WO1996039176A1 - Utilisation de la tolerance orale pour supprimer les reponses immunitaires de th1 et de th2 et pour supprimer la production d'anticorps - Google Patents

Utilisation de la tolerance orale pour supprimer les reponses immunitaires de th1 et de th2 et pour supprimer la production d'anticorps Download PDF

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WO1996039176A1
WO1996039176A1 PCT/US1996/010386 US9610386W WO9639176A1 WO 1996039176 A1 WO1996039176 A1 WO 1996039176A1 US 9610386 W US9610386 W US 9610386W WO 9639176 A1 WO9639176 A1 WO 9639176A1
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ova
mammal
antigen
disease
cells
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PCT/US1996/010386
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Youhai Chen
Howard L. Weiner
Aharon Friedman
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Brigham & Women's Hospital
Yissum Research Development Company Of The Hebrew University Of Jerusalem
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Priority to AU61138/96A priority Critical patent/AU6113896A/en
Publication of WO1996039176A1 publication Critical patent/WO1996039176A1/fr

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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
    • A61K39/0005—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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal

Definitions

  • This invention relates to methods for suppression of specific Th2 (as well as Thl) immune responses and antibody production and finds applicability in the treatment of antibody-mediated autoimmune diseases.
  • Th2 lympho ⁇ cytes Difficulties in generating tolerance of Th2 lymphocytes in vi tro (6-8) or of Th2-mediated antibody production in vivo have been encountered in other experimental systems in which tolerance was induced by intravenous (I.V.) or intraperitoneal (I.P.) administration of soluble antigens (9-12) .
  • I.V. intravenous
  • I.P. intraperitoneal
  • IL-2 and IFN ⁇ were not produced in cultures, and a diminished IgG2a antibody response was observed in vivo (tolerance of Thl responses) .
  • B-cells have been even more resistant to suppression by tolerization techniques.
  • Th2 lymphocytes play an important role in antibody production, successful suppression of Th2 responses would prove a useful tool in suppressing abnormal antibody-mediated immune responses.
  • Oral tolerance is a clinically attractive method to treat immune dysfunctions (such as autoimmune diseases) for several reasons:
  • Thl-mediated autoimmune disease oral tolerance results in active suppression, i.e., elicitation of antigen specific T-cells which are or include Th2 cells and which are targeted to the afflicted tissue and exert local suppressive effect.
  • active suppression i.e., elicitation of antigen specific T-cells which are or include Th2 cells and which are targeted to the afflicted tissue and exert local suppressive effect.
  • Methods and compositions useful in suppression of an immune response associated with a T-cell mediated or T-cell dependent autoimmune disease by orally induced tolerance (or by tolerance induced by inhalation) using daily or less frequent administration of autoantigens or more generally bystander antigens with and without enhancers have been described in various patents and patent applications by the present inventors and their co-workers: Ser. No. 07/843,752, filed February 28, 1992; Ser. No. 08/202,677, filed February 25, 1994; Ser. No.
  • immune dysfunctions are antibody-mediated autoimmune diseases, (for example certain aspects of systemic lupus erythematosus, autoimmune thyroiditis, myasthenia gravis, glomerulonephritis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pemphigus vulgaris, Grave's disease, insulin resistance (encountered in Type II diabetes) , and pernicious anemia) .
  • autoimmune diseases for example certain aspects of systemic lupus erythematosus, autoimmune thyroiditis, myasthenia gravis, glomerulonephritis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pemphigus vulgaris, Grave's disease, insulin resistance (encountered in Type II diabetes) , and pernicious anemia.
  • Another object is to devise methods and compositions for suppressing abnormal antibody-mediated immune responses.
  • a further object is to devise methods and compositions for treating antibody-mediated autoimmune responses, in order to treat antibody-mediated autoimmune diseases.
  • the present invention relates to a method for treating a mammal suffering from an antibody- mediated autoimmune disease comprising orally administering to said mammal at least one autoantigen (as defined below) specific for said disease; and continuing said administration for a period of time until a Th2 cell mediated autoimmune response associated with said disease is suppressed.
  • the amount of said antigen, the schedule (frequency) of said administration, and the period of time are selected to effect said suppression.
  • the present invention relates to a method for treating a mammal suffering from an antibody- mediated autoimmune disease comprising administering to said mammal via the oral route an autoantigen specific for said disease for a period of time sufficient to accomplish at least one of the following: reduce the number of autoreactive Th2 cells in said mammal recognizing said autoantigen; reduce the number of autoreactive antibodies in said mammal recognizing said autoantigen; and eliminate or decrease the severity of at least one clinical symptom or indicator associated with said disease.
  • the present invention relates to a method for treating a mammal suffering from an antibody- mediated autoimmune disease comprising parenterally administering to said mammal at least one autoantigen specific for said disease; and continuing said administration until a Th2 cell mediated autoimmune response associated with said disease is suppressed.
  • Th2 Th2
  • Thl Th2
  • Other aspects of the invention relate to suppression of a Th2 (or both Th2 and Thl) response associated with antibody-mediated autoimmune disease by oral administration of at least one autoantigen specific for the antibody-mediated autoimmune disease in an amount, a frequency of administration and for a period of time effective to suppress said response.
  • Figures 1A and IB are plots of spleen cell proliferation averages ⁇ SEM in absorbance units at 570-630nm against stimulating antigen concentration in vitro. Mice were continuously exposed to OVA in drinking water for 20 days and primed by OVA-CFA (open triangles) . Control mice were primed by OVA-CFA (filled circles) or CFA alone (open circles) . Pooled erythrocyte depleted spleen cells were prepared 10 days after immunization and stimulated by different concentrations of OVA (Fig. 1A) or PPD (Fig. IB) . Figures 2A. 2B.
  • 2C and 2D are plots of antibody titers (averages ⁇ SEM) from individual mice (for all isotypes) expressed in absorbance units at 405 nm against reciprocal serum dilution. Mice were continuously fed with OVA (open triangles) . Control mice were primed by OVA-CFA (filled circles) or by CFA alone (open circles) . Serum samples were collected 15 days after immunization and individually assayed for IgG2a (Fig. 2A) , IgG2b (Fig. 2B) , IgGl (Fig. 2C) and IgE (Fig. 2D) .
  • Figure 3 is a plot of OVA specific cytokine release or antibody titers v. amount of fed antigen. Mice were continuously fed with different dosages of OVA for 20 days and primed by OVA-CFA. Cytokine (circles) and antibody (triangles) secretion respectively are shown for IL-2 (solid circles) and IL-4 (open circles) . Cytokine results are averages ⁇ SEM of quadruplicate cultures and are expressed in absorbance units at 570-630nm. IgG2a (solid triangles) and IgGl (open triangles) are also shown. Antibody titers are averages ⁇ SEM of titers from individual mice and are expressed in absorbance units at 405nm.
  • Figure 4 is a plot of average antibody binding ⁇ SEM from individual mice at a serum dilution of 1:400, presented in absorbance units at 405 nm against time of exposure to anti- gen. IgGl are shown by open circles and IgG2a are shown by solid circles.
  • Figure 5A is a plot of the percentage of V ⁇ 8.2+ T- cells which are also CD4+ versus antigen (OVA) feedings over feeding frequency. Mice were fed 0.5 (open circles), 5 (filled squares) or 500 mg (filled circles) of OVA every other day for five feedings, and T-cells were harvested for analysis prior to, and 24 hours after, each feeding.
  • OVA antigen
  • Figures 5B and 5C are fluorescence contour plots showing the probability of incidence of T-cells of various subtypes; CD4+ cells are depicted in the upper right-hand quadran .
  • Figure 6A is a plot of the percentage of V38.2+ cells which are undergoing apoptosis versus antigen (OVA) feedings over feeding frequency.
  • V08.2+ cells from mice fed 0.5 (filled circles) , 5 (filled squares) , and 500 mg (closed circles) were analyzed for the presence of degraded DNA which indicates apoptosis by staining with acridine orange.
  • Figures 6B and 6C are Forward Angle Light Scatter (FALS) plots showing the incidence of acridine orange staining (indicating dying cells) for various T-cell subtypes.
  • FALS Forward Angle Light Scatter
  • Figure 7A is a plot of percentage of V/38.2+ cells which are actively dividing versus antigen (OVA) feeding frequency.
  • V/38.2+ cells from mice fed 0.5 (open circles), 5 (filled squares) , and 500 mg (filled circles) were analyzed for DNA content which indicates active division, by staining with propidium iodide.
  • Figures 7B and 7C are FALS plots showing the incidence of DNA content for various T-cell subtypes from control(B) and OVA-fed animals.
  • Figure 8A-8E are plots showing the concentrations of cytokines IL-2 (8A) , IL-4 (8B) , IL-10 (8C) , (all in pg/ml) and IFN-Y (8D) and TGF-0 (8E) (both in ng/ml) versus antigen (OVA) feeding frequency.
  • “Mammal” is defined herein as any warm-blooded higher vertebrate organism (including a human) having an immune system and being susceptible to an autoimmune disease.
  • Autoimmune disease is defined herein as a spontaneous or induced malfunction of the immune system of mammals, including humans, in which the immune system fails to distinguish between foreign immunogenic substances within the mammal and/or autologous substances and, as a result, treats autologous tissues and substances as if they were foreign and mounts an immune response against them.
  • the term includes human autoimmune diseases and animal models therefor.
  • Autoantigen is any substance or a portion thereof normally found within a mammal that invokes an immune response within an individual, i.e. that is recognized by activated T- cells of the mammal or by antibodies in the mammal.
  • an antigen may be or may become the primary (or a primary) target of attack by the immunoregulatory system.
  • the term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes peptidic subclasses consisting essentially of immunodominant epitopes or immunodominant epitope regions of autoantigens.
  • Immunodominant epitopes or regions in induced autoimmune conditions are fragments or portions of an autoantigen that can be used instead of the entire autoantigen to induce the disease.
  • immunodominant epitopes or regions are fragments or portions of antigens specific to the tissue or organ under autoimmune attack and recognized by a substantial percentage of autoimmune attack T-cells or antibodies from a patient or a group of patients. See, e.g., 08/426,784 for determination of T-cell immunodominant epitopes.
  • Autoantigens and their immunodominant epitopes that elicit antibodies can be identified by antibody binding tests, ELISA assays or dot blot analysis using whole antigens or peptide fragments of a particular autoantigen (overlapping peptide method) .
  • Treatment of an autoimmune disease is intended to include both treatment to prevent or delay the onset of an autoimmune disease (or to prevent or delay the manifestation of clinical or subclinical, e.g., histological, symptoms thereof) , as well as therapeutic suppression or alleviation of symptoms after the manifestation of such autoimmune disease. In either case, treatment is accomplished by abating autoimmune attack and preventing or slowing down autoimmune tissue destruction. "Abatement”, “suppression” or “reduction” of autoimmune attack or reaction encompasses partial reduction or amelioration of one or more symptoms of the attack or reaction, i.e. reduction in number of activated autoreactive T-cells or in number of autoreactive antibodies.
  • a “substantially” increased suppressive effect (or abatement or reduction) of autoimmune reaction means a significant decrease in one or more markers or histological or clinical indicators of autoimmune reaction or disease.
  • Nonlimiting examples of symptoms associated with various autoimmune diseases are given below.
  • an improvement in one or more symptoms reported by the patient (e.g. fatigue) or observed by the attending physician or determined by quantitative or semiquantitative techniques can be used to assess efficacy of treatment according to the invention.
  • patient improvement can be assessed by assay to determine whether there has been a significant reduction in the frequency of autoreactive T-cells; or in the frequency of autoreactive antibodies, or both.
  • Oral administration includes oral, enteral or intragastric administration, and more generally any administration of an active ingredient that brings the ingredient in contact with the immune system at the gut-associated lymphoid tissue.
  • Parenteral administration includes subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal or intrathecal administration. Intravenous administration is preferred. Parenteral administration must be free of co- stimulatory substances which might cause an undesirable immune reaction.
  • the present inventors conceived that it should be possible to suppress both Thl and Th2 abnormal immune responses (whether these responses were directed against an external antigen or a self-antigen) via orally or parenterally induced tolerance.
  • the present inventors observed that maintenance of exposure to antigen appeared to be important for the persistence of tolerance in various different contexts: orally induced tolerance to OVA antigen in mice immunized with OVA
  • the simplest system for testing this was to make defined quantities of antigen available to the experimental animals throughout their daily activity period while being able to assess antigen consumption as well as its frequency. It was therefore decided to add the antigen used for immunization to the animals' drinking water.
  • this oral tolerization treatment i.e. the treatment involving prolonged exposure to antigen through "multi-dose administration" as defined below
  • multi-dose administration as defined below
  • the T-cells of the subject react to an antigen they recognize regardless of whether that antigen is endogenous (as in autoimmunity) or not (as in the present experimental models) .
  • Multi-dose daily oral administration of antigen to a subject to be treated i.e. a subject that mounts an immune response to that antigen
  • Multi-dose administration or “multi-dose exposure” or “exposure to multiple doses” encompasses administration occurring at a plurality of spaced apart intervals during the same day as further described below, as well as continuous administration either by intragastric or parenteral infusion, or by ingestion of a sustained-release dosage form. The term thus refers to a schedule or frequency of administration.
  • Th2 lymphocyte responses entrained (and was confirmed by) profoundly suppressed numbers of in vivo secreted antigen- specific antibodies, including IgGl and IgE (which are controlled by Th2 cytokines, with IgE being considered to be exclusively Th2-controlled) as well as IgG2a and IgG2b (which are controlled by IFN- ⁇ , a Thl cytokine) .
  • IgGl and IgE which are controlled by Th2 cytokines, with IgE being considered to be exclusively Th2-controlled
  • IgG2a and IgG2b which are controlled by IFN- ⁇ , a Thl cytokine
  • Thl and Th2 responses to the multi- dose fed antigen were confirmed by: (i) failure of T-cells from animals fed antigen by multi-dose administration to proliferate in vitro to the fed antigen (which also had been used for immunization) , as well as by (ii) suppression of Thl and Th2 cytokine secretion and cytokine gene expression by the tolerized T-cells. In fact, suppression of cytokines was demonstrated by ELISA, CT.4S cell proliferation and RT-PCR (reverse transcriptase polymerase chain reaction) . These tests demonstrate tolerization of Th2 lymphocytes.
  • the present inventors were the first to achieve complete tolerization of Th2 lymphocytes as demonstrated both in vitro and in vivo.
  • the present inventors determined further that even larger amounts of antigen orally administered also bring about suppression of Th2 (and Thl) responses even when not administered in multiple daily doses. Specifically, in experiments involving transgenic mice expressing essentially only a T-cell receptor specific to OVA (Vc ⁇ l3/VS8.2TcR) , considerable suppression of both Th2 and Thl responses was achieved by feeding large amounts of antigen intermittently in single doses. The mechanism of suppression is substantially through deletion of antigen-specific Th2 and Thl cells. The inventors concluded that this approach can be used to induce tolerance in Th2-mediated (antibody-mediated) responses associated with autoimmune disease.
  • This "high-dose” oral tolerization in which antigen can be administered only once daily or according to a less frequent administration schedule, as described below) can be used as an alternative or an adjunct to multi-dose oral tolerization.
  • subjects to be treated will be administered both high doses of antigens and multiple daily doses.
  • the animal model used involved animals that have only T-cells that are reactive with the same antigen as that used for immunization (OVA) .
  • OVA immunization
  • Multi-dose daily autoantigen administration achieves suppression of Th2 as well as Thl responses.
  • Thl Th2 as well as Thl responses.
  • suppression of only Thl requires only relatively modest amounts of antigen and intermittent administration: for example, in humans, rheumatoid arthritis symptoms have been suppressed with as little as 0.1 mg of collagen II administered once a day, for one month, followed by administration of 0.5 mg collagen II administered daily for two months.
  • mice need to be fed with a multi-dose daily regimen requiring relatively large amounts of antigen (see infra) for more than 15 and preferably at least 20 days.
  • Th2 responses can also be achieved in mice by single dose intermittent (e.g., once daily, every other day or twice a week) administration of even higher doses of antigen. Again, continuing the administration over a period of time increases suppression.
  • the amount of antigen effective to suppress Th2 responses to that antigen depends partially on the schedule (frequency) of its administration. Suppression, even complete suppression of Th2 responses to a particular antigen (OVA) can be achieved by administering e.g. 4 mg of antigen in several divided daily doses to mice whereas the same amount (4 mg) of OVA, if administered once a day, achieved only a slight suppression of IL-2 (see, e.g. Table III) .
  • a substantially higher amount of an antigen is needed to suppress Th2 responses if the antigen is administered once daily or intermittently. Suppression of Th2 responses is typically achieved after a number of feedings, in mice, typically three or more. Thus after three feedings of 5 or 50 mg of antigen on alternate days partial OVA-specific Th2 suppression (predominately T-cell deletion) was achieved in mice. Substantially more profound suppression resulted from feeding 500 mg of OVA according to the same schedule.
  • an autoantigen should be administered orally at least three and preferably at least five or six times a day at spaced apart intervals (e.g. with and/or between meals) .
  • the total daily dosage of multi-dose administration will be within the general range of 2.5 - 1,000 mgs of autoantigen depending on the autoantigen, preferably within the range of 15 - 500 mgs divided among several dosages (as stated above) . No maximum effective number of dosages or total daily intake of antigen has been discerned. Twelve daily doses provide a practical limit.
  • antigen When high-dose intermittent administration is used in humans, antigen could be administered less often: twice daily, once daily, three-times weekly, twice weekly or once a week. In that event, the amount of administered antigen should be within the range of 30 mg - 10 g per treatment depending on the antigen.
  • the duration of treatment in humans should be a minimum of two weeks, and typically three months, and may be continued indefinitely or as long as benefits persist.
  • the treatment may be discontinued if desired (in the judgment of the attending physician) and the patient monitored for signs of relapse. If clinical symptoms or other disease indicators show that the patient is relapsing, treatment may resume.
  • the dosage will vary with the disease, the antigen administered and may vary with the sex, age, and physical condition of the patient as well as with other concurrent treatments being administered. Consequently, adjustment and refinement of one or both of the dosages used and the administration schedules will preferably be determined based on these factors and especially on the patient's response to the treatment. Such determinations, however, require no more than routine experimentation, as illustrated in Examples A-C below.
  • Suitable antigens include autoantigens (as defined in the present detailed description) specific for a particular antibody-mediated autoimmune disease.
  • Nonlimiting examples of autoantigens for each of various antibody-mediated autoimmune diseases are:
  • Additional autoantigens can be identified by screening antigens for binding with antibodies or activated T- cells from the patient.
  • tissue extracts i.e., tissue lysates
  • Suitable formulations for use in tolerization of Th2- responses can be in any suitable orally administrable form.
  • a pill for example, a liquid, a capsule containing an effective amount of antigen.
  • Each oral formulation may additionally comprise inert constituents including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents and salts as is well-known in the art.
  • tablets may be formulated in accordance with conventional procedures implying solid carriers well-known in the art.
  • Capsules may be made from any pharmaceutically acceptable materials, such as gelatine or cellulose derivatives.
  • solid carriers include starch, sugar, bentonite, silica and other commonly used inert ingredients.
  • Diluents can include inter alia saline, syrup, dextrose and water.
  • the autoantigens used in the present invention can also be made up in liquid formulations or dosage forms such as, for example, suspensions or solutions in a physiologically acceptable aqueous liquid medium.
  • liquid media include water, or suitable beverages, such as fruit juice or tea which will be convenient for the patient to sip at spaced apart intervals throughout the day.
  • the antigen When given orally in liquid formulations the antigen may be dissolved or suspended in a physiologically acceptable liquid medium, and for this purpose the antigen may be solubilized by manipulation of its molecule
  • the antigen may be reduced to micronized form and suspended in a physiologically acceptable liquid medium.
  • the antigen should be administered in a solution.
  • sustained release oral dosage forms include those described in U.S. Patent No. 4,704,295 issued November 3, 1987; No. 4,556,552 issued December 3, 1985; No.4,309,404 issued January 5, 1982; No. 4,309,406 issued January 5, 1982; No. 5,405,619 issued April 10, 1995; WO 85/02092 published May 23, 1985; No. 5,416,071 issued May 16, 1995; No. 5,371,109 issued December
  • Sustained release oral dosage forms coated with bioadhesives are preferred.
  • Examples are compositions disclosed in EP 516,141; No. 4,226,848, Nagai et al., Oct. 1980; No. 4,713,243, Schiraldi et al., Dec. 1987; No. 4,940,587, Jenkins et al., July 1990; WO 85/02092; EPO 0 205 282; Smart, et al., J. Pharm. Pharmacol. 36:295-99. 1984; Sala et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 16_:420-21, 1989; Hunter et al., International Journal of Pharmaceutics 37:59-64, 1983; Bioadhesion - Possibilities and Future Trends, Kellaway, Course No. 470, May 22-24, 1989.
  • Preferred commercially available sustained release formulations and devices include those marketed by ALZA Corporation, Palo Alto, CA, under tradenames ALZET, INFUSET, IVOS, OROS, OSMET, or described in one or more U.S. patents: No. 5,284,660 issued Feb. 9, 1994; No. 5,141,750 issued Aug. 25, 1992; No. 5,110,597 issued May 5, 1992; No. 4,917,895 issued April 17, 1990; No. 4,837,027 issued June 6, 1989; No. 3,993,073 issued Nov. 23, 1976; No. 3,948,262 issued April 6, 1976; No. 3,944,064 issued March 16, 1976; No.3,699,963; PCT/US93/10077; PCT/US93/11660; EP 259013; and EP 354742.
  • Sustained release compositions and devices are particularly adapted for use in the present invention because they serve to prolong contact between the antigen and the gut- associated lymphoid tissue (GALT) and thus prolong contact between the antigen and the immune system.
  • sustained release compositions obviate the need for discrete multi-dose administration of the antigen and permit the required amount of antigen to be delivered to GALT in one or two daily doses. This substantially improves patient compliance. Parenteral Administration of Antigen -
  • An alternative method within the scope of the present invention for accomplishing tolerance of Th2 responses is a method comprising prolonged parenteral infusion of a subject to be treated with an autoantigen.
  • the duration of each treatment will be, for example, from about 1 hour to about 24 hours of continuous infusion repeated at intervals of
  • a preferred route of parenteral administration is intravenous administration.
  • Suitable parenterally deliverable amounts of antigen are within the range of about 2.5 to about 250 mg of antigen.
  • Suitable formulations include sterile solutions of antigen suitable for parenteral administration in an appropriate medium (e.g., saline in distilled water etc.). Buffers, emulsifiers, salts and other optional ingredients suitable for such preparations can be included.
  • Purified antigen should be administered without co-stimulatory factors (which would induce an immune response against the antigen) . Suppression is accomplished by anergy or clonal deletion.
  • mice Female BALB/C mice, 6-8 weeks of age, were used in all experiments. The mice were bred at the department of Animal Sciences, Hebrew University of Jerusalem, Rehovot, Israel, where they are periodically crossed with wild-type Balb/c mice. The animals were maintained in a temperature and light-controlled environment with free access to feed and water. During experiments mice fed with OVA intermittently several times during the day (see below) had free access to OVA solution in water, instead of water alone. Each experimental group contained no less than 5 mice.
  • OVA ovalbumin
  • mice were fed 20 intermittent boluses of OVA in water (4 mg OVA/feeding) over the period of 20 days.
  • Mice were immunized against OVA by injecting 20 ⁇ g OVA/mouse, either emulsified 1:1 in CFA, or absorbed by 1 mg (Al(OH) 3 .
  • Injections 100 ⁇ l/mouse were administered IP (for spleen and mesenteric lymph node [LN] analysis) or subcutaneously in hind foot-pads (for popliteal LN analysis) .
  • Presence of IgGl, IgG2a and IgG2b serum antibodies specific for OVA was tested by ELISA as described (4, 18) .
  • Serial two-fold dilutions of anti-OVA antisera were placed on ELISA plates (Nunc, Denmark) , previously coated with OVA, followed by biotinylated rat anti-mouse IgGl, IgG2a or IgG2b monoclonal antibodies (Pharmingen, San Diego, CA) , and finally by peroxidase-streptavidin (Kirkegaard & Perry Laboratories).
  • Spleen erythrocyte depleted
  • lymph node (LN) cell popliteal and mesenteric
  • Cultures contained pooled cells from no less than 5 mice.
  • the proliferation of T lymphocytes in spleen and LN cell cultures was assayed as described (14,19). Proliferation was measured by MTT oxidation (14,19) , and results are averages of quadruplicate cultures expressed in absorbance units (at 570- 630 nm) ⁇ SEM.
  • cytokine secretion 1 x 10 7 cells/well (in 1 mL) were cultured in 24 well plates (Nunc, Denmark) with or without OVA 1 mg/mL) . Cytokine secretion was determined temporally in supernatants collected from these cultures and was ascertained to be optimal after 9 hours culture for IL-4 detection, 20 hours for IL-2 detection, and 48 hours for IFN detection. Collected supernatants were frozen and stored at - 70°C till assayed (see below) . For the assay of cytokine gene expression, 5 x 10 7 spleen cells/ml were cultured with or without OVA (1 mg/mL) for 6 hours.
  • DMEM was used for all cultures, and was supplemented with 100 U/ml penicillin, 100 ⁇ g/mL streptomycin, 2 mM L- glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential a ino acids (all supplied by Biological Industries, Beit Haemek, Israel) , 5 x 10 _5 M 2-mercaptoethanol and 0.5% syngeneic serum.
  • IL-4, IL-2 and IFN ⁇ in supernatants were determined by capture ELISA as described (3,4,20) . Briefly, supernatants were added to microtiter plates, previously coated with rat anti-mouse IL-4, IL-2 or IFN ⁇ monoclonal antibodies
  • IL-4, IL-2 or IFN7 monoclonal antibodies (detecting antibodies, Pharmingen) were added and followed by peroxidase-labeled streptavidin. Bound cytokine was detected by the addition of ABTS (Kirkegaard & Perry) . Cytokine levels were calculated from a log-log plot of absorbance vs. concentration of reco binant cytokines (Pharmingen) , and results are expressed in pg/mL (for IL-4 and IL-2) or ng/mL (for IFN ⁇ ) .
  • Threshold sensitivities of ELISA assays were 5 pg/mL, 10 pg/mL and 2.5 ng/mL for IL-4, IL-2 and IFN ⁇ , respectively.
  • IL-2 and IL-4 levels were also determined by bio- assay using the CTLL-2 (IL-2 dependent) and CT.4S (IL-4 dependent; kindly provided by Dr. W.E. Paul, NIH, Bethesda, MD) cell lines as described (4) .
  • PCR using cytokine specific primers.
  • 3-actin, IL-2 and IFN ⁇ primer sequences were from Stratagene (La Jolla, CA) .
  • IL-4 sequences were as follows: IL-4 sense: 5'- CAGCTAGTTGTCATCCTGCTC-3' (76-97) and IL-4 antisense: 5'- CAGGAAGTCTTTCAGTGATGTGAA-3' (445-421). All primers used spanned genomic introns such that any contaminating genomic DNA was detected by a higher molecular weight band. Quantitative PCR conditions were first established for all sets of primers using either cDNA from Con A activated spleen cells or plasmid DNA.
  • Tested cDNA samples were amplified undiluted and with two additional two-fold dilutions with cytokine specific primers, and at 1/100, 1/200 and 1/400 serial dilutions with /S-actin primer sequences. ' The ratio of cytokine mRNA expression relative to j8-actin was obtained for each dilution and expressed as the mean ratio ⁇ SE (see all columns in Table III, except for the columns labeled "ratio" which show the ratio of the value of the column labeled OVA over the value of the column labeled "medium” and thus compares the OVA readings against the background) .
  • Example 1 Exposure of mice to multiple doses of OVA induces OVA-specific T lymphocyte unresponsiveness in vitro.
  • mice were exposed (orally tolerized) in their drinking water (ad libitum consumption) . Exposure was continued for 20 days, and then mice were immunized by OVA-CFA. Control mice were primed by OVA-CFA or
  • Fig. 1A T lymphocytes did not proliferate in response to OVA but exhibited a dose dependent response to PPD which was similar to that of the OVA- primed, non-tolerant control group (Fig. 1A-1D ; P ⁇ 0.05 for all antigen doses, representative of 6 experiments) .
  • Fig. 1A-1D P ⁇ 0.05 for all antigen doses, representative of 6 experiments.
  • Identical observations were made with LN (popliteal and mesenteric) cell cultures, with other protein antigens (human serum albumin [HSA] , and hen egg lysozyme (HEL) and with Al(OH) 3 as adjuvant (data not shown) .
  • HSA human serum albumin
  • HEL hen egg lysozyme
  • FIG. 1A and IB depict graphically the results of experiment involving multi-dose oral exposure to OVA and show that such exposure diminishes T lymphocyte proliferation response in vi tro.
  • Mice were multi-dose exposed to OVA in drinking water for 20 days and primed by OVA-CFA (open triangles) .
  • Control mice were primed by OVA-CFA (filled circles) or CFA alone (open circles) .
  • Control mice were primed by OVA-CFA (filled circles) or CFA alone (open circles) .
  • mice 5 mice, and are expressed in absorbance units at 750-630 nm.
  • Example 2 Determination of absence of both Thl and Th2 cytokine secretion in cultures derived from mice exposed to multiple doses of OVA in drinking water.
  • Table 1 the results in Table 1 were generated as follows: pooled, erythrocyte-depleted, spleen cells (10 x 10 6 /mL) were cultured 10 days after immunizations with or without OVA or PPD (1 mg/mL) . Cytokine secretion was determined by ELISA. Cytokine levels were calculated from a log-log plot of absorbance vs concentration of recombinant cytokines. Results are averages of quadruplicate cultures pooled from at least 5 mice ⁇ SEM. Values in bold lettering indicate significant cytokine secretion above threshold levels (P ⁇ 0.05) .
  • mice exposed to multiple doses of OVA did not secrete IL-4, IL-2 or IFN ⁇ in response to OVA (not at the designated time points and not at any other time point during a 48-hour culture period) , suggesting that both Th2 (IL-4 secretors) and Thl (IL-2 and IFN ⁇ secretors) OVA specific lymphocytes were tolerant.
  • Example 3 Determination that mice exposed to multi-doses of OVA in drinking water do not express Thl and Th2 cytokine ⁇ enes. To further evaluate the level of tolerance induced by exposing mice to multiple doses of OVA, we measured cytokine gene expression in response to OVA.
  • mice multi-dose fed or non-fed controls, were immunized by OVA-CFA; an additional non-fed group was primed by CFA alone.
  • Spleen cell cultures were prepared 10 days after immunization, incubated with or without OVA, and cells were collected to determine IL-4, IL-2 and IFN ⁇ mRNA expression by quantitative PCR (Table 2, representative of 3 experiments) .
  • the results in Table 2 were generated as follows: mice were exposed to multiple doses of OVA in drinking water for 20 days (1 mg/mL OVA in water) and then immunized I.P. by OVA-CFA. Control non-fed mice were primed by OVA-CFA.
  • erythrocyte-depleted spleen cells (5 x 10 7 /ml) were cultured 10 days after immunizations with or without OVA (1 mg/mL) for 6 hours. Cells were collected and total RNA was isolated. mRNA was reverse transcribed into cDNA and the expression levels of IL-4, IL-2, IFN ⁇ and ⁇ -actin messages were determined by quantitative PCR using cytokine specific primers, as described in Materials and Methods. Visualized bands were quantitated using a ⁇ -scope, and cytokine mRNA expression values are relative to those of S-actin mRNA used as an internal control ⁇ SEM. In addition, mRNA expression is presented as the ratio between mRNA expressed in response to OVA and that expressed in response to medium alone. Values in bold lettering indicate significant mRNA expression above levels expressed in response to medium alone (P ⁇ 0.05) .
  • mice were primed by OVA-CFA or CFA alone. Serum samples were collected temporally after immunization (15-60 days) and analyzed for OVA-specific IgGl, IgE, IgG2a and IgG2b secretion (Figs. 2A-2D, antibody titers in serum 15 days after immunization, representative of 5 experiments) .
  • Figure 2 depicts graphically the results of experiments in which mice were multi-dose fed with OVA for 20 days (1 mg/ml) and primed by OVA-CFA (open triangles) . Control mice were primed by OVA-CFA (filled circles) or by CFA alone
  • mice fed multiple doses of OVA did not produce any detectable antibody response to OVA; thus anti-OVA IgGl and IgE, as well as IgG2a and IgG2b, levels were completely diminished.
  • Example 5 Characterization of the tolerogenic signal required for tolerization of Th2 lymphocytes.
  • Thl lymphocytes Since selective tolerization of Thl lymphocytes was accomplished by means of a different feeding regimen (by a single or an intermittent feeding regimen; see e.g. ref. 4), it was of interest to determine comparative requirements for inducing Th2 lymphocyte tolerance.
  • Th2 lymphocytes The importance of the feeding regimen for tolerization of Th2 lymphocytes was studied by comparing the degree of tolerance generated by multi-dose exposure to OVA in drinking water for 20 days to that generated by an intermittent
  • mice were then immunized by OVA-CFA, and responses of both groups to OVA were compared 15 days after immunization (Table 3, representative of 4 experiments) .
  • mice were either exposed to multiple doses of OVA (1 mg/mL) or received daily boluses containing 4 mg/mL for 20 days. Details of cytokine measurement are as in Table 1; concentrations are pg/mL for IL-2 and IL-4, and ng/mL for IFN ⁇ . OVA specific responses are averages of quadruplicate cultures ⁇ SEM, and values in bold lettering indicate significant cytokine secretion above background threshold levels (see Table 1) (P ⁇ 0.05) .
  • Serum IgG2a and IgE were measured 15 days after immunization by isotype specific ELISA, and anti-OVA specific responses are expressed in absorbance units at 405 nm.
  • the values of IgG2a and IgE are from serum dilutions of 1:100 or 1:40 respectively, and are averages of at least 5 individual mice ⁇ SEM. Values in boldface indicate significant secretion as compared to levels in naive serum (mean absorbance 0.15 ⁇ 0.02) (P 0.05).
  • Thl and Th2 tolerance resulted from the multi-dose feeding regimen (both in vitro cytokine production and in vivo antibody secretion) , whereas the intermittent feeding regimen caused selective Thl tolerance while Th2 responses (cytokine and antibody) were unchanged, apart from a minor reduction in IL-4 secretion (P ⁇ 0.05) .
  • the method of antigen exposure multi-dose daily vs. intermittent
  • the minimal antigen dosage required for effective tolerization of Th2 lymphocytes was determined by multi-dose exposure of mice to different concentrations of OVA in drinking water (0-1 mg/mL) for 20 days.
  • OVA specific cytokine production in vitro and anti-OVA IgGl and IgG2a production were assayed 15 days after immunizing mice with OVA-CFA (Fig. 3, representative of 4 experiments) .
  • mice were multi-dose fed with different dosages of OVA for 20 days, and primed by OVA-CFA.
  • Spleens and serum samples were collected 15 days after immunization to determine cytokine (circles) and antibody (triangles) secretion respectively.
  • Erythrocyte- depleted spleen cells were cultured as detailed in the description of the experiment for Table 1, and supernatants were added to cultures of CTLL-2 or CT.4S cells for detection of IL-2 (solid circles) and IL-4 (open circles) respectively.
  • Results are averages ⁇ SEM of quadruplicate cultures and are expressed in absorbance units at 570-630 nm.
  • IgG2a solid triangles
  • IgGl open triangles
  • Antibody titers are averages of antibody binding ⁇ SEM from individual mice at a serum dilution of 1:400 and are expressed in absorbance units at 405nm. Each experimental group contained at least 5 mice.
  • Results in Fig. 3 show that tolerization of cytokine secretion in vi tro and antibody responses in vivo required multi-dose exposure to 1 mg/mL OVA (P ⁇ 0.05).
  • Thl and Th2 controlled responses required dosages of this antigen in excess of 0.1 mg/mL.
  • the minimal period required for effective tolerization of Th2 lymphocytes was determined by evaluating the temporal degree of tolerance generated in vivo by exposing mice to multiple doses of OVA (l mg/mL) . Mice were immunized temporally after feeding was initiated, and serum samples were collected, in each case, 15 days after immunization, and then assayed by ELISA for IgGl and IgG2a (Fig. 4, representative of 5 experiments) .
  • mice were exposed to multiple doses of OVA for different periods (1-20 days) . After each exposure period mice were immunized by OVA-CFA. Individual sera were prepared 15 days later and assayed by ELISA for anti-OVA IgGl (open circles) and IgG2a (solid circles) . Results are average antibody binding ⁇ SEM from individual mice at a serum dilution of 1:400, and are presented in absorbance units at 405nm. Each experimental group contained at least 5 mice. Similar binding patterns were seen at serum dilutions ranging from 1:10 up to 1:800; antibody activity attained background values (0.155) at a dilution of 1:1600. As shown in Fig.
  • Thl mediated responses were most sensitive to tolerance induction: a single-day period of multi- dose exposure r to OVA was sufficient to dramatically reduce specific IgG2a production (Thl-controlled) , which essentially ceased after 5 days of exposure.
  • Th2 controlled responses were highly resistant to tolerance induction and became so only after 20 days of multi-dose exposure to antigen: anti-OVA IgGl production gradually diminished with time of exposure to OVA and attained background values only after 15-20 days of exposure.
  • oral tolerization of Thl lymphocytes was achieved after a brief and intermittent exposure period to antigen.
  • Example 6 Frequency of CD4+, V»58.2+ T cells in Peyer's patch following antigen feeding.
  • Each data point in the Figure represents an average from 6-10 mice pooled from three independent experiments.
  • the statistical significance (chi square analysis) of frequency differences of CD4+, V08.2+ T cells among the various groups was as follows: for 0.5 mg fed, p ⁇ 0.001 versus 5 mg, 500 mg and unfed after 3 and 5 feedings; for 5 mg and 500 mg fed, p ⁇ 0.001 versus unfed after 3 and 5 feedings. '
  • CD4+,TcRV38.2+T cells of other lymphoid or nonlymphoid organs such as lung or liver (based on animals feed 500 mg OVA 3 - 5 times) .
  • Figures 5B and 5C are fluorescence (FACS) contour plots of Peyer's patches from animals fed 5 times with either PBS (Fig. 5B) or OVA (Fig. 5C) . These panels show the same data described above but illustrate pictorially the virtual absence of CD4+ cells from the upper right-hand quadrant of the FACS plot.
  • Example 7 Frequency of apoptosis of V38.2+ T cells in
  • Example 6 Peyer's patch cells as prepared in Example 6 were stained for V88.2 with FITC-conjugated MR5-2 mAb and for degraded DNA with acridine orange (Sigma, St. Louis, MO) . Fluorescence was analyzed as in Example 6, and data collection was gated on live V08.2+ cells. Data represent 10,000 events presented as probability (15%) contours. To demonstrate apoptosis, cells were stained with acridine orange using a modified method of Hardin et al. (J. Immunol . Meth. 154, 99-107
  • the statistical significance (chi square analysis) of the frequency differences of apoptotic VjS8.2+ T cells was as follows: for 500 mg fed, p ⁇ 0.001 after 2 feedings versus 5 mg., 0.5 mg, and unfed; for 5 mg p ⁇ 0.01 versus 0.5 mg and unfed after 3 and 5 feedings.
  • the Figure 6A graph indicates that feeding of 500 mg of OVA sharply increases the number of apoptotic V88.2+ T-cells after the second feeding, while 5 mg induces only a gradual apoptosis increase (to 8% by the 5th feeding), and 0.5 mg induces minimal apoptosis.
  • the increase of apoptotic cells seen in animals fed 500 mg returned to background levels by the 3rd feeding.
  • Example 8 Quantification of T cell activation in Peyer's patch following antigen feeding.
  • Peyer's patch cells as prepared in Example 6 were stained for VJ8.2 with FITC-conjugated MR5-2 mAb and for total DNA with propidium iodide (Noguchi in Current Protocols in Immunology, ed. Coligan et al., Wiley & Sons, Secaucus, N.J., 1994) . Stimulation is measured by determining the number of VS8.2+ cells with high DNA content through propidium iodide staining because such cells are actively dividing, that is, they are in the S/G2-M phase of the cell cycle. Fluorescence was analyzed as in Example 7.
  • V08.2+ cells in S/G2-M phase versus feeding over time is graphically represented in Figure 7 with each data point representing a pooled average from 6-10 mice in three separate experiments.
  • the statistical significance (chi square analysis) of the frequency differences of S/G2-M, V38.2+ T cells was as follows: for 500 mg fed, p ⁇ 0.001 after 1-3 feedings versus unfed; for 0.5 and 5 mg fed, p ⁇ 0.01 versus unfed at all time points.
  • the Fig. 7A graph shows that feeding of 500 mg of OVA induces an initial stimulation of V / 88.2+ cells which rapidly declines, presumably due to cell death, while stimulation induced by 5 mg and 0.5 mg feedings rise more slowly and tend to plateau at 4-5 feedings. Specifically, after one feeding of 500 mg OVA the percentage of activated cells rose to 7% and returned to 0 after five feedings. An increase in the percentage of activated cells also occurred in animals fed 0.5 and 5 mg and was maximal after 3 feedings although deletion was only observed in 5 mg fed animals.
  • Figs. 7B and 7C are FALS plots v. DNA content comparing unfed animals with animals fed 5 mg OVA once.
  • OVA-TcR transgenic mice were fed and sacrificed as described in Example 6.
  • Splenocytes 4 x 10 s cells/well, were cultured in 0.2 ml of serum-free medium containing various concentrations of OVA. Peyer's patches were not used because they contained an inadequate number of cells for multiple cytokine assays.
  • Culture supernatants were collected after 40 hrs (for IL-2, IL-4, IL-10, IFN- ⁇ ) or 72 hrs (for TCF-jS) . Cytokine concentration was determined by ELISA.
  • OVA initially enhances both Thl (IFN- ⁇ ) and Th2 (IL-4 and IL- 10) cytokines in the spleen which is completely lost with continued feeding, while IL-2 secretion decreases without prior enhancement. Feeding of 0.5 mg progressively enhances IL-4 and IL-10, with a minimal effect on the production of IL-2 and IFN- ⁇ . (These cytokine changes are consistent with the increase of CD4+,V08.2+ cells following low-dose feeding in Example 6 and the decrease of these cells in Example 7 following high- dose feeding.) This indicates that a minimum dose of antigen is required for suppression of a cytokine profile, and that this amount is higher for suppression of the Th2 cytokine profile.
  • TGF-3 all three feeding regimens enhanced the production of TGF-3 by OVA-specific T-cells. This indicates that antigen- specific T-cells secreting the nonspecific immunosuppressive Factor TGF- ⁇ are resistant to deletion by this tolerization regime. However, this is beneficial to treatment of autoimmune disease because TGF-J has the property of suppressing all immune responses in the vicinity of its release, including autoimmune responses.
  • Splenic T cell proliferative responses were greater than 90% suppressed in OVA fed vs. nonfed animals (3,432+.52 vs. 47,079 ⁇ 6,131 ⁇ CPM) measured by tritiated thymidine uptake and antibody responses (measured by ELISA) were suppressed by 50-75% in mice fed 500 mg OVA 5 times followed by subcutaneous immunization with 100 ⁇ g OVA/CFA.
  • Anti-OVA IgM titers measured by ELISA were reduced from 512 ⁇ 43 in non-fed to 128 ⁇ 21 in the fed mice; IgGl was reduced from 32 ⁇ 5 to 16 ⁇ 7; and IgG2a from 256 ⁇ 14 to 128 ⁇ 36.
  • mice There were few or no detectable anti-OVA antibodies in naive transgenic mice, which indicates that all the anti-OVA antibodies were induced by immunization with OVA. In addition to deletion, evidence of anergy was also observed in mice fed 500 mg OVA. Specifically, the reduced splenic T cell proliferative responses could be partially reversed (from 3,432 ⁇ 52 to 24,227+1468 ⁇ CPM) by preculture of cells with recombinant IL-2 indicating that a number of antigen-specific T-cells became unresponsive not due to apoptosis, but due to anergy.
  • An individual afflicted with myasthenia gravis is first orally administered 2.5 mg. of nicotinic acetylcholine receptor once a day for 1 week to eliminate the unlikely possibility of adverse reaction. Following this, the daily dosage is increased to 10 mg for a period of one to two weeks at the end of which the patient's antibody responses are measured using immunoassay (for reactivity to acetylcholine receptor) . If no improvement is seen the daily dosage is increased (progressively) to 25, 50 or 100 mg etc. (and the antibody responses are monitored weekly or every two weeks) until an effective dosage is determined.
  • Example B Autoimmune Thrombocytopenic Purpura An individual afflicted with this disease is subjected to the same regimen as in Example A except that platelet count is monitored (weekly) and preferably glycoprotein lib-Ilia are used as the orally administered autoantigen.
  • a dosage is effective when increased platelet counts are normal or approach normal but do not increase further with additional orally administered antigen.
  • an immunoassaymeasuring antibodies to platelets can be used to monitor the patient's progress.
  • Example C Autoimmune Thyrolditls An individual suffering from Hashimoto's disease is subjected to the same regimen as above but thyroglobulin is used as the antigen at the same amounts as in Example A. Effectiveness of a particular regimen, is assessed and the patient's progress is monitored by decrease in antithyro-globulin autoreactive antibody. Preferably, the patient should receive treatment at a particular dose for at least 3 - 4 weeks before effectiveness can be assessed. If indicated, the number of daily dosages can be increased.
  • TSHR would be the autoantigen orally administered and therapeutic progress will be monitored, e.g., by decrease in anti-TSHR autoreactive antibody titer, or TSH levels.
  • OVA Immunization IL-4 (9 h. pg/ml) IL-2 (20 h. pg/ml) IFN ⁇ (48 h. ng/ml) Fed OVA PPD OVA PPD OVA PPD

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Abstract

Procédés d'administration par voie orale d'auto-antigènes pour supprimer les réponses immunitaires spécifiques de Th2 ainsi que de Th1 et la production d'anticorps. La présente invention est destinée à être appliquée dans le traitement de maladies auto-immunes induites par des anticorps.
PCT/US1996/010386 1995-06-05 1996-06-05 Utilisation de la tolerance orale pour supprimer les reponses immunitaires de th1 et de th2 et pour supprimer la production d'anticorps WO1996039176A1 (fr)

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US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
US8613920B2 (en) 2007-07-27 2013-12-24 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
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