HUT74900A - Treatment of autoimmune disease using oral tolerization and/or type i interferon - Google Patents

Description

The present invention relates to a method of improving the treatment of autoimmune diseases, i.e., to improving the ability to reduce autoimmune reactions by oral tolerance. More particularly, the present invention relates to the oral administration of autoantigens or the administration of inactive antigens in combination with oral or parenteral administration of polypeptides having Type I interferon activity for the treatment of autoimmune diseases.

The present invention also relates to the oral use of interferons for the treatment of autoimmune diseases.

Autoimmune diseases are characterized by an abnormal immune response directed against normal autologous (self) cells.

Based on the type of immune response (or immune response) in question, the type of autoimmune response in mammals is generally classified into two distinct categories: cell-mediated (e.g., T-cell mediated) or antibody-mediated disorders. Examples of cell-mediated autoimmune responses include, but are not limited to, multiple sclerosis (MS), rheumatoid arthritis (RA), autoimmune thyroiditis (AT), autoimmune status of diabetes mellitus (juvenile or type 1). diabetes) and autoimmune uveoretinitis (AUR). Antibody-mediated autoimmune diseases include, but are not limited to, myasthenia gravis (MA, muscle weakness) and systemic lupus erythematosus (SLE).

Both categories of autoimmune diseases are currently treated with drugs that systemically, non-specifically suppress the immune response, i.e. drugs that are unable to selectively suppress the abnormal immune response. Such drugs include, but are not limited to, methotrexate, cyclophosphamide, lmuran (Azathioprine), and cyclosporin A. Steroids such as prednisone and methylprednisolone (which are also non-specific immunosuppressants) are used in many cases. . All currently used drugs have limited efficacy against autoimmune diseases caused by both cells and antibodies. Furthermore, these drugs have significant toxic and other side effects, and more importantly, they eventually induce "global" immunosuppression in the treated individual. In other words, long-term treatment with drugs suppresses the normal protective immune response against pathogens, thereby increasing the risk of infection. In addition, patients undergoing long-term global immunosuppressive therapy are at increased risk of developing serious medical complications such as cancer, renal failure and diabetes.

In their ongoing efforts to overcome the disadvantages of conventional treatment of autoimmune diseases, the present inventors and co-workers are well-suited to treating autoimmune diseases (and treating related T cell-mediated inflammatory diseases such as allograft rejection and retroviral neurological disease). medical preparations. These treatments are based on the concept of inducing tolerance, either orally or by inhalation, using autoantigens or idle antigens as a tolerant, or disease-suppressing fragments or analogs of autoantigens or idle antigens. The present inventors and co-workers have also designed methods and compositions for inducing tolerance by anergy by parenteral administration of immunodominant epitope peptides of autoantigens. This work is described in PCT / US93 / 0175, filed February 25, 1993, PCT / US91 / 01466, filed March 4, 1991, PCT / US90 / 07455, filed December 17, 1990 -I,

PCT / US90 / 03989, filed July 16, 1990, PCT / US91 / 07475, filed October 10, 1991, PCT / US93 / 07786, filed August 17, 1993,

PCT / US93 / 09113, filed September 24, 1993, PCT / US91 / 08143, filed October 31, 1991, PCT / US91 / 02218, filed March 29, 1991,

PCT / US93 / 03708, filed April 20, 1993 PCT / US93 / 03369, filed April 9, 1993, and PCT / US91 / 07542, filed October 15, 1991.

• · ······ · · · · · · · · · · ·

Autoantigens and idle antigens are defined below.

Intravenous administration of autoantigens, and preferably fragments thereof, which essentially contain epitope regions of the molecules, have been shown to induce immunosuppression through a mechanism called clonal anergy. Clonal anergy, or T-cell non-response, results in the deactivation of immune attack on T-cells specific to a particular antigen, resulting in a significant reduction in the immune response to said antigen. Thus, autoimmune T-cells specific to an autoantigen, such as myelin basic protein (MBP), once anergized, will no longer proliferate in response to that antigen. The inability of anergized T-cells to reproduce results in a decrease in immune response responses, leading to tissue damage responsible for the symptoms of autoimmune disease, such as the neural tissue damage observed in MS. There is also evidence that oral administration of autoantigens or immunodominant fragments thereof in a single dose and in substantially greater amounts than that which triggers active suppression can induce tolerance through anergy (or clonal deletion).

However, clonal anergy can only be induced if the administered antigen is a specific antigen recognized by immune attacking T cells (which we wish to anabolize) (completely inactive antigens do not induce tolerance through anergy). Thus, there are some limitations to the treatments that seek to achieve suppression through clonal anergy: the antigen may be unknown, or there may be many different types of immune-attacking cells specific to different antigens, or the antigens to which the immune attacking T cells are specific.

• ·

The present inventors and co-workers have developed a treatment regimen that utilizes autoantigens and acts by active suppression, i.e., by a mechanism other than clonal anergy. This method, which is thoroughly described in the related PCT application (PCT / US93 / 01705), involves the oral administration of auto-immune tissue specific antigens. These are called "inactive antigens" and are defined below. With this treatment, regulatory (suppressor) T cells are induced in intestinal lymphoid tissue (GLA) or, in the case of administration by co-inhalation, in mucosal lymphoid tissue (MALT). These regulatory cells enter the blood or lymphatic tissue, then migrate to the tissue or organ affected by the autoimmune disease and suppress the autoimmune attack on the affected organ or tissue. Inactivated antigen-induced T cells (which recognize at least one antigenic determinant of the inactive antigen used to induce them) target the site of autoimmune attack, where certain immunomodulatory factors and cytokines, such as transforming growth factor beta (TGF-β), interleukin-4 (IL-4) or interleukin-10 (IL-10). These suppressive substances make a difference and suppress all immune attack phenomena in the vicinity, regardless of the antigen that triggered them. (However, since oral tolerance to an inactive antigen triggers the release of these non-specific suppressive agents only in the vicinity of an autoimmune attack, there is no systemic immunosuppression.)

Serious efforts have been made by other researchers to treat autoimmune diseases, particularly MS, including parenteral administration of interferon beta (IFN-β), which is known to have immunomodulatory properties that can be exploited to combat other immune disorders. Interferon-β • • · ·········································•••••••••••••••••••••••

• · ·· · * ·· ··

- 6 studies have shown that interferon beta inhibits the activity of interferon v (IFN-γ). Interferon γ has been shown to exacerbate MS and to participate in the pathogenesis of MS lesions. Thus, interferon-β appears to have an effect, in part due to its ability to inhibit T-cell γ-interferon expression. The related property of the β-interferon polypeptide is also that it reduces II. Class II major histocompatibility complex (MHC) molecules on the surface of T cells, as well as their ability to enhance suppressor T cell activity, were thought to be responsible for the immunomodulatory properties of parenteral interferon beta. However, parenteral administration of only interferon beta has the disadvantage of requiring high doses to amplify known side effects of this substance.

Interferon beta-Multiple Sclerosis Study Group reported in April 1993 in Neurology that subcutaneous administration of either 1.6x10 ⁶ or 8x10 ⁶ units of β-interferon in patients with relapsing-remitting multiple sclerosis (compared with placebo) significantly reduces the severity of multiple sclerosis. and at higher doses, nuclear magnetic resonance imaging also reduces multiple sclerosis activity. The authors claim that the mechanism by which the interferon beta exerts its visible benefits is unknown. The polypeptide is neither an autoantigen nor an idle antigen. In addition, the effects of a substance on the immune system of a mammal (and the mechanism by which these effects are produced) are often different and depend on the amount of substance and / or route of administration. For example, subcutaneous administration of an alloantigen will induce an immune response to that antigen. Oral administration of the same alloantigen may induce tolerance by inducing the production of T cells that are specific to the orally administered antigen or may induce anergy (at higher doses and less frequently). Intravenous administration of the same alloantigen may induce tolerance by anergy.

To date, the use of interferon alone in the treatment of autoimmune disease has not been described. In addition, it has not been described that interferon can be administered orally or parenterally in combination with oral tolerance using autoantigens or idle antigens.

Accordingly, the present invention provides an improved and / or more convenient method for treating mammals suffering from autoimmune diseases.

The invention further relates to an improved method of treating mammals suffering from autoimmune diseases using only the oral route.

The invention also relates to a method of treating mammals suffering from autoimmune diseases by oral administration of interferons.

After oral tolerance to idle antigens in mammals suffering from an autoimmune disease, the present inventors and co-workers observed increased levels of TGF-β, interleukin-4 and interleukin-10 at the site of autoimmune attack [Chen, Y., et al., Science 265 1237-1240 (1994).

The present inventors and co-workers have recently discovered that oral or parenteral administration of polypeptides having the activity of type I interferon or type I interferon alone, or by co-inhalation of autoantigens or idle antigens, is beneficial in alleviating the symptoms of autoimmune diseases. The fact that even suboptimal doses of type 1 interferon potentiate the tolerating effect of autoantigens and idle antigens. This work is described in more detail in a priority document of this patent application. Interferon type I, in particular interferon beta, is known to have certain immunomodulatory properties, such as inhibiting the activity of interferon γ (· IFN-γ). Interferon γ has been shown to exacerbate MS and to participate in the pathogenesis of MS lesions. Thus, interferon-β appears to have an effect, in part due to its ability to inhibit T-cell γ-interferon expression. The related property of the β-interferon polypeptide is also that it reduces II. Class II major histocompatibility complex (MHC) molecules on the surface of T cells, as well as their ability to enhance suppressor T cell activity, were thought to be responsible for the immunomodulatory properties of parenteral interferon beta.

The mechanism by which orally administered interferon-β promotes tolerance, either alone or as a synergist when used in combination with an antigen, is not fully understood (for example, whether interferon-β is a Th2-enhancing cytokine; is not an object of the present invention). The polypeptide is neither an autoantigen nor an idle antigen. In addition, the effects of a substance on the immune system of a mammal (and the mechanism by which these effects are produced) are often different and depend on the amount of substance and / or route of administration. For example, subcutaneous administration of an alloantigen will induce an immune response to that antigen. Oral administration of the same alloantigen may induce tolerance by inducing the production of T cells that are specific to the orally administered antigen or may induce anergy (at higher doses and less frequently). Intravenous administration of the same alloantigen may induce tolerance by anergy.

To date, the oral (or parenteral) use of immunomodulatory (e.g., Th2-enhancing) cytokines, such as interleukin-4, in the treatment of autoimmune diseases has not been described. In addition, no Th2-enhancing cytokines have been described for oral (or

- 9 parenteral) for oral tolerance using autoantigens or idle antigens.

Accordingly, the present invention provides an improved and / or more convenient method for treating mammals suffering from autoimmune diseases.

The invention further relates to an improved method of treating mammals suffering from autoimmune diseases using only the oral route.

The invention also relates to a method of treating mammals suffering from autoimmune diseases by oral administration of immunomodulatory cytokines.

The authors of the invention made the following surprising discoveries:

oral or co-inhalation administration of various polypeptides having type I interferon activity has a beneficial effect in the treatment of autoimmune diseases;

A combination of (i) oral or co-inhalation administration of autoantigens or inactive antigens (or fragments thereof) and administration of polypeptides having type I interferon activity is significantly more effective than the use of treatment (i) or (ii) in the treatment of autoimmune diseases .

Other non-cytokine synergists such as bacterial lipopolysaccharides, Lipid A, cholera toxin β chain; etc also fits in with the previous combination as described below.

The figures in the appendix are briefly described below.

Figure 1 is a bar graph showing variations in EAE suppression (induced by the 71-90 MBP peptide) followed by oral (1A) or i.v. administration of the various MBP peptides. (1B).

Figure 2 (A) is a graph showing the effect of feeding an autoantigen (PLP) or an idle antigen (MBP) to an SLJ / J mouse.

Λ in which EAE was induced with a PLP peptide; (B) is a bar chart summarizing the data in Part (A).

Figure 3 is a bar graph showing suppression of EAE (induced by MBP peptides 71-90) in various guinea pigs either fed with MBP peptides alone or with soybean trypsin inhibitor (STI).

Figure 4 is a graphical illustration of part (AD) comparing how to suppress EAE in Lewis rats by oral administration of guinea pig MBP (AD), intraperitoneal administration of rat α / β interferon (A), and imitation rat interferon. (C) and combination therapy (B and D).

Figure 5 is a bar graph summarizing the data in Figure 4 (A-D).

Figure 6 is a comparative bar graph of a further experiment documenting suppression of EAE in Lewis rats, with a combination of orally administered guinea pig MBP and intraperitoneal α / β interferon or MBP or β-interferon alone.

Figure 7 (AC) is a graphical comparison of suppression of EAE in SJL / J mice by intraperitoneal administration of murine β-interferon (A), oral administration of bovine MBP (B), oral administration of bovine PLP (C), and a combination of both (B). and C).

Figure 8 is a bar graph showing suppression of bovine PLP-induced EAE in SJL / J mice with or without intraperitoneal administration of murine β-interferon; bovine MBP-induced EAE mouse with or without interferon beta; murine β-interferon only; and hen's egg with lysozyme (HEL) (control protein).

I I

Figure 9 shows the effect of collagen type II on oral γ-interferon or a combination of the two in inducing adjuvant arthritis in Lewis rats.

All patents, inventions and references cited herein are hereby incorporated by reference in their entirety. In the event of conflict, the description containing the definitions and interpretations of the present invention will prevail.

The following terms, when encountered in this description, have the following meanings:

(A) “Idle antigen or“ idle ”is a protein, protein fragment, peptide, glycoprotein, or any other immunogenic material (i.e., a substance capable of eliciting an immune response) that is (or is derived from) a component , which is specific for an organ or tissue under autoimmune attack; and (ii) triggers T-cells (which may be CD4 + or CD8 +) that regulate oral or enteral administration (which may be of the CD4 + or CD8 + type) and which target at least one antigen-specific immunosuppressive factor or immunoregulatory cytokine. (e.g., TGF-β, interleukin4 or interleukin-10), thereby suppressing immune-invasive cells that contribute to autoimmune destruction. The term includes, without limitation, autoantigens and fragments thereof that are involved in an autoimmune attack. It also includes antigens that are not normally exposed to the immune system but are exposed at the site of autoimmune attack, at the site of autoimmune tissue destruction. Examples are heat shock proteins, which, although not specific to a particular tissue, are generally protected from contact with the immune system. "Pure inactive antigen" means an inactive antigen that is not an autoantigen.

(B) "Idle suppression" means suppression of the site of autoimmune attack of cells contributing to autoimmune disruption; this suppression is mediated by the release of one or more immunosuppressive factors (including Th2-enhancing cytokines and Th1-inhibiting cytokines) from suppressor T cells induced by inactivation (or inhalation) of inactivated antigen and accumulated at the site of autoimmune disruption contributing cells. The result is an antigen-specific but locally restricted suppression of autoimmune responses responsible for tissue destruction.

(c) "Mammal" means an organism having an immune system and susceptible to autoimmune diseases.

(d) "Autoimmune disease", as used herein, means spontaneous or induced malfunction of the immune system of mammals, including humans, when the immune system is unable to distinguish between foreign immunogenic substances and / or autologous substances in the mammal, and consequently treating autologous tissues and substances as if they were foreign and eliciting an immune response against them. The term also applies to autoimmune diseases and their animal models.

(e) The term "autoantigen" means any substance or portion thereof normally found in a mammal but which is the primary (or primary) target of the immune regulatory system in an autoimmune disease. The term also refers to antigenic substances which, when administered to mammals, induce conditions having the characteristics of an autoimmune disease. In addition, the term also includes peptide subclasses that contain essentially immunomodulatory epitopes or immunodominant epitope regions of autoantigens. Immuno-dominant epitopes or regions under induced autoimmune conditions are an autoantigen • ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 13 fragments that can be used to induce disease instead of full autoantigen. In people with autoimmune disease, immune-dominant epitopes or regions are fragments of antigens specific to tissue or organ undergoing autoimmune attack and are recognized by a significant percentage (i.e., most but not necessarily absolute majority) of autoimmune attacking T cells.

(f) The term "treatment" refers to both prophylactic treatment, to prevent or delay the onset of an autoimmune disease (or to prevent the onset of clinical or subclinical, such as histological symptoms), and to suppress symptoms after the onset of such autoimmune diseases. relieving the immune system and preventing or slowing down autoimmune tissue damage.

(g) "Synergists" refers to substances that significantly enhance the suppression of clinical (and / or subclinical) manifestation of autoimmune diseases when used in combination with oral administration of an inactive antigen or parenteral administration of an autoantigen. In the preceding sentence, and hereinafter throughout the specification, co-administration means that the substance is administered substantially simultaneously with or shortly after oral (or co-inhalation) administration of inactive antigens. Of course, administration of a coupling agent should not precede the administration of an autoantigen or an inactive antigen to the extent that they are suppressed during the relevant effects of the first administration. Thus, the synergists are preferably administered 24 hours before or after the autoantigen or idle antigen, but most preferably one hour before or after the autoantigen or idle antigen. Polypeptides with type 1 interferon activity are examples of synergists. Other examples of synergists are given.

· (H) “Oral” administration means oral, enteral or oral. administration to the stomach. In addition, co-inhalation administration in aerosol form has the same tolerating effect and is equivalent to oral tolerance.

(i) "Parenteral" administration means subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal or intrathecal administration.

(j) An "attenuation", "suppression", or "reduction" of an autoimmune attack or reaction is a partial reduction or amelioration of one or more symptoms of an attack or reaction. The "substantially" increased suppression effect (or amelioration or reduction) of an autoimmune reaction is a significant reduction in one or more markers or histological, clinical indicators of the autoimmune reaction or disease. The reduction may be, for example, without limitation, a reduction of at least 1 unit in limb paralysis score or arthritis score or in the frequency of autoreactive T cells; a reduction of at least 0.5 units in the insulitis score (Zhang et al., 1991, Proceedings of the National Academy of Sciences, USA 88: 10252-10256).

animal Models

In the description of the present invention, reference is made to various model systems developed to study autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) has been studied in mice and other rodent species as a model for multiple sclerosis (MS). It will be obvious to those of ordinary skill in the art that much of the potential immunotherapy for MS will first be explored in this animal model. The disease is immunized with myelin basic protein (MBP) or proteolipid protein (PLP) and an adjuvant (such as Freund's complete kit). ·· ··

- with 15 adjuvants, "FCA". The antigen used to induce the disease is the autoantigen in the model. This treatment induces either a monophasic or a worsening / recurrent form of the demyelinating disease (depending on the type and species of rodent and well-known induction details) with either antigen. Many of the symptoms of induced disease are the same as those of MS autoimmune disease and can therefore be used as an animal model of MS. In addition, the successful treatment of EAE with oral tolerance and the parallel success in reducing the number of disease-inducing cells in humans and in many cases alleviating the symptoms of MS by oral administration of myelin demonstrate that EAE as a model system is suitable for the success of different oral tolerance treatments. anticipate.

Immunization with Mycobacterium tuberculosis or Freund's complete adjuvant (in oil) induces a disease that can be used as a model for human rheumatoid arthritis in the dorsal tail of sensitive mammals. Similarly, immunization with collagen type II and an adjuvant induces a disease (collagen-induced arthritis, or CIA) that serves as a model for human rheumatoid arthritis.

Immunization of Lewis rats with S antigen or IRBP antigen (InterPhotoReceptor Binding Protein) and an adjuvant induces uveoretinitis. Finally, a model of type 1 diabetes develops spontaneously in NODD mice and BB rats.

One or more of the aforementioned model systems may be well used to demonstrate the effectiveness of the improved treatment of the present invention. In fact, animal models are particularly suitable for testing therapies, including suppression with idle antigens, precisely because this suppression mechanism is antigen-specific. In the case of oral tolerance, this ·············································· · ··

As a result, the suppression of symptoms in the model is independent of many actual or potential differences between the human autoimmune disorder and its animal model. The same animal models can be used to test interferon-based therapies, since interferon is generally known to have the same activity in animal models as in humans.

Thus, the above animal models can be used to evaluate the applicability of the present invention to mammals (including humans). For example, a multiple sclerosis autoantigen, bovine myelin, has given humans a significant benefit over a large number of patients in a double blind study (Weiner et al., Science 259: 1321-1324 (1993)). In addition, symptoms of rheumatoid arthritis, such as tenderness of joints, swelling of AM, grip strength, etc. can be successfully suppressed in patients receiving oral collagen (0.1-0.5 mg once daily) (Trentham, D., et al., Science 261, 1727 (1993)). Finally, preliminary human studies with oral S-antigen have shown very encouraging results for uveoretinitis. Large-scale human studies are currently underway in multiple sclerosis, uveoretinitis, rheumatoid arthritis and diabetes. Each of these human studies confirms oral tolerance data in animal models using the appropriate disease model. Thus, the predictive value of animal models of oral tolerance to autoimmune diseases is strongly supported by these human clinical studies.

The individual treatments that are combined in the treatment methods of the present invention are described below. By individually describing the effects of each of the possible treatments, followed by the description of the combination treatment, the present invention enables one of ordinary skill in the art to understand the effectiveness of these treatments when combined. ···· * ·· ·· ·· ······

- reduction or elimination of tissue damage in autoimmune diseases.

Description of idle suppression - oral administration

In contrast to clonal anergy, suppression produced by administration of inactive antigens (oral or co-inhaled) is induced by the induction of targeted immune regulatory T cells that release one or more immunosuppressive factors, such as transforming growth factor beta (TGF-β); and / or interleukin-4; and / or interleukin-10 at the site of autoimmune attack. These regulatory T cells do not release large amounts of interleukin-2 or γ-interferon. Because regulatory T cells are generated, the mechanism of the processes involved is described as active suppression. Immunoregulatory cytokines evoked by regulatory regulatory cells are not specific for the antigen that initiates suppressor cells secreting them, even though these regulatory T cells only release immunosuppressive factors when administered orally (or inhaled). they excite them. The assembly of immune regulatory T cells within a mammal at a particular point where cells that are involved in autoimmune destruction of an organ or tissue are concentrated allows the release of immune regulatory substances in the vicinity of the autoimmune attack and suppresses all types of cells of the immune system , which is responsible for this attack.

Because T-suppressor cells are triggered by response to oral (or co-inhalation) tolerance to a tissue or organ specific antigen, suppressor T cells are targeted to cells of the organ or tissue under attack in a given autoimmune disease, where • ·

- IX are concentrated. Thus, an idle antigen may be an autoantigen or an immuno-dominant epitope of an autoantigen. Alternatively, the idle antigen may be another tissue-specific antigen that is not an autoantigen; thus, there is no need to identify the autoantigen (or autoantigens) involved.

More specifically, the active suppression mechanism of idle suppression on a tissue-specific (inactive) antigen is as follows: Once a tissue-specific inactive antigen is administered orally (or enterally, i.e. directly into the stomach), it enters the small intestine where it is exposed to the so-called Peyer's , which are groups of a large number of immunocytes located below the intestinal wall. These cells, in turn, communicate with the immune system, including the spleen and lymph gland. The result is that suppressor (CD8 + or CD4 +) T cells are induced, introduced into the blood or lymphatic system, and then accumulated at the site of autoimmune attack, where they cause the release of TGF-β and / or other immune regulatory agents that suppress the mammal. activated helper T cells directed against their own tissues; and B cells. Suppression induced in this manner is antigen-specific. However, the resulting tolerance is specific to the particular autoimmune disease, that is, to a particular tissue under autoimmune attack, due to the fact that the idle antigen is specific to the target tissue and suppressor cells induced by inactivation of the inactivated antigen suppress the immune attacking cells. in or near the lesion.

Inactive antigens and autoantigens (and fragments and analogs thereof) may be purified from natural sources (from tissue or organ normally found) and may be produced by recombinant DNA technology in bacterial, yeast, insect (e.g., baculovirus) and mammalian cells, employing techniques well known to those skilled in the art. The amino acid sequences of several potential and actual idle antigens are known (Hunt, C. et al., 1985, Proceedings of the National Academy of Sciences, USA 82, 6455-6459 (hsp70 heat shock protein); Burckhardt, H., et al., Eur. J. Immunol. 21, 49-54 (1991) (antigen collagen II epitope); Tuohy, V.K. et al., J. Immunol. 142, 1523-1527 (1989) (Encephalogenetic determinant of mouse PLP in mouse); Shinohara, T., et al., Progress in Retinal Research. Osborne.N and Chader, J., Pergamon Press, 1989, 51-55 (S-antigen); Donoso, L.A. et al., J. Immunol. 143: 79-83 (1989) (IRBP); Borst, D.E. et al., 1989, Journal of Biological Chemistry 264, 115-1123 (IRBP); Yamaki, K., et al .; FEBS 234, 39-43 (1988) (S antigen); Donoso,

L.A. et al., Eye Slit. 7, 1087 (1988) (IRBP); Wyborski, R.J. and others: Mole. Brain Slit. 8, 193-198 (1990) (GAD)].

Cattle and mouse PLP; MBP for cattle, human, chimpanzee, rat, mouse, pig, rabbit, guinea pig; human and bovine collagen alfa-1 (II) and bovine collagen alfa-1 (I); and the amino acid sequence of human insulin is well known and published; these antigens may be synthesized by recombinant DNA techniques in a manner known to one of ordinary skill in the art. Fragments of these antigens may be synthesized by chemical and recombinant DNA techniques as known to those of ordinary skill in the art.

Some tissue-specific antigens are commercially available: e.g., insulin, glucagon, myelin basic protein, collagen I, collagen II, proteolipid protein, and the like.

Idle antigens can be determined by routine experimentation. Any antigen from the tissue affected is a potential idle antigen. The potential idle antigen can be fed to mammals, and spleen cells from these mammals, or circulating T cells from blood or brain water, for example, can be removed in EAE or MS and stimulated in vitro with the same antigen. With Stimulation ···· • · «

- The induced T cells can be purified and the supernatants assayed for the amount and / or quality of TGF-β, interleukin-4, interleukin-10 or other immunoregulatory agents. More specifically, TGF-β can be measured quantitatively or qualitatively by ELISA, preferably using a suitable commercially available polyclonal or monoclonal antibody to TGF-β (R&D Systems, Minneapolis, MN; Celtrix Pharmaceuticals, Santa Clara, CA). Another known method can be used for the detection of TGF-β, such as that described in Example 2 below, using a commercial otter using a lung epithelial cell line. If the idle antigen elicits T-suppressor cells that do not release TGF-β, then T-cells may be assayed similarly for interleukin-4 or interleukin-10 secretion (antibodies to interleukin-4 and interleukin-10). commercially available, such as from Pharmigen, San Diego, CA). Tissue-specific antigens are ineffective inactive antigens that have been segregated from the inflammatory site (or site of autoimmune attack) such that the released immune regulatory cytokines are too far removed from the site of inflammation to exert suppressive activity.

The efficacy of orally induced idle suppression can be estimated, for example, by the disappearance of certain inflammatory markers (e.g., the number of activated T-cell clones directed against an organ or tissue subject to autoimmune attack); a decrease in interleukin-2 or γ-interferon levels at the same site; histological evaluation of the organ or tissue concerned (for example, by biopsy or nuclear magnetic resonance imaging); or a reduction in the number and / or severity of clinical symptoms associated with an autoimmune disease.

• t · »·« · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 21 Using Inactive Antigens - Doses

Tolerance induced by the inactive antigens of the present invention is dose dependent over a wide range of oral (or enteral) or inhalation doses. However, there is a minimum and maximum effective dose. In other words, active suppression of the clinical and histological symptoms of an autoimmune disease occurs within a specific dosage range which, however, varies from disease to disease, from mammal to mute, from idle antigen to idle antigen. For example, if the disease is in PLP-induced EAE mice, when MBP is used as an inactive antigen, the suppressive dose will vary from 0.1 to 1 mg / mouse / feed (fed approximately every other day, e.g. 5-7 feeds). there is a 10-14 day period). The most preferred dose is 0.25 mg / mouse / feed. To suppress the same disease in rats, the suppressive dose of MBP ranges from 0.5 to 2 mg / rat / feed, with the most preferred dose being 1 mg / rat / feed. In humans, the effective dose for multiple sclerosis when MBP is used as an oral tolerant is between 1 and 100 mg / day, preferably about 1-50 mg MBP / day (every day or every other day for a few months to several years). period), the optimum is 30 mg / day.

In rheumatoid arthritis, an effective dose for humans receiving type I or type II or type II collagen is about 0.1 to 1 mg / day, preferably 0.1 to 0.5 mg / day. In mice, in the case of adjuvant arthritis, the effective dose of collagen ranges from 3 to 30 mg / feed, using the same feeding regimen as in EAE.

Monitoring of the patient may be necessary to optimize the dose and frequency of administration. The exact amount and frequency of administration for a patient may vary with the condition of the patient, the frequency of manifestation, and the physical condition of the patient, as experienced in the art. ···· · - W * «*. • ··· · ·> »· • · · · · 4 · · · · · · · · · 4

Ismert well known in the art. This optimization is preferably performed occasionally. Optimizing the dosage required for immunosuppression is only routine experimentation as described below.

The severity of the disease can be estimated in a manner well known to those skilled in the art, depending on the type of disease. These methods include, but are not limited to, the following:

MS: Number and severity of attacks over a period of time; progressive accumulation of the disease (as measured, for example, on the Extended Disease Status Scale); the number and extent of lesions in the brain (as detected, for example, by nuclear magnetic resonance imaging); and the frequency of autoreactive T cells.

EAE: paralysis of the limbs, which can be classified as follows: 0 = no disease; 1 = reduced activity, weak tail; 2 = mild paralysis, insecure gait; 3 = mild paraperesis, limbs fluttering; 4 = tetraplegia; and 5 = death.

RA: joint swelling, joint tenderness, morning stiffness, grip strength, joint imaging techniques.

AUR: Visual acuity; the number of T cells in the eye and "cloudiness" in the eye.

Type 1 Diabetes: Pancreatic beta cell function (as assessed, for example, by the OGTT glucose tolerance test).

FEMALE MODEL: Insulitis and Delay in Diabetes.

CIA: Arthritis value, which is determined by the affected joints in the four paws and is plotted on the following 1-4 arbitrary scale: 0 = normal, 1 = redness only; 2 = redness plus swelling; 3 - severe swelling; 4 = joint deformation. Total arthritis value is the sum of all paw values. The maximum arthri • · · tis value is the highest value in an animal observed during the disease. According to the grading method, the highest possible arthritis value is 16 (four paws x 4 point per paw).

Symptom stabilization, under conditions where control patients or animals experience worsening of symptoms, is an indicator of the effectiveness of suppressive therapy. Another measure of improvement is the reduction or elimination of the administration of other drugs such as steroids or other anti-inflammatory drugs and biological reaction modifiers such as methotrexate, subcutaneous interferon and the like.

For an inactive antigen, the optimum dose having the maximum beneficial effect is as described above.

An effective dose that results in at least a statistically or clinically significant attenuation of at least one of the markers, symptoms, or histological evidence of a disease treated as described above.

When combined with interleukin-β treatment, the dose of idle antigen is preferably equivalent to that which would be used if only the idle antigen was administered orally or enterally, except that the combination is more effective in suppressing the autoimmune reaction. However, it is possible to reduce the amount of autoantigen or idle antigen by coupling type 1 interferon to oral idle antigen therapy. It is also possible to use a suboptimal amount of interferon beta in the concomitant therapy. The suboptimal amount is that amount of interferon beta which, although substantially inactive when administered alone, enhances the antigen tolerance, i.e. potentiation, of the concomitant therapy.

Determining the effective dose range and optimal amount of an idle antigen is a simple task for those skilled in the art. For example • · · ·

- 2-1 mammalian and human dose determination begins with a relatively low dose (e.g., 1 pg) which progressively (e.g., logarithmically) increases and measures the secretion of TGF-beta (and / or interleukin-4 or interleukin-10) cells and / or an estimate of the number of immune attacking T cells in the blood (e.g., by dilutional assay and reproductive capacity) and / or estimating the severity of the disease as described above. The optimal dose will be that which produces the maximum amount of suppressive cytokines in the blood and / or causes the greatest reduction in the symptoms of the disease. An effective dosage range that at least results in a statistically or clinically significant relief of a symptom specific to the disease being treated.

The maximum effective dose of an inactive antigen can be determined by examining progressively increasing doses in animals and then extrapolating to humans. For example, based on the doses given above for rodents, the maximum effective dose of MBP in humans has been determined between 50 and 100 mg / meal. Similarly, an effective amount of type II collagen in humans is about 1 mg / day.

The present invention can advantageously be used to prevent the onset of an autoimmune disease in susceptible individuals at risk of autoimmune disease. For example, methods of identifying patients at risk for developing type 1 diabetes are extensive and reliable and have recently been validated by the American Diabetes Association (ADA). Various assays have been developed which (particularly in combination) can provide very good predictions of susceptibility to type 1 diabetes (Diabetes Care 13: 762-775 (1990)). Details of one preferred screening method are available to one of ordinary skill in the art (Bonifacio, E., et al., The Chain, 335: 147-149 (1990)).

· · · · ······ · · · · · · · · · · · · · · · · · · · · · · · · ·

- 25 From a practical point of view, the prevention of the onset of autoimmune diseases is of prime importance for diabetes. Other autoimmune diseases, such as MS, RA, AT, and AUR, manifest themselves at an earlier stage of tissue damage before significant tissue damage occurs; consequently, the preventive treatment of these diseases is not as important as diabetes. In diabetes, it would be best to have preventive treatment given before substantially all of the pancreatic islet cells undergo significant destruction. After the islet cells have been destroyed, the treatment is no longer effective.

A non-limiting list of tissue or organ-specific, amplified or potential inactive antigens used in the treatment of autoimmune diseases and those effective in the treatment of these diseases is given in Table 1 below. Administration of combinations of antigens listed for each disease (alone or in combination with interleukin-4) is also expected to be effective in treating the disease.

Inactive antigens and interferon type I may also be administered by inhalation. The amount of inactive antigen to be inhaled is less than that to be administered orally. Likewise, the amount of type 1 interferon administered by inhalation is expected to be less. Effective amounts of inhalation therapy may be determined by the methods described above.

• · · • · · ·

- 16 • · · · · · · t «························. spreadsheet

Type cleaning cleaning cleaning cDNA cleaning cDNA Source J.Chromatogr. Biomed.Appl. 526-535 (90) J.Immunoi.Meth 121-219 (89) 151-177 (92) Exp.Eye Slit. 56: 463 (93) Eur.J.Pharm. 172: 231 (89) Bioi.Repród. 43: 559 (1990). Inactive antigen glucagon insulin glutamic acid decarboxylase (GAD) MBP, MBP fragments PLP, PLP fragments myelin-related glycoprotein myelin, oligodendrocyte glycoprotein, heat shock protein collagen I, II or III; RO / SS-A RO / SS-B-LA is a heat shock protein S-antigen IRBP recoverin acetylcholine receptor heat shock protein NASP (post-acrosomal sperm protein) Affected tissue Pancreatic beta cells myelinated neurons connective tissue eye muscle semen Autoimmune disease Type 1 diabetes Multiple sclerosis Rheumatoid arthritis Autoimmune uveitis Myasthenia Gravis Male infertility

. Continue Table

Type cleaning cDNA Source Bioi.Chem.H-S. 368: 531 (1987). Eur.J.Cell. Biol. 55: 200 (91) Inactive antigen Jo-1 antigen heat shock protein desmoglein factor XIII Affected tissue muscle Skin Autoimmune disease myositis pemphigus

• · ·· · · · ·

In any autoimmune disease, extracts from appropriate tissue as well as specific inactive antigens or fragments thereof may be used as an oral tolerant. In other words, the idle antigen does not need to be purified. For example, myelin (from another species) has been used for multiple sclerosis, pancreatic cell extracts have been used for type 1 diabetes, spleen cell extracts have been used to prevent allograft rejection (which is strictly not an autoimmune process), and muscle extracts have been used. However, administration of one or more antigens or antigen fragments is preferred.

Thus, according to the present invention, when treating type 1 diabetes, an effective amount of glucagon (as defined above) may be administered orally. Glucagon is specifically found in the pancreas. However, it is clear that glucagon is not an autoantigen because it is not expressed in beta cells of the pancreas, which are broken down in type 1 diabetes (glucagon is found only in another cell type, alpha). So glucagon is a "pure" idle antigen: apparently has no autoantigen activity. (The inactive activity of glucagon is probably due to its high local concentration in the intercellular space of the pancreas as it is secreted by alpha cells.)

Insulin has inactive activity in type 1 diabetes. It is currently unknown whether insulin is also an autoantigen. However, regardless of its mechanism of action, oral, enteral or inhaled insulin formulations suppresses IES diabetes and prevent autoimmune discrimination of pancreatic beta cells in animal models.

Multiple sclerosis and animal models modeling it have both disease-inducing and non-disease-causing fragments of MBP (i.e., · · · · · · · · · · · · · · · · · · · · ·· ···

- 2 ⁽ ), for example, a peptide containing 21-40 amino acids of guinea pig MBP known to induce EAE in mice and rats) has inactive activity not only in MBP-induced disease but also in PLP-induced disease too. In rats, feeding of idle antigen mainly produces CD8 + suppressor cells, which are restricted to class I, whereas mice produce both CD8 + and CD4 + regulatory cells (these CD4 + regulatory cells are probably restricted to class II).

In rheumatoid arthritis and animal models, collagen type I, type II and type III have inactive activity.

In the uveoretinitis and animal model (AUR), S-antigen and IRBP and fragments thereof have inactive activity.

Fragments of idle antigens may also be used. Useful fragments can be identified by the overlapping peptide method in Example 3 (a general technique, although described specifically in Example 3 for the identification of a non-inducing fragment of MBP). T cells from fed animals can be assayed for secretion of TGF- [beta] and / or interleukin-4 and / or interleukin-10 and their subtype can be determined (CD8 + and / or CD4 +).

Orally administered autoantigens and idle antigens induce the production of regulatory T cells, thereby inducing the production and / or release of TGF-β and / or interleukin-4 and interleukin-10. Such a T cell was identified as an CD4 + suppressor T cell in orally tolerated mice against EAE, and a CD8 + suppressor cell was identified in rats. Even immunodominant epitopes of autoantigens, such as MBP, are capable of inducing such regulatory T cells. Further such epitopes can be identified by inserting an inactive antigen into the food of a mammal and then isolating T-cells from the mammal that recognize a fragment of the antigen (and thus suppressive

30 fragments) or T-cells are identified from an animal fed with an inactive antigen that is adoptively capable of transferring protection to untreated animals.

Inactive antigens may be administered alone or in combination with autoantigens. The administration of autoantigens is as described in the following PCT applications: PCT / US93 / 01705 filed February 25, 1993; PCT / US91 / 01466, filed March 4, 1991, PCT / US90 / 07455, filed December 17, 1009,

PCT / US90 / 03989, filed July 16, 1990, PCT / US91 / 07475, filed October 10, 1991, PCT / US93 / 07786, filed August 17, 1993,

PCT / US93 / 09113, filed September 24, 1993, PCT / US91 / 08143, filed October 31, 1991, PCT / US91 / 02218, filed March 29, 1991,

PCT / US93 / 03708, filed April 9, 1993; and PCT / US91 / 07542, filed October 15, 1991, mentioned above. It is expected that at least two inactive antigens (either of which may or may not be autoantigens) will likewise be effective suppressors of autoimmune diseases.

In addition, other synergists may be used together in the treatment to enhance the effectiveness of the former. Non-interferon synergists useful in the present invention include, but are not limited to, bacterial lipopolysaccharides from many different Gram-negative bacteria, such as various subtypes of Escherichia coli and Salmonella (LPS, Sigma Chemical Co., St. Louis). , MO; Difco, Detroit, MI; BIOMOL Res. Labs., Plymouth, PA), Lipid A (Sigma Chemical Co., St. Louis, MO; ICN Biochemicals, Cleveland, OH; Polysciences, Inc., Warrington, PA). ); immunoregulatory lipoproteins, such as peptides covalently bound to tripalmitoyl-S-glycarylcysteinyl-seryl-serine (P ₃ C55), the preparation of which has been described in the literature (Deres, K. et al., Naturre 342: 561-564 (1989)); or the "Braun's" lipoprotein Escherichia coli, which may be prepared as described by Braun et al., (1976), Braun, V. Biochimica et Biophysica Acta 435, 335-337. For use in the present invention, LPS can be extracted from gram-negative bacteria and purified by the method of Galanes et al., Eur. J. Biochem. 9, 245 (1969); Skelly, RR, et al., Infect. Immun. 1979, 23, 287]. An effective dosage of non-interferon synergists in mammals is from about 15 pg to about 15 mg per kilogram body weight, preferably from 300 pg to 12 mg per kilogram body weight. Any other material that has the property of shifting immune responses toward the Th2-type response can be used.

Interferons useful in the present invention

The interferon family consists of proteins with different properties that are secreted by virtually every cell type in the body in response to a variety of different inducers. Interferons have three different types: α, β, and γ, which can be induced in lymphocytes, fibroblasts (or other non-leukocyte cells), or lymphocytes stimulated with antigen or mitogen. Interferons are known to influence immune responses at several different cellular targets by binding to cellular receptors. Both interferon α and interferon β are type I interferons.

The term human α-interferon refers to a group of at least 14 structurally related polypeptides transcribed from a multigenic family. Human interferon beta refers to two subtypes of interferon that are structurally related to interferon beta; interferon is the major subtype. A single receptor binds both interferon-β and interferon-β [Wierenga et al., “Lymphokines and ·

32 Cytokines of the Immune System, Comprehensive Medical Chemistry, Vol. 3, Chapter 15, 1101-1128 (1990). The sequences of human and murine interferons α and β have been published. In addition, a number of polypeptides having type I interferon activity have been published [4,569,908; 4793.995; 4,914,033; 4,769,233; 4,753,795; 4,738,845; 4,738,844; U.S. Patent Nos. 4,914,031 and 4,652,639]. U.S. Patent No. 4,652,639 relates to the interferon sequence of the Amgen, Inc. consensus. Many polypeptides of human and animal origin with type I interferon activity are commercially available.

Treatment with interferon type I

Clinical trials of parenteral treatment with interferon beta to suppress or reduce multiple sclerosis, an autoimmune disease, have resulted in a significant reduction in the frequency of attack (Jacobs et al., Science 214: 1026-1028 (1981)). In this study, 1 million units of interferon was administered intrathecally once or twice a week, followed by monthly doses. More recently, clinical trials of parenteral treatment with recombinant interferon beta have also shown that the frequency of attacks is significantly reduced (Johnson et al., 1990, Neurology 40 (Suppl. 1), 261). Various initial doses were used in this study, but the initial dose was followed by subcutaneous administration of 45 million units three times a week. The interferon beta used in this assay was different from the native protein when the cysteine residue at position 17 was changed to serine (IFN? -Ser-17, BETASERON, Berlex Laboratories). This mutation significantly enhances protein stability but does not alter its specific activity. Based on the promising results of Johnson et al. (1990, Neurology 40 (Suppl. 1), 261), a large multicenter study is currently underway. In addition, another large study is underway using glycosylated recombinant [?] Interferon (BIOFERON) produced in mammalian cells.

Although the mechanism of action of interferon beta is not yet clear, interferon γ is likely to be an activator of disease activity and interferon beta reduces the symptoms of the disease by suppressing the effects of interferon γ. Interferons, as a class, are known to be able to influence the immune system through the induction of MHC class I (HLA-A, B and C) and class II MHC (HLA-DR, DQ and DP) cell surface molecules. . These cell surface molecules are essential for basic immune functions, such as self / foreign, to present antigen to T cells, and are therefore thought to play a central role in the development of autoimmune disease.

Antigens associated with class I molecules are recognized by CD8 + (suppressor / cytotoxic) T cells, while antigens associated with class II molecules are recognized by CD4 + (helper / inducer) T cells. Class I molecules are constitutively expressed in almost all cells and each type of interferon enhances their expression. Class II molecules are normally restricted to certain cell types in which expression of interferon γ is regulated (McFarlin, Allergy Clinics of North America 8: 210-212 (1988)); Basham et al., J. of Immunol. 130: 1492-1494 (1983); Stein et al., J. Clin. Invest. 73: 556-565 (1984)]. This regulation is known to occur through the induction of intracellular molecules which, in turn, influence the expression of the MHC molecule (Vanguri et al., 1990, Journal of Biological Chemistry 265, 15409-15057). Interferon γ · · · · · · · · · · · · · · · · · · · · · · · ···

It also activates macrophages which act as effector cells in autoimmune attacks, such as demyelination in multiple sclerosis [Bever et al., Springer Sem. in Immunopathol. 8: 235-250 (1985); Prineas et al., Ann. of Neurol. 10: 149-158 (1981)]. Macrophages also synthesize proteinases that are capable of degrading autoantigens, such as MBP [Bever: Transact. of the Am. Soc. Neurochem. 25, 208 (1991)]. In addition,? -Interferon is capable of inducing adhesion molecules that regulate the delivery of lymphocytes to the site of inflammation, as seen in autoimmune diseases such as the CNS in Multiple Sclerosis [Male et al., Cell. Immunol. 1271-11 (1990)]. Thus, this cytokine is involved in many aspects of autoimmune disease.

The immune-activating role of γ-interferon is influenced by other cytokines. For example, interleukin-4, corticosteroids, prostaglandins, α-fetoprotein, TGF-β, and noradrenaline have been shown to downregulate class II expression in various systems [Cowan et al., J. of Neuroimmunol. 33: 17-28 (1991); Frohman et al. (1988) Proceedings of the National Academy of Sciences USA 85: 1292-1296; Racke et al., J. of Immunol. 146: 3012-2017 (1991); Ransohoff: Slit. in Immunize. 140: 202-207 (1989)]. Interestingly, class II molecules stimulated by γ-interferon can also be down-regulated by interferon beta, which has been shown to interfere with the transcription of class II-specific mRNA in many systems [Fertsch et al., J. of Immunol. 139: 244-249 (1987); Ransohoff et al., J. of Neuroimmunol. 33: 103-112 (1991)]. It has been shown in T lymphocytes that interferon beta directly suppresses the synthesis of interferon γ, which may explain its effect on the transcription of class II genes (Noronha et al., 1991, Neurology 41 (Suppl. 1), 219; Pantich et al.

···· • ··· · · · · · · · · · · · · · · · · · ·

- 3 5 Ann. of Neurol. 22: 139 (1987). Because class II molecules are known to play a central role in the recognition of cells as foreign, they are almost certainly involved in the pathology of autoimmune diseases.

Administration of γ-interferon to multiple sclerosis patients may result in acute exacerbation [Pantich et al., Ann. of Neurol. 22: 139 (1987). Interferon γ is also found in association with class II antigens in active sclerosis multiplex plaques [Traugott et al., N.Y. Acad. Sci. 340: 309-311 (1988)]. In addition, interferon beta has been shown to enhance the suppressor function of T cells from both multiple sclerosis patients and control subjects, presumably through the regulation of suppressor T-cell γ-interferon synthesis [Noronha et al., Ann. of Neurol. 27: 207-210 (1990); Panitch et al., J. of Neuroimmunol. (1992)]. Thus, down-regulation of γ-interferon with systemic interferon-β may be an effective means of preventing or controlling the severity of autoimmune attacks.

This approach is supported by the work on animal models. Specifically, in EAE, a model of multiple sclerosis discussed above, systemic administration of interferon beta prevented the development of the disease (Abreu, Immunological Communications 11, 1-7 (1982)). Adaptive transfer of disease, i.e., induction of disease by transferring MBP-sensitized T cells from rats with EAE to normal rats, can be prevented by treating the cells with β-interferon prior to transfer [Abreu: Internl. Arch. of Allergy and Appl. 76: 302-207 (1985). In both cases, these effects are likely due to downregulation of the rat la molecule (corresponding to human class II MHC), although this has not yet been demonstrated.

···· ···· ·· ·· ·· * ♦ · · · · • · · · · · · · · · ····· "···· ·· ·· · · ·

Oral use of type 1 interferon in the present invention

Orally administered type 1 interferons are effective in suppressing autoimmune diseases. In fact, interferon beta has been shown to be as effective as insulin in suppressing diabetes in NODD mice. This is a surprising fact and cannot be inferred from parenteral administration of interferon, since the mechanism by which interferon suppresses the disease is unknown.

Oral doses in rats and mice were found to be between 1,000 and 150,000 units and no maximal effective dose could be observed. This is in contrast to the response to idle antigens, which is reduced above the maximum effective dose. Doses administered with interferon alone are expected to be similar to those used with idle antigens, except that suboptimal doses of interferon type I may be used in concomitant treatment. Unlike the initial interferon, oral interferon has no side effects.

Parenteral IFN Doses (Combination Therapy)

The reduction observed with type 1 interferon alone is effective in the symptoms of autoimmune disease over a wide range of parenteral doses. In other words, suppression of the clinical and histological symptoms of an autoimmune disease occurs within a specific dosage range, but varies from disease to disease, from mammal to mammal, and depending on the form and activity of the interferon. For example, if the disease is induced in PLA or MBP-induced EAE mice, the suppressive dose for interferon beta is between 10,000 and 1 million units (treatments are given every other day, e.g.

5-7 treatments over a 10-14 day period). The most preferred dose is 69,000 units / mouse / treatment. To suppress the same disease in rats, ··········································································· · * *

The suppressive dose of '7 interferon beta ranges from about 5,000 to about 1 million units / rat / treatment, with the most preferred dose being 15,000 units / rat / treatment. The effective dose in humans with multiple sclerosis is between about 1 million and about 75 million units. preferably about 15 to 60 million units per dose, with a minimum frequency of once every month and a maximum frequency of injection every other day.

Determining the effective dosage range and the optimum amount for a combination therapy is not a problem for those skilled in the art. For example, mammalian doses and human doses may be determined by starting with a relatively low dose (e.g., 5000 units), incrementally increasing (e.g., logarithmically), and measuring the biological response to the treatment, such as the number of class II cell surface markers. and / or assess the severity of the disease according to well-known scoring methods (e.g., a scale of 1 to 5, measuring attack number, joint swelling, grip strength, swelling, visual acuity, interruption, etc. depending on the type of disease). The optimum dose will be the one that has the greatest effect on the measured biological phenomenon, i.e., which causes the greatest reduction in the number of class II molecules on the surface of T cells and / or which results in the greatest amelioration of the symptoms of the disease. An effective dose will be that which produces at least a statistically or clinically significant reduction in at least one symptom of the disease being treated, as described above. Again, suboptimal doses of parenteral interferon may be used in coupled therapy.

The parenteral route may be the subcutaneous, intramuscular, or intraperitoneal route, but the subcutaneous route is preferred when the route of administration is the following: · · · · · · · · · · · · · · · · · ···

- A parenteral route of 38 gels was selected. For parenteral administration, the interferon may be formulated in sterile saline or other carrier well known in the art and may contain excipients and stabilizers which are standard in the art.

Combination therapy

Surprisingly, it has been found that oral (or inhalation) administration of an inactive antigen in combination with oral or co-inhalation administration of type 1 interferon results in a treatment that is synergistic in its effect on autoimmune disease when compared to the individual treatments.

This combination treatment was tested in mice and rats using the animal model of MS EAE, the animal model of RA (IA), and the animal model of type 1 diabetes (NOD). Experimental protocols for these assays are described in the examples below. As shown in Figure 4, oral treatment of Lewis rats with guinea pig MBP after induction of EAE delays and at the same time reduces the clinical score of the disease (see A-D). Intraperitoneal administration of rat interferon alone did not appear to have any effect on the course and severity of the disease (see 8A). This treatment is directly comparable to the effect of administration of rat β-interferon mimicking (see 8C), which is a control substance and is prepared by injecting cells under the same growth conditions as for the production of interferon beta, but without inducer.

However, the combination of intraperitoneal rat β-interferon and oral tolerance to MBP reduces the clinical score of induced EAE (see te ······························································ · You · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 39 8B). This is an unexpected result, especially when compared to the combination of intraperitoneal rat imitation of interferon and oral tolerance (MBP). Rat interferon beta-MBP / MBP combinations have a moderate effect at best when compared to PBS control treatment and oral tolerance using MBP alone. Thus, the combination of intraperitoneal β-interferon and MBP oral tolerance has a synergistic suppressive effect on the observed clinical score of EAE. In other words, interferon potentiates the ability of the inactive antigen to tolerate. These treatments appear to have a much greater effect on the clinical score when used in combination therapy than when either treatment is used alone. The synergistic interaction is best seen in Figure 5, where the difference between the third column (GP-MBP (PO) + rat interferon (IP)) and the top of the figure and the sixth column (PBS (PO)) is greater than the sum of the differences. between the fourth (rat interferon (IP)) and the sixth column (PBS (PO)) and between the fifth (GP-MBP (PO)) and the sixth column ((PBS (PO)). The third column is also surprisingly smaller as the first column showing direct control of the combination treatment.

The synergizing or potentiating effect is further illustrated in Figure 6. Again, the effect on the clinical score of the combination therapy is greater than the sum of the effects of these treatments alone.

Similar results are obtained when treating EAE in mice using PLP as an inactive antigen instead of BMP. As shown in Figure 7, treatment with bovine MBP or bovine PLP suppresses the clinical score of immunized animals (see B and C). Administration of mouse murine interferon only intraperitoneally has no effect on the disease (see A).

···· '· ·· ···· «· • · · ·»

Λ “9. 1 **. ♦ * · * · • · · · · «·· * 4 ···· ··

However, the combination of β-interferon treatment with oral tolerance using either bovine MBP or bovine PLP results in a significant reduction in clinical scores (see B and C). Combination treatment appears to reduce the duration of EAE in mice (note the clinical value of 0, especially in combination with bovine MBP (B)). As a result, the synergistic suppressive effect observed in rats is also evident in the mouse EAE system. The effect is more illustrated by the bar graph in Figure 8. The difference between the first and sixth columns is clearly greater than the difference between the second and sixth columns, or between the fifth and sixth columns, indicating that combination therapy is much more effective than oral tolerance or interferon beta treatment. itself. Furthermore, when the effect of bovine PLP on the clinical score is added to the effect of murine interferon beta on the clinical score, the amount of the decrease is even less than the reduction in the clinical score obtained with the combination treatment. Thus, the combination has a synergistic effect on clinical score relative to each treatment.

The combination of oral administration of type 1 interferon with oral (inactive) tolerance also shows synergism or potentiation.

Finally, the administration of NON-BREAKING HYPHEN (8209) mice to interferon only orally indicates that interferon beta is as effective in suppressing the disease as orally administered insulin.

The following examples illustrate the present invention but do not limit its scope.

The following materials and methods were used in the experiments described below:

• ·

Animals: 6-8 weeks old Lewis rats were purchased from Harlan-Sprague Dawley Inc., Indianapolis, IN. 8-week old SJL / J mice were purchased from Jackson Laboratories (Bar Harbor, ME). The animals are maintained on standard laboratory diets and water is given as desired. Animals are maintained according to the guidelines of the Committee on Care of Animals (Pub. #DHEW: NIH, 85-23, corrected in 1985).

Antigens and Reagents

Guinea pig MBP and mouse MBP are purified from brain tissue by modifying the method of Deibler et al., Prep. Biochem. 2, 139 (1972)]. Protein content and purity are assayed by gel electrophoresis and amino acid analysis. Concanavalin A and histone were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). Peptides were synthesized on a peptide apparatus from Center for Neurologic Disease, Brigham and Women's Hospital, and purified by HPLC. The amino acid sequences of MBP peptides synthesized for use are as follows: 21-40

MDHARHGFLPRHRDTGILDS (immunosuppressive epitope region when administered orally to rats)

71-90

SLPQKSQRSQDENPVVHF (immunodominant encephalogen region in rats); 151-170

GTLSKIFKKLGGRDSRS.

interferons

Rat, rat imitation, and mouse α / β interferon were purchased from Cytimmune, Lee Biomolecular Research (San Diego, CA). Consensus interferon is available from Amgen Ltd.

• · · ·

• ·

Induction of tolerance

For oral tolerance or active suppression, rats were fed 1 mg MBP in PBS or MBP alone, by gastric intubation with a stainless steel 18-gauge needle (Thomas Scientific, Swedesboro, NJ). The animals are fed five times at 2-3 day intervals, with the last feeding being given two days before immunization.

Induction of EAE

For active-induced disease, Lewis rats were immunized in the left foot with 25 pg guinea-pig MBP in 50 µl PBS, which was eluted with an equal volume of Freund's adjuvant (CFA) containing 4 mg / ml Mycobacterium tuberculosis (Difco). For adoptively transmitted EAE, an MBP active T-cell line is generated from rats immunized with MBP dissolved in CFA and raised and maintained as described by Ben-Nun et al., Eur. J. Immunol. 11, 195 (1982)]. Enkephalitogenic cells were harvested after activation by culturing with 2 mg / ml Concanavalin A (ConA) using irradiated thymocytes from animals immunized as antigen presenting cells (APC). Cells were harvested from cultures with a Ficoll Hypaque gradient (Hypaque 1077, Sigma). then washed twice with phosphate buffered saline before transfer. 5x10 ⁶ encephalogenic cells were injected intraperitoneally into 0.1 ml of PBS irradiated (750 rad, 24 hours earlier) recipient rats. The viability of both modulator and encephalogen cells was determined to be greater than 90% by trypan blue exclusion.

Clinical evaluation

The animals are evaluated for the occurrence of EAE daily in a blinded manner. Clinical symptoms of EAE are evaluated using the following system: 0 = no disease; 1 = flabby tail; 2 = paralysis of hind limbs: 3 = bilateral paralysis of hind limbs,

- Γ 'urine drainage; 4 = paralysis of four limbs: and 5 = death. The duration of the disease is measured by the number of days since disease onset (10 to 11 days after active immunization and 3 to 5 days after adoptive transfer of the disease) until complete recovery of each animal.

Delayed Type Hypersensitivity (DTH) test

DTH is assayed by injecting 25 µg MBP in PBS subcutaneously in the ear. The thickness was measured with a visual obstruction observer 48 hours before and after treatment using a micrometer (Mitutoyo, Japan). For each animal, the thickness of the ear is recorded before and after treatment, and the results are expressed as the mean (± SEM) of each experimental group.

Histology

Histological analysis of pathological changes was performed in rats with adoptively transferred EAE. Ten days after adoptive transfer (or induction of disease), the spinal cord is removed and fixed in 10% neutral buffered formalin. Paraffin sections were prepared and stained with Luxol fast bluehematoxylin and eosin using standard procedures [Sobel et al., J. Immunol. 132: 2393 (1984)]. Spinal cord tissue is sampled in the same way in each animal and evaluated in a blinded manner with the number of focal points per section (> 20 or more aggregated inflammatory cells) in the parenchyma and meninges [Sobel et al., J. Immunol. 132: 2393 (1984)].

Statistical analysis

Clinical scales were evaluated using Wilcoxon bivariate tests on value samples, chi-square analyzes were performed to compare the incidence of disease across groups, and the means were compared using Student's · · · ··· ······ · ·

- Perform 11 T-tests. In general, five animals per day are used for each experiment.

Example 1

Investigation of TGF-β induction

Measurement of TGF-β activity in serum-free culture supernatant

As described above, serum-free culture supernatants were collected from antigen-tolerated rats (Kehri et al., 1986, J. Exp. Med. 163: 1037-1050; Wahl et al., J. Immunol. 145: 2514-2419 (1990)]. Briefly, modulator cells are first cultured with antigen (50 μΙ / ml) for 8 hours in growth medium. The cells are then washed three times and resuspended in serum-free medium for the remainder of the 72-hour culture, then harvested and frozen until assayed. Determination of TGF-? Content and isoform type in supernatants was performed using a pulmonary epithelial cell line (American Type Culture Collection, Bethesda, MD # CCL-64) as determined by Danielpour et al., J. Cell. . Physiol. 138: 79-86 (1989)] and then determined by sandwich enzyme-linked immunosorbent assay (SELISA) as described previously (Danielpour et al., Growth Factors 2, 61-71 (1989)). The percentage activity of TGF-β is determined prior to assay without prior acid activation of the sample.

This assay can be adopted for testing any antigen that may be used as an inactive antigen. The antigens, antigen fragments and / or antigen amounts that cause the highest concentration of TGF-β, as measured by this assay, are considered to be the most suitable antigen and / or amount of the present invention.

- 15 Lesi methods. Alternatively, a transwell culture system, described below, may be used to indicate the level of TGF-β produced. This culture system measures TGF-? Production as a function of suppressing cell proliferation.

Transwell cultures

A two-chamber transwell culture system (Costar, Cambridge, MA), 24.5 mm in diameter, was used, consisting of two sections separated by a semipermeable polycarbonate membrane. pore size 0.4 Lim. The two chambers are 1 mm apart, allowing the cells to incubate together at close proximity without directly contacting each other. To measure the suppression of proliferative responses in vitro, cells from 5x10 ⁴ antigen lines grown and maintained, for example, as described above (Ben-Nun, A. et al., Eur. J. Immunol. 11, 195 (1981)] 10 ⁶ irradiated (2500 rad) thymocytes were cultured in 600 µl growth medium in the lower chamber. Spleen cells from orally tolerized rats or control rats (fed BSA) were added to the upper chamber (5 χ 10 ⁵ cells in 200 μΙ). The spleen cells were removed 7-14 days after the final feeding and a single cell suspension was prepared by passing the spleen through a stainless steel sieve. The antigen (50 pg / ml) was added in a volume of 20 µl. Because modulator cells are separated from reacting cells by a semipermeable membrane. so there is no need for irradiation. In some experiments, modulator cells are added together with the reacting cells, and in these cases, the modulator cells are irradiated immediately (1250 onto you) before being placed into the culture. The composition of the growth medium was RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 2 x 10 ° M β-mercaptoethanol, 1% sodium · · · · · · · · · · · · · · · · · · · · · ·· ·· ···· * pyruvate, 1% penicillin and streptomycin, 1% glutamine, 1% HEPES buffer, 1% non-essential amino acid and 1% autologous serum. Each transwell is prepared in four replicates. The transwells were incubated at 37 ° C in a humidified atmosphere of 6% CO ₂ and 94% air for 72 hours. After 54 hours of culture, each lower cell was pulsed with 4 µCi of [ ³ H] thymidine, then after 72 hours of culture, plated and placed in a well of a 96-well round bottom plate (Costar), and recovered on glass fiber filters and standard liquid. Percent suppression is calculated using the formula:

100x (responding cell cultured with 1-Acpm modulators / Acpm responding cells).

Example 2

Identification of immunosuppressive epitopes of guinea pig MBP by oral route

To examine oral vs. The mechanism of IV tolerance is that MBP peptides covering both the encephalogenic and non-encephalogenic regions are administered orally and intravenously prior to immunization for active-induced disease. The guinea pig MBP 71-90 peptide is encapsulated in Lewis rats [Swanborg et al., J. of Immunol. 114, 191 (1975)]. As shown in Figure 1B. As shown in FIG. 6A, suppression of EAE by intravenous tolerance can only be achieved with whole MBP and the encephalogen peptide 71-90, but not with the guinea pig MBP peptide 21-40. However, oral tolerance to 21-40 effectively suppresses EAE (Fig. 1A). Guinea pig peptide 21-40 was chosen because it has been shown in previous experiments that it produces splenocytes which release TGF-? . Guinea pig control peptides 131-150 were not suppressed when administered orally or intravenously (Fig. 1A). It should be noted that in addition to suppression by intravenous administration, the encephalogenic MBP 71-90 peptide also suppresses when administered orally. These results show that peptides derived from an immunodominant domain of an MBP against a particular host can suppress T cell function when administered orally or intravenously, but by another mechanism, depending on the treatment and administration protocol.

The results of these experiments indicate that there are fundamental differences in the mechanism of EAE suppression when MBP is administered orally or parenterally (i.e., intravenously). These results suggest that the oral antigen acts mainly by generating active suppression, whereas the parenterally administered antigen acts via clonal anergy. This conclusion is specifically supported by. that spleen cells from intravenously tolerated animals are unable to suppress adoptively transmitted EAE. In addition, different fragments of MBP show different abilities to suppress EAE, depending on the route of administration (see, for example, Figure 1).

The transwell system mentioned in Example 1 is used to identify epitopes on guinea pig MBP that induce TGF-β release from suppressor T cells.

MBP disease-inducing fragments (autoimmune response epitopes) are first amplified as follows: Guinea pig MBP overlap peptides are commercially available or synthesized using well-known techniques using a commercially available peptide synthesizer (Applied Biosystem) according to the manufacturer's instructions. . Total MBP is then fed to animals, and lymph glands from orally tolerated animals are induced with MBP peptides. Started cells kill T cells • ·

The ability of IS to kill is then quantified using a proliferation assay as described in Example 4, and by testing the ability of proliferating cells to transmit disease.

The guinea pig MBP 71-90 peptide was by far the most potent inducer of killer T cells. and therefore the most potent disease-causing fragment of MBP. Thus, this region of guinea pig MBP corresponds to the immunomodulatory epitope of the protein.

When spleen cells obtained from MBP-fed and immunized with MBP / FCA (see Example 1) were co-cultured in the transwell system with spleen cells isolated from OVA-fed animals, guinea-pig MBP 21-40, 51-70 and peptides corresponding to amino acids 101120 and 101120 to the modulator well, it appears that each is capable of triggering suppression of OVA-fed line proliferation. However, unlike experiments in other animals, the immunodominant epitope of guinea pig MBP in rat (corresponding to amino acid residues 71-90) is ineffective in inducing suppression in the transwell system. The peptides corresponding to the guinea pig MBP residues 151-170 and 161-178 inhibit the proliferation of the OVA (response) line, but this effect is non-specific and is likely due to the toxicity induced by these peptides in vitro, since the same peptides inhibit the proliferation of spleen cells isolated from OVA-fed animals when co-cultured with control (non-MBP-fed) modulator cells (data not shown).

Such a system can be readily adapted by one of ordinary skill in the art to identify peptides of interest to the antigen of interest in this mode of treatment.

Ι <) • · · · · · · · · · · · · · · · · · · · · · · · · · ···

Example 3

Oral tolerance using bovine PLP or mouse MBP

To demonstrate suppression with idle antigen, groups of 5-6 seven-week-old SJL / L mouse females (Jackson Labs, Bar Harbor, ME) were immunized with mouse PLP peptide 140-160 on days 0 and 7, followed by we give them the following treatments:

Groups:

1. Histone feeding (0.25 mg / mouse)

2. Mouse MBP Nutrition (0.25 mg / mouse)

3. Bovine PLP Nutrition (0.25 mg / mouse) (autoantigen suppression)

Each group was treated every other day for 7 days. In the intravenous group, the substance is injected into the vascular plexus. The PLP peptide used is the bovine PLP 140-160 disease-inducing fragment. The amino acid sequence of this peptide is COOH-PLAYTIGVFKDPHGLWKGLCNH ₂ , which represents the aforementioned amino acids.

As shown in Figure 2, both murine MBP and bovine PLP were equally effective in downregulating PLP-induced EAE when administered orally. An unspecific protein, a histone, was ineffective in suppressing EAE when given orally. Thus, an inactive antigen, in this case the mouse MBP, effectively suppressed EAE when administered orally to bovine PLP-induced EAE.

Guinea-pig MBP 71-90 for feeding various peptides. (the major immuno-dominant epitope of guinea pig MBP in rats, as shown in Example 1 above), we also examined the effects on EAE-induced Lewis rats.

EAE was induced by inducing 0.25 mg of peptide containing amino acids MBP 71-90 in complete Freund's adjuvant, and then investigating the effect of various marine MBP peptides on EAE.

As shown in Figure 3, orally administered whole guinea pig MBP and a peptide 21-40 of guinea pig MBP were as effective in suppressing 71-90 induced EAE in guinea pig MBP as in orally administered 71-90 itself. . Guinea pig MBP 131-150 was ineffective in inducing tolerance. The peptides were fed with STI, which prevents their degradation by gastric juice and enhances their biological activity. Total DTH responses to MBP (as a result of injection of 25 Ltg MBP in PBS) are suppressed by feeding MBP or by feeding any of the MBP peptides 21-40 or 71-90. However, DTH responses to guinea pig MBP 71-90 were suppressed only when either total MBP or guinea pig 71-90 peptide was fed and not affected by guinea pig MBP 21-40. This is consistent with the observation that MBP7190 fragment is not involved in idle suppression when administered to mice in which the disease is induced by peptide 71-90.

Example 4

Suppression of EAE in rats by a combination of oral tolerance to intraperitoneal [Hinterferon] with seafood (GP) MBP

EAE was induced in thirty female Lewis rats (ranging in weight from 175 to 200 g) by immunization with 100 l of emulsion on day 0 containing 0.25 mg of guinea-pig (GP) MBP and 100 pg of Mycobacterium tuberculosis (mt) as adjuvants. These rats were divided into five groups and given the following treatments on days 10, 12, and 14:

Groups:

1. PBS fed (1 ml / rat)

2. Guinea pig fed with MBP (1 mg / rat)

3. Rat (injected with-/ β-interferon (15,000 units / rat)

4. Guinea-pig MBP (1 mg / rat) fed + rat ^ / ((- injected with interferon (150,000 units / rat)

5. Guinea pig MBP (1 mg / rat) fed + ((injected with -interferon-imitated (0.20 IRU / rat)

6. ((-Injected with -interferon (0.20 IRU / rat)

Each injection is administered intraperitoneally. Following treatment, the clinical evaluation (as described above) is recorded daily for each rat and, for each group, the atlages clinical score gives the individual data points on the days following immunization.

As shown in Figures 4 and 5, oral tolerance to MBP has the expected effect on suppression of clinical scores (see Figure 4, A-D). Interferon α / β alone had very little effect on the disease at the dose administered, and was comparable to the PBS controls and the β-ßηterferon mimic control (see Figure 4, C and D). However, the combination of feeding guinea pig MBP and ((-interferon injection) significantly reduces the clinical score in treated animals. This is particularly significant when we see the ability of the β-interferon mimic formulation to suppress EAE suppressing MBP ( see Figure 4, D) In summary, the mean clinical score in this experiment is as follows:

PBS α / β IFN MBP «/ {(IFN + MBP IFN imitation IFN imitation + MBP 3.0 2.4 2.0 0.6 2.6 2.4

• 9 • · * 9 α «··. * · ··· <> ·· ♦ «·« · · · · · · · · · ·

Α Figure 6 shows similar data from an experiment shown in Figures -4, -2. and on day 0, female Lewis rats (150200 g) fed with 1 mg MBP and / or injected with 150,000 units / rat interferon α / β followed by immunization with 25 mg GP-MBP.

The combination of oral GP MBP has a synergistic suppressive effect on intraperitoneal β-interferon in rat EAE. This assertion is based on the comparison between the level when each treatment is applied separately and the superadditive level of suppression seen with combination therapy. Suppression with the combination is more than the sum of the two separate levels of suppression achieved with each individual treatment. The above results are determined by delayed-type hypersensitivity assays. In addition, in vitro measurement of cytokines produced by lymphocytes from nourished animals in response to culture-specific antigens suggests that synergistic effects may be associated with increased production of TGF-β and interleukin-4 or interleukin-10. Thus, interferon beta acts as a synergist to enhance oral tolerance to EAE.

Example 5

Suppression of EAE in mice by a combination of oral tolerance to MBP or PLP and intraperitoneal? -Interferon

EAE was induced in 35 female SJL / J mice by immunizing the mice on day 0 and 7 with 0.2 ml of a suspension containing 200 Lig of bovine PLP and 200 mg / ml Mycobacterium tuberculosis (MT). The mice were divided into 7 groups, receiving the following treatments on days 5, 8 and 10:

"·" * • • · · · · · · · · · · · ········ ········· _ν »> X

Groups:

1. Chick lysozyme (HEL) feeding (0.25 mg / mouse)

2. Mouse [(-interferon injection (69,000 units / mouse)

3. MBP feeding (0.25 mg / mouse)

4. Bovine MBP feeding (0.25 mg / mouse). plus [(-interferon injection (69,000 units / mouse)

5. Feeding of bovine PLP (0.25 mg / mouse)

6. Bovine PLP Feeding (0.25 mg / mouse) plus [(-interferon injection (69,000 units / mouse)

All injections are given intraperitoneally. Clinical scores were recorded daily for each mouse after treatment, and the mean clinical score for each group gives the data point for each day post-immunization.

The combination of oral bovine MBP with intraperitoneal [(-interferon and oral PLP) also has a synergistic suppressive effect on EAE in mice (see Figures 7 and 8). This claim is based on a comparison between The suppressive effect of the combination is more than the sum of the two separate levels of suppression achieved by the individual treatments.

• ·

Suppression of EAE in rats by oral MBP and oral γ-interferon · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Example 6 with a combination

Female Lewis rats (150-200 g) were fed with 1 mg of Myelin Basic Protein (MBP), various doses of rat <RTI ID = 0.0> <i> / "> - </RTI> or a combination of MBP and interferon. Oral proteins were administered a total of seven times each day with 4 preimmunizations and 3 postimmunizations with 25 µg of gpMBP to induce EAE. Alleles were scored for signs of paralysis from day 9 on a scale of 0 to 5. The results are reported below:

Groups fed Occurrence Starting day Average max value HEL 1.0 mg 5/5 12.0 2.0 β-interferon 20000 E 4/5 13 1.0 Interferon-j 10000 N 5/5 ........ 12 1.4 ...... β-interferon 5000 E 5/5 12.6 1.0 gpMBP 1.0 mg 5/5 12.4 1.0 gpMBP 1.0 mg + β- -interferon 20000 E 4/5 13.2 0.8

Example 7

Synergism of oral β-interferon and oral MBP in mouse EAE suppression

Female SJL mice are fed with 0.25 mg of bovine brain protein Myelin

Prior to immunization with Basic Protein (MBP) and 200 µg PLP and 200 µg MT, 3 times 5000 units of murine interferon β are added or *>

no, to induce EAE. Animals were evaluated for signs of paralysis from day 9 on a scale of 0 to 5. The results are reported below:

Groups fed Occurrence Starting day Average max. lis value HEL 1.0 mg 5/5 15.8 2.2 |) -interferon 5000 E 4/5 17.2 2.1 gpMBP 0.25 mg 4/5 18.8 1.6 gpMBP 0.25 mg + β- -interferon 5000 E 2/5 17.5 0.9

Example 8

Effect of oral γ-interferon on the induction of adjuvant arthritis in rats

Female Lewis rats (120-140 g) were fed with collagen type II (Cll) at the indicated doses and / or 5000 units of interferon beta, starting every other day, starting 10 days prior to immunization. On day 0, the animals are injected with 1 mg / 0.1 ml of Mycobacterium tuberculosis (MT). A +10. starting on day 2, the animals are scored on a scale of 0 to 4 for signs of arthritis. The arthritis score for each animal is the sum of the scores for each of the four paws.

The results are shown in Figure 9.

Claims (13)

PATENT CLAIMS • · A method of treating a mammal having a diagnosis of T cell mediated or T cell dependent autoimmune disease, comprising the steps of: the mammal is administered orally or enterally: (i) an amount of inactive antigen coupled to (ii) an interferon type I activity, the amount of (i) and (ii) in combination effective to suppress the autoimmune response in said mammal. The process of claim 1, wherein the amounts of (i) and (ii) are synergistically effective in suppressing said reaction when compared to administering said (i) and (ii) alone. 3. The method of claim 1, wherein said polypeptide is interferon beta and is administered parenterally. 4. The method of claim 1, wherein said polypeptide is administered interferon beta and orally. 5. The method of claim 1, wherein said mammal is a rodent and said disease is a rodent model of multiple sclerosis. 6. The method of claim 1, wherein said mammal is a human and said disease is multiple sclerosis. The method of claim 5, wherein said inactive antigen is selected from the group consisting of myelin basic protein (MBP), proteolipid protein (PLP), fragments thereof, and at least two of the above. ···· The method of claim 6, wherein said inactive antigen is selected from the group consisting of myelin basic protein (MBP), proteolipid protein (PLP), fragments thereof, and a combination of at least two of the foregoing . 9. The method of claim 7, wherein said interferon beta is from the same species as said mammal. 10. The method of claim 8, wherein said interferon beta polypeptide is derived from human interferon beta. 11. The method of claim 1, wherein said disease is selected from the group consisting of rheumatoid arthritis and an animal model thereof, and said idle antigen is selected from the group consisting of collagen type I, type II collagen. collagen type III, collagen type III, fragments thereof, and combinations of two or more of the foregoing. 12. The method of claim 1, wherein said disease is type 1 diabetes and an animal model thereof, and said idle antigen is selected from the group consisting of: glucagon, insulin, fragments thereof, and the foregoing. a combination of two or more of the foregoing. 13. The method of claim 1, wherein said disease is selected from the group consisting of uveoretinitis and animal models thereof, said inactive antigen is selected from the group consisting of S antigen, an interferon receptor retinoid binding protein (S). IBRP), fragments thereof, and combinations of two or more of the foregoing.