EP0681589A1 - Utilisation d'une composition pharmaceutique comprenant une dose efficace d'interleukine-10, d'un analogue et/ou d'un agoniste d'interleukine-10 - Google Patents

Utilisation d'une composition pharmaceutique comprenant une dose efficace d'interleukine-10, d'un analogue et/ou d'un agoniste d'interleukine-10

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Publication number
EP0681589A1
EP0681589A1 EP94906191A EP94906191A EP0681589A1 EP 0681589 A1 EP0681589 A1 EP 0681589A1 EP 94906191 A EP94906191 A EP 94906191A EP 94906191 A EP94906191 A EP 94906191A EP 0681589 A1 EP0681589 A1 EP 0681589A1
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Prior art keywords
cells
medicament
prevention
diseases
manufacture
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EP94906191A
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German (de)
English (en)
Inventor
Michel Goldman
Thierry Velu
Daniel Abramowicz
Catherine Bruyns
Paul Capel
Anne Delvaux
Vincent Donckier
Catherine Gerard
Arnaud Marchant
Olivier Pradier
Liliane Schandene
Fabienne Willems
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Universite Libre de Bruxelles ULB
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Universite Libre de Bruxelles ULB
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Priority to EP94906191A priority Critical patent/EP0681589A1/fr
Publication of EP0681589A1 publication Critical patent/EP0681589A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • 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]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention concerns the use of a pharmaceutical composition comprising an effective amount of interleukin-10, an analog and/or an agonist of interleukin-10, for the manufacture of a medicament intended for treating mammals, particularly humans, in order to cure or prevent a disease chosen among the group consisting of the diseases induced by an excessive amount of interleukin-5, an excessive T-cell stimulation through B7/CD28 and/or ICAM-1/LFA-I molecular interactions and/or an excessive monocyte procoagulant activity.
  • a pharmaceutical composition comprising an effective amount of interleukin-10, an analog and/or an agonist of interleukin-10, for the manufacture of a medicament intended for treating mammals, particularly humans, in order to cure or prevent a disease chosen among the group consisting of the diseases induced by an excessive amount of interleukin-5, an excessive T-cell stimulation through B7/CD28 and/or ICAM-1/LFA-I molecular interactions and/or an excessive monocyte procoagulant activity.
  • Human IL-10 is a cytokine which, in its mature form, has 160 amino acids and contains 2 intramolecular disulphide bridges. It also contains a glycosylation site, but is not glycosylated.
  • IL-10 is produced in the body by a number of different cell types.
  • Cells involved in the immune system namely lymphocytes T and B and monocytes/ macrophages represent important sources of IL-10.
  • keratinocytes and certain tumour cells are also capable of producing IL-10.
  • the factors controlling IL-10 synthesis are not completely understood.
  • IL-10 originally known as Cytokine Synthesis Inhibitory Factor (CSIF) . IL-10, originally known as Cytokine Synthesis Inhibitory Factor (CSIF) . IL-10, originally known as Cytokine Synthesis Inhibitory Factor (CSIF) . IL-10, originally known as Cytokine Synthesis Inhibitory Factor (CSIF) . IL-10, originally known as Cytokine Synthesis Inhibitory Factor (CSIF) . IL-10, originally known as Cytokine Synthesis Inhibitory Factor (CSIF) .
  • CIF Cytokine Synthesis Inhibitory Factor
  • IL-10 TH1 cytokine production
  • APC Antigen Presenting Cells
  • IL-10 acts mainly indirectly on T-lymphocytes by interfering with the activation signals presented to them by the APC. It is thought that inhibition of the expression of the Class II Major Histocompatibility Complex (MHC) on the APC surface may be one of the mechanisms involved.
  • MHC Class II Major Histocompatibility Complex
  • IL-10 as a mammalian cytokine inhibiting TH1 lymphocytes, controlling cell-mediated immune responses.
  • CCF IL-10
  • IL-10 for suppressing Graft-vs-Host disease or tissue rejection.
  • the main mechanism of action of IL-10 in this setting is described as being the down-regulation of MHC Class II expression on monocytes.
  • Down-regulation of ICAM-1 and B7 expression on monocytes is explicitly ruled out in this document as a mechanism of action of IL-10. Consequently, the use of IL-10 in diseases involving activation of T- lymphocytes by the B7/CD28 and ICAM-l/LFA-1 pathway are not described or suggested.
  • IL-10 International Patent Application WO 93/02693, published on 18th February 1993, (prior art according to A.54 (3) EPC) describes the use of IL-10 or an analog or agonist or antogonist thereof, for the treatment or prevention of Septic Shock and Toxic Shock.
  • IL-10 is administered either simultaneously with, or after, exposure to the LPS or superantigen. No suggestion is made that administration of IL-10 prior to endotoxin or superantigen exposure can prevent morbidity and mortality associated with gram-negative or gram- positive sepsis.
  • compositions suitable for use in the treatment of acute or chronic inflammation which comprise IL-10 in association with IL-4. Use of IL-10 alone is not described.
  • the technical problem underlying the present invention was : i) to identify an efficient means of treating and preventing septic shock, preferably before exposure to endotoxin or superantigen ; ii) to identify a treatment for diseases involving activation of the coagulation system by induction of tissue factor expression at the surface of monocytes ; iii) to identify, in clinical situations involving alloreactive responses, a means of blocking all T-lymphocyte activation pathways to allow treatment of patients presenting signs of rejection resistant to standard treatments, and ; iv) to identify a treatment for diseases involving excessive IL-5 production by T-lymphocytes.
  • IL-10 is an efficient inhibitor of TNF- ⁇ production by LPS-stimulated monocytes ; ii) IL-10 prevents the induction of monocyte procoagulant activity ; iii) IL-10 inhibits B7 and ICAM-1 expression on monocytes, and ; iv) IL-10 inhibits IL-5 production by T- lymphocytes.
  • the invention relates to pharmaceutical compositions comprising an effective amount of IL-10, an analog or an agonist of IL-10, for the treatment of conditions involving septic shock, over-production of IL-5 by T-lymphocytes ; excessive T-cell stimulation by B7/CD28 or ICAM-l/LFA-1 interactions or monocyte procoagulant activity.
  • the invention also relates to methods of preventing or controlling or suppressing these conditions in an individual, said method comprising the step of administering to the individual the above-described pharmaceutical compositions.
  • IL-10 is meant pure mammalian, particularly human, interleukin 10 as purified from natural sources or preferably as produced by expression in a suitable host of a recombinant DNA sequence encoding interleukin 10.
  • the IL-10 of this invention also includes the viral form of interleukin 10, chimeric proteins composed of sequences of human and viral IL-10 origin, provided that the properties of IL-10 as required by the present invention are maintained.
  • IL-10 also englobes analogs and peptides of interleukin 10 as described, for example, in PCT publication WO 91/00349 as a cytokine synthesis inhibitory factor.
  • Retroviral vectors are introduced in mammalian cells (for example, CHO cells) .
  • the recombinant proteins obtained in the supernatant is similar to the native protein expressed in mammals.
  • retroviral particles carrying the IL-10 gene are produced in the supernatant of packaging cells transfected by these constructions. These particles can be used to infect mammalian cells, and thereby to induce the expression of IL-10 by these cells.
  • Beculoviral vectors are used to produce larger quantities of recombinant protein, compared to retroviral vectors. Although this system of expression yields recombinant protein with glycosylation and disulfide-bridge, it induces altered posttranslational modifications and different glycosylations.
  • IL-10 an amount sufficient at least to ameliorate or prevent a symptom of one of the aforementioned conditions.
  • the effective amount for a particular patient may vary depending on such factors as the state of the condition being treated, the overall health of the patient, the method of administration, the severity of side-effects, and the like.
  • Analogs and/or agonists of IL-10 may be molecules which mimic IL-10 interaction with its receptors. Such may be analogs or fragments of IL-10, or antibodies against ligand binding side epitopes of the IL-10 receptors, or anti-idiotypic antibodies against particular antibodies which bind to receptor- interacting portions of IL-10.
  • Antibodies can be raised to the IL-10 cytokine, fragments, and analogs, both in their naturally occuring forms and in their recombinant forms. Additionally, antibodies can be raised to IL-10 in either its active forms or in its inactive forms, the difference being that antibodies to the active cytokine are more likely to recognize epitopes which are only present in the active conformation. Anti- idiotypic antibodies are also contemplated in these methods, and could be potential IL-10 agonists.
  • IL-10 is administered as a pharmaceutical composition comprising an effect amount of IL-10 and a pharmaceutical carrier.
  • a pharmaceutical carrier can be any compatible non-toxic substance suitable for delivering the compositions of the invention to a patient.
  • compositions useful for parenteral administration of such drugs are well known, see e.g. : Remington's Pharmaceutical Science, 15th Ed. (Mack Publishing Company, Easton, PA 1980, U.S.A.) .
  • compositions of the invention can be introduced into a patient's body by implantable or injectable drug delivery system, see e.g. : URQUHART et al. (1984) Ann. Rev. Pharmacol. Toxicol . 24 ; 199-236, Lewis, ed. Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981) ; U.S. patent 3,773,919, and U.S. patent 3,270,960,
  • the IL-10 When administered parenterally, the IL-10 is formulated in a unit dosage injection form (e.g., solution, suspension or emulsion) in association with a pharmaceutical carrier.
  • a pharmaceutical carrier examples include normal saline, Ringer's solution, dextrose solution, and Hank's solution.
  • Nonaqueous carriers such as fixed oils and ethyl oleate, can also be used.
  • a preferred carrier is 5 % dextrose/saline.
  • the carrier can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g. , buffers and preservatives.
  • the IL-10 is preferably formulated in a purified form, substantially free of aggregates and other proteins, at a concentration in the range of about 10,000 - 100,000 U/ml .
  • a standard unit [U] of activity is the amount of IL-10 necessary to produce a half-maximal response by the MC/9 mast cell line rTHOMPSON-SNIPES et al (1991) J. of Exp. Med. 173:507-5101.
  • the IL-10 can be administered in most cases by continuous infusion to the patient, so that an amount in the range of about 100,000 - 100,000,000 U is delivered per day (i.e., about 1,500 - 150,000 U/kg/day) .
  • the daily infusion rate can be varied based on monitoring of side-effects and blood cell counts of the patent. However, in some cases (e. g. , for preventing side- effects from anti-CD3 treatment) , 11-10 is preferably given as a single injection of about 50,000 5,000,000 U.
  • IL-10 administration for the prevention and/or treatment of allograft rejection in accordance with this invention, it is possible to use either IL-10 administration, incubation of cells with IL-10, or IL-10 gene transfer. It is believed that such gene transfer can be carried out in a conventional manner, preferably in one of the following ways:
  • the gene coding for IL-10 is transferred in vitro into cells of the donor, which cells are subsequently injected into the patient in order to induce chimerism and allograft tolerance.
  • the transfer can be performed using different vectors, such as retroviral vectors (Anderson (1992) Science 256 : 808-813 ; Rosenberg et al (1990) New England Journal of Medecine 323 : 570-578) .
  • the gene coding for IL-10 is transferred in vitro into autologous and alloreactive lymphocytes isolated from the patient. These modified cells are subsequently reinjected into the patient in order to induce allograft tolerance. The transfer can be performed using different vectors, such as retroviral vectors. 3) The gene coding for IL-10 is transferred into the allografted cells by perfusion of the graft with a medium containing IL-10 viral particles. The transfer can be performed using different vectors, such as vectors derived from adenovirus (Rosenfeld et al (1991) Science 252 : 431-434) .
  • Endotoxin or lipopolysacharide (LPS) from gram- negative bacteria is a major causative agent in the pathogenesis of septic shock (1) .
  • a shock-like state can indeed be. induced experimentally by a single injection of LPS into animals.
  • These toxic effects of LPS are mostly related to macrophage activation, leading to the release of multiple inflammatory mediators.
  • tumor necrosis factor (TNF) appears to play a crucial role, as indicated by the prevention of LPS toxicity by the administration of neutralizing anti-TNF antibodies (2-5) .
  • IL-10 is effective in inhibiting TNF release and associated toxicity in a mouse model of septic shock. IL-10 can therefore be used to prevent the morbidity and the mortality associated with gram-negative sepsis, either alone or in combination with conventional therapeuties (26) .
  • IL-10 pretreatment with IL-10 prevents LPS-induced toxicity.
  • IL-10 can be used preventively in situations where bacterial sepsis and related septic shock are likely to be encountered, for example before surgery, particularly field surgery, and other situations where bacterial infection is likely.
  • IL-10 is administered before potential exposure to the bacterial micro-organism.
  • IL-10 can also be used to prevent cerebral malaria (neuromalaria) , since : a) adhesion molecules (especially ICAM-1) expression is playing a major role in the induction of cytoadherence of infected erythrocytes to the brain microvascular endothelium ; b) the expression of these adhesion molecules is stimulated by TNF which is induced by malaria antigen and ; c) malaria antigen-induced TNF production can also be inhibited by IL-10 administration. In this case, IL-10 can thus inhibit the development of the pathological entity, either alone or in combination.
  • IL-10 can also be used according to this aspect of the invention, in the treatment or prevention of toxic shock syndrome.
  • This syndrome is related to the T cell and monocyte activation induced by toxins from gram-positive bacteria (e. g. , Staphylocci) .
  • IL-10 efficiently blocks this process and can therefore be used therapeutically for this purpose, either alone or in combination with conventional therapies (26, 27) .
  • the present inventors have demonstrated, for the first time, that IL-10 inhibits monocyte procoagulant activity.
  • IL-10 can be used effectively in acute diseases characterized by activation of coagulation system involving the induction of tissue factor expression at the surface of monocytes, such as septic shock, meningococcemia, promonocytic leukemia, first dose reactions induced by anti-CD3 monoclonal antibodies, etc... and all diseases characterised by thrombotic processes such as antiphospholipid syndrome, hemolytic uremia syndrome, allograft rejection, disseminated intravascular coagulation, and high-risk surgery.
  • coagulation system involving the induction of tissue factor expression at the surface of monocytes, such as septic shock, meningococcemia, promonocytic leukemia, first dose reactions induced by anti-CD3 monoclonal antibodies, etc... and all diseases characterised by thrombotic processes such as antiphospholipid syndrome, hemolytic uremia syndrome, allograft rejection, disseminated intravascular coagulation, and high-risk surgery.
  • IL-10 can be used as sole active principal or in combination with other treatments acting on the extrinsic pathway of the coagulation system.
  • IL-10 is the sole cytokine present in the pharmaceutical composition.
  • Tissue Factor at the surface of human monocytes, leading to induction of procoagulant activity, can be induced by bacterial LPS (resulting in septic shock) , by anti-CD3 monoclonal antibodies, or by inflammatory mediators such as TNF or IL-1.
  • IL-10 is used in the preparation of a medicament for treatment or prevention of anti-CD3-induced shock syndrome.
  • IL-10 has a dual function in this application and acts both by inhibiting cytokine synthesis (TNF- and IL-1) by monocytes and lymphocytes, and by inhibiting the procoagulant activity.
  • IL-10 can be administered prophylactically before the first injecion of 0KT3 in allograft recipients, either alone or in combination with conventional therapies.
  • IL-10 inhibits not only Class II MHC expression but also B7 and ICAM-1 expression on human monocytes. This finding is also surprising in view of the observations by DING et al (J. Immunol., 151, 1224-1234, August 1993) who report, in mice, that IL-10 inhibits B7 up-regulation on mouse macrophages, but did not modify the expression of ICAM-1 on these cells.
  • B7 and ICAM-1 Intercellular Adhesion Molecule-1 are "accessory molecules" expressed on the membrane of Antigen Presenting Cells (APC's), which interact with their counterpart receptors (CD28 and LFA-1, respectively) on the surface of the T-lymphocyte, and which are crucial for the co-stimulation of the T- lymphocyte. Therefore, IL-10 inhibits T-lymphocyte activation by interfering with both the MHCII/T cell receptors interaction, and the B7/CD28, and ICAM-l/LFA-1 interactions. The latter effects are especially relevant to transplantation immunity. Indeed, antigen-presenting cells (APC) deficient in these costimulatory molecules induce T cell unresponsiveness (T cell anergy) in vitro.
  • APC Antigen-presenting cells
  • IL-10 inhibits within the T lymphocyte the B7/CD28 activation pathway.
  • the dual action of IL-10 on this pathway indicates that IL-10 can be administered to promote tolerance to allografts.
  • IL-10 can be used in the treatment or prevention of diseases involving activation of T-lymphocytes through the B7/CD28 or ICAM-l/LFA-1 pathway, particularly diseases associated with alloreactive responses, such as allograft rejection and Graft versus Host Disease. Diseases involving Host versus Graft reactions are particularly preferred.
  • IL-10 may be used either alone or in combination with cellular suspensions (blood cells, spleen cells, bone marrow cells, bone marrow stem cells...) from the donor to prevent or treat rejection of solid allograft.
  • IL-10 can be injected in the graft before transplantation in order to inhibit donor passenger leucocytes and/or in the recipient by parenteral route.
  • IL-10 can also be added to donor cell suspensions before their infusion in the recipient in order to induce transplantation tolerance.
  • IL-10 can be used in the prevention or treatment of acute graft versus host disease after bone marrow transplantation.
  • IL-10 is added to donor bone marrow cells before their infusion in the recipient or injected in the recipient after the bone marrow transplantation.
  • IL-10 Since, contrary to previous findings, IL-10 has now been found to block the ICAM-l/LFA-1 pathway as well as the B7/CD28 pathway, known to be cyclosporin-A resistant, IL-10 can either be used as sole active principal or in association with cyclosporin-A or other related immunosuppressive agents such as FK 506 or rapamycin. Therefore, it may be advantageous to add IL-10 to conventional treatments in order to prevent or treat allograft rejection. IL-10 may be particularly useful in patients in whom conventional treatments, including cyclosporin-A fail to prevent rejection.
  • IL-10 presents further advantages in comparison to standard treatments, for example it can prevent or treat allograft rejection when administered for only a short time, without requiring continuous administration. Also, as will be demonstrated below, the inventors found that IL-10 inhibits IL-5 production by T-lymphocytes. Since eosinophils are induced by IL-5 and are involved in some forms of graft rejection, the beneficial effect of IL-10 in transplantation settings will also be related to the inhibition of IL-5 production. Furthermore, IL-10 might also be used in association with anti-CD3 mAb. In this latter setting, IL-10 will enhance the immunosuppressive effect of the mAb and also prevent its side effects related to cytokine release.
  • IL-10 which is a cytokine produced by TH2 lymphocytes, has been described to date as a potent inhibitor of TH1 cytokines. There have been no reports of IL-10 as an inhibitor of TH2 cytokines.
  • the present inventors have found that the inhibitory effect of IL-10 within T-cells does not specifically affect the production of TH1 cytokines by these cells and that IL-10 is indeed able to inhibit the production of IL-5, a cytokine typically produced by TH2 lymphocytes.
  • IL-10 inhibits production of IL-5 by T cells when these cells are costimulated by B7/CD28 signaling, but not when they are stimulated by PMA and A23187 calcium ionophore.
  • IL-10 can be of therapeutic value in diseases involving excessive IL-5 production by T-lymphocytes.
  • IL-10 is of therapeutic value in eosinophilic diseases (for which no satisfactory treatments are available) such as atopic diseases including allergic rhinitis and bronchial asthma, atopic dermatitis and other cutaneous diseases associated with eosinophil infiltration (including chronic eczema, erythema multiforme, dermatitis herpetiformis, mastocytosis, bullous disorders) , Crohn' s disease, broncho-pulmonary aspergillosis, Spanish toxic oil syndrome, myalgia-eosinophilia syndrome induced by L-tryptophane preparations, certain drug allergic reactions, tropical eosinophilia, helminthic diseases, Schulman syndrome (eosinophilic fasciitis) , Churg-Strauss syndrome and other vasculitides with eosinophilia, Loeffler syndrome, chronic eos
  • atopic diseases including allergic rhinitis and bronchial asthma,
  • rIL-10 should preferably be given either alone or in combination with interferon- alpha or interferon-gamma. Also, since hyper ⁇ eosinophilic syndromes are characterised by pulmonary involvement with interstitial infiltrates, an appropriate route of IL-10 administration is through the use of aerosols, as well as the routes mentioned earlier. Topical applications including ointments are also suitable.
  • anti-IL-10 antibodies can be used in therapy or prevention of diseases involving insufficient production of IL-5 from T-lymphocytes.
  • FIG. 1 Construction of the murine IL-10 retroviral vector.
  • Plasmid pTGlH-mIL-10 was derived from the Harvey murine sarcoma virus (Ha-MuSV) cloned in pBR322 (pC06-HX) .
  • the entire coding region of v- ras H (SacII-XhoI) was removed and replaced by the 560 bp fragment coding for the murine IL-10 cDNA.
  • Ha- MuSV sequences are shown in bold lines ; pBR322, in broken lines. Restriction endonucleases sites : H, Hindlll ; S, SacII ; X, Xhol ; B, BamHI.
  • Black box coding sequences of IL-10 ; open box : coding sequences of v-ras H ; and cross-hatched boxes ; Ha- MuSV LTRs ;
  • mice with no pretreatment mice with no pretreatment
  • Two tailed p value has been determined with the Fisher's exact test.
  • cDNA's used in the examples are the following :
  • mIL-10 (mouse IL-10) cDNA
  • example 2 The cloning of mIL-10 (mouse IL-10) cDNA is described in detail in example 2 as well as in the article entitled "Interleukin-10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia", by C. Gerard et al., Journal of Experimental Medicine 177 : 547-550, 1993.
  • the sequence of this cDNA is identical to the published sequence (patent application WO 91/00349, page 29, lines 15-26), except for one nucleotide located #247 where the present inventors found a "C” and not a "T” as described in WO 91/00349.
  • hIL-10 human IL-10
  • cDNA The cloning of hIL-10 (human IL-10) cDNA is described in the paper entitled "Interleukin-10 inhibits the induction of monocyte procoagulant activity by bacterial lipopolysaccharide", by 0. Pradier et al. , European Journal of Immunology 23 : 2700-2703, 1993.
  • the sequence of this cDNA is identical to the published sequence (patent application WO 91/00349) .
  • VIL-10 (viral IL-10) DNA (Viera P. et al. , P.N.A.S. 88 : 1172-1176, 1991) has been cloned by performing polymerase chain reaction (PCR) using specific oligonucleotides for the viral IL-10 DNA from the Epstein-Barr virus (EBV) .
  • PCR polymerase chain reaction
  • EBV Epstein-Barr virus
  • Retroviral vectors
  • the same vector is used to insert the human and viral IL-10 cDNA's described above.
  • a second retroviral vector (pC07-FX) is derived from the sequences of both Harvey Murine Sarcoma Virus and Friend Murine Leukemia Virus. This vector allows higher expression and higher viral titer compared to tne first vector described above. It has been published by Feld an et al. in Journal of Virology 63 : 5469-5474, 1989.
  • the IL-10 cDNA's were inserted in the SacII and Xhol sites of the pC07-FX vector.
  • mice Animals. 10-15 week-old BALB/c mice were from the KUL Proef Dieren Centrum (Leuven, Belgium) .
  • LPS from E. coli was from Sigma Chemical Co. (St. Louis MO, U.S.A.).
  • JESS-2A5 mAb a neutralizing rat anti-mouse IL-10 IgGl mAb, was from Tim Mosmann (Department of Immunology, University of Alberta, Edmonton, Canada) (9) .
  • LO-DNP mAb a rat IgGl antibody used as control, was from H. Bazin (Experimental Immunology Unit, Universite Catholique de Louvain, Brussels, Belgium) .
  • Mouse recombinant IL-10 cloning and expression. Specific oligonucleotides for the murine IL-10 cDNA were synthesized according to the IL-10 sequence (10) . Restriction sites were included at their 5* end for subcloning: HincII/SacII for the sense primer 5'- CTCCATCATGCCTGGCTCA-3 » (nucleotide 69-87) and Smal/Xhol for the antisense primer 5'- TACACACTGCAGGTGTTTTAGC-3 • (nucleotide 608-629).
  • RNA was prepared from the spleen of mice injected with the hamster anti-CD3 mAb 145-2C11 as a nonspecific stimulator of cytokine transcription (11) .
  • 1 ⁇ g RNA was reverse transcribed using the antisense oligonucleotide (1 ⁇ g) as primer and 200 U Mo-MuLV reverse transcriptase (RT) (Promega Corp, Madison, WI, U.S.A.) in RT buffer (50mM KC1, 20mM Tris HCl, pH 8.3, 2.5mM MgCl 2 , 0.1 mg/ml acetylated BSA, dNTPs 2.5 mM each, RNasin 20 U [Promega Corp.]) in a final volume of 20 ⁇ l. To this were added 2.5 U Taq DNA polymerase, and 1 ⁇ g of each sense/antisense primer, in the same buffer, and in a total volume of 100 ⁇ l.
  • a PCR was performed using a DNA thermal
  • Nucleotides 322, 465-468, 522 and 523 were different from those published (10) , but the amino acids remained unchanged.
  • the SacII-XhoI restriction fragment, containing the murine IL-10 cDNA, was inserted in the Harvey murine sarcoma virus-derived retroviral vector pC06-HX in place of the coding sequence of p21 ras (13) . In this construction, called
  • IL-10 cDNA is the only functional gene.
  • IL-10 pTGlH-mlLlO was cotransfected with the plasmid pSV2-neo, which contains the neomycin resistance gene as a selectable marker. Transfection was performed in the CH0-K1 (Chinese hamster ovarian Kl) cell line using a modified calcium phosphate method, except that no carrier DNA was present (14) . Selection for transfectants was initiated by adding 400 ⁇ g/ml G418
  • IL-10 concentrations were determined by reference to a standard curve of recombinant IL-10 expressed in COS cell supernatant (470 U/ml)
  • Serum TNF Levels Blood samples were obtained from individual mice by retroorbital puncture. Serum levels of TNF were estimated by a cytotoxicity assay on actinomycin-D-treated WEHI-164 clone 13 cells (15) . Results were expressed in pg/ml in reference to the cytotoxic activity of a standardized (NBSB, UK) preparation of recombinant murine TNF expressed in E. coli (16) . This preparation had a specific biological activity of 2.25 x 10 8 IU/mg, with 1 IU representing about 4 pg murine TNF. IL-10 was found not to be interfering with this bioassay.
  • mice were injected intravenously with 100 ⁇ g of LPS 30 min after an intraperitoneal administration of either CHO-IL-10 or mock supernatants. Serum TNF levels were determined 1.5, 3, and 6 h after LPS challenge, whereas rectal temperatures were measured at regular intervals with a digital thermometer. In another series of experiments, the effects were evaluated of IL-10 on the lethality induced by a single dose of 500 ⁇ g LPS.
  • mice were pretreated ei t her t he medium used for culturing CHO-Kl cells, or the mock superna Serum levels oi biologically active TNF (mean ⁇ SEM) were determined 1.5h 3h after LPS challenge.
  • the anti-IL-10 completely abrogated the protective effect of IL-10 on hypothermia whereas the control rat mAb had no effect.
  • mice were challenged with 500 ⁇ g LPS, a dose which is lethal within 72 h in 50 % of the animals. All mice pretreated with 1,000 U IL-10 survived the LPS injection, whereas pretreatment with supernatant from mock-transfected CHO-Kl cells did not modify the LPS-induced lethality (Fig.3) .
  • IL-10 prevents the toxicity of LPS in a murine model of endotoxin shock.
  • the beneficial effect of IL-10 could be due at least in part to the reduction of TNF synthesis or release. Indeed, a similar level of protection against LPS-induced lethality has been obtained by the use of neutralizing anti-TNF antibodies (2-5) .
  • IL-10 might also inhibit or prevent the synthesis and/or release of other monocyte/macrophage-derived cytokines involved in the pathogenesis of septic shock, especially IL-1(1, 6, 19, 20).
  • IL-10 can therefore be added to the potential immunointervention strategies for the prevention and/or treatment of septic shock, which already include anti-TNF mAb (2-5) , IL-1 receptor antagonist (21-23) , differenciation factor/leukemia inhibitory factor (24) , and G-CSF (25) . Because of its deactivation effect on macrophages, IL-10 therapy could be of particular interest, possibly in combination with some of these other biological agents.
  • EXAMPLE 3 USE OF IL-10 IN PROTHROMBOTIC STATES : INHIBITION OF PROCOAGULANT ACTIVITY OF MONOCYTES
  • LPS has been shown to induce the expression of a procoagulant activity (PCA) due mainly to the expression of tissue factor (TF) on monocytes and endothelial cells.
  • PCA procoagulant activity
  • TF tissue factor
  • Inflammatory mediators such as TNF or interleukin 1 (IL-1)
  • LPS could also induce on endothelial cells the expression of TF. Therefore, the effects of IL-10 on this prothrombotic mechanism was investigated, using cultured human umbilical vein cells (HUVEC) and peripheral blood mononuclear cells (PBMC) .
  • HUVEC human umbilical vein cells
  • PBMC peripheral blood mononuclear cells
  • PCA and TF expression induced on monocytes by LPS (1 ⁇ g/ l) was neutralized by more than 80% when human recombinant IL-10 (1 to 2.5 U/ml) was preincubated overnight with the PBMC prior to the adjunction of LPS. At least 6 hours of monocytes-preincubation with IL-10 was necessary to obtain maximal PCA inhibition. No influence of IL-10 on LPS-induced tissue factor expression by EC was found.
  • the human recombinant IL-10 (hIL-10) was obtained as follows. Total RNA from human PBMC stimulated in vitro with an anti-CD3 monoclonal antibody, 0KT3, was isolated and reverse transcribed using Mo-MuLV reverse transcriptase. A polymerase chain reaction (PCR) was performed using specific oligonucleotides for the human IL-10 cDNA: sense primer 5'- AAGGCATGCACAGCTCAGCACTGCTC-3 ' (nucleotides 26-51) and anti-sense primer 5'-CCACCCTGATGTCTCAGTTTCGTATC-3 ' (nucleotides 555-580) .
  • a PCR product with the predicted size of 554 and the predicted restriction pattern was cloned in a retroviral vector and cotransfected in CHO-Kl cells with the plasmid pSV2- neo which contains the neomycin resistance gene. After 10 days in selective medium (400 ⁇ g/ml G418) , individual resistant colonies were isolated, and their supernatants collected after 24 hours culture were tested for their ability to inhibit the OKT3-induced IFN- 7 production by human PBMC.
  • Control (mock) supernatant from CHO-Kl cells transfected with the control plasmid and with pSV2-neo has been selected and collected similarly.
  • LPS from E.coli 055.B5 was obtained from Sigma (St Louis, Mo) .
  • PBMC peripheral blood mononuclear cells
  • PBMC from healthy volunteers were prepared from blood anticoagulated with citrate phosphate dextrose adenine (CPDA) . After centrifugation of buffy coats on a Ficoll Hypaque gradient followed by washing with Hank's balanced salt solution (HBSS) without calcium and magnesium, PBMC were cultured at 3*10 6 / ⁇ l in one ml culture medium consisting of RPMI 1640 supplemented with Hepes (20 mM) , glutamine, and 10% fetal calf serum (FCS) .
  • CPDA citrate phosphate dextrose adenine
  • Final PBMC preparations typically contained 22% to 30% monocytes, 70% to 78% lymphocytes, less than 1% neutrophils and less than 20.000 platelets/ml as estimated with a STK-S cell counter and cell identification using May Grunwald Giemsa staining. All reagents and the culture medium contained less than 10 pg/ml endotoxin as measured using a Limulus Amoebocytes Lysate assay.
  • Human umbilical vein endothelial cells were cultured using M199 medium supplemented with 20% pooled human serum, essential amino-acids, endothelial cell growth factor (ECGF - 40 ⁇ g/ml) , heparin (100 ⁇ g/ml) , penicillin and streptomycin. Cells from the second passage were transfered into 96 wells plates and used at confluence after 24 h in culture medium without ECGF and heparin.
  • PCA Procoagulant activity
  • PCA on the surface of PBMC stimulated for 6 h with 1 ⁇ g/ml LPS was determined by a single stage clotting assay. The number of monocytes among the PBMC suspension was adjusted to 5*10 5 monocytes/ml. This cell suspension was then incubated 1 min with 100 ⁇ l of 25 mM CaCl 2 before the initiation of clotting by the addition of 100 ⁇ l pooled normal citrated plasma. Clotting time was recorded with a KC10 apparatus and interpolated into U/ml of PCA by reference to a standard curve generated with serial dilutions of a commercial thromboplastin. One unit corresponds to the amount of thromboplastin giving a normal clotting time of 12.4 sec.
  • tissue factor/factor VII pathway normal human plasma was replaced in some experiments by a factor Vll-deficient plasma or by normal plasma pre-incubated for 30 min with 12D10, a neutralizing mouse anti factor VII MoAb, or the cells were incubated with a neutralizing mouse IgG 1 anti-tissue factor MoAb (4507) .
  • thrombin generation was measured after sequential addition of calcium (100 ⁇ l, 30 mM CaCl 2 , 2 minutes at 37°C), normal human citrated plasma (45 seconds, 37°C) and a chromogenic substrat (S2238, 0.7 mM, 100 ⁇ l, 20 min at 37°C). The reaction was stopped with acetic- acid (50 ⁇ l) and the absorbance read at 405 mm. The amount of thrombin generated in this system was calculated using a standard curve obtained with purified thrombin and was expressed per 1*10 5 HUVEC.
  • TF molecules at the surface of cultured PBMC was quantified by flow cytometry after staining with a fluoresceinated mouse IgGl anti-tissue factor MoAb. After culture, cells were washed with PBS supplemented with 0.5% BSA, and 6*10 5 cells were incubated for 1 h at 4°C with the anti-tissue factor MoAb or with an irrelevant IgGl MoAb as control. After washing and fixation with 1% formalin, cells were analysed with a FACScan flow cytometer. Monocytes were gated using side scatter and forward light scatter properties. In this gate more than 70% cells expressed the TF antigen and 95% of cells bearing the TF antigen coexpress the CD14 molecule. Flow cytometry standardisation icrobeads coated with known amounts of fluorescein were used to establish a calibration curve allowing the determination of mean equivalents of soluble fluorescence (MESF) .
  • EMF mean equivalents of soluble fluorescence
  • tissue factor mRNA expression by reverse PCR.
  • IL-10 inhibits LPS-induced PCA on PBMC
  • IL-10 inhibits the LPS-induced tissue factor expression on monocytes :
  • IL-10 was incubated for 18 hours with PBMC before the 6 hours challenge with LPS. Incubation with IL-10 for 6 to 24 h before LPS addition also leads to optimal prevention both of PCA and of TF expression on monocytes. The inhibitory effects of IL-10 steadily diminished when preincubation was reduced to 3 and 1.5 h. No or minimal inhibition ( ⁇ 20%) of PCA or TF expression was seen when IL-10 was added together with LPS. IL-10 fails to inhibit LPS-induced PCA on endothelial cells :
  • hIL-10 inhibits the LPS- induced monocytes procoagulant activity. This direct anticoagulant effect, associated with the hIL-10's ability to suppress inflammatory mediator release, suggests that exogenous administration of hIL-10 could provide a new therapeutic strategy for septic shock.
  • EXAMPLE 4 INTERLEUKIN-10 MODULATES THE RELEASE OF CYTOKINES AND REDUCES THE SHOCK SYNDROME INDUCED BY ANTI-CD3 MONOCLONAL ANTIBODY IN MICE
  • IL-10 decreases the secretion of interferon- 7 and TNF- ⁇ by activated TH1 cells and macrophages.
  • hIL-10 Recombinant human IL-10 (hIL-10) was applied to PBMC simulated _in vitro with OKT3.
  • Supernatants of unstimulated and of 0KT3-stimulated PBMC contained 150 pg/ml and 8000 pg/ml of TNF- ⁇ (measured by IRMA) respectively.
  • hIL-10 decreased TNF- ⁇ secretion in a dose-dependent manner, with the highest hIL-10 concentration resulting in a 85% inhibition of TNF secretion.
  • hIL-10 v/as also able to supress by more than 90% the release of INF- 7 induced by 0KT3.
  • TNF- ⁇ serum levels rose from undetectable ( ⁇ 50 IU/ml) to (mean+sem) 795+150 IU/ml 90 min after injection of 145-2C11 mAb.
  • Intraperitoneal injection of 1000 U of mIL-10 30 min before anti-CD3 challenge allowed for a significant reduction of serum TNF- ⁇ levels (at 90 min: 327+36 IU/ml, p ⁇ 0.05 as compared to mice injected with 145-2C11 alone) .
  • the hamster mAb 145-2C11 directed against the mouse CD3 complex was produced as ascites in nude mice.
  • the neutralizing anti-mouse IL-10 mAb JES5-2A5 was a kind gift of Dr T. Mosmann (University of Alberta, Edmonton, Canada) .
  • Murine recombinant IL-10 (mIL-10) was obtained as culture supernatants from CHO-Kl cells stably transfected with the corresponding cDNA, as previously described. Supernatants collected from mock-transfected cells were used as control.
  • the endotoxin levels of the mAb preparations and CHO-Kl cell supernatants were below 1 ng/ml as determined by a Limulus amoebocyte lysate assay.
  • Serum levels of TNF were measured according to the method of example 1.
  • IFN- 7 was quantitated by two-site ELISA using Db-1 and Fl rat anti-mouse IFN- 7 Abs.
  • IL-10 serum levels were determined by ELISA using the JES5-2A5 and SXC1 mAbs purchased from Pharmingen.
  • IL-6 serum levels were measured using the 11-6 dependent 7TD1 cell line and the hexosaminidase method. The lower limits of detection of TNF, IFN- 7 , IL-10 and IL-6 were 2 U/ml, 5 U/ml, 2 U/ml and 5 pg/ml respectively.
  • mice were injected ip with 200 ⁇ l of either 1000 U mIL-10 or mock supernatant, followed 30 min later by iv injection of 10 ⁇ g of the 145-2C11 mAb.
  • This dose of IL-10 was chosen because it was previously found optimal in a model of endotoxemia. Rectal temperature was monitored with a digital thermometer during the next 24 h. Blood samples were obtained by retrooribtal puncture at various times after the anti-CD3 mab injection for determination of serum cytokine levels. Blood glucose levels were measured at 4 h using a standard micromethod with Glucostix strips and Glucometer II M.
  • mice were sensitized by an ip injection of 10 mg of D-galactosamine given 90 min before the administration of 200 ⁇ g of the mAb.
  • cyclosporine A was injected ip (50 mg/kg for each dose) 18 and 3 h before anti-CD3 challenge, a protocol previously shown to inhibit the systemic release of cytokines.
  • m-ILlO inhibits the systemic release of IFN- 7 and TNF but not of IL-6 after injection of 145-2C11 mAb :
  • hILlO differentially regulates the release of IFN- 7 and IL-10 induced by the 145-2C11 mAb :
  • hIL-10 pretreatment did not reduce the endogenous release of IL-10, as assessed by peak serum levels of IL-10 4 h after 145-2C11 mAb injection.
  • the resistance of.:iL-10 to the inhibitory effects of 11-10 contrasts with the effect of CsA which blocked equally IFN- 7 , TNF and 11-10 release.
  • mIL-10 reduces the acute toxicity of the 145-2C11 Ab :
  • hypothermia and hypoglycemia are sensitive parameters of the shock syndrome induced by the 145-2C11 mab.
  • IL-10 significantly reduced the drop in rectal temperature consecutive to 145-2C11 mAb injection.
  • the hypoglycemia following 145-2C11 mAb injection was not modified by mIL-10 pretreatment.
  • the protective potential of IL-10 was further investigated in a lethal model obtained in mice sensitized with D-Gal, 30% of D-Gal-sensitized mice died after 145-2C11 mAb and pretreatment with mll-10 reduced this lethality to 5%. These results show that the cytokine release and the in vivo toxicity induced by anti-CD3 mAbs are reduced by IL-10.
  • IL-10 was found to inhibit IL-5 production in a dose-dependent manner in T-cells activated with either anti-CD3 mAb cross linked on B7/CD32 - transfected mouse fibroblasts, or by p.m.A in conjunction with anti-CD28 mAb.
  • IL-10 Human IL-10 (rIL-10) was expressed in a baculoviral system using the IL-10 cDNA clone described previously (Pradier, O., C. Gerard, A. Delvaux, M. Lybin, D. Abramowicz, F. Capel, T. Velu and M. Goldman, 1993, Eur. J. Immunol., 23 : 2700).
  • the IL-10 preparation was semi-purified by ionic separation chromatography followed by gel filtration. In some experiments, we verified that the activity of this preparation was similar to that of commercially available rIL-10.
  • mice fibroblast cell lines 3T6 expressing human CD32 (FC7RII) either alone or in association with the B7/BB1 antigen (referred to as B7) were obtained and maintained in culture. Transfected cells were incubated for 5 hours with 10 ⁇ g/ml mitomycin-C, washed three times and then used in coculture with T cells.
  • PBMC peripheral blood mononuclear cells
  • HBSS Hanks' balanced salt solution
  • T lymphocytes were purified using one cycle of Lymphokwik-T treatment. The T lymphocytes were further incubated with anti-CD56, -CD19, -DR, -CD14 and anti- CDllb mAb for 30 min at 4*C, washed and incubated with goat anti-mouse lgG-coated magnetic beads.
  • T cells preparations contained more than 98% CD3+ CD28+ DR- T cells without detectable CD14+ monocytes.
  • CD4+ T lymphocytes were further purified by magnetic immunodepletion using anti-CD8 coated Dynabeads.
  • T cells were seeded at 2.10 5 / w e_.l in the experiments using transfectant cells and at 1.10 5 /well in the other conditions. After different times of incubation, culture supernatants were collected and stored at -20°C until assayed for cytokine determinations.
  • IL-5 levels were determined by sandwich ELISA using the following anti-human-IL-5 mAbs : H30 rat anti-human IL-5 mAb IgG2b as coating mAb and mAb7 mouse anti-human IL-5 mAb IgGl as second mAb.
  • anti-human-IL-5 mAbs H30 rat anti-human IL-5 mAb IgG2b as coating mAb and mAb7 mouse anti-human IL-5 mAb IgGl as second mAb.
  • rIL-10 did not interfer with the immunoenzymatic detection of rIL-5.
  • Commercially available kits were used for determination of IL-2 and IFN- 7 . Lower limits of detection were 10 pg/ml for IL-5, 1 IU/ml for 11-2, and 10 U/ml for IFN-7.
  • RNA from purified T cells was extracted using the guanidium thiocyanate method and analysed for IL-5 mRNA and hyposanthine phosphoribosyl transferase (hprt) RNA by a reverse PCR method. Briefly, 1 ⁇ g of RNA was reversed-transcribed using Moloney murine leukemia virus (MoMuLV) reverse transcriptase (200 U/assay) . The resulting cDNA was subjected to 32 PCR cycles. Each cycle was performed at 93°C for 1 min, 53 °C for 2 min and 70°C for 3 min. 11-5 and HPRT primers were synthetized according to the human cDNA sequences.
  • MoMuLV Moloney murine leukemia virus
  • 5' sense primer was 5'- GCTTCTGCATTTGAGTTTGCTAGCT-3 ' and 3 ' antisense primer was 5'TGGCCGTCAATGTATTTCTTTATTAAG-3 ' .
  • PCR amplification using these primers resulted in a mRNA- specific 291 bp fragment (16) .
  • Costimulation provided by the B7 molecule is known to dramatically enhance the responses of human resting T cells to anti-CD3 mAb (reviewed in Annu. Rev. Immunol., 1993, 11 : 191, Linsley P.S. and J.A. Ledbetter, the role of the CD28 receptor during T cell responses to antigen) .
  • the production of IL-5 by resting T cells stimulated by the anti-CD3 mAb in presence of B7/CD32 transfected fibroblasts (B7/CD32 transfectants) was evaluated, using transfected fibroblasts expressing CD32 alone (CD32 transfectants) as control.
  • the CD32 molecule (FC 7 RII) allows binding of the mAb to transfectants which is required for efficient crosslinking of the TCR/CD3 complex. While IL-5 was undetectable in presence of cells transfected with CD32 alone, significant levels IL-5 were produced together with IL-2 when B7/CD32 transfectants were used for costimulation. Peak levels of IL-2 were detected at 24 h while maximal IL-5 levels were achieved at 48 h. To determine whether TCR/CD3 engagement is required for IL-5 induction, T cells were stimulated with PMA together with B7/CD32 transfectants or anti-CD28 mAb. IL-5 was produced in both systems while PMA alone was inefficient to induce significant cytokine secretion.
  • IL-5 production in response to PMA and calcium ionophore A23187 was always much lower than in response to PMA and B7/CD28 signaling. In these systems too, maximal IL-5 levels were higher at 48 h than at 24 h while IL-2 was maximal at 24 h.
  • IL-5 production by resting T cells stimulated with anti-CD3 mAb or PMA and B7/CD28 signaling was observed in 7 independent experiments with 7 different donors. As expected, CD4+ cells were the major source of IL-5 as depletion of CD8+ cells did not influence IL-5 production.
  • rIL-10 inhibits IL-5 production by human resting T cells costimulated by B7/CD28 signaling :
  • rIL-10 The effects of rIL-10 on IL-5 production were first analyzed in the activation system based on anti-CD3 mAb in conjunction with B7/CD32 transfectants. As shown in figure 4, rIL-10 inhibited in a dose-dependent manner the production of IL-5 in this setting. In parallel, we confirmed previous data indicating that rIL-10 inhibits IL-2 but no IFN- 7 production by purified T cells. In control experiments, rIL-10 modified neither B7 expression by transfected fibroblasts nor CD28 expression by purified T cells. As IL-2 has been shown in other systems to be involved in the induction of IL-5, it was verified that the effect of rIL-10 on 11-5 was not due to IL-2 inhibition.
  • rIL-10 reduces 11-5 mRNA accumulation in T cells activated by PMA and anti-CD28 mAb :
  • IL-10 interfered with IL-5 gene expression
  • resting T cells were stimulated for 24 h with PMA and anti-CD28 mAb in absence of presence of rIL-10 (50 U/ml) before IL-5 mRNA analysis by reverse PCR.
  • rIL-10 clearly inhibited IL-5 mRNA accumulation whereas it had no detectable effect on the expression of HPRT housekeeping gene.
  • rIL-10 does not inhibit IL-5 production in response to PMA and A23187 calcium ionophore :
  • rIL-10 did not reduce and even enhanced IL-5 production induced by PMA + A23187. In contrast, rIL-10 dramatically reduced the levels of IL-5 produced in response to PMA + anti-CD28 mAb. The degree of inhibition was similar (around 70%) in healthy individuals and in the patient with Omenn's syndrome.
  • Endogenous IL-10 down-regulates IL-5 production by resting T cells :
  • EXAMPLE 6 IL-10 INHIBITS B7 AND ICAM-1 - EXPRESSION ON HUMAN MONOCYTES
  • This example demonstrates modulation by IL-10 of the expression of the accessory molecules B7 and ICAM-1 on monocytes. This property may contribute to the immunosuppressive properties of IL-10.
  • Recombinant cytokines was obtained from supernatant of CHO (Chinese Hamster Ovary-Kl) cells transfected with an expression vector containing the human IL-10 cDNA. Control (mock) supernatant was collected from CHO-kl cells transfected with a similar vector without inserted IL-10 gene.
  • PBMC Peripheral blood mononuclear cells
  • mAb primary murine monoclonal antibody
  • Primary mAbs were F(ab) '2 fragments of the B7-24 anti-B7 mAb (18) , anti-ICAM-1 mAb, anti-HLA-DR mAb or irrelevant mouse IgG mAb. The binding of these antibodies was revealed by incubation for 30 min at 4°C with FITC-coupled F(ab) '2 fragments of a rabbit antimouse immunoglobulin antiserum.
  • IL-10 inhibits ICAM-1 expression on monocytes :
  • IL-10 decreased the expression of MHC class II molecules (HLA-DR) on monocytes both at the basal state and after IFN- 7 stimulation was first confirmed.
  • HLA-DR MHC class II molecules
  • the influence of IL-10 on ICAM-1 expression on those cells was then examined.
  • the FACS histogram showed that IL-10 reduced the expression of ICAM-1 on resting monocytes.
  • the percentage of inhibition of basal ICAM-1 expression ranged between 50 and 65%.
  • the lack of influence of mock supernatant demonstrated the specificity of the IL-10 preparation.
  • IFN- 7 stimulation led to a clear upregulation of ICAM-1 expression on monocytes as compared to medium alone while it did not change the isotype control staining.
  • IL-10 3 U/ml
  • IFN- 7 -induced ICAM-1 upregulation at the surface of monocytes.
  • the inhibitory effect of the IL-10 preparation was dose-dependant and specific, since no inhibition was observed with the mock supernatant.
  • IL-10 inhibits B7 upregulation on IFN- 7 stimulated monocytes :
  • EXAMPLE 7 SYSTEMIC ADMINISTRATION OF rIL-10 INHIBITS EDEMA FORMATION AND IFN- 7 SYNTHESIS DURING ALLOREACTIVE RESPONSE IN VIVO
  • IL-10 down-regulates the expression of B7 molecules and of ICAM-1 on macrophages or monocytes. These properties of IL-10 suggest that it might inhibit the induction as well as the effector phase of acute cellular transplant rejection and possibly promote allograft tolerance. As a first approach to this question, we analyzed the effects of systemic administration of rIL-10 in an in vivo model of alloreactivity induced by localized injection of allogeneic cells.
  • Mouse rIL-10 was cloned as previously described (Example 1) and produced in Sf9 insect cells using the baculoviral expression vector pBlueBac2 from Invitrogen Corp. (San Diego, CA) . rIL-10 was affinity purified using the JE ⁇ 5-2A5 anti-IL-10 monoclonal antibody obtained from Pharmingen. For in vivo administration, rIL-10 was diluted in saline buffer containing 0.1% mouse serum (rIL-10 vehicle) to obtain a final concentration of 10.000 U/ml as determined by ELISA.
  • mice The right rear footpads of BALB/c mice were injected on day 1 with a suspension of 2.5x10° syngeneic (BALB/c) or semi-allogeneic (A/JxBALB/c) Fl spleen cells in 0.1 ml of RPMI 1640. From day 0 to day 5, mice received every 8 h an intraperitoneal injection of either 1000 U rIL-10 or control vehicle. This dose of IL-10 was chosen because it was found effective in inhibiting cytokine release in two different in vivo models, namely endotoxin shock and polyclonal T cell activation induced by anti-CD3 monoclonal antibody. On day 5, left and right lymph nodes were surgically removed from the popliteal fossa and weighed. Results were expressed as delta values (in mg) representing the difference in weight between the right (injected side) and left (uninjected side) lymph nodes.
  • delta values in mg
  • lymph node cells were counted and lymph node cells from each group were pooled, stained with phycoerythrin-conjugated anti-CD4 (L3T4) or FITC-conjugated anti-CD8 monoclonal antibody (Lyt2) obtained from Becton Dickinson and percentages of CD4+ and CD8+ cells were determined by flow cytometry.
  • L3T4 phycoerythrin-conjugated anti-CD4
  • Lyt2 FITC-conjugated anti-CD8 monoclonal antibody
  • RNAs from popliteal lymph node cells were extracted using the Micro-Fast TrackTM mRNA isolation kit from Invitrogen Corp. 1 ⁇ g of each RNA sample was reverse transcribed into cDNA using oligo dT primers. PCR amplification was then performed using primers specific of IFN-7 or hypoxanthine-phosphorybosyl transferase (HPRT) . Amplified products were separated on 2% agarose gels and visualized by ethidium bromide staining.
  • rIL-10 inhibits draining lymph node swelling after subcutaneous injection of allogeneic cells :
  • IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147: 3815.

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Abstract

L'invention se rapporte à l'utilisation de l'interleukine-10 (IL-10) ou d'analogues ou d'agonistes de celle-ci, pour la préparation d'un médicament destiné au traitement ou à la prévention de maladies impliquant (i) la production d'interleukine-5 par des lymphocytes-T ou (ii) l'activation de lymphocytes-T par l'intermédiaire de la voie de B7/CD 28 ou d'ICAM-1 (molécule d'adhésion intercellulaire-1)/LFA-1 ou (iii) l'activation de l'activité procoagulante de monocytes et (iv) des maladies infectieuses graves impliquant le facteur de nécrose tumorale (TNF).
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JPH08510719A (ja) 1996-11-12

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