CA2185353A1 - Treatment of autoimmune disease using oral tolerization and/or type i interferon - Google Patents

Abstract

The invention is directed to methods for treating autoimmune diseases such as multiple sclerosis by orally administering a bystander antigen such as myelin basic protein or proteolipid protein in conjunction with a polypeptide having Type I interferon activity in such a manner as to induce oral tolerance to the bystander antigen resulting in suppression of the autoimmune response.

Description

~ WO 95127499 i2 ~ 8~ 3~ 12(~
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TT~T~I-'I'Ml~T OF A~TONE DISEASE
rT~TT~ Tv~r, Tr~T.r!T~T~ oN ~Nn/oR TYPE I ~ h ~ r' FT~rn OF T~ ~ ~v~ ~_ This invention pertains to an;, ~,v~ t in the treatment of ~l~tO1 A diseases, i.e. in the ability to suppre_s autoimmune reactions by uge of oral tol~r;~At1--n.
More specifically, the invention is directed to the oral 5 administration of autoantigens or bystander antigens in comhination with oral or parenteral administration of a polypeptide having Type I interferon activity for the treatment of ~lt-; ^ dlseases.
Yet another aspect of the invention pertains to oral 10 use of interferons in the treatment of autoimmune diseases.
R~v( Ic.:It. Al ~ IF ~R TNVV~TTI0~1 Autoimmune diseases are ~hArA~AtPrized by an Ahn~A,rr~l immune response directed against normal autologous ~self ) 15 tissues.
Based on the type of immune response (or immune reaction) involved, aut-; A diseases in mammals can generally be ~'lAaA;f;ed in one of two different categories.
cell-mediated (i.e., T-cell~ tPd) or antihody-mediated 20 disorders. Non-limiting examples of cell-- ~l;At~ A~ltO;
diseases lnclude multiple sclerosis (M~ t~ ;~al arthritis (RA), autoimmune thyroiditis (AT), the Alltt~; A stage of diabeteA mellitus (juvenile-onset or Type 1 diabete~3) and A1~t~ 1nc~ uveoretinitis (AUR). Antibody-mediated AlltO;
25 diseases include without limitation myasthenia gravis (~) and systemic lupug eryth t~ A11A (SL~) .

Wo 95n7499 2 ~ 8 5 3 5 l . }~ 120 ~
Both categories of autoimmune diseases are currently being treated with drugs that su~press immune responses systemically in a non-specific~m~iner, i.e., drugg ;nr~r~hle of selectively :~u~ ssing the~-hnnrr-l immune response. Non-5 limiting ~ of such ~ drugs include methotrexate,cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
Steroid r~ _ ~ul~ds such as prednisone and methylprednisolone (also non-,2Fer~f1r; - L t88ants) are also employed in many instances. All of these currently employed drugs have 10 limited efficacy against both cell- and antibody-mediated autn; - diseases. rurthermore, such drugs have 8ignificant toxic and other side effects and, more important, eventually induce "global"; ~ ~ 88ion in the 8ubject being treated.
In other words, prolonged treatment with the drugs 15 downregulates the normal protective immune response against pathogens thereby increasing the rigk of ; nf~rt; nn . In addition, r~t;-nt- subjected to prolonged global immuno-suppression have an increased risk of developing severe medical complications from the treatment, such as r-l;gn~nr;e~, kidney 20 failure and diabetes.
In a rnnt; nll; n~-J ef f ort to overcome the drawbacks of conv~nt;nn~l treatments for ~to; - disease, the present inventors and their CU..JLh~L~ have devised methods and rhArr~rellt;r~l f, l~t;ons useful for treating _lltO;
25 diseases (and related T-cell - ~;At-fl inflammatory disorders such as allograft rejection and retroviral-associated neurological disease). These tr.-- ,, are based on the concept of ;nrlllr;ng tolerance, orally or by ;nh~l~t;onl using as the tolerizers -lltn~nt;gens or bystander antigens or 30 disease-suppressive fragments or analogs of p~ltr~Ant;gens or bystander antigens. The present inventors and their co-workers have also devised method~ and f l~t;ons for ;n~ r;n~
tolerance via anergy by the parenteral administration of n~, n~nt epito~ic peptides of ~ltoAAnt;gens. This body 35 of work has been described in PCT Application Nos.
PCT/US93/01705 filed February 25, 1993, PCT/US91/01466 filed March 4, 1991, PCT/US90/07455 filed December 17, 1990, PCT/US90/039~9 filed July 16, 1990, PCT/US91/07475 filed ~ Wo 9s/27499 2 ~ 8 5 ~ 5 ~ /.J . c tl20 October 10, 1991, PCT/US93/07786 filed August 17, 1993, PCT/US93/09113 filed Septem~er-24, 1993, PCT/US91/08143 filed October 31, 1991, PCT/US91/02218 filed March 29, 1991, PCT/US93/03708 filedApril 20, 1993, PCT/US93/03369 filedApril 5 9, 1993, and PCT/US91/07542 filed October 15, 1991.
Autoantigens and bystander antigens are def ined below .
Intravenous administration of autoantigens and preferably ~r~S A thereof consisting essentially of 10 ; ~A"lnm; nAnt epitopic regions of their molecules has been found to induce immune Au~ ion through a -h~n; om called clonal anergy. Clonal anergy, or T-cell nonresponsiveness, causes deactivation of immune attack T-cells specific to a particular antigen, the result being a significant reduction 15 in the immune response to this antigen. Thug, the ~lltn;
response-promoting T-cells specific to an autoantiyen, such as myelin basic protein (Mi3P), once anergized, no longer prolif erate in response to that antigen. me inability of the anergized T-cell~ to proli~erate results in a rA~lllctinn of the 20 immune attack reactions that cause the tissue damage respon-sible f or the ~ t n; - disease symptoms, such as the neural tissue damage observed in M~. There iB also evidence that oral administration o~ autoantigens or ; n~l( ' nFlnt r , Ar thereof in a single dose and in subst~nt;Ally larger amounts 25 than those that trigger active 2~u~L~ ion may also induce tolerance through anergy, or clonal deletion.
Clonal anergy, however, can be induced only when the administered antigen is the specific antigen recognized by the immune attack T- cells sought to be anergized (pure bystander 30 antigens do not induce tolerance through anergy). Thus, regimes that rely on clonal anergy to achieve suppression have certain limitations: the autoantigen may not be known, or there may be several ty~es o~ immune attack T- cells specif ic to dif~erent antigens, or the antigens to which the immune attack 35 T- cells are speci~ic may change over time .
- The present ~ inventors and their co-workers have developed a method o~ ~ treatment that uses A~ltn~ntlgens and proceeds by active suppression, a different mArh~n;o~ than W0 95l274sg 2 1 8 ~ 3 ~ / L /~ l 120 ~

clonal anergy. This met~od, discussed extensively in the related PCT Application PCT/US93/01705, involves the oral administration of antigens speci_ic to the tissue under autoimnune attack, called "bystander antigens" and defined 5 below. This treatment a~uses regulatory (6uppression) T- cells to be induced in the gut-associated lymphoid tissue (GALT) or, in the case of by-inhalation administration, in the mycosa associated lymphoid tissue (MAIT). These regulatory cells are released in the blood or lymphatic circulation and then migrate 10 to the organ or tissue afflicted with the ~ o; - disease and suppress ~lltn; ? attack of the Affl i rt~ organ or tissue . The T- cells elicited by the bystander antigen rero~n; ~e at least one antigenic det~n; nAnt of the bystander antigen used to elicit them and are targeted to the locus of 15 autoimmune attack where they mediate the release of fiuppressive factors and cytokines, such as trans~orming growth factor beta (TGF-~) interleukin-4 (IL-4) or interleukin-10 ~IL-10). These suppressive substances are indiscriminate and l'lUIJ~JLe~l:i all immune attack rh~nl - at that vicinity regardless of the 20 antigen that triggers them. (However, because oral tolerization with a bystander antigen causes the release of these nonspecific suppressive substances only in the vicinity of ~lltn; A attack, no systemic immuno.iu~ L~suion ensues. ) Recently, e_forts have also been made by third parties in the treatment o_ autn; - disease, speclfically MS, involving the parenteral use of the cytokine ,~-interferon (IFN-,~), which is known to have; ~ lAtnry properties that can be used to advantage in combatting another immune 3 o disease . Studies of IEN- ,~ have shown that it tends to inhibit the activity of ~y-interferon (IFN-~y). IFN-~y has been shown to exacerbate ~S, and may be involved in the pathogenesis of MS
lesionfi Thus, IFN-,B appears to have an effect due in part to its ability to inhibit IFN-~y expression by T- cells . The related ability of IFN-,B polypeptide to reduce expression of class II ma~or histocompatibility complex (~IC) molecules on T-cell surfaces, as well as the ability to increase activity of suppressor T- cells, are also thought to be responsible f or 2i853 3 woss/2749s - J r~ 20 the tolerance-promoting; rmr,fll-lAtory properties of IF~
~owever, parenteral use of IFN-,B alone has the disadvantage of reyuiring high doses, which amplify the cnowl side-effects of this substance.
In N~ olo~A~y 43:655-661, April 1993, the IFN-~7 Multiple Sclerosis Study Group reported that sllhc~ltAnprll7R
administration of either 1.6 X 106 or 8 X 10; units of IFN-,~ in patients with relapsing-remitting multiple sclerosi_ significantly reduced PYArPrh~A~tir~nR of MS compared to a placebo group and, at the higher dose, also decreased .~S activity assessed by magnetic resonance imaging. The authors stated that the AhAn; pm by which IFN-,~ exerted its apparent beneficial effect is not snown. IFN-~ is neither an autoantigen nor a bystander antigen Furth~ ~, the effect of a substance on the immune system of a mammal (as well as the ~7~1An; I by which these effects are brought about) is often different fl~p=Anfl;nrJ on the amount and/or route of administration of that suhstance. For example, R~lhAIltAn~oilA
administration of an alloantigen induces an immune response to that antigen. Oral administration of the same substance may induce tolerance by eliciting T-suppressor cells that are specific to the orally administered antigen or (with higher doses and infreriuent administration) may induce anergy.
Intravenous administration of the same alloantigenic substance may induce tolerance by way of anergy.
To date t~ere haR been no teaching of oral use of interf eron alone in the treatment of autoimmune disease .
Further, there has been no teaching of oral or parenteral use of interferon in conjunction with oral tr,l~ri7Ation employing autoantigens or bystanders.
Accordingly, one object of the present invention is to provide an improved and/or more convenient method f or treating m~mmals suffering from Alltr; ^ diseases.
An additional obj ect of the present invention is an ~ ~_uv~d method for treating mammals suffering from autoimmune diseases exclusively via the oral route.

~8~35~
Wo 95/27499 1 ~"~ s C 1120 A third object o,f~ the invention i9 a method for treating mammals sufferi~g Erom autoi~cnune diseases through the oral administration of interf erons .
5 ~Y OF ~R lNV 'IL_ It has now been f ound that:
- oral or by-;nl~Al~t;nn administration of various polypeptides having Type I interferon activity is of benefit in the treatment of ~l~to; - diseases;
- a , ;n~t;nn of (i) oral or by-;n~ t~on administration of ~ n~nt;gens or bystander antigens (or fragments of them) and (ii) oral or parenteral administration of polypeptides having Type I interferon activity is subst~nt;~lly more effective in the treatment of 2luto; ?
diseases, than administration of (i) or (ii) alone.
U6e of other synergists, such as h~ctf~r;~l lipopolysaccharide, Lipid A, chol~r;ltn~; n ~ chain etc ., as described below, can be con~oined to the foregoing, ;n~tion.
R17T~F n~r~TPTION OF T~ r Figure l is a bar graph showing the variation in suppression of EAE (induced by MBP peptide 71-90) following oral (lA) or i.v. (lB) administration of different MBP
peptides .
Figure 2 (A) is a graph depicting the effect of feeding an ~lto~nt; gen (PLP) or a bystander antigen (MBP) on EAE induced in S~/J mice with a Pl,P-peptide; (B) is a bar graph summarizing the data of (A).
Figure 3 is a bar graph showing the suppression o~
EAE (induced with MBP-peptide 71-90) by feeding various guinea pig MBP peptides alone or in cn~in~tion with soybean trypsin inhibitor (STI).
Figure 4 (A-D) are graphic ~ _ r; ~nn~ of the suppression o~ EAE in ~ewis rats with oral administration of guinea pig MBP (A-D), intraperitoneal administration of rat a~
IFN (A), rat mock IFN (C) and combination treatments (B and D) .
Figure 5 is a bar graph summary of the data of Figure 4 (A-D) .

~ wo gs/27499 2 1 8 5 3 ~ 20 Figure 6 i8 a ,- , ~t;ve bar graph o~ an additional ~,o~l do~ t;nj the suppres~ion o~ EAE in Lewis rats with rn~-'n~t;nn of orally administered guinea pig MBP and ;ntr~rPritoneally administered rat ~Y/,B IFN, a~ well as with MBP
5 or IFN-,B alone.
Firure 7 (A-C) are graphic comparisons of the suppression of EAE in SJI/J mice with ;ntr~rPritoneal administration of mouse IFN- ~ (A), oral administration of bovine ~P (B), oral administration of bovine PLP (C), and l0 combinations thereof (B and C).
Figure 8 is a bar graph of the 5u~ lon o~ bovine P~P-induced EAE in SJL/J mice with or without intraperitoneal administration of mouse~ IFN-,~; bovine ~3P-induced EAE with and without mouse ~-interferon; mouse IFN-,B alone; and hen egg 15 lysozyme (HEL) (control protein).
Figure 9 is a graphic illustration of the effect of Type II Collagen, or oral IFN-,~, or the c ;n~t;on thereof, on the ;nfll~rt~ nn of ad~uvant arthritis in Bewis rats.

2 0 T~T~T~TT.T.`T) ,T~T.!~rT TPTION OF ~ ~V~TIQ~
All patent applications, patents, an~ literature references cited in this specification are hereby incorporated by re~erence in their :entirety. In case of conflict, the description including the definitions and interpretations of 25 the present disclosure will prevail.
.

218~353 Wo gs127499 - 1 _ I I L ~ 120 Defin; t; nnc The following tè~ms, when used in this disclosure, shall have the - ;ngQ ascribed to them below:
(a) '`Bystander antigen" or "by8tander" is a protein, 5 protein frAs t; peptide, glycoprotein, or any other ~ nunogenic substance (i.e. a substance capable of eliciting an immune response) that (i) i8 or is derived from a --,t specific to an organ or tisgue under Alltn; ~ attack; and (ii) upon oral or enteral administration elicits regulatory lO (suppressor) T-cells (which can be of the D4+ or CD8+ type) that are targeted to the organ or tissue under attack where they cause at least one immunoregulatory cytokine or immunoregulatory factor (such as II.-4, I~-lO, or TGF-~) to be released and thereby ~u~Lesq immune attack cells that 15 cnntr;hllte to Alltn; - destruction. The destructive cells are suppressed even though they may be specific to a different immunogenic substance from that used to elicit the regulatory cells. The term includes i~ut is not limited to A~tnAnt;gens (defined below) and frA,3 q thereof involved in Al~to;
20 attack. In Afl~l;tinn~ the term includes antigens normally not exposed to the immune system, which become exposed in the locus of autolmmune attack as a result of Allto; - destruction of overlying tissue. An example is heatshock proteins, which although nonspecific to a particular tissue are normally 25 shielded form contact with the immune system. "Pure bystander"
is a bystander antigen that is not an Allt~-Ant;gen.
(b) "Bystander suppre8sion" is ~u~L~sion at the locus of Al~to; - attack of cells that rnntr; hllt~ to Alltn;~1n- destruction; this suppression is _t_d by the 30 release of one or more; - ~, ~ssive factors from suppresgor T-cellg elicited by the ingegtion or ;nh~ t;nn of a bystander antigen and recruited to the site where cells contributing to autn; ~ de8tructio~ are found. The result is nonspecific but locally restricted downregulation of the 35 autoimmune responses r~.Qpnnc;hl e for tissue destruction.
(c) "Mammal" is defined herein as any organism having an immune system and being susceptible to an Al~t~
disease .
F

~ Wo gsl27499 ~ ~ 8~; 3 ~) 3 r ~ ~/u.. 5 ~120 (d) "Autoimmune disease" is defined herein a~ a gpf~ntAnPflllq or induced r~-l fllncti n~ of the immune system of mammals, includiny humans, in which the immune system fails to distinguish between foreign immunogenic substances within the mammal and/or autologous substances and, as a result, treats autologous tissues and substance8 as if they were foreign and mounts an immune response against them. The term 1 nf 1 llflf~R
human aUtQ; A diseases and animal models therefor.
(e) "Autoantigen" is any substance or a portion lO thereof normally found within a mammal that, in an autoimmune disease, becomes the primary (or a primary) target of attack by the immunoregulatory system. The term also includes antigenic substances that induce conditions having the characteristics of an Al~to; ~A disease when administered to 15 mammals. Additionally, the term includes peptide substances consisting essentially of ; fI nAnt epitopes or ~ nnfl~A~;nAnt epitope regiong of autoantigeng. T jA,fl, 'nAnt epitopes or regions In induced ~AlltO; - conditions are fragments of an Al~tnAnf;gen that can be used instead of the 20 entire A-lt~AAnt;gen to induce the disease. In humans afflicted with an AlltQ; - diseage, 1 ~ ~nAnt epitopes or regions are r~y~ Ls of antigens specific to the organ or tissue under autoimmune attack and rf-cogn; 7Pfl by a substantial percentage (e.g. a majority though not npf-~RR~rily an absolute majority) 25 of ~17tf~; ^ attack T- cell8 .
(f) "Treatment" is ;nt_nflP~fl to include both the prophylactic treatment to prevent an AlltOi A di8ease (or to prevent the manifestation of flin;cAl or subfl;n;rAl, e.g., histological , symptoms thereof ), as well as the therapeutic 30 suppression or alleviation of symptoms after the manifestation of such autoimmune disease, by abating AlltA; A attack and preventing or slowing down autoimmune tissue destruction.
(g) ~Synergists" are defined herein as substances that augment or enhance sub8t~ntiAl ly the ~u~lesAion of the 3~ rl ;n; AAl (and/or gubcLinical) manifestation of autoimmune diseases when administered in conjunction with the oral administration of a bystander antigen or parenteral administration of an autoantigen. As used in the preceding 2~85353 Wo95/Z7499 I~ / 1120 0 sentence, and elsewhere in this specification, ~in conjunction with~ (or "in association with~) me~ns before, substantially simultaneously with, or afte~r~ oral (or by-inhalation) administration of bystander àntigens. Naturally, administra-5tion of the con~oined substance should not precede nor follow administration of the ~ to~nt;gen or bystander antigen by so long an interval of time that the relevant ef f ects of the substance administered first have worn off. Therefore, the synergists should pref erably be administered within about 24 10hours before or after the ~lt~nt;gen or bystander antigen, and most preferably within about one hour before or after administration of the antigen. Polypeptides having Type I
interferon activity are examples of synergists. ~xamples of other synergists are given below.
(h) "Oral" administration ;nrl~rl~q oral, enteral or intragastric administration. In addition, by ;nh ll At; nn administration in aerosol form ~rr, ~ l; qh~q the same tolerizing ef~ect and i5 e~auivalent to oral tr~l ~r; 7~tion.
(i) rParenteral" administration includes 20subcnt~n,~r,llc, intradermal, tnt qcular, intravenous, intraperitoneal or intrathecal administration.
(~) n~h~t Il, "~u~r~,iion~ or "rPtlllrt~nn" of autoimmune attack or reaction ,~n~ ~ qses partial reduction or ~m~l; r~ration of one or more symptoms of the attack or reaction.
25A llsubst~nt;~llyll increased suppressive effect (or abatement or reduction) o~ ~lltOi ^ reaction means a si~n; f; c~nt decrease in one or more markers or histological or clinical indicators of ~1ltr,; ^ reaction or disease. Nonlimiting examples are a r~lllrti/ n by at least 1 unit in limb paralysis 30score or in arthritis score or a sign;f;r~nt r~rl11rt;r,n in the fresluency of autoreactive T-cells; a ro~l-lct;r,n of at least about 0.5 units in insulitis scoring (measured e.g. as described in Zhang et al., PNAS, 1991,88:10252-10256).
~n; m~ l M,~ l q Throughout the present sper;f;r~t;rn, reference is made to various model systems that have been developed f or studying i:llltO; ^ diseases. Experimental ~utr~ 1n~
PnrPph~l omyelitis (EAE) has been studied in mice and other W095l27499 2~ 8~3-S3 1~1,. ,120 rodent specie5 as a model for M~lltiple Sclerosis (MS). Ti~03e of ordinary skill in the art recognize that many potential imm.une theraples for MS are first tested in this animal model system. The disease is induced by; ; 7;~t; on with myelin basic protein (M3P) or proteolipid protein (PLP) and an adjuvant (such as Freund' s Complete Adjuvant, FCA) . The antigen that i8 used to induce the disease is the autoantigen in the model. This treatment, with either antigen, induces either a mnnnrh~qic or an ~cf~rh~t;ng/remitting form of demye-l ;n~t;n~ disease (depending on the type and species of rodent and well-known details of in~ tinn) The induced disease has many of the characteristics of the autoimmune disease MS and serves as an animal model therefor. Furth~ ~c:, the successful treatment of 13AE by oral tolerization, and the parallel success in decreasing the fre~uency of disease-;nrlllo;n~ cells in humans, and, in any cases, ameliorating the symptoms of MS, using oral administration of myelin, v~l ;t9At~-q the use of EAE as a model system for predicting the success of dif f erent oral tolerization regimens .
T ; 7i:1t; on with Myco}:acteri tl~herc~-l osiq or with Freund' s Complete Ad~uvant in oil into the dorsal root tail of susceptible mamm~als induces a disease u8ed as a model for human rh~ollm~to~ r~ arthritis . In like manner, ; ; 7~tion with Type II collagen with an adjuvant will also induce a disease (collagen-induced arthritis or "CIA") that serves as a model f or human rheumatoid arthriti3 .
T ;7:1t;on of ~ewis rats with S-antigen or IR3P-antigen (InterPhotoReceptor ~inding Protein) and an adjuvant induces ~lltn; ^ uveoretinitis. Finally, a model ~or Type I diabetes develops ~rnnt~n~ollqly in the NOD Mouse and the B~3 Rat .
One or more of the above disclosed model systems may be employed to demonstrate the efficacy and improved treatment provided by the present invention. In fact, the animal models are particularly suitable for testing th~r~r;,oq involving bystander ~u~L~,sion, precisely ~ecause this suppression -hiln;qm is antigen-r~onspecific. In the case of oral tol~r;7~t;nn, therefore, the suppression of symptoms obtained wO 95n7499 2 ~ 8 5 3 ~:3 12 P~ S/~ 1120 in the model i8 independent of many of the actual or potential differences between a human autn; ~ disorder and an animal model therefor. The same animal models are suitable for testing thPr~r;PR based on use of interferon because interferon iB generally known to have the same activities in animal models as in humans.
The above animal models can be thus used to establish the utility of the present invention in mammals (;nrlllrl;ng humans) . For example, a multiple sclerosis autoantigen, bovine myelin, orally administered to humans in a double-blind study conferred a rnnR;~or~hle benefit to a signi$icant patient subset (Weiner, H. et al. ScieIlce 259:1321-1324, 1993). In addition, rheumatoid arthritis symptoms, such as joint tF~nrl~rn~RR, aM gtiffnegg, grip strength, etc., were successfully suppressed in humans receiving oral collagen_(0.1-0 . 5 mg single dose daily) . (Trentham, D . et al ., Science 251:1727. 1993.) Finally, preliminary human trials with oral S-antigen showed very encouraging results for uveoretinitis.
Large scale human trials are presently conducted _or multiple sclerosis, uveoretinitis, rh~ tni~l arthritis and Type I
diabetes. All of these human trials now validate the animal data on oral tolerization using the appropriate disease model.
Thus, the predictive value of animal models f or = oral tol,or; 7At; r,n treatment of ~lltn; - diseases hab now been substantially supported by these human clinical studies.
What follows is a description of the individual tr''~tm~ntR that have now been r( ;ned in the treatment method of the present invention. By describing the effect of each of the possible tr~ R individually, followed by a discussion of the c ' ;n:lt;nn treatment, the present specification allows one of ordinary skill to understand the efficacy of these treatments, when rnmh;no~l, to reduce or ~l;m;n~te tissue damage in auto; m-A 1nP disease .
r)escri~tion of Bystander Surpression--Oral ~tlm;n;qtration In contrast to clonal anergy, suppression ~ tf~rl by oral (or by-;nh~l~t;on) administration of bystander antigens is brought about by elicitation of targetable regulatory T-cells that release one or more nnnRrer; f; r ; n~ ~l~l~ ~t:ssive Wo ss/27499 - ~ 1 8 ~ ~ ~ 3 F~ ~ >_ 1I2O

factors, such as transforming growth factor-beta (TGF-,~) and/or interleukin 4 (I~-4) and/or ;ntPriPllk;n 10 (IL-10) at the locus of the imm~une attack. These regulatory T-cells do not release II, - 2 or ~y - IFN. Because regulatory T- cells are elicited, the - 5 t~hCn; cm at work is known as active ~uL L L~sYion. The immunc,~LLLL~sive substances releasedby the elicited cells are not specific for the antigen triggering the suppressor cells that release them, even though these regulatory T-cells relea~e immunosuppressive factors only when triggered by the orally administered (or inhaled) antigen. Recruitment of the regulatory T- cells to a locus within a mammal where cells contributing to the autoimmune destruction of an organ or tissue are ~nnnpntr~ted allows for the release of n~ L~L Le88ive 8ubstanceæ in the vicinity of the autoimmune attack and 8uppresses all types of immune 8ystem cells responsible for such attack.
Becau8e the T-suppres80r cells have been elicited in response to oral (or by-;nh~l~tion) toleri~ation with a tissue-or organ-specific antigen, the target for the r ULJLJL~:880r T-cells i9 the organ or tissue under immune attack in the particular ~llto; e disease where the destructive cells will be concentrated. Thus, the bystander antigen may be an ~ltnuntigen or an; n~l~ n~nt epitope of an ~ltn~nt;gen~
Alternatively, the bystander may be another tis~ue-specific antigen that is not an ~llto~nt;gen; hence, the ~lltn~nt;gen (or autoantigens) involved need not be ;~lPnt;f;e~l In more detail, the active ~:~ULLLe~sion -h~n~^ of bys~ander~lLLL~s,iionforatissue-specific (bystander) antigen is as follows: After a~tissue-speci~ic bystander antigen is administered orally (or PntPr;l1 ly, i.e., directly into the stomach) it passes into the small intestine, where it comes into contact with the 80-called Peyer's patches, which are collection8 of immunocytes located under the intestinal wall.
These cells, in turn, are in communication with the immune sy8tem, including the spleen and lymph node8. The result is that YULJ~L~8UOL (CD8+ or CD4+) T-cells are induced and recruited to the area of: autoimmune attack, where t~ey cause the relea8e of TGF-,~ and/or another immunoregulatory sub8tances 8~353 w095/27499 ~ r~l,. 5~0~120 0 that downregulate the B-cells as well as the activated helper T- cells directed against the mammal ' s own tissues . Suppression induced in this manner is antigen-nonspecific However, the resulting tolerance is specific for the ~to~ ~ disease by 5virtue of the fact that the bystander antigen i_ specific for the tissue under attack and ~ju~ sse_ the immune attack cells that are found at or near the tissue being damaged Bystander antigens and ~to~nt1gens (as well a_ fL__ 'R and analogs of any of them) can be purified frQm 10natural sources (the tissue or organ where they normally occur) and can also be obtained using Le:_ ' ;n;~nt DNA technology, in bacterial, yeast, insect (e.g. baculovirus) and mammalian cells using techniques well-known to those of ordinary skill in the art . Amino acid sPrl ~PnrPq for many potential and actual 15bystander antigens are known : See , e . g ., Hunt , C . et al ~USA), 82:6455-6459, 1985 (heat shock protein hsp70);
Burkhardt, H., et al., Eur. I. Immunol. 21:49-54, 1991 (antigenic collagen II epitope); Tuohy, V.K., et al., J.
Imm~a~l- 142:1523-1527, 1989 (PnrPrh~l1togenic detPrm;n~nt of 20mouse PLP in mice); ~h; nnh~ra, T. et al ., In PrQr~rPR~ in Ret;n~l Research, Osborne, N. & Chader, J. Eds, Pergamon Press 1939, pp. 51-55 (S-antigen); Donoso, L.A., et al , J. Irranunol.
;L~:79-83, 1989 (IR?3P); Borst, D.E., et al., J. Biol. Chem.
~Ç~:115-1123, 1989 (IR?3P); Yamaki, K. et al., E~ 2~:39-~3, 251988 (S-antigen); Donoso, J.A. et al., Eye Res. 7:1087, 1988 (IRBP); Wyborski, R.J., et al., ~. ~ ~. 8:193-198, 1990 (GAD) .
The amino acid sequences for bovine and mouse PI,P;
bovine, human, rh; ~ 7PP, rat, mouse, pig, rabbit, guinea pig 30MBP; human and bovine collagen alpha-l(II) and bovine collagen alpha-l(I); and human insulin are well-known and p1lhl;RhPd and these antigens can be synthPR; 7ecl by rernmh;nAnt techniques, as is well-known in the art ~r:q3 ~ R of these antigens can be chemically synthPc; 7P~1 or also sy~thPR; 7P~l by rPC n~nt 35terhn; rl,lpR, Some tissue- specif ic antigens are commercially available: e.g. insulin, glucagon, myelin, myelin basic protein, protPol 1rid protein, collagen I, collagen II, etc.

4 ,', 2~8~353 W0 95127499 I ~ 20 ~ystander antigens can be ;flPnt;f;~d with routine expPr;-- ti~ti~n Any antigen from the afflicted tissue is a potential bystander. The potential bystander can be fed to mammals, and spleen cells or circulating T-cells from, e.g. the 5 blood or c,:L~u~inal fluid in the case of EAE or MS, from these mammals can be removed and st; l~tefl ;n vitro with the same antigen. T-cells elicited by 8t; 1iqt;~n can be purified and sUpPrni~ti~nt~ can be te8ted for their content of TGF-~, IL-4, IL-10 or other ~ - eyulatory substances quantitatively 10 andJor gualitatively. In particular, TGF-I~ can be mea8ured ~auantitatively or qualitatively, by ELISA using a 8uitable commercially available polyclonal or preferably - ~l rmAl antibody raised against TGF-,B (e.g. one available from RSD
Systems, ~inneapolis, MN or Celtrix Phi~r~ P-lt;~Al~, Santa 15 Clara, CA). Alternatively, another known assay for TGF-,~
detection can be employed, such as that described in Example 2 below using a commercially available mink lung epithelial cell line. If the bystander antigen elicits T-suppressor cells that do not release TGF-,~, the T-cells can be similarly tested 20 for secretion of I~-4 or IL-10. (Antibodies to IL-4 and IL-10 are commercially available, e.g. from Pharmigen, San Diego, CA.) Tissue-specific antigens that are not effective bystander antigen3 are those go segregated from the ;nfli tnry locug so that the immunoregulatory factors or cytokines released will 25 be too far removed from~ the locus of ;nfli tion to exert a substantial suppressive effect. ~fficacy of interferon or conj oint therapy can be assessed using the same methods .
The efficacy of bystander suppression induced orally or by ;nh~ t;on can be assessed, e.g., by: fl;m;nllt;on in 30 certain ;nfli t;on markerg, guch ag the number of activated T-cell clones directed against the organ or tissue that is the target of iiltlto1 ^ attack; decrease ln I~-2 or IFN-~y levels at the same locus; histological evaluation of the afflicted organ or tissue (e . g ., by biopsy or magnetic resonance 35 imaging); or rpfl~lctl ~n in the number and/or severity of clinical symptoms as80ciated with an autoimmune diseai~e.

W09s/27499 2 ~ 8 S 3 5 3 P~ 101120 ~
Use of Bvstander Antiqonc - Do9aqes The tolerance induced :by the bystander antigens of this invention is dose~ rpnrl~nt over a broad range of oral (or enteral) or ;nh~1~hle dosages.~ ~owever, there are minimum and 5 maximum effective dosages~ In other words, active suppression of the clinical and histological gymptomg of an aut n;
disease occurs within a specific dosage range, which, however, varies from disease to disease, mammal to mammal, and bystander antigen to bystander antigen. For example, when the disease 10 is PlBP-induced EAE in mice, the ffu~effffive dosage range when MBP is used as the bystander is from about 0 . l to about mg/mouse/feeding (with feedings occurring about every other day e.g., 5-7 feedings over a 10-14-day period) . A most preferred dosage is 0.25 mg/mouse/feeding. For suppression of the same 15 disease in rats, the MBP suppressive dosage range is from about 0.5 to about 2 mg/rat/feeding and the most preferred dosage is l mg/rat/feeding. The effective dosage range for humans with MS, when MBP is used as the oral tolerizer, is between about l and about lO0, preferably between about l and about 50 mg MBP
20 per day (administered every day or on alternate days for a period of time ranging from several months to several years) with the optimum being about 30 mg/day.
For rh~11r-tn;rl arthritis, the effective dosage range f or humans receiving either Type I or II collagen is about 0 . l 25 to about l mg/day, preferably 0.1-0.5 mg/day. For ad~uvant-induced arthritis in mice the ef~ective collagen dosage range is about 3 to about 30 micrograms/feeding with the same feeding schedule as for EAE.
Monitoring of the patient is desirable in order to 30 optimize the dosage and frequency of administration. The exact amount and frequency of administration to a patient may vary depending on the stage, frequency of manifestation and severity of the patient ' 8 disease and the physical condition of the patient, as is well-appreciated in the art. Such opt;m;7~t;nn 35 is preferably effected on a case-by-case bagig. Opt;m;7~t;nn of the dosage nF~ ry for immune suppression involves no more than routine exper; ~tinn, given the gll;rl~l ;n~.q disclosed herein .

Wo 9~/27499 2 1 8 5 3 ~ 3 ~ ' 1120 Assessment ~ of the disease severity can be Arc~ hPC7 according to well-known methods ~7epl~nr7;ng on the type of disease. Such methods include without limitation:
MS: severity and number of attacks over a period of time; ~1LUy r bY~iive ;ir~ 1 ,At j nn of fl~ cAh; l; ty (which can be measured e . g . on the ~An~7e~7 nicAh;llty Status Scale); number and extent of lesions in the brain (as revealed, e.g., by magnetic resonance imaging); and ~requency of autoreactive T- cells .
EAE: limb paralysis which can be scored as follows:
0-no disease; l-decreased activity, limp tail;
2-mild paralysis, unsteady gait; 3-moderate paraparesis, lih7b~ splayed apart; 4-tetraplegia; and 5-death.
RA: joint swelling, ~oint t~n~7Prn~cc, morning st;ffnecR~ grip strength, joint imaging techniques .
A~R: visual acuity; nuh7ber of T-cells in the eye and ''r701l-7;n~'cc~l in the eye.
Type I Diabetes: pancreatic beta cell function (assessed; e.g., by OGTT glucose tolerance test ) .
NOD Model: insulitis and delay of diabetes onset.
CIA: Arthritis score based on number of affected joints in each of four paws and grading each on an arbitrary scale of 1-4 as follows:
0=normal; l=redness only; 2=redness plus swelling; 3=severe swelling; and 4=joint deformity. The total arthritis score is the sum of the scores for all paws. Maximum arthritis score is the highest score for an animal over the course of the disease.
According to this grading method the highest arthritis score possible is 16 (4 paws X 4 score-per-paw) . StAh; 1; 7Ation of symptoms, under conditions wherein control patients or animals experience a worsening of symptoms, is WO 95l27499 ~! 1 8 S 3 5 '~ sl20 1~
one indicator of efficacy of a auLJ~l~asive treatment .
Another measure of i _uv~ is the dose reduction or disrr~nt;nllAnre of other medications, e.g., steroids or other 5 anti-;nfl tory l[~edications, and biologic response modifiers such as methotrexate, subrut~nP~I~q interferon and the like.
The optimum dosage of a bystander antigen will be the one g~n~rAt;ng the maximum beneficial effect assessed as described above.
An effective dosage will be one that causes at least a statistically clinical signif icant attenuation of at least one marker, symptom or histological evidence characteristic of the disease being treated.
When ;nPfl with IFN-~B treatment, the dosage of 15 bystander antigen should be equal to that which would have been used if oral or enteral administration of the bystander antigen was used alone, except that the ~ ' ;nAt;~n is more effective in abating autoimmune reaction, and thus au~ ssing disease.
However, it is poRR;hlo to decrease the amount of Allt~Ant;gen 20 or bystander antigen when Type I interferon is conjoined to oral bystander therapy Furthermore, it is possible to use suboptimal amounts of interf eron ~ in conj oined therapy . A
suboptimal amount is an amount of IFN-~B which although substAnt;Al ly ineffective when administered alone still 25 enhances the tol~l-; 7;ng ability of the antigen in conjoint therapy, i.e. has a pot~nt;At;ng effect.
Ascertaining the effective dosage range as well as the optimum amount of bystander antigen is well within the skill in the art. For example, dosages for mammals and human 30 dosages can be detF~r~n;n~rl by beginning with a relatively low dose (e.g., 1 microgram), progressively increasing it (e.g.
logarithmically) and measuring the number of TGF-beta (and/or IL-4 and/or IL-10) secreting cells and/or assessing the number and activation of immune attack T- cells in the blood (e . g . by 35 limiting dilution analysis and ability to proliferate) and/or ACIS~RA;n~ the disease seve}ity, as described above The optimum dosage will be the the maximum amount of suppressive cytokines in the blood and/or~causing the ~185353 WO 95/27499 1~ 7n greatest decrease in disease symptoms An effective dosage range will be one that causes at least a statistically or clinically sign; f; oçlnt attenuation of at least one symptom characteristic of the disease being treated The maximum effeative dosage of a bystander can be ascertained by testing ~luyL~ ively higher dosages in animals and then ~rtr~rnl~t;nrJ to humans. ~or example, based on the dosages given above, the maximum effective dose of MRp for humans has been estimated at between about 50 and 100 mg.
Similarly, the maximum effective Collagen II dose for humans is estimated at about 1 mg/day.
The present invention can also be advantageously used to prevent the onset of an autoimmune disease in susceptible individuals at risk for an ;slltn; ~ disease. For example, metbods for the identification of patients who are at risk for developing Type 1 ~;~het~R are extant and reliable and have been recently endorsed by the American Diabetes Asaociation (ADA). Various assay systems have been developed which (P~rer;~lly in c ;n~t;~n) have a high predictive value assessing 8usceptibility to Type 1 diabetes (D;~het~q (-~re 1~:
762-775, 1990. Details of one preferred screening test are available to those of ordinary skill in the art (Rnn;fAr;n, E.
et al., The T~nrrt 335: 147-149, 1990).
Prom a practical point of view, preventing the onset of most ;~utr; - diseases is of most importance in the case of diabetes. Other ~lltr,; ^ diseases MS, RA, AT and AUR are declared at an earlier stage of tissue destruction, before substantial tissue damage has taken place; therefore preventive treatment of these diseases is not as important as in the case 3 0 of diabetes . In diabetes, it i8 important to intervene with an effective treatment prior to the substantial destruction of subst~nt;~lly all of the pancreatic islet cells. After the islet cells are destroyed, the treatment would not be ef f ective .
A non-limiting list of autoimmune di8eases and tissue- or organ-specific r~nf; ' or potential bystander antigens ef f ective in the treatment of these diseases when administered in an oral or ;nh~l~hle form are set forth in Wo gs/27499 2 ~ 8 ~ 3 5 3 ~ o ~120 0 Table l below. Administration~ of cQrnbinations of antigens listed for each individual disease (with or without conjunction with Type I interferon) is alsQ expected to be effective in treating the disease . ~ ~ `
~ 3ystander antigens can be administered by inhalation and so can Type I interferon. The bystander amounts that need to be inhaled are generally smaller than those f or oral administration. It is Ant;~lr~tpfl that the Type I interferon arnounts that need to be administered by ;nhAlAtlOn will be lO likewise smaller. Effective amounts can be assessed using the same hnr~nl ogieg provided above.

~ wo g5l27~L99 2 1 8 5 3 S 3 ~ 0 ,l20 ~ ' m tn ~ o I CO
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,~
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W0 9St27499 2 1 8 5 3 5 ~ 0 ~l20 ~

,~
' n . .
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LL
~-~.. U7 ; ,-2~8S3~3 wos5n7499 ~ 3~ ~1120 For any ~11tn; ~ disease, extracts of the relevant tissue, as well as specific bystander antigeng or frt_ -~R
thereof, can be used as oral tolerizers. In other words, the - 5 bystander need not be purified. For example, myelin has been used for ~qS, pancreatic ce,ll_extracts have been u5ed for Type 1 diabetes, splenic cell ~tr~ct~ have been used to prevent allograft rejection (which is not, strictly sr~Ak;nJ, an llltni A rh~nl ) and muscle ~Ytr~rtR h-ave been used to treat myositis. However, administration of one or more individual antigens or fragment8 is preferred.
Thus, according to the present invention, when treating Type 1 fl~hetPR, an effective amount (det~orrn;n~fl as described above) of glucagon can be administered orally.
Glucagon is sperif;r~l1y present in the pancreas. Glucagon, however, is clearly not an autoantige~ because it is not expressed in pancreatic beta cells which are destroyed in the course of Type l diabetes (glucagon is found exclusively in alpha cells, a different cell type). Thus, glucagon is a "pure" bystander: it does not appear to have any ~ltnAnt;gen activity. (Presumably, the bystander activity of glucagon results from its high local cnnr~ontr~tion in the pancreatic intercellular milieu due to its secretion from alpha cells.) Insulin has bystander activity for Type l diabetes.
It is not at present known whether insulin is also an tn~ntlgen~ However, whatever the mechanism of action, oral, enteral or; nhill;~hl e insulin preparations are effective in 8uppressing Type l fl;~hAt~R and animal models therefor by preventing ~11to; ? distinction of pancreatic beta cells.
For multiple sclerosis and its animal models, both disea8e-;nfl11r;n~ and non-;nfl11r;n~ fr~ntR of M~3P (e.g. a peptide comprising guinea pig MBP amino acids 21-40 which is known not to induce 3~ in mice or rats) have by8tander activity not only for ~3P-induced disease but also for PLP-induced disease. In ratg, feeding of by8tander g~n~-r;ltf~R
mostly CD8+ Yu~ Yor cells which are Cla8s I restricted whereas in mice both CD8+ and CD4+ regulatory cells are g~nAr~ted (thege CD4+ cellg are probably Class II restricted) .

2~L8~353 W0 95/27499 P~ 5 ~120 O

For rh, tn;~l arthritis and animal models therefor, Type-I, Type-II and Type-II~ collagen are known to have bystander activity.
For uveoretinitis and its animal model AUR, S-antigen 5 and IR~3P and f r~,; ~ c thereof h~aye bystander activity .
Fr~3 ~ of bystander antigens can also be employed.
Useful fr?~s ~ can be ;rl~nt;fied using the ovorl~rr;ng peptide method of E~Qmple 3 (which is a general techni~ue although in E~cample 3 it is described specifically with respect 10 to identification of ~ n;n-lllr;n~ fr~s cl of ~3P). T-cells from fed animals can be tested for secretion of TGF-~, and/or Il.-4 and/or IL-10 and can further be identified by subtype (CD8+ and/or CD4+).
Orally administered ~ltn~nt;gens and bystander 15 antigens elicit regulatory T-cells and thereby induce the production and/or release of TGF-~B and/or IL-4 and IL-10. One such T- cell has been identif ied in mice orally tolerized against EAB as a CD4+ ffU~ 8ffOI T-cell, and a CD8+ uu~L~ssor T-cell has been identified in rats. Bven; n~l~ 'n;lnt 20 epitopes of autoantigens, e.g. ~3P, are capable of ;n~ r;
such regulatory T- cells . Additional such epitopes can be ;rlPnt;f;ed by feeding a bystander antigen to a mammal and isolating from the mammal T-cells that rero~n;7e a fragment of the antigen (and thus identifying suppressive fr 1~3 ~), or 25 by identifying T-cells from a }~ystander fed mammal that can adoptively transfer protection to naive (not-fed) animals.
The bystander antigens can be administered individually or in r~ ' inut;nn~ of at least two. ~lltn~nt jgen and bystander administration is carried out as disclosed in PCT
Applications PCT/US93/01705 filed February 25, 1993, PCT/US91/01466 filed March 4, 1991, PCT/US90/07455 filed December 17, 1990, 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 ; nn~ above. It is anticipated that .

Wo 95l27499 2 ~ 8 5 ~ S 3 r~ 20 administration of at least Swo bystander antigens (either or both of which may but need not be an ~lltn~ntigen) will also result in effective suppression of autoimmune disease.
In addition, other synergists can be conjoined in the 5 treatment to enhance the ef f ectivèness of the above . Non-limiting examples of ~nn;nt-~rferon synergists for use in the present invention include bacterial lipopolysacc_arides f rom a wide variety of gram negative bacteria such as various subtypes of ~. ~oli and ~;3l ^ll~ (LPS, Sigrna Chemical Co., 10 St. I,ouis, MO; Difco, Detroit, MI; BIOMOL Res. ~abs., Plymouth, PA), Lipid A (Sigma Chemical Co., St. ~ouis, MO; ICN
Biochemicals, Cleveland, O~; Polysciences, Inc., Warrington, PA) and immunoregulatory lipoproteins, such as peptides covalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-15 serine (P3 C55) which can be obtained as disclosed in Deres, R.et al. (Nature, 342:561-564, 1989) or ~Brauns" lipoprotein from E. s~i which can be obtained as disclosed in Braun, V., Bionh;Tn. BioT~hys. ~ 435:335-337, 1976. ~PS for use in the present invention can be ~rtr~l~t,-fl from gram-negative bacteria 20 and purified using the method of Galaneg et al- (~LE. J.
Biolh~m 9:2~5, 1969) and Skelly, R.R., et al. (Infect. ~L
23:287, 1979). The effective do8age range for nnn~nt~rferon sy~nergists for mammals is from about 15 ~g to about 15 mg per kg weight and preferably 300 ,ug - 12 mg per kg weight. Any 25 other substance which = _as the ~LU~UIeL l y of biasing i-m-mune responses towards Th2 - type response can be used .
Interfernnq U8eful ;n the Prespnt Invent;on Interferons (IFNs) are a diverse family of proteins that are secreted by virtually every cell type within the body 30 in response to a variety of ;nflllcPrF~ T_ere are three ma~or species of IFN: a, ~, and ~y, which are respectively induced from lymphocyte8, from fibroblast8 (or other nonleukocyte cells), or from lymphocyte8 t_at have been st; l ~tPfl with antigen or mitogen . Interf erons are known to af ~ect 35 immunological re8ponse8 at multiple cellular targets through binding to cellular reCeptors. IFN a and ,~ are both Type I
interf eron .

W095l27499 2~85353 26 ~ U~ 'CI120 ~
Human IFN-cY refers~ to a group of at least 14 structurally related polypeptides that are transcribed from a multigene family. Human IFN-,B refers to two IFN subtypes, which are structurally related to IFN-~, and IFN-~Bl is the 5 ma~or subtype. A~single receptor binds both IFN-~ and IFN-,B.
(Wierenga et al. "Ly ~hnk;nP~ and Cytokines of the Immune System" Ch. 15, Com~rehensive Medicinal fhPm;qtry, vol. 3, pp.
1101-1128, 1990) . The am.ino acld sP~PnrPq of human and rodent O! and ,~ interferons have been pl-hl; qhprl. Also p -hl i qhpd are 10 a number of polypeptides that have Type I interferon activity.
See, e.g. U.S. Patent Nos. 4,569,908; 4,793,995; 4,914,033;
4,769,233; 4,753,795; 4,738,845; 4,738,844; 4,914,031 and 4,652,639 (consen~us interferon from Amogen, Inc.). A number of human and animal polypeptides having Type I interf eron 15 activity are commercially available.
Trea with TyDe I Interf er~on Clinical trials for parenteral treatment with IFN-~of relapsing and remitting MS, an ~to; - disease, resulted in a signi~icant decrease in attack rate (Jacobs et al., 20 ScienCe 214:1026-1028, 1981). In this trial, 1 million units of IFN-,B were administered ;ntr~thpr;~lly once or twice a week, followed by monthly dosages. More recently, clinical trials with recnmh;n~nt IFN-,~ also administered parenterally have also resulted in si~n;f;r~ntly lower attack rates (Johnson et al., 25 N~llroloqy 40 (Suppl. 1): 261,1990). This trial Ut;1;7P~l various initial dosages, but followed the initial dose with subcutaneous administration of 45 million units three times a week. The rPr, ; n~nt IFN-~ used in this trial had been altered from the native protein in that the cysteine at 30 position 17 had been changed to serine (IFN-~-ser-17, BETA~3ERON~, Berlex Laboratories). This mutation greatly increases the stahility of the protein, yet does not alter the specif ic activity . A large multicenter trial based on the ~L~ ~;ng results of Johnson et al. is currently in progress.
35 An ~ ; t; nn~l large trial using a glycolsylated re~ ` n~nt IFN-~ produced in l ;~n cells (BIOFERON~) is also ongoing.
Although the m~rh~n; ~m of action of IFN-,B is still uncertain, it is probable that IFN-~y is an activator of disease ~ WO 951~7499 2 1 8 5 3 ~ 3 J~ 120 activity and IFN-~ reduces disease symptom~s through ~uppression of its effects. It is known jthat IFNs as a class can ~ l~Ate the immune system through $n-11lrt; nn of MHC class I (H~A-A, B, and C) and class II ~ (H~A-DR, DQ, and DP) cell surface 5 molecules. These surface molecules are essential for basic immune fllnrt; nnR such as self/nonself discrimination and antigen pres~ntAtinn to T-cells and thus are thought to play a central role in autoimmune disease development.
Antigens associated with class I molecules are 10 recognized by CD8+ (suppregsor/cytotoxic) T-cells, whereas antigens associated with class II molecules are rf~cosn; 7ed by CD4+ (helper/inducer) T-cells. Class I molecules are constitutively expressed on nearly all cells, and all IFN types augment thelr expression. Class II molecules are normally 15 restricted to certain cell types, where IFN-1~ regulates expression (McFarlin, Allerqv Clln;cs of NQrth r ica ~:210-212,1988; Basham et al., ~J. oE T nl, ~Q:1492-1494,1983;
Sztein et al, ~J. Cl;n. Inv~ t. 73:556-565,1984). This regulation is thought to occur through ;nf~llrt;nn of 20 intrArPl 1111 Ar proteins which, in turn, mediate MHC molecule expression (Vanguri et al. ,J. of Biol. f'hem j~:15409-15057, 1990) . IFN- y also activates marcrophages, which act as effector cells in autn~mmlln,o attacks, such as the demyelination seen in MS (Bever et al., Sl?r;nq~r Sem. in T n,l7Athnl . 8:235-25 250,1985; Prineas et al., Ann. of N~llrol. 0:149-158, 1981).
Macrophages also synthesize proteinases that can degrade A11tnAnt;gens, such as MBP (Bever, TrAnRArt. of th~ ~m. SOC.
Nellrorh~m. j~:208,1991). IFN-~y can also induce A~h~Rjnn molecules which regulate homing of lymphocytes to sites of 30 inflammation as is seen in A1ltO; - disease, for example, the CNS in MS (Male et al., Cell. I nl. 1271-11,1990). Thus, this cytokine has been implicated in several aspects of autoimmune disease.
The immune activating effects of IFN--y are mntll1lAt~d 35 by other cytokines. For example, interleukin-4, corticosteroids, prostA~lAnrl;nc, cY-fetoprotein, TGF-,B, and noradrenaline have all been shown to downregulate class II
exE~ession in various experimental systems (Cowan et al., J.
.

Wo gs/27499 2 ~ 8 ~ 3 5 3 . ~ s ll20 of Neuro~ nl. 33: 17-28,1991; Frohman et al., Proc. Natl.
Acad. Sci. USA 85:1292-1296,1988; Racke et al., ,J. of Immunol.
146:3012-2017,1991; Ransohoff, Res. in Immunol. 140:202-207,1989) . Interestingly, IFN- y stimulated class II molecules 5 can also be downregulated by IFN-,(~/ which has been shown to interfere with transcription of class II-specific mRNA in several systems (Fertsch et al., IJ. of Immunol. 139:244-249,1987; Ransohoff et al., ~. of Neuroimmunol. 33:103-112,1991). It has been shown in T lymphocytes that IFN-,~
10 directly suppresses the synthesis of IFN-~y, which may explain its e~f ect on class II gene transcription (Noronha et al ., Neuroloqy 41 (Suppl. 1) :219,1991; Pantich et al., An~. of Neurol. 2~:139,1987). As class II molecules are known to be central to the recognition of cells as foreign, they almost 15 certainly play a major role in the pathology of ~tn; ?
disease .
Administration of IFN-~ to MS patients can precipitate acute P~-~r~rh~t;nn~ (Pantitch et al., su~ra) . IFN-y iS also present in association with class II antigens ln 20 active MS plataues (Traugott et al., N.Y. Acad. Sci. ~:309-311,1988). Further, rec ;n~nt IFN-,~ has been shown to improve suppressor function of T-cells from ~oth MS patients and control subjects, possibly through a regulation of IFN- r synthesis by the suppressor T- cells (Noronha et al ., Ann . o~
25 Neurol. ~Z: 207-210,1990; Panitch et al., ~. of ~ rn; nl.
1992) . Downregulation of IFN- y by systemic IFN-,~ could thus be an effective means of preventing or modulating the severity of ~l~tO; ? attacks.
This view has also been supported by work in animal 30 models. Specifically, in EAF, the model for MS discussed above, systemic administration of IFN-~ has prevented the development of the disease (Abreu, Immunolor~ir~ ; catin 1~ :1-7, 1982) . Adoptive transfer of the disease, that is, ;n(91lr;n~ the disease through the transfer of M~3P-sensitized T-35 cells from rats suffering from ~AE to normal rats, is preventedif the cells are incubated with IFN-,B before transfer (Abreu, Int~rnl . l~rch. of Pllerr~y and ~ p. T -1. 76:302-207, 1985) .
In both cases it is likely that these effects are due to ~ W095/~7499 2 ~ 8 ~ 3 S 3 ~ 1120 downregulation of the rat Ia molecule, which corresponds to human class II ~C, although this has not been proven.
Or~L U~e of T~rPe I IPN Alon~ in the Pre~ent Tnvention Type I interferons orally administered are effective 5 in suppre5sing autoimmune disease. In fact, IFN-~B has been as effective as insulin in ~iu~Lessing diabetes in NOD mice. This fact is aurprising and cannot be inferred from parenteral use of IFN-,B as the -^h~n; r~ by which interferon suppresses disease is unknown.
For rats and mice, oral dosages that have been found effective range between 1,000 and 150,000 units with no maximum effective dosage having been discerned. This contrasts with the response to bystander antigens, which ~ ; n~ above a maximum effective dose. It is expected that the dosages 15 employed with IFN alone are similar to those employed in ' ;n~tlon with bystander antigens, except that suboptimal dosages of Type I interferon can also be used in conjoint therapy. Unlike parenteral interferon, there are no side effects with oral interferon.
20 P~re~te~Al IEN Do~a~e~ (r 'nntlon Trea ~
The reduction of Al~to; - disease symptoms seen with the administration. of Type I IFN alone i8 effective over a broad range of parenteral dosages. In other words, suppression of the clinical and histological symptoms of an 25 auto; ~ disease occurs within a specific dosage range, which, however, varies from disease to disease, mammal to mammal and the ~orm and activity of IFN. For example, when the disease is PLP- or ~3P-induced EAE in mice, the ~U~Lt:s~iVe dosage range when mice IFN-,~ is used is from about 10,000 to 30 1 million units (with tr~ occurring about every other day (e.g., 5-7 trF~ t~ over a 10-14-day period). A most preferred dosage is ~9,000 units/mouse/treatment. For suppression of the same disease in rats, the IFN-,~ suppressive dosage range is from about 5, 000 to aLhout 1 million 35 units/rat/treatment and the most preferred dosage is 15, 000 units/rat/treatment. The effective dosage range for humans with MS is between about 1 million units and about 75 million units, preferably be~ween about 15 and about 50 million units W095/27~99 21 85353 F~1/~ 1120 ~

per dose, administered as infre~luently as monthly and as f re~auently as every other day .
Ascertaining the effective dosage range for con~oint therapy as well as the optimum amount i8 well within the skill 5 in the art. For eXample, do8age,~`~0r mammals and human dosages can be detèrntined by begi~ tg with a relatively low dose (e.g., 5, 000 units), progressively increasing it (e.g.
logarith~; r~ 1 ~ y) and measuring a biological reaction to the treatment, for eYample reduction in class II surface markers lO on circulating T-cells and/or by scoring the disease severity, according to well-known scoring methods (e.g., on a scale of l to 5, or by measuring the number of attacks, or by measuring joint swelling, grip 8trength, stiffness, visual acuity, ability to reduce or disrnnt;nllp medication, etc. rl~r~nrl;ng on 15 the type of disease). The optimum dosage will be the one having the greatest influence on the biological rh~n~ nn being mea8ured, such as that which causes the greatest reduction in class II molecules on the T-cell surface, and/or that which causes the greatest decrease in disease symptom8.
2 0 An ef f ective dosage range will be one t_at causes at least a statistically or clinically 8ignificant att~ml~t;nn of at least one symptom characteristic of the disease being treated, as discussed hereinabove. Again, suboptimal dosages of parenteral interf eron can be used in conj oint therapy .
Parenteral administration may be via subr11t~3nl~n11q, intramuscular, or ;ntr~r~ritoneal~ routes, with subrmtt;lnon11~
being preferred for treatment ~u,~oses if the parenteral route is selected. In the case of parenteral administration, 30 interferoLt may be f, ~ rl in sterile 8aline or other carriers well known in the art, and may include ~Yr;r;~nt~ and st~h; l; 7~r~ that are standard in the art.
nA~m Ther~17Y
It has been surprisingly discovered that the oral (or 35 by ;nh~l~t;nn) administration of a bystander antigen in conjunction wlth oral or parenteral administration of Type I
IFN, results in a treatment which is synergistic in its effect ~ wo 95,2749g 2 ~ 8 5 ~ S 3 ~ ~ "1 l?n on autoimmune disease, when compared to the effect of the two trl:~A ' A ~PrA rA t~l y .
This treatment has been studied both in rat5 and mice, using the animal model for MS, EA~, the animal model for 5 RA (L~), and the animal model~ for Type I diabetes, NOD. The experimental protocol for these; studies is disclosed in the 13xamples below. A8 seen in Figure 4, treatment of Lewis rats with orally administered guinea pig MBP after induction of 13AE
both delays the onset and reduces the clinical score of the 10 disease (see A-D). The intraperitoneal administration of rat IFN-~ alone appears to have almost no effect on the timing or severity of the diaease (see 8A). This treatment is directly ~rAhl e the effect of administration of mock rat IFN-,~ (see 8C), a control substance which is produced by subjecting cells 15 to the same growth conditions as those producing IFN-~ but in the absence of an inducer.
However, tr~eatment with a ~ ;nAt1nn of ;ntrAroritoneal rat IFN-,~ and oral tolerization using MBP
greatly reduces the rl;n;c~l score of the induced EAE (see 8B) .
2C This result is unexpected, especially when contrasted to the effect of a ~_ ;n~tion of ;ntr~qrf~ritoneal mock rat IFN-~ and oral tolerization using MBP. The mock rat IFN-~/MBP
rrml~;n~t;on ig at begt of ;nt,~rm~rl;At~ effect as compared to the PBS control treatment and oral tolerization with MBP alone.
25 Thus, the combination treatment of ;ntr~r~ritoneal IFN-,~ and oral tolerization with MBP shows a synergistic ~u~L~ ive effect on the cli'nical score of the ~AE seen. In other words, the interferon potentiates the trl~r;~;nrj ability of the bystander. These treatme~ts clearly have a much greater effect 30 on clinical score when used in conjunction compared to the ef f ect achieved with either treatment alone . The synergistic interaction is be8t represented by Figure 5, where the di~ference between the third bar (GP-MBP(PO)~RAT IFN(IP)) from the top of the graph and the sixth bar (PBS (PO) ) is greater 35 than the addition of tha dif f erences between the f ourth (RAT
IFN(IP)) and 8ixthbar (PBS(PO)) and the fifth (GP-M}3P(PO)) and sixth bar (PBS (PO) ) . The third bar is also strikingly smaller 21~3~i3 w0 95/27499 ~ r~ tl20 than the first bar, a direct control of the combination treatment .
This synergistic o~ potPnt;~t;n, effect i8 further illustrated in the data of Figure 6. Again, the effect on the 5 clinical score of the ~ ' ;n~t;nn treatment is greater than the addition of the effects of eac~h~i.treatment alone.
Similar results hav`ë been achieved with the treatment of EAE in mice when P~P is used as the bystander antigen instead of MBP. As seen in Figure 7, treatment with either 10 bovine ~3P or bovine PI P suppressed the clinical score of the ; 7Pd animals ~see B and C) . Intraperitoneal administration of mouse IFN-,B alone had no effect on the disease (see A).
Xowever, a combination of the IFN-~ treatment with 15 oral tolerization using either bovine MBP or bovine P3~P showed a significant reduction in clinical score (see B and C, respectively). The r ' ~n:~t;nn treatment appears to decrease the duration o~ EAE in mice (note return to clinical score of 0, especially for c, ' n~t;nn with bovine MBP (B) ) . Therefore, 20 the synergistic ~uL,~L~ive effect seen in rats i6 also seen in the mouse EAE system. This effect is better illustrated in the bar graph of Figure ~. The difference between the first and sixth bar is clearly greater than the difference between the second and sixth bar or the dif f erence between the f if th 25 and sixth bar, indicating the r ;n~t;nn therapy is more effective than either the oral tolerization or IFN-~B treatment alone. Further, if the effect on rl;n;r~3l score of bovine PLP
alone is added to the effect on clinical score of mouse IFN-~alone, the total rP~lllrt; nn is still less than the reduction of 30 clinical score achieved by the combination treatment. Thus, the combination has a synergistic effect on rl;n;ri:ll score, as compared to the effects of each treatment method alone.
The result of experiments with oral Type I IFN in combination with oral (bystander) toleration therapy also show 35 synergy, or potPnt1~t;
Finally, experiments with orally-administered IFN-~alone in NOD mouse show IFN-~ to be as effective in suppressing disease as orally-administered insulin.

21~3:~
w0 ss~74ss , ~ l20 The following PY~r~rl Pq are illustrative of the present invention and do not limit the scope of the invention.
In the e~cperiments described below the following materials and methods were used.
Animal8. Female Lewis rats 6-8 weeks of age were obtained from Harlan-Sprague Dawley Inc. (Tnfl;AnArrl;c, IN). SJL/~T
mice, 8 weeks of age were obtained from Jackson Laboratories, Bar Harbor, ME. Animals were ~-intA;nP~9 on standard laboratorY
chow and water ad libitum. Animals were ~-;nt~;npd in accordance with the gll;~lPl ;nPC for the Committee on Care of TIAl~rAtnry Animals of the Laboratory Research Council (Pub.
#DIIEW:NIII, -85-23, revised 1985).
I~ntt ;~ n~l Reacre~tg. Guinea pig MBP was purified ~rom brain tissue by the ';f;P~ method of Deibler et al. (Prep.
1~ ~3iochem. 2:139, 1972). Protein content and purity were monitored by gel eleCtL~)~hUL ::sis and amino acid analysis .
Concanavalin A and histone were obtained from Sigma (St. Louis, MO). Peptides were synthesized in the peptide facility of the Center for Neurologic Disease, Brigham and Women's }~ospital, and purified on HPLC. The amino acid ser~uences of the peptides synthesized are: 21-40, MD~IARHGFLPR~RDTGILDS
(immun~yu~ule~yive epitope region when orally administered to rats ); 71- 9 0, S~PQRSQRSQDENPWHF ( immunodominant PnrPrhAlitogenic region in rats); 151-170, GTLsRIFKT~ T~nc~.
Interfernn~. Rat, mock rat, and mouse IFN-s!/~l were obtained from Cytimmune, Lee Biomolecular Research, San Diego, CA. Consensus interferon (CON 1) is available ~rom Amgen, Inc.
Induation of Tnl~r~nn~. For oral tolerance or active suppression, rats were fed 1 mg of MBP dissolved in 1 ml PBS, or PBS alone, by gastric ;ntllh~At;nn with a 18-gauge stainless steel animal feeding needle (Thomas Scientific, Swede8boro, NJ). Animals were fed five times at interyals of 2-3 days with the last feeding two days before; ;zAt;on.
IuductloII of ~ . For actively induced disease, Lewis rats were immunized in the left foot pad with 25 ~g of guinea pig ~D3P in 50 ILl of PBS emulsified in an er~ual volume of complete Freund's adjuvant (CFA) rnntA;n;ng 4 mg/ml of MYcobacter;-~m tllhprrlll nc; c (Difco) . For adoptively transferred woss/27499 2~g~3~ r~ J, 5'Cll21 BAE, an MBP active T cell line was estAhl; qhPd from rats ;7P~l with MBP in CFA, rai8ed and r-;ntA;nPd according to the method of Ben-Nun et al. (Euro. J. Immunol. ~ 195, lga2) .
RnrPrhAl ' togenic cells were collected after activation by culture with Concahavalin A (ConA)t (2 ~m/ml) using irradiated thymocytes from; ' 7Prl an~s as antigen presenting cells (APCs) . Cells were harvested from cultures via a ficol hypariue gradient (~ypariue 1077, Sigma) and washed twice in PBS prior to transfer. 5xlO~ PnrPrhAl; togenic cells were injected intraperitoneally in 0.1 ml PBS into ;rrA~l;Ated (750 rada, 24 hrs . earlier), rer~ riPnt rats . Cell viability of both --- l Atnr and PnrPrh;~l; togenir cells was determined by trypan blue exclusion and was greater than 90~.
n~rnl eV~ ntion. Animals were evaluated in a blind fashion every day for evidence of EAE. Clinical severity of EAE was scored as follows: 0, no disease; 1 limp tail; 2, hind limb paralysis; 3, hind limb paraplegia, ;nrnnt;nPnrP; 4, tetraplegia; and 5 death. Duration of disease was measured by counting the total number of days from disease onset (usually days 10 or 11 after active; ; 7Atir~n and 3-5 days after adoptive transfer of disease) until complete recovery for each animal .
DelaYed tYDe hY~er~n~ tiVltY (DTII) te~tinq. DTH was tested by injecting 25 ~g of MBP in PBS sllhr~tAn~o1lqly in the ear. Thickness was measured by a blinded observer, before and 48 hours after challenge, using micrometer calipers (~itutoyo, Japan) . The dif f erence of ear thickness bef ore and af ter challenge was recorded for each animal, and the result was expressed as the mean for each experimental group + SBM.
3 o TT~ ~tolorY. Histologic analysis of pathological changes was performed in rats with adoptively transferred EAR.
Spinal cords were removed on day 15 after adoptive transfer (or disease ;nflllrt;nn) and fixed with 109~ neutral buffered formalin. Paraffin sections were prepared and stained with ~uxol fast blue-hematoxylin and eosin, by standard procedures (Sobel et al., J. ImmunQl. ~2:2393, 1984). Spinal cord tissue was sampled in an identical manner for each animal and numbers of ;nfl tory foci per section (clusters of ~20 or more ~ wO9S127499 2~8~ 3 . . ~ tll20 aggregated infl; torY cells), in parenchyma and meninges were scored in a blinded fashion (Sobel et al., ~,).
Statist~r~l ~n~Y~is. Clinical scales were analyzed with a two-tailed Wilcoxon rank sum test for score samples, chi 5 s~auare analysis was used in~comparing the ;nr;rlrnre of disease be~ween groups, and comparison of means was performed by using the Student's t-test. For individual experiments, 5 animals were generally used per group.
E~AMPLE 1: A~saY for T~i;F-~ Tnr~ t~r~n M~AI ~ Of TGF-~ Activity ~n Senlm-Free CUlt~lre SUT~f'rnAtAntA. Serum free culture sUpGrnAtAnt~ were collected from the antigen-tolerized animals as previously described (I~ehri, et al. J. R~n.Med.163: 1037-1050, 1986; Wahl, et al.
15 ,J,T nl,l45: 2514-2419,1990). Briefly, ' l~tnr cells were first cultured for 8 hours with the antigen (50 ~l/ml) in proliferation medium. Thereafter cells were washed three times and r~All~p~Qn~ l in serum-free medium for the l~ ln~l~r of the 72 hour culture, collected, then frozen until assayed.
20 Det~rminAt;nn of TGF-,~ content and isoform type in supernatants was performed using a mink lung epithelial cell line (American Type Culture Collection, Bethesda, MD #CCL-64) according to Danielpour et al. (Danielpour, D., et al. J. Cell. Pb~Ysiol.
138: 79-86,1989).), and rnnf; '9 by a Sandwich Enzyme linked 25 T~l7nn~orhent Assay (SE~ISA) assay as previously described (Danielpour et al. Growth Factors 2: 61-71,1989). The percent active TGF-,B was ~lrt~rm;nP~l by assay without prior acid activation of the samples.
This assay can be adapted to test any antigen which 30 is a r~n~ te for use as a bystander. Those antigens, antigen fras A and/or amounts of antigen which produce the highest rnnrF~ntration of TGF-,B as measured by this assay can be considered those antigens and/or amounts most suitable for use in the treatment method of the present invention.
35 Alternatively, a transwell culture system, described below, can be used to indicate the level of TGF-,~ which is being produced.
This culture system measures the prn8~1rt; nn of TGF-,B as a function of suppression of cell proliferation.

~18~3~'3 wo 95/27499 ~ 120 ~ r~ 1 r~ e~. A dual chamber transwell culture system (Costar, Cambridge, MA), which i8 24.5 mm in diameter and consists of two compartments separated by a semi-p~ hle polyr~rhnn~te me~mbrane, with a-pore size of 0.4 ~m, was used.
5 The two ~ are 1 mm ~a~pa~rt, allowing cells to be coin-cubated in close proximity without direct cell-to-cell contact.
To measure in vitro ~lu~l~s~ion o~ proliferative ~ vllses in transwell cultures, 5 x 10~ antigen line cells, raised and r-;ntAin~ ~or example, as previously described (Ben-Nun, A.
10 et al., Eur. J. I rl. 11:195, 1981), were cultured with 106 ;rr~ t~od (2,500 rad) thymocytes, in 600 ~11 of proliferation media in the lower well. Spleen cells ~rom orally tolerized rats or controls (fed BSA) were added to t_e upper well (5 x 105 cells in 200 ~Ll). Spleen cells were removed 7-14 days 15 after the last feeding, and a single cell suspension was prepared by pressing t~e spleens through a stainless steel mesh. The antigen (50 ,ug/ml) is added in a volume of 20 ~Ll.
Because modulator cells are separated from ~ UIl~eL cells by a semi-permeable membrane, they do not require irradiation.
20 In some experiments, l~t~r cell8 were added in the lower well together with responder cells, and in these instances tr~r cells were irradiated (1,250 rad); ~ t-~ly before being placed in culture. Proli~eration media consisted of RPMI
1640 (Gibco Laboratories, Grand Island, NY) supplemented with 25 2 x 105 M 2-mercaptoethanol, 1% sodium pyruvate, 1~ penicillin and streptomycin, 196 gl~lt~m1nP, 1 ~ XEPES buffer, 1~6 r~nnGqqPnt;;ll amino acidg, and 196 autologous serum. Each transwell was performed in quadruplicate. The transwells were incubated at 37C in a ' cl~fied 6% CO2 and 94~ air ai ,Jh~re 30 for 72 hours. After 54 hours o~ culture, each lower well was pulsed with 4 ~Ci of [3X] thymidine and at 72 hours split and reseeded to three wells in a round-bottomed 96-well plate (Costar) for harvesting onto fiberglag8 filters and ro11nt;ng using standard litauid sr;nt;ll~t;rn techni~ues. Percent 35 suppression = 100 x (1 - ~ cpm r~qpr,nfl~rq cultured with modula-tors/~ cpm of r~ fl~

wo ssl274ss ~ ; 3 5 3 R~MPLE 2: I~nl ~f~r~;nn of T - _, e~ive E~ito~e~ o~ Gulne~ Pia N13P bY Oral Route To investigate the ` -; P~ of oral vs. IV
tolerance, MBP peptides Pnl ~ q~ing both Pnr~rh~l; togenic and - 5 non- PnrPrh~ l; togenic regions of MBP were administered both orally and intravenously prior to; ~tion ~or actively induced disease M~3P peptide 71- 9 0 of guinea pig MBP is Pnrprh~l itogenic in Lewis rats (Swanborg et al., J. Immunol.
191, 1975). As shOWn in Figure lB, Yul~LesYion of EAE via IV trlPr;7~t;on only occurred with whole MBP and Pnr~rh~l;togenic peptide 71-90, but not with guinea pig M~3P
peptide 21-40. Oral tolPr;7~t;nn with 21-40, however, was effective in Y~ ;n~ EAE (Fig. lA) . Guinea-pig peptide 21-40 was chosen as previous experiments have demonstrated that it caused spleen cells of rats orally tolerized to whole MBP
to release TGF-B Miller, A. et al. FA~3EB 6:1686, 1992.
Control guinea pig M~3P peptide 131-150 did not yu~yLess when administered either orally or intravenously (Fig. lA) . Of note is that in addition to ~u~ Leg~iing via the IV route, PnrPph~l-itogenic MBP peptide 71-90 also ~uppressed when given orally.
This result 1n1;r~tP~ that peptides derived from the immuno-~11 n;lnt domain of a given MBP towards a given host can suppress T-cell function when they are orally or intravenously administrated, but do 80 by different -h~n;l ~lPrPn~l;n~ on the route and protocol of administration.
me results of these experiments show that there are basic differences in the mPrh~n;P~ of suppression of EAE between orally and parPntPr~lly (e.g. intravenously) administered MBP.
me results suggest that orally administered antigen acts prPtlt-m;n~ntly via the gPnPr~t;rn of active suppression, whereas parPntPr~lly administered antigen acts via clonal anergy.
Sper;fir~lly supporting~ this conclusion is the inability of spleen cells from IV tolerized animals to ~u~Less adoptively-transferred EAE. Additionally, different fr~5 ~ of MBP
displayed different abilities to suppressing EAF clPrpn~l;n~ on the routes of administration (see, for example, Figure 1).

w0 9s/27499 ~ 1 $ ~ ~ 5 3 . ~ . '0 1120 The transwell System of Example 1 above was used to identify the epitopes present on guinea pig MBP which induce the release of TGF-~B from _ui~L~ssor T-cells.
The disease-;n~llr;nr ~ c (AlltO; ~ response 5 epitopes) of ~3P w-ere first rnnf; ' as follows: Overlapping peptides of guinea pig M3P were ; obtained f rom commercial sources or synthoC; 70d in accordance with well-known techniques, specifically using a commercial peptide synthesis ~rp~r~tllC (from Applied ~3iosystems) and following the 10 r-nllf~rtllrer' S instructions . Whole ~!BP was then fed to rats and lymph node cells from the orally tolerized animals were triggered with the M~3P-peptides. me ability of the triggered cells to induce killer T- cells was then quantitatively (lPtorm;notl by a proliferation assay, as described in Example 15 4, and by testing the ability of the proliferating cells to transf er the disease .
A peptide spanning residues 71-90 of guinea pig M3P
was by far the most efficient inducer of killer T-cells and therefore the most potent disease-promoting fr~_ t of MBP.
20 This region of guinea pig rOEIP therefore ~ur ~ ~ cl,.",flc to the r~ ' n~nt epitope of the protein.
When spleen celis obtained from animals fed r~3P and ;7ecl with M13P/CFA (as described above in Example 1) were co-cultured in the transwell system with spleen cells isolated 25 from OVA-fed animals, peptides corresponding to guinea pig M3P
amino acid residues 21-40, 51-70 and 101-120 added to the mr,rllll ~tr,r well were all capable of triggering ~uL~rt~ion of proliferation of the OVA-fed line. ~ut, contrary to other experiments with different animals, the; "~, 'ncnt epitope 30 of ,uinea pig M3P in rats, (corrocprinrl;n~ to amino acid residue nos. 71-90) wa~ ineffective in triggering ~uy~~ iOn in the transwell system. Peptides coll ~ ; nrj to guinea pig ~BP
residue nos. 151-170 and 161-178 inhibited proliferation of the OVA (responder) line but this effect was non-specific, and may 35 have been due to toxicity induced I ~ by these peptides, as these same peptides inhibited proliferation of spIeen cells isolated from OVA-fed animals when co-cultured with control (non-M~3P-fed) l~t~r cells (data not shown).

~ W0 9S127499 2 1 8 ~ 3 S 3 ~ ,. C ~120 Such a system can be easily adapted by one of ordinary skill such that peptides o~ the antigen of interest which are effective in the present treatment method can be ;(l~nt;f;~

R~ pr~ 3: Oral Tcl~ An~ ing - Bo~rlne - PLP or ~ BP
In order to demonstrate bystander suppression, groups of 5-6 female, 7 week old, S TL/J mice (Jackson Labs, Bar 10 Harbor, ME) were; ~ 7e~i with P~P peptide 1~0-160 on days 0 and 7 and received the following tr-~A c GROUPS : -1. Fed Histone (0.25 mg/mouse) 2 . Fed Mouse MBP ( 0 . 25 mg/mouse) 3. Fed Bovine PLP (0.25 mg/mouse) (~llto~nt;gen suppression) Each group was treated every other day f or 7 days .
In the intravenous group, the m-tP~ was injected into the eye plexus. The PLP peptide used was the disease inrlll~;n~
fragment 140-160 of bovine PLP. This peptide has the amino acid sequence cooH-pLA~ll~v~ pT~rT~T~ representing the f oregoing amino acid residues .
As shown in Figure 2, both mouse Mi3P and bovine PLP
were e~ually effective in down-regulating PLP-peptide-induced EAE when orally administered. A non-specific protein, histone, was ineffective in suppressing EAE when administered orally.
Thus, a bystander antigen, in this case mouse Mi3P, effectively suppressed EAE when orally administered to animals induced for 3 0 EAE with bovine PLP .
The effects of feeding various peptides to Lewis rats induced for EAE by guinea-pig M~3P residue nos. 71-90 (the major nc~ ' n~nt epitope of guinea pig ~3P as shown in Example 1 above) were also studied.
EAE was induced by; ;7;ng with 0.25 mg of guinea pig M;3P amino acid residue nos 71-90 in Complete Freund's Adjuvant and the effect of feeding various guinea pig M~3P
peptides on EAE was ~m; nP~q, 2~S~53 WO 9sl27499 ~ 20 10 ~"
As shown in Fig. 3, orally administered whole guinea plg MBP and a 21-40 guinea piy peptide were eQually effective in downregulating ~3AE induced by guinea pig MBP 71- 9 0 as was orally administered 71-90 itself. Guinea pig MBP peptide 131-5 150 was lneffective in conferring tolerance. Peptides werealso f ed with STI which prevents their breakdown by gastric juices and F.nh;~nr~a their biological effect. DTH responses to whole MBP were ~u~y~essed by feeding MBP or any one of the MBP-peptides 21-40, or 71-90. IIowever, DTII responses to guinea pig 10 MBP peptide 71-90 were only suppressed by feeding either whole MBP or yuinea pig peptide 71-90 and were not affected by guinea pig MBP peptide 21-40 (Fig. 3). This is consistent with the conclusion that MBP fri~j ' 71-90 does not participate in bystander ~u~- ession when fed to mice in which disease had 15 been induced with peptide 71-90.
FY~ J.R 4: S..",t,. e3Bion of ~ in Rats wlth a combination o~ Or~ rolerization using Guine~ pig (GP) NBP and Intr~eritQneal IEN-I~

EAE was induced in thirty female Lewis rats, weighing between 175 and 200g, by; ; 7;n~ on day 0 with 100~1 of an emulsion of 0 25 mg of GP MBP and 100mg of ~ycobacterium 25 ~lherrlll osi~ (Mt), as an adjuvant . These rats were divided into five groups and were given the following tr~ ~ on days 10, 12, and 14:
Grou~s 1. Fed PBS (lml/rat) 302. Fed GP MBP (lmg/rat) 3 . Injected rat IFN-a/,B (15, 000 unit8/rat) 4. Fed GP MBP (lmg/rat) + in~ected rat IFN-a/~ 0,000 units/rat) 5. Fed GP MBP (lmg/rat) + injected mock IFN-~B (.20 IRU/rat 356. Injected mock rat IFN-,~ (.20 IRU/rat) All injections were done intraperitoneally.
Following treatment, clinical evaluations, as described above, were recorded daily for each rat, with the average clinical 40 score for each group providiny the data point for each day post-; ; 7~t; nn, .

~ Wo gs/27499 2 ~ ~ ~ 3 ~ ~3 ~ 0 ~120 As shown in Figures 4 and 5, oral tolerization with MBP had its expected suppressive effect on clinical score (see Figure 4, A-D). At the amount administered, IFN~ alone had very little effect on the diseaae, and is comparable to both 5 the PBS controls as welI as the mock IFN-,B control (see Figure 4, C and D). However, the c~ ;n~t;nn of feeding GP MBP and intrap~r;tnn.o~l injection of IFN-,B significantly reduces the ~l;n;-~l 8core seen in the treated rats. This is ~pet;~lly significant given the ~apparent ability of the mock IFN-,B
10 preparation to counter the ability of MBP to suppress EAE (see Figure 4, D). In summary, the mean maximum clinical score in this experiment was:
PBS (Y/,B IFN MBP ~/,B IFN + mock IFN mock IFN
MBP + MBP
153.0 2.4 2.0 0.6 2.6 2.4 Figure 6 relates similar data from an ~ r;
which was done on female I,ewis rats (150-200g) fed with lmg of MBP, and.or in~ected with 150,000 units rat o~ -IFN on days -4, 20 -2 and 0 and; ; ~Pd with 25 mg GP-MBP.
The, n~t; nn of oral GP MBP with intraperitoneal IFN-~ has a synergistic suppressive effect of BAB in rats.
This assertion is based on the comparison between the level of suppression seen with each treatment sep~r~t~ol y, and the 25 superadditive level of suppression seen with the c -n~t;on treatment. The suppression seen with the c in~t;nn is in excess of the addition of the two levels of ~iu~L~ssion achieved with each separate treatment. The above results were cnnf; ' by delayed- type hypersensitivity experiments . In 30 addition, mea; uL~..~ ~s of the in vitro production of cytokines by lymphocytes from fed animals in response to specific antigen in culture, showed that the synergistic effect may be related to ~n~ n~c9 production of TGF,B and IL4 or IB10. ,~IFN thus acts as a synergist to enhance oral tolerance to BAB.

FY7`~PLE 5: Suppression of EAE in ~ice wlth a Combination of Oral Tolerization uslng MBP or PLP ~"d ltoneal T~N- B

Wo9s/27499 2~8~3~3 ~ 4120 EAE was induced in 35 SJL/J, 8 week old, female mice by; ~;n~ on day 0 and day 7 with 0.2 ml of an ~ 1R;nn rnnt~;n;nJ 200mg of bovine PI,P and 200 mg of Mt. These mice were divided into 7 groups and received the f ollowing 5 trP~ R on days 5, 8, and 10:
GROUPS
1. Fed hen egg lysozyme (~IEL) (0.25mg/mouse) 2. Injected mouse IFN-,B (69,000 units/mouse) 10 3. Fed bovine MBP (0.25 mg/mouse) 4. Fed bovine M3P (0.25 mg/mouse) + in~ected mou8e IFN-,i~
t69, 000 units/mouse) 5. Fed bovine P~P (0.25 mg/mouse) 6. Fed bovine PLP (0.25 mg/mouse) + injected mouse IFN-~
15 (69, 000 units/mouse) All inj ections were done ; ntrArpritoneally .Following treatment, rl;n;r~l evaluations, as described above, were recorded daily for each mouse, with the average clinical 20 score for each group providing the data point for each day post-; ; 7~tion.
me ;nAtinn of oral bovine MsP with intraperitoneal IFN- ,B and the c ; n~ t; on of oral bovine PI-P
with intraperitoneal IFN-~ both have a synergistic ~"L'l-" ~Rsive 25 effect of EAE in mice (see Figures 7 and 8). This assertion is based on the comparison between the level of suppression seen with each treatment separately, and the superadditive level of ~,uL~L~Yion seen with the c~ n~t;nn treatment. The ~ rPr~ ;tive effect attributable to the cnm~;n~tinn is the 3 0 excess of the sum of the ef f ect achieved with each separate treatment .
T~:Y7'`'PT~F~ 6: Synergy of Oral IFN~ nd Oral MBP in ~nt EAE SuDDre~lolL _ Female ~ewis rats (150 to 200 g) were fed with 1 mg 35 of Myelin sasic Protein (MBP), varying doses of rat o~/~
Interferon, or a ~ '; n~t j nn of Msp and inter~eron. Oral protein~ were given a total of seven times every other day, 4 prP; 7~t;on and 3 post; i7~t;nn with 25 ,lLg gpMsP for the ;n~urt;nn of EAl~. Animalg were gcored for signs of paralysis 40 beginning on day 9 on a scale of 0 to 5.
The results were as follows:

WO 95l27499 ~ ~ ~ S 3 ~ o 1120 GRO~PS DAY OF lOEAN MAX

HEL
1.0mg 5/5 12.0 2.0 5~IFN
20000 U 4/5 13 1 . 0 IPN
10000 U 5/5 12 1.4 ~ IFN
105000 U 5/5 12 . 6 1 . 0 gpMBP
1.0 mg 5/5 12.4 1.0 gpMBP 1. 0 mg +~ IFN 20000 U 4/5 13.2 0.8 Ex~Dle 7: Synergy of Oral ,t~-IFN and Oral MPP in Mouse E~T Su~ ression Female S~ mice were fed with 0.25 mg the bovine brain protein Myelin Basic Protein (MBP) with or without 5000 20 Units of murine ~-lnterferon three times prior to; ;7~t;~m with 200 ~g PI~P and 200 ~g MT for the induction of EAE.
Animals were scored for~signs of paralysis beginning on day 9 on a scale of O to 5.
The results were as follows:

FED ONSET SCORE
H~
0 . 25 mg 5/5 15 . 8 2 . 2 ~IPN
305000 U 4/5 17.2 2.1 gpM3P
0.25 mg 4/5 18.8 1.6 gpMBP 0 . 25 mg +,BIFN 5000 U 2/5 17.5 0.9 r l^ 8: Effect of Or~Ll ,B-lnt~C~a~v~ on the Inductlon of Adluvsnt Ar~h lti~ in Rats Female Lewis Rats weighing 120-140 grams were fed 40 Collagen Type II (CII) at the inrl;~t~fl doses and/or 5000 Units of ,~Interferon every other day starting on day -10 before 218~353 W0 9~27499 ~ - II2O

i7~tion. On day 0, animals were lnjected with 1 mg/ 0.1 ml of Mycobacterium tuberculosis lMT) id. Beginning on day +10, animals were scored for signs of arthritis on a scale of O to 4. The arthritis score for each animal was the sum of the 5 score, for each of the four paws.
The results are in Fig. 9.
E 1 e ~: orA~ I ln 5~ ea~lon of NOD Dlab~tes Groups of NOD mice (3 animals per group) were treated 10 as follows:
1. control 2. ovalbumin fed (lmg/feeding/rnouse) 3. e~uine insulin fed (lmg/feeding/mouse) 4. Mouse IFN-~ (5000 u/feeding/mouse) 15 All mice were fed 10 times on alternate days. About 3 weeks af ter the experiment the f ollowing parameters were assessed =
insulitis, CD5 T-cells, CD4 T-cells, CD8 T-cells, macrophages, and various cytokines. The results are presented below:
20 Group Unred Fed Fod Insulin Fed ~-IFN
Ov~lbunl~ Alone llorphology massive massive mild to very little peri-i~let peri-islet moderate or no and some and some peri-islet inGulitis intra- islet intra- islet rwC
D3~C MI~C infiltr~tes infiltrates infiltrates CDS T-cells ~75t NNC ~75~ ~C ~75t M~C very few +
cells CD4 T-cells dense ~75'~ MNC ~75~ C very few gubset ;nf;l~r~t~ positive including iL cells islets, moder~te numoer of CD8+ cells 25 CD8 T-cell dense dense though some very few gubset infiltrates, very small positive but posltive though number of very small cells ialets CD4+ islet~ number of mostly mostiy CD4+ cells negative negative Uacroph~ges about -5t about -5'~ small numoer small number (F4/80) ~C l~C of adven- of ~Idven-including including titial a~d titial and intra-islet intra-islet peri-islet peri-islet cells cells cells cells =~
~ WO 9S1~7499 ~ 1 8 ~ 3 5 3 ~ U . ~zo Group ~n~ed Fed Fed In~ulin F--d ~-}FN
Ovalbu~in Alone IL-2R small number S-lOt ~C very few 1-2 cells/
of peri- including poeitive aection lelet ~C intra-ielete celle I~-2 amall small negative negative numbere of ~ numbere of peri- and peri- and intr~ let intra-ielet M~C ~C
IL-4 negative nogative emall number small number o~ peri- nd of peri- ~nd intra - i c~let intr~ - i elet r~c ~C
IL- 7 negative negative negative negative 5 II,-10 negative only a few emall number only a few celle/ of peri- ~md cell~/
section intra-ielet eection ~C
IFX-gamma >50% I!~C >SO~ hXC negative negative adj ~ cent or adj acent or in ielet~ in iulet IFr~ lpha >SO~ ~C >50% MXC negative negative adj~cent or adj~cent or in ielet~ in islete /

Claims (13)

WHAT IS CLAIMED:

1. A method f or treating a mammal diagnosed with a T-cell mediated or T-cell dependent autoimmune disease the method comprising the step of:
orally or enterally administering to said mammal an amount of (i) a bystander antigen in conjunction with an amount of (ii) a polypeptide having Type I interferon activity, the amounts of (i) and (ii) being effective in combination in suppressing autoimmune response in said mammal.

2. The method of claim 1 wherein the amounts of (i) and (ii) are synergistically effective in suppressing said response in combination compared to the sum of the suppressive effects achieved by administering each of (i) and (ii) alone

3. The method of claim 1 wherein said polypeptide is IFN-.beta. and is administered parenterally.

4. The method of claim 1 wherein said polypeptide is IFN-.beta. and is administered orally.

5. me method of claim 1 wherein said mammal is a rodent and said disease is a rodent model for multiple sclerosis.

6. The method of claim 1 wherein said mammal is a human and said disease is multiple sclerosis.

7. The method of claim 5 wherein said bystander antigen is selected from the group consisting of myelin basic protein (MBP), proteolipid protein (PLP), fragments thereof and combinations of at least two of the foregoing.

8. The method of claim 6 wherein said bystander antigen is selected from the group consisting of myelin basic protein (MBP), proteolipid protein (PLP), fragments thereof and combinations of at least two of the foregoing.

9. The method of claim 7 wherein said IFN-.beta.
polypeptide is derived from the same species as said mammal.

10. The method of claim 8 wherein said IFN-.beta.
polypeptide is derived from human IFN-.beta..

11. The method of claim 1 wherein said disease is selected from the group consisting of rheumatoid arthritis and animal models therefor and said bystander antigen is selected from the group consisting of Type I collagen, Type II collagen, Type III collagen, 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 I diabetes and animal models therefor and said bystander antigen is selected from the group consisting of glucagon, insulin, fragments thereof, and combinations 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 therefor and said bystander antigen is selected from the group consisting of S-antigen, interphotoreceptor retinoid binding protein (IRBP), fragments thereof, and combinations of two or more of the foregoing.