Note: Descriptions are shown in the official language in which they were submitted.
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
. . . , _ _ . . . _ _ _ _ , _ ,
21 87345
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
21 87345
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
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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
_ . _ .. . , .... , , . . . . _ _ _ ,
21 87345
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
11
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.
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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
13
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
14
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
16 O
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
17
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
2 1 87345
W095l27500 P~~ 01512
19
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.
WO 95.127500 ~ ! . 2 1 8 7 3 4 5 . ~I/.)~,51C Irl2
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WO 951~7~00 2 1 8 7 3 4 5 P~l/u~ ~4~1z
21
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WogsJ27~00 21 87345 F.IIIJ. 5'0l'l2
22 0
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
23
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
24 O
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
25
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
26 O
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
27
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
28 O
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
29
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
30 0
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
31
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
32
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
33
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
34
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
36
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
37
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
38 O
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.
