Note: Descriptions are shown in the official language in which they were submitted.
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COMBINED USE OF INTERLEUKIN-10 AND
CYCLOSPORIN FOR IMMUNOSU~ SION THERAPY
Field of the Invention
The invention relates to a method of su~pressi. ,g grafVtissue rejection,
graft-versus-host ~lise~-cie and autoimmune diseases. In particular, the
invention relates to the combined use of interleukin 10 and cyclosporin for
immunosu~",r~ssion therapy.
R.~ d of the l,~
Interleukin 10 (IL-10), a cytokine produced by T iymphocytes, was first
ider,li~ied by its ability to inhibit interferon gamma (IFN-~ and IL-2 synthesis
by mouse and human T Iymphocytes. riort, lli"o et al., 1989, J. Exp. Med.
170:2081-2089; Moore etal., 1990, Science248:1230-1252; Vieira etal.,
1991, Proc. Natl. Acad. Sci. USA 88: 1172-1177. IL-10 was subsequently
shown to be produced by B cells (O'Garra et al., 1990, Intemat Immunol.
2:821-828) and macrophages (riorelllillo etal., 1991, J. Immunol. 147:3815-
3822).
IL-10 exerts a wide range of effects on a variety of cell types. IL-10
inhibits the synthesis of a wide spectrum of cytokines produced by T cells and
monocytes. In addition to inhibiting the synthesis of IFN-~ and IL-2, IL-10 has
also been shown to inhibit production of the monokines IL-1 a, IL-1,B, IL-6 and
TNFa . de Waal etal., 1991, J. Exp. Med.174:1209-1217. IL-10 has growth
promoting effects on murine thymocytes and T cells (MacNeil et al., 1990,
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Immunol. 145:4167) and mast cells (Thompson-Snipes etal., 1991, J. Exp.Med.173:507-512), and it stimulates cytotoxic T-cell de~,elop",erlt (Chen and
Zlotnik, 1991, J. Immunol. 147:628-533).
Mouse and human IL-10 have high sequence similarity with a protein
encoded by an open reading frame in the Epstein-Barr Virus. The e~,.,ression
product of this open reading frame, named viral IL-10, also has the c~r~city to
inhibit cytokine synthesis. Moore et a/.,1990, .Sc;~nce 248:1230-1252; Vieira
etal., 1991, Proc. Natl. Acad. Sci. USA88:1172-1177.
Several cytokines, including IL-2, IFN-yand TNF-a, have been shown
to regulate the mixed Iymphocyte reaction (MLR). Shevach, 1985, Annu.
Rev. Immunol. 3:397; Fidelus et al., 1982, Transplantation 34:308; Tadmori et
al., 1985, J. Immunol. 134:4542-4550; Tadmori etal., 1986, J. Immunol.
136:1155-1162; Novelli etal., 1991,147:1445-1450; Landolfo ef al., 1985,
Science229:176-180; Shalaby etal., 1988, J. Immunol. 141:499-505. It has
been reported that IFN-~ may pay an important role in MLR graft rejection.
Novelli etal., 1991, J. Immunol. 147:1445-1450; Landolfo etaL, 1985,
Science 229:176-180. Antibodies to IFN-y orto TNF (Shalaby etaL,1988, J.
Immunol. 141:499-505) have been shown to block MLR-induced prolir~,~lion.
In these stllr~ies it was found that antibodies to IFN-~ su~,pr~ssed the MLR in
human systems as well as allograft reactivity in vitro and in vivo in the mouse.International ~pplic~ion Pul-lic~l;ol) No. WO 93/17698 discloses the
use of IL-10 to s- ~press tissue graft rejection. The use of both human IL-10
and viral IL-10 is desc-iL,ed.
Cyclosporin (also known as cyclosporin A; CSA), a cyclic peptide
produced by the fungus Tolypocl~ rn inflatum Gams and other fungi
i"lpe~re~;li, has cytokine il II.ibilio" ability. It has been found that inhibition of
IL-2 production by cyclosporin (Shevach, 1985, Annu. Rev. Immunol. 3:397;
Fidelus et al., 1982, Transplantation 34:308), or an antibody of CD2 (Tadmori
etal.,1985, J. Immunol.134:4542-4550) de,or~7sses T-cell prolir~rdlion
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induced bya MLR (Tadmori etal, 1986, J. Immunol. 136:1155-1162). CSA
suppresses in vivo and in vitro cell-mediated responses (Fidelus et al., 1982,
Transplai ,tation 34:308-311) and is currently being used in most organ
transplantation immunosu~,pr~ssive protocols. CSA p.~ ngs survival of
allogeneic l.d,-~ lants involving skin, heart kidney, pancreas, bone marrow
small i"te-~line and lung and is also known to su~press graft-versus-host
~~ise~se (GVHD) and delayed-type hype,~e"sili~ity. A problem with CSA,
however, is organ toxicity. High doses of CSA can cause profound and
irreversible neph-otoxicily as well as hep~toxicity and cardiotoxicily. There
thus exists a need for an immunosuppressant l,~dl",e-,l method that will allow
a.l~"i"i~l,dlion of lower levels of CSA, thereby reducing the toxic effects of this
agent.
S~ . y of the Invention
The current invention fills this need by providing such a method. More
particularly, this invention provides a method for su~ pressi. ~y tissue or organ
rejection cc~. ~.pri:.i"y administering an effective amount of interleukin 10 and
cyclosporin to a patient experiencing or at risk of tissue graft l~je~ tion.
This invention further provides a method for su~.press;. ~y graft-versus-
host ~~ise~se cGlllplisiny ad",i.,isLeri"g an effective amount of interleukin 10and cyclosporin to a patient a~ ted with or at risk for graft-versus-host
~ise~-se.
Still further this invention provides a method fortreating ~l~tci..,n,une
~lise~-ses co" " ri:,i- ,y administering an effective amount of interleukin 10 and
~ cyclosporin to a patient afflicted with an autoimmune ~ise~ce.
Pharm~ceutic~l CGIllposiliGl,s comprising a cGr, ~i.,dliGn of IL-10 and
CSA are also provided by this invention.
SlJ~STITlJT~ ~ R~L~ 26)
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Brief ~e s~L ;~ion of the C. .~ Figure
The accol"~al)ying drawing Figure .ler"G":jl,ates sy"ery;~ c
suppression of T cell prolireraliol) in MLR by a combination of human IL-10
and cyclosporin.
D~ e s ;,~tion of the l-.~r~.,li ~.,
All refer~nces cited herein are hereby incorporated in their entiretv by
,~ferel Ice.
A failure of major organs is a principal cause of ~ice~se and death in
mammals. Surgical replacement of a lli,eq-sed organ, by llansplantation with
a normal organ obtained from a,~olher ",a~"mal of the same sl~ecies, can be a
life saving procedure. Unfortunately, normal bodily immune defel Ise
mechanisms l ecoyl li~e such organ l, ~n~ lants as foreign and attack them,
resulting in graft failure and l~jectiGr). As such, a major impediment to
lla~ lanlaliG n of allogeneic tissues and organs is graft reje_liGI ~ by the
llalls~lanl recipient. The cell-mediated immune response of the reGi,~JiE.II, orhost, to the donor tissue plays an i~llpGIlanl role in the rejection process. This
response has two important phases: (i) recoy"iliol, of the donor cells or
tissue as ror~ , l" in the conle,~l of the major hi~locGm~ y complex
(MHC); and (ii) destruction of the foreign cells by the host cells. As part of this
process, a number of host cells undergo pr.l;~e,dlio,l and acquire c~loloxic;ly
-- that is, the ability to kill donor cells displaying the appropriate anliyeOs.Thus, cell-mediated immunity can be descriL,ed in temms of two measurable
full~;liGns: pr.l;~rdtion, and cytotoxic activity -- see Dubey etal., chapter 131
in Rose et a/., Editors, "Manual of Clinical La~oralory Immunology", 3d edition
(American Society of Mi~;l.Liology, Washington, D.C., 1986).
Dcv~ lo~l"enl of cell culture techniques has led to the e~ h")enl of
in vitro methods that mimic the in vivo immunization process, thus providing
SVBSTITUTE SHEET (RULE 26)
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measures forthe ~ssessl.,el~t of cell-mediated immunity in vitro. Of particular
utility in regard to l.~ plantation is the mixed Iymphocyte r~a.;lion (MLR), or
mixed Iymphocyte culture. R~sic~lly, MLR comprises co-culturing a sa"-ple of
responder cells and a sample of inactivated stimulator cells such that the
stimulator cells are allogeneic with respect to the respo, ..ler cells (i.e., the
stimulator cells are obtained from a different person from that from whom the
responder cells are taken), and measuring the proliferative response of the
responder cells. More specifically, MLR con~ ; of mixing responder
Iymphocytes (mimics host cells) in a s-~t-'-'P culture system with stimulator
Iymphocytes (mimics donor cells), the plulireralion and/or l-~,.scli~liol,
machinery of which has been disabled, e.g. by irradiation or treatment with a
DNA synthesis inhibitor (e.g., mitomycin C) or the like. The stimulator cells
are inactivated so that they can still carry out their stimulatory function but are
inhibited from any other functions that could obscure the ~es,c Gnse measured
from the responder cells, i.e., the stimulator cells are treated so that they are
incapable of r~plicalion, but their antigen processi. ,y machinery ,er ,ai- ,s
functional.
After the cells have been cultured for several days, a number of
dirr~r~,~l measurements can be made to quantify the degree of reactivity of
the responder cells to the stimulator cells. Usually, the response measured in
the responder cells is cellular proliferation. Proliferation of the responder cells
may be detemmined by the uptake of l,ilialed thymidine using standard
protocols. For example, from 2.5 - 10 x 104 stimulator cells are added to 2.4
x 104 allogeneic CD4+ (responder) cells in 96-well round-bottom tissue-
culture plates and are inc~ ~h~ted for 4 days in an a~,propriale medium. After
inc- ~h~tion~ the cells are pulsated with 1 ~LCi of l,ilia~ed thymidine for 6 hours,
and then they are harvested and measured for tritiated thymidine uptake, e.g.,
by sc;, llilldliGI~ counting.
SUBSTITUTE SHE~ ~RULE 26)
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It has unexpectedly been discovered that the combined/concurrent
admi, li~lrdliG~ I of IL-10 and CSA, or IL-10 and a CSA analogue, causes a
synery;~lic su~pression of T cell ,~rolir~raliGn. Surprisingly, this synergisticeffect is seen only when relatively low levels of these agents are used
together. While the invention will her~i. ,arler be discussed in terms of the
co",bi.,ed use of IL-10 and CSA, it is to be u"der~lood that an analogue of
CSA may also be cG,.lL:.,ed with IL-10 to cause synergistic su~pressio" of T
cell prolir~raliol1, and that such combinations are cG"Ie",plated for use in thepractice of this invention.
The cGm~i.,slion of IL-10 and CSA can advantageously be used in the
suppressicin of pathology ~-~soci~ted with T cell responses, in particular,
autoimmune ~i~e~ses, graft-versus-host fiise~ce (GVHD) and tissue graft
rejection. The invention can be used to su~,pr~ss cell-mediated .eacliGns
such as allograft rejection and GVHD. Moreover, cGn~i~le~i"g the diverse
biological activities of IL-10, the concurrent use of IL-10 and CSA may support
GVL (graft-versus-leukemia) in allogeneic bone marrow l,~nspla.lts.
The invention may be used to prevent the l~jeclion or prolong the
survival of allogeneic ~ns~-lanl~ of skin, heart, kidney, pancleas, bone
marrow, small i"le~li"e, lung, etc.; to treat ~l~tc:.",~,une ~ise~-ses such as, for
exa,nple, rheumatoid arthritis, lupus, diabetes mellitus, multiple s~'erosis andmyasthenia gravis; and to treat other ~ise~ces where CSA has been used,
such as psoriasis. Due to the activity of IL-10, CSA can by used in lower
amounts, thereby avoiding or reducing the serious side effects normally
~csoc~ e~l with the use of this drug.
Tra"s~,la"l .t:cipients may be recipients of kidney, liver, heart, heart-
lung, bone marrow, cornea transplant, etc. The tran~ lanled tissue itself is
typically human in origin but may also be from another species such as a
rhesus monkey, baboon or pig. As used herein, the term "tissue" includes
individual cells, such as blood cells, including prog6rlil0rs and precursors
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thereof, and pa, Icr~3alic cells, as well as solid organs and the like. The termsolid organ means a heart, skin, a liver, a lung, a kidney, a pancreas, an
i"~e:ili"e, endocrine glands, a bladder, skeletal m~scles, etc.
The methods of the invention can be used prophylactically or for
treatment of established autoimmune ~ise~se, GVHD or graft rejection.
Individuals suitable for treatment by the methods of the invention include any
individual at risk (predisposed) for dcvel~ . ,9 GVHD or tissue rejection, i.e., a
tra,~splalll patient, or an individual exhibiting clinical sy""~l~",s. Prophylactic
use el ,cG"~p~cses admini~ liGn prior to transplantation as well as post-
,lanlaliol1 admini~t,~lion in the absence of any clinical sy""~l~",s ofGVHD or graft l_jectio", to prevent or postpone onset of ~lise~se/r~;e~lion.
In the practice of the invention, IL-10 and CSA are to be "concurrently"
administered to a patient. Concurrently admil,i~l~rillg means the IL-10 and
CSA are administered to the subject either (a) simultaneously in time
(optionally by formulating the two together in a common carrier), or (b) at
diirrelenl times during the course of a co"""on l,cal",e"l schedule. In the
latter case, the two compounds are administered s--rricienlly close in time to
achieve the i"lencled effect. Typically, if one agent is admini~ler~d within
about the half-life of the first agent, the two agents are considered to be
concurrently admir,i~,~erl:d. The active agents may be ad~"i"islered together
in a single pharm~ce~tic~l composition or separately. Both active agents (i.e.,
IklO and CSA) should be present in the patient at sur o ~I co,-,L,i"ed levels
to be therapeutically effective. The routes of admini;,ll~liGI) of the IL-10 andCSA may be the same or dirrer~,.l.
Generally, IL-10 and CSA are administered as a pharm~ce~ ~tic~
cGI~"~osition cGr"p-i~;,-y an effective amount of IL-10 and CSA in a
pharmaceutical carrier. A phaml~ceutic~l carrier can be any compatible, non-
toxic s~ -ce suit~hle for delivering the composilions of the invention to a
patient.
SUBSTITUTE SHEET (RU~E 26)
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As used herein, interleukin 10 or IL-10 is defined as a protein which (a)
has an amino acid sequence s~ ,lially ide"lical to a known sequence of
mature (i.e., lacking a sec,~lory leader sequence) IL-10 as ~ closed in
l"le,.,dlional ~pplicAtion Pl~l~liGhlion No. 91/003249, and (b) has biological
activity that is con"~on to native IL-10. For the pUI ~.oses of this invention,
both glycosylated (e.g., produced in eukaryotic cells such as yeast or CHO
cells) and unglycosylated (e.g., cl,el" --lly sy"ll,esi ed or pro~ ce~ in E. colIL-10 are equivalent and can be used interchar,yeably Also included are
muteins and other analogs, including viral IL-10, which retain the biological
activity of IL-10.
IL-10 suitable for use in the invention can be obtained from a number
of sources. For e,~ar"ple, it can be isoldl~.l from culture media of activated
T-cells C~p~hlQ of secr~ling the protein. Additionally, the IL-10 or active
fray"~enl~ thereof can be chemically synthesized using standard techniques
known in the art. See, e.g., Mer,i~ielcl, 1986, Science 233:341-347 and
All ,e, l.", et al., Solid Phase Peptide SJ~"Il,esls, A Practical A~ ruach, 1989,
IRL Press, Oxford.
Preferably, the protein or polyl,e~lide is obtained by recombinant
tecl " ,i~ues using isol-ted nucleic acids encoding the IL-10 polypeptide.
General methods of molec~ r biology are cJesc, iL.ed, e.g., by Sa" ,br~,olc ef
al., 1989, Male~ rCloning, A Lal~ordlunyManual,, 2d Ed., Cold Spring
Harbor, New York and Ausubel et a/. (eds). Current P~,locols in Mol~c~ r
Biology, GreenNViley, New York (1987 and periodic snl.plQI~enl~). The
appropridle sequences can be obtained using standard tecl-"i~1ues from
either gencJI~ic or cDNA libraries. DNA constructs encoding IL-10 may also
be prepared synthetically by est~hlished standard methods, e.g., in an
automatic DNA synthesizer, and then purified, ar"~ealed, ligated and cloned in
SUlt::~hlQ vectors. Atherton et a/., 1989. Polymerase chain reaction (PCR)
SVBSTITUTE SHEET (.~ULE 26~
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techniques can be used. See e.g., PCR rl ~locols: A Guide to Mefhods and
Ar plicAtions~ 1990, Innis et al . (ed.), Academic Press, New York.
The DNA constructs may contain the entire native sequence of IL-10 or
a homoloyue thereof. The term "homologue" is intended to indicate a natural
variant of the DNA sequence encoding IL-10 or a variant or fragment
pro~h~ced by moc3i~icaliG" of the DNA sequence. Examples of suitable
mc~di~icaliGns of the DNA sequence are nucleotide sl~hstitutions which do not
give rise to another amino acid sequence or nl~c~o~tirle sllhstitlltions which do
give rise to a different amino acid sequence and therefore, possibly, a
di~r~nl protein structure. Other exa"" les of possible mo~i;ric~lio"s are
insertions of one or several nucleotides into the sequence, addition of one or
several nucleolides at either end of the sequence, or deletion of one or
several nucle~lides at either end or within the sequence. Any hG1ll0l;)9OUS
DNA sequence encoding a protein which exhibits IL-10 activity (e.g., with
respect suppr~ssion of T cell proliferation) similar to that of the naive protein
is cG"Ie""~lated for use in the claimed invention.
The nu 'sotirle sequences used to t,an:jr~ct the host cells can be
l"oJi~ied, as ~lescribecl above, to yield IL-10 muteins and fraylllellls with a
variety of desired properties. Such modified IL-10 can vary from the naturally-
occurring sequence at the primary level, e.g., by amino acid insertions,
sl~hstitutions, ~leliolls and fusions. Preferably, amino acid sllhstitutions will
be conservative; i.e., basic amino acid residues will be repl~~ed with other
basic amino acid residues, etc. These ,,,G.i;ric~liolls can be used in a
number of combinations to produce the final modified protein chain.
Amino acid sequence variants can be prepared with various objectives
in mind, including increasing serum half-life, f~r~ t~ y puli~icaliGIl or
preparation, improving therapeutic efficacy, and lesseni"g the severity or
occurrence of side effects during therapeutic use. The amino acid sequence
variants are usually predetermined variants not found in nature, although
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others may be post-translational variants, e.g., glycosylation variants or
proteins which are conjugated to polyethylene glycol (PEG), etc. Such
variants can be used in this invention as long as they retain the biological
activity of IL-10.
Preferably, human IL-10 is used for the treatment of humans, aithough
viral or mouse IL-10, or IL-10 from some otheml~ammalian species, could be
used instead. Most preferably, the IL-10 used is recombinant human IL-10.
Reco.n' .,a..l production of human IL-10 is descriLed in U.S. Patent No.
5,231,012. Preparation of human and mouse IL-10 has been desc.ibed in
Intemational Applicalion Publication No. WO 91/00349. The cloning and
ex~.r~ssiol, of viral IL-10 (BCRFI protein) from Epstein Barr virus has been
disclosed by Moore et al. (Science 248:1230,1990), and is clesc-ibed in EP 0
506 836.
CSA may be is administered in a manner as is conventionally
practiced. See, e.g., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 7th Ed,1985, p.1299. For example, CSA may be provided as
an oral solution of 100 mg/ml with 12.5% alcohol, and for intravenous
admini~ iol, as a solution of 50 mg/ml with 33% alcohol and 650 mg of
polyoxyell,lated castor oil. When administered intravenously, CSA may be
given as a dilute solution of 50 mg to 20 - 100 mg of normal saline or 5 %
de,xl,ose in water, by slow infusion over a period of several hours. The
intravenous dose is typically one third of the oral dose. Most p, er~raL,ly,
ad~..i..i~l.~liGn of CSA is orally, eitherin c~pslJ'e ortabletfomm. Such
formulations may be prepared by any suitable method of pharmacy which
includes the step of bringing into ~ssoci~tion the active compound and a
suitable carrier (which may contain one or more ~ccessory i. ~yl edie. ~ls). In
general, the formulations can be prepared by uniformly and intimately
ad~"i~i"y the active compound with a liquid or finely divided solid carrier, or
both, and then, if necessary, shaping the resulting mixture. For example, a
~UBS~ T~ ~HE~T ~IJL~ 26)
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tablet may be prepared by compr~ssi"g or molding a powder or granules
containing the active co",pound, o~.liol ,ally with one or more ~Gcessory
ingredients. Co" ,pressed tablets may be prepared by co" ~~,re~si. ,g, in a
suitable machine, the cGr"pound in a free-flowing fomm, such as a powder or
granules containing the active compound, optionally mixed with a binder,
lubricant, inert diluent, and/or surface active dispersing agent(s). Molded
tablets may be made by molding, in a slJi~hlQ machine, the powder~3d
G,..pound mGi~lened with an inert liquid binder. The preparation of CSA is
sclQsed in U.S. Patent No. 4,117,118. CSA which may be used in the
practice of the invention is cor"" ,ercially available under the name
SANDlMMUNE(g) from Sandoz Pham~ceutic~ls Col~,Grdlion.
Synergistic suppr~ssion of T cell proliferation may also be observed
using IL-10 and an analogue of CSA. As used herein, a "CSA analogue" is
meant to include synthetic analogues as well as any agent which exhibits the
same activity/mechanism of action as CSA. Such agents include, for
example, FK-506. FK-506 is a macrolide immunosuppressant isolated from
Stre,~ ces tsukubaenis no. 9993. EP 0 184 162 (Fujisawa).
Adl.,i"i~ lion of IL-10 is preferably parenteral by intraperitoneal
intravenous, s'~hc~t~neous or intramuscl~r i"je ~;o" of infusion or by an
other ~ccel,l~hle systemic method. Admini~ liol, by intramuscuhr or
subcutaneous injection is most pr~re,-~d. All~,.,ali-/ely, the IL-10 may be
admir.i~lered by an inplantable or injectable drug delivery system. See, e.g.,
Urquhart etal, 1984 Ann Rev. Pharacol. Toxicol24:199; Lewis, ed.,1981,
Controlled Release of Pesticides and Pharmaceuticals, Plenum Press, New
York, New York: U.S. Patent Nos. 3,773,919, and 3,270,960. Oral
adl..i,.isl.aliG" may also be carried out, using well known formulations which
protect the IL-10 from gac;l-ui. .le~li- .al ~r ,teases.
Cor"posilions useful for parenteral a.ll"i.,i;~ lion of such drugs are
well known. See, e.g., Remington's Pharmaceutical Science,11th Ed.,1990,
11
SU13STlTUTE SH~F~ (RllLE 2~
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Mack Publishing Co., Easton, PA. When administered parenterally, the IL-10
is typically formulated in a unit ~Jos~ge injectable form (solution, suspension,emulsion) in association with a phamm~ceutic~l carrier. Examples of such
carriers are nommal saline, Ringer's solution, dexl-use solution, and Hank's
solution. Non-aqueous carriers such as fixed oils and ethyl oleate may also
be used. A preferred carrier is 5% d~lluse/saline. The carrier may conl~il)
minor amounts of additives such as sub~lances that enl-arce isotonicity and
chemical stability, e.g., buffers and preservatives. The IL-10 is l,r~rerably
formulated in purified fomm s~hstPntially free of agy~eyales and other source
proteins at a CGI .ce"l.~lion in the range of about 5 to 20 llg/ml. Any of the
well known carrier protei. .s such as human serum albumin can also be added
if desired.
IL-10 can also be delivered by standard gene therapy tecl-ni~ es,
including e.g., direct DNA i.,jeclior into tisslJes, the use of r~cGIlll~ .lant viral
vectors or ,.~hos,uholipid and i.."~lanl~liol) of l.~ rected cells. See, e.g.,
Rose..bery~ 1992, J. Clin. Oncol. 10:180.
IL-10 and CSA are concurrently admir,;;,lert:~l to a human patient in an
amount effective to provide an immunosuppressive effect. As used herein
"effective amount" means an amount sufficient to reduce or prevent GVHD,
an aulci...mune .J;se~e or tissue lejec;liorl, and refers to the co.. ' i -ed effects
of the two agents working in co, .ce- l. One or both agents may, for e~dmple,
be used at a dose which, if used alone, would be considered suLopli.--al for
the i. .le. ~de~l purpose. The effective amount for a particular patient may vary
depending on such factors as the state, type, and amount of tissue
transplanted, the overall health and age of the patient, the route of
adl"i"i;,l.~.lion, the severity of observed side~effects, and the like. The
effective dose of IL-10 typically will range from about 0.1-2~ ,ug/kg/day,
preferably about 1-16 ~Lg/kg/day. The effective dose of CSA typically will
range of from about 1-14 mg/kg/day, more pr~r~rdbly from about 1-8
12
SU~SmU~ULE
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mg/kg/day. Preferably, ad",i"i~t.~lion is to begin simultaneously with
tral,spla, ~l~liol ~, or 2 to 4 hours before transplantation. Admi, l6lldliGI, may,
however, begin within the 24-hour period preceding transplantation or within
the 24-hour period following t,~n~pla"l~lion. It is also cGnte~",ulated that
admi, Yi~ lion can be started at any time after transplantation to r~ lace or
s~ le ,lel~L other compounds being administered to a patient to prevent graft
tejeclion. The length of admini~ " may vary and, in some cases, may
continue over the remaining lifetime of a patient, to control graft rejection
processes.
EXAMPLES
This invention can be illustrated by the following non-limiting examples:
General Materials and Methods
In the experiments described below, the rcll~w;.,~ protocols and
procedures were ~,211~vlcd:
Media
Cells were prepared and cultured in RPMI 1640 (JRH Biosciences,
Lenexa, KS), s~rpl~mented with 10% fetal calf serum (Hazleton Biologics,
Inc., Lenexa, KS), 2mM L-glutamine (JRH ''iosciel ,ces, Lenexa, KS), 80 ~lgml
yenldlllic;~l (Sigma Chemical Co., St. Louis, MO) or 100 u/ml penicillin-
sl-t:~.Lomycin (JRH r'iosciQnces, Lenexa, KS).
ELISA
ELISA kits for cytokine production detemmination were purchased from
R&D systems, Minne~polis, MN.
Human PBMC and Monocyte PLII;r;C~l;On
Human blood samples were coll~-cte~l in heparinized vacutainers. To
f~ri'it~te removal of red blood cells (RBC), 2 ml of 6% dextran in pl,osphate
buffered saline (PBS) (JRH Biosciences, Lenexa, KS) was added to each
vacutainer, mixed and allowed to stand at room te,.,perdlure for 30-45 min.
The top buffy coat layer was carefully removed and cells centrifuged (300 x 9,
13
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10 min., 4 ~C). The cells were washed 3 times in 15 ml PBS. The peripheral
blood mononuclear cells (PBMC) were isolated using a FICOLL-PAQUE
(Pharmacia, Piscalav,tay, NJ) gradient. Ten ml of cells were layered on top of
4 ml FICOLL-PAQUE, centrifuged (1400 x 9, 20 min., room te"~erdlure) and
the cells sedimenting at the interface were collected. These PBMC were
washed 3 times in PBS, enumerated and resuspended in ap~.ropri~le media
for use in MLR experiments. In some of the expe,i",en~s peripheral blood
monocytes were prepared by incubating PBMC in medium s~F~len,ented with
10% fetal bovine serum (FBS) and allowing adherence for 1 hour and 37 ~C in
5% CO2 atmosphere in T-75 flasks each containing 107 PBMC. After
removing the nonadherent cells, the flasks were extensively washed with
warm medium, then incl~h~ted with cold PBS on ice for 15 minutes. Adherent
monocytes were s~hseq~ ~ently recovered by rer)e~t~d pipetting, washed and
resuspended in co""~lete medium. Cell purity determined by staining with
CD14 mGnoclonal antibodies and flow microfluo,ul"el,ic (FMF) analysis was
92% CD14+. Viability determined by trypan blue PYcl~sion was >95%.
Mixed Lymphocyte Reaction
The stimulator PBMC were treated with 50 ~11 mitomycin C (Sigma
Chemical Co., St. Louis, MO) for 20 minutes at 37 ~C. The responder PBMC
and the stimulator cells were added to a 96-welH"ic,uliler plate (Becton
D jCk;nSGI " Lincoln Park, NJ) at 1 x 105 cells per well of each, along with
cytokines or a~ ,lil-od,es in a total volume of 200 ~LI in triplets. The cultures
were inc~ ~h~ted at 37 ~C with 5 % CO2 in air for 6 days. The cultures were
then pulsed with 1 ~Ci [3H]TdR (1 5.6CI/mmol, NEN, Boston, MA) per well for
16 hours. The cells were harvested onto a filter using a ~C wcll cell harvestor
(Skatron, Inc., Sterling, VA) and counted on a beta counter (Pharrrlacia LKB
Nuclear Inc., Gaithersburg, MD).
14
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W O 97/05896 PCT~US96/12538
Immunofluor~scence and Flow Cytometry
The su~ e" ~ald, ll from the MLR was removed and the non-adherent
PBMC harvested. The a-ll ,6r~nl cells were harvested by incl ~h~tion with 5
mM EDTA at 4 ~C for 20 minutes and gently scraped. Cells were combined,
centrifuged at 300 x g for 10 minutes and washed 3 times in ice-cold PBS.
The viable cells were enumerated by Trypan Blue exc'~-~ion using a
Ne~h~eur counting chamber, resu.spended to 1 x 107 celUml in PBS and 100
LI of human IgG (1 mg/mi) was added to each well and inc~h~ted on ice.
Thirty minutes later, 100 ~Uwell FBS was added and the plate centrifuged at
500 x 9 for 10 minutes. The su,u6",alant was removed and the cells were
resuspended in the appropriate amounts of mouse-anti-human monoclonal
antibodies coupled to FITC. For dual-stained samples, cells were
concurrently incllh~ted with 20 ,ul of mouse-anti-human monoclonal antibodies
to each surface marker. All volumes were made up to 40 ~11 with PBS.
Isotypic control a"libodies were used at the same c~"ce, It~ as the
specific markers. Samples were incl ~b~te~l for 30 minutes at 4 ~C, FBS was
added (100 ~ll/well), and the samples were centrifuged as previously
descl il,ed. Cells were washed 3 times in 200 ,~LI A~ uot-s of PBS, CGI ll~il lil l9
50% FBS, and resuspended in 1 ml of PBS containing 0.01% sodium azide.
Flow cytometric analysis was performed using a FACScan flow cytometer
(Becton Dichil.sc~ll Immunocytometry Systems (BDIS), San Jose, CA) and
viable cells gated, based upon prupi~;Jm iodide excl-lsion. Mean channel
fluGr~sc6l .ce and percent positives for specific markers were cJeter",i. ,ed
using LYSIS ll software (BDIS). For purifying B cells and l~-GnoCytes, PBMC
were respectively stained with CD20 or CD14 and sorted by positive
s~le~-liol1. Purity of the sorted cells ranged between 95 and 98% in three
experiments.
~;UBSTITUT~ SHE~ ULE 26~
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Depletion of Antigen P1~se"li-,a Cells from Human PBMC
Purified PBMC from human blood were collec;l~d in heparinated tubes
by FICOLL gradient. The blood was diluted 1:1 with PBS. 30 ml of the
diluted blood was laid on top of 20 ml Ficoll in a 50 ml conical tube and spun
at 2,000 rpm for 30 minutes. The interface cells were collsc: d and washed 3
times with medium (10% FCS/RPMI).
The plastic adl ,er~"l a, lliyel) pr~se, llir ,9 cells (APC) were removed by
resu~ el,ded PBMC in 1% human serum (Type AB) at 1X106 cells/l"l and
inc~lh~ted at 37 ~C for 30 min. The cells which floated were coll _~d and
placed in media (10% FCS/RPMI).
Nylon wool adherent APC were removed as follows:
A 10 ml syringe was packed with 1.5 g of nylon wool (Poly~ciences
Inc., Cat. # 18369) and ~otocl~ved. A 3-way ~lol,cock and a 22-gauge needle
were attached to the syringe, and the column was washed with 50 ml of
prewarmed medium. A cell suspen:,iol, of 5x107 cells/ml of plastic adherent
APC was prepared in prewammed medium. Just before adding the cell
suspension, the wool was rinsed with 5 ml of pre ~vdr",ed medium the column
was left to run dry, and the sl-.pcoGk was closed. Cells (5-10x107) were
added. The cells were allowed to peu~ le the column, and the s~ cock
was closed. An ~ Gl ,al 0.5 ml of medium was added and the column
maintained at 37 ~C for45 minutes. Nonadl,er~nt cells were colle tecl into a
tube by washing the column with 20 ml of prewammed medium, eluted at 1
drop per second.
Demon~ liG" of Immunosu~,pr2ssive Effects
A major property of IL-10 is the ability to inhibit cell-" ,e- l; led immunity
by blocking production of cytokines of the Th 1 type (IL-2, IFN) and
."onocytes (TNF-a). Since these cytokines are known to be the major
regulators of graft acceplar,ce and graft versus host ~ise~-ce (GVHD), the
Sl 3E~STITUTE SHEE~ ~ULE 26)
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effect of human IL-10 (hlL-10) on the allo$~eneic stimulation of T cells was
invesliy,~lPd
IL-10 Su,upresses MLR-lnduced Proliferation
To ~-~sess the effect of IL-10 on T-cell pr~,l;f~r~lion induced by
alloantigen, the effect of IL-10 on the proliferative response evoked in the
primarv one-way MLR, where peripheral blood mononuclear cells (PBMC)
from one donor were co-cultured with mitomycin-treated PBMC of an
unrelated donor with and without IL-10 was examined.
In 20 independent experi,~e~ using PBMC from 16 unrelated donors,
IL-10 (100-200 U/ml) strongly su~ pr~ssed MLR-induced proliferation. In
these studies, the allogeneic stimulation induced a strong proliferative
response and the su~pr~ssio" by IL-10 ranged between 65% and 100%
regardless of the stimulation index of the MLR. Table 1 shows that a
monoclonal antibody to IL-10 neul,~ ed IL-1 0-induced su~",ressio,- of MLR.
The s~".pr~:ssion observed in these cultures was attrihnt~le to IL-10, since
the ~d~lition of ne~ J monoclonal antibody, but not its isotypic control,
reversed the IL-10 sl",pr~ssive effect .
TABLE 1
Culturea CPM (mean i SD)b
MLR 5127 i 935
MLR + IL-10 643 i 197
MLR + IL-10 + 19B1 5035 i 131
MLR + IL-10 + rat.lgG1 486 i 14427
aMLR cultures were set up as desc,il.ed above. Human IL-10
(100U/ml) 19B1 (a rat ~ ~onochnal antibody to IL-10) and its isotypic
control rat IgG1 were used at 2.5 U g/ml.
bData are 3HTdR uptake M i SD of t,, d~t~r" ,a~ion and are
,erl~dli~/e of S ~A~ h.
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Inability of IL-10 to inhibit prc,l;h~r~lion in MLR induced by B cell lines is
associatec~ with lack of inhibition of TNF-~c production
Earlier inve~liydlion of the mechanism of action of IL-10 revealed that it
suppresses activation of human T-cell clones induced by specific soluble
antigens presented on normal monocytes but not when the antigens were
pr~se"lad by EBV-LCL B-cell lines. de Waal et al.., 1991, J. Exp. Med.
174:915-924. In these studies it was not clear wl~U-er the inability of IL-10 tosuppress T-cell activation when EBV-LCLs were used was due to the fact
they were B cells or to the pos~i' .i'~{y that these cells rep.t:se"led a dirl~r~
subpopulation, and/or that they were EBV-transformed B cells. To address
this question further, the ability of IL-10 to s-""),~ss the proliferation in MLR
when allogeneic PBMC, purified B cells, monocytes or B cell lines were used
as stimulators was studied. Table 2 shows that IL-10 suppresses ,or~!;~rdlion
in MLR when normal B cells, but not B-cell lines, are used as stimulators.
TABLE 2
Addition to MLRa- CPM (MiSD)b
MLR Medium L-10 CSA
PBMC+B 3939 +100 182 i 47 ND
PBMC+JY 26222 + 6293 25182 + 7302 7144 i 263
PBMC+Daudi 37357 _ 4497 4~763 i 3089 14167 i 2762
a MLR cultures were set up between PBMC (lxlO5/well) and mitomycine treated B cell line
(lxlO5/well) or 4 x 104) purified B cells, po~itiiely selected CD20+ by FMF, as deso,ibed above.
Human IL-10 was used at 200 U/ml and C~-,iO-~ipGIill A 40 ng/ml.
b Data are (3)HTdR uptake M ~ SD of 1-, ' dt,l~r-- -alion and are ~ enldli-/e of 3
r~ "l:,.
ND = not done
As shown in Table 2, IL-10 strongly suppresses the MLR-induced
p~ lion when purified normal B cells (985% CD20+) or monocytes (98%
CD14+) were used as stimulators and PBMC as responder celis. However,
18
SUB~I~TE SHE~ (RUL~ 26~
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W O 97/05896 PCTAJS96/12538
IL-10 did not su~,pr~ss the reaction when B cell lines (Daudi or JY), were uses
as stimulators of the MLR. Higher doses of IL-10 up to 1000 U/ml did not
SU~ SS JY-induced MLR. These data suggest that the inability of Ik10 to
suppress MLR induced by B-cell lines is not attributable to the fact that they
are B cells but rather sucJgest the possibility that these cell lines may stimulate
MLR via dirrer~nl mecha";s",(s) from that used by norrnal B cells.
As can be seen in Table 2, CSA inhibited prolire,dliol, in the
JY-induced MLR.
To invesli~le the mechanism by which B cell line-induced MLR resists
suppression by IL-10, the effect of IL-10 on cytokine s~" nh esis in this MLR
was e~d".i. ,ed. The presence of IL-2, IFN~y and TNF-a in su~ e" ,dl~"ts of
MLR set up with and without IL-10 (200 U/ml) for 60 hours was d~l~"~;.,ed
using ELISA kits. Table 3 shows r~:~,nase,,taliv-e data from two ex~.eri",er"--n
TABLE 3
Cytokine conce, lbdlion (pg/ml)b
MLRa (R+ S) IFN-~ IL-2 TNF-o~
PBMC JY 5715 3006 154
PBMC + JY + IL-10 2960 1294 209
PBMC +JY+ CSA 509 720 84
PBMC + PBMC 200 120 c25
PBMC + PBMC + IL-10 c25 c50 c25
a The MLR cultures were set up as desclil,ed above using PBMC (1 x 106/ml) as
~e:.~,or,-l~r and mitomycin C-treated JY cells (5 x 105 cell/) or " - ~f ~eic PBMC ( 5 x 105)
as stimulators in a 24-well plate for 60 hours. IL-10 was used at 100 U/ml and CSA at 40
nglml.
bData are the means of l~i, ' dt:lelll - ,dlion (SD C15%) of cytokine conce~ dliol,s
d ~ ~ in the sl",er-ldldr,ts of these cultures. Results are l~r~sel,ldli~e of 2 or 3
t:A"er" "ents. Detection limits of the co"" "e,~.idl ELISA kits were 25 pglml for IFN-~ and
TNF-oc, and 50 pg/ml for IL-2.
~.
Table 3 shows that high levels of cytokines were clele~l~ le in the
supernatants of MLR induced by JY. In particular, this data shows that levels
of IL-2 and IFN-y but not that of TNF-a, are depr~ssed in the supernatants of
MLR set up with IL-10. In contrast, CSA inhibited TNF-a production.
19
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CA 02228379 1998-01-30
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Since supematants of cultures of JY cells alone (l,ealed or u"ll~dted
with mitomycin C) did not contai.. ~4ter,t~hle TNF-a, the source of TNF-a in
these MLRs could be attributed to the activated .~ .G.,der PBMC. Together,
the data su~gest that the inability of IL-10 to inhibit prolifel dLiol I in MLR
between PM BC and JY may be due to the inability of IL-10 to inhibit TNF-a
synthesis, which allow this cytokine to sy.,eryi~e with the resid~lAl lL-2 in
these cultures and to overcome the IL-10-induced su~ ssio". This
conclusion is supported by the finding that CSA which, unlike IL-10, inhibits
pl.li~r~liol~ in the JY-induced MLR (Table 2), also inhibited TNF-a production
in MLR (Table 3). The ability of CSA, but not IL-10 to s~ n2ss pr l;'a.~lion in
the B-cell line-induced MLR, indicates a distinct n~eché~ .l, of action by IL-10verses CSA on alloge..eic immune rl3s,l~0l)ses.
Comparison of the rolel .~;y of IL-1 Q and CSA
Since CSA also has a cytokine-sy..ll,esis inhibition ability and currently
being used in most immunosu~rt:ssive pr~locols in organ tldnsplanlaliol),
the activity of CSA was cGl "par~d to that of IL-10.
Using an in vitro eA~ e,i",ental model for allostimulation, the one-way
mixed Iy""ul)G~yte reaction (MLR), hlL-10 was found to be a more potent
i, lhil,i~or than CSA (IC50: 8pM and 4nM respec~ively). Moreover, it was found
that the ~d~ition of combi l~liol ,s of hlL-10 and CSA at low doses caused
synergistic supp~ssiGn of T cell ,~rulirer~liGl, in MLR.
As can be seen in the Figure, the ~d~ition of comb ,aliol ,s of hlL-10
and CSA at certain doses (IL-10 and CSA: 0.5-40 r~ic~"olar of each) to MLR
cultures caused syne,y;;,lic su~ ,r~ssiol, of T cell prc,l;fer~liGI ~ in MLR. Athigher concel Ill~lions of each agent (360 pM) the synergy was lost.
Sy"er~;~lic su~,pr~ssio,) between IL-10 and CSA has been observed in in vivo
studies.
IL-10 and CSA sy~ler~J;-;iCAlly inhibited T cell activation in MLR. This
synergy was observed at lower doses of hlL-10 and CSA. From this data it
SU~TlTl~E SHEE~ 2~)
CA 02228379 1998-01-30
WO 97/05896 PCT~US96/12538
can be observed that the ad~ lrdliol) of a cGIllbilldlion of IL-10 and CSA is
a more effective suppressive therapy than either IL-10 or CSA individually.
Effect of IL-10 and CSA on Direct and Indirect Allostimulation
Allostimulation of T cell can be exerted via two palh~rdys; direct
allostimulation and indirect allostimulation. As used herein, "direct
allostimulation" means the respol-se of the recipient T cell to alloanlige"s as
intact MHC r~olee~les on the surface of ~l'.,yeneic stimulator cells in the graft.
Direct allostimulation is the cause of acute graft reieuliGn and is the p, i"~ ,al
cor.l.ibutor to dl ILiy~d~l cytotoxic T cell r~SpGrlSe Illed;dlil ~y early leje~,tiGn
epi~odes. "Indirect allostimulation," as used herein means the response of
the recipient T cell graft major l.islocolnpatibility (MHC) alloanli-Jens that have
been prucess6d and presenled by the recipients' APC. Indirect
allostimulation the cause of ch,unic allograft rt:jeuliGn and antigraft anliLodyproduction, xenoyldrl rejection, and induction of unr~,wl,siveness
(tolerance).
The effect of hlL-10 and CSA on these two ~dlll~l~ys of allostimulation
was studied. Direct allostimulation which results from the il ll~ liGn of the T
cell with allo MHC ",clec~'es on the stimulator cells is thought to be
respGnsiLla for the early acute graft rejection. Indirect allostimulation which
results from T cell i"lerd-;liGrl with shed alloantigen of the stimulator cells
pr~senled by the recipient (APC) is thought to be ,~ onsiL)le for the late graftr~3jEcliol ,. To examine the effect of IL-10 and CSA on the direct
allostimulation, a one way primary MLR was set up in which PBMCs of the
respo".ler were ciepletecl of APC's and cultured with PBMCs of the stimulator.
Addition of IL-10 to these cultures c~nsecl more su~pr~ssio" than CSA.
Similar results were obtained when CD4+ or CD8+ cells were used as
responders in these cultures.
21
SUBSTITUTE SHEET (~ULE ~6
CA 02228379 1998-01-30
W O 97/05896 PCT~US96/12538
Forthe indirect allo~li"~ulation, the APC's were dsFI~: d from the
stimulator popl ll~tion and cultured with PBMCs of the responder hlL-10 was
also sllul ,!Jer inhibitor to the indirect allostimulation than CSA.
Human IL-10 was found to be more effective than CSA in SUppl~355;~ ~y
both the direct allostimulation and the indirect allostimulation of T cells. Since
hlL-10 su~ pr~sses both the direct and the indirect T cell allostimulation, a
comLi. ,alio" of IL-10 and CSA can be used to prevent acute graft ~eje~,tiGn
(,ne.J; ~19d by the direct allostimulation), and c;hr~ni;: graft and xe, log,~tl~jedcliGI) (",ed;dted by the indirect ~o ,li"-ulation).
Many modi~icdlior,s and va~i~liGns of this invention can be made
without departing from its spirit and scope, as will be apparent to those skilled
in the art. The speciric embodiments descriL ed herein are offered by way of
example only, and the invention is to be limited only by the terrns of the
ap~.e".Jed claims, along with the full scope of equivalents to which such
claims are entitled.
22
SUBSTITUTE SHEE~ (RULE 26)
