HUT76101A - Treatment of autoimmune disease using oral tolerization and/or th2-enhancing cytokines - Google Patents

Description

The present invention relates to a method for improving the ability to reduce the response to an autoimmune reaction by oral tolerance. More particularly, the present invention relates to the oral (or inhalation) administration of autoantigens, or more generally, the administration of inactive antigens in combination with the oral administration of polypeptides having Th2-enhancing cytokine activity to reduce autoimmune reactions or responses.

The present invention also relates to the oral use of Th2-enhancing cytokines (i.e., cytokines that divert the immune system toward a Th2 response or by enhancing Th2 responses or inhibiting Th1 responses) to reduce autoimmune responses.

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. These include, but are not limited to, these medicines.

We would limit ourselves, methotrexate, cyclophosphamide, lmuran (Azathioprine) and cyclosporine A. Steroids such as prednisone and methylprednisolone (also non-specific immunosuppressants) are also 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 have well-established methods and therapeutics for treating autoimmune diseases (as well as related Y-cell-mediated inflammatory diseases such as allograft rejection and retroviral neurological disease). formulations were designed. 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. This work is described in dependent patent application PCT / US93 / 0175, filed February 25, 1993, PCT / US91 / 01466, filed March 4, 1991, PCT / US90 / 07455, filed December 1009. 17, 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 1991 October 31, PCT / US91 / 02218, filed March 29, 1991, PCT / US93 / 03708, 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 via clonal fc V '- 5 anergy: the antigen may not be known, or there may be many different types of immune-attacking cells specific to different antigens, or they may change over time. antigens to which 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). Among these, TGF-β is an antigen-specific immunosuppressive factor, provided that all immune responses are suppressed, regardless of the antigen that triggered them. (However, since oral tolerance to an inactive antigen only induces TGF-β release in the immediate vicinity of the autoimmune attack, there is no systemic immunosuppression.) Interleukin-4 and interleukin-10 are also antigen-specific • · · ·

r - 6 immune regulatory cytokines. In particular, interleukin-4 enhances the Th2 response, i.e. acts on T-cell precursors and induces them to differentiate most into Th2 cells. Interleukin-4 is also an indirect inhibitor of Th1 enhancement. Interleukin-10 is a direct inhibitor of Th1 responses.

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, particularly interferon beta, is known to possess certain immunomodulatory properties, such as inhibiting the activity of γ-interferon (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. of histocompatibility complex (MHC) molecules on the surface of T-cells, as well as its ability to enhance suppressor T-cell activity,

. _Λ ......

• · · · · · ··· · · ·.

·· ·· * · * '·· is responsible for the immunomodulatory properties of parenteral interferon-β tolerance.

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, oral (or parenteral) use of Th2-enhancing cytokines for oral tolerance using autoantigens or idle antigens has not been described.

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 present inventors have made the following surprising discoveries:

oral or co-inhalation of peptides or peptide fragments having interleukin-4 and its Th2-enhancing cytokine activity is advantageous in suppressing autoimmune reactions associated with autoimmune disease;

a combination of (i) oral or co-inhaled administration of autoantigens or inactive antigens (or fragments thereof), and (ii) administration of polypeptides having Th2-enhancing cytokine activity is significantly more effective than using autoantigens or inactive antigens alone (or Th2-enhancing cytokines alone). use alone) in suppressing autoimmune reactions associated with autoimmune diseases.

In addition, parenteral administration of interleukin-4 has a suppressive effect on the autoimmune response associated with autoimmune disease and enhances the suppressive effect of idle antigens.

The use of other non-cytokine synergists is also compatible with the previous combination.

All patent applications, patent and literature references cited in this application are hereby incorporated by reference. In case of conflict, the definitions and interpretations of the present invention apply.

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

"Th2-Enhancing Cytokines" refers to naturally occurring antigen-specific immunoregulatory agents that: (i) are normally secreted or induced by regulatory immune system cells and (ii) enhance the frequency of Th2 cells (and / or inhibit Th1 cells).

Surprisingly, it has been found that oral administration of Th2-enhancing cytokines, alone or in combination with inactive antigens (see below), is beneficial in reducing autoimmune reactions or responses. Such cytokines include, but are not limited to, interleukin-4 as well as fragments thereof that retain their Th2-enhancing activity. It should be noted, however, that it is currently unknown whether the tolerance effect observed with oral administration of interleukin-4 and the enhancement effect observed when interleukin-4 is co-administered with an inactive antigen are due to the Th2-enhancing properties of interleukin-4.

An "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 that specific to an organ or tissue under autoimmune attack; and (ii) oral or enteral administration elicits regulatory (suppressor) T cells (which may be of the CD4 + or CD8 + type) directed against the target organ or tissue, wherein at least one antigen-specific immunosuppressive factor or immunoregulatory cytokine is present. (such as TGF-β, interleukin-4, or interleukin-10), thereby suppressing immune attacking 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.

"Idle suppression" is suppression of the site of autoimmune attack of cells contributing to autoimmune destruction; this suppression is mediated by the release of one or more immunosuppressive factors (including Th2-enhancing cytokines and Th1 inhibitory cytokines) from suppressor T cells induced by inactivation (or inhalation) of inactive antigen and where cells that contribute to autoimmune destruction are found. The result is an antigen-specific but locally restricted suppression of autoimmune responses responsible for tissue destruction.

"Mammal" means an organism that has an immune system and is susceptible to autoimmune diseases.

"Autoimmune disease", as used herein, means spontaneous or induced malfunctioning 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 the autologous tissue. and treating substances as if they were aliens and eliciting an immune response against them. The term also applies to autoimmune diseases and their animal models.

The term "autoantigen" refers to any substance or portion thereof normally found in a mammal, but in an autoimmune disease, it is the primary (or primary) target of the immune regulatory system. 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 are fragments of an autoantigen under induced autoimmune conditions 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.

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 or alleviate symptoms after the onset of such autoimmune diseases. attacking the immune system and preventing or slowing down autoimmune tissue damage. Suppressing or diminishing an autoimmune attack or reaction involves partially reducing or ameliorating one or more symptoms of the attack or reaction. A substantially enhanced suppressive effect (or amelioration or reduction) of an autoimmune reaction is a significant reduction in one or more markers or histological or clinical indicators of the autoimmune reaction or disease. Examples include, but are not limited to, a reduction of at least 1 unit in limb paralysis score or arthritis score, or a significant decrease in autoreactive T cell frequency; a reduction of at least 0.5 units in the insulitis score (Measured in Zhang et al., 1991, Proc.

As used herein, the use of a Th2 enhancer cytokine "in association with (or" in association with "inactive antigens) means that the inactive antigens are administered substantially simultaneously with, prior to or after oral (or co-inhalation) administration. . The term "substantially at the same time" means within 24 hours, and preferably within one hour, before, or after, administration of an inactive antigen.

By "oral" administration is meant oral, enteral or gastric administration. In addition, co-inhalation administration in aerosol form has the same tolerating effect and is equivalent to oral tolerance.

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

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 initiated by immunization with myelin basic protein (MBP) or proteolipid protein (PLP) and an adjuvant (such as Freund's Complete Adjuvant, "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 details of the induction) 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. These animal models also provide a good predictor of successful oral tolerance to idle antigens.

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.

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. Therefore, in the case of oral tolerance, the suppression of symptoms obtained 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 Th2-enhancing cytokine-based therapies, since cytokines generally have the same or similar 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 effect 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, in reducing or eliminating 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 Th2-enhancing cytokines such as 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. As regulatory T cells are generated, the mechanism of the processes involved is described as active suppression. Immunoregulatory cytokines evoked by regulatory regulatory cells are antigen-specific, even though these regulatory T cells only release (or induce release) immune regulatory cytokines when they are excited by the same antigenic determinant as orally administered (or inhaled) antigen. . 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 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: After a tissue-specific (inactive) antigen is administered orally (or enterally, i.e. directly into the stomach), it enters the small intestine where it is contacted by the so-called Peyer. patches and pauses, which are groups of large numbers of immunocytes located beneath 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 circulation, and then assemble at the site of autoimmune attack, where they cause the release of TGF-β and / or other immune regulatory agents that suppress activated helper T cells directed against the mammalian own tissues as well as B cells (Chen, Y. et al., Science 265: 1237-1240 (1994)). 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, 51-55 (1989) (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)].

• · · · • ··· «

.. · · .. · ·. ·

- 17 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 (II); and the amino acid sequence of human insulin is well known and published. These fragments can be synthesized by recombinant DNA techniques, as is well known to those skilled 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. Stimulation-induced T cells can be purified and the supernatants assayed for 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) [Miller, A. et al., J. Immunol. 148: 1106 (1992)]. 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 for interleukin-4 or interleukin-10 secretion in a similar manner ···· ......

· · · · · · · · · · · ·

· .. · .. · · .. · ·.

- 18 aspects (anti-interleukin-4 and anti-interleukin-10 antibodies are commercially available, e.g., from Pharmingen, 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.

Use of idle antigens - doses

Tolerance induced by the inactive antigens of the present invention is dose dependent over a wide range of oral (or enteral) or inhaled 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 is 0.5 to 2 mg / rat / feed

varies, 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 people receiving type I or type II or type III collagen is about 0.1 to 1 mg / day, preferably 0.1 to 0.5 mg / day. In rats, in the case of adjuvant arthritis, the effective dose of collagen ranges from 3 to 30 mg / feed, following the same diet regimen

For UAE.

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 is well known to those skilled 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 sensitivity, morning stiffness, grip strength, joint imaging techniques.

AUR: Visual acuity; number of T cells in the eye and "cloudiness" in the eye, type 1 diabetes: pancreatic beta cell function (such as

OGTT glucose tolerance test).

FEMALE MODEL: Insulitis and Delayed 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 arthritis 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. (Clinically significant attenuation is what is observed by a clinician with expertise in a particular autoimmune disease.)

When combined with interleukin-4 treatment, the dose of idle antigen is preferably equivalent to that which would be used if only the inactive antigen was administered orally, except that the combination is more effective in suppressing the autoimmune reaction. However, the level of interleukin-4 may be the same as that used when only interleukin-4 is administered, or it may be suboptimal (i.e., an amount that would not be sufficient if only interleukin-4 was used, but enough to potentiate the tolerating effect of idle antigen).

Determining the effective dose range and optimal amount of an idle antigen is a simple task for those skilled in the art. For example, mammalian and human dose determination begins with a relatively low dose (e.g., 1 pg) which progressively (e.g., logarithmically) increases and measures TGF-beta (and / or interleukin-4 or interleukin-10) secreting cells and / or by estimating the number of immune attacking T cells in the blood (e.g., by limiting dilution 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)).

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 below.

table. 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 Th2-enhancing cytokines may also be administered by inhalation. The amount of inactive antigen to be inhaled is less than that to be given orally. Similarly, the amount of Th2-enhancing cytokines administered by inhalation is expected to be less. Effective amounts of inhalation therapy may be determined by the methods described above.

 spreadsheet

Type cleaning cleaning cleaning cDNA Source j J.Chromatogr. Biomed.Appl. 526-535 (90) J.Immunoi.Meth.h1 21-219 (89) 151-177 (92) Exp.Eye Slit. 56, 463 (93) Inactive antigen glucagon insulin GAD MBP, MBP fragments PLP, PLP fragments myelin related GP myelin oligodendrocyte glycoprotein; heat shock protein collagen I, II or III; RO / SS-A RO / SS-B-LA heat shock protein S-antigen IRBP recoverin Affected tissue Pancreatic beta cells myelinated neurons connective tissue eye Autoimmune disease Type 1 diabetes Multiple sclerosis Rheumatoid arthritis Autoimmune uveitis

2F. Continue Table

cleaning cDNA cleaning cDNA Eur.J.Pharm. 172, 231 (89) Bioi.Repród. 43, 559 (1990). Bioi.Chem.H-S. 368, 531 (1987). Eur.J.Cell. Biol. 55, 200 (91) acetylcholine receptor heat shock protein NASP (post-acrosomal sperm protein) Jo-1 antigen heat shock protein desmoglein Factor XIII muscle semen muscle Skin Myasthenia Gravis Male infertility myositis pemphigus

- AQ • ·

For any autoimmune disease, extracts from appropriate tissue as well as specific inactive antigens or fragments thereof may be used as oral tolerants. 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) can 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, although anti-insulin autoantibodies have been found in type 1 patients. However, whatever its mechanism of action, oral, enteral or inhaled insulin formulations suppress type 1 diabetes and prevent autoimmune destruction of pancreatic beta cells in animal models.

In multiple sclerosis and in animal models of this model, both disease-inducing and non-disease-causing fragments of MBP have inactive activity, not only in MBP-induced disease but also in PLP-induced disease. In rats, feeding of idle antigen mainly produces CD8 + suppressor cells, which are restricted to class I, whereas mice produce both CD8 + suppressors and CD4 + regulatory cells (the latter probably being restricted to class 11) [Chen, Y., et al. : Science 265: 1237-1240 (1994)].

In rheumatoid arthritis and animal models, collagen type I, type II and type IIIas have oral toilizer activity. Other collagens are probably similarly active.

In the uveoretinitis and animal model, 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 and T cells from fed animals can be tested for secretion of TGF-β-ß and / or interleukin-4 and / or interleukin-10 and their subtype can also 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. Immuno-dominant epitopes of autoantigens, such as MBP, are capable of inducing such regulatory T cells. Other such epitopes can be identified by inserting an idle 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 »· • ·

28 fragments) or T-cells from an animal fed with an inactive antigen are capable of adoptively 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 March 1991 October 10, 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 the co-administration of at least two autoantigens (and / or fragments of autoantigens) or, more broadly, at least two other inactive antigens (and or fragments of inactive antigens) will likewise be effective suppressors of autoimmune diseases.

In addition, other cytokine and non-cytokine synergists may be used in combination to enhance the efficacy of oral tolerance using inactive antigens alone or using inactive antigens plus Th2-enhancing cytokines. The use of other cytokine synergists (type I interferons) is described in U.S. Patent Application Serial No. 08 / 225,372. Non-cytokine 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, Ml; BIOMOL Rés. Labs., Plymouth, PA), A Lipid A • ·

- 29 (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 "Braun's" lipoprotein from Escherichia coli, which may be prepared as described by Braun et al., (1976), Braun, V. Biochimica et Biophysica Acta 435, 335-337; and the cholera toxin β chain (CTB), whose synergistic ability has been described (albeit not in relation to immune suppression) (Sun et al., 1994, Proceedings of the National Academy of Sciences, USA 91). but especially Lipid A is particularly preferred for use in the present invention because it is less toxic than the entire LPS molecule. For use in the present invention, LPS can be extracted from Gram-negative bacteria and Galanes et al. (Galanes et al., Eur. J. Biochem. 9, 245 (1969); Skelly, RR et al., Infect. Immun. 23, 287 (1979)). An effective dose of non-cytokine synergists in mammals is about to about mg / kg body weight, preferably 300 pg to 12 mg / kg body weight An effective dose of oral Type 1 interferon is in mammals 1000-150,000 units, no maximum effective dose was observed.

Oral use of Th2-enhancing cytokines alone in the present invention

In accordance with the present invention, oral, enteral or co-inhaled administration of Th2-enhancing cytokines is used to suppress autoimmune diseases. An example of a Th2 enhancer cytokine is interleukin-4.

Interleukin-4 can be purified from natural sources (e.g., T cells that normally produce it) and can also be produced by recombinant DNA technology using bacterial, yeast, insect and mammalian cells using techniques well known to those skilled in the art. Interleukin-4 is also commercially available. The human interleukin-4 DNA sequence is disclosed by Yokota et al., 1986, in Yokota et al., Proceedings of the National Academy of Sciences USA 83: 5894.

According to the present invention, the mode of administration of interleukin-4 is preferably oral or enteral. Preferred oral or enteral pharmaceutical formulations include pills, liquids, capsules containing an effective amount of interleukin-4.

Each of the oral (or enteral) formulations of the present invention may further contain inert components, including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents, and salts, as are well known to those skilled in the art. For example, tablets may be prepared by conventional techniques using solid carriers well known to those skilled in the art. The capsules used in the present invention may be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives. Controlled release oral delivery systems and / or enteric coatings for oral dosage forms may also be contemplated, such as those disclosed in U.S. Patent No. 4,704,295, issued November 3, 1987; No. 4,556,552, filed December 3, 1985; No. 4,309,404, filed January 5, 1982; and 4,309,406, filed January 5, 1982.

Solid carriers include starch, sugar, bentonite, silicate, and other commonly used carriers. Carriers and diluents include, but are not limited to, saline, syrup, dextrose, and water, which may be used in the compositions of the present invention.

It will be appreciated that the amount of active ingredient or additives in a single dose or in each dosage form need not necessarily be effective on its own, since the required amount can be achieved by administering multiple unit doses (i.e., capsules, tablets, or combinations thereof).

Generally, when administered orally or enterally, interleukin-4 may be administered in a single dose or in multiple doses.

Suppression of the clinical and histological symptoms of an autoimmune disease occurs after a specific minimum dose, but varies depending on the disease, the mammalian species and the cytokine. The effective dose range for oral interleukin-4 in humans is between 2000 and 50,000 international units per day. The maximum dose is best determined experimentally. It is expected that higher doses are conceivable but not necessary. It is not necessary that a dose of interleukin-4 alone be active when interleukin-4 (or other Th2 enhancer cytokine) is used in combination with an autoantigen or an idle antigen. Suboptimal doses of a Th2 enhancer cytokine that potentiate the idle antigen or the autoantigen may thus be used.

Determining the effective dosage range as well as the optimum amount is straightforward for those skilled in the art from the information in this section. For example, mammalian and human doses may be determined by starting with a relatively low dose of cytokine (i.e., 500 units of interleukin-4, progressively), increasing (e.g., logarithmically), and measuring the biological response of the treatment, such as induction of regulatory cells (CD4 + and / or CD8 +) as described by Chen et al., Chen, Y., et al., Science.

32 265: 1237-1240 (1994)], a reduction in the number of class II cell surface markers on circulating T cells, and / or a measure of the severity of the disease, using well-known scoring methods (e.g., a 1 to 5 scale). , or by measuring the number of attacks or by measuring swelling of the joints, grip strength, stiffness, degree of visual acuity, or the ability to reduce or stop medication, etc., depending on the type of disease. The optimum dose will be the one that has the greatest influence on the biological phenomenon being measured, i.e., which causes the greatest induction of regulatory T cells, or which causes the greatest reduction in immune attacking cells, and / or which produces the strongest reduction in the symptoms of the disease. There will be an effective dose that results in at least a statistically significant improvement in at least one symptom of the disease being treated, or a significant change in a marker (i.e., the frequency of regulatory or activated T cells).

Interleukin-4 may be administered once daily for a period of from 30 days to several months (e.g. 3-6 months) or even years (e.g. 2-6 years). In fact, therapy can continue indefinitely (unless the resulting benefit stops), since oral administration has a lower risk of side effects.

Protease inhibitors (i.e., soybean trypsin inhibitor, aprotinin, antipain) can be added to oral dosage forms containing interleukin-4 to increase the amount absorbed. In this case, the dose of interleukin-4 may be reduced.

Parenteral administration of interleukin-4 may be used alone or as an adjuvant to oral tolerance therapy, but oral interleukin-4 is preferred because of the systemic action of parenteral interleukin-4. However, parenteral interleukin-4 is quite effective in suppressing autoimmune diseases, as illustrated below. Parenteral dosage form in mammals • · • ·

The size of 33 generally ranges from 500 international units of interleukin-4 to about 1,000,000 international units, although the upper limit of this range can be experimentally determined. It is expected that the upper limit is the amount at which the maximum suppressive effect of parenteral interleukin-4 can be observed (i.e., efficacy at higher doses is not lost but may be unnecessary). Parenteral administration may be carried out subcutaneously, usually once every other day (without limitation), in single or divided doses.

Combination therapy

Surprisingly, it has been found that oral (or inhalation) administration of an inactive antigen in combination with oral, parenteral, or co-inhalation administration of interleukin-4 results in a treatment that suppresses the autoimmune response and that the effect of the combination therapy is greatly enhanced. compared to its separate application.

This combination treatment was investigated in rodents using MS and EAE animal models. Experimental protocols for these assays are described in the examples below.

Treatment of mice with 1000 international units of interleukin-4 alone, administered orally five times prior to induction of EAE, reduces the incidence of the disease and all decreases in clinical score are also observed.

Treatment with a combination of oral administration of rat interleukin-4 (1000 units) and oral tolerance to MBP reduces both the incidence and clinical severity of the disease and delays the onset of the disease. In fact, the delay in disease onset was greater (30 days) with combination therapy than when either interleukin-4 or MBP was administered alone (21 or 22 days).

The combination of intraperitoneal interleukin-4 and oral tolerance to an idle antigen is expected to have a synergistic suppressive effect on the clinical score of EAE, which is observed when both the inactivated antigen and the Th2 enhancer are decreased to reveal this effect. . It is clear that these treatments have a significantly stronger suppressive effect when used in combination than when either treatment is used alone.

In the practice of the present invention, the idle antigen is administered essentially as described above. Interleukin-4 is co-administered with the idle antigen. Oral or enteral administration of interleukin-4 can be achieved as described above.

The parenteral route may be the subcutaneous, intramuscular or intraperitoneal route, although the therapeutic goals are best suited for the subcutaneous route (although the intraperitoneal route is used in the examples below). For parenteral administration, interleukin-4 may be formulated in sterile saline or other carriers well known to those of ordinary skill in the art and may contain excipients and stabilizers generally accepted in the art.

Determining the optimal delivery range for both inactive antigen and Th2-enhancing cytokines is a straightforward task for those skilled in the art, in light of the information described below. Just as when inactive antigens or Th2-enhancing cytokines are administered separately, routine variations in dosages, combinations, and duration of treatment may be performed under conditions in which the autoimmune reaction occurs.

- 35 severity can be measured. Useful dose and administration parameters that result in a decrease in the autoimmune response, including a decrease in the number of autoreactive T cells or a decrease in the incidence or severity of at least one clinical or histological symptom of the disease.

It is preferable to use cytokines from the same species as the species being treated.

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

Anvaqs and Methods

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

Animals: 6-8 week old female 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. Histone, chicken lysozyme and ovalbumin were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). The peptides were synthesized on a Center Forum Neurologic Disease, Brigham and Women's Hospital peptide apparatus and then HPLC

- 36 cleaned. The amino acid sequences of MBP peptides synthesized for use are as follows:

71-90

SLPQKSQRSQDENPVVHF (immunodominant encephalitogenic region in rats); amino acid residues 140-160 of murine PLP peptide (disease-inducing epitope in rats);

amino acids 139-153 of the murine PLP peptide (disease-inducing epitope in mice).

Mouse interleukin-4 was obtained from Collaborative Biomedical Products, Bedford, MA.

Induction of tolerance

For oral tolerance or active suppression, rats were fed 1 mg MBP in PBS or MBP alone, by gastric intubation with an 18 stainless steel feed 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. When mice are used in the experiments, the range of tolerance is substantially the same except that 0.5 mg MBP and / or 1000 or 5000 units of interleukin-4 are used for the tolerance. Details of the mouse experiments are given below.

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). When mice were used in the experiments, 400 pg MBP dissolved in 0.1 ml PBS / CFA containing 4 mg / ml Mycobacterium tuberculosis was used.

• ·

- 37 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 the hind limb; 3 = bilateral paralysis of hind limbs, urine drainage; 4 = paralysis of four limbs; and 5 = death. Disease duration is measured by the number of days since disease onset (typically 10-11 days after active immunization in control rats and 9 days after immunization in control mice) until complete recovery (or death) of each animal.

Histology

Histological analysis of pathological changes is performed in animals with induced 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 blue hematoxylin 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 for each site of inflammation. (> 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 analysis to compare disease incidence across groups, and means compared with Student's T-test. In general, five animals per day are used for each experiment.

Example 1

Investigation of TGF-β induction

Measurement of TGF-S 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: 10371050; 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 lung epithelial cell line (American Type Culture Collection, Bethesda, MD # CCL-64) as described 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)). Percentage activity of TGF-β was determined prior to assay, prior to acidic 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 amounts of antigen that cause the highest concentration of TGF-β, as measured by this assay, are considered to be the most suitable antigen and / or amount for the treatment method of the present invention. Alternatively, a transwell culture system, described below, can be used to detect the level of TGF-β produced.

* «' ³⁹ '

This culture system measures TGF-β production as a function of suppressing cell proliferation.

The presence of interleukin-4 and / or interleukin-10 in the supernatant of antigen-stimulated cells may also indicate that the antigen is an inactive antigen. Interleukin-4, interleukin-10 (and TGF-β) can be assayed using a commercially available antibody to each polypeptide as described by Chen et al., Science 265: 1237-1240. 1994)].

Transwell cultures

A two-chamber transwell culture system (Costar, Cambridge, MA), 24.5 mm in diameter and divided into two portions separated by a semipermeable polycarbonate membrane, has a pore size of 0.4 µm. The two chambers are 1 mm apart, allowing the cells to incubate together at close proximity without directly contacting each other. To measure in vitro suppression of proliferative responses, cells derived from 5 to 10 ⁴ antigen lines grown and maintained, for example, as described above (Ben-Nun, A. et al., Eur. J. Immunol. 1_1_, 195 (1981)] were cultured with 10 ⁶ irradiated (2500 rad) thymocytes in 600 µl of propagation 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 l). 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. Since modulator cells are separated from reacting cells by a semipermeable membrane, there is no need for irradiation. In some experiments, modulator cells are added together with the reactive cells, and in these experiments, the modulator cells are added.

- In 40 cases, the modulator cells were irradiated immediately (1250 onto you) before being placed in culture. Composition of the growth medium was RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 2 x 10 ⁵ M β-mercaptoethanol, 1% sodium pyruvate, % 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 μθ [ ^{2 3} H] thymidine, then after 72 hours of culture, plated and placed in a well of a round bottom 96-well 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

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) ···· • ·

The PLP peptide used is a disease-causing fragment of bovine PLP 140-160. The amino acid sequence of this peptide is COOHPLAYTIGVFKDPHGLWKGLCNH ₂ , which represents the aforementioned amino acids.

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, 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 the guinea pig MBP 71-90 peptide in complete Freund's adjuvant, and then investigating the effect of various guinea pig MBP peptides on EAE.

Orally administered whole guinea pig MBP and a 2140 peptide of guinea pig MBP were as effective in suppressing guinea pig MBP 71-90-induced EAE as did oral 71-90 itself. Guinea pig MBP 131-150 was ineffective in inducing tolerance in rats. The peptides were fed with STI, which prevents their degradation by gastric juice and enhances their biological activity. Total DTH responses against MBP (25 μg MBP by injection in PBS) were suppressed by MBP feeding or by feeding any of 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 ··· ·· ·· · · · · · «· ♦ * · - · ·

peptide. This coincides with the observation that MBP fragment 71-90 is not involved in idle suppression when administered to mice in which the disease is induced by peptide 71-90 (Chen, Y., et al., Science 265; 12371240 (1994).

In the following experiment, an assay system for determining whether an orally administered antigen acts as an inactive suppressor is shown. The whole antigen could have been used instead of the fragments.

Example 3

Suppression of EAE in mice by a combination of oral tolerance to MBP and oral interleukin-4

EAE was induced in eight-week old female SJL / J mice immunized with 400 pg mouse MBP on day 0 in 0.1 ml suspension containing 4 mg / ml Mycobacterium tuberculosis (MT) followed by an injection of pertussis toxin ( 100 ng / mouse) on days 0 and 2. A -10., -8., -6., -4. and -2. days, different groups of mice were treated as follows:

Groups

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

2. Interleukin-4 feeding (1000 IU / mouse)

3. MBP feeding (0.5 mg / mouse)

4. MBP feeding (as in group 3) plus interleukin-4 (as in group 2).

The animals were observed for 35 days for disease onset. The animals were scored daily for signs of disease, starting at day 9, using a 0-5 scale.

The results are as follows: Substantially simultaneous administration of interleukin-4 with MBP clearly enhanced the suppressive effect of MBP on the incidence and delay of the disease. At 1000 IU / mouse / feed, interleukin-4 alone had a similar suppressive effect on disease incidence as when MBP alone was fed.

This type of experiment can be used to evaluate the efficacy of treatment with Th2 enhancer cytokine alone or combination therapy with Th2 enhancer and an inactive antigen. The dose may be adjusted by monitoring the effect of the dose variations.

Example 4

Suppression of EAE in mice by a combination of oral tolerance to MBP and oral interleukin-4

EAE was induced in eight-week old female SJL / J mice by immunization with 400 pg mouse MBP on day 0 in 0.1 ml suspension containing 4 mg / ml Mycobacterium tuberculosis (MT) followed by injection of a pertussis toxin ( 100 ng / mouse) on days 0 and 2. A -10., -8., -6., -4. and -2. days, different groups of mice were treated as follows:

Groups

1. Mouse Interleukin-4 Feeding (5000 International Units / Mouse)

2. Mouse MBP Feeding (0.5 mg / mouse)

3. Mouse MBP feeding (as in group 2) plus interleukin-4 (as in group 1).

The animals were observed for 35 days for the appearance of the disease. Animals were evaluated daily for symptoms of disease, starting at day 9, using a 0-5 scale.

This experiment demonstrates that administration of interleukin-4 alone at this dose significantly delays the onset of disease, reduces mortality, and / or reduces mean and maximum clinical counts. Furthermore, feeding interleukin-4 at said dose in combination with MBP significantly enhances the suppressive effect that can be observed when MBP is administered alone.

Example 5

PLP-induced EAE suppression in mice with a combination of oral tolerance using MBP and oral interleukin-4

EAE was induced in eight-week-old female SJL / J mice immunized with 100 pg mouse PLP 139-151 peptide on day 0 in 0.1 ml suspension containing 4 mg / ml Mycobacterium tuberculosis (MT) followed by a pertussis toxin injection (100 ng / mouse) on days 0 and 2. A -10., -8., -6., -4. and -2. days, different groups of mice were treated as follows:

Groups

1. Mouse Interleukin-4 Feeding (5000 International Units / Mouse)

2. Mouse MBP Feeding (0.5 mg / mouse)

3. Mouse MBP feeding (as in group 2) plus interleukin-4 (as in group 1).

The animals were observed for 35 days for the appearance of the disease. Animals were evaluated daily for symptoms of disease, starting at day 9, using a 0-5 scale.

This experiment demonstrates that administration of interleukin-4 alone at this dose significantly delays the onset of PLP-induced disease, decreases mortality, and / or reduces mean and maximum clinical numbers. Further, when interleukin-4 is fed at said dose in combination with pure idle antigen MBP, it significantly enhances the suppressive effect that can be observed when MBP alone is fed.

Example 6

Suppression of adjuvant arthritis in mice by combination of oral tolerance with collagen type 11 and interleukin-4

Arthritis was induced in male DBA / 1 Lac J mice by 300 μg bovine Type II collagen subdermal immunization in a suspension containing 4 mg / ml Mycobacterium tuberculosis. A -14, -12, -10, -8, -6, -4. and -2. days, different groups of mice are treated as follows:

Groups

1. Mouse Interleukin-4 Feeding (5000 International Units / Mouse)

2. Mouse Type II Collagen Feeding (0.5 mg / mouse)

3. Mouse type II collagen feeding (0.5 mg / mouse) (as in group 2) plus interleukin-4 feeding (as in group 1).

The animals were observed for arthritis for 60 days. A +10. from day 1, the animals were evaluated for signs of arthritis on a scale of 0 to 4. The arthritis value for each animal is the sum of the values for the four paws.

This experiment demonstrates that, at the above-mentioned dose, if only interleukin-4 is fed, it significantly delays the onset of disease symptoms and / or reduces the mean and maximum clinical values. In addition, when interleukin-4 is fed at the above-mentioned dose in combination with collagen type II, it significantly enhances the suppressive effect obtained when fed with collagen alone.

• · · · • · · ·

Example 7

Suppression of diabetes in diabetes in combination with oral interleukin-4 and insulin alone

Groups of four-week non-obese diabetic (NOD) mice (3 animals per group) were treated as follows:

1. Ovalbumin feeding (1 mg / mouse)

2. Horse insulin feeding (1 mg / mouse)

3. Mouse Interleukin-4 Feeding (5000 International Units / Mouse)

4. Insulin feeding (as in group 2) and interleukin-4 feeding (as in group 3).

Mice are fed ten times, alternating days. Approximately 3 weeks after the experiment, the following parameters are estimated: time of onset of insulin and / or diabetes.

This experiment demonstrates that feeding the interleukin-4 alone at the above-mentioned dose significantly reduces the above clinical indications. In addition, when interleukin-4 is fed in combination with insulin, it significantly enhances the suppressive effect that can be observed when only insulin is administered.

Example 8

Suppression of EAE in mice by a combination of oral tolerance to MBP and intraperitoneal administration of interleukin-4

EAE was induced in eight-week-old female SJL / J mice immunized with 400 pg mouse MBP on day 0 in 0.1 ml suspension containing 4 mg / ml Mycobacterium tuberculosisA (MT) followed by an injection of pertussis toxin (100 ng). / mouse) on days 0 and 2. Different groups of mice were treated as follows:

· «·· ·· · · · · ·

Groups

1. Mouse Interleukin-4 (5000 International Units / mouse / injection) is administered intraperitoneally on days 0 and 3.

2. Mouse MBP (0.5 mg / mouse) was fed at -10, -8, -6, -4. and -2. days.

3. Mouse MBP is fed (as in group 2) plus interleukin-4 (as in group 1).

The animals are monitored for the presence of the disease for 35 days. Animals were scored daily from day 9 on the basis of disease symptoms on a scale of 0 to 5.

In this experiment, it was demonstrated that, at the doses described above, injecting only interleukin-4 would delay the onset of the disease and reduce clinical values. The magnitude of this effect is such that it is impossible to judge whether parenteral administration of interleukin-4 can enhance the protection afforded by oral administration of MBP, which also showed comparable suppression of the autoimmune response (as measured by the indicators described above).

Example 9

Suppression of EAE in mice by combination oral tolerance using MBP and intraperitoneal administration of interleukin-4

EAE was induced in eight-week-old female SJL / J mice immunized with 400 μg mouse MBP on day 0 in 0.1 ml suspension containing 4 mg / ml Mycobacterium tuberculosis (MT) followed by an injection of pertussis toxin ( 100 ng / mouse) on days 0 and 2. Different groups of mice were treated as follows:

·· ··. ·. '··· :: :::: ......' ·. · ·

- 48 *

Groups

1. Mouse MBP feeding (0.3 mg / mouse) at -10, -8, -6, -4. and -2. days.

2. Intraperitoneal injection of mice with interleukin-4 (500 International Units / mouse).

3. Intraperitoneal injection of mice with interleukin-4 (1000 International Units / mouse).

4. Intraperitoneal injection of mice with interleukin-4 (2000 IU / mouse).

5. Mouse MBP feeding (as in group 1) plus interleukin-4 (as in group 2).

6. Mouse MBP feeding (as in group 1) plus interleukin-4 (as in group 3).

7. Mouse MBP feeding (as in group 1) plus interleukin-4 (as in group 4).

Interleukin-4 injections were administered as described in Example 8.

The animals are monitored for disease for 35 days. Animals are evaluated daily from day 9 on the basis of disease symptoms on a scale of 0 to 5.

This experiment demonstrates that, at the indicated reduced doses, parenteral administration of interleukin4 in combination with MBP significantly enhances the suppressive effect that can be observed when MBP is administered alone.

Claims (17)

A method of suppressing an autoimmune reaction in a mammal suffering from a T-cell mediated or T-cell dependent autoimmune disease, characterized in that the mammal is administered: (i) an inactive amount of antigen, by oral or enteral route; (ii) an amount of a non-interferon polypeptide having Th2-enhancing cytokine activity, said antigen and the amount of said polypeptide being substantially more effective in combination than administering said antigen or said cytokine alone. The method of claim 1, wherein the amount of said antigen and said polypeptide in combination is substantially more effective in suppressing said reaction than when said antigen and said polypeptide are administered alone. 3. The method of claim 1, wherein said polypeptide is selected from the group consisting of interleukin-4 and fragments thereof having Th2-enhancing cytokine activity. 4. The method of claim 1, wherein said polypeptide is interleukin-4. The method of claim 4, wherein said interleukin-4 is derived from the same species as said mammal. 6. The method of claim 1, wherein said polypeptide is administered orally. 7. The method of claim 1, wherein said mammal is a rodent and said disease is a rodent model of multiple sclerosis. • · - 50 8. The method of claim 1, wherein said mammal is a human and said disease is multiple sclerosis. 9. The method of claim 7, wherein said idle antigen is selected from the group consisting of milein, myelin basic protein (MBP), proteolipid protein (PLP), tolerogenic fragments thereof, and the foregoing. at least two. 10. The method of claim 8, wherein said inactive antigen is selected from the group consisting of myelin, myelin basic protein (MBP), proteolipid protein (PLP), tolerant fragments thereof, and at least two. 11. The method of claim 1, wherein said disease is selected from the group consisting of rheumatoid arthritis, collagen-induced arthritis and adjuvant-induced arthritis, and said idle antigen is selected from the group consisting of type I antigens. collagen, collagen type II, collagen type III, tolerogenic fragments thereof, and combinations of two or more of the foregoing. 12. The method of claim 1, wherein said disease is selected from the group consisting of type 1 diabetes and said idle antigen selected from the group consisting of GAD, glucagon, insulin, tolerogenic fragments thereof, and the foregoing. a combination of two or more. 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. 14. A method of suppressing an autoimmune reaction in a mammal having a diagnosis of T cell-mediated or T cell dependent autoimmune disease, comprising the steps of: administering to said mammal a non-interferon polypeptide having Th2-enhancing cytokine activity in an amount effective to suppress at least one of the clinical or histological symptoms of said disease. 15. The method of claim 14, wherein the polypeptide is selected from the group consisting of interleukin-4 and fragments thereof having Th2-enhancing cytokine activity. 16. The method of claim 14, wherein said mammal is a human. 17. The method of claim 14, wherein said polypeptide is administered interleukin-4 by oral or enteral route.