WO2000053214A1 - Utilisation de l'interleukine 11 pour prevenir une cytotoxicite d'origine immunologique - Google Patents

Utilisation de l'interleukine 11 pour prevenir une cytotoxicite d'origine immunologique Download PDF

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WO2000053214A1
WO2000053214A1 PCT/US2000/006369 US0006369W WO0053214A1 WO 2000053214 A1 WO2000053214 A1 WO 2000053214A1 US 0006369 W US0006369 W US 0006369W WO 0053214 A1 WO0053214 A1 WO 0053214A1
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cells
interleukin
immune
cell
mediated
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PCT/US2000/006369
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English (en)
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James Keith
Joseph M. Carroll
Jordan S. Pober
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Genetics Institute, Inc.
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Priority to AU37383/00A priority Critical patent/AU3738300A/en
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    • 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]
    • A61K38/2073—IL-11

Definitions

  • the present invention relates to the field of prevention and treatment of immune-mediated disorders using interleukin-11. More particularly, the present invention relates to preventing or treating graft-versus-host disease and CTL- and/or complement-dependent rejection of organ or tissue transplants using interleukin-11.
  • MHC proteins major histocompatibility complex proteins
  • Additional minor histocompatibility proteins exist which can also contribute to immunological recognition events.
  • the individual mammal's immune system recognizes its own MHC proteins, or those of its identical twin, as self and thus does not destroy its own cells or those of its identical twin.
  • Members of the same species may share major and/or minor histocompatibility antigens, and thus an individual may not recognize the cells of another member of its species as non-self, depending on the degree of the differences between the MHC proteins of the two individuals.
  • the first individual's immune system may proceed to destroy the cells of the second individual.
  • the major histocompatibility proteins are known as "HLA" antigens.
  • transplantation patients are also subjected to immunologic recognition in the opposite direction, that is, the donor tissue may contain immunologically competent cells which proceed to destroy the recipient's cells, a condition termed "graft-versus-host disease" or "GVHD".
  • Graft-versus-host disease can develop when bone marrow, blood products, or solid organs containing immunocompetent cells are transferred from a donor to a recipient.
  • the recipient is at risk for the development of graft-versus-host disease.
  • Graft-versus-host disease may also develop when there are antigenic differences between donor and recipient for the minor histocompatibility antigens.
  • graft-versus-host disease can also develop between MHC-matched persons.
  • surgery patients who receive directed blood transfusion for example, transfusion of blood from an HLA homozygous child to a heterozygous parent, may also develop graft-versus-host disease.
  • immunocompetent donor cells for example, by in vitro manipulation of the donor tissue.
  • immunocompetent T cells may be removed from donor bone marrow through physical separation such as by lectin agglutination, or by treatment of the bone marrow with monoclonal antibodies directed to T cells.
  • use of bone marrow depleted of T cells is associated with a higher rate of graft failure, which is frequently fatal.
  • Use of T cell depleted bone marrow grafts is also associated with an increased incidence of relapse among the recipients, particularly recipients having chronic myelocytic leukemia.
  • Another approach to preventing immune-mediated injury is to interrupt the complement cascade (e.g., by depleting C3 with cobra venom factor or by inhibiting the C3 convertase with recombinant soluble CR1).
  • antibody depletion has unacceptable risks of over- immunosuppression (i.e. nfectjon), and experimental studies of inhibition of the complement cascade with cobra venom factor or sCR1 show incomplete inhibition.
  • An additional drawback to the use of cobra venom is the prospect of systemic effects due to the large amounts of vasoactive and chemotactic C3a and C5a produced.
  • immunosuppressive therapy Another common practice for inhibiting immune-mediated disorders is to subject the recipient to immunosuppressive therapy after transplantation. Such immunosuppression may occur by use of glucocorticoids, cyclosporin, methotrexate, or combinations of such drugs. However, immunosuppression also results in increased incidence of infection, and even when immunosuppressant drugs are used, immune-mediated cytotoxicity may still occur.
  • IL-11 demonstrates the ability to protect endothelial cells from immune-mediated injury.
  • Cells pretreated with IL-11 demonstrate a significant decrease in both cytotoxic T cells (CTL) and complement-mediated cytotoxicity over cells which are not pretreated with IL-11.
  • CTL cytotoxic T cells
  • IL-11 vascular endothelial cells
  • STAT transcription protein
  • MAPK mitogen activated protein kinase
  • graft versus host disease GVHD
  • non-immune-mediated necrotic injuries such as localized tissue or cell injury caused by loss of blood supply, corrosion, burning, or the local lesion of a disease.
  • IL-11 analogs, and derivatives thereof, are administered to patients, either prophylactically or at the onset of symptoms associated with the aforementioned disorders.
  • IL-11 can be administered in suitable pharmaceutically acceptable carriers either alone or in combination with other conventional agents useful in alleviating the symptoms associated with the aforementioned disorders.
  • the invention comprises a method of preventing an immune-mediated disease which comprises administering to a mammal, prior to exposure to foreign cell surface proteins, a therapeutically effective amount of interleukin-11.
  • the invention comprises a method of ameliorating an immune- mediated disease which comprises administering to a mammal, at the time of exposure to foreign cell surface proteins, a therapeutically effective amount of interleukin-11.
  • the invention comprises a method of treating an immune-mediated disease which comprises administering to a mammal experiencing an immune-mediated disease a therapeutically effective amount of interleukin-11.
  • the therapeutic dose is effective to prevent, ameliorate or treat an immune-mediated disease resulting from exposure to foreign cell surface proteins, such as donor major and/or minor histocompatibility antigens.
  • the therapeutically effective amount of interleukin-11 comprises 1 to 100 ⁇ g/kg body weight.
  • Methods are also provided for preventing non-immune mediated necrotic disorders which comprises administering to a mammal, prior to or at the time of local tissue injury, a therapeutically effective amount of interleukin-11.
  • the therapeutically effective amount of interleukin-11 comprises 1 to 100 ⁇ g/kg body weight.
  • the therapeutic dose is also effective to prevent, ameliorate or treat non-immune mediated disorders resulting from such injury.
  • FIGURES Figure 1 is a graphic illustration showing the results of the experiment described in Example 7.
  • Figure 2 is a graphic illustration showing the results of the experiment described in Example 8.
  • Figure 3 is a graphic illustration showing the results of the experiment described in Example 9.
  • graft-versus-host disease GVHD
  • endothelial cell EC
  • human umbilical vein endothelial cell HBVEC
  • interleukin-11 IL-11
  • rhlL-11 recombinant human IL-11
  • activators of transcription protein STAT
  • interleukin-12 IL-12
  • tumor necrosis factor TNF
  • mitogen-activated protein kinase MAPK
  • nuclear factor- ⁇ B nuclear factor- ⁇ B
  • NF- ⁇ B nuclear factor- ⁇ B
  • ICM-1 intracellular adhesion molecule-1
  • MHC major histocompatibility complex
  • CTL cytotoxic T lymphocytes
  • JK Janus kinase
  • P-STAT1 P-STAT1
  • P-STAT3 P-STAT3
  • kits for treating disorders where protection against CTL and/or complement-mediated cytotoxicity are shown to be beneficial including, without limitation, GVHD, and rejection of organ or tissue transplants.
  • methods of treating non-immune-mediated necrotic injuries such as localized tissue or cell injury caused by loss of blood supply, corrosion, burning, or the local lesion of a disease.
  • IL-11 is a stromal cell-derived pleiotropic cytokine which interacts with a variety of hematopoietic and non-hematopoietic cell types. Recombinant human IL-11 stimulates megakaryocytopoiesis in vitro and in vivo. Weich, N. S., et al. (1997) Blood 90:3893-3902; and Orazi, A., et al. (1996) Exp. Hematol. 24:1289-1297. IL-11 also stimulates erythropoiesis and regulates macrophage proliferation and differentiation, de Haan, G., et al. (1995) Br. J. Haematol. 90:783-790. Due to its thrombopoietic activities in vivo, IL-11 is used to treat chemotherapy-induced thrombocytopenia. Kaye, J. A. (1996) Curr. Opin. Hematol. 3:209-215.
  • IL-11 In addition to its hematopoietic effects, IL-11 also protects against various forms of mucosal epithelial cell injury. For example, IL-11 has been shown to protect small intestinal cells from combined radiation, chemotherapy, and ischemia (Du, X., et al. (1997) Am. J. Physiol. 272:G545- G552; Orazi, A., et al. (1996) Lab. Invest. 75:33-42; andKeith, J. C, Jr., et al. (1994) Stem. Cells. (Dayt). 1(12)79-89); reduce experimental colitis induced by trinitrobenzene sulfonic acid in rat (Qiu, B. S., etal.
  • IL-11 has also been shown to improve survival and decrease TNF production after radiation-induced thoracic injury. Redlich, C. A., et al. (1996) J. Immunol. 157:1705-1710. Human IL-11, expressed as atransgene in bronchial mucosa, reduces mortality associated with hyperoxia in mice. Waxman, A. B., et al. (1998) J. Clin. Invest. 101:1970-1982. This enhanced murine survival may result from reduced lung injury, including alveolar-capillary protein leak, endothelial and epithelial cell membrane injury, lipid peroxidation, pulmonary neutrophil recruitment, IL-12 and TNF production, and DNA fragmentation.
  • IL-11 protects mucosal membranes
  • IL-11's anti-inflammatory effects are believed to result, at least in part, from down-regulation of various proinflammatory cytokines. Leng, S. X. and J. A. Elias (1997) J. Immunol.159:2161 -2168; Trepicchio, W. L, et al. (1997) J. Immunol. 159:5661-5670; and Trepicchio, W. L, et al. (1996) J. Immunol. 157:3627-3634.
  • IL-11 may also cause immune deviation from a T H 1-like to a T H 2-like phenotype, thereby alleviating immune-mediated injury. Hill, supra.
  • IL-11 belongs to the interleukin-6 (IL-6) family of cytokines, all of which use gp130 as a critical component for signal transduction.
  • IL-6 interleukin-6
  • Taga, T. and T. Kishimoto (1997) Annu. Rev. Immunol. 15:797-819; Zhang, X. G., et al. (1994) J. Exp. Med. 179:1337-1342; and Yang, Y. C. and T. Yin (1995) Ann. N.Y. Acad. Sci. 762:31-40.
  • IL-11 initiates signaling via binding to a unique IL-11- receptor- ⁇ (IL-11R ⁇ ) chain. Nandurkar, H. H., et al.
  • Activated JAKs phosphorylate tyrosine residues within the cytopiasmic region of gp130 which then serve as docking sites for signal transducer and activators of transcription proteins, STAT3 and STAT1.
  • the activated JAKs subsequently phosphorylate tyrosine residues within the bound STAT proteins, causing the STATs to dissociate from gp130, dimerize, and enter the nucleus to act as transcriptional activators of target genes.
  • STAT dimers may be additionally phosphorylated on serine or threonine residues by mitogen activated protein kinases (MAPKs) that are also activated in response to cytokine binding to the receptor.
  • MAPKs mitogen activated protein kinases
  • IL-11 is described in detail in International Application PCT/US90/06803, published May 30, 1991; as well as in U.S. Patent No. 5,215,895; issued June 1, 1993.
  • a cloned human IL-11 was previously deposited with the ATCC, 10801 University Boulevard, Manassa, VA 20110-2209, on March 30, 1990 under ATCC No. 68284.
  • IL-11 may also be produced recombinantly as a fusion protein with another protein.
  • IL-11 can be produced in a variety of host cells by resort to now conventional genetic engineering techniques.
  • IL-11 can be obtained from various cell lines, for example, the human lung fibroblast cell line, MRC-5 (ATCC Accession No. CCL 171) and Paul et al., the human trophoblastic cell line, TPA30-1 (ATCC Accession No. CRL 1583). Described in Proc Natl Acad Sci USA 87:7512 (1990) is a cDNA encoding human IL-11 as well as the deduced amino acid sequence (amino acids 1 to 199).
  • U.S. Patent No. 5,292,646, supra describes a des-Pro form of IL-11 in which the N-terminal praline of the mature form of IL-11 (amino acids 22-199) has been removed (amino acids 23-199).
  • any form of IL-11 which retains IL-11 activity, is useful according to the present invention.
  • IL-11 may also be produced by known conventional chemical synthesis.
  • Methods for constructing the polypeptides useful in the present invention by synthetic means are known to those of skill in the art.
  • the synthetically constructed cytokine polypeptide sequences by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with the natural cytokine polypeptides are anticipated to possess biological activities in common therewith.
  • Such synthetically constructed cytokine polypeptide sequences or fragments thereof, which duplicate or partially duplicate the functionality thereof may also be used in the method of this invention. Thus, they may be employed as biologically active or immunological substitutes for the natural, purified cytokines useful in the present invention.
  • Modifications in the protein, peptide or DNA sequences of these cytokines or active fragments thereof may also produce proteins which may be employed in the methods of this invention.
  • Such modified cytokines can be made by one skilled in the art using known techniques.
  • Modifications of interest in the cytokine sequences, e.g., the IL-11 sequence may include the replacement, insertion or deletion of one or more selected amino acid residues in the coding sequences. Mutagenic techniques for such replacement insertion or deletion are well known to one skilled in the art. (See, e.g., U. S. Patent No. 4,518,584.)
  • sequences of the cytokine polypeptides which may be useful therapeutically as described herein may involve, e.g., the insertion of one or more glycosylation sites.
  • An asparagine-linked glycosylation recognition site can be inserted into the sequence by the deletion, substitution or addition of amino acids into the peptide sequence or nucleotides into the DNA sequence.
  • Such changes may be made at any site of the molecule that is modified by addition of O-linked carbohydrate. Expression of such altered nucleotide or peptide sequences produces variants which may be glycosylated at those sites.
  • Additional analogs and derivatives of the sequence of the selected cytokine which would be expected to retain or prolong its activity in whole or in part, and which are expected to be useful in the present method, may also be easily made by one of skill in the art.
  • One such modification may be the attachment of polyethylene glycol (PEG) onto existing lysine residues in the cytokine sequence or the insertion of one or more lysine residues or other amino acid residues that can react with PEG or PEG derivatives into the sequence by conventional techniques to enable the attachment of PEG moieties.
  • PEG polyethylene glycol
  • Additional analogs of these selected cytokines may also be characterized by allelic variations in the DNA sequences encoding them, or induced variations in the DNA sequences encoding them. It is anticipated that all analogs disclosed in the above-referenced publications, including those characterized by DNA sequences capable of hybridizing to the disclosed cytokine sequences under stringent hybridization conditions or non-stringent conditions (Sambrook et al., Molecular Cloning. A Laboratory Manual, 2d edit, Cold Spring Harbor Laboratory, New York (1989)) will be similarly useful in this invention.
  • fusion molecules prepared by fusing the sequence or a biologically active fragment of the sequence of one cytokine to another cytokine or proteinaceous therapeutic agent, e.g., IL-11 fused to IL-6 (see, e.g., methods for fusion described in PCT/US91/06186 (WO92/04455), published March 19, 1992).
  • IL-11 a biologically active fragment of the sequence of one cytokine
  • IL-6 e.g., IL-11 fused to IL-6
  • combinations of the cytokines may be administered together according to the method.
  • IL-11 encompasses the protein produced by the sequences presently disclosed in the art, as well as proteins characterized by the modifications described above yet which retain substantially similar activity. Standard laboratory tests are utilized to monitor progress of the treatment Decreased symptomatology could also be used to monitor the effectiveness of treatment as is well known to physicians skilled in the art of treating such disorders.
  • an immune-mediated disorder may result when a tissue transplant is donated by an individual whose genetic characteristics differ from those of the recipient, especially as regards the MHC and minor histocompatibility antigens expressed on the surfaces of each individual's cells. If the recipient's immune system recognizes the donor's cells as non-self, the recipient's immune system will destroy the donor's cells.
  • An immune-mediated disorder or disease may also result from immunologic recognition in the opposite direction, that is, when the donor tissue contains immunologically competent cells which proceed to destroy the recipient's cells, such as in GVHD.
  • the present invention also contemplates the administration of a therapeutically effective amount of IL-11 to a mammal at risk of developing a non-immune-mediated disease or disorder.
  • non-immune-mediated disorder and “non-immune-mediated disease” are used interchangeably to refer to a condition characterized by necrotic injury, such as the localized tissue or cell injury caused by loss of blood supply, corrosion, burning, or the local lesion of a disease.
  • the term "complement” means a complex group of proteins and glycoproteins found in the blood of vertebrates. These proteins function in the production of inflammation, opsonize foreign materials for phagocytosis, and mediate direct cytotoxicity against various cells.
  • Complement action against cells proceeds by activation of a protease called C3 convertase via one of two pathways: the classic pathway, where binding to an antigen-antibody complex involving IgG or IgM activates C1 which cleaves C2 and C4 to produce a protease that activates C3 by cleaving it to produce C3b; or the alternative pathway, where C3b is produced by a C3 converting protease formed from other complement factors, including Factors B, D, and P, activated by other activators, such as bacterial endotoxin, certain polysaccharides or complexes of antigen with other antibodies.
  • C3 convertase via one of two pathways: the classic pathway, where binding to an antigen-antibody complex involving IgG or IgM activates C1 which cleaves C2 and C4 to produce a protease that activates C3 by cleaving it to produce C3b; or the alternative pathway, where C3b is produced by a C3
  • C3b in complex with activated C2 and C4 or with activated Factor B and P, cleaves C5 to produce C5b which combines sequentially with C6, C7, C8, and C9 to form the "membrane attack complex” (MAC) that is capable of damaging biological membranes.
  • MAC membrane attack complex
  • tissue means an aggregate of mammalian cells which may constitute a solid mass or a suspension of cells, such as blood cells, or a mammalian cell line.
  • the term 'therapeutically effective amount means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e.g., a reduction in the incidence or severity of acute or chronic graft- versus-host disease compared to that expected for a comparable group of patients not receiving interleukin-11, as determined by the attending physician.
  • a meaningful patient benefit e.g., a reduction in the incidence or severity of acute or chronic graft- versus-host disease compared to that expected for a comparable group of patients not receiving interleukin-11, as determined by the attending physician.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
  • a therapeutically effective amount of IL-11 is administered to a mammal at risk of developing an immune-mediated disorder.
  • the IL-11 may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing T cell-depleted autologous or syngeneic bone marrow, immunosuppressive drugs, cytokines, lymphokines, or other hematopoietjc factors.
  • the IL-11 When co-administered with T-cell-depleted autologous or syngeneic bone marrow, immunosuppressive drugs, one or more cytokines, lymphokines, or other hematopoietjc factors, the IL-11 may be administered either simultaneously with the T-cell-depleted autologous or syngeneic bone marrow, immunosuppressive drugs, cytokine(s), lymphokine(s), other hematopoietic factor(s), or sequentially.
  • the attending physician will decide on the appropriate sequence of administering the IL-11 in combination with the T-cell depleted autologous or syngeneic bone marrow, immunosuppressive drugs, cytokine(s), lymphokine(s), and other hematopoietjc factor(s).
  • Interleukin-11 used to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous or subcutaneous administration to the patient is preferred.
  • the interleukin-11 When a therapeutically effective amount of interleukin-11 is administered orally, the interleukin-11 will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule and powder contain from about five to 95% inte ⁇ ieukin-11 , preferably from about 25-90% interleukin-11.
  • a liquid carrier such as water, petroleum, oils of animal or plant origins such as peanut oil, mineral oil, soy bean oil, or sesame oil, or synthetic oils, may be added.
  • the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose, or other saccharide solutions, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains about 0.5 to 90% by weight of interleukin-11 and preferably from about 1 to 50% interleukin-11.
  • interleukin-11 When a therapeutically effective amount of interleukin-11 is administered by intravenous, cutaneous or subcutaneous injection, the interleukin-11 will be in the form of a pyrogen-free, parenterally-acceptable aqueous solution.
  • parenterally-acceptable protein solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to interleukin-11, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art
  • the pharmaceutical composition for use in the present method may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those with skill in the art. It is contemplated that the pharmaceutical composition used to practice the method of the present invention should contain about 0.1 pg to about 100 mg of interleukin-11 per ml of solution, preferably about 0.1 mg of interleukin-11 per ml of solution.
  • the duration of the application of IL-11 will be in the range of 12-48 hours of continuous or intermittent subcutaneous or intravenous administration, beginning prior to or at the time of tissue transplantation.
  • "at the time of tissue transplantation” is defined as being during the 1 hour period before or the 1 to 24 hour period after the transplantation.
  • IL-11 is administered beginning at least 24 hours prior to the time of tissue transplantation.
  • a method for preventing or ameliorating graft-versus-host disease preferably 1 ⁇ g/kg to 100 ⁇ g/kg of IL-11 may be administered daily to the mammal, more preferably 1 ⁇ g/kg to 75 ⁇ g/kg of IL-11 may be administered daily to the mammal, and most preferably 1 ⁇ g/kg to 15 ⁇ g/kg may be administered daily to the mammal.
  • the first dose of IL-11 is given one hour after tissue transplantation and two more doses are given on days one and two post- transplant
  • Alternative treatment regimens may be appropriate for individual patients and will be determined by the attending physician, taking into account the nature and severity of the condition being treated, and the nature of the prior treatments which the patient has undergone.
  • Modifications of the treatment regimen set forth above for prevention or ameliorating an immune-mediated disease may be made for treatment of ongoing acute or chronic disease.
  • acute disease is defined as occurring during the time period from three days to 100 days post transplantation in humans; and "chronic" disease is defined as occurring at any time after 100 days post-transplantation in humans.
  • chronic disease is defined as occurring at any time after 100 days post-transplantation in humans.
  • 1 ⁇ g/kg to 100 ⁇ g/kg may be administered daily to a mammal experiencing acute or chronic graft-versus-host disease, until improvement or remission of the symptoms of acute or chronic graft-versus-host disease is observed.
  • the attending physician will decide on the appropriate duration of subcutaneous or intravenous therapy using the pharmaceutical composition of IL-11 in the method of the present invention.
  • IL-11 has a direct effect on vascular EC.
  • Cultured HUVECs express IL-11 R ⁇ as well as gp130, and stimulation of HUVECs with IL-11 induces rapid phosphorylation of gp130, STAT3, STAT1, and p42/p44 MAPKs.
  • IL-11 pretreatment induces HUVECs to become resistant to injury mediated by CTL or by antibody plus complement.
  • IL-11 does not inhibit proinflammatory response of HUVECs to TNF, such as NF- ⁇ B activation or adhesion molecule expression.
  • IL-6 Functional receptor complexes for IL-6 family of cytokines including IL-11, oncostatin M, and IL-6 share gp130 as a component critical for signal transduction.
  • IL-11 binds to IL-11R ⁇ chain on the cell surface, and IL-11/IL-11R complex then associate with gp130, causing it cluster. This is essentially the same mechanism of action that has been observed for IL- 6/IL-6R signaling.
  • Oncostatin M differs from IL-11 and IL-6 in that it directly binds to gp130 and signals through either gp130/leukemia inhibitory factor receptor ⁇ or gp130/oncostatin M receptor heterodimers. Renne, C, et al. (1998) J. Biol. Chem. 273:27213-27219; and Auguste, P., et al. (1997). Because gp130 is ubiquitously expressed, the responsiveness of cells to a particular cytokine of IL-6 family is determined by the relative expression of other receptor component. Among receptors for IL-6 family of cytokines, endothelial cells lack IL-6R ⁇ chain (Romano, M., et al. (1997) Immunity.
  • oncostatin M This greater potency of oncostatin M may also explain why oncostatin M but not IL-11 appears to activate pro-inflammatory functions of HUVECs. Modur, V., et al. (1997) J. Clin. Invest. 100:158-168; Yao, L, et al. (1996) J. Exp. Med. 184:81-92. It also does not appear to be mitogen for HUVECs (unpublished observations). Thus, not all gp130 signaling cytokines induce the same biological responses in EC, and some differences may relate to signal strength.
  • cytoprotection of HUVECs is protein-synthesis dependent.
  • IL-11 induces new or increases expression of a cytoprotective protein.
  • Activation of transcription factors STAT and MAPKs may play a role in the induction of these cytoprotective proteins.
  • concentrations of IL-11 that confer the most significant cytoprotection activate STAT3 and MAPKs but not STAT1 in HUVECs.
  • low doses of IL-11 produce primarily STAT3 heterodimers which translocate into the nucleus and mediate the cytoprotective effects of IL-11.
  • STAT1 activation by high dose of IL-11 may antagonize the IL-11 -mediated cytoprotection, by formation of STAT3/STAT1 heterodimes.
  • Cytotoxic CD8 + T lymphocytes can recognize and kill appropriate allogenic targets by direct contact. Shresta, S., et al. (1998) Curr. Opin. Immunol. 10:581-587.
  • Target cell killing by CTL involves specific recognition of Class I MHC-peptide complex on the target cell by T cell receptor. LFA3 and ICAM-1 or ICAM-2 expressed on the target cells facilitate CTL-target cell recognition by enhancing cell-cell contact. CTL can not efficiently recognize and kill target cells which do not express class I MHC, LFA3, or ICAM-1. As shown in the Examples below, IL-11 significantly protects HUVECs against lysis by allospecific CTLs without effecting the expression of either class I MHC or ICAM-1 on these cells.
  • LFA3 levels are also unaffected (unpublished observations). Thus the mechanism of protection probably follows conjugate formation. Bound CTL deliver a lethal hit by two different mechanisms: granule exocytosis, also known as the perforin/granzyme B pathway, and Fas/Fas ligand (FasL) signaling. Shresta, S., et al. (1998) Curr. Opin. Immunol. 10:581-587. HUVECs can not be killed through Fas engagement by Fas ligand. Richardson, B.C., (1994) Eur. J. Immunol. 24:2640-2645; unpublished observations.
  • Recombinant human IL-11, neutralizing antibody (Ab) to IL-11, and anti-IL-11 receptor ⁇ chain Ab were provided by Genetics Institute (Andover, MA).
  • Recombinant human oncostatin M and recombinant human IFN- ⁇ were purchased from R&D Systems (Minneapolis, MN).
  • Anti-human gp130 and anti-phosphotyrosine Abs were purchased from Upstate Biotechnology (Lake Placid, NY) and anti-l ⁇ B ⁇ Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Anti-class I MHC antibody (W6/32) was prepared as ascites in our laboratory from a clone provided by Dr. Jack Strominger (Harvard University, Cambridge, MA), and FITC- conjugated anti-human class I MHC mAb (W6/32) was purchased from Serotech (Oxford, England).
  • Mouse anti-human-E-selectin mAb (clone H14/18) and non-blocking (K16/16) Ab were made as ascites in our laboratory.
  • Mouse anti-human-ICAM1 mAb (E16) was a gift from Dr. Dario Altieri (Yale University, New Haven, CT).
  • FITC-conjugated goat anti-mouse Ab was purchased from Boehringer Mannheim (Indianapolis, IN).
  • Baby rabbit complement was purchased from Pel-Freez (Brown Dear, Wl) Cell isolation:
  • K562 cells were obtained from the American Type Culture Collection (Rockville, MD, Accession number: CCL-243) and cultured at 37EC in 5% C0 2 -humidified air in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FBS, 2 mM L- glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • RPA Ribonuclease Protection Assay
  • GAPDH glyceraldehyde phosphate dehydrogenase
  • lysates were clarified by centrifugation at 10,000 x g for 15 min, and protein concentrations of the supernatant were determined by using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Lysates were prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by adding an equal volume of 2X SDS-PAGE sample buffer (100 mM Tris-CI, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and heating the mixture in a boiling water bath for 3 min.
  • 2X SDS-PAGE sample buffer 100 mM Tris-CI, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol
  • HRP horse raddish peroxidase
  • ECL enhanced chemiluminescence
  • HUVECs were suspended by washing with Hanks Buffered Saline Solution and incubated for 1 min with Trypsin/EDTA. Detached cells were collected and washed twice with ice cold PBS containing 1% BSA, and incubated with specific primary mAb (either anti-E-selectin, anti-ICAM1, or FITC-conjugated anti-class 1 MHC) for 30 min at 4EC. Replicate aliquots were incubated with non-binding isotype control mAb. Labeled cells were washed twice with PBS/1% BSA were fixed with 2% paraformaldehyde before being analyzed.
  • primary mAb either anti-E-selectin, anti-ICAM1, or FITC-conjugated anti-class 1 MHC
  • Transient transfection of HUVECs were performed using a DEAE-dextran protocol as described previously.
  • Karmann, K., et al. (1996) J. Exp. Med. 184:173-182.
  • Cell were transfected with both a ⁇ B-luciferase promoter reporter gene, which contains two KB sites from Ig kappa enhancer (Min, W., et al. (1996) Mol. Cell Biol. 16:359-368) and a constitutively active ⁇ - galactosidase expression construct (Promega, Madison, Wl).
  • Cell lysates were assayed for luciferase and ⁇ -galactosidase activities using Promega reporter assay system (Promega, Madison, Wl).
  • Luciferase activity was measured using a Berthold (Schwarzwald, Germany) model LB9501 luminometer, and ⁇ -galactosidase was measured spectrophotometrically (at 420 nm). Luciferase values in relative light units were normalized to ⁇ -galactosidase units to control for transfection efficiency.
  • Complement mediated lysis :
  • Target HUVECs were grown in 96-well plates to confluence and incubated with 20 ⁇ M calcein-AM (Molecular Probes, Eugene, OR) in Medium 199 and 5 mM HEPES for 30 min at 37 EC. The medium was replaced by complete EC growth medium and cells were rested overnight. IL-11 in media or media alone were added for an addition of 6 h, after which cells were washed twice with Medium 199, 5% FBS, 5 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin. Where indicated, 20 ⁇ g/ml of cycloheximide were included during the incubation with IL-11.
  • 20 ⁇ g/ml of cycloheximide were included during the incubation with IL-11.
  • Percent specific lysis was calculated as [(sample release- spontaneous release)/(maximal release -spontaneous release)] X 100%.
  • Percent cytoprotection was calculated as [(percent specific lysis in the present of IL-11/percent specific lysis in the absence of IL-11) X 100. Spontaneous release was generally ⁇ 25%.
  • Alloreactive class l-restricted CD8 + T cells lines were produced as described elsewhere from peripheral blood CD8 + T cells. Biedermann, B.C. and J.S. Pober (1998) [In Process Citation]. J. Immunol. 161 :4679-4687. To determine percent lysis, target HUVECs were loaded calcein as described above and incubated overnight. IL-11 in media or media alone were added for an addition of 6 h, after which cells were washed twice with Medium 199, 5% FBS, 5 mM HEPES, 2 mM L- glutamine, 100 u/ml penicillin, 100 ⁇ g/ml streptomycin.
  • Effector CTL cells were added in a total volume of 150 ⁇ l/well at a predetermined effector/target ratio of 30:1.
  • Replicate wells were incubated with lysis buffer (50 mM sodium borate, 0.1% Triton X-100, pH 9.0) to determine maximum release or without CTL to determine spontaneous release. After 4 h incubation at 37EC, released calcein was measured as described above. Percent cytoprotection was calculated as described above. Spontaneous release was generally ⁇ 25%.
  • RNA from cultured HUVECs, CACO-2 or K562 cells was incubated over night with probes for human IL-11R ⁇ chain and GAPDH genes.
  • Samples were digested with RnbaseA/T1 and protected fragments (321 bp for IL-11 R ⁇ and 96 bp for GAPDH were resolved on a 6% acylamide/TBE-Urea gel. Lysates from either HUVECs or K562 cells were resolved on SDS-PAGE as described above and immunoblotted with specific antibody to IL-11 receptor ⁇ chain. The IL-11 receptor ⁇ chain was detected in HUVECs and K562 cells at about MW 83 kD.
  • HUVECs were either untreated (control), treated with IL-11 (100 ng/ml), or oncostatin M (20 ng/ml) for 2 min and 10 min.
  • Cell lysates were immunoprecipitated with specific antibody to gp130. Immune complexes were resolved on SDS-PAGE and immunoblotted with a phosphotyrosine specific antibody as described above. The results indicate that IL-11 tyrosine phosphorylates gp130 in HUVECs.
  • HUVECs were untreated (control), treated with increasing concentrations of IL-11 (0.1, 0.3, 1.0, 3.0, 10.0, 30.0, 90.0, 270.0 ng ml) or treated with increasing concentrations of oncostatin M (60, 125, 250, 500, 1000 ng/ml) for 10 min. Lysates were resolved on SDS-PAGE and immunoblotted with specific antibody to either phospho- STAT3 (P-STAT3), STAT3, phospho-STAT1 (P-STAT1), or STAT1. The results indicate that IL-11 exhibits a dose-dependent phosphorylation of STAT3 and STAT1 in HUVECs.
  • HUVECs were either untreated (control), treated with IL-11 (100 ng/ml), or treated with oncostatin M (200 pg/ml) for 2.5 min, 5 min, 10 min 20 min, 40 min and 60 min. Lysates were resolved on SDS-PAGE and immunoblotted with specific antibody to either phospho-STAT3 (P-STAT3) or phospho-STAT1 (P-STAT1). The results indicated that IL-11 phosphorylation is time-dependent.
  • HUVECs were cultured in a M199 media containing 1 % FCS for 17 h, after which the media was removed and cells were rested for 2 h in the M199 media containing no FCS.
  • HUVECs were either untreated (control) or treated with increasing concentrations of IL-11 (0.3, 1.0, 10.0, 30.0, 90.0, 270.0, 400.0 ng/ml) for 10 min.
  • HUVECs were either untreated (control) or treated with IL-11 (200 ng/ml) for 5 min, 15 min, 30 min and 40 min, or treated with oncostatin M (OnM) (1 ng/ml) for 40 min. Lysates were resolved on SDS-PAGE and immunoblotted with specific antibody to either phospho-p44/p44 antibody (P-42/44) or p42/p44 antibody (p42/p44). The results indicate that IL-11 had the ability to phosphorylate p43 and p44 MAP kinases in a dose- and time-dependent manner.
  • HUVECs were treated with either 10 ng/ml or 100 ng/ml of IL-11 or with 10 U/ml TNF for 5 and 15 min. Other HUVECs were pretreated with media alone (control) or media containing various doses of IL-11 (0.5, 50, 250, 500 or 1000 ng/ml). After 4 h, cells were stimulated without or with TNF (10 U/ml) for 15 min. Lysates were resolved on 10 % SDS-PAGE and immunoblotted with specific antibody to l ⁇ B ⁇ .
  • Example 2 The IL- 11 receptor alpha chain is expressed on B and T lymphocytes.
  • Murine B Cells and CD4+ and CD8+ T cells were purified from Balb/c spleens using positive selection with MACS (Magnetic Cell Sorting) Microbeads conjugated to anti-mouse B220, CD4 or CD8 antibodies, as per manufacturers protocol (Miltenyi Biotec, Sunnyvale, CA).
  • RNA was extracted from purified cell populations as described above using RNA Stat-60 (Tel-test, Inc., TX) as per manufacturer's protocol.
  • RNA was DNase treated (RQ1 DNase, Promega, Madison, Wl) for 30 min at 37°C, then heat inactivated for 5 min at 75°C.
  • RT-PCR was performed (GeneAmp RNA PCR Kit, Perkin Elmer) using 40ng RNA (10ng for GAPDH) and oligo pairs specific for murine GAPDH, IL-11 receptor a chain, IL-10 receptor, IL-6 receptor or gp130, and visualized on an ethidium-stained agarose gel. PCR reactions on RNA samples in the absence of reverse transcriptase were negative for each oligo pair, and served as a control for DNA contamination. The IL-11 receptor alpha chain mRNA was detected in highly purified populations of CD4+, CD8+ and B220+ lymphocytes.
  • Example 3 IL-11 1ncreases Antigen Specific Cytolytic Activity in Bulk Spleen Cultures.
  • % specific lysis (% lysis on NP peptide pulsed target cells)-(% lysis of control target cells).
  • the example indicates that IL-11 can significantly enhance the % specific lytic activity of NP peptide-specific cytotoxic T cells.
  • a 250% enhancement in % specific lysis is observed in cells treated with IL-2 and IL-11 versus cells treated with IL-2 alone.
  • Example 5 IL-11 Increases the Number of Influenza Specific IFN ⁇ - Producing Cells.
  • Sterile flat bottom 96 well plates (Costar) were coated with an anti-IFN ⁇ antibody (R46A2, 10 Fg/ml, 50 ul/well) at 37°C for 2 hrs and blocked with PBS+1 %BSA-+ .05% Tween 20 at 37°C for 1 hour and washed with PBS.
  • Serial dilutions of splenic cells diluted in RPMI+10% FCS were added to the plates and incubated for 16 hr at 37°C, and then washed 6 times.
  • Biotinylated anti-IFNy antibody XGM.1 (1.19 Fg/ml) was added to each well and incubated 90 min at room temperature.
  • Example 6 IL-11 1ncreases IFN ⁇ Production from Influenza Specific CTLs.
  • ELISA plates EIA capture plates, Costar
  • an anti-IFNy antibody R46A2, 3 Fg/ml, 50 ul/well
  • THSG Tris high salt gelatin
  • Serial dilutions of IFN ⁇ control supematants and of unknown samples diluted in PBS+10% FCS were added to the plates and incubated for 2 hr at room temperature, and then washed 4 times.
  • Biotinylated antibody XGM.1 (1.19 Fg/ml) was added to each well and incubated 90 min at room temperature.
  • IL-11 can enhance the amount of IFN-g produced from influenza-specific cytotoxic T cells. 2-4 fold elevated levels of IFN-g were detected in the conditioned medium of cells treated with IL-2 and either 10 ng/ml or 100 ng/ml IL-11.
  • Example 7 IL-11 does not inhibit TNF-mediated ⁇ B-luciferase promoter-reporter gene activity.
  • HUVECs were transiently cotransfected with ⁇ B-luciferase promoter-reporter gene and a ⁇ -galactosidase expression construct.
  • Cell were treated with different doses of IL-11 as indicated in Figure 1.
  • IL-11 As indicated in Figure 1.
  • Luciferase activity was expressed as light units normalized to ⁇ -galactosidase activity. Data are presented as the mean • +/- s.d. of triplicate in each group from one experiment. Shown in Figure 1 is one of the three different experiments with similar outcome.
  • Example 8 IL-11 treated HUVECs acquire resistance against complement-mediated cytolysis.
  • HUVECs Confluent pooled HUVECs were loaded with calcein and treated 6 h with IL-11 at the indicated concentrations. HUVECs were washed and incubated with anti-W6/32 Ab (2.5 ⁇ g/ml) followed by addition of baby rabbit complement (25 %). After 1 h, supernatant was harvested and released calcein was measured. Percent cytoprotection was calculated as described in Material and
  • Example 9 Cytoprotection of HUVECs by pretreatment with IL-11 is protein synthesis dependent.
  • HUVECs were loaded with calcein, chased and pretreated for 6 hours without (open circles) or with 0.5 ng/ml IL-11 (closed circles) in the presence (panel B) or absence (panel A) of cycloheximide.
  • Cells were washed and incubated with W6/32 (2.5 ⁇ g/ml) for 30 min before complement was added at the indicated concentrations. After 1 h, supernatant was harvested and calcein release measured. Percent specific lysis was calculated (see Material and Methods). Maximal and spontaneous release was significantly different for cy cloheximide treated cells and controls. These values were therefore determined for the 2 groups separately, and sample release was normalized to these values. Shown is one of the three different experiments with similar outcome.
  • Example 10 Effect of IL-11 treatment on E-selectin and ICAM-1 expression.
  • HUVECs were treated with either TNF or IL-11 in the amounts indicated in Table 1.
  • Surface levels of E-selectin and ICAM-1 were quantified by indirect immunofluorescence using H4/18 and E16 antibody, respectively. The results are shown in Table 1.
  • the numbers are expressed as corrected mean fluorescence intensity (cMFl) calculated by subtracting the mean fluorescence value for isotype match control antibody (k16/16) from the mean fluorsescence value for either H4/18 or E16 antibody. Results from 2 independent experiments are shown. 11-11 had very little effect on E-selectin or ICAM-1 expression.
  • Example 11 Effect of IL-11 treatment on CTL-mediated cytolysis.
  • Example 12 Effect of IL-11 on class I MHC expression.
  • HUVECs were trypsinized and stained for class I MHC. The results are shown in Table 3. Results from 2 separate experiments are shown. IL-11 does not affect class I MHC expression on HUVECs.
  • Interleukin-11 prevents apoptosis and accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation. Lab. Invest. 75:33-42.
  • IL-11 a pleiotropic cytokine: exciting new effects of IL-11 on gastrointestinal mucosal biology. Stem. Cells. (Dayt). 12 Suppl 1:79-89.
  • Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. J. Exp. Med. 179:1337-1342.
  • Stat3 a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264:95- 98.
  • Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules. J. Clin. Invest. 100:158-168.
  • Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase. J. Immunol. 149:1676-1682.

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Abstract

l'utilisation de l'interleukine 11 pour prévenir, améliorer et traiter une maladie d'origine immunologique chez un mammifère nécessitant un tel traitement.
PCT/US2000/006369 1999-03-11 2000-03-10 Utilisation de l'interleukine 11 pour prevenir une cytotoxicite d'origine immunologique WO2000053214A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854028A (en) * 1989-11-22 1998-12-29 Genetics Institute, Inc. Compositions comprising IL-11 and methods of making and using IL-11

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5854028A (en) * 1989-11-22 1998-12-29 Genetics Institute, Inc. Compositions comprising IL-11 and methods of making and using IL-11

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