that the activity of Axokine® is retained even after simultaneous removal of N- and C-terminal amino acids (Breusing K. Generierung and Charakterisierung eines verbesserten Ciliary Neurotrophic Factors (CNTF). Inauguraldissertation zur Erlangung der Doktorwürde der Medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel 2007).
The CNTF analog Axokine®, developed by the company Regeneron, has already been tested as a potential therapeutic for adiposity. However, in clinical studies the induction of neutralizing antibodies against CNTF has been observed in numerous patients and the substance did not enter the market.
The therapeutic use of CNTF or CNTF analogs, respectively, has been proven problematic so far due to the severe side effects which are associated with their systematic administration. The fact that CNTF in high concentrations is able not only to bind to its original receptor, but also the IL-6R can possibly explain some of the IL-6R-mediated side effects of systemically administered CNTF, such as fever (Shapiro L, Zhang X X, Rupp R G, Wolff S M, Dinarello C A. Ciliary neurotrophic factor is an endogenous pyrogen. Proc Natl Acad Sci USA 1993; 90(18):8614-8) or the induction of an acute phase response in the liver (Dittrich F, Thoenen H, Sendtner M. Ciliary neurotrophic factor: pharmaeokinetics and acute-phase response in rat. Ann Neurol 1994; 35(2):151-63). These effects may be avoided by a monospecifity for the CNTFR.
The induction of neutralizing antibodies against CNTF observed in clinical studies is another so far very problematic aspect when using CNTF as a medicament. These antibodies can both jeopardize the therapeutic effect of the medicament and interfere with the body's own CNTF which is possibly associated with severe consequences. Therefore not only the company Regeneron but also the companies Xencor and Merck have dealt with the mitigation of CNTF immunogenicity (see WO 02/070698 A2; US 2005/0064555 A1 and US 2005/0069987 A1).
Due to the activation of IL-6R CNTF exhibits a B cell and therefore immune stimulating activity. It is hence possible that the pronounced antibody formation that was observed in response to B cell stimulation can be explained by the capability of administered CNTF to bind and activate IL-6R. A CNTF variant which is not able to activate the IL-6R would possibly no longer possess this property. Since on the other hand B cells do not express CNTFR, such a CNTF variant should not be capable of stimulating B cells. It is therefore obvious that the antibody formation against the CNTF variant is prevented in this way.
There is an urgent need for new therapeutics for adiposity and neurological diseases. CNTF variants without affinity or with only low affinity to IL-6R which render it possible to prevent IL-6R-associated side effect are interesting candidates for a new generation of CNTF medicaments for neurological diseases and for the regulation of body weight.
It is therefore an object of the present invention to provide new CNTF variants without affinity or with low affinity to the IL-6R which can be used as therapeutics without IL-6R-associated side effects.
According to the invention, this object is achieved by CNTF variants having the features of claim 1. The dependent claims relate to advantageous embodiments of the invention.
The CNTF variants according to the invention are CNTF molecules without affinity or with low affinity to the IL-6R so that these molecules will bind to the IL-6R with a lower affinity than the CNTF. This loss in activity is surprisingly achieved by a single amino acid substitution, namely the replacement of the arginine residue in position 28 of human wild-type CNTF (SEQ ID NO: 7) by a non-positively charged amino acid residue. In this way, IL-6R-mediated side effects of systemically administered CNTF, such as fever or the induction of an acute-phase response in the liver, can be prevented by the affinity loss to the IL-6R achieved by the invention. The CNTF variants according to the invention should moreover not be able to stimulate B cells so that the formation of antibodies against these CNTF molecules might possibly be prevented. Owing to the possibility of avoiding IL-6R-associated side effects the CNTF variants according to the invention are particularly suitable as CNTF therapeutics.
The invention will be illustrated in more detail in the figures. The following is shown:
Subject of the present invention are CNTF variants which recruit the CNTFR as α-receptor but, in contrast to wild-type CNTF, do not interact with the IL-6R or only to a slight extent. This is achieved according to the invention by the substitution of the arginine residue in position 28 of the human wild-type CNTF (SEQ ID NO: 7) by a non-positively charged amino acid residue.
According to the invention, the non-positively charged amino acid residue preferably is an alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine or valine residue. According to the invention, the replacement in position 28 of human wild-type CNTF (SEQ ID NO: 7) does not occur by the following positively charged amino acid residues: a histidine or lysine residue.
In a specific embodiment of the invention, the amino acid substitution according the invention occurs in the CNTF variant mutCNTF (SEQ ID NO: 3 and 4). SEQ ID NO: 8 represents the general sequence of the CNTF variants according to the invention based on mutCNTF, in which the replacement of the arginine residue by non-positively charged amino acid residue occurs at position 15. In a preferred embodiment of the invention the arginine (R) in position 28 of human wild-type CNTF (position 15 of mutCNTF) is replaced by glutamic acid (E) (NNTF-1; SEQ ID NO: 5 and SEQ ID NO: 6).
As described in the examples, the inventors have expressed the CNTF variant mutCNTF (SEQ ID NO: 3 and 4) and the R28E-CNTF variant according to the invention (NNTF-1; SEQ ID NO: 5 and 6) in the form of inclusion bodies using a bacterial system, followed by purification and subsequent refolding. Then, the activity was tested on two different BAF/3 cell lines which, apart from the required β subunits, express either CNTFR or the IL-6R, thereby providing evidence for specificity for the CNTF variant. The loss in activity is illustrated by the fact that only the interaction of an interleukin with a suitable receptor setting can promote cell proliferation.
The cDNA of mutCNTF (SEQ ID NO: 3) in the vector pPCR-Script-mutCNTF served as a template for the generation of the NNTF-1 cDNA. Using the primer pairs
The two C-terminal c-myc and His-tagged recombinant proteins mutCNTF and NNTF-1 were produced as described in example 2.
The activity testing of the proteins mutCNTF and NNTF-1 was performed on the murine cell lines BAF/3 CNTFR-gp130-LIFR and BAF/3 IL-6R-gp130-LIFR, respectively (example 3). Due to a stable transfection these cell lines express, apart from the β-receptors gp130 and LIFR, either the CNTFR or the IL-6R as an α-receptor, so that only an interleukin which is capable of interacting with the corresponding receptor setting can promote their proliferation.
The proliferation assays shown make clear that on the one hand mutCNTF and NNTF-1 are able to stimulate the proliferation of the BAF/3 CNTFR-gp130-LIFR cells, whereas on the other hand only mutCNTF but not NNTF-1 exerts an activity on BAF/3 IL-6R-gp130-LIFR cells. As expected, IL-6 could only stimulate proliferation of BAF/3 IL-6R-gp130-LIFR cells (
In
To examine whether the desired loss in interaction with the IL-6R can be achieved with NNTF-1, this cytokine variant according to the invention was tested in comparison with the two cytokines IL-6 and mutCNTF on the cell lines BAF/3 CNTFR-gp130-LIFR and BAF/3 IL-6R-gp130-LIFR. IL-6 showed the known characteristic of inducing proliferation of BAF/3 IL-6R-gp130-LIFR cells. MutCNTF also had this effect, however, only at higher concentrations. From this, it can be concluded that mutCNTF has affinity to the IL-6R, although this affinity is weak. By contrast, NNTF-1 did not show such an effect even at high concentrations: proliferation and survival of the BAF/3 IL-6R-gp130-LIFR cells could not be stimulated anymore by NNTF-1. Experiments with BAF/3 CNTFR-gp130-LIFR cells showed that these cells expectedly reacted to mutCNTF by proliferation. From these results it can be concluded that NNTF-1, but not mutCNTF, is not able to bind the IL-6R anymore. This could be demonstrated by cell proliferation which only occurs if the ligand binds to its receptor and thereby initiates the intracellular signal cascade. The binding characteristics to CNTFR were not detectably influenced by the amino acid replacement R28E in NNTF-1. Surprisingly, the replacement of a single amino acid is sufficient to reduce the affinity of mutCNTF to the IL-6R such that no cell proliferation is possible anymore. In this way, a central role of the arginine residue 28 of human CNTF in the interaction between the cytokine and the receptor is implied.
Subject of the present invention are therefore nucleic acid molecules that encode CNTF variants, which comprise a non-positively charged amino acid residue instead of the arginine residue in position 28 of human wild-type CNTF (SEQ ID NO: 7).
Apart from the described CNTF peptides, which comprise a non-positively charged amino acid residue instead of the arginine residue in position 28 of human wild-type CNTF (SEQ ID NO: 7), biologically active fragments thereof are also subject of the invention. Biologically active means that the fragments do not have affinity to the IL-6R in the proliferation assay provided in the examples or in any other detection method known to the skilled person and are therefore specific for the CNTFR, just as the underlying full-length peptides. Preferably, they are derivatives which lack one or more amino acids at the N- or C-terminal. However, amino acids from the sequence can also be deleted. Such fragments preferably do not have more than 30% of the amino acids deleted.
Furthermore, CNTF peptides in which single amino acids have been replaced are also subject of the invention. Preferably, the replacements are conservative replacements; i.e. amino acids with similar characteristics are replaces, e.g. alanine by serine, leucine by isoleucine, etc. It is preferred that not more than 10% of the amino acids within the peptides are replaced.
Moreover, single amino acids can also be replaced by non-naturally occurring amino acids, i.e. by amino acids which carry additional functional groups, such as hydroxyproline, methylthreonine, homocysteine, etc. Also in this case, it is preferred that not more than 10% of the amino acids are modified in this manner. Further, the peptides can carry derivatisations, which means that they can be, e.g., amidated, glycosylated, acetylated, sulphated or phosphorylated.
The present invention moreover provides a production method for the peptides of the invention. Apart from genetic engineering of the peptides, the total synthesis on common solid phases in the sense of the Merrifield synthesis or a liquid phase synthesis are likewise possible. The synthesis strategy and the composition of the peptides and derivatives derived therefrom with correspondingly protected amino acids are known to the skilled person.
The present invention also relates to a vector which is characterized by a nucleic acid molecule of the invention, as well as to a host cell which is characterized by a vector of the invention and which is able to express a peptide of the invention.
Host cell, characterized by the vector of claim 5.
The present invention also relates to a medicament comprising the peptide of the invention for different therapeutic indications. For this purpose the peptides can be used as highly purified compounds or—if sufficient for the particular use—they can be used in the form of a partially purified peptide mixture or as a mixture of several peptides of the invention.
Another subject of the invention is a medicament comprising a peptide of the invention for treating a disorder or disease associated with CNTF.
Subject of the present invention is therefore also a medicament comprising a peptide of the invention for treating adiposity and diseases which are associated with adiposity, such as adiposity-associated insulin resistance, glucose intolerance, diabetes, hyperglycemia, or hyperinsulinemia and/or for the reduction of body weight.
Subject of the present invention is also a medicament comprising a peptide of the invention for the treatment of neurological disorders or diseases or differentiation disorders or diseases or nerve damage.
Neurological disorders or diseases or differentiation disorders or diseases or nerve damages which are associated with CNTF comprise degenerative diseases, such as retinal degeneration, diseases or disorders which comprise the spinal chord, cholinergic neurons or hippocampus neurons, or diseases or disorders which comprise motoneurons, such as amyotrophic lateral sclerosis or those of the Nervus facialis, such as facial palsy. Other diseases or disorders that can be treated comprise: peripheral neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's chorea or muscular atrophy, which e.g. result from denervation, chronic abuse, metabolic burden and malnutrition, or from a condition such as muscular dystrophy syndrome, congestive myopathy, an inflammatory muscular disease, toxic myopathy, nerve trauma, peripheral neuropathy, medicament- or toxin-induced damage or motoneuropathy. The present invention also relates to diseases or disorders which result from damage of the nerve system, wherein such damage can be caused by a trauma, a surgical intervention, an infarction, an infection, and a malignancy or by exposure to a toxic agent.
Additionally, the invention relates to a medicament for the treatment of adiposity and diseases associated with adiposity and/or for the reduction of body weight and for the treatment of a neurological disorder or disease or a differentiation disorder or disease or nerve damage associated with CNTF comprising a peptide of the invention as at least one of its active ingredients and a pharmacological acceptable carrier.
The invention will be described in more detail in the following examples.
The SOE-PCR for the generating a point mutation is performed in several steps. In addition to the terminal primers of a normal PCR, two primers are designed which are located at exactly complementary positions of the template DNA and embrace the position of the base triplet to be replaced. Thus, the template is not completely amplified, but instead is amplified in two parts, each with a terminal 5′ primer and the 3′ primer which comprises the mutation and vice versa. In this way two DNA fragments are generated which can be joined with one another in a final ligation PCR owing to their complementary ends (
The amplification of the DNA was carried out according to the following scheme:
The ligation PCR was carried out using the following conditions:
Primer:
For protein expression of the inserts NNTF-1 and mutCNTF, which had been cloned in the expression vector pET-23a (+) (Novagen, Gibbstown, USA), the E. coli strain BL21 (DE3) pLysS (Novagen, Madison, USA) was used. The expression was performed in 1 liter LB medium, wherein said medium had been previously autoclaved and a selection antibiotic had been added. In a first step, colonies of transformed bacteria were picked from an agar plate and incubated overnight in a small volume of 50 ml. Subsequently, 10 ml of this volume were transferred to the expression batch, and the batch was agitated at 180 rpm and 37° C. in an incubator until an OD600 of 0.8 was reached. Then, expression was induced with the lactose analog isopropyl β-D-thiogalactopyranoside (IPTG) in a final concentration of 1 mM. After 4 hours the batch was centrifuged for 20 minutes at 4,000 rpm and the pellets were stored at −20° C. in 50 ml centrifugation tubes until purification. The sedimented expression batch was resuspended in a series of 5 repetitions in 100 ml 1×PBS each (initially with the addition of 0.1% (v/v) Tween®20), sonicated on ice three times for one minute using the homogenizer SONOPULS HD 2200 (BANDELIN, Berlin, Deutschland) (30% power) and once again centrifuged for 20 minutes at 15,000 rpm, wherein the supernatant could be discarded. After the last centrifugation step the pellets were resuspended in a total of 12 ml denaturing buffer 1 (6 M GuHCl; 50 mM Tris-HCl pH 8.0). After incubation overnight at room temperature on a roller table the protein solution was centrifuged for 10 minutes at 10,000 rpm and the supernatant was stored at −20° C. until its further processing.
An additional purification was achieved by metal chelate affinity chromatography. 2 ml of Ni-NTA agarose (Qiagen, Hilden, Germany) were used as a supporting material which was washed twice with 10 ml 1×PBS, equilibrated with 10 ml buffer 1 and subsequently loaded with 10 ml protein solution, and the content of the column was incubated on a roller table for 10 minutes at room temperature. After flow through two washing steps were carried out, each with 10 ml buffer 1, and the elution of the proteins was performed with 3×4 ml buffer 2 (6 M GuHCl; 50 mM Tris-HCl pH 8.0; 250 mM imidazole). Successful purification was monitored by SDS-PAGE.
Since the proteins which had been dissolved in GuHCl were present in denatured form, a dialysis was performed in which protein solution [1 mg/ml] was subjected to dialysis in a volume ratio of 1:1000 twice for 12 hours against 50 mM CAPS pH 11.0 at 4° C. The correct folding was verified by CD spectroscopy.
Finally, the FPLC system ÄKTAexplorer™ (Pharmacia Biotech, Uppsala, Sweden) was used to separate monomeric proteins from aggregated multimeric proteins. After washing the Hiload 16/60 Superdex 75 pg (column volume 120 ml), which was connected to the explorer system, with 120 ml 0.5 M NaOH equilibration was carried out with 240 ml degassed and sterile-filtered 50 mM CAPS pH 11.0, and the column was loaded with 2 ml dialyzed protein solution. During the separation process the proteins were detected at 280 nm. Subsequently, the fractionated elution volume was analyzed by SDS-PAGE and concentrated using centrifugal concentrators (Vivaspin 20, MWCO 10,000; Sartorius, Aubagne, France) to 0.5 to 1 mg/ml.
The two proteins mutCNTF and NNTF-1 were presented as monomers. IL-6 was prepared as described (Fischer, M, Goldschmitt, J, Peschel, C, Brakenhoff, J P, Kallen, K J, Wollmer, A, Grotzinger, J and Rose-John, S I. A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat Biotechnol 1997; 15: 142-145).
The study of biological activity of NNTF-1 and mutCNTF was performed on two BAF/3 cell lines (48), which had been transfected with the required receptor components:
Cell line: BAF/3 IL-6R-gp130-LIFR
Origin: murine pro-B cells, stably transfected with cDNA for gp130, IL-6R, LIFR
Culturing: in suspension, DMEM+10% FCS+1% P/S, IL-6 [10 ng/ml]
Cell line: BAF/3 CNTFR-gp130-LIFR
Origin: murine pro-B cells, stably transfected with cDNA for gp130, CNTFR, LIFR
Culturing: in suspension, DMEM+10% FCS+1% P/S, CNTF [10 ng/ml]
The cells were washed three times with sterile PBS, counted and resuspended in DMEM (+/+) in a concentration of 5×103/100 μl. After addition of the respective cytokines mutCNTF or NNTF-1, 100 μl/well were each pipetted into a 96 well plate and cultured for two days with an increasing concentration series starting with 1 pg/ml and ending with 1 μl/ml cytokine. Then, the proliferation assays were performed. The cells were cultured at 37° C. and a CO2 content of 5%. Proliferation of the cells was measured using the CellTiter-Blue Cell Viability Assay (Promega GmbH, Mannheim) in accordance with the manufacturer's protocol, and the absorption at 570 nm was determined photometrically as the measure for cell proliferation. The absorption is directly proportional to the number of viable cells. All values were determined in triplicate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2011 104 822.0 | Jun 2011 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/061143 | 6/13/2012 | WO | 00 | 2/26/2014 |
