WO1998016248A1 - Methodes de stimulation de la tolerance orale et de traitements d'une maladie auto-immune a l'aide d'inhibiteurs d'interleukine-12 - Google Patents

Methodes de stimulation de la tolerance orale et de traitements d'une maladie auto-immune a l'aide d'inhibiteurs d'interleukine-12 Download PDF

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WO1998016248A1
WO1998016248A1 PCT/US1996/016007 US9616007W WO9816248A1 WO 1998016248 A1 WO1998016248 A1 WO 1998016248A1 US 9616007 W US9616007 W US 9616007W WO 9816248 A1 WO9816248 A1 WO 9816248A1
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disease
autoimmune disease
autoimmune
cells
antigen
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PCT/US1996/016007
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English (en)
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Warren Strober
Brian L. Kelsall
Thomas Marth
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The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services filed Critical The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority to PCT/US1996/016007 priority Critical patent/WO1998016248A1/fr
Priority to AU72576/96A priority patent/AU7257696A/en
Publication of WO1998016248A1 publication Critical patent/WO1998016248A1/fr
Priority to US11/450,503 priority patent/US20060228360A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • the present invention relates to a method for enhancing oral tolerance in a subject with an autoimmune disease, allergic disease, graft-versus-host (gvH) disease or transplantation rejection.
  • the present invention provides a method for enhancing oral tolerance in a subject to treat an autoimmune disease, allergic disease, GvH disease or transplantation rejection comprising orally administering an antigen associated with the autoimmune disease, allergic disease, GvH disease or transplantation rejection to be treated and administering an inhibitor of interleukin-12 (IL-12) in amounts sufficient to enhance oral tolerance in the subject and treat or prevent autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • IL-12 interleukin-12
  • TGF ⁇ transforming growth factor beta
  • cytokines down regulate T cell responses not only to specific (fed) antigen, but also to other antigens (15, 16).
  • Such "bystander" suppression is the basis for possible treatment of autoimmune diseases with the oral administration of autoantigens that may have a minor role in disease induction (e.g. oral insulin for diabetes mellitus) as well as for the possible treatment of allergic disease, graft-versus-host (GvH) disease or transplant rejection.
  • diseases induction e.g. oral insulin for diabetes mellitus
  • GvH graft-versus-host
  • Interleukin-12 is a recently characterized cytokine with unique structure and pleiotropic effects (40-43). It consists of two disulfide-linked subunits, p40 and p35, that form functionally active p40/p35 heterodimers or inhibitory p40 homodimers.
  • IL- 12 is produced mainly by macrophages/ monocytes and can be efficiently induced by intracellular parasites, bacteria and bacterial products. Functional studies have shown that IL-12 enhances cytolytic activity of natural killer (NK) cells and macrophages and induces, in synergism with the B7/CD28 interaction, cytokine production and proliferation of activated NK cells and T cells (44).
  • IL-12 plays a pivotal role in Thl T cell differentiation and induces naive T cells to produce IFN- ⁇ .
  • administration of IL-12 has been shown to be an effective treatment of mice with established parasitic infections, which elicit a Th2 T cell response (45,46).
  • Th2 T cell response 45,46
  • enhancement of the therapeutic effect could be critical to the ultimate clinical usefulness of oral tolerance to treat autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • the present invention overcomes previous shortcomings in the clinical use of oral tolerance for treating or preventing autoimmune disease, allergic disease, GvH disease or transplantation rejection by providing a method for enhancing oral tolerance in a subject comprising orally administering an oral tolerance-inducing antigen and administering an inhibitor of IL-12.
  • the present invention provides a method for enhancing oral tolerance to an antigen associated with an autoimmune disease in a subject having the autoimmune disease comprising orally administering to the subject an antigen associated with the autoimmune disease and administering an inhibitor of IL-12 in amounts sufficient to enhance oral tolerance.
  • Also provided in the present invention is a method for treating or preventing an autoimmune disease in a subject comprising orally administering to the subject an antigen associated with the autoimmune disease and administering an inhibitor of IL-12 in amounts sufficient to treat or prevent the autoimmune disease, thereby treating or preventing the autoimmune disease.
  • the present invention provides a method for enhancing oral tolerance to an antigen associated with an autoimmune disease, an allergic disease, GvH disease or transplantation rejection in a subject having any of these diseases comprising orally administering to the subject an antigen associated with the autoimmune disease, allergic disease, GvH disease or transplantation rejection and administering an inhibitor of IL- 12 in amounts sufficient to enhance oral tolerance.
  • Any animal which is subject to autoimmune disease, allergic disease, GvH disease or transplantation rejection can be treated by this method although humans are the primary therapeutic target.
  • nonhuman subjects that can be treated by the methods taught herein can include, but are not limited to, mice, rats, rabbits, dogs, cats, non-human primates, as well as any other species now known or later discovered to manifest an autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • oral tolerance refers to an antigen-induced immune response initiated by the oral administration of a soluble protein antigen and resulting in systemic unresponsiveness to the administered antigen. Such unresponsiveness may be specific for the administered antigen or may be antigen-non-specific as a result of production of an antigen-non-specific suppressor substance such as TGF ⁇ .
  • enhancement of oral tolerance means increasing systemic unresponsiveness induced by oral antigen.
  • the enhancement of oral tolerance induced by an oral antigen can be by any means by which clonal deletion or the production of TGF ⁇ is augmented (1,2, 65).
  • Such means can also include, but are not limited to, administering TGF ⁇ directly to the subject; administering an inhibitor of interferon- gamma to the subject; administering an antibody to the interferon-gamma receptor, which prevents binding of interferon-gamma, to the subject; administering an inhibitor of tumor necrosis factor- alpha to the subject; administering an antibody to the tumor necrosis factor-alpha receptor, which prevents binding of tumor necrosis factor-alpha, to the subject; administering an inhibitor of interferon-alpha to the subject; administering an antibody to the interferon-alpha receptor, which prevents binding of interferon-alpha, to the subject; administering interleukin-10 (IL-10) to the subject; or any combination of these means, as well as other means or combinations of means now known or identified in the future to augment clonal deletion or the production of TGF ⁇ in a subject to enhance oral tolerance induced by an oral antigen.
  • IL-10 interleukin-10
  • autoimmune disease describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effects. This may include production of B cells which produce antibodies with specificity for all antigens, allergens or major histocompatibility (MHC) antigens or production of T cells bearing receptors recognizing self components and producing cytokines that cause inflammation.
  • MHC major histocompatibility
  • autoimmune diseases include, but are not limited to, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, diabetes mellitus, pernicious anemia, autoimmune gastritis, psoriasis, Bechet's disease, idiopathic thrombocytopenic purpura, Wegener's granulomatosis, autoimmune thyroiditis, autoimmune oophoritis, bullous pemphigoid, pemphigus, polyendocrinopathies, Still's disease, Lambert-Eaton myasthenia syndrome, myasthenia gravis, Goodposture's syndrome, autoimmune orchitis, autoimmune uveitis, systemic lupus erythematosus, Sjogren's syndrome and ankylosing spondylitis (1,2,69,70,73-77,85-89), as well as any other autoimmune disease now known or discovered in the future.
  • allergic disease describes a disease state or syndrome whereby the body produces a dysfunctional immune response composed of immunoglobulin E (IgE) antibodies to environmental antigens and which evoke allergic symptoms.
  • allergic diseases include, but are not limited to, asthma, ragweed pollen hayfever, allergy to food substances and allergic reactions (54-60,78- 80).
  • GvH disease describes a disease state or syndrome whereby an immune response is initiated by grafted cells and is directed against the subject's body with adverse effects. Examples of GvH disease include, but are not limited to, acute and chronic GvH disease following bone marrow transplant (64).
  • Transplantation rejection describes a disease state or syndrome whereby the transplant recipient's body produces an immune response against the engrafted tissue, resulting in rejection. Transplantation rejection can occur, for example, with kidney, heart, lung or liver transplants as well as with any other transplanted tissue (61-63, 81-84).
  • An antigen associated with an autoimmune disease is a self antigen or an antigen that cross reacts with a self antigen to which the body produces a dysfunctional immune response that causes disease.
  • An antigen associated with an allergic disease is an environmental antigen (allergen) which induces an individual with an appropriate genetic/environmental background an IgE response which mediates mast cell/basophil activation, causing allergic symptoms.
  • An antigen associated with GvH disease or transplantation rejection comprises an MHC to which grafted cells or host cells react and cause adverse effects.
  • antigens can be, but are not limited to, myelin basic protein, collagens, allergens, insulin, Fel-d- 1, cells expressing MHC antigens (54-64, 78-89), as well as any other antigen now known or identified in the future to be functional in inducing or enhancing oral tolerance in a subject.
  • the antigen of this invention can be a single type of antigen or a combination of types of antigens.
  • the antigen of this invention can either be from the species to which it is administered or it can be a homologous antigen from a different species.
  • the inhibitor of IL-12 can be an antibody, either polyclonal or monoclonal, that is specifically reactive with interleukin-12 or a receptor which binds IL-12.
  • Other inhibitors of IL-12 can include, but are not limited to, p40 IL-12 homodimers, inhibitors of IL-12 production, such as IL-10, TGF ⁇ , dimerized complement components, C 3 B, C 3 Bi, antibodies to CD46 or complement receptor (CR3), as well as any other compound or reagent now known or later discovered which acts to inhibit the activity or production of IL-12.
  • the antibodies of this invention can be from any source.
  • antibodies are preferably of human origin or are antibodies generated in other species and "humanized” for administration in humans as described in the Examples provided herein.
  • the antibodies of this invention can be fragments which retain the ability to bind their specific antigens are also contemplated for this invention.
  • fragments of antibodies which maintain IL-12 binding activity are included within the meaning of the term "antibody.”
  • Such antibodies and fragments can be made by techniques known in the art and screened for specificity and activity according to the methods set forth in the Examples herein. For example, general methods for producing antibodies can be found in Harlow and Lane (48).
  • the present invention also provides a method for treating or preventing an autoimmune disease, allergic disease, GvH disease or transplantation rejection in a subject comprising orally administering to the subject an antigen associated with the autoimmune disease, allergic disease, GvH disease or transplantation rejection and administering an inhibitor of interleukin-12 in amounts sufficient to treat or prevent the autoimmune disease, allergic disease, GvH disease or transplantation rejection, thereby treating or preventing the autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • the autoimmune disease, allergic disease, GvH disease or transplantation rejection can be treated or prevented by inducing oral tolerance in a subject in which the administration of an oral antigen alone may not be sufficient to induce oral tolerance or by enhancing an oral tolerance already induced by the administration of an oral antigen.
  • Administration of the oral antigen includes the subject simply ingesting (eating) the antigen or the antigen can be provided.
  • the antigen associated with an autoimmune disease is orally administered to the subject in a pharmaceutically acceptable carrier.
  • Suitable carriers for oral administration of the antigen include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants.
  • Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
  • Suitable liquid carriers may be water, pyrogen free saline, pharmaceutically accepted oils, or a mixture of any of these.
  • the liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents.
  • suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel.
  • the antigen may be contained in enteric coated capsules that release the antigen into the intestine to avoid gastric breakdown.
  • the antigen associated with an autoimmune disease, allergic disease, GvH disease or transplantation rejection and the inhibitor of IL-12 can be administered to the subject in amounts sufficient to enhance oral tolerance and to treat or prevent autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • Optimal dosages used will vary according to the individual being treated and antigen being used.
  • antigen associated with an autoimmune disease, allergic disease, GvH disease or transplantation rejection would be administered orally in a dosage range between 0.0001 and 1.5 mg/kg body weight/day with a preferred dosage range of 0.01-0.5 mg/kg/day and most preferred dosage or 0.1 mg/kg/day.
  • Antigens can be administered every other day for from one week to several years.
  • Administration of the antigen can be stopped completely following a prolonged remission or stabilization of disease signs and symptoms and readministered following a worsening of either the signs or symptoms of the disease, or following a significant change in immune status, as determined by routine follow-up immunological studies well known to a clinician in this field (e.g., a return to significant reactivity of immune cells to a particular suspected or known disease- causing antigen or to a particular tolerogen (71-89).
  • the inhibitor of IL-12 can be orally or parenterally administered in a carrier pharmaceutically acceptable to human subjects.
  • Suitable carriers for oral administration of the inhibitor of IL-12 can include one or more of the substances described above for oral administration of antigen.
  • a sterile solution or suspension is prepared in saline that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, for example, into subcutaneous or intramuscular tissues, as well as intravenously.
  • the inhibitor of IL-12 may be contained in enteric coated capsules that release the inhibitor into the intestine to avoid gastric breakdown.
  • the inhibitor of IL-12 may be microencapsulated with either a natural or a synthetic polymer into microparticles 4- 8 ⁇ m in diameter, which target intestinal lymphoid tissues and produce a sustained release of inhibitor for up to four weeks (47,53).
  • antibodies to IL-12 in soluble form, would typically be administered parenterally in a single dosage of between 1 mg and 100 mg/kg of body weight, with a preferred dosage range of 5-50 mg/kg and most preferred dosage of between 10 and 20 mg/kg.
  • Subjects can be given antibodies to IL-12 as a single injection each week for between one and 52 weeks.
  • 500 mg to 1000 mg of antibodies to IL-12 can be given P.O.
  • 500 mg to 1000 mg can be microencapsulated as described for slow release over a four to eight week period.
  • the amount of oral antigen and inhibitor of IL-12 administered will vary among individuals on the basis of age, size, weight, condition, etc.
  • dosages are best optimized by the practicing physician and methods for determining dosage are described, for example, in Remington 's Pharmaceutical Sciences (49).
  • the efficacy of administration of a particular dose of an oral antigen and an inhibitor of IL-12 in enhancing oral tolerance in a human subject having an autoimmune disease, allergic disease, GvH disease or transplantation rejection can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory test that have a documented utility in evaluating disease activity. These signs, symptoms and objective laboratory tests will vary depending on the particular disease being treated or prevented as will be well known to any clinician in this field. Examples of such methods include, but are not limited to the following:
  • Autoimmune disease e.g., multiple sclerosis: The severity and number of attacks, or for continuously progressive disease, the worsening of symptoms and signs, the cumulative development of disability, the number or extent of brain lesions as determined by magnetic resonance imaging and the use of immunosuppressive medications (76,77).
  • Allergic disease e.g., asthma: The number and severity of attacks as determined by symptoms of wheezing, shortness of breath and coughing. The measurement of airway resistance by the use of respiratory spirometry, the extent of disability and the dependence on immunosuppressive medications or bronchodilators (78-80).
  • GvH disease or transplant rejection e.g., rejection of lung transplant
  • the signs of acute and chronic rejection can include symptoms such as, for example, shortness of breath or decreased exercise tolerance.
  • Other parameters for determining efficacy can be measurement of arterial blood gases, determination of A-a gradients, evaluation of chest X-rays and dependence on immunosuppressive medications (81-84).
  • the efficacy of administration of a particular dose of an oral antigen and an inhibitor of IL-12 in treating an autoimmune disease, allergic disease, GvH disease or transplantation rejection in a subject diagnosed as having an autoimmune disease, allergic disease, GvH disease or transplantation can be determined by standard methods of evaluation of the particular signs, symptoms and objective laboratory test for a particular disease, as known in the art (75-89). If 1) a subject's frequency or severity of recurrences is shown to be improved, 2) the progression of the disease is shown to be stabilized, or 3) the need for use of other immunosuppressive medications is lessened, based on a comparison with an appropriate control group and knowledge of the normal progression of disease in the general population or the particular individual, then a particular treatment will be considered efficacious.
  • the efficacy of administration of a particular dose of an oral antigen and an inhibitor of IL-12 in preventing an autoimmune disease, allergic disease, GvH disease or transplantation rejection in a subject not known to have an autoimmune disease, allergic disease, GvH disease or transplantation rejection but known to be at risk of developing an autoimmune disease, allergic disease, GvH disease or transplantation rejection can be determined by evaluating standard signs, symptoms and objective laboratory tests, as would be known to one of skill in the art, over time. This time interval may be large, with respect to the development of autoimmune or allergic diseases (years/decades) or short (weeks/months) with respect to the development of GvH disease or transplantation rejection.
  • OVA-TCR transgenic mice in a BALB/c background clone DO11.10, that recognizes the 323-339 peptide fragment of OVA in the context of IA d (17), were kindly provided by Dennis Y. Loh (Howard Hughes Medical Institute, Washington University, St. Louis, MO). Syngeneic BALB/c mice were obtained from the National Cancer Institute (Bethesda, MD). Transgenic and BALB/c mice were housed in the NIAID Twinbrook II animal facility (Rockville, MD) and in the NIH Building 10A animal facility, respectively, under standard animal housing conditions.
  • mice 6 to 12 weeks of age were fed 10 mg, 100 mg, or 250 mg of OVA protein (Sigma, St. Louis, MO) dissolved in 0.5 ml phosphate buffered saline (PBS) or PBS alone once or every other day for three doses by intragastric intubation. Mice were sacrificed three days after the last feeding. Control mice were fed PBS since initial studies demonstrated that cells from mice fed PBS and from mice fed high doses of control protein (human serum albumin, Sigma) showed identical proliferation and cytokine responses after OVA restimulation in vitro.
  • PBS phosphate buffered saline
  • mice were given subcutaneous injections of 50 ⁇ g OVA [dissolved in 25 ⁇ l PBS and emulsified in the same volume of complete Freund's adjuvant (CFA, Sigma)] into the right hind footpad five days after the last feeding and sacrificed six days after the injection.
  • Footpad swelling was determined by measuring the specific increment in footpad thickness with a Mitutoyo micrometer (specified minimum reading 0.001 inches and defined closing pressure at reading point; Thomas Scientific, Swedesboro, NJ).
  • mice receiving three doses of 250 mg OVA orally mice fed PBS three times, and unfed mice were given intravenous injection of 0.75 mg of a rat monoclonal antibody to murine IL-12 or, as a control, 0.75 mg of a rat anti-mouse IgG (Sigma).
  • Neutralizing anti-IL-12 antibodies were purified from ascites of nude mice injected intraperitoneally with the C17.8 hybridoma cell line (kindly provided by Dr. G. Trinchieri, The Wistar Institute, Philadelphia, PA) using the E-Z-Sep antibody purification procedure (Pharmacia, Piscataway, NJ).
  • the treatment protocol was as follows: day 0 and 4: injection of anti-IL-12; day 1, 3 and 5: feeding of 250 mg of OVA; day 8: sacrifice of mice and cell isolation.
  • cRPMI Media Cell culture medium
  • NCTC-109 media Gibco, Grand Island, NY
  • Whittaker heat-inactivated fetal calf serum
  • Spleen or PP cells enriched for T cells were obtained using a negative immunoaffinity selection technique, in which cells were passed over an anti-mouse Ig coated glass bead column (Isocell mouse T cell column, Pierce, Rockford, IL) according to manufacturer's instructions; the yield of CD3 positive cells when analyzed by flow cytometry was approximately 85 % .
  • Spleen cells from BALB/c mice enriched for dendritic cells were prepared as previously described via plastic adhesion (18).
  • splenic tissue was digested with collagenase and DNASE I in cIMDM, and plated in cRPMI on a plastic tissue culture dish (Falcon) for 60 min at 37°C.
  • Non-adherent cells were removed by washing with warm PBS.
  • the adherent DC-enriched population was cultured and antigen-pulsed in cRPMI supplemented with submaximal doses of antigen (1 mg OVA/ml) for approximately 20 hours. This was followed by washing off the non-adherent, DC-enriched cell population with warm PBS.
  • the DC population thus obtained was > 70 % CD lie positive as determined by flow cytometric analysis.
  • CD3 clone 2C11
  • CD4 RM4-4
  • CD45R B220; RA3-6B2
  • clonotypic antibody KJ1-26 kindly provided by Drs. K. Nakayama and D. Loh, Howard Hughes Medical Institute, St. Louis, MO
  • anti-CDllc N418) monoclonal antibody
  • PE-labeled goat F(ab') 2 anti-hamster IgG Caltag, San Francisco, CA.
  • the analysis was done on a Becton Dickinson (San Jose, CA) FACScan flow cytometer in association with Lysis II or Cellquest software.
  • Total TGF ⁇ released from cells in culture was assayed by the following method: Cells were cultured for 72 h in serum free media consisting of RPMI supplemented with 2mM glutamine and Nutridoma SP [1 % (vol/vol), Boehringer Mannheim].
  • Total, activated TGF ⁇ (19) was measured by sandwich ELISA in lOO ⁇ l of culture supernatant after acidification with 10 ⁇ l IM HC1 for 60 min at 4°C and neutralization with 10 ⁇ l of IM NaOH.
  • sandwich ELISA a monoclonal anti- TGF ⁇ 1, 2, 3 antibody (Genzyme) and a polyclonal chicken anti-TGF ⁇ antibody (R+D Systems) were used as capture and secondary antibody, respectively.
  • Recombinant human TGF ⁇ l (Genzyme) was used as standard.
  • organs were dissected, placed in freezing media (OCT, Miles, Elkhart, IN) and frozen in 2-Methylbutane and dry ice. 10 ⁇ m sections of the tissue were placed on silanated slides, dried, and fixed in 10% neutral formalin. Tissue sections were placed in PBS containing 0.1 % bovine serum albumin (Sigma), and endogenous peroxidase activity was quenched by 5 min incubation with 2% H 2 O 2 .
  • freezing media OCT, Miles, Elkhart, IN
  • 2-Methylbutane and dry ice 10 ⁇ m sections of the tissue were placed on silanated slides, dried, and fixed in 10% neutral formalin. Tissue sections were placed in PBS containing 0.1 % bovine serum albumin (Sigma), and endogenous peroxidase activity was quenched by 5 min incubation with 2% H 2 O 2 .
  • the labeling of degraded DNA specific for apoptotic cells was performed using a modification of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique by applying the Oncor ApopTagTM peroxidase system (Oncor, Gaithersburg, MD) according to the manufacturer's instructions.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
  • Results represent mean values (+/-) standard deviations (S.D.) where applicable. Statistical significance of differences was determined by the Student's t- test.
  • OVA-TCR transgenic mice were fed OVA protein (10, 100, or 250 mg) either once or three times, and three days after the last feeding, proliferative responses of cell populations from the spleen, peripheral (inguinal) lymph nodes, mesenteric lymph nodes (MLN), or PP were compared to those of control mice.
  • Proliferative responses of whole spleen cells from mice fed 10 or 100 mg of antigen were similar to those from control mice, but spleen cell responses from mice fed 250 mg were significantly reduced.
  • cytokines IL-2, IFN- ⁇ , and IL-4
  • IL-2, IFN- ⁇ , and IL-4 cytokines
  • IL-2, IFN- ⁇ , and IL-4 production by whole spleen cells and spleen T cells was shown to be suppressed when compared to unfed controls.
  • IL-2 and IL-4 responses in the PP were similar to controls and the IFN- ⁇ response was significantly elevated (5- fold) above controls. Cytokine production by whole MLN cells was very low or undetectable for every feeding regimen.
  • Antibodies to IL-12 were administered intravenously to OVA-TCR-transgenic mice simultaneous with antigen feeding (3 x 250 mg OVA) and proliferative responses of cells were determined over time in culture. The proliferative responses vary depending on what time point in culture is chosen for comparison. In the first 96 hours in culture, the PP cells from the fed, fed + anti-IL-12 and the control mice all demonstrate increases in proliferation over that time period. At later time points in culture, i.e. after 96 hours, there is a decline in proliferation of cells from all groups and from all organ systems in culture. However, a more rapid and significant decrease in proliferation in PP and MLN cells was found in mice fed OVA and given systemic anti-IL-12.
  • mice were either fed PBS or OVA (3 x 250 mg), or fed PBS or OVA (3 x 250 mg) plus systemically administered anti-IL-12 as in previous protocols.
  • OVA 50 ug
  • CFA complete Freund's adjuvant
  • Anti-IL-12 treatment of OVA-fed mice is associated with increased TGF ⁇ production
  • cells from the PP and spleen were examined for the ability to produce cytokines, particularly inhibitory cytokines that might mediate oral tolerance.
  • OVA-TCR transgenic mice were fed PBS or OVA (3 x 250 mg), or fed PBS or OVA (3 x 250 mg) and treated systemically with anti-IL-12.
  • IFN- ⁇ secretion was highly suppressed in all cell populations in the fed plus anti-IL-12 treated mice.
  • IL- 4 secretion was similar in spleen and PP cell cultures.
  • IL-4 secretion was increased when compared to OVA feeding alone, but this level was not higher than in control (unfed) mice.
  • Anti-IL-12 treatment also affected IL-10 secretion.
  • IL-10 secretion was considerably increased compared to fed-only mice.
  • the fact that IL-10 was not increased in the cultures of purified spleen T cells suggests that non-T cells were the source of the IL-10.
  • TGF ⁇ production spleen and PP whole cells and purified T cells from OVA-fed plus anti-IL-12 treated mice produced increased amounts of TGF ⁇ compared to OVA-fed-only and control mice; this effect was particularly evident in PP cells, where it was clear that cells from OVA-fed-only mice produced no more TGF ⁇ than controls, but cells from OVA-fed plus anti-IL-12 treated mice produced very high levels of TGF ⁇ .
  • the levels of TGF ⁇ produced by purified T cell populations from both spleen and PP were elevated to similar or higher levels than those with whole cells, suggesting that T cells and not non-T cells were the source of the TGF ⁇ .
  • TGF ⁇ is produced relatively late (peak levels after 72-120 hours) in culture (22), proliferation levels were measured after 116 hours in culture, a point when PP responses were suppressed by anti-IL-12 treatment.
  • Cells from both the spleen and the PP of the fed plus anti-IL-12 treated mice were found to induce a marked reduction in the proliferative responses to OVA when mixed in a 1: 1 ratio with cells of control mice. Such suppression, however, was not observed when cells of the fed mice were mixed with cells of the control mice.
  • the suppressed proliferation in the mixed cell cultures (19,940 + 1299 cpm) was reversed to the values expected (74,249+ 8107 cpm) if no suppression was occurring, i.e. values similar to the mean proliferation of the two cell populations alone.
  • the number of apoptotic PP cells after OVA feeding was observed to be increased when compared to PBS-fed mice.
  • An increased number of apoptotic cells after feeding and simultaneous administration of anti-IL-12 was also shown.
  • the overall number of apoptotic cells in the spleen was demonstrated to be lower than in the PP and no discernable difference could be seen between the treatment groups.
  • the degree of apoptosis was lower than that in the PP; nevertheless, the spleen contained a similar increase in KJ1-26+ apoptotic cells in the OVA-fed plus anti-IL-12 treated group, as compared with the fed-only group.
  • the TUNEL technique was also performed on cultured cells, demonstrating that the percentage of apoptotic PP and spleen cells was indeed negatively correlated with the proliferative responses (Table II).
  • oral antigen and antibodies to IL-12 to a human subject having an autoimmune disease, allergic disease, GvH disease or transplantation rejection.
  • 0.01 - 100 mg/kg of antigen can be administered orally to the subject and 1-100 mg/kg of antibodies to IL-12 can be administered parenterally one time each week over a two year period or until clinical parameters, i.e, signs, symptoms and objective laboratory tests with which clinicians in this field will be familiar, indicate prolonged remission, stabilization or improvement.
  • antibodies to IL-12 For oral administration of antibodies to IL-12, 500 to 1000 mg of antibodies to IL-12 can be administered in a single dose each day for up to two years or until various clinical parameters known to field clinicians in this field, i.e., signs, symptoms and objective laboratory tests, indicate prolonged remission, stabilization or improvement.
  • Rodent monoclonal or polyclonal antibodies can be modified according to the protocols set forth in Junghans et al. (50), Brown et al. (51) and Kettleborough et al. (52). Specifically, rodent antibodies can be modified for human administration by constructing, through recombinant DNA protocols known to one of skill in the art, a chimeric rodent-human antibody composed of rodent variable regions and human heavy and light chain constant regions. Another approach to humanizing rodent antibodies is to graft rodent complementarity-determining regions (CDRs) from the rodent variable regions into human variable regions. By using either of these approaches, rodent antibodies can be humanized for administration into human subjects.
  • CDRs complementarity-determining regions
  • T helper type 1 development of naive CD4 + T cells requires the coordinate action of interleukin-12 and interferon- ⁇ and is inhibited by transforming growth factor- ⁇ . Eur. J. Immunol. 24:793-798.
  • NKSF Natural Killer Cell stimulatory factor
  • NKSF natural killer cell stimulatory factor
  • IL-12 endogenous interleukin 12 regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL-12 both inhibits and prophylactically immunizes against egg pathology. J. Exp. Med. 179:1551-1561.
  • Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis. J. Exp. Med. 181 :387-391.
  • Table II Quantitation of apoptotic cells from the PP and the spleen by TUNEL technique
  • Cells from the PP and the spleen were prepared as described from control mice, mice fed OVA (3 x 250 mg), and mice fed plus treated with anti-IL-12. Dual color flow cytometry using clonotypic mAb KJ1-26 or mAb to B cells (B220) followed by fluorescent anti-digoxigenin antibody (which detects degraded DNA typical for apoptotic cells), or the latter antibody alone (“all cells”) was performed on freshly isolated or cultured cells (stimulated with 1 mg/ml OVA in vitro). Data represent the mean of duplicates of percentage apoptotic cells.

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Abstract

La présente invention concerne une méthode de stimulation de la tolérance orale à un antigène associé à une maladie auto-immune chez un sujet présentant ladite maladie auto-immune et consistant à lui administrer par voie orale un antigène associé à ladite maladie auto-immune ainsi qu'un inhibiteur d'interleukine-12 en des doses suffisantes pour stimuler la tolérance orale. L'invention concerne également une méthode de traitement ou de prévention d'une maladie auto-immune chez un sujet consistant à lui administrer par voie orale un antigène associé à la maladie auto-immune et un inhibiteur d'interleukine-12 en des doses suffisantes pour traiter ou prévenir ladite maladie, traitant ou prévenant ainsi la maladie auto-immune.
PCT/US1996/016007 1996-10-11 1996-10-11 Methodes de stimulation de la tolerance orale et de traitements d'une maladie auto-immune a l'aide d'inhibiteurs d'interleukine-12 WO1998016248A1 (fr)

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AU72576/96A AU7257696A (en) 1996-10-11 1996-10-11 Methods for enhancing oral tolerance and treating autoimmune disease using inhibitors of interleukin-12
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US6706264B1 (en) 1994-03-14 2004-03-16 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of conditions promoted by an increase in levels of IFN-y
US7041296B1 (en) 1999-11-12 2006-05-09 The United States Of America As Represented By The Department Of Health And Human Services Methods of treating inflammatory bowel disease using cholera toxin B subunit
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US8178092B2 (en) 2008-03-18 2012-05-15 Abbott Laboratories Methods of treating psoriasis by administration of antibodies to the p40 subunit of IL-12 and/or IL-23
US8557239B2 (en) 2009-09-14 2013-10-15 Abbvie Inc. Methods for treating psoriasis using antibodies that bind to the P40 subunit of IL-12 and/or IL-23
US8940873B2 (en) 2007-03-29 2015-01-27 Abbvie Inc. Crystalline anti-human IL-12 antibodies
US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
US9683255B2 (en) 2005-09-09 2017-06-20 Qiagen Gmbh Method for activating a nucleic acid for a polymerase reaction

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US6830751B1 (en) 1994-03-14 2004-12-14 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of rheumatoid arthritis
US8012475B2 (en) 1994-03-14 2011-09-06 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US7534430B2 (en) 1994-03-14 2009-05-19 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US7138115B2 (en) 1994-03-14 2006-11-21 Genetics Institute, Llc Use of IL-12 and IL-12 antagonists in the treatment of autoimmune diseases
US6706264B1 (en) 1994-03-14 2004-03-16 Genetics Institute, Llc Use of IL-12 antagonists in the treatment of conditions promoted by an increase in levels of IFN-y
US9072725B2 (en) 1998-12-09 2015-07-07 Abbvie Biotherapeutics, Inc. Method for treating psoriasis
EP1137766A4 (fr) * 1998-12-09 2002-08-14 Protein Design Labs Inc Modele animal du psoriasis destine a la prevention et au traitement du psoriasis humain
US9078876B2 (en) 1998-12-09 2015-07-14 Abbvie Biotherapeutics, Inc. Method for treating psoriasis
EP1137766A1 (fr) * 1998-12-09 2001-10-04 Protein Design Labs, Inc. Modele animal du psoriasis destine a la prevention et au traitement du psoriasis humain
US8765918B2 (en) 1999-03-25 2014-07-01 Abbott Gmbh & Co., Kg Human antibodies that bind human interleukin-12
US9035030B2 (en) 1999-03-25 2015-05-19 AbbVie Deutschland GmbH & Co. KG Human antibodies that bind the P40 subunit of human IL-12 and methods for using the same
US7504485B2 (en) 1999-03-25 2009-03-17 Abbott Gmbh & Co., Kg Human antibodies that bind human IL-12
US8865174B2 (en) 1999-03-25 2014-10-21 Abbvie Inc. Methods of treatment using human antibodies that bind IL-12
US7883704B2 (en) 1999-03-25 2011-02-08 Abbott Gmbh & Co. Kg Methods for inhibiting the activity of the P40 subunit of human IL-12
US7041296B1 (en) 1999-11-12 2006-05-09 The United States Of America As Represented By The Department Of Health And Human Services Methods of treating inflammatory bowel disease using cholera toxin B subunit
WO2001034175A2 (fr) * 1999-11-12 2001-05-17 The Government Of The United States Of America, Asrepresented By The Secretary, Dept. Of Health And Human Services Methodes de traitement de la maladie intestinale inflammatoire utilisant les sous-unites b de la toxine du cholera
WO2001034175A3 (fr) * 1999-11-12 2002-01-17 Us Health Methodes de traitement de la maladie intestinale inflammatoire utilisant les sous-unites b de la toxine du cholera
US7279459B2 (en) 2001-01-04 2007-10-09 Vascular Biogenics Ltd. Methods employing and compositions containing plaque associated molecules for prevention and treatment of atherosclerosis
US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
US9683255B2 (en) 2005-09-09 2017-06-20 Qiagen Gmbh Method for activating a nucleic acid for a polymerase reaction
US7776331B1 (en) 2007-01-16 2010-08-17 Abbott Laboratories Methods of treating plaque psoriasis
US9051368B2 (en) 2007-01-16 2015-06-09 Abbvie, Inc. Methods for treating psoriasis by administering an antibody which binds an epitope of the p40 subunit of IL-12 and/or IL-23
US8940873B2 (en) 2007-03-29 2015-01-27 Abbvie Inc. Crystalline anti-human IL-12 antibodies
US8178092B2 (en) 2008-03-18 2012-05-15 Abbott Laboratories Methods of treating psoriasis by administration of antibodies to the p40 subunit of IL-12 and/or IL-23
US8945545B2 (en) 2008-03-18 2015-02-03 Abbvie Inc. Methods of treating psoriasis by administration of antibodies to the p40 subunit of IL-12 and/or IL-23
US8557239B2 (en) 2009-09-14 2013-10-15 Abbvie Inc. Methods for treating psoriasis using antibodies that bind to the P40 subunit of IL-12 and/or IL-23

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