WO2002064158A2

WO2002064158A2 - Use of a conjugate of il-6 and the receptor thereof for inducing cellular expression of scf and flt-3l

Info

Publication number: WO2002064158A2
Application number: PCT/DE2002/000535
Authority: WO
Inventors: Stefan Rose-John; Malte Peters
Original assignee: Stefan Rose-John; Wobus Anna M
Priority date: 2001-02-14
Filing date: 2002-02-14
Publication date: 2002-08-22
Also published as: DE10106827A1; WO2002064158A3; AU2002244625A1

Abstract

Disclosed is the use of a conjugate of IL-6 and the receptor thereof, especially an IL-6/sIL-6R fusion protein, for inducing the cellular expression of SCF and Flt-3L. Preferably, the conjugate is used in the ex vivo expansion of stem cells or precursor cells, especially those of the hematopoetic system.

Description

Use of a conjugate of interleukin-6 (IL-6) and its receptor for inducing the cellular expression of stem cell factor (SCF) and flat-3 ligand (FI.-3L)

The present invention relates to the use of a conjugate of (IL-6) and its receptor for inducing the cellular expression of SCF and FI.-3L. This use is preferably used in the ex vivo expansion of stem or progenitor cells, in particular those of the hematopoietic system , used.

The possibility of ex vivo expansion of stem or progenitor cells, especially those of the hematopoietic system, has for many areas of research and medicine, e.g. the regeneration or replacement of tissues and organs, is of great importance. It is known that e.g. B. hematopoietic stem or progenitor cells can be stimulated differently according to the stage of their differentiation, which leads to functional changes in the hematopoietic system during development. Hematopoietic stem or progenitor cells from different organ compartments show considerable differences in their capacity for proliferation and self-renewal. The ability for proliferation and self-renewal can be found e.g. in fetal liver cells, umbilical cord blood cells and in adult bone marrow cells (to an increasing extent). The functional difference between these cell types is most likely represented by a developmentally regulated expression pattern of the cytokine / growth factor receptors. However, the ex vivo expansion of stem or progenitor cells, especially those of the hematopoietic system, has been associated with a number of difficulties in practice. For example, factors such as SCF and FI-3L had to be added to the culture media used to expand the cells, which was often difficult and costly because of their availability.

Thus, the present invention addresses the technical problem of overcoming these disadvantages of the methods used in the prior art, i.e. To provide means that allow simple and efficient ex vivo expansion of stem or progenitor cells.

According to the invention, this is achieved by providing the embodiments characterized in the patent claims. Surprisingly, it was found that stem or progenitor cells, in particular those of the hematopoietic system, can be expanded easily and efficiently, when a conjugate of IL-6 and its receptor, in particular an IL-6 / slL-6R fusion protein, is added to the culture medium.

IL-6 is a pleiotropic cytokine that interacts with a wide range of target cells. IL-6 is primarily a mediator of cell growth and differentiation, e.g. that of the hematopoietic system. IL-6 acts on the target cells via a receptor complex consisting of a specific IL-6 binding protein (IL-6R) and a signal-transmitting molecule (gp 130). The IL-6 / IL-6R complex induces the homodimerization of two gp 130 molecules, which in turn initiates intracellular signaling. Soluble forms of IL-6R (slL-6R) are found in different areas of the body and are increased in patients with various chronic diseases associated with inflammation and haematological diseases. In humans, the cleavage of IL-6R to slL-6R is triggered in response to acute phase proteins and bacterial toxins. The IL-6 / slL-6R fusion protein (Hyper-IL-6) is a fusion polypeptide that is a human slL-6R polypeptide, i.e. comprises the extracellular or soluble subunit of an IL-6 receptor, and a human IL-6 peptide, the two polypeptides being linked to one another via a polypeptide linker. The structure of "Hyper-IL-6" and its amino acid sequence are described in DE 196 08 813 C2.

In the experiments leading to the present invention, it was found, for example, that intracellular expression of an IL-6 / slL-6R fusion protein (Hyper-IL-6) and subsequent secretion or addition of this fusion protein to a growth medium resulted in the cellular expression of SCF and can be induced by FI.-3L. In the experiments, it could be shown by immunohistochemical studies that the cellular expression of SCF and FI.-3L in the liver and spleen was not induced by double-transgenic mice (ie mice which expressed both IL-6 and slL-6R) however in individually transgenic mice for IL-6 or slL-6R. It was also shown that stimulation with Hyper-IL-6 also led to induction of cellular expression of SCF and FI.-3L on murine NIH / 3T3 fibroblasts and human prepuce fibroblasts. When human fibroblasts (HTB 158) were stimulated with Hyper-IL-6 and then co-cultivated with umbilical cord blood CD34 ⁺ cells, a significant upregulation of the colony growth of the umbilical cord blood CD34 ⁺ cells was observed.

The present invention thus relates to the use of a conjugate of IL-6 and its receptor, in particular an IL-6 / slL-6R fusion protein (Hyper-IL-6) for inducing the cellular expression of SCF and FI.-3L

The term "conjugate of IL-6 and its receptor" includes any conjugate in which IL-6 and its receptor or parts thereof can be covalently linked to one another. For example, the conjugate can be one in which IL-6 and its receptor or parts thereof are linked to one another via a linker, such as a disulfide bridge, a peptide or a chemical agent. The conjugate is preferably a fusion protein, in particular an IL-6 / slL-6R fusion protein (Hyper-IL-6), as described in DE 196 08 813 C2 or a fusion protein that is different from the fusion protein described in DE 196 08 813 C2 distinguishes that it has deletions, additions and / or substitutions of one or more amino acids and / or (a) modified amino acid (s) in the IL-6, slL-6R and / or linker portion, the biological activity not is significantly influenced. Whether such a fusion protein still has the desired biological properties can be determined using conventional methods, e.g. by means of the methods described in the examples below.

In the use according to the invention, it may be expedient to bring the cells into contact with the conjugate by culturing them in a medium containing the conjugate or by transforming them with a vector which leads to intracellular expression of the conjugate and subsequent secretion. The person skilled in the art knows suitable transformation systems and vectors, e.g. for gene therapy, and in this connection reference is also made to DE 196 08 813 C2 (see also Examples 1 to 4 below). The person skilled in the art is able to decide which procedure is most advantageous in the respective case.

The person skilled in the art is also familiar with suitable cultivation methods and media in order to be able to cultivate mammalian cells, for example hematopoietic stem or precursor cells; see, for example, DE 19608 813 C2, EP-B1 0695351 or Examples 1, 5 and 6 below. The person skilled in the art can also determine the optimum concentration of the conjugate for the desired purpose using simple experiments; see for example DE 196 08 813 C2. The basic medium, which contains the conjugate, among others, can be any medium which is usually used for the cultivation of mammalian cells. In a preferred embodiment of the medium according to the invention, the basic medium in which the conjugate is contained is DMEM (Life Technologies, Karlsruhe, Germany). The desired cells are in the above medium under suitable conditions, optionally with (partial) renewal of the medium in suitable Intervals, bred. Suitable conditions, for example with regard to suitable containers, temperature, relative air humidity, 0 ₂ content and CO ₂ content of the gas phase, are known to the person skilled in the art. Preferably, the cells are cultured in the above medium under the following conditions: (a) 37 ° C, (b) 100% rel. Humidity and (c) 5% C0 ₂ .

The conjugate as an additive to a medium is prepared in accordance with customary methods, the conjugate preferably being produced recombinantly as described in Example 2 of DE 196 08 813 C2 using the DNA sequence described in Example 1 of DE 196 08 813 C2.

If the conjugate is not used directly, e.g. used as an additive to a medium, but if it is made available to the cell by intracellular expression and subsequent secretion, the DNA sequence coding for the conjugate is inserted into a suitable vector, preferably expression vector, under the control of a suitable promoter, suitable promoters are known to the expert. The vector containing the DNA sequences can be transfected in various ways: a) Lipid or liposome-packed DNA or RNA, e.g. generally described by Nabel et al., Proc. Natl. Acad. Be. USA 93, (1996), 15388-15393 (see also Nair et al., Nature Biotechnology 16 (1998), 364 ff; b) With a bacterium as a transport vehicle for the expression vector. Suitable bacteria are e.g. (attenuated) listeria [e.g. Listeria monocytogenes], Salmonella strains [e.g. Salmonella spp.]. In general, this technique is described by Medina et al., Eur. J. Immunol. 29 (1999), 693-699, and Guzman et al., Eur. J. Immunol. 28: 1807-1814 (1998). Furthermore, WO 96/14087; Weiskirch et al., Immunological Reviews 158 (1997), 159-169 and US-A-5,830,702; c) Using a virus as a vector (Kim et al., J. of Immunotherapy 20 (4) (1997), 276-286; d) Using Gene-Gun (Williams et al., Proc. Natl. Acad. Sei. USA 88: 2726-2730 (1991).

The vector is preferably a virus, for example an adenovirus, vaccinia virus or an AAV virus. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV as well as fowlpox virus, canarypox virus, influenza virus or Sindbis virus.

General methods known in the art can be used to construct vectors or Plasmids containing the above DNA sequences and suitable control sequences can be used. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY ( 1989).

A preferred use relates to the use of a conjugate for the ex vivo expansion of stem or progenitor cells, in particular those of the hematopoietic system. The cells are grown or kept as described above or in Examples 1, 5 and 6 below, preferably using a medium containing the conjugate. Examples of suitable stem or progenitor cells are those of the mesenchymal or hematopoietic system, with stem or progenitor cells of the latter, such as CD34 ⁺ cells, being particularly preferred. The ex vivo expansion of stem or progenitor cells, in particular of CD34 ⁺ cells, is preferably carried out by co-cultivation with feeder cells as cell turf, in particular human fibroblasts, preferably HTB-158 fibroblasts, which had previously been treated with the conjugate, for example as in the Examples 1, 5 and 6 below.

Another preferred use relates to the in vivo use of a conjugate for the treatment of diseases which are associated with a reduced expression of SCF and Ftl-3L or in which an increase in the expression or concentration of SCF and F.I-3L is desirable. Examples of such diseases are tumor or degenerative diseases.

Other preferred uses of a conjugate are as follows:

(a) Use for improving the reconstitution and purification of stem or progenitor cell transplants contaminated with tumor cells, whereby an expansion of stem or progenitor cells, in particular those of the hematopoietic system, which ultimately allows a more efficient purge.

(b) Use to improve the mobilization of stem or progenitor cells, in particular those of the hematopoietic system to peripheral blood, whereby an expansion of stem or progenitor cells takes place. (c) Use to improve gene transfer in stem or progenitor cells, in particular those of the hematopoietic system, with an expansion of stem or progenitor cells.

The conjugate is preferably used as a component of a medium in a concentration of 5 to 100 ng / ml.

Brief description of the figures

FIG. 1: Cellular SCF expression in the liver of mice transgenic with respect to IL-6 and IL-6 / slL-6R. Immunohistochemical analysis of SCF expression in the liver from 4 (A), 8 (B) and 16 (C) weeks old, double-transgenic mice for IL-6 / slL-6R, a 16-week-old mouse (D) which was individually transgenic for IL-6, a 16-week-old mouse (E) which was individually transgenic for slL-6R and a non-transgenic mouse Mouse as a control (F). The bars correspond to 100 μ.

Figure 2: SCF-mRNA expression in the liver of transgenic mice and control mice. Total RNA from the liver was prepared and a Northem blot analysis was carried out with it. Lanes 1-7 show the mRNA of 4 to 16 week old mice transgenic for IL-6 / slL-6R, lanes 8-13 show the mRNA of individually transgenic mice for IL-6 aged 4 to 16 16 weeks and in lanes 14 and 15 the mRNA from a 4 week or 16 week old control mouse. In the lower part of the figure, the RNA gel stained with ethidium bromide is shown for the detection of the same sample amounts for all lanes.

FIG. 3: Cellular SCF expression in the spleen of mice transgenic for IL-6 and IL-6 / slL-6R. Immunohistochemical analysis of the SCF expression in the spleen of 6 weeks (AC) and 16 weeks (A'-C) transgenic mice that expressed both IL-6 and slL-6R (A, A '), only slL-6R (B, B') or only IL-6 (C, C) in the liver. The bars correspond to 100 tm.

FIG. 4: Immunoprecipitation of the membrane-bound and soluble form of SCF-2 from the spleen of mice transgenic for IL-6 / slL-6R and single transgenic mice. Splenic tissue from transgenic and non-transgenic mice (as indicated in the figure) was homogenized and immunoprecipitation using a polyclonal rabbit anti murine SCF antibody subjected. The immunoprecipitates were loaded on an SDS-polyacrylamide gel and then a Western blot was carried out using the polyclonal rabbit anti-murine SCF antibody. A strong band at approximately 29 kD and a weaker band at approximately 20 kD appear only in immunoprecipitates from the spleen of mice transgenic for IL-6 / slL-6R. These bands most likely correspond to the membrane-bound and soluble form of SCF-2 (KL-2).

FIG. 5: Flt-3 expression in the liver of mice transgenic for IL-6 and IL-6 / slL-6R. Immunohistochemical analysis of Flt-3L expression in the spleen of 6 weeks (AC) and 16 weeks (A'- C) old transgenic mice expressing either both IL-6 and slL-6R (A, A '), only slL-6R (B, B') or only IL-6 (C, C) in the liver. The bars correspond to 100 μm.

Figure 6: SCF and Flt-3L expression on murine NIH / 3T3 cells and primary human prepuce fibroblasts

NIH / 3T3 cells (AC, A'-C) and primary human prepuce fibroblasts (A "-C", A "'- C") were seeded on gelatin-coated glass chamber supports and in DMEM in a water-saturated atmosphere at 5% CO ₂ grown. The cells were stimulated with the corresponding cytokines for 24 hours: only with medium (A-A '"), with 10 ng / ml IL-6 (BB"') or with 10 ng / ml Hyper-IL-6 (CC " An analysis by immunofluorescence was then carried out and primary antibodies against SCF (AC, A "-C") and FI.-3L (A'-C, A '"- C") were used as described in Example 1.

Figure 7: Expansion of human umbilical cord blood CD34 ⁺ cells by co-cultivation with human fibroblasts.

Human CD34 ⁺ cells obtained from umbilical cord blood were purified by means of immunomagnetic beads (Dynabeads) which were coated with an onoclonal anti-CD34 antibody. (A, B) 10,000 cells were seeded on human HTB-158 fibroblasts which, as indicated in Example 1, with IL-6 (50 ng / ml) or Hyper-IL-6 (10 ng / ml) in 3 ml culture medium had been treated. After two weeks, the cells were counted and seeded in soft agar in the presence of SCF (100 ng / ml) IL-3, IL-6, EPO (10 U / ml) and G-CSF (200 ng / ml). ml). After a further two weeks, the colonies were stained and counted. (C, D) 10,000 cells were seeded in 3 ml culture medium as indicated and either treated with medium or with IL-6 (50 ng / ml) or Hyper-IL-6 (10 ng / ml). After two weeks the cells were counted and in the presence of SCF (100ng / ml), IL-3, IL-6, EPO (10th U / ml) and G-CSF (200 ng / ml) sown in soft agar (1000 cells / ml). After a further two weeks, the colonies were stained and counted.

The following examples illustrate the invention.

Example 1 General procedures

(A) Generation of transoen mice

The generation of mice transgenic for human slL-6R and human IL-6 is described in Peters et al., J.Exp.Med. 183 (1996), 1399 and Fattori et al., Blood 83 (1994), 2570. By crossing homozygous IL-6R and IL-6 mice, hemizygous double transgenic mice (IL-6 / SIL-6R mice) were obtained, which both co-express transgenes (Peters et al., J.Exp.Med. 185 (1997) , 755). The expression of the slL-6R transgene is under the control of the neonatal PEPCK promoter, which is transcriptionally activated 3 days after birth and has no intra-uterine developmental effects, the IL-6 transgene is under the control of the murine metallothionein-1 - Promotors (Fattori et al., 83 (1994), 2570).

(B) Antibodies

The following antibodies were used: Anti-Gr-1 (RB6-8C5), Mac-1 (M1 / 70), B220 (RA3-6B2), CD4 (H129.19), and CD8a (53-6.7) (all of Pharmingen, San Diego, USA; these monoclonal antibodies were used as line-specific markers), goat anti-human SCF-IgG polyclonal antibody (R + D, Wiesbaden, Germany), rabbit polyclonal anti-mouse SCF- IgG (Genzyme, Cambridge, USA) and polyclonal goat anti-mouse / human-Ftl-3L-IgG (Santa Cruz, USA). The following secondary antibodies were used: FITC-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) and FITC-conjugated mouse anti-goat IgG (Dianova).

(C cell culture

Murine fibroblasts (NIH / 3T3) and primary prepuce fibroblasts were grown in DMEM at 5% CO ₂ in a water-saturated atmosphere. All cell culture media were supplemented with 10% fetal calf serum (FCS), 100 mg / L streptomycin and 60 mg / L penicillin. (D. Isolation and cultivation of human CD34 ⁺ cells

Human CD34 ⁺ cells from umbilical cord blood were purified using immunomagnetic beads (Dynabeads) coated with a monoclonal anti-CD34 ⁺ antibody. The liquid culture and assays for hematopoietic progenitor cells were carried out as described by Fischer et al. (Nature Biotech. 15 (1997), 142). For this purpose, 10,000 cells were seeded on human HTB-158 fibroblasts treated with IL-6 (50 ng / ml) or hyper-IL-6 (10 ng / ml) in a 3 ml culture as indicated in the legend to FIG. 7. After two weeks, the cells were counted and seeded in soft agar (500 cells.) In the presence of SCF (100 ng / ml), IL-3, IL-6, EPO (10 U / ml) and G-CSF (200 ng / ml) per ml). After a further two weeks, colonies were stained and counted. In a control experiment, 10,000 cells were seeded in 3 ml cultures without fibroblasts that did not contain either cytokine, IL-6 or Hyper-IL-6. The experiment was then carried out as described above, except that 1000 cells were sown in the soft agar.

(E) Immunohistochemical studies and immunofluorescence

Immunohistochemical studies were performed using the "ABC" method. Frozen sections of murine liver and spleen from transgenic and non-transgenic animals were analyzed. Polyclonal rabbit anti-mouse SCF-IgG (Genzyme, Cambridge, USA) and polyclonal goat anti-mouse / human-Flt-3L-IgG (Santa Cruz, USA) was used as the primary antibody and this was used in a dilution of 1:50 at 4 ° C. as the secondary antibody was biotin conjugated goat -anti-rat-IgG (Dianova, Hamburg, Germany). The signal was developed with horseradish-peroxidase-coupled streptavidin and diaminobenzidine as chromogen. The sections were counter-stained with Mayer's haemalum and eosin. The sections were viewed microscopically and microphotographs were taken with taken with an Olympus "NHH" microscope.

Murine NIH / 3T3 cells, human HUT-158 cells and human primary prepuce fibroblasts were grown in DMEM in glass chamber carriers (LabTeks; Nunc Nalgene, Nasperville, USA) until 80% confluency was reached. The cells were stimulated with the appropriate cytokines for 24 hours. After stimulation, the cells were washed twice with PBS and fixed in 50% methanol / 50% acetone for 15 min at -20 ° C. The slides were air dried and incubated for 30 min in PBS / 5% bovine serum albumin. The cells were washed at 4 ° C with the polyclonal rabbit anti-mouse SCF antibody (Genzyme, USA) at a dilution of 1:50 in PBS / 1% bovine serum albumin. incubated. A FITC-conjugated goat anti-rabbit IgG antibody (Dianova, Germany) at a dilution of 1: 100 in PBS / 1% bovine serum albumin. The carriers were evaluated using fluorescence microscopy.

(F) Extraction of total mRNA from liver and detection of SCF mRNA via Northern blots. The mice were sacrificed by cervical dislocation and total RNA from the liver was isolated using the phenol / chloroform method (Rose-John, Carcinogenesis 9 (5) (1988), 831). 5 μg of heat-denatured RNA per sample were separated on a 1% agarose gel which contained 7% formaldehyde. The separated RNA was transferred to GenScreen-Plus ™ membranes (Dupont-New England Nuclear, Dreieich, Germany) according to the manufacturer's instructions. The filters were prehybridized for 2 hours at 68 ° C. in 10% dextran sulfate, 1 M NaCl, 1% SDS and incubated in the same solution with cDNA fragments which had been labeled with [ ³² P] -ATP by random priming , The mouse SCF cDNA fragment was obtained by reverse transcription using an "RT-PCR" kit (Invitrogen, USA) of total mRNA obtained from murine NIH / 3T3 fibroblasts that were exposed to 10 ng for 24 hours / ml IL had been stimulated. The following primers were used: 5'-GTC AAA ACC AAG GAG ATC TGC-3 '(sense) and 5'-AGG GAG CTG GCT GCA ACA GGG-3' (antisense). The fragment obtained (577 bp) was cloned into a pBS ⁺ vector (Stratagene, la Jolla, USA) and the correctness of the construct was confirmed by sequencing.

(G. Immunoprecipitation of murine SCF from liver and spleen

Spleen homogenates were prepared from transgenic and non-transgenic mice. The supernatants of the homogenates were pre-clarified with 100 μg Protein A-Sepharose ™ (20 mg / ml; Pharmacia, USA) for 4 hours at 4 ° C. The samples were then immunoprecipitated at 4 ° C for a further 4 hours using a polyclonal rabbit anti-murine SCF antibody (Genzyme, Cambridge, MA, USA) at a concentration of 2 / tg / ml. After adding 100 // I Protein A-Sepharose ™ (20 mg / ml) and incubating at 4 ° C. for a further 2 hours, the Sepharose-bound SCF was obtained by centrifugation, once with TNET buffer (Tris / HCl, 10 mM , pH 7.4; Na deoxycholate, 0.4%; EDTA, 60 mM; Triton-x-100, 1%) and washed twice with PBS / 0.05% Tween, then resuspended in 2x Laemmli buffer and over SDS-PAGE separated. The separated proteins were electrically blotted on nitrocellulose membranes and finally a western blot analysis was carried out using rabbit anti-murine polyclonal SCF antibody at a concentration of 2 yg / ml. A peroxidase-conjugated goat anti-rabbit IgG antibody (Dianova, Hamburg, Germany) was used as the secondary antibody. The "enhanced chemiluminescence System "(ECL) from Amersham (Arlington Heights, USA).

Example 2

The expression of SCF-mRNA is double transgenic in the liver for IL-6 / slL-6R

Mice highly upregulated, but not in individually transgenic animals

The cellular expression of SCF in double transgenic mice (IL-6 / slL-6R) was analyzed by means of immunohistochemical examinations of the liver of transgenic and non-transgenic mice. FIG. 1 shows that the expression of SCF in the liver of 4 to 16 week old mice for IL-6 / slL-6R double transgenic mice is increased as a function of age (FIG. 1A-C). In contrast, no cellular expression of SCF could be detected by this method in the liver of 16-week-old animals that were individually transgenic for IL-6 (FIG. 1D) or slL-6R (FIG. 1E) or in control animals (FIG. 1F) ,

SCF mRNA expression in the liver was also examined using Northem blot analysis. To this end, total RNA from the liver of IL-6 / slL-6R double-transgenic mice (FIG. 2, lanes 1-7), of IL-6 single-transgenic mice (FIG. 2, lanes 8-13) or of control animals was used (Figure 2, tracks 14 and 15) prepared at the age levels indicated in the figure. Consistent with the data obtained from immunohistochemical studies, an age-dependent increase in SCF-mRNA expression could only be detected in the IL-6 / slL-6R double-transgenic mice. In the animals that were individually transgenic for IL-6, some (basal) expression of SCF was shown in the 4 and 8 week old animals, but not in the 16 week old animals. A weak band was detected in a 4-week-old control mouse, but no expression was found in a 16-week-old mouse. These results show that the presence of both IL-6 and slL-6R, but not exclusively IL-6, leads to an increased expression of SCF-mRNA and an increased concentration of the protein in the liver.

Example 3

Cellular SCF expression is upregulated in the spleen of mice transgenic for IL-6 / slL-6R, but not in single transgenic mice

n The cellular SCF expression in the spleen of transgenic and non-transgenic, 6-week-old (FIG. 3A-C) and 16-week-old mice (FIG. 3A'-C) was analyzed by means of immunohistochemical studies. A basal expression of SCF could be demonstrated in the spleens of all analyzed animals with an age of 6 weeks (FIG. 3A, IL-6 / sl L-6R-double transgenic; FIG. 3B, slL-6R-single transgenic; FIG. 3C, IL-6 single transgene). In the older animals at 16 weeks of age, SCF expression increased significantly in the double transgenic mice for IL-6 / slL-6R (FIG. 3A '), but not in the single transgenic mice for slL-6R or IL-6 (Figure 3B 'and 3C). SCF immunoreactivity was strongest in megakaryocytes with peripheral (sub) membrane accentuation, but was also detectable in earlier stages of granulopoiesis. No specific signal could be detected in erythropoietic cells, neutrophil granulocytes and mature lymphocytes. These results show that the co-expression of IL-6 and slL-6R, but not only IL-6, leads to an increased expression of SCF in the spleen.

SCF was then immunoprecipitated from the spleen of transgenic and non-transgenic animals from a litter. As shown in Figure 4, significant amounts of SCF protein can only be immunoprecipitated from the spleen of mice transgenic for IL-6 / slL-6R, but not from mice transgenic for IL-6 or slL-6R or from non-transgenic Mice of a litter. A strong band at approximately 26 kD and a weaker band at approximately 20 kD appeared in immunoprecipitates from the spleen of mice transgenic for IL-6 / slL-6R. These bands most likely represent the membrane-bound and soluble forms of SCF-2 (KL-2). Two alternatively spliced SCF transcripts encode the two membrane-bound proteins SCF-1 and SCF-2. The expression of SCF-2 predominates predominantly in the spleen, due to the lack of sequences which comprise the main cleavage site in exon 6, compared to proteolytic cleavage is resistant. Due to a secondary cleavage site, presumably located in exon 7, SCF-2 is processed into soluble SCF-2, but not as efficiently as SCF-1

Example 4

FIt-3L expression is upregulated in the liver of mice transgenic for IL-6 / slL-6R, but not in single transgenic mice

There was Flt-3L expression in the liver of transgenic mice at 6 weeks of age (Figure 5A-C) and 16 weeks (Figure 5A'-C) analyzed. In 6-week-old animals, basal Fit-3L expression was found in mice transgenic for IL-6 / slL-6R (FIG. 5A), in mice transgenic for slL-6R (FIG. 5B) and in individually transgenic for IL-6 Mice (Figure 5C) observed. In older mice (16 weeks old) the Flt-3L expression was increased only in mice transgenic for IL-6 / slL-6R (FIG. 5A '), not in slL-6R (FIG. 5B') and in for IL-6 single transgenic animals (Figure 5C). Thus, the presence of IL-6 together with slL-6R, but not only IL-6, led to an upregulation of the cellular Flt-3L protein in the spleen.

Example 5 Studies on the induction of cellular SCF and Flt-3L protein on NIH / 3T3 cells and primary human prepuce fibroblasts

In order to investigate the effects of IL-6 and IL-6 / slL-6R in vitro, NIH / 3T3 cells (FIG. 6A-C, A'-C), human HTB-158 fibroblasts and primary human prepuce fibroblasts were used (Figure 6A "-C", A '"- C") 24 hours without cytokines (Figure 6A-A "'), only with IL-6 (Figure 6B-B '") or with the fusion protein Hyper-IL-6 (FIG. 6C-C "), in which IL-6 is covalently linked to the soluble receptor via a flexible amino acid linker. In all three cell types, treatment with IL-6 only led to a very weak upregulation of the expression of SCF and FI .-3L (Figure 6B-B '"). However, treatment with Hyper-IL-6 led to a greatly increased expression of SCF and FI.-3L (FIG. 6C-C "). Thus, in murine and human fibroblast cell lines and human primary fibroblasts, co-stimulation with IL- 6 and slL-6R for a strong expression of the hematopoietic cytokines SCF and FI.-3L

Example 6 Studies on the expansion of human umbilical cord blood CD34 ⁺ cells on human fibroblasts stimulated with Hyper-IL-6

It was examined whether the observed upregulation of SCF and FI.-3L in response to Hyper-IL-6 is sufficient for the expansion of human umbilical cord blood Cd34 ⁺ cells in vitro. Given the species specificity between human and murine cytokines and their receptors and the fact that Both human and murine fibroblast cell lines SCF and FI.-3L expressed equally well after stimulation with IL-6 or Hyper-IL-6, the human fibroblast cell line HTB-158 was used for the co-cultivation experiments. HTB-158 cells were treated with IL-6 (50 ng / ml) or Hyper-IL-6 (10 ng / ml) in 3 ml culture medium. The fibroblasts were then cultured together with human umbilical cord blood CD34 ⁺ cells for two weeks. In addition to the total cell number (FIG. 7A), the frequency of colony-forming units was also determined after a period of 14 days in order to estimate the expansion of immature progenitor cells. As can be seen from FIG. 7B, the pre-stimulation with IL-6 only led to a very slight increase in the number of colonies after 14 days. However, pre-stimulation with Hyper-IL-6 resulted in double the colony count after 14 days compared to the control treatment. The experiment was repeated in the absence of fibroblasts to demonstrate that the observed effects on colony growth were actually dependent on the presence of human fibroblasts (Figures 7C and 7D). It was shown that stimulation of CD34 ⁺ cells with IL-6 or Hyper-IL-6 did not lead to an increase in colony formation after 14 days (FIG. 7D). Thus, this experiment shows that in vitro stimulation of human fibroblasts with Hyper-IL-6 increases the expansion of human hematopoietic progenitor cells when co-cultivated.

Claims

claims

1. Use of a conjugate of IL-6 and its receptor for inducing the cellular expression of SCF and FI.-3L

2. Use according to claim 1, wherein the receptor is present as slL-6R.

3. Use according to claim 1 or 2, wherein the conjugate is a fusion protein.

4. Use according to any one of claims 1-3 for the treatment of diseases which are associated with a reduced expression of SCF and FI.-3L or in which an increase in the expression or concentration of SCF and F.I-3L is desirable.

5. Use according to claim 4, wherein the disease is a tumor or degenerative disease.

6. Use according to any one of claims 1-3 for ex vivo expansion of stem or progenitor cells.

7. Use according to claim 6, wherein the conjugate feeder cells is added as a cell lawn for the ex vivo expansion of stem or progenitor cells.

8. Use according to claim 7, wherein the feeder cells are fibroblasts.

9. Use according to any one of claims 6 to 8, wherein the stem or progenitor cells are those of the hematopoietic system.

10. Use according to any one of claims 1-3 to improve the reconstitution and purification of stem or progenitor cell transplants contaminated with tumor cells.

11. Use according to any one of claims 1-3 to improve the mobilization of stem or progenitor cells.

12. Use according to claim 11, wherein the stem or progenitor cells are those of hematopoietic system are.

13. Use according to any one of claims 1-3 to improve gene transfer in stem or progenitor cells.

14. Use according to claim 13, wherein the stem or precursor cells are those of the hematopoietic system.