Method and compositions for suppressing allograft rejection in mammals

Disclosed herein are methods for suppressing allograft rejection in mammals comprising administering to a mammal about to undergo or having undergone allograft surgery an agent selected from the group consisting of splenic tissue from an allograft donor, splenic extracts, cultured lymphocytes from an allograft donor, extracts of said cultured lymphocytes, MHC antigens, transplantation rejection suppressive fragments and analogs of MHC antigens in an oral or aerosol form. Also disclosed herein are pharmaceutical formulations and dosage forms for use in said methods.

FIELD OF THE INVENTION 
This invention relates to methods and compositions for suppressing the 
immune response in animals. More particularly, but not by way of 
limitation, the present invention is directed to pharmaceutical 
formulations and methods for suppressing and controlling the immune 
response of mammals against the introduction of foreign tissue. The 
invention also includes methods for prolonging the survival of 
transplanted organs and tissues. 
BACKGROUND OF THE INVENTION 
The success of surgical transplantation of organs and tissue is largely 
dependent on the ability of the clinician to modulate the immune response 
of the transplant recipient. Specifically the immunological response 
directed against the transplanted foreign tissue must be controlled if the 
tissue is to survive and function. Currently, skin, kidney, liver, 
pancreas and heart are the major organs or tissues with which allogeneic 
transplantations are performed. It has long been known that the normally 
functioning immune system of the transplant recipient recognizes the 
transplanted organ as "non-self" tissue and thereafter mounts an immune 
response to the presence of the transplanted organ. Left unchecked, the 
immune response will generate a plurality of cells and proteins that will 
ultimately result in the loss of biological functioning or the death of 
the transplanted organ. 
Tissue and organ transplant recipients are customarily treated with one or 
more cytotoxic agents in an effort to suppress the transplant recipient's 
immune response against the transplanted organ or tissue. For example, 
cyclosporine (cyclosporin A), a cyclic polypeptide consisting of 11 amino 
acid residues and produced by the fungus species Tolypocladium inflatum 
Gams, is currently the drug of choice for administration to the recipients 
of allogeneic kidney, liver, pancreas and heart (i.e., wherein donor and 
recipient are of the same species of meals) transplants. However, 
administration of cyclosporine is not without drawbacks as the drug can 
cause kidney and liver toxicity as well as hypertension. Moreover, use of 
cyclosporine can lead to malignancies (such as lymphoma) and lead to 
opportunistic infection due to the "global" nature of the 
immunosuppression it induces in patients receiving long term treatment 
with the drug, i.e., the hosts normal protective immune response to 
pathogenic microorganisms is downregulated thereby increasing the risk of 
infections caused by these agents. 
Preliminary results have shown FK-506 (which has a similar mode of action 
as cyclosporine) to be as potent as cyclosporine in its immunosuppressive 
qualities and to have fewer toxic side effects than cyclosporine. However, 
because studies on FK-506 are only in the early stages, it is not 
available to the general population. Hence, the use of this agent is 
limited. 
Other drugs and/or therapies which are currently administered (either in 
conjunction with cyclosporine or alone) to suppress the rejection of 
allogeneic grafts or allografts are also non-specific immunosuppressive 
drugs or therapies. Steroids, such as prednisone and methylprednisalone, 
and Azathioprine (an analog of 6-mercaptopurine) are among the 
non-specific immunosuppressive drugs used to prolong allograft survival in 
transplantation recipients. 
OKT3 monoclonal antibodies, directed against the CD3 antigen present on 
T-cells, have also been employed as non-specific immunosuppressive 
therapeutic agents in allograft recipients. However, OKT3 monoclonal 
antibodies are of murine origin and the patients to whom such monoclonal 
antibodies are given mount an immune response against these foreign 
proteins. Thus the usefulness of such materials is limited. 
Another drawback to the above-mentioned drugs and antibodies is that they 
must be administered indefinitely to suppress allogeneic graft rejection, 
and tolerance to the foreign tissue does not develop. 
Total lymphoid irradiation (TLI) is yet another form of non-specific 
immunosuppressive therapy that has been used clinically and experimentally 
to prolong allograft survival. The radiation exposure and treatment 
schedule for TLI were developed for the treatment of Hodgkin's disease and 
were subsequently found to be immunosuppressive. Although, TLI induces 
production of the "global" immunosuppression mentioned above and has the 
same limitations of other global immunosuppressive therapies, it is the 
only form of immunosuppression currently in use which appears to induce a 
specific tolerance to allogeneic tissue. However, TLI is cumbersome to 
administer and is in an early stage of development, and thus its 
usefulness is limited. 
The oral and aerosol administration of antigens has also been recognized as 
an effective way to suppress the immune response in meals to these 
antigens. The advantages of administering antigens via the oral route 
include: the simplicity of the techniques involved; the convenience of 
such techniques since many of the methods can be developed in-situ at the 
research or treatment facility; the safe, non-toxic effects of the 
ingestion route; and the specificity that can be provided with the 
antigens. 
Recent studies on several autoimmune disease models have demonstrated that 
the oral administration of antigens can suppress at least the portion of 
the immune response that is directed against autoantigens and also protect 
the treated animals from the induction of specific autoimmune diseases. 
For example, various animal models are available for the study of Type 1 
diabetes as an autoimmune disorder. These include the BB rat (Nakbookda, 
A. F., et al., Diabetologic 14: 199-207, 1978) and the NOD (non-obese 
diabetic) mouse in which diabetes develops spontaneously (Prochazka et al. 
Science 237:286, 1987). Islet-cell specific, CD4- and CD8-positive 
T-lymphocytes have been implicated as the causative agents responsible for 
damage to islet beta cells, as demonstrated by transfer of lymphocytes 
from affected adults to newborn animals (J. Exp. Med. 166:823, 1987). 
Experimental allergic encephalomyelitis (EAE) is an induced T-cell mediated 
autoimmune disease directed against myelin basic protein (MBP) that is 
widely used as an animal model for the human disease Multiple Sclerosis 
(MS). EAE can be induced in small mammals by intravenous administration of 
MBP and a strong adjuvant, such as Freund's complete adjuvant. This 
treatment induces an acute, monophasic autoimmune disease with the 
characteristics of MS. 
Weiner et al., U.S. Patent Application entitled Method Of Treating Or 
Preventing Type 1 Diabetes By Oral Administration Of Insulin Ser. No. 
595,468, filed Oct. 10, 1990, now abandoned and replaced by Ser. No. 
08/472,016, pending, a continuation of 08/235,121, in turn a continuation 
of 08/070,020, in turn a continuation of 07/896,484, in turn a 
continuation of 07/595,468. Ser. No. 07/595,468 discloses oral and aerosol 
compositions and pharmaceutical formulations containing insulin which are 
useful for treating mammals suffering from or at risk for autoimmune 
diseases having the characteristics of Type 1 diabetes. 
Weiner et al., U.S. patent application Ser. No. 460,852 filed Feb. 21, 
1990, (the national stage of PCT Application No. PCT/US88/02139, filed 
Jun. 24, 1988), which is a continuation-in-part application of U.S. patent 
application Ser. No. 065,734 filed Jun. 24, 1987, generally discloses the 
treatment of autoimmune diseases by oral administration of autoantigens. 
Ser. No. 460,852 is now abandoned and replaced and replaced by its 
continuation, Ser. No. 08/279,275. 
Weiner et al., U.S. patent application Ser. No. 454,806 filed Dec. 20, 
1989, now abandoned and replaced by 08/419,502 pending, a continuation of 
08/053,306 (Ser. No. 08/480,188, a continuation of 08/419,502, has been 
allowed and the issue fee has been paid) discloses the aerosol 
administration of autoantigens, disease-suppressive fragments of said 
autoantigens and analogs thereof as an effective method for treating 
T-cell mediated autoimmune diseases. 
Weiner et al., U.S. patent application Ser. No. 487,732, filed Mar. 2, 
1990, now abandoned, and replaced by 08/202,677, pending, a continuation 
of 07/945,443 discloses synergists (enhancers) for use with oral 
administration of autoantigens, disease suppressive fragments and analogs 
thereof as effective treatments for T-cell mediated autoimmune diseases. 
Weiner et al., U.S. patent application Ser. No. 551,632 filed Jul. 10, 
1990, a continuation-in-part of U.S. patent application Ser. No. 379,778, 
filed Jul. 14, 1989, now both abandoned. Ser. No. 551,632 has been 
replaced by 08/419,505, a continuation of 08/324,368, in turn a 
continuation of 08/178,461, in turn a continuation of 08/052,826, in turn 
a continuatoin of 08/928,881, in turn a continuation of 08/809,206, in 
turn a continuation of 551,632. Ser. No. 551,632 discloses methods of 
preventing or treating uveoretinitis in mammals by oral administration of 
purified S antigen, Interphotoreceptor Retinoid Binding Protein (IRBP) 
antigen or disease suppressive fragments thereof. 
Nagler-Anderson, et al., (Proc. Natl. Acad. Sci. (U.S.A.) 83: 7443-7446, 
1986), describe the oral administration of collagen to suppress 
collagen-induced arthritis in a mouse model. 
However, the above-mentioned references do not disclose the use of antigens 
to suppress the mammalian graft rejection mechanism because it has not 
been shown that the principle of oral administration of transplantation 
antigens could prevent allograft rejection. 
The present invention proposes the clinical administration to mammalian 
graft recipients of alloantigens via oral and aerosol routes to induce a 
tolerance to foreign tissue grafts. The invention will be primarily useful 
in the field of organ transplantation including bone marrow. Although 
previous studies have shown that alloantigens injected intravenously to 
recipients can prolong the survival of renal transplants (Transplantation 
39:56, 1985; J. Immunol. 121:1480, 1978; J. Exp. Med. 149:1042, 1979), no 
disclosure or suggestion of introducing these antigens orally or in an 
aerosol form was made therein. 
It is, therefore, an object of the present invention to provide agents and 
methods for suppressing the detrimental immune response in mammals to the 
grafting or transplantation of foreign (or "non-self") tissues and organs. 
Another object of the present invention is to provide pharmaceutical 
formulations and preparations that may be administered to mammals to 
suppress the immune rejection of surgically transplanted tissues. 
A still further object of the invention is to provide synthetic 
compositions and pharmaceutical formulations that may be administrated to 
mammals via the oral or aerosol route to suppress the mammalian immune 
response to the presence of transplanted tissue or organs. 
These and other objects of the present invention will become apparent to 
those of ordinary skill in the art in light of the following. 
SUMMARY OF THE INVENTION 
It has now been unexpectedly discovered that compositions comprising 
specific antigenic agents, including by way of non-limiting example 
allogeneic spleen tissue and cultured lymphocytes and specific Major 
Histocompatibility Complex (MHC) antigens can be administered to mammals 
via the oral or aerosol route to suppress the mammalian immune response to 
surgically transplanted "non-self" organs or tissues. Because the effect 
is dependent upon MHC molecules present on the surface of spleen cells, 
which differ between the tissue donor and the recipient, administration of 
these antigens alone is expected to be effective. 
Orally administered allogeneic splenocytes can suppress the immune response 
of a host mammal which normally occurs shortly after transplant surgery 
against surgically transplanted "non-self" tissue in an antigen-specific 
manner. It has also been found that oral ingestion of allogeneic spleen 
tissue preparations depresses the delayed type hypersensitivity reaction 
and mixed lymphocyte reaction in mammals. Compositions and pharmaceutical 
formulations for oral administration of allogeneic splenocytes may be 
prepared from natural allogeneic tissue. For administration to humans such 
compositions comprise synthetic derivatives of antigens i.e., peptide 
fragments of MHC antigens. 
In practicing the method of the present invention, pharmaceutical 
formulations containing synthetic antigens or natural allogeneic splenic 
or lymphocyte tissue or cell derivatives are prepared and orally 
administered to mammalian subjects some time prior to organ or tissue 
transplant surgery. 
Additionally, an aerosol delivery system can be prepared with essentially 
the dosages of splenocyte derivatives or MHC antigens as above and a 
pharmaceutically suitable carrier or diluent. The aerosol formulations can 
also be administered sometime prior to transplant surgery via the aerosol 
route. These and other improvements will be described in the following 
descriptions, drawings and appended claims.

DETAILED DESCRIPTION OF THE INVENTION 
The contents of all patent applications, patents and literature references 
referred to in this specification are hereby incorporated by reference in 
their entirety. 
The present invention addresses the need for an alternate to existing 
methods for suppressing the immune response directed against foreign 
tissue transplants, as for example, post-transplant surgery. In addition, 
the methods of the present invention provide for prolonged survival of 
organ and tissue allogeneic grafts (i.e. transplants from individuals of 
the same species) in a mammal in need of such treatment. 
Thus, the present invention provides means whereby the rejection of tissue 
allografts can be prevented, thus prolonging the survival of transplanted 
tissue and organs. 
It has now been unexpectedly discovered that oral administration of 
allogeneic splenocytes or synthetic MHC antigens (or immune suppressive 
fragments or analogs thereof) is effective for suppressing the in vitro 
mixed lymphocyte reaction which is a model system for the graft rejection 
response in post-transplant mammalian recipients. 
Without wishing to be bound to any particular theory of operation or 
mechanism of action for the invention it is believed that the oral 
administration of allogeneic splenocytes or derivatives of MHC antigens 
pursuant to the present invention affects the immunological mechanisms of 
graft rejection, i.e. the activation of helper T-cells is decreased by the 
induction of specific suppressor T-cells. 
In the following discussions the following terms shall have the meaning 
ascribed to them below. 
"Oral administration" shall mean both oral administration and enteral 
administration (delivery directly into the stomach). 
"Mammal" shall mean any organism having an immune system and therefore 
susceptible to allogeneic graft rejection. 
"Aerosol" refers to finely divided solid or liquid particles that may be 
created using a pressurized system such as a nebulizer. The liquid or 
solid source material contains MHC antigens and/or disease suppressive 
fragments and analogs thereof as defined herein. 
The "aerosol route" of administration means delivery of an aerosol 
formulation to a host via the nasal or oral airway. 
"Major Histocompatibility Complex" (MHC) is defined as a complex series of 
mammalian cell surface proteins. The MHC plays a central role in many 
aspects of immunity both in presenting histocompatibility (or 
transplantation) antigens and in regulating the immune response against 
conventional (foreign) antigens. There are two types of MHC protein 
molecules, Class I and Class II. Class I MHC proteins are present on 
virtually all tissues and Class II MHC proteins are present on the surface 
of activated T-cells, macrophages and other immune system cells. The human 
MHC genes (the HLA genetic locus) are located on human chromosome 6, the 
mouse MHC genes are located in the H-2 genetic locus on mouse chromosome 
17 the analogous rat MHC genes are referred to as RTI. 
"Class I MHC antigens" are defined as membrane glycoproteins present on the 
surface of all nucleated cells and play a key role in antigen recognition 
by CD8+ cytotoxic T-cells. 
"Class II MHC molecules" are membrane glycoproteins that form part of the 
MHC and are most important in the initiation of immune responses. Class II 
MHC molecules are found mainly on cells of the immune system including 
B-cells, macrophages, brain astrocytes, epidermal Langerhan's cells, 
dendritic cells, thymic epithelium and helper T-cells. Class II MHC 
molecules are involved in regulating the immune response during tissue 
graft rejection, stimulation of antibody production, graft-versus-host 
reactions and in the recognition of "self" (or autologous) antigens, among 
other phenomena. 
"MHC antigens" are defined herein as Class I and/or Class II MHC antigens. 
MHC antigens of the present invention include both Class I and Class II, 
either alone or in combination. 
"Allogeneic tissue extracts" are defined as splenocyte, splenic tissue or 
cultured lymphocyte extracts obtained from an allogeneic transplant donor 
and prepared as described below. 
"Immune suppressive fragments" means any peptide or polypeptide containing 
partial amino acid sequences or moieties of analogs of the relevant MHC 
antigens possessing the ability to induce suppression of the hosts immune 
response against organ or tissue allogeneic grafts. Such fragments need 
not possess the alloantigeneic properties of the entire MHC molecule. 
"Analogs" of immune suppressive fragments refers to compounds that are 
structurally related to suppressive fragments of MHC antigens thereof 
which possess the same biologic activity, i.e., the ability to suppress a 
mammalian host's response against a transplanted organ or tissue. The term 
includes peptides having amino acid sequences which differ from the amino 
acid sequence of the relevant MHC antigens of the potential graft 
recipient by one or more amino acid residues. 
Disease suppressive fragments and analogs for use in the present invention 
can be synthesized using well known solid phase synthesis techniques 
(Merrifield, R. B. Fed. Proc. Am. Soc. Ex. Biol. 21: 412, 1962 and J. Am. 
Chem. Soc. 85: 2149, 1963; Mitchel, A. R. et al., J. Am. Chem. Soc. 98: 
7357, 1976; Tam, J. et al., J. Am. Chem. Soc. 105: 6442, 1983). Analogs 
can be constructed by identifying an equivalent amino acid sequence and 
using the peptide synthesis techniques disclosed above. 
Analogs can be provided using the known amino acid sequence of MHC antigens 
as disclosed in Immunogenetics 29:231-234, 1989. 
Disease-suppressive analogs and fragments can also be obtained using 
recombinant DNA techniques that are well-known in the art. 
Disease suppressive fragments of MHC antigens and analogs thereof can be 
identified using routine experimentation using suitable in vivo systems 
such as those of Examples 1-4 below. 
T-lymphocytes can be obtained from a potential allograft donor using 
methods well known in the art and cultured as described in Transplantation 
41:549, 1986 and Transplantation 48:639, 1989 and administered to a mammal 
about to undergo or having undergone (as described below) an organ or 
tissue allograft. 
Extracts (or lysates) of splenic tissue or cultured lymphocytes can be 
prepared using techniques well known in the art such as those described in 
Example 1 below. 
In accordance with the present invention, conventional tissue typing, 
well-known in the art and routinely conducted on all transplant donors and 
recipients, is performed on a potential transplant donor to determine the 
MHC phenotype of the donor tissue or organ. Synthetic MHC antigens, 
disease suppressive fragments or their analogs can then be synthesized 
using the techniques described above. These antigens and/or fragments may 
be administered to mammals, especially humans, who are to receive a 
transplant, or to patients that have already received transplanted 
"non-self" tissue. The methods and compositions of the present invention 
may be used to treat mammals that have previously received "non-self" 
organ or tissue transplants and are beginning to display the initial 
symptoms of allograft rejection (such as fever, tenderness of the 
transplanted organ or loss of function thereof). The method and 
compositions of the invention are useful to preserve the organ or tissue 
and damp down or shut off that portion of the immune response of the 
recipient that is directed against the transplanted tissue or organ. To be 
effective the compositions and methods of the present invention must be 
administered before total rejection occurs. 
Pursuant to the present invention, MHC antigens or transplantation 
rejection suppressive fragments or their analogs are ingested by a mammal 
that is to receive, or has already received a "non-self" organ or tissue 
transplant via the oral or enteral route, in an amount of between about 
0.1 mg per kg body weight and about 10 mg per kg of body weight per day. 
The pharmaceutical compositions of the invention may be administered as a 
single dose or in multiple dose form via the oral or enteral route. 
Preferably, the is administered in an amount between about 1 mg and about 
5 mg per kg body weight of said mammal per day. The exact amount to be 
administered will vary depending on the severity and stage of a patient's 
disease and the physical condition of the patient. 
When administering splenic cells, cultured lymphocytes or extracts thereof, 
between about 10.sup.6 and about 10.sup.9 cell equivalents per kg body 
weight per day may be administered in single or divided doses. 
The timing of such treatments shall be such that, if possible, the 
pharmaceutical formulations or dosage forms of the present invention are 
administered between about 7 and about 14 days before the transplantation 
is performed. The treatment is preferably continued for at least about 6 
months after the transplanted organ or tissue has been introduced into the 
host (recipient) organism and may be continued indefinately if necessary 
or desirable. 
In addition, if a transplant recipient (either already receiving the 
compositions of the invention or not) begins to manifest symptoms of 
rejection, the pharmaceutical formulations of the present invention may be 
administered in increased amounts and/or frequency. 
The present invention also is directed to oral dosage forms and 
pharmaceutical formulations for administration to mammals in order to 
prolong the survival of or suppress the rejection of a transplanted organ 
or tissue. It will be understood that any statistically significant 
prolongation in graft survival pursuant to the treatment of the present 
invention is within the scope of the invention. 
The oral pharmaceutical formulations of the present invention may also 
contain inert constituents including pharmaceutically acceptable carriers, 
diluents, fillers, solubilizing or emulsifying agents and salts of the 
type that are well-known in the art. For example, tablets and caplets may 
be formulated in accordance with conventional procedures employing solid 
carriers, such as starch and bentonite, that are well-known in the art. 
Examples of solid carriers include bentonite, silica, dextrose and other 
commonly used carriers. Further non-limiting examples of carriers and 
diluents which may be used in the formulations of the present invention 
include saline and any physiologically buffered saline solution such as 
phosphate buffered saline, pH 7-8 and water. 
Capsules employed in the present invention may be made from any 
pharmaceutically acceptable material such as gelatin or cellulose 
derivatives. The active biological materials of the invention may be 
administered in the form of sustained release oral delivery systems and/or 
enteric coated oral dosage forms such as those described in U.S. Pat. No. 
4,704,292 issued Nov. 3, 1987, U.S. Pat. No. 4,309,404 issued Jan. 5, 1982 
and U.S. Pat. No. 4,309,406 issued Jan. 5, 1982. 
It will be appreciated that the unit content of active ingredient or 
ingredients contained in an individual dose of each dosage form need not 
in itself constitute an effective amount for suppressing graft rejection 
since the necessary effective amount can be reached by administration of a 
plurality of dosage units. 
The preferred route of administration of the dosage forms of the present 
invention is orally or enterally. Preferred oral or enteral pharmaceutical 
formulations or dosage forms may comprise for example, between about 70 mg 
and about 500 mg of MHC antigens, disease suppressive fragments or analogs 
thereof or between about 10.sup.7 -10.sup.10 cell equivalents when using 
allogenic cells or extracts thereof. 
In an alternative embodiment of the present invention the pharmaceutical 
formulations of the present invention are administered to mammals in 
aerosol form. It is anticipated that smaller quantities of the allogeneic 
tissue extracts or MHC antigens, disease suppressive fragments or their 
analogs will be required to achieve suppression of graft rejection when 
using the aerosol form of administration. This has been found to be the 
case in treating experimental allergic encephalomyelitis (EAE) with myelin 
basic protein (MBP), and also in treating adjuvant arthritis with collagen 
as disclosed in the co-pending U.S. patent application of Weiner et al. 
Ser. No. 454,806 filed Dec. 20, 1989. The quantity of MHC antigens, 
disease suppressive fragments or the analogs of such meterials which may 
be administered in an aerosol dosage form would be between about 0.01 mg 
and 10 mg per kg body weight of a mammal per day. The aerosol dosage forms 
of the present invention may be administered to a patient via the aerosol 
route in a single dosage form or multiple dosage forms. The exact amount 
to be administered will vary depending on the state and severity of a 
patient's disease, the activity of the patients immune system and the 
physical condition of the patient. 
When administering splenic cells, cultured lymphocytes or extracts thereof, 
between about 10.sup.5 and about 10.sup.9 cell equivalents per kg body 
weight per day may be administered in single or divided doses in an 
aerosol form. 
The aerosol pharmaceutical formulations of the present invention may 
include, as optional ingredients, pharmaceutically acceptable carriers, 
diluents, solubilizing or emulsifying agents, and salts of the type that 
are well-known in the art. Specific non-limiting examples of the carriers 
and/or diluents that are useful in the aerosol pharmaceutical formulations 
of the present invention include water, normal saline and 
physiologically-acceptable buffered saline solutions such as phosphate 
buffered saline solutions, pH 7.0-8.0. 
Examples of useful solubilizing and emulsifying agents are physiologically 
balanced salt solutions, phosphate buffered saline and isotonic saline. 
The salts that may be employed in preparing the aerosol dosage forms of 
the invention include the pharmaceutically acceptable salts of sodium and 
potassium. 
The route of administration of allogeneic spleen cells, cultured 
lymphocytes extracts thereof or MHC antigen or disease suppressive 
fragments or their analogs according to this alternate embodiment of the 
present invention is in an aerosol or inhaled form. The aerosol 
compositions of the present invention can be administered as a dry powder 
or in an aqueous solution. Preferred aerosol pharmaceutical formulations 
may comprise, for example, a physiologically-acceptable buffered saline 
solution containing between about 7 mg and about 700 mg of the 
compositions of the present invention, disease suppressive fragments or 
analogs thereof. 
Dry aerosol in the form of finely divided solid particles of tissue 
extracts from spleen cells, MHC antigens disease suppressive fragments or 
analogs thereof that are not dissolved or suspended in a liquid are also 
useful in the practice of the present invention. The compositions of the 
present invention may be in the form of dusting powders and comprise 
finely divided particles having an average particle size of between about 
1 and 5 microns, preferably between 2 and 3 microns. Finely divided 
particles may be prepared by pulverization and screen filtration using 
conventional techniques that are well known to those skilled in the art. 
The particles may be administered by inhaling a predetermined quantity of 
the finely divided material, which can be in the form of a dry atomized 
powder. 
The pharmaceutical formulations of the present invention may be 
administered via the aerosol route by means of a nebulizer, as an example 
those described in U.S. Pat. Nos. 4,624,251 issued Nov. 25, 1986; 
3,703,173 issued Nov. 21, 1972; 3,561,444 issued Feb. 9, 1971 and 
4,635,627 issued Jan. 13, 1971. The aerosol material is inhaled by the 
subject to be treated. 
Other systems of aerosol delivery, including for example the pressurized 
metered dose inhaler (MDI) and the dry powder inhaler as disclosed in 
Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. 
pp. 197-224, Butterworths, London, England, 1984 can be used in 
conjunction with the method of the present invention. 
Aerosol delivery systems of the type disclosed herein are available from 
numerous commercial sources including Fisons Corporation (Bedford, Mass.), 
Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co., (Valencia, 
Calif.). 
In accordance with the present invention, experiments were performed in 
which the effects of oral administration of allogeneic splenocytes to 
Lewis rats were studied, with particular attention being given to the 
effects on the immune response of the transplant recipient. To this end, 
the in vitro mixed lymphocyte response (MLR), the delayed type 
hypersensitivity (DTH) reaction, and the in vivo accelerated cardiac 
allograft rejection techniques were utilized. In each case, the oral 
administration (to the recipient of a "non-self" tissue transplant) of 
splenocyte cells from a donor animal resulted in suppression of these 
T-cell mediated immune reactions. As T-cells have been implicated as the 
major mediators of allograft rejection, the results of these tests 
establish the practical efficacy of the methods and pharmaceutical 
formulations of the present invention. 
The present invention is illustrated in specific working examples presented 
below which are intended to illustrate the present invention without 
limiting the scope thereof. 
EXAMPLE 1 
PREATION OF MATERIALS AND TEST SUBJECTS 
1. Subject Mammals 
The test population was comprised of male rats of the Lewis (LEW), Wistar 
Furth (WF) and Brown Norway (BN) variety (obtained from Earlan Sprague 
Dawley Inc., Indianapolis, Ind.). The rats in the experiments described 
below were approximately 8-10 weeks old, and were bred under careful 
observation. 
2. Preparation of splenocytes for oral administration 
Fresh splenic tissue was obtained from syngeneic (same species, same 
strain) or allogeneic (same species, different strain) animals shortly 
prior to oral administration. Single cell splenocyte suspensions were 
prepared by mashing the fresh spleen through a standard stainless steel 
mesh (2 inches by 2 inches). Red blood cells were specifically lysed with 
Tris-ammonium chloride buffer according to standard proecudres well known 
in the art, washed twice with Hank's Balanced Salt Solution (HBSS) and 
resuspended into various concentrations as described below before use. 
3. Preparation of splenocyte lysate 
Splenocytes prepared as in the above method were lysed by repetitive 
freeze-thawing in the following manner: 
(a) Cells were quick frozen at -70.degree. C. for 30 minutes; 
(b) Quick frozen splenocytes were then thawed at 37.degree. C.; 
(c) This freeze-thaw cycle was repeated one more time. 
The resulting materials were used for oral administration. 
EXAMPLE 2 
ORAL ADMINISTRATION OF PREED SPLENOCYTE SUSPENSION 
A one milliter dose of the cell suspension as prepared in Example 1, was 
orally introduced to each test rat with a syringe having a ball-tipped 
feeding needle (Thomas Scientific, Swedesboro, N.J.). 
The following laboratory immunological and pathological procedures were 
conducted on the lymphatic organs of the test rats. 
EXAMPLE 3 
MIXED LYMPHOCYTE REACTION 
Cervical lymph nodes were taken from the responder (LEW) and the stimulator 
(WF or BN) rats. The excised nodes were then pressed through stainless 
steel mesh as above and suspended in Phosphate Buffered Saline. 
The isolated lymph node cells were then washed twice and resuspended into 
RPMI 1640 medium, containing 10% fetal calf serum (FCS), 1% penicillin and 
streptomycin (Microbiological Associates, Walkersville, Mass.) 
2.times.10.sup.-5 M 2-mercaptoethanol, and 5 mM HEPES, at a concentration 
of 6.times.10.sup.6 cells/ml. Responder cells were seeded into a 96-well 
flat-bottomed culture plates (Costar Cambridge, Mass.) at 50 microliters 
per well, with or without irradiated stimulator cells (3000 Rads gamma 
irradiated using a Shepherd irradiator, Model 143-45 and a Cesium-137 
source) of the same volume. 5 The treated cells were then cultured at 
37.degree. C. with 5% CO.sub.2 for four days before they were pulsed for 6 
hours with .sup.3 H-thymidine (1 microCi/well, obtained from NEN Dupont, 
Boston, Mass.). Cell proliferation was monitored by incorporation of 
.sup.3 H-thymidine measured by a Beckman liquid scintillation counter. 
SUPPRESSOR ASSAY 
Obtained lymph node cells were irradiated (1000 Rads of gamma radiation) 
and added to a test MLR at concentrations varying from 5 to 20% of total 
cells per well (experimental wells). Control wells were set up with no 
modulators while background wells had only responder cells. These cultures 
were incubated at 37.degree. C. and in 5% CO.sub.2 for 96 hours. 
Proliferation was assayed by pulsing the plates with 1 microCi/well .sup.3 
H-thymidine for the last 6 hours of culture. The plates were then 
harvested as described above. 
DELAYED TYPE HYPERSENSITIVITY DTH REACTIONS 
Rats of each group were immunized subcutaneously in the footpad with 10 
million gamma irradiated (3000 RAD) allogeneic splenocytes. Ten days 
later, they were injected again with the same dosage in the ear lobe. The 
responses were determined as the changes in the ear thickness before and 
48 hours after the challenge. 
CELL TYPING 
The phenotypes of the extracted lymphocytes were tested by indirect 
immunofluorescent staining and with a fluorescence-activated cell sorter 
(FACS). The lymph cells were first incubated for 1 hour with primary 
monoclonal antibodies against the cell surface markers CD4 or CD8, or 
mouse immunoglobulin (Organon-Teknica, Westchester, Pa.) and washed twice 
with PBS containing 0.02% sodium azide. They were then further incubated 
with FITC-conjugated goat-anti-mouse IgG (1:40) (Organon Teknica) in the 
dark for 30 minutes and in the presence of 15% autologous normal rat 
serum. The cells were thoroughly washed and fixed with 1% formaldehyde 
before testing. 
Additionally, surgical transplant methods of the type described in the 
following example were performed. 
EXAMPLE 4 
CARDIAC ALLOGRAFT 
LEW rats were subjected to surgical transplant procedures. An accelerated 
rejection model was used wherein LEW strain rats were pre-sensitized with 
BN strain full-thickness skin grafts seven days before the cardiac 
allograft, with and without oral ingestion of splenocyte preparations. 
Seven days later, a (LEWxBN)Fl strain test vascularized cardiac allograft 
was performed on each pre-treated rat. The cardiac grafts were anastomosed 
to the infra-renal abdominal aorta. Rejection was defined as complete 
cessation of heart beat as determined by daily palpation of the 
recipient's flank. 
The above-described methods were used to obtain the following results: 
I. SUPPRESSION OF THE MIXED LYMPHOCYTE REACTION (MLR) BY ORAL 
ADMINISTRATION OF ALLOGENEIC SPLENOCYTE PREATIONS 
Splenocytes from WF rats were freshly prepared and were administered orally 
to LEW rats two, five or ten times over a 1-2 week period. 
The individual dosages were 50 million cells per oral administration. 
Seven days following the last oral administration, lymph nodes were taken 
from both a control group and those given oral splenocytes for MLR studies 
using WF or BN stimulators. As shown in FIG. 1, LEW rats which had 
ingested allogeneic splenocytes showed significantly reduced reaction 
against the lymphocytes from the WF strain. This phenomenon was observed 
in all three feeding protocols (i.e., 2, 5 or 10 times). However, only the 
group that received ten feedings showed suppression against the BN strain, 
the third party control. 
These results indicate that limited ingestion of allogeneic splenocyte 
preparations induced antigen specific suppression of the MLR. 
II. COMISON OF SUPPRESSION OF MLR BY ORAL ADMINISTRATION OF ALLOGENEIC 
VERSUS SYNGENEIC SPLENOCYTE PREATIONS 
A dose response study was subsequently conducted to determine the effect of 
feeding syngeneic versus allogeneic cells. LEW rats were fed twice with 1, 
5, 25 or 50 million splenocytes from either LEW or WF strains. The results 
are set forth in Table I below. 
TABLE I 
______________________________________ 
The Effect of Feeding Syngeneic and Allogeneic Splenocytes on MLR 
Strains used 
Dosage MLR Relative 
for feeding 
cells/feeding 
delta/CPM Response (%) 
______________________________________ 
-- -- 115015 .+-. 7707 
100 
LEW 1 .times. 10.sup.6 
128520 .+-. 8338 
112 
5 .times. 10.sup.6 
54391 .+-. 10988 
47 
25 .times. 10.sup.6 
39088 .+-. 7294 
34 
50 .times. 10.sup.6 
81329 .+-. 8013 
71 
WF 1 .times. 10.sup.6 
71135 .+-. 13721 
62 
5 .times. 10.sup.6 
79011 .+-. 13721 
68 
25 .times. 10.sup.6 
56196 .+-. 15254 
49 
50 .times. 10.sup.6 
73541 .+-. 11636 
64 
______________________________________ 
Feeding at the lowest dosage (1 million) of syngeneic cells did not induce 
suppression; all other doses, both syngeneic and allogeneic cells, show 
some suppression to varying degrees. 
III. EFFECT OF INGESTED LYSATE OF ALLOGENEIC SPLENOCYTE PREATIONS ON MLR 
The effect of ingested lysate alone on MLR was next studied to determine 
whether live splenocytes were required for the orally induced tolerance. 
Rats were given two separate oral doses of either live splenocytes or the 
corresponding lysate prepared by the repetitive freeze and thaw method 
(described above) and the effect of these treatments were compared. FIG. 2 
shows that cell lysate alone was sufficient in suppressing the MLR, 
indicating that a subcellular fragment was involved in suppressing the 
cell-mediated immunity. 
IV. KINETICS OF MLR SUPPRESSION BY ORAL ADMINISTRATION OF ALLOANTIGENS 
The kinetics of the orally induced tolerance to alloantigen was studied by 
giving two oral doses of splenocytes to separate LEW rat groups, 14 days, 
7 days, 3 days, and 1 day before the MLR was performed. As shown in FIG. 
3, the groups which were given oral doses 1 day or 3 days before MLR was 
performed did not induce suppression. The groups with 7-day and 14-day 
intervals between the last oral ingestion and MLR showed dramatic 
reduction of proliferation in MLR, indicating that more than 4 days were 
required for the induction of oral unresponsiveness to alloantigens. 
V. SUPPRESSION OF DTH RESPONSE AGAINST ALLOANTIGENS 
In addition to the in vitro MLR, the effect of ingesting allogeneic 
splenocytes on the delayed type hypersensitivity (DTH) response, in vivo, 
in LEW rats was examined. LEW rats were orally administered 10 feedings of 
50 million splenocytes from either syngeneic or allogeneic (WF) animals. 
After the last oral ingestion, the test for DTH was initiated with the 
animals being immunized subcutaneously in their foot pads. The same 
animals were injected again 10 days later in the ear lobes. The DTH was 
measured as the changes in the ear thickness before and 48 hours after the 
challenge. The results are shown in FIG. 4. 
Approximately 50% decrease in DTH response to WF was observed in rats fed 
with cells of the same strain, but not in those fed with syngeneic LEW 
splenocytes. The DTH response against BN was not affected by the 
pre-treatment, indicating that the DTH suppression was antigen specific. 
VI. ACTIVE SUPPRESSION IS INVOLVED IN MEDIATING DECREASED PROLIFERATION IN 
THE MLR 
In order to study the mechanism of inhibition of MLR proliferation in the 
fed animals, a suppressor cell assay was performed to determine if CDS+ 
suppressor, cells were involved in mediating the observed effects. 
Lymphocytes from either control or pre-fed animals were irradiated with 
1000 RADS of gamma radiation before being added to a primary MLR, serving 
as modulators. 
Lewis rats (3/group) were pre-treated 10 times orally with varying dosages 
(as indicated in the Table) of WF splenocytes. One week later, their 
cervical lymph nodes were taken and the cells served as modulator after 
being irradiated 1000 Rad of gamma radiation. The primary LEW anti-WF and 
LEW anti-BN MLR and Con-A stimulation cultures were set up as described 
above. Modulator cells were added to the primary cultures at a 1/5 ratio. 
The results are set forth in Table II below. 
TABLE II 
__________________________________________________________________________ 
SUPPRESSION OF PRIMARY MLR BY LYMPHOCYTES FROM 
ALLOGENEIC SPLENOCYTES FED RATS 
anti-WF anti-BN Con A 
Source of 
CPM CPM CPM 
modulator 
(.times.10.sup.-3) 
% Supp. 
(.times.10.sup.-3) 
% Supp. 
(.times.10.sup.-3) 
% Supp. 
__________________________________________________________________________ 
-- 112 .+-. 21 
405 .+-. 78 
280 .+-. 4.7 
control 
104 .+-. 11 
7 464 .+-. 15 
0 276 .+-. 6.6 
1.4 
fed 10 .times. 10.sup.6 
106 .+-. 17 
5.3 334 .+-. 120 
17 277 .+-. 34 
1 
fed 25 .times. 10.sup.6 
84 .+-. 15 
25 443 .+-. 17 
0 305 .+-. 3.9 
0 
fed 50 .times. 10.sup.6 
1 .+-. 0.9 
99 84 .+-. 7.1 
79 199 .+-. 12 
29 
__________________________________________________________________________ 
The results in Table II show that adding 20% of modulators from pre-fed 
animals, but not from the control animals, suppressed the primary 
LEW-anti-WF MLR. This suggests that suppressor cells were induced after 
feeding and these in turn mediated suppression of the MLR. 
VII. PHENOTYPE OF LYMPH NODE CELLS FROM FROM ANIMALS INGESTING SPLENOCYTES 
Cervical lymphocytes from either control or fed animals were cultured with 
irradiated WF stimulators for 5 days, then sorted for CD4+ or CD8+ cells 
by indirect immunoflouresence staining. The results shown in FIG. 5 show 
that pre-feeding rats with allogeneic splenocytes resulted in an increase 
in CD8+ (suppressor T-cells) cells and a decrease in CD4+ (helper T-cells) 
cells when compared to controls. 
VIII. ORAL ADMINISTRATION OF SPLENOCYTES PREVENTS ACCELERATED CARDIAC 
ALLOGRAFT REJECTION 
To demonstrate the prevention of allograft rejection, an accelerated 
rejection transplantation model, as described above, was used. LEW rats 
were pre-sensitized with BN skin grafts 7 days before challenge with 
vascularized BN test cardiac allografts, to study the effects of feeding 
allogeneic donor splenocytes on test graft survival. 
While unsensitized controls rejected their cardiac allografts on the 6th 
through the 8th day, all sensitized control animals hyperacutely rejected 
their cardiac allografts within 36 hours. Test animals fed 5-10 feedings 
of 50 million splenocytes, 7 days prior to the skin graft, or even on the 
day of the skin graft, exhibited increased test cardiac allograft 
survival, to 7.62.+-.0.5 days. 
These results show that feeding allogeneic splenocytes prevents 
sensitization and converts accelerated rejection into an acute form. 
The specificity of this phenomenon was examined as described below. 
Cardiac recipient LEW rats were either unfed (n=10), fed LEW (syngeneic) 
lymphocytes (n=4), fed BN splenocytes (but received a WF cardiac 
allograft, n=6) or were fed BN splenocytes (and received a BN cardiac 
allograft, n=8). All fed animals received 5-10 feedings of 
50.times.10.sup.6 splenocytes. The results are shown in FIG. 6. 
As can be seen in FIG. 6, only the rats which were fed allogeneic 
splenocytes showed cardiac allograft survival beyond day 3. LEW rats fed 
third party (BN) lympocytes but receiving a WF graft did not demonstrate 
enhanced cardiac allograft survival, demonstrating the specificity of this 
reaction. 
In a preliminary attempt to study the mechanism of graft prolongation, the 
MLR of cervical lymph node cells from control and fed sensitized LEW rats 
were examined at 48 hours after the cardiac transplant. The results are 
shown in FIG. 7. 
There was a suppression of the MLR in the fed animals as compared to the 
control (FIG. 7). These data are consistent with the previous MLR findings 
in the naive animal model.