WO2002066052A1 - Use of a conjugate of il-6 and an il-6 receptor in tumor therapy - Google Patents

Use of a conjugate of il-6 and an il-6 receptor in tumor therapy Download PDF

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WO2002066052A1
WO2002066052A1 PCT/DE2002/000533 DE0200533W WO02066052A1 WO 2002066052 A1 WO2002066052 A1 WO 2002066052A1 DE 0200533 W DE0200533 W DE 0200533W WO 02066052 A1 WO02066052 A1 WO 02066052A1
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cells
mice
conjugate
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receptor
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Stefan Rose-John
Andrzej Mackiewicz
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Stefan Rose-John
Andrzej Mackiewicz
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of a conjugate of IL-6 and an I -6 receptor, preferably Hyper-IL-6, for the treatment of a tumor.
  • the tumor is preferably melanoma, kidney or pancreatic carcinoma.
  • cytokine genes by appropriately transfected tumor cells has so far been tried as a strategy for increasing the immune responses to various types of cancer.
  • cytokines e.g. Tumor cells expressing TNF- ⁇ , IFN-gamma, I -2, IL-4, IL-6, IL-12, IL-18, or GM-CSF could be shown to promote or not to promote the formation of tumor-specific T lymphocytes.
  • T cell-mediated mechanisms of tumor cell killing can induce, for example Granulocyte inflammatory reactions.
  • genetically modified tumor vaccines (GMTV) based on these cytokines are in clinical investigation as specific immunotherapeutics for cancer treatment.
  • Allogeneic or autologous tumor cells were modified with the corresponding cytokine genes in order to be able to reach high concentrations of the cytokines acting as adjuvants at the vaccination site and to keep the systemic concentration low.
  • the results achieved with this approach so far do not allow a definitive conclusion to be drawn as to whether they can actually represent a sufficiently effective therapeutic approach to cancer treatment.
  • the technical problem underlying the present invention is therefore to provide a specific immunotherapeutic agent, that allows effective cancer therapy.
  • the corresponding secreted p84 glycoprotein could be detected in the supernatant of transfected cells and showed full activity on BAF / gpl30 cells which respond to IL-6 / sIL-6R but not to IL-6 alone.
  • the administration of recombinant H-IL-6 to C57BL / 6 mice led to a prolonged expression of an acute phase protein, which indicates a long systemic residence time of the fusion protein.
  • Transfected Bl ⁇ cells (B16 / H-IL-6 cells) showed morphological changes together with a dramatic growth inhibition in vitro.
  • mice The subcutaneous injection into C57BL / 6 mice resulted in a virtually complete rejection of the B16 / H-IL-6 cells, this effect being partially reversed in mice transgenic for a GM-CSF receptor antagonist. This indicates a GM-CSF-dependent rejection of cells transfected with H-IL-6.
  • APC antigen presenting cells
  • the present invention thus relates to the use of a conjugate of IL-6 and an 11-6 receptor or a DNA sequence encoding this conjugate for the treatment of a tumor.
  • conjugate of IL-6 and an IL-6 receptor refers to a polypeptide, the IL-6 (or the biologically active part of IL-6) and the IL-6 receptor (or for the ligand Binding responsible part of the receptor).
  • This polypeptide can be a polypeptide in which, for example, the two partners are linked to one another via covalent or non-covalent bonds in accordance with routine methods known to the person skilled in the art.
  • the two polypeptides can, for example, be linked to one another via a disulfide bridge.
  • the conjugate is preferably a fusion protein composed of IL-6 and an IL-6 receptor.
  • the conjugate preferably contains the soluble part of the IL-6 receptor (sIL-6R), ie the extracellular or soluble subunit of the interleukin-6 receptor.
  • sIL-6R soluble part of the IL-6 receptor
  • the two polypeptides of the fusion protein are linked via a peptide linker.
  • the conjugate is an IL-6 / SIL-6R fusion protein
  • Hyper-IL-6 i.e. a fusion polypeptide that contains a human sIL
  • IL-6 / sIL-6R fusion protein used in the present application includes the fusion protein with the amino acid sequence shown in DE 196 08 813 C2, as well as a fusion protein which is different from that in DE 196 08 813 C2 disclosed fusion protein in that it has deletions, additions or substitutions of one or more amino acids and / or (a) modified amino acid (s), wherein the biological activity is not significantly affected. Whether such a fusion protein still has the desired biological properties can be investigated using customary methods, for example using the method described in the examples below become .
  • the conjugate preferably Hyper-IL-6
  • an immunotherapeutic agent i.e. in the form of allogeneic or autologous tumor cells which are transformed with a DNA sequence which encodes the polypeptide, preferably Hyper-IL-6, and which is expressed.
  • suitable transformation systems and vectors e.g. for gene therapy, and in this connection reference is made to DE 196 08 813 C2 and the examples below.
  • the e.g. Hyper-IL-6 coding DNA sequence inserted into a vector suitable for gene therapy e.g. a vector based on 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. Other suitable viruses are fowlpox virus, canarypox virus, influenza virus or Sindbis virus as the basis of a vaccine.
  • the DNA sequence encoding Hyper-IL-6 can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).
  • An allogeneic melanoma cell line (eg Mich-1 or Mich-2) is transduced with eg Hyper-IL-6 cDNA, preferably MSCV-based dicistronic retroviral double copy vectors being used. Two proteins are read from a transcript, this being achieved by using an "internal ribosome entry" site. After expansion, the grown cells are trypsinized, irradiated (for example with 100 Gy using Co-60 or 6 MV photons), aliquoted and frozen in liquid nitrogen. Before the
  • the cells are frozen with regard to contamination
  • Vaccination thawed The vaccination of the patients can be done subcutaneously (for example with 5 x 10 7 cells), with the patients initially being immunized four times at intervals of two weeks, then once a month for a year and then at intervals of two months. If the disease progresses, vaccination can take place more often, for example eight times in the beginning
  • conjugate defined above for the treatment of melanoma, kidney or pancreatic carcinoma.
  • the immune response against B16 cells induced by H-IL-6 depends on the presence of GM-CSF, it can be advantageous for the use according to the invention not only the IL- 6 / IL-6R conjugate but also to administer GM-CSF (eg as a substance or via transfection of the cells with a DNA sequence encoding GM-CSF).
  • GM-CSF eg as a substance or via transfection of the cells with a DNA sequence encoding GM-CSF.
  • the present invention thus also relates to the use of a conjugate of IL-6 and an 11-6 receptor in combination with GM-CSF.
  • the fusion protein cDNA contains a 39 bp stretch encoding a flexible linker between SIL-6R and IL-6.
  • H-IL-6 10 7 Bl6 / H-IL-6 cells were incubated for 24 hours in 5 ml of medium containing 0.1 FCS. 100 ul supernatant was used for the Western blot analysis with an anti-IL-6 antibody. H-IL-6 could only be detected as a protein with a relative molecular mass of 84 kD in the supernatant of transfected B16 cells. The right lanes were loaded with 200 ng recombinant IL-6 or SIL-6R.
  • Figure 2 Biological activity of Hyper-IL-6 (a) proliferation of BAF / gpl30 cells in response to increasing amounts of H-IL-6 and IL-6
  • Figure 3 Growth inhibition and morphological changes of H-IL-6 transfected Bl6 melanoma cells
  • Bl6 / H-IL-6 cells show an elongated morphology and tend to form cell protrusions.
  • FIG. 4 MHC-1 expression in unchanged Bl6 / H-IL-6 cells.
  • the surface expression of MHC-1 molecules K b and D ° B16 / H-IL-6 cells and control cells were determined by FACS using an anti-H-2K b D b -MAK.
  • FIG. 5 Tumor growth in mice to which H-IL-6 transfected, sham-transfected or parental Bl6 melanoma cells s.c. had been injected
  • Figure 6 Reduced growth inhibition of Bl6 / H-IL-6 cells in mice transgenic for a GM-CSFR antagonist
  • 5 ⁇ 10 5 parental Bl6 cells were injected into transgenic mice and wild-type mice.
  • the H-IL-6 cDNA was prepared using the vectors pCDM8-sIL-6R and pCDM8-IL-6, of which the sIL-6R and IL-6 cDNAs were generated by PCR.
  • the linker cDNA was introduced using Xhol sites at the C-terminus of SIL-6R and at the N-terminus of IL-6.
  • H-IL-6 cDNA was re-inserted into the vector pCDM8 using an N-terminal Mot1 and a C- terminal Hindlll site ligated.
  • the cell line B16 / H-IL-6 was transfected with 20 ⁇ g pCDM8-H-IL-6 DNA together with 2 ⁇ g pSV2Neo plasmid DNA (Southern and Berg, J.Mol .Appl .Genet. 1 (1982) , 327-341) (coprecipitation with calcium phosphate).
  • C Cell Culture and Assavs The cells were grown in DMEM at 5% CO 2 in a water-saturated atmosphere. All cell culture media were supplemented with 10% FCS, 100 mg / ml streptomycin and 60 mg / 1 penicillin. The proliferation of B16 and B16 / H-IL-6 cells was shown in
  • the density of the living cells was determined after 24, 48, 72, 96 and 120 hours using the MTT (3 - [4, 5 - dimethyththiazol - 2 - yl] - 2, 5 - diphenyltetrazolium bromide) assays the mitochrondrial
  • mice were used for the tumor-inducing experiments and FI crossings from mice transgenic for the FVB / N GM-CSF receptor antagonist and C57BL / 6 mice. The experiments were carried out with the tg2 strain.
  • flow cytometry analyzes were carried out using a "FACScan” device (Becton Dickinson, Mountain View, CA, USA). Debris and dead cells were separated and living cells were plotted using the "FACScan Lysis II" software (Becton Dickinson). As a control, the same experiment was carried out with an antibody specific for IgG2.
  • Bl6 cells were grown with a plasmid containing the cDNA for the fusion protein H-IL-6 under the control of the CMV promoter
  • Neo-resistant clones were evaluated for the expression of the H-
  • Il-6 protein screened by testing the activity of cell supernatants from BAF3 / gpl30 cells. For this example, two positive clones were selected and one sham-transfected clone that contained only the pSV2-neo plasmid.
  • Figure lb shows the expression of the 84 kD glycoprotein of a positive transfectant, detected by immunoprecipitation of the radioactively labeled protein from the supernatant via an anti-IL-6 antibody. Negative transfectants and parental Bl6 cells did not secrete H-IL-6 into the medium.
  • Figure lc shows a Western blot analysis of the medium of transfected and parental B16 cells after a tenfold concentration.
  • the IL-6 / sIL-6R fusion protein was detected with the anti-IL-6 antibody as efficiently as recombinant IL-6. There was no cross-reaction of the anti-IL-6 MAK with SIL6R (right lane).
  • the H-IL-6 levels in the supernatant of transfected cells were determined by measuring the biological Activity of the fusion protein determined on a BAF3 cell line (BAF3 / gpl30) transfected with gpl30 (FIG. 2b). Untransfected BAF3 cells did not express gpl30 and IL-6R and therefore do not respond to IL-6 or H-IL-6.
  • FIG. 2b Cell supernatants from transfected B16 cells were harvested after 24 hours and a dose-dependent proliferation of the BAF3 / gpl30 cells was observed.
  • the EC 50 concentration was determined to be 0.2 ⁇ l / ml (FIG. 2b). This corresponds to H-IL-6 levels of 500 pg / ml / 10 7 cells / 24 hours (FIG. 2a).
  • the biological activity of H-IL-6 in vivo and its stability were tested by injection (ip) of 2 ⁇ g recombinant H-IL-6 in C57BL / 6 mice.
  • the expression of the acute phase protein haptoglobin was analyzed after 72 hours by determining the serum level by means of Western blot analyzes (FIG. 2c).
  • H-IL-6 can induce acute phase proteins in mice at much lower doses than IL-6.
  • Figure 3a shows the proliferation of parental B16 and transfected Bl6 / H-IL-6 cells, which was measured over a period of five days at different times. Cell growth was significantly inhibited in Bl6 / H-IL-6 cells and cell densities were approximately 10% compared to the parental B16 cells at the end of the time series.
  • transfected Bl6 cells showed an altered morphology, which was caused by prolonged cell bulges, loss of Pigmentation and a contiguity in long rows was marked, which indicates a differentiation status (Figure 3b).
  • mice which had received parental B16 cells were injected ip three times a week with 3 ⁇ g recombinant H-IL-6 , After two weeks, the mice were sacrificed and the tumor weights were determined. This systemic Treatment with recombinant H-IL-6 reduced tumor growth by about 40 to 50%.
  • GM-CSF has been thought to play a key role in the rejection of tumor cells by the immune system.
  • transfected and parental Bl6 cells were injected into mice transgenic for the mutated K14E / E21K GM-CSF protein.
  • This GM-CSF mutein has recently been shown to bind to the GM-CSF receptor ⁇ chain, but not to stimulate the signal-transducing ⁇ unit of the GM-CSF receptor complex.
  • the K14E / E21K-GM-CSF protein behaves as a GM-CSF receptor antagonist.
  • mice transgenic for the K14E / E21K-GM-CSF antagonist showed levels of 2 to 15 ng / ml in the serum (FIG. 6a).
  • Transgenic and non-transgenic control mice were sacrificed after two weeks and the tumor volumes were determined as described above.
  • the tumor growth of the Bl6 / H-IL-6 cells in wild-type mice FVB / N x C57BL / 6 was comparable to that in C57BL / 6 mice with an average tumor volume of 50 to 100 mm 3 .
  • transgenic GM-CSF Kl4E / E21K mice were an order of magnitude higher than in the non-transgenic control mice (900 mm 3 ), which indicates that the rejection of the transfected tumor cells was dependent on the presence of biologically active GM-CSF ( Figure 6b).
  • Tumors produced by parental B16 cells in both transgenic and wild-type mice were comparable to those in C57BL / 6 mice.

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Abstract

The invention relates to the use of a conjugate of IL-6 and an IL-6 receptor, preferably hyper-IL-6, for treating tumors. The tumors treated are preferably melanomas, renal or pancreatic carcinomas.

Description

Verwendung eines Konjugats aus IL-6 und einem IL-6 Rezeptor zur Tumortherapie Use of a conjugate of IL-6 and an IL-6 receptor for tumor therapy
Die vorliegende Erfindung betrifft die Verwendung eines Konjugats aus IL-6 und einem I -6 Rezeptor, vorzugsweise Hyper-IL-6, zur Behandlung eines Tumors. Bei dem Tumor handelt es sich vorzugsweise um ein Melanom, ein Nieren- oder ein Pankreas-Karzinom.The present invention relates to the use of a conjugate of IL-6 and an I -6 receptor, preferably Hyper-IL-6, for the treatment of a tumor. The tumor is preferably melanoma, kidney or pancreatic carcinoma.
Die Expression von Zytokingenen durch entsprechend transfizierte Tumorzellen wurde bisher als eine Strategie zur Erhöhung der Immunantworten gegenüber verschiedenen Krebsarten erprobt. Für Zytokine, z.B. TNF-α, IFN-gamma, I -2, IL-4, IL- 6, IL-12, IL-18 oder GM-CSF, exprimierende Tumorzellen konnte gezeigt werden, daß sie Bildung von tumorspezifischen T- Lymphozyten fördern oder nicht-T-Zell-vermittelte Mechanismen der Tumorzellenabtötung induzieren können, z.B. Granulozyten- Entzündungsreaktionen. Inzwischen befinden sich auf diesen Zytokinen basierende genetisch modifizierte Tumorvakzine (GMTV) als spezifische Immuntherapeutika zur Krebsbehandlung in der klinischen Untersuchung. Dabei wurden allogene oder autologe Tumorzellen mit den entsprechenden Zytokingenen modifiziert, um so einerseits hohe Konzentrationen der als Adjuvans wirkenden Zytokine an der Impfstelle erreichen zu können, andererseits aber die systemische Konzentration niedrig halten zu können. Allerdings lassen die bisher mit dieser Vorgehensweise erreichten Ergebnisse noch keine endgültige Schlußfolgerung darüber zu, ob sie tatsächlich einen ausreichend wirksamen therapeutischen Ansatz zur Krebsbehandlung darstellen können.The expression of cytokine genes by appropriately transfected tumor cells has so far been tried as a strategy for increasing the immune responses to various types of cancer. For cytokines, e.g. Tumor cells expressing TNF-α, IFN-gamma, I -2, IL-4, IL-6, IL-12, IL-18, or GM-CSF could be shown to promote or not to promote the formation of tumor-specific T lymphocytes. T cell-mediated mechanisms of tumor cell killing can induce, for example Granulocyte inflammatory reactions. In the meantime, genetically modified tumor vaccines (GMTV) based on these cytokines are in clinical investigation as specific immunotherapeutics for cancer treatment. Allogeneic or autologous tumor cells were modified with the corresponding cytokine genes in order to be able to reach high concentrations of the cytokines acting as adjuvants at the vaccination site and to keep the systemic concentration low. However, the results achieved with this approach so far do not allow a definitive conclusion to be drawn as to whether they can actually represent a sufficiently effective therapeutic approach to cancer treatment.
Somit liegt der vorliegenden Erfindung das technische Problem zugrunde, ein spezifisches Immuntherapeutikum bereitzustellen, das eine wirksame Krebstherapie erlaubt.The technical problem underlying the present invention is therefore to provide a specific immunotherapeutic agent, that allows effective cancer therapy.
Die Lösung dieses technischen Problems erfolgte durch die Bereitstellung der in den Patentansprüchen gekennzeichneten Ausführungsformen. Es wurde überraschenderweise gefunden, daß das vorstehende technische Problem durch die Verwendung eines Konjugats aus IL-6 und einem 11-6 Rezeptor, vorzugsweise Hyper-IL-6, gelöst werden kann. Bei den zu der vorliegenden Erfindung führenden Untersuchungen wurde die niedrig- immunogene murine Melano zellinie B16 mit einem Säugerexpressionsvektor transfiziert, der die cDNA für ein sIL-6R/IL-6-Fusionsprotein (mit der Bezeichnung Hyper-Il-6 oder H-IL-6) enthielt. Das entsprechende sezernierte p84- Glykoprotein konnte im Überstand von transfizierten Zellen nachgewiesen werden und es zeigte auf BAF/gpl30-Zellen, die auf IL-6/sIL-6R reagieren nicht jedoch auf IL-6 alleine, volle Aktivität. Die Verabreichung von rekombinantem H-IL-6 an C57BL/6-Mäuse führte zur einer verlängerten Expression eines Akutphaseproteins, was auf eine lange systemische Verweildauer des Fusionsprotein hinweist. Transfizierte Blβ-Zellen (B16/H- IL-6-Zellen) zeigten morphologische Veränderungen zusammen mit einer dramatischen Wachstumshemmung in vitro. Die subkutane Injektion in C57BL/ 6-Mäuse führte zu einer praktisch vollständigen Abstoßung der B16/H-IL-6-Zellen, wobei dieser Effekt in für einen GM-CSF-Rezeptorantagonisten transgenen Mäusen zum Teil wieder aufgehoben wurde. Diese deutet auf eine GM-CSF-abhängige Abstoßung von mit H-IL-6 transfizierten Zellen hin. Die systemische Behandlung von Mäusen mit Tumoren mit rekombinantem H-IL-6 verringerte das Tumorwachstum um etwa 40 bis 60%. Diese Ergebnisse zeigen, daß H-IL-6 (vor allem in Kombination mit GM-CSF) ein hohes antitumorales Potential aufweist und daß es auch zu einer gesteigerten Bildung von antigenpräsentierenden Zellen (APC) , z.B. dentritischen Zellen, kommt, was auch ex vivo bestätigt werden konnte.This technical problem was solved by providing the embodiments characterized in the patent claims. It has surprisingly been found that the above technical problem can be solved by using a conjugate of IL-6 and an 11-6 receptor, preferably Hyper-IL-6. In the investigations leading to the present invention, the low-immunogenic murine melanoma cell line B16 was transfected with a mammalian expression vector which isolated the cDNA for an sIL-6R / IL-6 fusion protein (designated Hyper-Il-6 or H-IL- 6) contained. The corresponding secreted p84 glycoprotein could be detected in the supernatant of transfected cells and showed full activity on BAF / gpl30 cells which respond to IL-6 / sIL-6R but not to IL-6 alone. The administration of recombinant H-IL-6 to C57BL / 6 mice led to a prolonged expression of an acute phase protein, which indicates a long systemic residence time of the fusion protein. Transfected Blβ cells (B16 / H-IL-6 cells) showed morphological changes together with a dramatic growth inhibition in vitro. The subcutaneous injection into C57BL / 6 mice resulted in a virtually complete rejection of the B16 / H-IL-6 cells, this effect being partially reversed in mice transgenic for a GM-CSF receptor antagonist. This indicates a GM-CSF-dependent rejection of cells transfected with H-IL-6. Systemic treatment of mice with tumors with recombinant H-IL-6 reduced tumor growth by approximately 40 to 60%. These results show that H-IL-6 (especially in combination with GM-CSF) has a high antitumor potential and that it also leads to an increased formation of antigen presenting cells (APC), e.g. dentritic cells, which could also be confirmed ex vivo.
Die vorliegende Erfindung betrifft somit die Verwendung eines Konjugats aus IL-6 und einem 11-6 Rezeptor oder einer dieses Konjugat kodierenden DNA-Sequenz zur Behandlung eines Tumors. Der hier verwendete Begriff "Konjugat aus IL-6 und einem IL-6 Rezeptor" betrifft ein Polypetid, das IL-6 (oder den biologisch aktiven Teil von IL-6) und den IL-6-Rezeptor (oder den für die Liganden-Bindung verantwortlichen Anteil des Rezeptors) umfaßt. Bei diesem Polypeptid kann es sich um ein Polypetid handeln, bei dem z.B. die beiden Partner über kovalente oder nicht-kovalente Bindungen gemäß dem Fachmann bekannten Routineverfahren miteinander verknüpft sind. Die beiden Polypeptide können beispielsweise über eine Disulfidbrücke miteinander verknüpft sein. Vorzugsweise handelt es sich bei dem Konjugat um ein Fusionsprotein aus IL- 6 und einem IL-6-Rezeptor . Vorzugsweise enthält das Konjugat den löslichen Anteil des IL-6-Rezeptors (sIL-6R) , d.h. die extrazelluläre bzw. lösliche Untereinheit des Interleukin-6- Rezeptors. In einer besonders bevorzugten Ausführungsform sind die beiden Polypeptide des Fusionproteins über einen Peptidlinker verbunden.The present invention thus relates to the use of a conjugate of IL-6 and an 11-6 receptor or a DNA sequence encoding this conjugate for the treatment of a tumor. The term "conjugate of IL-6 and an IL-6 receptor" as used herein refers to a polypeptide, the IL-6 (or the biologically active part of IL-6) and the IL-6 receptor (or for the ligand Binding responsible part of the receptor). This polypeptide can be a polypeptide in which, for example, the two partners are linked to one another via covalent or non-covalent bonds in accordance with routine methods known to the person skilled in the art. The two polypeptides can, for example, be linked to one another via a disulfide bridge. The conjugate is preferably a fusion protein composed of IL-6 and an IL-6 receptor. The conjugate preferably contains the soluble part of the IL-6 receptor (sIL-6R), ie the extracellular or soluble subunit of the interleukin-6 receptor. In a particularly preferred embodiment, the two polypeptides of the fusion protein are linked via a peptide linker.
In einer noch mehr bevorzugten Ausführungsform handelt es sich bei dem Konjugat um ein IL-6/SIL-6R FusionsproteinIn an even more preferred embodiment, the conjugate is an IL-6 / SIL-6R fusion protein
(Hyper-IL-6) , d.h. ein Fusionspolypeptid, das ein humanes sIL-(Hyper-IL-6), i.e. a fusion polypeptide that contains a human sIL
6R-Polypeptid und ein humanes IL-6-Polypeptid umfaßt, wobei die beiden Polypeptide über einen Polypeptidlinker miteinander verknüpft sind. Die Struktur von "Hyper-IL-6" sowie dessen Aminosäuresequenz sind in DE 196 08 813 C2 beschrieben sowie in Peters et al . , J. Immunol .161 (1998), 3575-3581. Der in der vorliegenden Anmeldung verwendete Ausdruck "IL-6/sIL-6R Fusionsprotein (Hyper-IL-6) " umfaßt dabei das Fusionsprotein mit der in DE 196 08 813 C2 gezeigten Aminosäuresequenz, sowie ein Fusionsprotein, das sich gegenüber dem in DE 196 08 813 C2 offenbarten Fusionsprotein dadurch unterscheidet, daß es Deletionen, Additionen oder Substitutionen von einer oder mehreren Aminosäuren und/oder (eine) modifizierte Aminosäure (n) aufweist, wobei die biologische Aktivität nicht wesentlich beeinflußt wird. Ob ein solches Fusionsprotein noch die gewünschten biologischen Eigenschaften aufweist, kann mittels üblicher Verfahren, z.B. mittels des in den nachstehenden Beispielen beschriebenen Verfahren untersucht werden .6R polypeptide and a human IL-6 polypeptide, wherein the two polypeptides are linked via a polypeptide linker. The structure of "Hyper-IL-6" and its amino acid sequence are described in DE 196 08 813 C2 and in Peters et al. , 1998, J. Immunol. 161: 3575-3581. The expression "IL-6 / sIL-6R fusion protein (Hyper-IL-6)" used in the present application includes the fusion protein with the amino acid sequence shown in DE 196 08 813 C2, as well as a fusion protein which is different from that in DE 196 08 813 C2 disclosed fusion protein in that it has deletions, additions or substitutions of one or more amino acids and / or (a) modified amino acid (s), wherein the biological activity is not significantly affected. Whether such a fusion protein still has the desired biological properties can be investigated using customary methods, for example using the method described in the examples below become .
Je nach Ausgestaltung der erfindungsgemäßen Verwendung kann es zweckmäßig sein, das Konjugat, vorzugsweise Hyper-IL-6, als Immuntherapeutikum zu verabreichen, d.h. in Form von allogenen oder autologen Tumorzellen, die mit einer DNA-Sequenz transformiert sind, die das Polypeptid, vorzugsweise Hyper-IL- 6, kodiert, und wobei dieses exprimiert wird. Der Fachmann kennt geeignete Transformationssysteme und Vektoren, z.B. zur Gentherapie, und in diesem Zusammenhang wird auf DE 196 08 813 C2 und die nachstehenden Beispiele verwiesen.Depending on the design of the use according to the invention, it may be expedient to administer the conjugate, preferably Hyper-IL-6, as an immunotherapeutic agent, i.e. in the form of allogeneic or autologous tumor cells which are transformed with a DNA sequence which encodes the polypeptide, preferably Hyper-IL-6, and which is expressed. The person skilled in the art knows suitable transformation systems and vectors, e.g. for gene therapy, and in this connection reference is made to DE 196 08 813 C2 and the examples below.
Bei Verwendung des Konjugats, vorzugsweise Hyper-IL-6, als Immuntherapeutikum, ist die z.B. Hyper-IL-6 kodierende DNA- Sequenz in einen für die Gentherapie geeigneten Vektor inseriert, z.B. einen Vektor, der auf einem Virus basiert, beispielsweise ein Adenovirus, Vaccinia-Virus oder ein AAV- Virus . Besonders bevorzugt sind Retroviren. Beispiele für geeignete Retroviren sind MoMuLV, HaMuSV, MuMTV, RSV oder GaLV. Weitere geeignete Viren sind Fowlpox-Virus, Canarypox- Virus, Influenza-Virus oder Sindbis-Virus auch als Basis einer Vakzine. Für Zwecke der Gentherapie kann die Hyper-IL-6 kodierende DNA-Sequenz auch in Form von kolloidalen Dispersionen zu den Zielzellen transportiert werden. Dazu zählen beispielsweise Liposomen oder Lipoplexe (Mannino et al., Biotechniques 6 (1988), 682).When using the conjugate, preferably Hyper-IL-6, as an immunotherapeutic agent, the e.g. Hyper-IL-6 coding DNA sequence inserted into a vector suitable for gene therapy, e.g. a vector based on 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. Other suitable viruses are fowlpox virus, canarypox virus, influenza virus or Sindbis virus as the basis of a vaccine. For the purposes of gene therapy, the DNA sequence encoding Hyper-IL-6 can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).
Die für die Anwendung als Immuntherapeutikum geeigneten Bedingungen sind dem Fachmann bekannt. So kann beispielsweise folgendermaßen vorgegangen werden:The conditions suitable for use as an immunotherapeutic are known to the person skilled in the art. For example, you can do the following:
Eine allogene Melanomzellinie (z.B. Mich-1 oder Mich-2) wird mit z.B. Hyper-IL-6 cDNA transduziert, wobei vorzugsweise MSCV-basierte dicistronische retrovirale Doppelkopie-Vektoren verwendet werden. Hierbei werden von einem Transkript zwei Proteine abgelesen, wobei dies durch die Verwendung einer "Internal Ribosome Entry" Site erreicht wird. Nach Expansion werden die gezüchteten Zellen trypsiniert, bestrahlt (z.B. mit 100 Gy unter Verwendung von Co-60 oder 6 MV-Photonen) , aliquotiert und in flüssigem Stickstoff eingefroren. Vor demAn allogeneic melanoma cell line (eg Mich-1 or Mich-2) is transduced with eg Hyper-IL-6 cDNA, preferably MSCV-based dicistronic retroviral double copy vectors being used. Two proteins are read from a transcript, this being achieved by using an "internal ribosome entry" site. After expansion, the grown cells are trypsinized, irradiated (for example with 100 Gy using Co-60 or 6 MV photons), aliquoted and frozen in liquid nitrogen. Before the
Eingefrieren werden die Zellen hinsichtlich KontaminationenThe cells are frozen with regard to contamination
(Bakterien, Pilze oder Mycoplasmen) getestet und kurz vor der(Bacteria, fungi or mycoplasma) tested and shortly before
Vakzinierung aufgetaut. Die Vakzinierung der Patienten kann subkutan (z.B. mit 5 x 107 Zellen) erfolgen, wobei anfänglich die Patienten viermal in Abständen von zwei Wochen immunisiert, dann ein Jahr lang jeweils einmal pro Monat und danach in Abständen von zwei Monaten immunisiert werden können. Im Fall des Fortschreitens der Erkrankung kann die Vakzinierung öfter erfolgen, z.B. anfänglich achtmal inVaccination thawed. The vaccination of the patients can be done subcutaneously (for example with 5 x 10 7 cells), with the patients initially being immunized four times at intervals of two weeks, then once a month for a year and then at intervals of two months. If the disease progresses, vaccination can take place more often, for example eight times in the beginning
Abständen von zwei Wochen und dann einmal pro Monat.Every two weeks and then once a month.
Besonders bevorzugt ist die Verwendung des vorstehend definierten Konjugats zur Behandlung eines Melanoms, eines Nieren- oder eines Pankreaskarzinoms .It is particularly preferred to use the conjugate defined above for the treatment of melanoma, kidney or pancreatic carcinoma.
Da es sich in den zu der vorliegenden Erfindung führenden Untersuchungen zeigte, daß die durch H-IL-6 induzierte Immunantwort gegen B16-Zellen von der Anwesenheit von GM-CSF abhängt, kann es für die erfindungsgemäße Verwendung vorteilhaft sein, nicht nur das IL-6/IL-6R-Konjugat sondern auch GM-CSF zu verabreichen (z.B. als Substanz oder über Transfektion der Zellen mit einer GM-CSF kodierenden DNA- Sequenz) . Somit betrifft die vorliegende Erfindung auch die Verwendung eines Konjugats aus IL-6 und einem 11-6 Rezeptor in Kombination mit GM-CSF.Since it was found in the investigations leading to the present invention that the immune response against B16 cells induced by H-IL-6 depends on the presence of GM-CSF, it can be advantageous for the use according to the invention not only the IL- 6 / IL-6R conjugate but also to administer GM-CSF (eg as a substance or via transfection of the cells with a DNA sequence encoding GM-CSF). The present invention thus also relates to the use of a conjugate of IL-6 and an 11-6 receptor in combination with GM-CSF.
Kurze Beschreibung der FigurenBrief description of the figures
Figur 1: Design und Expression von Hvx>er-IL-6Figure 1: Design and expression of Hvx> er-IL-6
(a) Schematische Darstellung des für die Expression und Sezernierung von H-IL-6 in Säugerzellen entworfenen Vektors PCDM8(a) Schematic representation of the vector PCDM8 designed for the expression and secretion of H-IL-6 in mammalian cells
Die cDNA für das Fusionsprotein enthält eine Strecke von 39 bp, die einen flexiblen Linker zwischen SIL-6R und IL-6 kodieren.The fusion protein cDNA contains a 39 bp stretch encoding a flexible linker between SIL-6R and IL-6.
(b) Nachweis von H-IL-6 im Überstand von transfizierten B16- Zellen Bl6/H-IL-6-Zellen wurden ü.N. radioaktiv markiert und H-IL-6 wurde vom Überstand mittels eines anti-IL-6-Antikörpers immunpräzipitiert . In nicht-transfizierten Zellen konnte kein H-IL-6 nachgewiesen werden. (c) Nachweis von H-IL-6 mittels Westernblot-Analvsen(b) Detection of H-IL-6 in the supernatant from transfected B16 cells Bl6 / H-IL-6 cells were brought over radiolabelled and H-IL-6 was immunoprecipitated from the supernatant using an anti-IL-6 antibody. No H-IL-6 could be detected in non-transfected cells. (c) Detection of H-IL-6 by Western blot analysis
107 Bl6/H-IL-6-Zellen wurden 24 Stunden in 5ml 0,1 FCS enthaltendem Medium inkubiert. 100 μl Überstand wurden für die Westernblot-Analysen mit einem anti-IL-6-Antikörper verwendet. H-IL-6 konnte als Protein mit einer relativen molekularen Masse von 84 kD nur im Überstand von transfizierten B16-Zellen nachgewiesen werden. Die rechten Spuren wurden mit 200 ng rekombinantem IL-6 bzw. SIL-6R beladen.10 7 Bl6 / H-IL-6 cells were incubated for 24 hours in 5 ml of medium containing 0.1 FCS. 100 ul supernatant was used for the Western blot analysis with an anti-IL-6 antibody. H-IL-6 could only be detected as a protein with a relative molecular mass of 84 kD in the supernatant of transfected B16 cells. The right lanes were loaded with 200 ng recombinant IL-6 or SIL-6R.
Figur 2: Biologische Aktivität von Hyper-IL-6 (a) Proliferation von BAF/gpl30-Zellen als Reaktion auf ansteigende Mengen an H-IL-6 und IL-6Figure 2: Biological activity of Hyper-IL-6 (a) proliferation of BAF / gpl30 cells in response to increasing amounts of H-IL-6 and IL-6
(b) Das gleiche Experiment wie (a) . jedoch mit ansteigenden Mengen an Überständen von Bl6/H-IL-6-Zellen (siehe Figur lc) Die Daten stellen Mittelwerte ± Standardabweichung aus drei parallelen Experimenten dar.(b) The same experiment as (a). but with increasing amounts of supernatants from Bl6 / H-IL-6 cells (see FIG. 1c). The data represent mean values ± standard deviation from three parallel experiments.
(c) Induktion der Haptoglobin-Svnthese in vivo C57BL/6-Mäuse (neun Tiere) wurden mit 2 μg rekombinantem H-IL- 6 behandelt und 72 Stunden nach der Injektion getötet. Haptoglobin wurde in den Seren der Mäuse durch Westernblot- Analysen nachgewiesen.(c) Induction of haptoglobin synthesis in vivo C57BL / 6 mice (nine animals) were treated with 2 μg of recombinant H-IL-6 and sacrificed 72 hours after the injection. Haptoglobin was detected in the sera of the mice by Western blot analysis.
Figur 3 : Wachstumshemmung und morphologische Veränderungen von mit H-IL-6 transfizierten Bl6-MelanomzellenFigure 3: Growth inhibition and morphological changes of H-IL-6 transfected Bl6 melanoma cells
(a) Proliferation von B16- und Bl6/H-IL-6-Zellen nachgewiesen über MTT-Assay.(a) B16 and Bl6 / H-IL-6 cell proliferation detected by MTT assay.
(b) Bl6/H-IL-6-Zellen zeigen eine verlängerte Morphologie und neigen zur Ausbildung von Zellprotrusionen.(b) Bl6 / H-IL-6 cells show an elongated morphology and tend to form cell protrusions.
Untere Abbildung: Kontrollzellen; obere Abbildung: Mit H-IL-6 transfizierte Zellen. Vergrößerung: 200fachLower figure: control cells; upper picture: cells transfected with H-IL-6. Magnification: 200 times
Figur 4: MHC-1-Expression in unveränderten Bl6/H-IL-6-Zellen Die Oberflächenexpression von MHC-1-Molekülen Kb und D° auf B16/H-IL-6-Zellen und Kontrollzellen wurde mittels FACS unter Verwendung eines anti-H-2KbDb-MAK bestimmt.Figure 4: MHC-1 expression in unchanged Bl6 / H-IL-6 cells. The surface expression of MHC-1 molecules K b and D ° B16 / H-IL-6 cells and control cells were determined by FACS using an anti-H-2K b D b -MAK.
Figur 5: Tumorwachstum in Mäusen, denen H-IL-6 transfizierte, scheintransfizierte oder parentale Bl6-Melanomzellen s.c. injiziert worden warenFigure 5: Tumor growth in mice to which H-IL-6 transfected, sham-transfected or parental Bl6 melanoma cells s.c. had been injected
(a) C57BL/6-Mäusen wurden 5 x 103 H-IL-6-transfizierte, scheintransfizierte oder parentale Bl6-Zellen s.c. injiziert. Nach zwei Wochen wurden die Tiere getötet und die Tumorvolumina nach Exzision bestimmt. Das Tumorwachstum war in Tieren, die Bl6/H-IL-6-Zellen erhielten, drastisch verringert . Jeder Punkt in der Figur entspricht einem Tier.(a) C57BL / 6 mice were sc injected with 5 x 10 3 H-IL-6 transfected, sham transfected or parental Bl6 cells. After two weeks, the animals were sacrificed and the tumor volumes after excision were determined. Tumor growth was drastically reduced in animals receiving Bl6 / H-IL-6 cells. Each point in the figure corresponds to an animal.
(b) Überleben von Mäusen, denen 5 x 105 H-IL-6-transfizierte oder parentale Bl6-Zellen s.c. injiziert worden waren.(b) Survival of mice sc injected with 5 x 10 5 H-IL-6 transfected or parental Bl6 cells.
Figur 6: Reduzierte Wachstumshemmung von Bl6/H-IL-6-Zellen in für einen GM-CSFR-Antagonisten transgenen Mäusen (a) Serumspiegel des K14E/E21K GM-CSF- Pro t eins in Wildtypmäusen und transgenen Mäusen nachgewiesen über ELISA. (b) Acht transgenen Mäusen (Stamm tg2) und acht Wildtyp- FvB/BL76-Mäusen wurden s.c. 5 x 105 B16/H-IL-6-Zellen injiziert und das Tumorwachstum wurde nach zwei Wochen bestimmt. Als Kontrolle wurden transgenen Mäusen und Wildtypmäusen 5 x 105 parentale Bl6-Zellen injiziert.Figure 6: Reduced growth inhibition of Bl6 / H-IL-6 cells in mice transgenic for a GM-CSFR antagonist (a) Serum levels of the K14E / E21K GM-CSF-Pro one in wild-type mice and transgenic mice detected by ELISA. (b) Eight transgenic mice (strain tg2) and eight wild-type FvB / BL76 mice were injected with sc 5 x 10 5 B16 / H-IL-6 cells and tumor growth was determined after two weeks. As a control, 5 × 10 5 parental Bl6 cells were injected into transgenic mice and wild-type mice.
Die nachstehenden Beispiele erläutern die Erfindung.The following examples illustrate the invention.
Beispiel 1 Allgemeine VerfahrenExample 1 General procedures
(A) Herstellung der Plasmide und Transfektionsverfahren Die H-IL-6-cDNA wurde unter Verwendung der Vektoren pCDM8-sIL- 6R und pCDM8-IL-6 hergestellt, von denen die sIL-6R und IL-6 cDNAs über PCR erzeugt wurden. Die Linker-cDNA wurde unter Verwendung von Xhol-Stellen am C-Terminus von SIL-6R und am N- Terminus von IL-6 eingeführt. H-IL-6 cDNA wurde erneut in den Vektor pCDM8 mittels einer N-terminalen Motl- und einer C- terminalen Hindlll-Stelle ligiert. Bezüglich der genaueren Schritte zur Erzeugung der H-IL-6-cDNA wird auf DE 196 08 813 C2 verwiesen. Die Zellinie B16/H-IL-6 wurde durch Transfektion mit 20 μg pCDM8-H-IL-6-DNA zusammen mit 2 μg pSV2Neo-Plasmid- DNA (Southern und Berg, J.Mol .Appl .Genet . 1 (1982), 327- 341) (Copräzipitation mit Calciumphosphat) erzeugt.(A) Preparation of the plasmids and transfection methods. The H-IL-6 cDNA was prepared using the vectors pCDM8-sIL-6R and pCDM8-IL-6, of which the sIL-6R and IL-6 cDNAs were generated by PCR. The linker cDNA was introduced using Xhol sites at the C-terminus of SIL-6R and at the N-terminus of IL-6. H-IL-6 cDNA was re-inserted into the vector pCDM8 using an N-terminal Mot1 and a C- terminal Hindlll site ligated. With regard to the more precise steps for generating the H-IL-6 cDNA, reference is made to DE 196 08 813 C2. The cell line B16 / H-IL-6 was transfected with 20 μg pCDM8-H-IL-6 DNA together with 2 μg pSV2Neo plasmid DNA (Southern and Berg, J.Mol .Appl .Genet. 1 (1982) , 327-341) (coprecipitation with calcium phosphate).
(B) Nachweis und biologische Aktivität von H-IL-6 im Überstand von Bl6/H-IL-6-Zellen 107 Bl6/H-IL-6-Zellen wurden ü.N. mit 50 μg(B) Detection and biological activity of H-IL-6 in the supernatant of Bl6 / H-IL-6 cells 10 7 Bl6 / H-IL-6 cells were brought to sea level. with 50 μg
[35S]Methionin/Cystein in Methionin/Cystein-freiem Medium metabolisch markiert und H-IL-6 wurde mit einem an Protein A-[ 35 S] Methionine / cysteine was metabolically labeled in methionine / cysteine-free medium and H-IL-6 was treated with an
Sepharose gebundenen anti-IL-6-Antikörper immunpräzipitiert .Sepharose bound anti-IL-6 antibody immunoprecipitated.
Für einen Western-Blot wurden 107 Bl6/H-IL-6-Zellen 24 Stunden mit 5 ml 0,1% FCS enthaltendem Medium inkubiert. Der Überstand wurde entfernt und zehnfach konzentriert. Für die Western- Blot-Analysen mit dem anti-IL-6-Antikörper mAB-8 (CLB, Amerstam, Niederlande) wurden 10 μg des konzentrierten Überstands verwendet.For a Western blot, 10 7 Bl6 / H-IL-6 cells were incubated with 5 ml of medium containing 0.1% FCS for 24 hours. The supernatant was removed and concentrated ten times. For the Western blot analyzes with the anti-IL-6 antibody mAB-8 (CLB, Amerstam, The Netherlands), 10 μg of the concentrated supernatant were used.
(C) Zellkultur und Assavs Die Zellen wurden in DMEM bei 5% C02 in einer wassergesättigten Atmosphäre gezüchtet. Alle Zellkulturmedien waren mit 10% FCS, 100 mg/ml Streptomycin und 60 mg/1 Penicillin supplementiert . Die Proliferation der B16- und B16/H-IL-6-Zellen wurde in(C) Cell Culture and Assavs The cells were grown in DMEM at 5% CO 2 in a water-saturated atmosphere. All cell culture media were supplemented with 10% FCS, 100 mg / ml streptomycin and 60 mg / 1 penicillin. The proliferation of B16 and B16 / H-IL-6 cells was shown in
Mi kr o t i t erp 1 a 11 en mit 96 Vertiefungen ( 10.000 Zellen/Vertiefung/O, 1 ml) gemessen. Die Dichte der lebenden Zellen wurde nach 24, 48, 72, 96 und 120 Stunden mittels des MTT ( 3 - [ 4 , 5 - D i me thy l - th i a z o l - 2 - yl ] - 2 , 5 - diphenyltetrazoliumbromid) -Assays der mitochrondrialenMi cr i t erp 1 a 11 s with 96 wells (10,000 cells / well / O, 1 ml) measured. The density of the living cells was determined after 24, 48, 72, 96 and 120 hours using the MTT (3 - [4, 5 - dimethyththiazol - 2 - yl] - 2, 5 - diphenyltetrazolium bromide) assays the mitochrondrial
Succinatdehydrogenase bestimmt (Boehringer Mannheim, Deutschland) . Morphologische Veränderungen der B16/H-I1-6- Zellen wurden nach Züchtung der Zellen auf Kammerträgern (NUNC, Naperville, IL, USA) für 24 Stunden bestimmt. Mikroaufnahmen wurden bei einer 200fachen Vergrößerung in einem CID ( "dif f erential interference contrast" ) -Modus aufgenommen. (D) Tiere C57BL/6-Mäuse wurden in einem Zyklus von 12 Stunden Licht und 12 Stunden Dunkelheit gehalten und mit Futter und Wasser nach Bedarf versorgt .Succinate dehydrogenase determined (Boehringer Mannheim, Germany). Morphological changes in the B16 / H-I1-6 cells were determined after culturing the cells on ventricular supports (NUNC, Naperville, IL, USA) for 24 hours. Micrographs were taken at 200X magnification in a differential interference contrast (CID) mode. (D) Animals C57BL / 6 mice were kept on a cycle of 12 hours light and 12 hours dark and were fed food and water as needed.
(E) Erzeugung von hinsichtlich des GM-CSF-Rezeptors transgenen Mäusen Die für murines GM-CSF kodierende cDNA (Gough et al . , Nature 309 (1984), 763-767) wurde in Übereinstimmung mit den veröffentlichten Aminosäureänderungen K14E und E12K (Altmann und Kastelein, J.Biol.Chem. 270 (1995), 2233-2240) modifiziert. Diese cDNA wurde in den Keratin 10-(E) Generation of Mice Transgenic for the GM-CSF Receptor The cDNA coding for murine GM-CSF (Gough et al., Nature 309 (1984), 763-767) was developed in accordance with the published amino acid changes K14E and E12K (Altmann and Kastelein, J.Biol.Chem. 270 (1995), 2233-2240). This cDNA was
Expressionsvektor (Blessing et al . , J.Cell.Biol. 135Expression vector (Blessing et al., J. Cell.Biol. 135
( 1996 ), 227-239 ) inseriert und für die pronukleäre Mikroinjektion von befruchteten Eiern von Mäusen des Stamms FVB/N wie in Hogan et al . (Manipulating the Mouse Embryo. A Laboratory Manual., Cold Spring Harbor, NY (1986)) beschrieben verwendet. An den Nachkommen wurden an Ohren und Schwänzen Biopsien vorgenommen und diese wurden mittels PCR unter Verwendung eines Rinderkeratin 10-spezifischen Primers (5'-TAA CAC ATG TGG GAT ACA CCC-3 ' ) und eines murinen GM-CSF- spezifischen Oligonukleotids (5 '-CTG GCT GTC ATG TTC AAG GCG- 3'; Position 1021-1042; GenBank-Zugangsnummer X05906) analysiert. Die für die tumorinduzierenden Experimente verwendeten Mäuse waren FI-Kreuzungen von für den FVB/N GM- CSF-Rezeptorantagonisten transgenen Mäusen und C57BL/6-Mäusen. Die Experimente wurden mit dem Stamm tg2 durchgeführt.(1996), 227-239) and for the pronuclear microinjection of fertilized eggs from mice of the strain FVB / N as described in Hogan et al. (Manipulating the Mouse Embryo. A Laboratory Manual., Cold Spring Harbor, NY (1986)). Biopsies were performed on the offspring's ears and tails and these were analyzed by PCR using a bovine keratin 10-specific primer (5'-TAA CAC ATG TGG GAT ACA CCC-3 ') and a murine GM-CSF-specific oligonucleotide (5' -CTG GCT GTC ATG TTC AAG GCG- 3 '; position 1021-1042; GenBank accession number X05906) analyzed. The mice used for the tumor-inducing experiments were FI crossings from mice transgenic for the FVB / N GM-CSF receptor antagonist and C57BL / 6 mice. The experiments were carried out with the tg2 strain.
(F) Subkutane "Challenge" Experimente Die Mäuse wurden am Rücken rasiert und subkutan wurden 105 B16 oder Bl6/H-IL-6-Zellen injiziert. Das Tumorwachstum wurde durch Messung der senkrechten Durchmesser überwacht. Die Mäuse wurden nach zwei Wochen getötet oder wenn die Tumore eine schwere Nekrose zeigten oder eine Größe von 300 mm2 erreicht hatten. Alle Tumore wurden exzisiert und durch Bestimmung des Gewichts und Volumens bewertet. (G) Durchflußzvtometrie-Analvsen B16- und Bl6/H-IL-6-Zellen wurden zweimal mit PBS (0,1% Tween) gewaschen und eine indirekte Immunfluoreszenzfärbung wurde durch Inkubation der Zellen (4°C, 30 Min.) mit einem FITC- gekoppelten monoklonalen anti-H-2KHDH-Antikörper (Cedar Lane, Hernby, Ontario, Kanada) durchgeführt. Nach zweimaligem Waschen der Zellen mit PBS (0,1% Tween) wurden Durchflußzytometrie-Analysen mittels eines "FACScan"-Geräts (Becton Dickinson, Mountain View, CA, USA) durchgeführt. Debris und tote Zellen wurden ausgesondert und lebende Zellen mittels der "FACScan Lysis II"-Software (Becton Dickinson) geplottet. Zur Kontrolle wurde das gleiche Experiment mit einem für IgG2 spezifischen Antikörper durchgeführt.(F) Subcutaneous "Challenge" experiments The mice were shaved on the back and 10 5 B16 or Bl6 / H-IL-6 cells were injected subcutaneously. Tumor growth was monitored by measuring the vertical diameter. The mice were sacrificed after two weeks or when the tumors showed severe necrosis or had reached 300 mm 2 . All tumors were excised and evaluated by weight and volume. (G) Flow analysis B16 and Bl6 / H-IL-6 cells were washed twice with PBS (0.1% Tween) and indirect immunofluorescence staining was performed by incubating the cells (4 ° C, 30 min) with a FITC-coupled anti-H-2KHDH monoclonal antibody (Cedar Lane, Hernby, Ontario, Canada). After washing the cells twice with PBS (0.1% Tween), flow cytometry analyzes were carried out using a "FACScan" device (Becton Dickinson, Mountain View, CA, USA). Debris and dead cells were separated and living cells were plotted using the "FACScan Lysis II" software (Becton Dickinson). As a control, the same experiment was carried out with an antibody specific for IgG2.
Beispiel 2 Transfektion von H-IL-6-cDNA in Bl6-MelanomzellenExample 2 Transfection of H-IL-6 cDNA in Bl6 Melanoma Cells
Bl6-Zellen wurden mit einem die cDNA für das Fusionsprotein H- IL-6 unter Kontrolle des CMV-Promotors enthaltenden PlasmidBl6 cells were grown with a plasmid containing the cDNA for the fusion protein H-IL-6 under the control of the CMV promoter
(Figur la) und mit dem pSV2-neo-Plasmid cotransfiziert . 30(Figure la) and co-transfected with the pSV2-neo plasmid. 30
Neo-resistente Klone wurden hinsichtlich der Expression des H-Neo-resistant clones were evaluated for the expression of the H-
Il-6-Proteins durch Testen der Aktivität von Zellüberständen von BAF3/gpl30-Zellen gescreent. Für dieses Beispiel wurden zwei positive Klone ausgewählt und ein scheintransfizierter Klon, der nur das pSV2-neo-Plasmid enthielt. Figur lb zeigt die Expression des 84 kD Glykoproteins einer positiven Transfektante, nachgewiesen mittels Immunpräzipitation des radioaktiv markierten Proteins aus dem Überstand über einen anti-IL-6-Antikörper. Negative Transfektanten und parentale Bl6-Zellen sezernierten nicht H-IL-6 ins Medium. Figur lc zeigt eine Westernblot-Analyse des Mediums von transfizierten und parentalen B16-Zellen nach einer zehnfachen Konzentrierung. Das IL-6/sIL-6R-Fusionsprotein wurde mit dem anti-IL-6-Antikörper genauso effizient nachgewiesen wie rekombinantes IL-6. Es gab keine Kreuzreaktion des anti-IL-6- MAK mit SIL6R (rechte Spur) . Die H-IL-6-Spiegel im Überstand transfizierter Zellen wurden durch Messung der biologischen Aktivität des Fusionsproteins auf einer mit gpl30 transfizierten BAF3-Zellinie (BAF3/gpl30) bestimmt (Figur 2b) . Untransfizierte BAF3-Zellen exprimierten gpl30 und IL-6R nicht, antworten daher nicht auf IL-6 oder H-IL-6. Zeilüberstände transfizierter B16-Zellen wurden nach 24 Stunden geerntet und es konnte eine dosisabhängige Proliferation der BAF3/gpl30-Zellen beobachtet werden. Die EC50-Konzentration wurde mit 0,2 μl/ml bestimmt (Figur 2b). Dies entspricht H-IL-6-Spiegeln von 500 pg/ml/107 Zellen/24 Stunden (Figur 2a) . Die biologische Aktivität von H-IL-6 in vivo und dessen Stabilität wurden durch Injektion (i.p.) von 2 μg rekombinantem H-IL-6 in C57BL/6-Mäuse getestet. Die Expression des Akutphase-Proteins Haptoglobin wurde nach 72 Stunden durch Bestimmung der Serumspiegel mittels Westernblot- Analysen analysiert (Figur 2c) . Nach IL-6-Injektion konnten nach 72 Stunden keine erhöhten Haptoglobin-Spiegel nachgewiesen werden. Acht von neun getesteten Mäusen zeigten deutlich erhöhte Haptoglobin-Spiegel im Vergleich zu Kontrolltieren, die PBS erhielten. Somit kann H-IL-6 bei wesentlich geringeren Dosen als IL-6 Akutphase-Proteine in Mäusen induzieren.Il-6 protein screened by testing the activity of cell supernatants from BAF3 / gpl30 cells. For this example, two positive clones were selected and one sham-transfected clone that contained only the pSV2-neo plasmid. Figure lb shows the expression of the 84 kD glycoprotein of a positive transfectant, detected by immunoprecipitation of the radioactively labeled protein from the supernatant via an anti-IL-6 antibody. Negative transfectants and parental Bl6 cells did not secrete H-IL-6 into the medium. Figure lc shows a Western blot analysis of the medium of transfected and parental B16 cells after a tenfold concentration. The IL-6 / sIL-6R fusion protein was detected with the anti-IL-6 antibody as efficiently as recombinant IL-6. There was no cross-reaction of the anti-IL-6 MAK with SIL6R (right lane). The H-IL-6 levels in the supernatant of transfected cells were determined by measuring the biological Activity of the fusion protein determined on a BAF3 cell line (BAF3 / gpl30) transfected with gpl30 (FIG. 2b). Untransfected BAF3 cells did not express gpl30 and IL-6R and therefore do not respond to IL-6 or H-IL-6. Cell supernatants from transfected B16 cells were harvested after 24 hours and a dose-dependent proliferation of the BAF3 / gpl30 cells was observed. The EC 50 concentration was determined to be 0.2 μl / ml (FIG. 2b). This corresponds to H-IL-6 levels of 500 pg / ml / 10 7 cells / 24 hours (FIG. 2a). The biological activity of H-IL-6 in vivo and its stability were tested by injection (ip) of 2 μg recombinant H-IL-6 in C57BL / 6 mice. The expression of the acute phase protein haptoglobin was analyzed after 72 hours by determining the serum level by means of Western blot analyzes (FIG. 2c). No elevated haptoglobin levels were detected after 72 hours after IL-6 injection. Eight out of nine mice tested showed significantly increased haptoglobin levels compared to control animals that received PBS. Thus, H-IL-6 can induce acute phase proteins in mice at much lower doses than IL-6.
Beispiel 3 Untersuchung der in vitro-Eigenschaften von parentalen B16-Example 3 Investigation of the In Vitro Properties of Parental B16
Zellen und TransfektantenCells and transfectants
Um zu untersuchen ob H-IL-6-Transfektion das Zellwachstum in vitro beeinflußt wurden Proliferationsassays durchgeführt und die Zelldichten mittels eines kolorimetrischen Assays mit MTT bestimmt. Figur 3a zeigt die Proliferation von parentalen B16- und transfizierten Bl6/H-IL-6-Zellen, die über einen Zeitraum von fünf Tagen zu verschiedenen Zeitpunkten gemessen wurde. In Bl6/H-IL-6-Zellen war das Zellwachstum deutlich gehemmt und die Zelldichten betrugen etwa 10% im Vergleich zu den parentalen B16-Zellen am Ende der Zeitreihe. Außerdem zeigten transfizierte Bl6-Zellen eine veränderte Morhologie, die durch verlängerte Zellauswölbungen, Verlust der Pigmentierung und ein Aneinanderliegen in langen Reihen gekennzeichnet war, was auf einen veränderten Differenzierungsstatus hinweist (Figur 3b) . Ähnliche Effekte konnten beobachtet werden, wenn Bl6-Zellen mit rekombinantem IL-6/sIL-6R-Fusionsprotein behandelt worden waren. Außerdem ergab die Messung von MHC-1 keinen Unterschied zwischen parentalen Bl6-Zellen und Transfektanten, was darauf hinweist, daß der niedrige immunogene Status aufgrund der geringen MHC- 1-Expression von transfizierten Bl6-Zellen nicht durch das Transfektionsverfahren und die Expression von H-IL-6 veränderte wurde (Figur 4) .In order to investigate whether H-IL-6 transfection influences cell growth in vitro, proliferation assays were carried out and the cell densities were determined using a colorimetric assay with MTT. Figure 3a shows the proliferation of parental B16 and transfected Bl6 / H-IL-6 cells, which was measured over a period of five days at different times. Cell growth was significantly inhibited in Bl6 / H-IL-6 cells and cell densities were approximately 10% compared to the parental B16 cells at the end of the time series. In addition, transfected Bl6 cells showed an altered morphology, which was caused by prolonged cell bulges, loss of Pigmentation and a contiguity in long rows was marked, which indicates a differentiation status (Figure 3b). Similar effects could be observed when Bl6 cells were treated with recombinant IL-6 / sIL-6R fusion protein. In addition, measurement of MHC-1 revealed no difference between parental Bl6 cells and transfectants, indicating that the low immunogenic status due to the low MHC-1 expression of transfected Bl6 cells was not affected by the transfection method and the expression of H- IL-6 was changed (Figure 4).
Beispiel 4 Untersuchungen zum TumorwachstumExample 4 Studies on tumor growth
Die Tumorigenizität der H-IL-6-Transfektanten wurde durch s.c. Injektion in syngene C57BL/6-Mäuse untersucht. Figur 5a zeigt, daß Bl6/H-lL-6-Zellen im Vergleich zu parentalen oder scheintransfizierten Bl6-Zellen wesentlich kleinere Tumore bildeten. Sechs von acht Tieren in der Bl6/H-IL-6-Gruppe zeigten nach zwei Wochen eine vollständige Abstoßung der Tumorzellen. Das mittlere Volumen der etablierten Tumore in dieser Gruppe betrug 50 bis 100 mm3, während die parentalen oder scheininfizierten B16-Zellen Tumore mit 1800 bzw. 1900 mm3 ausbildeten. In einem parallelen Experiment wurde das Überleben von Mäusen beobachtet, denen s.c. parentale oder transfizierte Bl6-Zellen injiziert worden waren (Figur 5b) . 50% der Tiere, die Bl6-Zellen erhalten hatten, waren moribund und mußten nach drei Wochen getötet werden. Kein Tier dieser Gruppe überlebte 33 Tage nach Injektion, während 8 von 10 Mäusen, die Bl6/H-IL-6-Zellen erhalten hatten, nach 89 Tagen überlebt hatten. Um zu untersuchen, ob ein durch rekombinantes H-IL-6 induziertes verringertes Wachstum von Bl6-Zellen in vivo beobachtet werden kann, wurden Mäusen, die parentale B16- Zellen erhalten hatten, dreimal pro Woche 2 μg rekombinantes H-IL-6 i.p. injiziert. Nach zwei Wochen wurden die Mäuse getötet und die Tumorgewichte bestimmt. Diese systemische Behandlung mi t rekombinantem H- IL- 6 verringerte das Tumorwachstum um etwa 40 bis 50% .The tumorigenicity of the H-IL-6 transfectants was examined by sc injection into syngeneic C57BL / 6 mice. Figure 5a shows that Bl6 / H-IL-6 cells formed much smaller tumors compared to parental or sham-transfected Bl6 cells. Six of eight animals in the Bl6 / H-IL-6 group showed complete rejection of the tumor cells after two weeks. The mean volume of the established tumors in this group was 50 to 100 mm 3 , while the parental or sham infected B16 cells developed tumors of 1800 and 1900 mm 3, respectively. In a parallel experiment, the survival of mice which had been injected with parental or transfected Bl6 cells was observed (FIG. 5b). 50% of the animals that received Bl6 cells were moribund and had to be killed after three weeks. No animal in this group survived 33 days after injection, whereas 8 out of 10 mice that received Bl6 / H-IL-6 cells survived after 89 days. In order to investigate whether a reduced growth of Bl6 cells induced by recombinant H-IL-6 can be observed in vivo, mice which had received parental B16 cells were injected ip three times a week with 3 μg recombinant H-IL-6 , After two weeks, the mice were sacrificed and the tumor weights were determined. This systemic Treatment with recombinant H-IL-6 reduced tumor growth by about 40 to 50%.
Beispiel 5Example 5
Die Abstoßung von Bl6/H-IL-6-Zellen ist in für einen GM-CSFR- Antagonisten transgenen Mäusen beeinträchtigtThe rejection of Bl6 / H-IL-6 cells is impaired in mice transgenic for a GM-CSFR antagonist
Es wurde davon ausgegangen, daß GM-CSF eine Schlüsselrolle bei der Abstoßung von Tumorzellen durch das Immunsystem einnimmt. Um die Rolle von GM-CSF bei der Abstoßung von H-IL-6 sezernierenden Bl6-Melanomzellen zu untersuchen wurden transfizierte und parentale Bl6-Zellen in für das mutierte K14E/E21K GM-CSF-Protein transgene Mäuse injiziert. Für dieses GM-CSF-Mutein konnte kürzlich gezeigt werden, daß es zwar an die GM-CSF-Rezeptor-α-Kette binden, nicht jedoch die signaltransduzierende ß-Einheit des GM-CSF-Rezeptorkomplexes stimulieren konnte. Somit verhält sich das K14E/E21K-GM-CSF- Protein als ein GM-CSF-Rezeptorantagonist . Für den K14E/E21K- GM-CSF-Antagonisten transgene Mäuse zeigten Spiegel von 2 bis 15 ng/ml im Serum (Figur 6a) . Transgene und nicht-transgene Kontrollmäuse wurden nach zwei Wochen getötet und die Tumorvolumina wurden wie vorstehend beschrieben bestimmt. Das Tumorwachstum der Bl6/H-IL-6-Zellen in Wildtypmäusen FVB/N x C57BL/6 war mit dem in C57BL/6-Mäusen vergleichbar mit einem durchschnittlichen Tumorvolumen von 50 bis 100 mm3. Die Tumorvolumina in transgenen GM-CSF Kl4E/E21K-Mäusen war eine Größenordnung höher als in den nicht-transgenen Kontrollmäusen (900 mm3) , was darauf hindeutet, daß die Abstoßung der transfizierten Tumorzellen von der Anwesenheit von biologisch aktivem GM-CSF abhing (Figur 6b) . Von parentalen B16-Zellen sowohl in transgenen als auch Wildtyp-Mäusen produzierte Tumore waren mit denen in C57BL/6-Mäusen vergleichbar. GM-CSF has been thought to play a key role in the rejection of tumor cells by the immune system. In order to investigate the role of GM-CSF in the rejection of Bl6 melanoma cells secreting H-IL-6, transfected and parental Bl6 cells were injected into mice transgenic for the mutated K14E / E21K GM-CSF protein. This GM-CSF mutein has recently been shown to bind to the GM-CSF receptor α chain, but not to stimulate the signal-transducing β unit of the GM-CSF receptor complex. Thus the K14E / E21K-GM-CSF protein behaves as a GM-CSF receptor antagonist. Mice transgenic for the K14E / E21K-GM-CSF antagonist showed levels of 2 to 15 ng / ml in the serum (FIG. 6a). Transgenic and non-transgenic control mice were sacrificed after two weeks and the tumor volumes were determined as described above. The tumor growth of the Bl6 / H-IL-6 cells in wild-type mice FVB / N x C57BL / 6 was comparable to that in C57BL / 6 mice with an average tumor volume of 50 to 100 mm 3 . The tumor volumes in transgenic GM-CSF Kl4E / E21K mice were an order of magnitude higher than in the non-transgenic control mice (900 mm 3 ), which indicates that the rejection of the transfected tumor cells was dependent on the presence of biologically active GM-CSF ( Figure 6b). Tumors produced by parental B16 cells in both transgenic and wild-type mice were comparable to those in C57BL / 6 mice.

Claims

Patentansprüche claims
1. Verwendung eines Konjugats aus IL-6 und einem IL-6 Rezeptor oder einer dieses Konjugat kodierenden DNA-Sequenz zur1. Use of a conjugate of IL-6 and an IL-6 receptor or a DNA sequence encoding this conjugate for
Behandlung eines Tumors .Treatment of a tumor.
2. Verwendung nach Anspruch 1, wobei das Konjugat ein Fusionsprotein aus IL-6 und dem löslichen Anteil des IL-6- Rezeptors (sIL-6R) ist.2. Use according to claim 1, wherein the conjugate is a fusion protein of IL-6 and the soluble portion of the IL-6 receptor (sIL-6R).
3. Verwendung nach Anspruch 2, wobei das Fusionsprotein Hyper- IL-6 ist.3. Use according to claim 2, wherein the fusion protein is hyper-IL-6.
4. Verwendung nach einem der Ansprüche 1 bis 3, wobei das Konjugat in Form eines Immuntherapeutikums verwendet wird.4. Use according to any one of claims 1 to 3, wherein the conjugate is used in the form of an immunotherapeutic.
5. Verwendung nach einem der Ansprüche 1 bis 4, wobei der Tumor ein Melanom, ein Nieren- oder ein Pankreaskarzinom ist.5. Use according to one of claims 1 to 4, wherein the tumor is a melanoma, kidney or pancreatic carcinoma.
6. Verwendung nach einem der Ansprüche 1 bis 5, wobei außerdem GM-CSF verabreicht wird. 6. Use according to any one of claims 1 to 5, wherein GM-CSF is also administered.
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