CA2264748A1 - Muc-1 as an immunosuppressive therapeutic agent for the treatment of inflammatory and autoimmune conditions - Google Patents

Abstract

The invention relates to novel methods of treatment and pharmaceutical compositions comprising the mucin MUC-1 and its derivatives. Preferred MUC-1 derivatives comprise multiple tandem repeats of the MUC-1 core sequence. In fact, the figure shows that lower serum MUC-1 levels correlate with longer survival time. The disclosed compositions and methods are particularly useful in treating autoimmune disorders, inflammatory disorders, organ transplant rejection and graft versus host disease.

Description

1015202530CA 02264748 1999-03-01W0 98/ 10783 PCT/US97/15928MUC-1 AS AN IMMUNOSUPPRESSIVE THERAPEUTIC AGENT FORTHE TREATMENT OF INFLAMMATORY AND AU'I'OIMMUNE CONDITIONSBACKGROUND OF THE INVENTIONAutomimmune disorders represent.a diverse collectionof disorders, unrelated save for their commoninflammatory etiology. Current treatments focus on thisetiology and utilize a wide variety of medicaments,including non-steroidal antiinflammatories,corticosteroids, and even cytoablative agents.Unfortunately, neither the existing medicaments nortreatments which utilize them are wholly satisfactory.Likewise, similar dissatisfaction exists with respect tomany inflammatory disorders, organ transplant rejectionand graft—versus host disease. Thus, there exists a needfor new medicaments and new methods of treatment forthese disorders.SUMMARY OF THE INVENTIONIt is, therefore, an object of the present inventionto provide new methods for treating autoimmune disorders,inflammatory disorders, organ transplant rejection andgraft versus host disease. According to this object,methods are provided which comprise administering apharmaceutically effective amount of MUC-1, a MUC-1derivative or a MUC-1 carbohydrate derivative to apatient in need of said treatment.It is yet another object of the invention to providenovel pharmaceutical compositions of new and effectivemedicaments to implement methods for treating autoimmunedisorders, inflammatory disorders, organ transplantrejection and graft versus host disease. According tothis object of the invention pharmaceutical compositionsare provided which comprise an amount of MUC—1, a MUC-1derivative or a MUC-1 carbohydrate derivative effectivefor treating an autoimmune disorder, inflammatorydisorder, organ transplant rejection or graft versus host101520253035CA 02264748 1999-03-01W0 93/10733 PCT/US97/15928disease, in combination with a pharmaceutically effectivevehicle.In preferred methods and compositions, the MUC-1derivative comprises multiple tandem repeats of the MUC-1core sequence .BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 shows expression of MUC-1 at the surface ofcultured murine (410.4) breast carcinoma cellstransfected with the human MUC-1 gene as ascertained byflow cytometry using the antibody B27.29. Note the highMUC-1 expression (fluorescence intensity) cnf MUC—1Hi,which is approximately 2.5-fold higher than that of MUC-1Lo, whereas the wild-type 410.4 cell line does notexpress MUC-1.Figure 2 shows binding of MCF-7 cells to 4 hr stimulatedHUVEC is inhibited most efficiently by anti-E selectin(Serotec 1.2B6) when endothelial monolayers arepreincubated.with monoclonal antibodies, and.by anti-sLeW“(CSLEX & B67.4 sLeU when MCF-7 cells are pretreated withmonoclonal antibodies (Figure 2a). Binding of MCF-7cells to 24 hr stimulated HUVEC is inhibited by anti-ICAM-1 (Serotec 84H10) (endothelial pretreatment) and byanti-MUC-1 (B27.29) (MCF-7 pretreatment) (Figure 2b).Adhesion was assessed by a standard static endothelialadhesion assay described in Berry, et al. Br. J. Cancer51:179 (1985). Results are expressed as a percentage offluorescence signal from the total number of cells addedto each well; each value represents the mean of threereplicates +/- SD (error bars); " denotes wells coatedwith stimulated monolayers. The experiments shown arethe best of three independent experiments of each type.Figure 3 shows high MUC-1 expressing transfectant(GZHi), which shows approximately four fold higherbinding to 24 hr stimulated HUVEC than that of wild type(410.4), and approximately two fold higher binding than101520253035CA 02264748 1999-03-01W0 93/10733 PCT/US97/ 15928that of the low MUC-1 expressor (GZLO) (Figure 3a) whichis inhibitable by anti-MUC-1 (B27.29) and anti-ICAM-1(18E3D) (Figure 3b). Adhesion was measured and theresults are expressed as described in the description ofFigure 2. The experiments shown are the best of threeindependent experiments of each type.Figure 4a shows determination of serum MUC-1 levels bysandwich radioimmunoassay. Figure 4b shows that lowerserum MUC-1 levels correlate with longer survival time.Figure 5 shows high MUC—1 expressing cells (GZHi), whichbind to immobilized recombinant soluble ICAM-1-Ig fusionprotein and are inhibited by anti—MUC-1 (B27.29), anti?ICAM-1 (18E3D), and soluble MUC-1. Adhesion was measuredas described in Takada, et al. Cancer Res. 53:354 (1993).The values shown are the means of three replicates +/-SD; “ denotes ICAM-1 coated wells. The experiment shownis the best of four independent experiments.Figure 6 shows high MUC-1-expressing cells (MUC-1 Hi)bind optimally to recombinant ICAM-1 (rhICAM-1) at 37°Ca.This adhesion mechanism is susceptible to almost completeinhibition at 4°C. Adhesion was measured and the resultsare expressed as described in the description to Figure3. The experiment shown is representative of threeindependent experiments. bars, standard deviation (SD).Figure 7 shows that addition of purified human MUC—1 tothe in vitro human T cell culture inhibits T cellproliferative:response against strong allogenic stimulus.Figure 8, panel a, shows the inhibition of T cellproliferation by the addition of 10 ug/ml of MUC-1 or aMUC—1 derivative, as compared to OSM or culture mediumcontrols. Panels b and c show the abrogation of mucin-mediated T cell inhibition by anti CD28 Mab and IL-2,respectively.101520253035W0 98/ 10783CA 02264748 1999-03-01PCT/U S97/ 15928Figure 9 shows the direct relationship between the numberof MUC-1 tandem core repeats and the inhibition of T cellproliferation.DETAILED DESCRIPTION OF THE INVENTIONThe present invention relates to the use of MUC-1and its derivatives to treat inflammatory or autoimmunedisorders, based on the ability of MUC-1 to bind to ICAM-that MUC-1 hasimmunosuppressive effects in tumor models. The presentMUC—1 and itsderivatives to suppress or prevent transplant rejectionIt is known that bothinflammatory and autoimmune disorders are associated withThus,employed as1 and on evidence suggestinginvention also relates to use ofand graft versus host reactions.a hyperreactive, or overreactive, immune response.MUC-1 and itsimmunosuppressive agents to treat these disorders byderivatives may besuppressing the overreactive immune response.BackgroundMucins are a family of glycoproteins, greater than200kDa.molecules with an extended extracellular domain composedSome mucins, such as MUC-1, are membrane boundof tandem repeats of amino acid (aa) sequences whichcontain sites.Devine, et al. BioEssays 14: 619 (1992).studiesnumerous potential 0-glycosylationNumerous clinical have suggested thatmucinous tumor antigens, both expressed on the cellsurface of tumor cells and shed from the surface of tumorcells, are associated with a poor prognosis of a varietyof cancer types. See, for example Itzkowitz, et al.Cancer 66: 1960 (1990).Over ninety percent of breast cancers show anincreased expression of a membrane bound molecule, MUC-1(Episialin, Epithelial Membrane Antigen, PolymorphicEpithelial Mucin, Human Milk Fat Globule Membrane antigenBerry, et al. Br. J. Cancer 512179 (1985). MUC—1is a well—characterized tumor cell surface mucin which isetc.).101520253035W0 98/ 10783CA 02264748 1999-03-01PCT/US97/15928shed into the serum and carries repeating sialyl-Tnepitopes. See Cancer Research 47:5476 (1987) andReddish, et al. Glycoconjugate Journal 13:1 (1996). MUC-1 is a highly glycosylated mucin type glycoproteinpresent on the luminal surface of most glandularepithelia and is often found increased over the entiresurface of many carcinoma cells. This mucin extends farabove the cell surface making it easily available forinteractions with other cells. MUC-1 is expressed innormal breast and in approximately 90% of breast cancers.MUC-1 is present as both a transmembrane form as well asin a secreted form. It is not known if both show similarpatterns of glycosylation.Mucins are known to lubricate mucosal surfacesproviding protection, and cell surface mucins are thoughtto participate in cel1—cell interactions. See Samuel andLongenecker gg Vaccine Design: The Subunit and AdjuvantApproach, Powell, et al., eds. (Plenum Press, N.Y. pp.875-890 (1995)). Some investigators support an anti-adhesive function for MUC-1 through steric hindrance andnegatively charged 0-linked sialic acid residues.Ligtenberg, et al. Cancer Res. 52:2318 (1992).The extracellular and cytoplasmic domains cansupport intercellular adhesion. The extracellular domainis composed of 30 to 90.tandem repeats of a highlyglycosylated twenty aa sequence. The presence ofnumerous highly glycosylated tandem repeats allows MUC-1to extend 200-500 nm above the cell surface far beyondthe surrounding 10-30 nm glycocalyx. This places it inan ideal position to enhance adhesion with those cellsSialylatedLewis“’residues, which are also carried by MUC-1, mediatepossessing the appropriate receptor(s).the adhesion of malignant colonic cells to E-Selectin.Sawada, et al. Int. J. Cancer 57: 901 (1994), Zhang, etal. Int. J. Cancer 59: 823 (1994). The 69 aa cytoplasmicdomain of MUC-1 is linked to microtubules of thecytoskeleton, which is similar to the structure of otheradhesion moieties. This interaction with the101520253035CA 02264748 1999-03-01WO 98/10783 PCT/US97/15928submembraneous actin cytoskeletal filaments is thought toaccount for the preferential expression of MUC—1 on freeor unattached membrane surfaces. Parry, et al. Exp. CellRes. 188: 302 (1990).The presence of membrane bound MUC—1 couldfacilitate tumor cell interactions with stimulatedendothelial cells during the process of blood bornemetastases. Several carcinoma cell lines adhere in vitroto activated endothelial cells using molecules similar tothose employed by normal extravasating inflammatorycells. Takada, supra, Carlos, et al. Blood 84:2068(1994). In this process, high affinity interactionsbetween endothelial cell immunoglobulin superfamilymembers such as ICAM-1 or VCAM are thought to involveselective interactions with members of the Integrinfamily. Carlos, supra.ICAM-1 (Intracellular Adhesion Molecule) isexpressed on B cells and T-cells and antigen presentingcells (APCs) where it participates in cell-cellinteractions important for the induction of an immuneresponse. It is also present in high levels onendothelial cells. ICAM-1 (CD 54) is found on most cellsof the immune system, particularly on cytokine-activatedcells, of both hemolytic and non—hemolytic originfibroblasts and keratinocytes.Recently CD43, a. highly glycosylated. mucin. typeglycoprotein with no structural resemblance to the beta2 integrins (CD 11a/b with CD18) has been reported tobind ICAM-1. Rosenstein, et al. Nature 354: 233 (1991).This suggests that there is pliancy in the receptor-ligand recognition so that members of different receptorclasses could interact. Thus, it is possible that MUC-1mediates adverse effects through an interaction withadhesion molecules, such as ICAM-1.Support for an ICAM—1/MUC-1 interaction can be drawnfrom studies of cytotoxic T lymphocytes and MUC-1transfected cells. These interactions are shown to benon-MHC restricted and are ICAM—1 dependent. van de Weil-101520253035CA 02264748 1999-03-01W0 93/ 10783 PCT/US97/15928van Kemmenade, et al. Immunology 151: 767 (1993). Theyalso are inhibited by SM3, an antibody against the aasequence DTRP of the MUC-1 tandem repeat. Jerome, et al.Immunology 151: 1654 (1993). This suggests that theICAM—1 binding site on MUC—1 lies within the peptidecore, which is uniquely exposed by the cancer-associatedunderglycosylation of MUC—1.In breast cancer, the amount of membrane—bound MUC—1is increased. The mucin is also altered with fewercarbohydrate residues, thereby exposing usually crypticepitopes on the protein core and interior carbohydrates.This cancer—associated configuration forms the basis foranticancer immunotherapy. Longenecker, et al. EweImmunologist 1:89 (1993), Agrawal, et al. Cancer Res.55:2257 (1995). In advanced breast cancer, MUC—1 is shedfrom tumor cells and is thus elevated in the serum, whereit correlates with an unfavorable prognosis and possiblyimmunosuppression through the induction of T-cell anergy.Reddish et al. Cancer Immunol Immunother. 42:303-O9(1995).Because soluble MUC—1 can competitively inhibitadhesive interactions of MUC-1-positive cells with ICAM—1(Figure 5), it is possible that serum MUC—1 is inducingthe anergic state by occupying ICAM—1 receptors oncytotoxic T-cells (CTLs). By occupying peritumoralendothelial cell ICAM—1 receptors, serum MUC-1 could alsoinhibit adhesive interactions of migrating cells with theendothelium and thus (a) cause decreased recruitment ofinflammatory cells to the tumor site and (b) facilitatetumor cell escape and metastasis from the primary mass.Preparations of MUC-1 and MUC—1 DerivativesThe MUC-1 used in the compositions and methods ofthis invention may be purified from sources such ascancer cell lines secreting MUC—1 and pleural effusionsor ascites fluid from cancer patients. See Example 8.The MUC—1 used in the methods of this invention may alsobe obtained by recombinant DNA techniques that are well 101520253035CA 02264748 1999-03-01W0 98I10783 PCT/US97/15928known to those of skill in the art. See, for exampleGendler, et al., J. Biol Chem. 265:15286 (1990).As used herein a "MUC—1 derivative" is used to referto a peptide that is structurally and/or functionallyrelated to MUC—1. Such derivatives may retain some orall of the functional characteristics of MUC—1, inparticular, the immunosuppressive function of MUC—1. Theimmunosuppressive function easily may be measured usingthe assays set for below in the Examples.A MUC—1 derivative may be partially deglycosylatedor completely unglycosylated MUC—1 protein.Deglycosylation of MUC-1 protein can be carried out usingtechniques that are well known to the skilled artisan.A MUC—1 derivative may be a fragment of the MUC-1protein. Such fragments may be glycosylated orunglycosylated. In accordance with the presentinvention, fragments within the invention can be obtainedfrom purified MUC-1 or MUC—1 produced by recombinant DNAmethodology by methods that include digestion withenzymes such as pepsin or papain. Alternatively, MUC—1fragments encompassed by the present invention can besynthesized using an automated peptide synthesizer suchas those supplied commercially by Applied Biosystems,Multiple Peptide Systems and others, or they may beproduced manually, using techniques well known in theart. See Geysen et al., J. Immunol. Methods 102: 259(1978).MUC—1 derivatives also include glycosylated or non-glycosylated synthetic peptides. In addition, MUC—1derivatives within the present invention includeproteolytic cleavage-resistant MUC-1 fragments or MUC—1fragments containing one or more non-natural amino acids,such as D—amino acids.In another embodiment, the MUC-1 derivative wouldinclude the extracellular tandem repeat region of MUC—1,which includes repeats of the amino acid sequence DTRP(Asp-Thr-Arg-Pro). Preferrably these tandem repeats101520253035CA 02264748 1999-03-01W0 98/ 10783 PCT/US97/l5928include the sequence SAPDTRP (Ser-Ala-Pro—Asp-Thr—Arg—Pro).some preferred MUC-1 derivatives comprise at leastone peptide core repeat of the MUC-1 mucin. A MUC-1peptide core repeat in the native MUC-1 moleculecomprises the 20 amino acid sequence PDTRPAPGSTAPPAHGVTSA(Pro-Asp-Arg—Thr-Pro—Ala-Pro-Gly-Ser-Thr-Ala-Pro-Pro—Ala-His-Gly-Val—Thr—Ser-Ala). Useful synthetic derivativesinclude permutations of this sequence, for example,GVTSAPDTRPAPGSTAPPAH, where the repeat merely begins withGVTS rather than PDTR. Other, similar permutations arealso possible.Moreover, one or more amino acids of the coresequence may be altered, preferrably in a conservativemanner known in the art, such that the requisiteimmunosuppressive activity is maintained. other MUC-1derivatives comprise at least one truncated peptide corerepeat of the MUC-1 mucin, for example, GVTSAPDTRPAPGSTA.of course any combination of core sequence permutation,alteration or truncation may be linked together to formmultiple, and especially tandem multiple repeats.Data presented below in Examples 8 and 9 demonstratethat fewer than 1 core repeat fails to exhibit the MUC-1immunosuppressive function. But, where more than 1repeat is present, it is observed that the degree ofimmunosuppression increases linearly with the number ofrepeats present. Thus, although an upper limit to thenumber of repeats that effectively can be employed islikely, MUC-1 derivatives containing very large numbersof repeats are contemplated. For example, the nativeMUC-1 contains 60-100 of such repeats.Some embodiments contemplate from about 2 to about100 core repeats. However, more preferred MUC-1derivatives comprise from 2-20 repeats of the MUC-1 core.Most preferred MUC-1 derivatives comprise from 3-6repeats of the MUC-1 core and the repeats preferably arearranged in tandem.101520253035CA 02264748 1999-03-01wo 98/10783 PCT/US97/15928.-10-of course, as described above, these preferred MUC-1derivatives may be glycosylated or partially glycolysatedaccording to methods known in the art. Moreover, it iscontemplated that MUC—1 and MUC-1 derivatives can bemodified with large molecular weight polymers, such aspolyethylene glycols.Also illustrative of an MUC-1 derivative within thepresent invention is a non-peptide "mimetic," i.e., acompound that mimics one or more functionalcharacteristics of the MUC-1 protein. Mimetics aregenerally water-soluble, resistant to proteolysis, andnon-immunogenic. Conformationally restricted, cyclicorganic peptides which mimic MUC-1 can be produced inaccordance with known methods described, for example, bySaragovi, et al., Science 253: 792 (1991)."MUC—1 carbohydrate derivatives" are alsocontemplated. Such a derivative, as used herein, refersto a glycopeptide which retains at least one functionalcharacteristic of MUC-1, such as immunosuppression. Sucha carbohydrate derivative may include all or part of thecarbohydrate that is attached to the MUC-1 protein.Mimetics that mimic at least one property of MUC-1carbohydrate may also be used. IDesiqninq other MUG-1 DerivativesIn another embodiment, MUC—1 derivatives ‘may bedesigned to block ICAM-1-mediated cell interactions toeffect immunosuppression. In antigen specific T-cellresponses, T-cells interact via T-cell receptors (TCRs),with antigen—presenting cells (APCs). In addition tointeraction of TCR with MHC complex molecules duringantigen presentation to T-cells, a number of otherinteractions between APCs and TCR are required, usingvarious accessory molecules (co—receptors). Interactionof accessory molecules is important in a T-cell responsebecause these interactions (1) increase the avidity ofinteraction between the APC and the T-cell and (2) inducevarious intracellular signal pathways in the T—cells.101520253035CA 02264748 1999-03-01W0 93/10733 PCT/US97/15928-11-Interaction of ICAM-1 with its ligand is importantin conjunction with TCR ligation to produce an efficientT-cell stimulatory signal. However, when the ICAM-1interaction with its ligand and cross—linking occurs inthe absence of TCR ligation, the T-cells become anergic(non-responsive). Therefore, MUC-1 or its derivativescan be used as immunosuppressive agents: when MUC—1 orits derivatives interacts with and cross-links ICAM-1molecules in the absence of an antigenic stimulus, thisresults in nonresponsiveness (immunosuppression) of theT-cells.Thus, in this embodiment, MUC—1 derivatives may bedesigned to block MUC~1/ICAM-1 interactions which willreduce the ICAM-1—mediated cellular immune response. Inone embodiment, such blocking MUC—1 derivatives may bedesigned by using MUC-1 derivative binding to recombinanthuman (rh) ICAM-1-immunoglobulin (Ig) fusion protein. Inthis method, the fusion protein is immobilized in thesolid phase. Libraries of synthetic MUC-1 peptides,glycopeptides and glycoconjugates based on the MUC—1tandem repeat can be screened for blocking activity.Those compounds that partially or completely block MUC—1binding to ICAM-1 may be screened for therapeuticeffectiveness.The multiple copies. of the MUC-1 tandem repeatcontained in the MUC-1 protein may be able to cross-linkseveral ICAM-1 molecules, leading to immune suppression.The "libraries" of synthetic peptides containing a singlecopy of the tandem repeat should not produce this effect.Similarly, those peptides that contain less than anentire tandem repeat should not produce this effect. Thedata presented below in Examples 8 and 9 support thisview.Once potential immunosuppressive MUC-1 derivativeshave been identified using the ICAM-1/MUC-1 bindingassay, the derivatives can be further evaluated using aT-cell based immune suppression system. Such a system _ ,.........................w... M. ..... . ..101520253035CA 02264748 1999-03-01W0 98/ 10783PCTIU S97/ 15928-12-determines the ability of a derivative to prevent invitro T—cell induction.Because MUC—1 interacts with ICAM-1, and is expectedto react with other adhesion molecules such as ICAM-3,various MUC—1 derivatives should be useful as drugs thatblock the MUC-1/adhesion molecule interaction. Suchadhesion inhibitors could be used to treat cancerpatients who are immunosuppressed by MUC—1 mucin.Pharmaceutical Formulations‘The pharmaceutical compositions of the inventiongenerally contain a pharmaceutically effective amount ofMUC-1, a MUC—1 derivative or a MUC—1 carbohydratederivative. Preferably, MUC—1, a MUC—1 derivative or aMUC—1 carbohydrate derivative is admixed with a .pharmaceutically effective vehicle (excipient).A suitable formulation will depend on the nature ofthe disorder to be treated, the nature of the medicamentchosen, the route of administration desired and thejudgment of the attending physician. Suitableformulations and pharmaceutically effective vehicles, canbe found, for example, ix: REMINGTON'S PHARMACEUTICALSCIENCES, chapters 83-92, pages 1519-1714 (MackPublishing Company 1990) (Remington's), which are herebyincorporated by reference.Methods of the InventionMUC-1, a MUC—1 derivative or a MUC—1 carbohydratederivative may be used to treat autoimmune disorders andinflammatory disorders. MUC—1 may also be used toprevent or suppress organ transplantation rejection andgraft versus host disease in bone marrow transplantation.Thus, the methods of the invention typically compriseadministering a pharmaceutically effective amount of MUC-1, a MUC—1 derivative or a MUC—1 carbohydrate derivativeto a patient in need of treatment. The patient may be ahuman or non—human animal.101520253035W0 98/10783 "1 3 "CA 02264748 1999-03-01As used herein, an "inflammatory disorder" refers toany of the many inflammatory disorders that are wellknown to those of skill in the art. These disordersinclude, but are not limited to, the following disorders:inflammatory arthritis such as rheumatoid arthritis,psoriasis, allergies such as allergic contact dermatitis,and ankylosing spondylitis.As used herein, an "autoimmune disorder" refers toeany of the many autoimmune disorders that are well knownto those of skill in the art. These disorders include,but are not limited to, the following disorders:myasthenia gravis, systemic lupus erythematosus,polyarteritis nodosa, Goodpastures syndrome, isopathicthrombocytopenic purpura, autoimmune hemolytic anemia,‘Grave's disease, rheumatic fever, pernicious anemia,insulin-resistant diabetes mellitus, bullous pemphigold,pemphigus vulgaris, viral myocarditis (Cocksakie B virusresponse), autoimmune thyroiditis (Hashimoto's disease),male infertility (autoimmune), sarcoidosis, allergicencephalomyelitis, multiple sclerosis, Sjorgens disease,Reiter's disease, Celiaczdisease, sympathetic ophthalmia,and primary biliary cirrhosis.Intracapsular, intravenous, intrathecal, andintraperitoneal routes of administration of MUC—1 and itsderivatives may be employed. The skilled artisan willrecognize that the route of administration will varydepending on the disorder to-be treated. For example,intracapsular administration may be used when treatingarthritis. Injection into the hepatic portal vein may beemployed when treating inflammatory hepatitis. Intra-organ injection of the thyroid may be used when treatingthyroiditis.Eitherintravenouscn:intraperitonealadministrationmay be used when treating autoimmune diseases of thegastrointestinal tract, such as pancreatitis or colitis.Intrathecal administration may be appropriate whentreating autoimmune encephalitis.PCT/US97/15928101520253035CA 02264748 1999-03-01WO 98/10783 PCT/US97/15928Intravenous or intra-organ injections may beemployed to prevent or suppress transplant rejections,such as kidney transplants.The term "treating" in its various grammatical formsin relation to the present invention refers topreventing, curing, reversing, attenuating, alleviating,minimizing, suppressing or halting the deleteriouseffects of a disease state, disease progression, diseasecausative agent (e.g., bacteria or viruses) or otherabnormal condition.Determining a pharmaceutically effective amount ofMUC-1, a MUC-1 derivative or a MUC-1 carbohydratederivative is well within the purview of the skilledclinician and largely will depend on the exact identityof the inventive compound, particular patientcharacteristics, route of administration and the natureof the disorder being treated. General guidance can befound, for example, in the publications of theInternational Conference on Harmonisation and inREMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28,pp. 484-528 (Mack Publishing Company 1990).Determining a pharmaceutically effective amountspecifically will depend on such factors as toxicity andefficacy of the medicament. Toxicity may be determinedusing methods well known in the art and found in theforegoing references. Efficacy may be determinedutilizing the same guidance in conjunction with themethods described below, for example, in Examples 8 and9. A pharmaceutically effective amount, therefore, is anamount that is deemed by the clinician to betoxicologically tolerable, yet efficacious.The foregoing discussion and following examples arepresented merely for illustrative purposes and are notmeant to be limiting. Thus, one skilled in the art willreadily recognize additional embodiments within the scopeof the invention that are not specifically exemplified.EXAMPLES101520253035CA 02264748 1999-03-01W0 98/10783 PCT/US97/15928-15-Example 1 _This example shows that mice bearing MUC—1transfected tumor cells (which secrete MUC-1 into theserum) who develop the highest serum MUC—1 levelsfollowing tumor transplantation are the mice showing theshortest survival time compared to mice who had non-detectable levels of serum MUC-1 following tumortransplantation.MUC—1 Hi cells were transplanted subcutaneously andtumors developed in over 90% of the mice within 6-8weeks. Mice were followed for survival and serum MUC—1levels were measured at various times following tumortransplant. Figure 4a shows that the 8 mice that diedbefore day 63 had higher serum MUC—1 levels than thosethat survived to day 63. Figure 4b shows that of the 8mice who died before day 63, there was a significantdirect association between MUC—1 level and survival time.Example 2This example shows that intravenous injection ofMUC—1 mucin made mice more susceptible to the tumortransplant of human MUC—1 transfected tumor cells. Theprocedures employed in this example are similar to thosedescribed in Fung, et al. Cancer Research 51: 1170-1176(1991), which is hereby incorporated by reference.However, in the present example, the human MUC—1 systemwas used instead of the epiglycanin system.CB6 mice were injected intravenously with MAb B27.29affinity purified MUC—1 mucin (20 micrograms eachtreatment or approximately 400 units) prior to (day-2)and after (days +2 and +6) an attempted tumor challenge.Affinity purified mucin had been prepared by affinityadsorption on a B 27.29 CnBr-Sepharose matrix from apleural effusion fluid obtained from. a patient withadenocarcinoma of the ovaries. MUC—1 mucin treated andcontrol mice were challenged with 1x10°GZ-Hi tumor cellsinjected into the subcutaneous space along the flank.Mice were observed by palpation and visual inspection at101520253035CA 02264748 1999-03-01W0 98/ 10783 PCT/U S97! 15928-15-the injection site for the occurrence of tumors over aperiod of 8 weeks.Results:Experimental Group % Tumor TakeControls 60%MUC-1 pretreated 100%Example 3This example shows that adding purified human MUC-1mucin to human T-cell cultures strongly inhibits T—cellproliferation against a strong allo-antigenic stimulus invitro.The mixed lymphocyte reaction is conducted by mixingthe lymphocytes of HLA disparate individuals in in vitrotissue cultures. The "responder population" in thisexperiment is purified T—cells from one population, whilethe "stimulator" population in this experiment is theadherent antigen presenting cells obtained from an HLAmismatched individual donor. The two cell populationswere mixed and cultured either in the presence or absenceof various doses of B27.29 affinity purified MUC-1 mucinthat was purified from a pleural effusion fluid. Theresults of this experiment are presented in Figure 7.Example 4This example shows that MUC-1 mucin binds to ICAM-1.Tumor cells that are transfected with human MUC-1 boundto ICAM—1 on endothelial cells. MUC-1 appears to be aligand for Intercellular Adhesion Molecule (ICAM-1).Antibodies to ICAM-1 and MUC-1 inhibited adhesion of MUC-1 positive cells to human umbilical vein endothelial cell(HUVEC) monolayers in a manner directly related to thelevel of MUC-1 expression. Similar antibody inhibitionof adhesion of MUC-1 positive cells was found using ICAM-l transfected cells and immobilized recombinant humanICAM-1-Ig fusion protein. These results suggest thatICAM—1 binds to the peptide core of the tandem repeat101520253035CA 02264748 1999-03-01W0 98/ 10733 PCT/US97/15928-17-sequence of the MUC—l molecule thereby implicating MUC—lin breast cancer metastases and immune suppression.MUC-1 preferentially localizes to free membranesurfaces including a peripheral ensheathing pattern ofstaining on the surface of intravascular tumor emboli.Tumor emboli stained for MUC-1 using the DABimmunoperoxidase technique showed intense peripheralstaining in contrast to solid tumor nests which show adiffuse cytoplasmic staining pattern. There was unevenstaining along the endothelial surface, which probablyrepresents shed antigen.Several mucins have recently been described asvascular ligands. Berg, et al. Nature 366:695 (1993).Thus, the staining pattern of MUC-1 suggested that MUC-1may be involved in endothelial cell adhesion.Tumor cell - endothelial cell adhesion assay:Endothelial cell monolayers were grown in 24 well tissueculture plates and stimulated with 20 U/mL TNFa + 20 U/mLIL-1B (Phariningen) for 4 or 24 hr. To these platesBCECF AM ester (Molecular Probes) fluorescent dye labeledtumor cells (1.5 x 10U500uL/well) were added andincubated for 25 min. at 37 C. Non -— specificallyadherent cells were removed by vigorous agitation (shakerat 175 rpm) and peripheral aspiration. Remainingadherent cells were then lysed with detergent (NP-40) for30 min. and the dye signal was quantitated using a SPEXfluorimeter and compared to the signal from the totalnumber of cells added to each well.Tumor cell - immobilized ICAM-1 adhesion assay: 96well tissue culture plates were coated with 50 uL of asolution with 20 ug/mL recombinant soluble ICAM-1 in PBSfor 1 hr at room temperature. Wells were then blockedwith 1% BSA for 2 hr at 37 C and washed four times withPBS. Antibodies (10 ug/mL) (anti—ICAM-1 164B, 18E3D,84Hl0, anti-E selectin l.2B6) and soluble MUC-1 (10ug/mL) were added to the wells for 90 min. at roomtemperature. Wells were then washed again beforeaddition of cells. BCECF labeled tumor cells were101520253035CA 02264748 1999-03-01W0 93/10783 PCT/US97/15928pretreated +/- 20 ug/mL of anti—MUC-1 (B27.29, or DF3P)and were then added to the appropriate wells for 40 min.at 37 C. Wells were then washed and percent adhesion wasthen determined as described in the tumor cell -endothelial cell adhesion assay.Results: A series of monoclonal antibodies werescreened for their ability to inhibit the binding of ahuman breast carcinoma cell line (MCF—7) to humanumbilical vein endothelial cell (HUVEC) monolayers whichhad been stimulated with IL-IB and TNF-a. Consistentwith other studies of epithelial cancer cell lines,following four hour cytokine stimulation of the HUVECs,antibodies to E selectin or its ligands (SLe“U showed thegreatest inhibition of adhesion of the MCF—7's to theHUVECs (Fig. 2a). Antibody to ICAM-1 (84H10) also showedinhibition but much less than that obtained by anti—Eselectin (1.2B6). An antibody to MUC—1 (B27.29), had noeffect. The adhesion molecule profile of cytokinestimulated HUVEC's varies over time. See Iwai, et al.,Int'l Journal of Cancer 54: 972 (1993). E-selectin isexpressed early with a maximum peak at 4 hours and isgradually lost by 22 hours. ICAM-1 is also expressed at4 hours but it remains highly expressed after 22 hours.Thornhill, et al. Scand. J. Immunology 38: 279 (1993).Therefore, the adhesion assay was repeated afterprolonging endothelial stimulation to 24 hours. Underthese conditions antibodies to E selectin and sLe“ nowshowed very little inhibition (Fig. 2b). Consistent withthe adhesion molecule profile at 24 hours, antibodies toICAM-1 showed substantial inhibition of tumor cell-HUVECadhesion. Interestingly, the MUC-1 antibody (B27.29)also showed quite potent inhibition of the MCF-7 cells tothe HUVEC's (Fig. 2b)., This suggested that MUC-1 couldbe an additional mucin type ligand for ICAM-1.To isolate MUC—1 as the relevant adhesion molecule,the 24 hour adhesion assay was repeated using theparental murine 410.4 mammary adenocarcinoma cells aswell as two derivative cell lines transfected with the101520253035WO 98/10783CA 02264748 1999-03-01PCT/US97/ 15928-19-human MUC-1 gene but showing either low (GZLo) or high(GZHi) expression of MUC-1. The level of MUC-1expression was quantitated by concurrent flow cytometricanalysis as well as by immunohistochemistry. Fig. 3ashows an increase in adhesion with increasing MUC-1expression with GzHi showing a two fold increase inadhesion over GZLo and a four fold increase over the wildtype (410.4). This adhesion was inhibited by antibodiesto MUC-1 (B27.29) and ICAM-1 (18E3D) while anti—Eselectin and control antibodies (CD31) showed no effect(Fig. 3b).To exclude the possibility that adhesion was beingmediated by endothelial molecules other than ICAM-1, theadhesion of the MUC-1 transfectants was studied usingimmobilized recombinant human ICAM-1-Ig fusion protein.Adhesion was compared to Bovine Serum Albumin and410.4 and GZLo cellsdisplayed similarly low binding to 1% BSA and ICAM-1coated wells. GZHi however, showed approximately 3.5fold higher binding to ICAM-1 than did 410.4 and GZLo,while its binding to 1% BSA was comparable to that of410.4 and GZLo (Fig. 5). In addition, anti-MUC-1(B27.29) and anti—ICAM-1 (18E3D) antibodies successfullyabrogated the increased adhesion of GZHi to ICAM-1. Theseantibodies did not, however, inhibit adhesion of highCollagen type I as controls.MUC-1 expressing cells to a collagen type I control in- Pretreatment of the ICAM-1coated wells with soluble MUC-1 was equally effective atblocking adhesion of GZHi to ICAM-1.These results show that MUC-1 can selectively bindsubsequent experiments.to the immunoglobulin superfamily member, ICAM-1. Apossible binding site on MUC-1 for ICAM-1 is suggested bythe inhibition of the interaction by B27.29. Thisantibody recognizes a limited sequence of the MUC-1 corepeptide tandem repeat which encompasses that recognizedby SM3. Reddish, et al. Tumor Marker One 7: 19 (1992).This implicates the core peptide sequence of the tandemrepeat as the binding site for ICAM-1.101520253035CA 02264748 1999-03-01W0 98/ 10733 PCT/US97/15928-20-Example 5A molecule with considerable sequence homology toICAM—l is ICAM—3. This adhesion molecule shows a uniquepattern of expression and is found typically only in thenew vasculature of solid tumors and in certain vascularhyperplasias. Patey, et al. Am. J. Pathol. 148: 465-472(1996).Using the procedures described in Example 4, theability of MUC-1 to bind to ICAM-3 is assessed. Once ithas been determined that ICAM—3 binds to MUC-1, varioustreatment regimens can be developed based on the ICAM-3/MUC—1 binding.Using the procedures described in Example 4. theability of MUC-l to bind to other adhesion molecules isassessed. Once adhesion molecules have been identifiedthat bind to MUC-1, various treatment regimens can bedeveloped.Example 6one example of an autoimmune disease is myastheniagravis. In this disease a patient produces antibodies tothe acetylcholine receptor at the neuromuscular junction.This production of antibodies is dependant upon theactive participation of CD+helper T cells. These T cellsare activated by antigen presenting cells that expressICAM-1 which interacts with 1? cell LFA—1 forming anadhesion between APC and T cells. Blocking of thisinteraction will prevent T cell activation and thesubsequent T cell help needed for the disease manifestingantibody response. Patients with symptoms ofmyasthenia gravis are treated by the systemicadministration of a pharmaceutically acceptable amount ofMUC-l, a MUC-1 derivative or a MUC-1 carbohydratederivative, thereby down regulating the production od CD4T cell helpers which will then turn off the production ofthe specific antibody producing B cells that aredependant upon T cell help. Antibody previously produced10152025W0 98/10783CA 02264748 1999-03-01PCT/U S97/ 15928-21-will clear from the circulation in the normal time coursefor IgG half—life clearance leading to a relief ofsymptoms of the crippling symptoms of this disease.Example 7In the case of inflammatory arthritis a CD4+T cellpopulation is known to invade the synovium of effectedjoints and to produce the pro-inflammatory cytokines TNFand interferon gamma. These cytokines then induceresident synovial cells to produce collagenase and otherhydrolytic enzymes that degrade of collagen and destroytendons cartilage and ligaments. The activation of theseCD4 T cells requires ICAM-1 interaction with antigenpresenting cells. This interaction could be blocked bythe intrasynovial (or systemic) administration of apharmaceutically effective amount of MUC-1, a MUC-1derivative or a MUC-1 carbohydrate derivative, therebyblocking the T cell activation and subsequent productionof the pro-inflammatory cytokine mediators of thiscrippling disease.Exam e 8This example demonstrates the ability of syntheticpeptides, having multiple tandem repeats of the MUC—1core, to inhibit T cell proliferation.10152025CA 02264748 1999-03-01W0 98/ 10783 PCT/US97/15928_-22-Mucins:MUC-1 was purified from ascites fluid obtained fromovarian cancer patients. 2M sodium acetate at pH 5 wasadded ‘to the ascites fluids and centrifuged for 30minutes at 20krpm. After filtration through a 0.45micron cellulose acetate filter, the solution was mixedwith B27.29 Mab (Reddish et al., J. Tumor Marker Oncol.7:19-27 (1992)) CNBr coupled to sepharose 4B overnight,followed by washing with 1M NaCl/PBS. The affinity boundMUC—1 mucin was eluted with 50 mM diethanolamine (Fisherpurified) in 150 mM Nacl at pH11. The eluant wasneutralized with 2M sodium acetate at pH 5. The affinitypurified material .was dialyzed against PBS and thensterile filtered with Nalgene 0.2 micron celluloseacetate syringe filter. The affinity purified MUC-1mucin was quantitated by using Truquant BR RIA assay(Biomira Diagnostics Inc., Roxdale, ON, Canada). For thecalculation of amount of MUC—1 mucin, the conversionformula 1 BR unit as approximately 50 ng of MUC-1 mucin,was used.Synthetic MUC—1 derivatives contained 1, 3, 4, 5 or6 tandem repeats of the MUC-1 core and were approximately16, 60, 80, 100 and 120 amino acids in length; The 16-mer contained the sequence GVTSAPDTRPAPGSTA. The otherderivatives contained. tandem :repeats of the sequenceTAPPAHGVTSAPDTRPAPGS.Ovine submaxillary mucin (OSM) was employed as acontrol.101520253035CA 02264748 1999-03-01W0 93/10733 PCTIU S97/ 15928-23-T cell cultures:Enriched T cell populations were purified from buffycoats obtained from normal red cross donors using nylonwool columns by previously reported procedures. See,e.g., Agrawal et al., J. Immunol. 157: 2089-95 (1996) andAgrawal et al., J. Immunol. 157: 3229-34 (1996). For theallo MLR, mitomycin C treated allogeneic PBLs werecocultured with purified T cells in the presence orabsence of affinity purified MUC-1 mucin or control OSM.In most of the experiments, the T cells were cultured 6-7days in AIM V medium in the absence or presence of MUC-1,MUC-1 derivative or OSM at the indicated concentration.After this time, the T cells were harvested, washed andcultured as indicated.Proliferation assay:Purified T cells (1WVml) were cultured in AIM Vmedium with allo PBLs in the absence or presence of MUC-1, MUC-1 derivative or OSM 10 ug/ml for 6-7 days. Tcells were harvested and plated in 96 well flat bottomplates at 105/well with allo PBLs (105/well), in thepresence or absence of affinity purified MUC-1, MUC-1derivative or OSM. Control cultures were treated witheither 50 U/ml IL-2 or 1 ug/ml anti—CD28 Mab. After 4days of culture, 3H—thymidine (1 uCi/well) was added.The cells were harvested on the fifth day' and 3H-thymidine incorporation was measured by liquidscintillation.Results:As seen in Figure 8, synthetic peptides containing3-6 tandem repeats of the MUC-1 core significantlyreduced the level of T cell proliferation relative tocontrol. This effect was not observed with a peptidecontaining a single repeat. Moreover, this effect wasreversed by treatment with IL-2 or CD28 Mab. As seen inFigure 9, inhibition of T cell proliferation was directlyproportional to the number of MUC-1 core repeats present.10152025303540CA 02264748 1999-03-01W0 93/ 10733 PCT/US97/15928-24-Table 1. demonstrates the. statistical significance ofthese data as compared to the medium control.Table 1Sample n3 repeats =0.00094 repeats =0.00075 repeats <0.00016 repeats <0.0o01Table 2 demonstrates the statistical significance ofthese data compared to the OSM control."Table 2Sample D3 repeats =0.0364 repeats =o.0035 repeats <0.00016 repeats <0.0001Example 9This example illustrates the direct correlation ofnumber of tandem repeats with the inhibit T cellproliferation. The methods used are described in Example8.Synthetic peptides, as described in Example 8,corresponding to 3, 4, 5 or 6 MUC-1 repeats, were addedat 10 ug/ml to allo cultures and T cell proliferation wasmeasured at 14 days from the start of culture. Becausethe same amount of each peptide was present in each well,all samples contained the same total number of individualrepeats. Thus, the only difference was the number ofrepeats tandemly joined in the peptide. In other words,any effect observed is a result of the oligomeric natureof the repeats, not the absolute number of them.As seen in Figure 9, the number of tandemly joinedrepeats directly correlated with inhibition of T cellproliferation. The percent reduction in T cellproliferation was directly proportional to the number oftandem repeats of MUC-1 added to the culture (R = 0.85,p < 0.0001).

Claims (19)

We Claim:

1. A method of treatment, comprising administering a pharmaceutically effective amount of MUC-1, a MUC-1 derivative or a MUC-1 carbohydrate derivative to a patient suffering from an autoimmune disorder.

2. The method of claim 1, wherein said patient is suffering from an autoimmune disorder selected from the group consisting of inflammatory arthritis, rheumatoid arthritis, psoriasis, allergies, allergic contact dermatitis, and ankylosing spondylitis.

3. The method of claim 1, wherein said patient is suffering from an autoimmune disorder selected from the group consisting of myasthenia gravis, systemic lupus erythematosus, polyarteritis nodosa, Goodpastures syndrome, isopathic thrombocytopenic purpura, auto;mmllne hemolytic anemia, Grave's disease, rheumatic fever, pernicious anemia, insulin-resistant diabetes mellitus, bullous pemphigold, pemphigus vulgaris, viral myocarditis (Cocksakie B virus response), autoimmune thyroiditis (Hashimoto's disease), male infertility (autoimmune), sarcoidosis, allergic encephalomyelitis, multiple sclerosis, Sjorgens disease, Reiter's disease, Celiac disease, sympathetic ophthalmia, and primary biliary cirrhosis.

4. The method of claim 1, wherein said patient is suffering from organ transplant rejection or graft versus host disease.

5. The method of claim 1, wherein said MUC-1 derivative comprises more than one MUC-1 core repeat.

6. The method of claim 1, wherein said MUC-1 derivative comprises from about 2 to about 100 MUC-1 core repeats.

7. The method of claim 6, wherein said MUC-1 derivative comprises from about 2 to about 20 MUC-1 core repeats.

8. The method of claim 7, wherein said MUC-1 derivative comprises from about 3 to about 6 MUC-1 core repeats.

9. The method of claim 5, wherein said core repeats are arranged tandemly.

10. The method of claim 5, wherein said MUC-1 derivative is glycosylated.

11. A pharmaceutical composition comprising a pharmaceutically effective amount of MUC-1, a MUC-1 derivative or a MUC-1 carbohydrate derivative, in combination with a pharmaceutically effective vehicle.

12. The composition of claim 11, wherein said MUC-1 derivative comprises more than one MUC-1 core repeat.

13. The composition of claim 12, wherein said MUC-1 derivative comprises from about 2 to about 100 MUC-1 core repeats.

14. The composition of claim 13, wherein said MUC-1 derivative comprises from about 2 to about 20 MUC-1 core repeats.

15. The composition of claim 14, wherein said MUC-1 derivative comprises from about 3 to about 6 MUC-1 core repeats.

16. The composition of claim 12, wherein said core repeats are arranged tandemly.

17. The method of claim 12, wherein said MUC-1 derivative is glycosylated.

18. The method of claim 11, wherein said pharmaceutically effective amount is an amount effective for treating an autoimmune disorder or an inflammatory disorder.

19. The method of claim 11, wherein said pharmaceutically effective amount is an amount effective for treating organ transplant rejection or graft versus host disease.