CA2187345A1 - Treatment of autoimmune disease using oral tolerization and/or th2-enhancing cytokines - 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 non-interferon polypeptide having Th2-enhancing cytokine activity in such a manner as to induce oral tolerance to the bystander antigen resulting in suppression of the autoimmune response.

Description

WO 95127500 2 1 8 7 3 4 5 r~ m 5.04 l2 .

'TRF7~ OF AUL~ DISEASE
~JSING ~ T. T~T~oT7~ oN ~n/OR Th;~ Tt'T~
FT1:!T.n OF l~E 1JYVI L~
This invention pertains to an 1 uv~ t in the ability to reduce autoimmune reactiong by use of oral tolerization. More sper;f;r~lly, the invention i8 directed to the oral (or by-;nh~1~t;nn) administration of ~lto~nt;gens or, 5 more generally, bystander antige~s in rn~l ;n~t;nn with oral administration of polypeptides having Th2-F~nh;~nrin~ cytoki~e activity for reducing autn; - reactions or L~ 8eS.
Another aspect of the invention pertains to oral use of Th2-Pnh~nr;n~ cytokines (i.e. cytokines that bias the immune 10 aystem towards a Th-2 responge either by increasing Th-2 responses or by inhibiting Th- l responses ) in reducing autoimmune rP~rt; on~.
~aC~[~~. OF TIIE ~Y~I._ .
~llto; ? diseases are char~rtPr; ~Pd by an ;~hnnrr-l immune response directed against normal autologous (self ) tissues .
3ased on the type of immune response (or immune reaction) involved, autoimmune diseases in mammals can 20 generally be rl~ ed in one of two different categories:
cell- l;~tP~ (i.e., T-cell-r~ tPd) or antibody- ~;;3tP~9 disorders. Non-limiting e~camples of cell-mediated ;l~lto;~ln~
diseases include multiple sclerosis (MS), rheumatoid arthritis (RA), allto; ~ thyroiditis (AT), the autoimmune stage of 25 diabetes mellitus (juvenile-onset or Type 1 diabetes) and autoimmune uveoretinitis (AUR). Antibody-mediated autoimmune -W0 95/27500 i 2 1 8 7 3 4 5 . ~ o ~512

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diseases include without limitation myasthenia gravis (MG) and systemic lupus erythematosus (SLE).
Both categories of autoimmune diseases are currently being treated with drugs that suppress immune responses 5 systemically in a non-specific manner, i.e., drugs in- ~r~hle of selectively suppressing t~e ~hno~n~l immune response. Non-limiting P~r~mrl ~c of such drugs include methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
Steroid compounds such as prednisone and methylprednisolone 10 (also non-specific immunosuppressants) are also employed in many instances. All of these currently employed drugs have limited efficacy against both cell- and antibody~ ted autr; ^ diseases. Furth~ ~, such drugs have significant toxic and other side effects and, more important, eventually 15 induce "global" immuno~u~ s~ion in the subject being treated.
In other words, prolonged treatment with the drugs downregulates the normal protective immune response against pathogens thereby increasing the risk of inf ection . In addition, p~t~nt~ subjected to prolonged global immuno-20 suppression have an increased risk of developing severe medicalcomplications from the treatment, such as mali~n~nr;~, kidney f ailure and diabetes .
In a rnnt;nll;nS effort to overcome the ~r:lwhFlrkq of conv~nt' nnAl trl~tm~nt~ for ~lltO; ? disease, the present 25 inventors and their cc,.i~LkelY have devised methods and rh~^r~ltical f, ll~tions useful for treating ;~1ltnl diseases (and related T-cell ~ t~l infl: tory disorders such as allograft rejection and retroviral-associated neurological disease) . These tr~ t c are based on the 30 concept of intlllr;ng tolerance, orally or by inhalation, using as the tolerizers autoantigens or bystander antigens or disease-suppressive fragments or analogE of autoantigens or bystander antigens. This body of work has been described in co-pending PCT Patent Applications Nos. PCT/US93/01705 filed February 25, 1993, PCT/US91/01466 filed ~qarch 4, 1991, PCT/US90/07455 filed December 17, 1990, PCT/US90/039B9 filed July 16, 1990, PCT/US91/07475 filed October 10, 1991, PCT/US93/07786 filed August 17, 1993, PCT/US93/09113 filed . . . , _ _ . . . _ _ _ _ , _ , wo gsl27s00 r~ 412

3 S ept ember 2 4, 19 9 3, PCT/ US 91/ 0 814 3 f i l ed Oct ober 31, 19 91, 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.
~lltn;lnt;geng and bystander antigens are defined below .
Intravenous administration of ~lto~nt; gens and preferably fragments thereof consisting essentially of n~q( ' n~nt epitopic regions of their molecules has been found to induce immune suppression through a mechanism called clonal anergy. Clonal anergy, or T-cell n~ ollsiveness, causes deactivation of immune attack T-cells specific to a particular antigen, the result being a signif icant rPrlllct i nn in the immune response to this antigen. Thus, the autoimmune response-promoting T-cells specific to an ~l1tn~nt;gen, such as myelin basic protein (M~3P), once anergized, no longer proliferate in response to that antigen. The inability of the anergized T-cells to proliferate results in a ro~l~lftinn of the immune attack reactions that cause the tissue damage respon-sible for the ;llltn; ^ disease symptoms, such as the neural tissue damage observed in MS. There is also evidence that oral administration of autoantigens or ~ ~ n;tnt fragments thereof in a single dose and in subst~nt;~lly larger amounts than those that trigger active suppression 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 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 types of im~mune attack T- cells specif ic to different a~tigens, or the antigens to which the immune attack T-cells are specific may change over time.
35 The present inventors and their co-workers have developed a method of treatment that uses autoantigens and proceeds by active suppression, a different ----h~n; ~m than clonal anergy. This method, discussed extensively in the Wo95l27500 ' ` ~ 2 1 87345 P~".,~ ,';G1512

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related PCT Application PCT/US93/01705, involves the oral administration of antigens specific to the tissue under autoimmune attack. These are called "bystander antigens " and are def ined below . This treatment causes regulatory

5 (suppressor) T-cells to be induced in the gut-associated lymphoid tissue ~GA~T), or, in the cage of by-inh~ t;~^,n administration, mucosa associated lymphoid tissue (MALT).
These regulatory cells are released in the blood or lymphatic tissue and then migrate to the organ or tissue afflicted with 10 the ~llt^; ? disease and which suppress autoimmune attack of the afflicted oryan or tissue. The T-cells ~ ^;t~l by the bystander antigen (which recognize at least one antigenic detPrm;n~nt of the bystander antigen used to elicit them) are targeted to the locus of ^llto; ^ attack where they mediate 15 the local release of certain ; , - l ~tory factors and cytokines, such as transforming growth factor beta (TGF-,B) interleukin-4 (IL-4) or interleukin-10 (IL-10) . Of these, TGF-,~ is an antigen-nonspecific i ^sllrpressive factor in that it ~U~Le:ggeg all immune attack rh~-- re^g~rfql ~A~ of the 20 antigen that triggers thege rh~n~ . (However, because oral tolerization with a bystander antigen causes the release of TGF-,~ only in the vicinity of aut^i ^ attack, no systemic immunosuppression ensues. ) Ih-4 and IL-10 are also antigen-nonspecific; ~ulatory cytokines. IL-4 in particular 25 ~nh:~n^~ Th2 response, i.e. acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells.
IL-4 also indirectly inhibits Thl ~Y~ rh~tion IL-10 is a direct inhibitor of Thl responses.
After orally tolerizing mammals afflicted with an 30 Al~toi ^ disease conditions with bystander antigens, the present inventors and their co-workers observed increased levels of TGF-~B, II--4 and IL-10 at the locus of autoimmune attack. Chen, Y. et al., Science, 265:1237-1240, 1994.
Recently, the present inventors and their C~J..JLh~L~
35 have found that oral or parenteral administration of Type I
interferon, or polypeptides having Type I interferon activity, either alone or in coniunction with oral or by-;nh^l~t;on administration of autoantigens or bystander antigens, is W~J 95~27500 ~ U~ 0 1512 beneficial in reducing the symptoms of aut; - disease. In fact, even suboptimal doses of Type I interferon potentiate the tolerizing effect of the ~ t~-nt;gens and bystander antigen.
This work has been described in more detail in one of the 5 priority documents of this application. Type I interferon, especially ,B-IFN, i- known to have certain ;mm11n~ ~~fllll_tory properties e . g . inhibition of the activity of ~y - interf eron tIFN-~y). IFN-~y has been shown to -Y~-_rh~t_ MS, and may be involved in the pathogenesis ,of ~MS lesions. Thus, IFN-~B
lO appears to have an effect due in part to its ability to inhibit IFN-~y expression by T-cells. The related ability of IFN-~polypeptide to reduce expression of class II ma~ or histo- ~~;-at;h;l;ty complex (ME~C) molecules on T-cell surfaces, as well as the ability to increase activity of suppressor T-15 cells, are~ also thought to be responsible for the tolerance-promoting ; ~ tory properties of IFN-~ used par~nt--rAl 1y The -h~nl r-~m by which orally administered IFN-,B
promotes tolerance, either alone or as a synergist when used 20 in conjunction with an antigen, is not well understood (for example it is not known whether interferon-~ i8 a Th2-~nh-n~-;ng cytokine; in any event it is not the subject of the present invention). The polypeptide is neither an -11tQ~nt;gen nor a bystander antigen. Furthermore, the effect of a substance on 25 the immune system of a mammal (ag well a8 the m_-h~n; 1 by which these effects are brought about) is often different depending on the amount and/or route of administration of that substance. For example, subr11t~nP~ administration of an alloantigen induces an immune response to that antigen. Oral 30 administration of the same alloantigen may induce tolerance by eliciting T- suppressor cells that are specif ic to the orally administered antigen or (with higher doses and inf reyuent administration) may induce anergy. Intravenous administration of the same alloantigenic substance m~ay induce tolerance by way 35 of anergy To date there has been no t~ h; ng of oral (or parenteral) use of; , - 1~tory (e.g. Th2-~nh~n~-;ng) cytokines such as IIJ-4 in the treatment of ~lltv; ^ disease.

Wo 95/27500 ~ ` ` 2 1 8 7 3 4 5 ~_111J,.,51G 1'12

6 0 Further, there has been no teaching of oral (or parenteral) use of Th2 -^nhAn^; ng cytokines in conjunction with oral tolerization employing autoantigens or bystanders antigens.
Accordingly, one object of the present invention is to provide an irnproved and/or more convenient method f or treating mammals suffering from ~lt^i ^ diseases.
An additional obj ect of the present invention is an improved method for treating mamma1S guffering from ~--toi diseases exclusively via the oral route.
A third object of the invention is a method for treating mammals suffering from autoimmune disea8es through the oral administration of ~mmlln~ tnry cytokines.
~TrA"~T~Y OF ~JR lluV~
It has now been surprisingly found that:
- oral or by-;nhAl~ti~An= administration of IL-4 and peptide or polypeptide fragments thereof having Th2-enhancing cytokine activity is of benefit in the abatement (suppression) Of ^~t,^.1 ^ r^A^t;~^nA associated with aut^1 ^ disease;
~ a combination of (i) oral or by-inhalation administration of allto^ntigens or bystander antigens (or fragments of them) and (ii) administration of polypeptides having Th2-enhancing cytokine activity is subs~ntl~l ly more e~f ective than the use o~ autoantigens or bystander antigens alone (or of the Th2-^nh~n^ing cytokines alone) in suppressing autoimmune reaction associated with autoimmune diseases.
In addition, parenteral administration of IL-4 both has a suppressing effect on ~tQ; ^ reaction associated with ~--t~ n^ disease and Pnh~nr^A the suppressive effect of 3 0 bystander antigens .
Use of other noncytokine synergists can be further conjoined to the foregoing combination.
llTi!T~TT.Rn ~b!~ T~e,ll,_ C~F T~ lNV~ -~lUN
All patent applications, patents, and literature references cited in this sp^~^if;~A~tion are hereby incorporated by reference in their entirety. In case of conflict, the wo gS1~7500 218 7 3 4 5 r~ o 1'12

7 description including the definitions and interi,rP~ ^n~ of the present disclosure will prevail.
Def initions The following terms, when used in this disclosure, 5 shall have the meanings ascribed to them below:
"Th2-PnhAnr;nrJ cytokines" are naturally occurring antigen-nonspecific immunoregulatory substances that: (i) are normally secreted or induced by regulatory immune system cells and (ii) enhance the fre~uency of Th2 cells (and/or inhibit Thl 10 cells ) .
It has now surprisingly been found that oral administration of Th2-f~nh~nr;nj cytokines, either alone or in conjunction with bystander antigens (see below), is beneficial in reducing Allto; ^ reactions or responses. ~on-limiting 15 examples of such cytokines include IL- 4, and f ragments thereof that retain Th2-PnhAnr;nrJ activity. It should be noted however, that it is not at this time known whether the tolerizing effect observed by oral administration of I~-4 and the tolPr; 7Ati~^n PnhAnr;ng effect observed by administering I~-20 4 in ~conjunction with a bystander antigen are due to the Th2-PnhAnr; nrJ properties of IL-4 .
"Bystander antigen" or ~bystander~ is a protein, protein fragment, peptide, glycoprotein, or any other immunogenic substance (i.e. a substance capAble of eliciting 25 an immune response) that (i) is, or is derived from, a component specific to the organ or tissue under Allto;
attack; and (ii) upon oral or enteral administration elicits regulatory (suppressor) T- cells (which can be of the CD4+ or CD8+ type) that are targeted to the organ or tissue under 30 attack where they cause at least one antigen-nonspecific immunosuppressive factor or immunoregulatory cytokine (such as TGF-,~, IL-4 or IL-10) to be released and thereby suppress immune attack cells that contribute to A~lto;~mlnP destruction.
The term includes but is not limited to autoantigens and 35 frAgmPntA thereof involved in Alltol attack. In addition, the term includes antigen_ normally not exposed to the immune system which become exposed in the locus of A~ltoi InP attack as a result of autoimmune tissue destruction. An example is Wo 95l27500 - 2 t 8 7 3 4 5 PcrluS95/04512

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heatshock proteins, which although not specific to a particular tissue are normally shielded Erom contact with the immune system .
"Bystander suppression" is suppression at the locus 5 of autoimmune attack of cells that contribute to autoimmune destruction; this suppression is mG~ tGd by the release of one or more immunosuppressive factorg (including Th2-Gnh~nc;ng cytokines and Thl-1nh;hit;ng cytokines) from G~ly~L~88or T-cells elicited by the ingestion (or ;nh~1~t;nn) of a bystander lO antigen and recruited to the site where cells contributing to :~lltn; ? destruction are found. The result is antigen-nonspecific but locally restricted downregulation of the autoimmune response8 responsible for tissue destruction.
"Mammal" is defined herein as any organism having an 15 immune system and being susceptible to an ~l~to; ~ disease.
"~llto;mmllnG disease~ is defined herein as a spnnt~nGoll~ or induced malfunction of the immune system of mammals, including humans, in which the immune system fails to distinguish between foreign; ,._.~ic substances within the 20 mammal and/or autologous substances and, as a result, treats autologous tissues and substances as if they were foreign and mounts an immune re8ponge again8t them. The term ; n~ G~
human autoimmune diseases and animal models therefor.
"Autoantigen" is any substance or a portion thereof 25 normally found within a ma~m,mal that, in an ~ltoi ^ disease, becomes the primary (or a primary) target of attack by the immunoregulatory system. The term also ; nt~ G~ antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals.
30 Additionally, the term includes peptic subclasses consisting essentially of immllnn~lnm;n~nt epitopes or t ~nmin~nt epitope regions of ~3lltn;lnt;gen8~ T~ ;n;~nt epitopes or resions in i~duced ~tnimmllnG conditions are f~~nt~ of an ~ltn~ntigen that can be used instead of the entire ~lltn~nt;gen 35 to induce the disease. In humans afflicted with an ~lltQ~mmllnG
disease,; n~1nm;ni~nt epltopeg or regions are fragments of antigens specific to the tissue or organ under illltC);mmllnG
attack and recognized by a substantial percentage (e . g . a _ . _ .. . , .... , , . . . . _ _ _ , W0 9sl27500 r~ 2 g majority though not n~rPcici~rily an absolute majority) of autoimmune attack T- cells .
"Treatment" is ;ntPnrifc`i to include both the prophylactic treatment to prevent or delay the onset of an A1itn;mmllnf~ disease (or to prevent the manifegtation of clinical or subclinical, e.g., histological, symptoms thereof), as well as the therapeutic suppression or alleviation of symptoms a~ter the manifestation of such autci ^ disease, by abating autoimmune attack and preventing or slowing down autoimmune tissue destruction. "Abatementn, "suppression" or "reduction"
of ;i-lto;mm~ln~ attack or reaction en~nmr;iqcies partial reduction or amelioration of one or more symptoms of the attack or reaction. A "subst;int; illy" increased suppressive effect (or abetment or re~ t; nn) Of ;i~ltO; - reaction means a significant decrease in one or more markers or histological or clinical indicators of autoimmune reaction or disease.
Nonlimiting examples are a reduction by at least 1 unit in limb paralysis score or in arthritis score or a signif icant r~ lctinn in the frequency of autoreactive T-cell8; a rP~ t;nn o~ at~least about 0.5 units in ;neilll;t;q scoring (measured e.g.
as described in Zhang et al., PNAS, 1991, 83:10252-10256).
As used in the present specification, administration of a Th2--~nh;in-;ng cytokine "in conjunction with" (or "in association with" ) bystander antigens means before, subst2nt;~lly simultaneously with, or after oral (or by-;nhAl~tinn) administration of bystander antigens.
"Subst~nt;~lly simult;inl~oliqly" means within the same 24-hour period, or preferably within one hour before or after bystander administration .
nOral" administration includes oral, enteral or ;ntrAg;~citric administration. In addition, by-;nh;il~t;nn administration in aerosol form acf ~ hPfi the game tolerizing effect and is equivalent to oral tolerization.
"Parenteral" administration includeg subc~lt~slnc~ollcil ;ntr~r`i~r~ l, ;nt cicular, intravenous, ;ntr~rPritoneal or intrathecal administration.

W0 95/27500 ~ 2 1 8 7 3 4 5 l ~ '0 ''17.

~n ~ m~ 1 MQ~1P1 ~
Throughout the present specification, reference i9 made to various model systems that have been developed for studying autn;mml~ne diseases. Experimental autoimmune 5 encephalomyelitis~ (EAE) has been studied in mice and other rodent species as a model for Multiple Sclerosis (MS). Those of ordinary skill in the art recognize tnat many of the potential immune therapies for MS are first tested in this animal model system. The disease is induced by; ; 7~tion 10 with myelin basic protein (MBP) or protenl ;r;~l protein (P~P) and an adjuvant (such as Freund's Complete Ad~uvant, "FCAn).
The antigen that is used to induce the disease is the ~tn~n~;~en in the model. This treatment, with either antigen, induces either a monophasic or an exacprhAt;n~/remitting form 1~ of demyelinating disease (depending on the type and species of rodent and well-known details of induction). The induced disease has many of the characteristics of the auto;
disease MS and serves as an animal model therefor. FUrth~
the successful treatment of EAE by oral tolerization, and the 20 parallel success in decreasing the frequency of disease-inducing cells in humans, and, in many cases,; -i;nrating the symptoms of MS, using oral administration of myelin, v~l;tlAtp~
the use of EAE as a model system ~or predicting the success of different oral tolerization regimens. T ' 7:~t;nn with 25 M~rnnh~cterillm tl-hPrc--lssi~ or with Freund's CQmplete Ad~uvant in oil into the dorsal root tail of susceptible mammals induces a disease used as a model for human rhl toid arthritis. In like manner, ~ ; 7~tion with Type II collagen with an adjuvant will also induce a disease (collagen-induced arthritis 30 or "CIA") t~at serves as a model for hum n rheumatoid arthritis. These animal models also serve as good predictors of successful oral tolerization using bystander antigens.
Tmmlln;7;0t1nn of Lewis rats with S-antigen or IR;3~-antigen (InterPhotoReceptor Binding Protein) and an adjuvant 35 induces autoimmune uveoretinitis. Finally, a model for Type I diaQetes develops spnnt~npoll~l y in the NOD Mouse.
One or more of the above disclosed model systems may be employed to demonstrate the efficacy and; ~_~,ved treatment _, . . . . .. .. . .

~1 87345 W0951~7500 P~,l/u., C~'12 provided by the present invention. In fact, the animal models are particularly suitable for testing therapies involving bystander suppression, precisely because this suppression mechanism is antigen-nonspecific. In the case of oral 5 tolerization, therefore, the suppression of symptoms obtained in the model i8 ;n~ r-~ntll~nt of many of the actual or potential differences between a human auto;mm~lnP disorder and an animal model therefor. The same animal models are suitable for testing th~rslr;f.c based on use of Th2-~nh~nr;n~ cytokines 10 because the cytokines generally have the same or similar 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 (including humans) . For example, a multiple sclerosis A~lto~n~;gen, bovine 15 myelin, orally administered to humans in a double-blind study conferred a conc;cllorable benefit to a significant patient subset (Weiner, H. et al. Science 259:1321-1324, 1993). In addition, rheumatoid arthritis symptoms, such as joint t~ntl~rn~ , AM stif fness, grip strength, etc ., were 20 successfully ~u~L~s~c:d in hu~mans receiving oral collagen (0.1-0 . 5 mg single dose daily) . (Trentham, D. et al ., Science 261:1727, 1993.) Finally, pr~l;min~ry human trials with oral S-antigen showed very encouraging results for uveoretinitis.
~arge scale human studies are presently conducted for multiple 25 sclerosis, uveoretinitis, rh~ to;d arthritis and ~;~hetoc.
All of these human trials now validate the animal data on oral tolerization using the a~u~Lu~Liate disease model. Thus, the predictive value of animal models for oral tolGrl7~tl-n treatment of autoimmune diseases is subst~nt;~lly supported by 30 these human clinical studies.
What follows is a description of the individual treatments that have now been c~ inP1 in the treatment method of the present invention By describing the effect of each of the possible tr~o~tm~ntc individually, followed by a discussion 35 of the combination treatment, the present specification allows one of ordinary skill to understand the efficacy of these treatments, when combined, to reduce or ~l;m~n~te tissue damage in autoimmune disease.

-~ 2 1 87345 Wo 95/27s00 I~.l.).. ' ~ IC12 Descri~otion of l~ys~nfl~r Sul~ression--or~l ~flminictr~tion In contrast to clonal anergy, ~u~L~s~ion mPfl;~tPfl by oral (or by-inhalation) administration of byætander antigens is brought ahout by elicitation of targetable immunoregulatory 5 T-cell8 that release one or more immunosuppressive factors, such as transforming growth factor-beta (TGF-~); and/or Th2-PnhAnr;n~ cytokineg, such as interleukin 4 ~IL-4); and/or interleukin 10 (IL-10) at the locus of the ~llto; ~ attack.
These rejulatory T-cells do not release high levels of IL-2 or 10 y-IErN. Because regulatory T-cells are elicited, the ~ h~n;cm at work is re~erred to as active suppression. The ~ ulatory cytokines released by the elicited regulatory cells are antigen-nonspecific, even though these regulatory T-cells release (or induce the release of ) im~munoregulatory 15 cytokines only when triggered by an antigenic detF~rm; nun~
identical to one o~l the orally ad~ministered (or inhaled) antigen. Recruitment of the immunoregulatory T-cells to a locus within a mammal where cells contributing to the P~ltni - destruction of an organ or tissue are cnnrpntrated 20 allows for the release of immunoregulatory suhstances in the vicinity of the ~ltO; ^ attack and suppresses all types of immune system cells rPPpon~ih1e for such attack.
Because the T-suppressor cells have been elicited in response to oral (or by-inh~l~t~nn) tolerization with a tissue-25 or organ-specific antigen, the target for the suppressor T-cells is the organ or tissue under immune attack in the particular autoimmune disease where the destructive cells will be rnnrPnt~ated. Thus, the bystander antigen may be an ;llltn~n~igen or an; ~ n~n~ epitope o~ an ~lltn~nt;gen.
30 Alternatively, the bystander may be another tissue-specific antigen that is not an autoantigen; hence, the ~lto~ntigen (or autoantigens ) involved need not be identif ied .
In more detail, an example of the active liU~L~iOn mP,-h~n;Pm Of bystander ~iu~ ion for a tissue-specific 35 (bystander) antigen is as follows: After a tissue-specific (bystander) antigen is administered orally (or Pn~Pr~l ly, i.e., directly into the stomach) it passes into the small intestine, where it comes into contact with the so-called Peyer's patches _ . _ . _ ..... _ _ . . .. ... .. ... .

wo ss~27500 2 1 8 7 3 4 5 } ~ -,; 1'12 and villi, which are collections of a large numoer of immunocytes located under the intestinal wall. These cells, in turn, are in co_munication with the immune syatem, including the spleen and lymph nodes. The result i9 that suppressor (CD8+ or CD4+) T-cells are induced, released into the blood or lymphatic cirr~ t ~ nn, and then recruited to the area of autoimmune attack, where they cause the release of TGF-,~ and/or other immunoregulatory substances that downregulate the activated helper T-cells as well I as the B-cells directed against the mammal's own tissues. Chen, Y. et al., Science, 1994 ~. Suppression induced in this manner is antigen-nonspecific. However, the resulting tolerance is specific for the particular autoimmune disease, i.e. for a particular tissue under autoimmune attack, by virtue of the fact that the bystander antigen is specific for the tissue under attack and suppressor cells elicited by ingestion of the bystander antigen suppress the immune attack cells that are found at or near the tissue being damaged.
Bystander antigens and ~tn~nt;gens (as well as fragments and analogs of any- of them) can be purified from natural sources (the tissue or organ where they normally occur) and can also be obtained using recombinant DNA technology, in bacterial, yeast, insect (e.g. baculovirus) and r-mm~ n cells using techniques well-known to those of ordinary skill in the art. Amino acid se~uences for many potential and actual bystander antigens are known: See, e.g., Hunt, C. et al (USA), ~:6455-6459, 1985 (heat shock protein hsp70);
Burkhardt, EI., et al., E~- ~. Immunol. ~1:49-54, 1991 (antigenic collagen II epitope); Tuohy, V.K., et al., ~J.
Immunol. 1~:1523-1527, 1989 (~nrGrh~l itogenic det~rm;n~nt of mouse PLP in mice); ~h;nnh~r~, T. et al., In Proqress in Ret~n~l Ro~P~rrh, Osborne, N. & Chader, J. Eds, Pergamon Press - 1989, pp. 51-55 (S-antigen); Donoso, L.A., et al., !l- ~-143:79-83, 1989 (IRBP); Borst, D.E., et al., J. B ol. Chem.
- 35 264:115-1123, 1989 (IRBP); Yamaki, K. et al., ~ 234:39-43, 1988 (S-antigen); Donoso, L.A. et al., ~ ~- 1:1087, 1988 (IRBP); Wyborski, R.J., et al., Mol. ~;a;i.n~. 8:193-198, 1990 (GAD) .

~ 21 87345 Wo 95/27500 1 ~".~,,,3'~ 12 0 The amino acid sequences f or bovine and mouse PLP;
bovine, human, rh;, ~:ee, rat, mouse, pig, rabbit, guinea pig MBP; human and bovine collagen alpha-l(II) and bovine collagen alpha-l(I~; and human insulin are well-known and published and 5 these antigens can be synthesized by rern-' ;n~nt techniques, as is well-known in the art. FrA ~c of these antigens can be chemically synthesized or also synthesized by recnmh; nAnt techniques .
Some tissue- specif ic antigens are commercially lO available: e.g. insulin, glucagon, myelin basic protein, myelin, collagen I, collagen II, prot~n-;r;d protein, etc.
Bystander antigens can be ~ dPnt; f; ~1 with routine exper; -tAt; nn . Any antigen from the a~flicted tissue is a potential bystander. The potential bystander can be fed to l5 mammals, and spleen cells or cirr11lAt;nrj T-cells from, e.g. the blood or cerebrospinal fluid in the case of EAE or MS, from these manLmals can be removed and st~ lAt~d in vitro with the same antigen. T-cells elicited by st;m11lAt;nn can be purified and sllr~rnAt~ntC can be tested for their content of TGF-,~, IL-20 4, IL-lO, or other iL~_..~oLt~ulatory substances. In particular, TGF-~ can be measured quantitatively and/or qualitatively by ELISA using preferably a suitable commercially available polyclonal or most preferably monoclonal antibody raised against TGF-~ (e.g. R~D Systems, M;nn~Arol;c, MN; Celtrix 25 phArr~re~1ticals, Santa Clara, CA). Miller, A. et al., J.
T nl., 148:1106, 1992. 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-30 suppressor cells that do not release TGF-,~, the T-cells can be similarly tested for secretion of IL-4 or IL-lO (Ant;ho~;~c for IL-4 and IL-lO are commercially available, e.g. from Pharmingen, San Diego, CA) . Tissue-specific antigens that are not effective bystanders are those so segregated from the 35 ;nflAmm~tory locus (of autoimmune attack) so that the immunoregulatory cytokines released will be too far removed from the locus of 1nfl: tion to exert a suppressive ei~fect.
_ _ _ _ _ _ ~ _ _ _ _ _ _ .. .. , . .... . .. _ _ . . .. . . .... , . . _ _ _ wossl~7500 218 7 3 4 5 r~ 0 1~12 The efficacy of orally induced bystander ~uppression can be assessed, e.g., by: tl;m;n1ltion in certain ~nfl. t;~A~n markers, such as the number of activated T-cell clones directed against the organ or tissue that is the target of ~llto1 A
5 attack; decrease in II,-2 or IFN--y levels at the same locus;
histological evaluation of the afflicted organ or tissue (e.g., by biopsy or magnetic resonance imaging); or reduction in the number and/or severity of clinical symptoms associated with an autoimmune disea8e.
10 Use of F~ystAnrlpr pntlqPnA - Dosaqes The tolerance induced by the bystander antigens of this invention is dose-dependent over a broad range of oral (or enteral) or ~nh~l~hle dosages. However, there are minimum and maximum effective dosages. In other words, active suppres8ion 15 of the clinical and histological symptoms of an autoimmune 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 is P~P-induced 3~AE in mice, the suppressive dosage range when 20 M3P is used as the ~ystander ig from about 0.1 to about 1 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 disease in rats, the ~!3~ suppressive dosage range is from about 25 0.5 to about 2 mg/rat/feeding and the most preferred dosage is 1 mg/rat/feeding. The effective dosage range for humans with MS, when ~3P is used as the oral tolerizer, is between about 1 and about 100, preferably between about 1 and about 50 mg M3P
per day (administered every day or on alternate days for a 30 period of time ranging from several months to several years) with the optimum being about 30 mg/day.
For rh, toid arthritis, the effective dosage range f or humans receiving either Type I or II or Typ,e III collagen is about 0.1 to about 1 mg/day, and preferably 0.1-0.5 mg/day.
35 For adjuvant-induced arthritis in rats, the effective collagen dosage range is about 3 to about 30 mi.:L~,yLdl..J/feeding with the same feeding schedule as for EA13.

WO9s/27500 21 87345 r~ o~rl2 Monitoring of the patient may be desirable in order ~o optimize the dosage and ~requency of administration. The exact amount and f requency of administration to a patient may vary fl~rPn~;n~ on the stage, frequency of manifestation and 5 severity of the patient ' 9 diseaEe and the physical condition of the patient, as is well-appreciated in the art. Such opt;rn; ~;~t;on is preferably effected on a case-by-case basi6.
Optimi~ation of the dosage n~r~RRAry for immune suppression involves no more than routine exper;-- ~t;nn, given the 10 g~ l; n-~R disclosed herein.
Assessment of the disease severity can be ~c ~1; RhPd according to well-known methods depending on the type of disease. Such methods include without limitation:
MS: severity and number of attacks over a period of time; progressive ~ tion of ~1; R~h; l 1 ty (which can be measured, e.g. on the R~?~nrl~d Disability Status Scale); number and extent of lesions in the brain (as revealed, e . g., by mag~etic resonance imaging); and f requency of ~ autoreactive T-cells.
E1~3: limb paralysis which can be scored as follows:
0-no disease; 1-decreased activity, limp tail;
2-mild paralysls, unsteady gait; 3-moderate paraparesis, limbs splayed apart; 4-tetraplegia; and 5-death.
RA: joint swelling, joint t~n~lPrn~Rsl morning stiffness, grip strength, joint imaging techniques .
AUR: visual acuity; number of T-cells in the eye and "cloudiness" in the eye.
Type I Diabetes: pancreatic beta cell function (assessed, e.g., by OGTT glucose tolerance test) .
NOD Model: insuliti~ and delay of diabetes onset.
CIA: Arthritis score based on number of affected ~oints in each of four paws and grading each on an arbitrary scale of 1-4 as follows:
0~normal; 12redness only; 2=redness plus _ _ . _ _ _ _ _ _ W095127500 ~ 2 1 87345 f~,l/u.,,~'0~'12 swelling; 3=severe swelling; and 4=joint def ormity . The total arthritis score is the sum of the scores for all paws. Maximum arthritis score i9 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) .
Stabilization of symptoms, under conditions wherein 10 control patients or animals experience a worsening of symptoms, is one indicator of efficacy of a ~u~Lt:ssive treatment.
Another measure of i~ LUV~ t is the ability to reduce or disrnnt;n~l~ other medications, e.g., steroids or other anti-;nfl: tory medications, and biologic response modifiers such as methotrexate, sllhr~lt~n~o1l~ interferon and the like. The optimum dosage of a bystander antigen will be the one g~nPr~t;nr~ the maximum beneficial effect aggessed as described above. An effective dosage will be one that causes at least a statistically or clinically significant atti~nll~t;nn of at least one marker, symptom or histological evidence characteristic of the disease being treated as described above.
(t~1;n;r;-lly significant-att~ml~tinn i5 one observed by a clinician of ordinary skill in the f ield of a particular autoimmune disease. ) When - ;n~rl with IL-4 treatment, the dosage of bystander antigen should be preferably equal to that which would have been used if oral or enteral administration of the bystander antigen was used alone, except that the cornbination is more effective in abating autc~; ~ reaction. ~Xowever, the level of I~-4 can be the same as that when IL-4 i~ used alone, or suboptimal (i.e. an amount which would not be effective if I~-4 were used alone but is nevertheless sufficient to potentiate the tolerizing effect of the bystande~ antigen.
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 msmmals and human dosages can be determined ~y beginning with a relatively low dose (e.g., 1 microgram), progressively increasing it (e.g.

Wo 95127500 2 1 8 7 3 4 5 r~ IC 1'12 18 {~
logarithmically) and measuring the number of TGF-beta land/or IL-4 or IL-l0) secreting cells and/or assessing the number and activation of immune attack T-cells in the blood (e.g. by limiting ~ n analysis and ability to proli~erate) and/or assessing the disease severity, as described above. The optimum dosage will be the one generating the maximum amount o~ suppressive cytokines in the blood and/or= causing the greatest decrease in disease symptoms. An effective dosage range will be one that causes at least a statistically or clinically significant attPn1l;ltlnn of at least one symptom characteristic of the disease being treated.
The maximum ef f ective dosage of a bystander can be ascertained by testing progressively higher dosages in animals and then ~tr~ t;ng to humans. For example, based on the dosages given above, for rodents, the maximum effective dose of ~3P for humans has been estimated between 50 and l00 mg/feeding. Similarly, the maximum effective amount of Collagen Type II f or humans has been estimated at about mg/day .
~ The present invention can also be advantageously used to prevent the onset of an ;~lltOi - disease in susceptible individuals at risk for an autot ^ disease. For example, methods for the identification of patients who are at risk for developing Type l diabetea are extant and reliable and have been recently endorsed by the American Diabetes A~3snr; ~t~, on (ADA). Various assay systems have been developed which (especially in inAt;r n) have a high predictive value assessing susceptibility to Type l diabetes (D~hetes Care 13:
762-775, l990). Details of one preferred screening test are available to those of ordinary skill in the art (~-~n~ o, ~.
et al., The ~ancet 33S: 147-149, l990) .
From a practical point of view, preventing the onset of most autoimmune diseases is of most importance in the case of diabetes. Other ~tQ1 ~ 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 of diabetes. In dlabetes, it would be best -o intervene with W095l27500 P~~ 01512 an effective treatment prior to the substantial destruction of substAnt;~l ly 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 diseases and tissue- or organ-specific confirmed or potential bystander antigens effective in the treatment of these diseases when admini8tered in an oral or ;nh~l~hle form are set forth in Table 1 below. Administration of comb;n~t;~nq of antigens lO listed for each individual disease (alone or in conjunction with I~-4) is also expected to be effective in treating the disease .
Bystander antigens and Th2-enhancing cytokines can be also administered by ;nh~l~t;on. The bystander amounts that 15 need to be inhaled are generally smaller than those f or oral adminigtration. It is anticipated that the amounts of Th2-f-nh~nc;n~ cytokineg administered by ;nhAl ~t; on will be likewise smaller. Effective amounts for ;nh~l~t-ion therapy can be assessed using the same methodologies provided above.

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WogsJ27~00 21 87345 F.IIIJ. 5'0l'l2 For any autr;mml~n~ disease, extracts of the relevant tissue, as well as specific bystander antigeng or frArJm~ntR
thereof, can be used as oral tolerizers. In other words, the bystander antigen need not be purified. For example, myelin 5 (which could be derived from dif~erer~t species) has been used for MS, pancreatic cell extracts have been used for Type l nl;Ahet~R, splenic cell ~ trArtR have been used to prevent allograft rejection (which is not, strictly speaking, an A1ltOi ^ rh~n, ), and muscle extracts have been used to 10 treat myositis. However, administration of one or more individual antigens or f ragments is pref erred .
Thus, according to the present invention, when treating Type l diabetes, a~ effective amount (nlpt~rm;n~d as described above) of glucagon can be administered orally.
15 Glucagon is Bper; f 1 rA l l y present in the pancreas . Glucagon, however, is clearly not an autoantigen because it is not expressed in pancreatic beta cells which are destroyed in the course of Type 1 n9; Ahet~o~ (glucagon is found exclusively in alpha cells, a different cell type). Thus, glucagon is a 20 "pure" bystander: it does not appear to have any autoantigen activity. (Presumably, the bystander activity of glucagon results from its high local concentration in the pancreatic interc~ l Ar milieu due to its secretion from alpha cells . ) I~sulin haR bystander activity for Type 1 diabetes.
25 It is not at present known whether insulin is also an ~llto~ntigen, although anti-insulin ~illtrAnt;hnA;es are found in Type l rAti,ontR. However, whatever the m~rhAn; l:m of action, oral, enteral or ;nhAlAhle insulin preparations are effective in suppressing Type l diabetes and animal models therefor by 30 preventing A~tn;~1no destruction of pancreatic beta cells.
For multiple sclerosis and animal models therefor, both disease ;n~l1lr;ng and noninducing fragments of Mi3~ have bystander activity not only for M~3P-induced disease but also for P~P-induced disease. In rats, feeding of bystander 3~ generates mostly CD8f suppressor cells which are class I
restricted, whereas in mice both CD8+ suppressors and cr4f regulatory cells are generated (the latter being probably Class II restricted). Chen, Y. et al. Science, 1994, supra.

wo ssl27500 2 1 8 7 3 4 5 P~ 4512 For rh~ toid arthritis and animal models therefor, Type-I, Type-II and Type-III collagen have activity as oral tolerizers. Other collagens are likely to be similarly active.
For uveoretinitis and its animal model, S-antigen and 5 I~3P and fLCL ~ thereof have bystander activity.
FLO.~ ' Fl of bystander antigens can also be employed.
Useful f ragments can be identif ied using the overlapping peptide method and T-cells from fed animals can be tested for secretion of TGF-,~, and/or IL-4 and!or II-10, and can further 10 be identified by subtype (CD8+ and/or CD4~).
Orally administered ~l~tn~ntigens and bystander antigens elicit regulatory T- cells and thereby induce the production and/or release of TGF-,~ and/or II-4 and IL-10. One such T-cell has been ;fl,ont;fled in mice orally tolerized 15 against EAE as a CD4+ suppressor T-cell, and a CD8+ suppressor T-cell has been ;fl~ont;f;ed in rats. Even 1 'nm;nAnt epitopes of ~l~lt~^^nt;gens, e.g. M3P are capable of inflll~^;ng such regulatory T-cells. Additional such epitopes can be identified by f eeding a bystander antigen to a mammal and isolating f rom 20 the mammal T-cells that recognize a fragment of the antigen (and thus identifying suppressive fragments), or by identifying T-cells from a bystander fed mammal that can adoptively trans~er protection to naive (not-fed) animals.
The bystander antigens can be administered alone or 25 in conjunction with autoantigens. Autoantigen 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 rn~nt;nn~^d above. It is - 35 anticipated that co-administration of at least two ^lltn~nt;gens (and/or fragments of autoantigens) or more broadly at least two other bystander antigeng (and/or bygtander frg_ ' cl) will also result in effective suppression of the ^~lto; ^ diseases.

} - 21 87345 Wo 9S/27500 PCrNSs5/04512 In addition, other cytokine and non-cytokine synergists can be conj oined in the treatment to enhance the ef ~ectiveness of oral tolerization using bystander antigens alone or bystander antigens plus Th2-Pnh~nr;n~AJ cytokines. Oral 5 use of other cytoklne synergists (Type I interferons) has bee~
described in co-pending U.S. Patent Application Serial No.
08/225,372. Non-limiting examples of non-cytokine synergists f or use in the preaent invention include bacterial lipopolysaccharides from a wide variety of gram negative 10 bacteria such as various gubtypeg of E. ~QLi and ~A~lmnn~
(LPS, Sigma Ch!emical Co., St. Louis, MO; Difco, Detroit, MI;
BIOMOL Res. Labs., Plymouth, PA), Lipid A (Sigma Chemical Co., St. Louis, MO; ICN Biochemicals, Cleveland, OH; Polysciences, Inc ., Warrington, PA); immunoregulatory lipoproteins , such as 15 peptides covalently linked to tripalmitoyl-S-glycarylcysteinyl-seryl-serine (P3 C55) which can be obtained as disclosed in Deres, K. et al. (~, ,~:561-564, 1989) or "Braun~s~
lipoprotein from ~ coli which can be obtained as di6closed in Braun, V., Biorhim. Bio~hys. ~L 435:335-337, 1976; and 20 chole~a toxin ,~-chain (CTB) the synergist ability of which have been described (though not in connection with Flh~t~ of ;~t~tQ;~mln~ reaction) by Sun, J-B et al., 1994 PNA~ (USA) 91, NJYI Ar 1994. LPS is preferred and Lipid A particularly preferred. Lipid A is particularly preferred for use in the 25 present invention because it is less toxic than the entire LPS
molecule . LPS f or use in the present invention can be extracted from gram-negative bacteria and purified using the method of Galanes et al~ ;L Biorh~m. 9:245, 1969) and Skelly, R R., et al. (Infect. Imm~ ;~:287, 1979). The 30 effective dosage range for noncytokine synergists for mammals is from about 15 ~Lg to about 15 mg per kg weight and preferaAhly 300 ~g - 12 mg per kg weight. The ef~ective dosage range for oral Type I interferon for mammals is from 1,000 - 150,000 units with no maximum effective dosage having been discerned.

W095/~7500 2 1 87345 P~l/u~ ~1512 Oral ~se of Th2-RnhAn~;n~ C5rtol~ine~ Alone ln the Present I~vention According to the present invention, oral, enteral, or by-inhalation administration of Th2-f~nh~nn;n~ cytokines i8 used to suppress ~autoimmune disease. An example of a Th2 ,~nh~nrors cytokine is IL-4.
IL-4 can be purified from natural sources (T-cells that normally produce it) and can also be obtained using re(~l ;n~nt DNA tec_nology, in bacterial, yeast, insect and l ;~n cells, using techniques well-known to those of ordinary skill in the art. In addition, IL-4 is commercially available. The DNA sequence Pnnorl;ng human IL-4 is disclosed in Yokota et al., Proc.Natl.Acad.Sci.USA 83:5894, 1986.
According to the present invention, the route of administration of IL-4 is preferably oral or enteral. The preferred oral or enteral rh~-n~ t;cal f~ t;nn~ may comprise, for example, a pill, a liquid or a capsule cnnt;~;n;ng an ef f ective amount of IL- 4 .
Each oral (or enteral) f, l ~tinn according to the prese~t invention may additionally comprise inert constituents including rh;~-c~tically acceptable carriers, diluents, f illers, solllh; l; ~; ng or emulsifying agents, and salts, as is well-known in the art. For example, tablets may be formulated in accordance with convPnt; nn~l ~L~,~edu~ ~:s employing solid carriers well-known in the art. Capsules employed in the present invention may be made from any ~h~r~utically acceptable material, such as gelatin, or cellulose derivatives.
Sustained release oral delivery systems and/or enteric co lt;
for orally administered dosage forms are also contemplated, such as those described in U.S. Patent No. 4,704,295, issued November 3, 1987; U.S. Patent No. 4,556,552, issued December 3, 1985; U.S. Patent No. 4,309,404, issued January 5, 1982; and U.S. Patent No. 4,309,406, issued January 5, 1982.
Examples of solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers. Further non-limiting ~ ~ l F'R of carriers and diluents which may be used in the form-l~t;~n~ of the present invention include saline, syrup, dextrose, and water.

Wo 95/27500 2 1 8 7 3 4 5 r~ l / ~b,,' ~01512 It will be appreciated that the unit content of active ingredient or ingredients rnnt~; n~ in an individual dose of each dosage form need not in itself constitute an effective amount, slnce the necessary effective amount can be 5 reached by administration of a plurality of dosage units (such as capsules or tablets or combinations thereof ) .
In general, when administered orally or ,~nt~ lly, IL-4 may be administered in single dosage form or multiple dosage forms.
Suppression of the clinical a~d histological symptoms of an autoimmune disease occurs af ter a specif ic minimum dosage, which, however, varies according to disease, species of m~mmal, and cytokine. For oral IL-4, the effective dose range for hllmans is between about 2,000 and 50,000 15 ;ntPrn~t;onal units per day, and preferably about 5,000 and about 20,000 ;ntf~rn~t;nn~l units per day. The maximum dosage is best ascertained by experimentation. It is anticipated that larger doses are permitted but 11nn~r~ Ary It is not necessary that a dose of IL-4 be effective by itself if IL-4 20 (or anPther Th2-~nh~nr;ng cytokine) is used in rrlmh;n~t; nn with an autoantigen or bystander. Suboptimal doses of Th-2 ,~nh~ncing cytokineg that would potentiate the effect of the bystander or autoantigen can thus be used.
Ascertaining the effective dosage range as well as 25 the optimum amount is well within the skill in the art in light of the infnrr-tinn given in this section. For example, dosages for m.~cmal8 and human dosages can be ~lPtGrm; n~A by beginning with a relatively low dose of cytokine (e.g. 500 units of I~-4, ~L~J~L~ ively) increasing it (e.g. logarithmically) and 30 measuring a biological reaction to the treatment, for example induction of regulatory cells (34+ and/or CD8+) as described in Chen, Y. et al., Science, 1994, ~;~, reduction in class II surface :markers on cirr~ t;ng T-cells, and/or by scoring the disease severity, according to well-known scoring methods 35 (e.g., on a scale of l to 5, or by measuring the num.~ber of attacks, or by measurillg joint. swelling, grip strength, st;ffn~R~, visual acuity, ability to reduce or~disrnnt;n-1P
medication, etc. d~l~on~l~n~ on the type of disease). The ... _ . .. _ . .... _ .. , .. ,,, . , . , , , ., _ _ _ _ _ _ . _ WO95117500 2 t 87345 .~I.,L ~ 1 .2 optimum dosage will be the one having the greatest influence on the biological rhPnn~rPnnn being measured, such as that which causes the greatest induction of regulatory T- cells or the greatest decrease in immune attack cells and/or that which 5 causes the greates~ decrease in disease symptoms. An effective dosage range will be one that causes at least a statistically or clinically significant attenuation of at least one symptom characteristic of the disease being treated, or a significant change of a marker (such as the frequency of regulatory or 10 activated T-cells).
Administration of I~-4 may be once daily for a period of time ranging from 30 days to several months (e.g. 3-6) or even years (e.g. 2-6). In fact, therapy may cont;nllP
1ntlPf;n;tPly (unless the obtained benefit does not persist) 15 given the low risk of side effects afforded by the oral route of administration.
Protease inhibitors (such as soybean trypsin inhibitor, aprotinin, Ant;rA;n) may be added to oral dosage forms cnntA;n;n~ I~-4 to increase the absorbed amount. In that 20 case, ~ the dosage of IL-4 may be decreased.
Parenteral administration of I~-~ may also be used alone or aa an adjunct to oral tolPr;~At;nn therapy but oral I~-4 is preferred because of the systemic effect of parenteral I~-4. Parenteral IL-4 however, is quite effective in 25 suppressing autoimmune disease, as illustrated below Parenteral dosage for man~nals generally can range from about 500 ;ntPrn~t;nnAl units of I~-4 to about 1,000,000 ;ntPrnAtinnAl units although the upper limit of this range can best be estAhl;chp~l by exper'-- At10n It is anticipated that 30 the upper limit will be an amount at which the maximum suppressive effect of parenteral I~-4 is observed (i.e.
efficacy will not be lost by using higher amounts but they may be unnecessary). Parenteral administration may take place subcl1tAnPn~l y typically once every other day (without 35 limitation) in a single or in divided doses.
~ ~n~tion Th~- Ar~y It has been surprisingly discovered that the oral (or by ;nhAlAtinn) administration of a bystander antigen in wo gsl27500 2 1 8 7 3 4 5 . ~11. c~o,rl2 conjunction with oral or parenteral or by-inhalation administration of IL-4, results in a treatment which suppresses autoimmune reaction, the effect of con~oint therapy being subst~nt;~11y augmented when compared to the effect of each 5 treatment separately.
This c ' 1n~t;nn treatment has been studied in rodents, using the animal model for MS, EA13. The experimental protocol for these studies is disclosed in the Examples below.
Treatment of mice with l000 international units of I~-4 alone administered orally 5 times before 1nrq1lrt1nn o~ EAE
has resulted in rPrl~1rt1nn in disease ;nri~lGnre and some reduction in maximum rl ;n;r~1 score.
Treatment with a ~ in~tlon of oral rat IL-4 (l000 15 units) and oral tol~or; ~t; nn using MBP reduces both disease onset and clinical score, and delays disease onaet. In fact, the delay in disease onset was substAnt;~11y greater (30 days) with the l_ ` ;n~t;nn treatment than with either IL-4 or M5P
alone (21 or 22 days respectively).
~ It is anticipated that the c n~t; nn treatment of intraperitoneal IL-4 and oral tolerization with a bystander antigen can show a synergistic suppressive effect on the clinical score of the 13AE seen if the doses of each of bystander and Th2 Pn~nr~r are reduced to reveal this effect.
These trP;~ ~ clearly have a subst~nt;~lly more prnnmlnrPtl suppressive ef ~ect when used in conjunction compared to the ef f ect achieved with either treatment alone .
In practicing the present invention, the bystander antigen is administered essentially as described above. I~-4 is administered in con~unction with the bystander antigen.
Oral or enteral administration of IL- 4 may be achieved as described above.
Parenteral administration may be via subcutaneous, intramuscular, or intraperitoneal, routeg, with gubrllt~n~-n~lc being preferred for treatment purposes (although intraperitoneal route was used in the examples below). In the case of parenteral administration, IL-4 may be f~ 1~te~ in sterile saline or other carriers well known in the art, and may .. . .., ... . _ _ _ _ _ . .
.. _ _ . . . _ ... .. . _ . . . .

w0 95/27500 2 1 8 7 3 4 5 P~ ' 0 1512 include excipients and stabilizers that are standard in the art .
Ascertaining the optimum regimen for administering both bystander antigens and Th2-~nhAn--;ng cytokines is well 5 within the skill ~ in the art in light o$ the information disclosed herein. As is the case with individually administering bystander antigens or Th2-~nhAn~;ng cytokines routine variation of dosages, c inAt;nnq, and duration of treatment is performed under circumstances wherein the severity 10 of autoimmune reaction can be measured. Useful dosage and administration parameters are those that result in reduction in autoimmune reaction, including a decrease in number of autoreactive T- cells, or in the occurrence or severity of at least one clinical or histological symptom of the disease.
It is preferred to utilize Th2-pnhAnc;ng cytokines derived f rom the same species as the species being treated.
The following examples are illustrative of the present invention and do not limit the scope of the invention.
MZ~TT~RTl~T..~ AND ~M~THODS
~ In the experiments described below the following materials and methods are used.
Animala . Female Lewis rats 6 - 8 weeks of age are obtained from Harlan-Sprague Dawley Inc. (Tn~l;AnAr~ , IN). SJL/J
mice, 8 weeks of age are obtained from Jackson Laboratories, Bar Harbor, ME. Animals are r-;ntA;nl~d on standard laboratory chow and water ad libitum. Animals are r-;ntA;n.or~ in accordance with the gll;A~l ;n~ for the Committee on Care of Laboratory Animals of the Laboratory Research Council (Pub.
#DHEW:NIH, 85-23, revised 1985).
r~nt~ n~ J~nA Rea~enta. Guinea pig MBP and mouse MBP is purified from brain tissue by the modified method of Deibler et al. (Pre~. Biochem. 2:139, 1972). Protein content and purity are monitored by gel electrophoresis and amino acid analysis. Histone, hen egg lysozyme and ovalbumin are obtained from Sigma (St. Louis, MO) Peptides are synthesized in the peptide facility of the Center for Neurologic Diseaae, Brigham and Women's Hospital, and purified on HPLC. The amino acid se auences of the ~3P peptides 8ynth~; 7Prl are: 71-90, Wo 9s/27500 ~ 2 1 8 7 3 4 5 SLPQRSQRS~ ;N~vv~ n-~dnm1n~nt Pnr~Prh;~l ;togenic region in rats); the mouse PLP peptides 140-160 (disease-inducing epitope in rats) and 139-153 (disease ~nAllr;ng epitope in mice) were also used.
Mouse IL:4 was obtained from rr~l 1 Ahnrative Biomedical Products, Bedford, MA.
,TnrlllrtioIl o~ Tnl Dr~n~ For oral tolerance or active suppression, rats are fed 1 mg of MBP dissolved in 1 ml PBS, or PBS alone, by gastric intllh~tinn with a 18-gauge stainless steel animal feeding needle (Thomas Sr;Pnt;fic, Swedesboro, NJ). Animals were fed five times at intervals of 2-3 days with the last feeding two days before; ; 7~tion. When mice are used for experiment8, the tolerization regime is subgt~nt;~lly the same except that 0 . 5 mg of MBP and/or 1000 or 5000 units of IL-4 are used for tolerization. Further details for mouse experiments are provided below.
I~ducl--lnn o~ ~r~ For actively induced disease, Lewis rats are; ; 7P~ in the left foot pad with 25 ~g of guinea pig MBP in 50 ~l of PBS emulsified in an eriual volume of cQmplete Freund's adjuvant (CFA) rnnt~n;n~ 4 mg/ml of MvcobactPri~lm tUhprrlll~ (Difco). When mice are used for experiments, 400 ,ug of MBP in 0.1 ml of PBS/CFA cnntA;n;n~ 4 mg/ml of Myco~acterium tuberculosi~.
rl In~,.Dl evAl~n~lnn Animals are evaluated in a blind fashion every day for evidence of EAE. rl;n;r~l severity of EAE is scored as follows: 0, no disease; 1 limp tail; 2, hind limb paralysis; 3, hind limb paraplegia, ;n~rnnt;n~nrP; 4, tetraplegia; and 5 death. Duration of disease is measured by rmlnt;n~ the total number of days from disease onset (for control rats usually days 10 or 11 after active; i 7~tion;
for control mice 9 days after ; ; 7~tion) until complete recovery ( or death) f or each ~ animal .
11~ stolo~y . Histologic analysis of ~ pathological changes can be performed in animals with induced EAE. Spinal cords are removed on day 15 after adoptive transfer (or disease induction) and fixed with 109~ neutral buffered formalin.
Paraffin ~ections are prepared and stained with Luxol fast blue-hematoxylin and eosin, by standard procedures (Sobel et .. . .. .. ~

wossl27500 21 87345 r~ 17 al., J. Immunol. 132:2393, 1984). Spinal cord tissue is sampled in an identical manner f or each animal and numbers of ;nflAmmAtory foci per section (clusters of ~20 or more aggregated ;nfli tory cells), in parenchyima and meninges are 5 scored in a blinded ~ashion (Sobel et al., supra).
Stati~tical i~ni~lYsi~. Clinical scales are analyzed with a two-tailed Wilcoxon rank sum test for score samples, chi square analysis is used in comparing the; nr; ~lPnr~ O_ disease between groups, and comparison of means is performed by using 10 the Student's t-test. For individual experiments, 5 animals are generally used per group.
EXAMPLE 1: A330y for TGF-,~ Tnd~ tion M~'A! t Of TGF-~ Activity Tn Sexum-Free t'lll tllre Su~ernatants. Serum free culture Sllr~rnAtAnt~ were collected from antigen-tolerized rats as previously described (Kehri, et al. ~. ~.Med.163: 1037-1050, 1986; Wahl, et al.
~.I~lln~l.l45: 2514-2419,1990). Briefly, ~ l~Atcr cells were 20 first ~ cultured for 8 hours with the antigen (50 ~l/ml) in proliferation medium. Thereafter cells were washed three times and res-lArPn~ 9 in serum-free medium for the l~ ;n~l~r of the 72 hour culture, collected, then frozen until assayed.
Det~rm;nAt;on of TGF-,~ content and isoform type in supernatants 25 was performed using a mink lung epithelial cell line (American Type Culture Collection, Bethesda, MD ~CCB-64) ACcortl;ng to Danielpour et al. (Danielpour, D., et al. J. Cell. Phvsiol.
138: 79-86,1989).), and confirmed by a Sandwich Enzyme Linked Immunosorbent Assay (SELISA) assay as previously described (Danielpour et al. Growth Factors 2: 61-71,1989) . The percent active TGF-,~ was detprm; n~tl by assay without prior acid activation of the samples.
This assay can be adapted to test any antigen which is a rAn~;~lAte for use as a bystander. Those antigens, antigen fragments and/or amounts of antigen which produce the highest conc~ntrAt;~ n of TGF-,~ 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.

Wogs/27500 ~ 21 87345 P ,/~ l'12 Alternatively, a transwell culture system, described below, can be used to indicate the level o~ TGF-~ which i9 being produced.
This culture system measures the production of TGF-S as a function of suppression of cell proliferation.
The appearance of II,-4 and/or I~-10 in culture 8up~rn; t;lntfl of antigen-st; lAt~rl cells may also serve as an indicator that the antigen is suitable for use as a bystander.
IL-4, I~-10 (and TGF-~) can be assayed by E~IZA using commercially available antibodies to each polypeptide as described in Chen, Y. et al, Science. 1994, su~ra.
~ n - 11 CU~ e~:. A dual chamber transwell culture system (Costar, Cambridge, ~A), which is 24.5 mm in diameter and consists of two compartments separated by a semi-p~ --hle polycarbonate membrane, with a pore size of 0 . 4 ~m, was used.
The two ~ el~ are 1 mm apart, allowing cells to be coin-cubated in close proximity without direct cell-to-cell contact.
To measure ;La Y~SL suppression of proliferative responses in transwell cultures, 5 x 104 antigen line cells, raised and ~-intA;n~l for example, as previously described (Ben-Nun, A.
et al, Eur. .J. T r~l. 11:195, 1981), were cultured with 106 irradiated (2,500 rad) thymocytes, in 600 ~l of proliferation media in the lower well . Spleen cells f rom orally tolerized rats or controls (fed BSA) were added to the upper well 15 x 105 cells in 200 ~l). Spleen cells were removed 7-14 days af ter the last ~eeding, and a single cell suspension was prepared by pressing the spleens through a stainless steel mesh. The antigen (50 ~g/ml) is added in a volume of 20 ,ul.
Because ~ l~tor cells are separated ~rom responder cells by a semi-permeable nf~, they do not require irrA~l~At;~n.
3 0 In some experiments, modulator cells were added in the lower well together with responder cells, and in these instances modulator cells were irradiated (1,250 rad) immediately before being placed in culture. Proliferation media consisted o~ RPMI
1640 (Gibco Laboratories, Grand Island, l~lY) supplemented with 2 x 105 M 2-mercaptoetharol, 196 sodium pyruvate, 1~ penicillin and streptomycin, 19~ glutamine, i % ~IEPES buffer, 196 nonessential amino acids, and 1~ autologous serum. Each transwell was per~ormed in quadruplicat~e. The trarswells were wo 95127500 2 1 8 7 3 4 5 ~ u~ Q4sl2 incubated at 37C in a humidified 6~ CO2 and 94% air atmosphere for 72 hours. A~ter 54 hours of culture, each lower well was pulsed with 4 /~ci of [3~I] thymidine and at 72 hours split and reseeded to three wells in a round-bottomed 96-well plate 5 (Costar) for harvesting onto fiberglags filters and ~ollnt;n~
using standard li~uid s-;nt;ll~t;on techniques. Percent suppression = 100 x (1 - ~ cpm responders cultured with modula-tors/~ cpm of rP~p~nr1-~rs).
;

?`''PLTZ 2: Oral Tol~rAn~e ~T~ing Bovine-PLP or Mouse NBP
In order to demonstrate bystander suppression, groups of 5-6 female, 7 week old, SJI,/ T mice (Jackson ~abs, Bar 15 Harbor, ME) were; ; ~Fd with mouse P~P peptide 140-160 on days 0 and 7 and received the following tr~tm~nt.
GROUPS
1. Fed Histone (0.25 mg/mouse) 2. Fed Mouse MBP (0.25 mg/mouse) 20 3. Fed Bovine PIP (0.25 mg/mouse) (autoantigen suppression) The P~P peptide used was the disease ; n~ ; n 7 fragment 140-160 of bovine PLP. This peptide has the amino acid sequence CooH-pI~ L~v~Ku~ T~t~2l L~L~:s~ ing the 25 foregoing amino acid residues.
Both mouse MBP and bovine P~P were equally effective in down- regulating PI,P-peptide- induced EAE when orally administered . A non - specif ic protein, histone, was inef f ective in suppressing EAE when administered Drally. Thus, a bystander 30 antigen, in this case mouse MBP, effectively suppressed EAE
when orally administered to animals induced for EAE with bovine P~P .
The effects of feeding various peptides to ~ewis rats induced for EAE by guinea pig MBP residue nos. 71-90 (the ma~or 35 ; ~ ml n~nt epitope of guinea pig MBP in rats as shown in Example 1 above) were also studied.
EAE was induced by immunizing with 0.25 mg of guinea pig MBP amino acid residue nos 71-90 in Complete Freund' s wo 95/27500 " 2 1 8 7 3 4 5 r~ ,5~ 12 Adjuvant and the effect of =feeding various guinea pig MBP
peptides on EAE was ~ mi nP~l, Orally administered whole guinea pig MBP and a 21-40 guinea pig peptide were equally effective in downregulating EAE
5 induced by guinea pig MBP 71-90 as was orally administered 71-90 itself. Guinea pig MBP peptide 131-150 was ineffective in conferring tolerance in rats. Peptides were also fed with STI
which prevents their breakdown by gastric juices and ~nhAnr~
their biological effect. DTH responses (to injection of 25 ,ug 10 of MBP in PBS) to whole MBP were suppressed by feeding MBP or any one o~ the MBP-peptides 21-40, or 71-90. Eowever, DTE~
responses to guinea pig MBP peptide 71-90 were only suppressed by feeding either whole MBP or ~guinea pig peptide 71-90 and were not affected by guinea pig MBP peptide 21-40. This is 15 consistent with the observation that MBP fragment 71-90 does not participate in bystander ~u~L~ssion when fed to mice in which disease had been induced with peptide 71-90. See, Chen, Y. et al., SCi~nr~ 1994, ~
The foregoing f~r; ' illustrates an assay system 20 for ~f~t~r~n;n;ng whether an orally administered antigen acts as a bystander suppressor. Whole antigens could have been used instead of fra~c~nt~
i~MPT~ 3: Su~re~sion of EAE in Miqe with a Combination 25 of Oral Tol~riz~tion u~ing M13P And Oral IL-4 EAE was induced in S~/J, 8 week old, female mice by ; 7; ng on day 0 with 400 ~g mouse MBP in 0 .1 ml of a suspension rr~nt~;n;n~ 4 mg/ml Mycobacterium tuberculosi~ (MT), followed by pertussis toxin injection (100 ng/mouse) on days 30 0 and 2. On days -10, -8, -6, -4, and -2, different groups of mice were treated as f ollows:
Grou~s 1. Ped hen egg lysozyme (~IE~) ~0.25 mg/mouse) 2. Fed mouse I~-4 ~1000 ;ntprn~t;onal units/mouse) 35 3. Fed mouse MBP (0.5 mg/mouse) 4. Fed mouse MBP (as in group 3) plus I~-4 (as in grouE~ 2).
Animals were monitored for disease onset for 35 days.
Animals were scored for signs of disease every day beginning 40 on day 9 on a scale of 0 to 5.
_ .. .. . .... . .. .. ..... . ... .. . .. .

w0 ss/27soo 2 1 8 7 3 4 5 r~ m /C 1512 The results were as followl3: Su}:st~nt;~11y simultaneous administration of oral II,-4 with M}3P clearly augmented the suppressive ef f ect of ~P in terms of disease ;nc;rq~nt e, and delay of onset. At lOOO ;nt~rn~tional 5 units/mouse/feeding, II-4 alone _ad a suppressive effect on disease ;ncid~nr~ similar to that of M~3P when fed alone.
This type of experiment can be used to assess efficacy of treatment with Th2-~nhAn~;n~ cytokine alone or ~- ;n~tion treatment with Th2 enh~nr~r and bystander. Dosages lO can be adjusted to observe the effect ¦8) of dosage variation.
EXAMP~ ~ 4 : s~ . eR,,lon of EAE in Mlce wlth ~ ColDbln~ ~lon of Oral Tolerlzatlon uslng M13P and Oral IL-4 EAE is induced in SJL/J, 8 week old, female mice by 15 ; n;~;ng on day O with 400 ,ug mouse ~3P in O.l ml of a suspension ccnt~;n;ng 4 mg/ml ~ycobacterium tuberculo8i~ (~qT), followed by pertussis toxin injection (lOO ng/mouse) on days O and 2. On days -lO, -8, -6, -4, and -2, different groups of mice are treated as f ollows:
2 0 ~Q~a l. Feed mouse IL-4 (5000 international units/mouse) 2. ~eed mouse M}3P (0.5 mg/mouse) 3 . Feed mouse M~3P (as in group 2 ) plus I~- 4 (as in group l ) .
Animals are monitored for disease onset for 35 days.
Animals are scored for signs of disease every day beginning on day 9 on a scale of O to 5.
This experiment will demonstrate that at the foregoing dose, feeding IL-4 alone significantly delays the onset of disease, decreases fatality, and/or reduces the mean and maximum clinical scores. Purthermore, feeding I~-4 at the foregoing dose, in combination with M~3P, significantly ;~ n the suppressive effect observed when ~3P alone is fed.
EXA~IPT~ 5: Suppre~lon of PLP-Induaed EAE ln Mice wlth a ~ombination of Oral Tolerization using ME~P And Oral IL - 4 EAE is induced in SJ~/J, 8 week old, female mice by ;~;ng on day O with lOO ~g mouse PLP 139-15l peptide in 40 O.l ml of a suspension c~nt~;n;ng 4 mg/ml Mycobacterium tuberculosiEI (MT), followed by pertussis toxin injection (lOO
. . .

Wo gs/27500 2 1 8 7 3 4 5 PCT/US95/04512 ng/mouse) on days 0 and 2. On aays -l0, -8, -6, -4, and -2, different groups of mice are treated as follows:
l . Feed mouse I~ - 4 ( 5 0 0 0 i n t prnA ~; nn~ 1 units/mouse ) 5 2 . Feed mouse MBP ( 0 . 5 mg/mouse) 3. Feed mouse M}3P ~(as in group 2~ plus I~-4 (as in group l).
Animals are monitored for disease onset for 35 days.
Animals are scored for signs of disease every day beginning on l0 day 9 on a scale of 0 to 5.
This experiment will demonstrate that at the foregoing dose, feeding II--4 alone significantly delays the onset of P~P-induced disease, decreases fatality, and/or reduces the mean and maximum clinical scores. Furthermore, 15 feeding II.-4 at the foregoing dose, in ~ ' ;n~t;nn with the pure bystander antigen MBP, significantly augments the suppressive effect observed when MBP alone is fed.
r le 6: Su~J ~s~lon of Ad~uv~nt Arthrltis in Mice with 2 0 a Combination of Oral Tolerization u~ing Type II
Col 1 J~g~n and IL-4 Arthritis is induced in male DBA/l ~ac ~ mice by 511h,~ l ; In; ~;~tion with 300 ~g bovine Type II Collagen in 25 a suspension cnnt~;n;n~ 4 mg/ml MT. On days -14, -12, -l0, -3, - 6, - 4, and - 2, aif f erent groups of mice are treated as f ollows:
Groul; 5 1. Feed mouse I~-4 (5000 internatiorlal units/mouse) 30 2. Feed mouse collagen type II (0.5 mg/mouse) 3. Feed mouse collagen type II :~as in group 2) plu8 IL-4 (as in group l ) .
Animals are monitored for o~set of arthritis for 60 days. Begimling on day +l0, animals are scored for signs of 35 arthritis on a scale of 0 to 4 . The arthritis score f or each animal is the sum of the score f or each of the f our paws .
This experiment will show that at the foregoing dose, feeding IL-4 alone significantly delays the onset of disease symptoms and/or reduces the mean and maximum clinical scores.
40 Furthermore, feeding Il,-4 at the foregoi~g dose, in combination with type II collagen, significantly ~ nt~ the suppressive ef f ect observed when collagen alone is f ed .

wo ssl27soo 2 1 8 7 3 4 5 1'~ 5,'0 1512 Exam~le 7: Oral IL-4 Alone and ln Combination with Insulin in Su~re~sion gf NOD Diab~tes Groups Qf 4-week-old non-obese diabetic ~NOD) mice (3 animals per group) are treated as follows:
1. Fed ovalbumin (lmg/mouse) 2. Fed eSIuine insulln (lmg/mouse) 3. Fed mouse I~-4 (5000 units/mouse) 4. Fed insulin (as in group 2) and IL-4 (as in group 3) Mice are fed 10 times on a1ternate days. About 3 weeks after the experiment the following pal ~rA are assessed: insulitis and/or time of diabetes onset.
This experiment will show that at the foregoing dose, feeding I~-4 alone significantly reduces the above clinical indicators. Furthermore, feeding IL-4 at the foregoing dose, in cornbination with insulin, gignificantly A115 ' ~ the suppressive effect observed when insulin alone is fed.

E~tAMPLE 8: Suppression of EAE in Mice with a Comblnation oi ~ Oral Tolerization using MBP and Intraperitoneal Administration of IL-4 EAE was induced in SJL/J, 8 week old, female mice by immunizing on day O with 400 ,ug mouse M3P in O.1 ml of a suspension ~nntA;n1n~ 4 mg/ml Mycobacterium tuberculosis (MT), followed by pertussis toxin injection (100 ng/mouse) on days O and 2. Different groups of mice are treated as follows:
3 0 ~Q~
1. Injected mouse IB-4 ~n~rAr~oritoneally (5000 ;n~Prn~tional units/mouse/injection) on day O and day 3.
2 Fed mouse M3P (0.5 mg/mouse) on days -10, -8, -6, -4, and -35 3. ~ed mouse M3P (as in group 2) plus in~ected IL-4 (as in group 1 ) .
Animals were monitored for disease onset for 35 days.
Animals were scored for signs of disease every day beginning 40 on day 9 on a scale of O to 5.
This experiment demonstrated that at the foregoing dose, in~ecting IL-4 alone significantly reduced the incidence, delayed the onset of disease and reduced the ,-l~nlrAl scores.
The magnitude of this effect was such that it was impossible _ _ _ _ _ _ _ _ _ _ _ _ WO 95/27500 2 1 8 7 3 4 5 r~ o ~512 to assess whether parenteral administration of I~-4 :could augment the protection afforded by oral M13P alone, which also showed comparable suppression of autoimmune reaction (as measured by the foregoing indicators).

RY~ ,R 9 Suppression of EA_ ln Mlce wlth a Comblnation of Oral Tolerizatlon uslng MBP and Tnt- ~r~ ltoneal Admlnistration of IL-4 EAE is induced in SJ~/J, 8 week old, female mice by immlln;~;ng on day 0 with 400 llg mouse M3P i~ 0.l ml of a suspension rnn~;nlng 4 mg/ml Mycobacterium tuberculo6i~ (MT), followed by pertussis toxin injection (100 ng/mouse) on days 0 and 2. Different groups of mice are treated as follows:
15 ~Q~L
Feed mouse ~3P (0.3 mg/mouse) on days -l0, -8, -6, -4, and -2. Inject mouse II,-4 intraperitoneally (500 ;n~Prn~t;nn~l units/mouse )0 3. Inject mouse IL-4 intraperitoneally (l000 ;ntorn~tinn~l units/mouse) 4. Inject mouse IL-4 intraperitoneally (2000 international units/mouse) 5. Feed mouse M13P (as in group l) plus IL-4 ~as in group 2).
6. Feèd mouse M}3P (as in group l) plus IL-4 (as in group 3).
7. Feed mouse M~3P (as in group 1) plus IL-4 (as in group 4).
IL-4 injections will be administered as in 3xample 8.
Animals are monitored ~or di~ease onset for 35 days.
Animals are scored for signs of disease every day beginning on day 9 on a scale of 0 to 5.
This experiment will demonstrate that at the ;n(9;~atod reduced doses, parenteral administration of I~-4, in combination with ~3P, significantly ~llgmont~ the suppressive effect observed with oral administration of M~P alone.

Claims (17)

WHAT IS CLAIMED:

1. A method for suppressing autoimmune reaction in a mammal diagnosed with a T-cell mediated or T-cell dependent autoimmune disease, the method comprising administering to said mammal: (i) via the oral or enteral route, an amount of a bystander antigen and (ii) an amount of a noninterferon polypeptide having Th2-enhancing cytokine activity, the amounts of said antigen and said polypeptide being effective in combination in reducing said autoimmune reaction substantially more than the amount of either said antigen or said cytokine administered alone.

2. The method of claim 1 wherein the amounts of said antigen and said polypeptide are substantially more effective in suppressing said reaction in combination compared to the suppressive effects achieved by administering each of said antigen and said polypeptide alone.

3. The method of claim 1 wherein said polypeptide is selected from the group consisting of IL-4 and fragments thereof having Th2-enhancing cytokine activity.

4. The method of claim 1, wherein said polypeptide is IL-4.

5. The method of claim 4, wherein said IL-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 for multiple sclerosis.

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 bystander antigen is selected from the group consisting of myelin, myelin basic protein (MBP), proteolipid protein (PLP), tolerogenic fragments thereof and combinations of at least two of the foregoing.

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

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 bystander antigen is selected from the group consisting of Type I collagen, Type II collagen, Type III collagen, 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 I diabetes and said bystander antigen is selected from the group consisting of GAD, glucagon, insulin, tolerogenic 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.

14. A method for reducing an autoimmune reaction in a mammal diagnosed with a T-cell mediated or T-cell dependent autoimmune disease, the method comprising:
administering to said mammal a noninterferon polypeptide having Th2-enhancing cytokine activity in an amount effective to reduce at least one clinical or histological symptom of said disease.

15. The method of claim 14 comprising administering a polypeptide selected from the group consisting of IL-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 IL-4 administered via the oral or enteral route.