[CANCER RESEARCH58. 315-321. January 15. 19981 Tolerance and Immunity to MUC1 in a Human MUC1 Transgenic Murine Model1

Gerald J. Rowse, Richard M. Tempero, Michelle L. VanLith, Michael A. Hollingsworth, and Sandra J. Genffler@

Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale. Scottsdale, Arizona 85259 (G. J. R., S. J. G.J. and Eppley Institute for Research in Cancer and Allied Diseases, University ofNebraska Medical Center, Omaha. Nebraska 68198 (R. M. T.. M. L V.. M. A. H.J

ABSTRACT Studies have shown that MUC1 is highly immunogenic in mice (8—10);this is most likely due to the low degree of homology in the The human epithelial , MUd, is a large transmembrane glyco TR region between MUC1 and its murine homologue, Mud (1 1). that is expressed on most simple epithelia. It is overexpressed and Mice immunized with peptides corresponding to the MUC1 TR pro aberrantly glycosylated on many human epithelial tumors, including more duce strong humoral immune responses and mice immunized with than 90% of human breast cancers. MUC1 is of interest as an immuno therapy target because patients with breast, ovarian, and pancreatic tumor cells that express MUC1 or with injections of MUC1 cDNA are cancers have T lymphocytes in their tumor-draininglymph nodes that can in most cases protected from subsequent tumor challenge by strong be induced to recognize and lyre MUC1-expressing tumor cells. We have humoral and cellular responses to MUC1 (12—14).The degree of produced a transgenic mouse model that expresses the human MUC1 dissimilarity between the sequence of human MUC1 and murine molecule on an inbred CS7BI/6 background to Investigate the effect of Muc 1 makes it likely that human MUC 1 is recognized as a foreign endogenous expression of MUC1 on the ability of mice to generate anti antigen by mice. Hence, it is not surprising that the murine immune tumorimmunitytoMUC1-expressingtumors.Transgenicmiceexpressed response to MUC1 provides effective protection against MUC1-ex the humantransgeneIna patternandlevelconsistentwiththatobserved pressing murine tumors because these responses are not produced in In humans. Transgenlc mice were tolerant to stimulation by MUC1 as the context of immunological tolerance. evidencedbytheabilityof MUC1-expressingtumorcellsto growin these Tolerance is a major hindrance to the development of effective mice, whereas MUC1-expressing cells were eliminated from wild-type mice. Moreover, transgenlc mice Immunized with MUC1 peptides failed to immune responses to tumors (15, 16). Tolerogenic mechanisms pro exhibit Immunoglobulin class switching to the IgG subtypes. These data tect against untoward autoimmune reactions under nonpathological suggest that endogenous expression of MUC1 protein by MUC1 tram circumstances (17—19)but may preclude effective immunotherapy for genic mice Induces T-cell tolerance to stimulation by MUC1. The tram tumors. In this report, we describe the production of an inbred syn genic mice will provide a useful model to Investigate the mechanisms that geneic C57BI/6 mouse strain transgenic for human MUC1 regulate Immunological tolerance to tumor antigens and will facilitate the (MUC1.Tg) and its initial characterization as a model system to Investigation of anti-MUC1 immunotherapy formulations. investigate parameters of immunological tolerance to MUC 1. Mice that are transgenic for a foreign protein develop B- and T-cell com INTRODUCTION partment tolerance and are refractory to immunization with the protein The human epithelial mucin MUC1 is a type I membrane-associ encoded by the transgene (20—22).Such immune tolerance is pre ated expressed on most simple epithelia. It has an ex dicted to be similar to that seen with endogenous self ; tensive extracellular domain that is composed primarily of a variable consequently, the correct temporal and spatial expression of MUC I number of TRs3 (1—3).The TR domain is a heavily O-glycosylated during development should have profound effects on the ability of the region rich in serine, threonine, and proline residues and consists of munne to provide effective anti-MUC 1 tumor immu repeating units of 20 amino acids with the sequence PDTRPAPG nity. A comparison of the immune response of the MUC1.Tg mice STAPPAHGVTSA MUC1 is frequently overexpressed in human with that of wt C57B1/6 mice will provide insight into the effect of tumors, including of the breast, lung, pancreas, colon, and endogenous expression of the MUCJ on immune responses and ovaries, Expression of MUC1 by tumors often results in loss of apical immune tolerance and facilitate the evaluation of anti-MUC1 localization and alterations in posttranslational modifications corn formulations in vivo within the context of an animal that expresses paredtocorrespondingnormalcelltypes(reviewedinRef.4). MUC1 Lymph nodes from patients with breast, ovarian, and pancreatic adenocarcinornas contain lymphocytes that recognize and lyse MUC 1-expressing tumor cells (5, 6), Recognition of MUC 1 by lyrn MATERIALSAND METHODS phocytes may be due to exposure of in the TR region of the Generationofthe MUC1,TgMice. Micewerehousedandmaintainedin protein backbone that normally are not exposed due to extensive microisolator cages under specific pathogen-free conditions. A 10.6-kb O@, These findings provide evidence for MUC 1-specific genomic SacII fragment containing the entire MUCI gene sequence, as well as stimulation of the immune system (@—7);however,cellular responses 1.5 kb of 5' sequence and 800 bases of 3' sequence, was isolated (23). DNA to MUC1 in humans have only been demonstrated by in vitro assays was diluted to a final concentration of I mg/mi for injection into the pronuclei following extensive stimulation of lymphocytes with antigen and of fertilized CS7B1/6mouse eggs, which were then transferred to pseudopreg cytokines (5, 6), nant CD1 females. Southern illot Analysis, Founder mice were identified by Southern blot analysis. Ten-gil aliquots (approximately 5 @.tgeach)of tail DNA were digested Received 7/16/97; accepted I 1/1 1/97. The costs of publication of this article were defrayed in part by the payment of page with I@coRI(NewEngland Biolabs, Beverly, MA) at 37°Cina total volume of charges. This article must therefore be hereby marked advertisement 10 accordance with 25 @1.TheDNA digestswereelectrophoresedthrougha0.7%agurosegeland 18 IJS.C. Section 1734 solely to indicate this fact. transferred to nylon membrane (Hybond N, Amersham Corp., Arlington I This work was supported by the Nat1on@l Cancer Institute, NIH, Grant R01-CA64389 and fundsfromthe MayoFoundationforliducationandResearch(toS.J. 0.); and by Heights, IL.). Subsequent hybridization and washing procedures were carried National Cancer Institute, NIH. Grants ROl-CA57362, R30 CA36727, and T32 CA09476 out according to the manufacturer's specifications. The human probe pMUC7 andGrantLB595fromthe NebraskaDepartmentofHealth(to M.A.H.). (2),correspondingto—@500bpof theTRdomain,waslabeledbyrandom 2 To whom requests for reprints should be addressed, at Department of Biochemistry priming in the presence of Ea-32PIdCTP (Amersham) to a specific activity of and MolecularBiology,MayoClinicScottsdale,13400li SheaBoulevard,Scottsdale, >1 X 10@dpm/@.Lg. AZ 85259. 3 The abbreviations used are: TR, tandem repeat; MUC1.Tg, MUCI transgenic; wt, PCR Screening,PCRwas usedto routinelyidentifyMUC1.Tgpositive wild-type. mice in the colony. PCR was carried out in a total volume of 50 @lin 315

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Perkin-Elmer Corp. Gene Amp tubes with the following reagents: 5 @llOX University of Pittsburgh School of Medicine, Pittsburgh, PA), which contains PCR buffer (Life Technologies, Inc., Grand Island, NY), 0.02% formamide, 1 5.25 copies of the TR. Peptides were synthesized by manual solid-phase mM MgCl2, 0.2 mM dNTPs, 100 nM 5'-CTTGCCAGCCATAGCACCAAG-3' synthesis using 9-fluorenylmethyloxycarbonyl NH2-terminal protected amino (bp 745-765; Ref. 1), and 100 flM 5'-CTCCACGTCGTGGACATfGATG-3' acids. Synthesis, purification, and characterization by mass spectroscopy of the (bp 1086—1065; Ref. I) primers, 10.25 units of Taq polymerase (Life Tech products are described elsewhere in detail (28, 29). nologies, Inc.), I @loftail DNA (approximately 500 ng), and reagent-quality PeptideImmunization.Freeze-driedpeptideswerereconstitutedinPBS, H20.The amplification program consisted of one cycle of 10 mm at 94°Cand pH 7.4, and mixed with equal volumes of RIB! Detox adjuvant (RIB! Immu 40 cycles of 30 s each at 94, 61, and 72°C.ThePCR product of each reaction noChem Research Inc., Hamilton, MT). The mixture was vortexed vigorously was analyzed by size fractionation through a I% agarose gel. Amplification of for 10 mm. Mice were injected i.p. with 50 @&gofpeptide and 50 @gof MUC1-positive DNA resulted in a 500-bp fragment. adjuvant in a total volume of 200 @l.Controlmice were immunized with 50 Cell Culture.Cell culturereagentswerepurchasedfromLifeTechnolo ,Lg of adjuvant alone. The immunizations were repeated at 3 and 6 weeks, and gies, Inc., unless noted otherwise. The B16-BL6 (Bl6) murine cell blood samples were obtained by tail vein bleeding 1week following the second line (24), kindly provided by J. Talmadge (University of Nebraska Medical and third immunizations. Center, Omaha, NE), was cultured in DMEM supplemented with 10% fetal ELISA. Detection of MUC1-specific antibodies was carried out by ELISA bovine serum, nonessential amino acids, vitamins, penicillin (50 units/mI), and using synthetic peptides corresponding to 1.2 or 5.25 copies of the MUC1 TR. streptomycin (50 @xg/ml)cocktailand 1mMsodium pyruvate (Sigma Chemical Briefly, 96-well microtiter plates (Immunlon 4, Dynatech, Chantilly, VA) were Co., St. Louis, MO). coated with 100 id/well of peptide (5 @g/mlinPBS, pH 7.4) and incubated Expression of -tagged MUC1. Epitope-tagged MUCI (MUC1F) overnight at room temperature. The plates were blocked with 200 s.d/well of cDNA was subcloned into the expression vector pH(3APr-l-neo (25) as de 5% skim milk in PBS for 2 h at room temperature. The plates were washed scribed previously (26). Bl6 cells were transfected with linearized plasmid once with 200 pAof buffer (PBS + 0.05% Tween 20). To determine antibody using the Lipofectin reagent according to the manufacturer's specifications. titers, 50 @lof dilute sera (1/100 to 1/12,800) was added per well in triplicate After transfection, single colonies were selected and expanded for MUC1F to the 96-well plates and incubated at 37°Cfor1 h. The plates were washed screening. Clonal cell lines were evaluated for MUC1F expression by immu four times with 200 @lofbuffer per well. Secondary antibodies specific for noblouing, flow cytometry, and analysis of surface immunofluoresence with different immunoglobulin subtypes (rabbit antimouse; Zymed) were added at the M2 antibody (International Biotechnologies Inc., Eastman Kodak Co., New 1/100 dilution, 50 s.d/well,and the plates were incubated for 1 h at 37°C.The Haven, CT). plates were washed four times with 200 @lofbuffer per well. The tertiary Immunoblotting. Cell lysates were prepared by scraping cells into 1 ml of antibody (peroxidase-conjugated goat antirabbit, Zymed) was added at 1/500 lysis buffer (10 mM Tris, 150 mM NaCI, I mrviphenylmethylsulfonyl fluoride, dilution (50 @.dJwell),andthe plates were incubated at 37°Cfor1 h. The plates 1% Triton X-lO0). After a 30-mm incubation at 4°C,lysateswere centrifuged were washed four times with 200 @lofbuffer per well. Plates were developed at 1000 X g (4°C)for 5 mm to remove cell debris. Supernatants were by the addition ofSO p1/wellof O-phenylene diamine (1 mg/ml; Sigma). Plates transferred to fresh tubes and stored at —20°C.Protein content of lysates was were allowed to develop for 15 mm, and absorbance was measured at A@5,,,@ determined by using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with in a plate reader (V,,...,@;Molecular Devices Co., Menlo Park, CA). BSA as standard.Celllysates wereresolvedon 6%denaturingpolyacrylamide Quantitative ELISA. All ELISA reagents were supplied by Zymed unless gels, transferred to polyvinylidene difluonde membranes electrophoretically, noted otherwise. Microtiter plates (96-well) were coated with 50 @lofpurified and blocked in Blouo [5% dry milk in TBS (0.9% NaCl, 10 mMTris, pH 7.4, IgGi, IgG2a, IgG2b, IgG3, or 1gM murine myeloma proteins (2 @g/ml-l ng/ml 0.5% MgCl2)] overnight. M2 or isotype control antibody (2 @gIml)in Blouo in PBS, pH 7.4) at 37°C for 2 h. Plates were incubated overnight with 200 @l were incubated for I h at 25°C.Followingthree 10 mm washes with Blotto, ofblocking solution (5% dry milk in PBS) at 4°C.PBS with 1% Tween 20 was horseradish peroxidase-conjugated secondary antibodies (Zymed Laboratories used for all scm dilutions, antibody dilutions, and washes. Diluted rem (50 p.1) Inc., San Francisco, CA) were diluted 1:2000 in Blotto and incubated for 1 h were added to the appropriate wells. Following four washes, 50 p.1of rabbit at 25°Cfollowedby three washes with Blono. ECL reagents were applied as antimouse isotype specific antibody at 1/300 dilution was incubated for 45 mm per the manufacturer's instruction, and the blots were exposed to ECL at 37°C.Followingfour washes, 50 p.1of goat antirabbit alkaline phosphatase sensitive film (Amersham). conjugated antibody at 1/1000 dilution was incubated for 45 mm at 37°C. Tumor Challenges.wt (TheJacksonLaboratory,BarHarbor,ME)and Following four washes, 50 p.1ofsubstrate buffer(lO% diethanolamine, pH 9.8) MUC1.Tg C57B1I6 mice were anesthetized with Metofane (Pitman-Moore, containing p-nitrophenyl phosphate (1 mg/ml) was added for color develop Mandelein, IL), and MUC1F-expressing B16 or control transfectants were ment. The reaction was stopped with the addition of 50 p.10.5 MNaOH, and injected s.c. between the scapulae. Tumor length (1) and width (2r) were color intensity was quantified at A,,@5nmMyeloma protein standards were used determined at the indicated days postinjection. Tumor volume was calculated to generate isotype-specific antibody binding curves. Within the linear range, using the formulas 4/3irr@ for spherical tumors and irr@lfor cylindrical tumors. the myeloma standards were used to determine the concentration of peptide . Tumors and normal tissues were obtained from specific antibody bound in diluted sera samples. wt and MUC1.Tg mice. Tissues were fixed in methacarn (60% methanol, 30% chloroform, 10% glacial acetic acid), paraffin embedded, and sectioned for immunohistochemical analysis. Briefly, endogenous peroxidase activity was RESULTS decreased by incubating the tissue sections with 0.6% H202 in PBS (pH 7.4) for 30 mm at 25°C.Afterwashing with PBS, pH 7.4, sections were blocked with Production of MUC1.Tg Mice. Three mice out of a total of 104 50% FCS and stained with HMFG-2 (IgGl) murine hybridoma (which recog mice screened were found positive by Southern blot analysis for the nizes the human MUC1 TR sequence but is not reactive with the mouse Mud MUCJ transgene. Of these founders, two lines transmitted the trans protein; Ref. 2), CT1 rabbit polyclonal antibody to the cytoplasmic tail of gene to their progeny. Both lines were fertile and healthy, and one line MUC1 (27), or antibody previously blocked with 5 mg/ml synthetic peptide (79.24) was selected for further studies. For immunohistochemical (undiluted) for 1 h at 25°C.Followingthree 5-mm washes with PBS, pH 7.4, analysis, the MUCJ transgene was moved onto the mouse Mud the sections were incubated with peroxidase-conjugated rabbit antimouse deficient background (30), and the pattern of expression was deter antibody (Dako Corp., Carpinteria, CA; diluted 1:100) for 1 h at 25°C. mined using an antibody to the MUC1 cytoplasmic tail (27). For Following three 5-mm washes, the substrate solution, consisting of 0.03% investigation of tumor growth and humoral immunity in MIJC1.Tg hydrogen peroxide in PBS and I mg/mI diaminobenzoate (Sigma), was added, and the color was allowed to develop for 5—8mm.Following color develop mice, transgenic animals with a mouse Mud-expressing background ment, the reaction was quenched by washing the slide in distilled water. The were used. tissue sections were counterstained lightly with hematoxylin. Immunohistochemical staining revealed that the level and pattern of SyntheticPeptides.Thepeptidesusedin thisstudywereTAPPA,a 24- MUC1 expression in cells and organs of the transgenic mice was amino acid peptide containing one complete 20-amino acid TR and 4 residues similar to that seen in humans, including the lung, mammary gland, of the adjoining repeat, and MUC1 105-mer (a generous gift of Dr. 0. Finn, and pancreas (Fig. 1) and the kidney (distal convoluted tubules and 316

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TOLERANCE IN MUC1 TRANSGENIC MICE

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MUCIexpression1.MUC1expressionintissuesoftheMUC1.Tgmouse.TissueswerestainedwithCT1antiserum,reactivewiththeMUCIcytoplasmictall(A.C,andE)todemonstrate aciniin or with peptide-blocked CF1 (B, D, and F) as a negative control. Specific staining was observed on the lumenal surface of bronchial in the lungs (A), the the pancreas (C), and the lumenal surface of mammary epithelial cells (E). Staining was not observed in peptide-blocked control sections (B, D, and F).

collecting ducts), gall bladder, salivary gland, stomach, and uterus not normally express Muc 1, such as colon, heart, liver, muscle, and (datanotshown).Asimilarpatternofstainingwasobservedusingthe spleen (Table 1). murine monoclonal antibody HMFG-2, although background staining Transfection of the B16 Murine Melanoma Cell Line. Bl6 is a was higher. The MUCJ transgene was not expressed in tissues that did well-characterized murine melanoma cell line derived from the C57B1/6 mouse strain (24). C57B1/6 mice developed progressively growing tumors after s.c. injection with Bl6 cells (24). An epitope miceTissuesTable1 TissueexpressionofMUCIin transgenic tagged MUCI (MUC1F) cDNA was subcloned into the BamHI site of Cl]antibody.Positivefrom MUC1.tg mice were stained with CFI antibody or peptide-blocked the expression vector pHf3APr-1-neo, which has previously been used tissuesGallbladder tissues Negative for establishing stable expression of MUC1 in a number of human and —Kidney +a Brain rodent cell lines (26), and transfected into the B 16 cell line. Several —Collecting Colon clonal lines expressing MUC1F were derived. The immunoblot shown —Distal ducts + Heart in Fig. 2 demonstrates MUC1F expression in three B16 clones, -Lung convoluted tubules + Liver —Acini Muscle designated Bl6.2, Bl6.5, and B16.9. Flow cytometric analysis was -Bronchi - Skin performed to determine MUC1F expression on cells of the B 16.2, —Type + Small intestine —MammaryII pneumocyte + Spleen B16.5, and B16.9 transfectant lines. B16.2, B16.5, and Bl6.9 lines +Pancreas gland expressed detectable levels of MUC1F on 80—92%of cells (data not +Stomach shown). Control Bl6 transfectants were produced by transfecting Bl6 +Salivary(Bninner's glands) glandAcini cells with the pH@APr-1-neo vector alone. -Collecting MUC1.Tg Mice Develop Progressive MUC1-positive Tumors; +Uterus ducts +a (virgin) MUC1-negative Variants Develop In wt Mice. To determine whether endogenous MUC1 expression would affect the growth rate @ stainednegativelytissues that stained positively for MUC1 expression; —, tissues that for MUC1 expression. of MUC1-expressing tumors, groups of seven wt and MUCI.Tg mice 317

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TOLERANCE IN MUCI TRANSGENIC MICE

Isotype control M2 shown). Interestingly, substantial differences in humoral responses were observed when mice were immunized with the 105-amino acid peptide comprising 5.25 MUC1 TR (105-mer). wt mice produced lrt O@ C@1 @1) O@ strong antibody responses to the MUC1 TR in 1gM and all IgG @@ @,o @1á ‘@o @.o @‘c ‘@o — — — — — — — — isotypes (Fig. 6). In contrast, MUC1.Tg mice exhibited a strong 1gM response but only weak IgG subtype responses (Fig. 6). The differ ences in humoral responses of the wt and MUC1 .Tg mice to the l05-mer peptide were highly significant (P < 0.05).

@@p• iØ@ 250 kDa DISCUSSION

The mechanisms that regulate immunological tolerance to tumor @4—205kDa antigens are formidable obstacles that prevent effective tumor immu notherapy. An inbred C57B1/6 MUC1 .Tg mouse has been developed Fig. 2. MUCIF expression in three Bl6 melanoma clones, Bl6.2, Bl6.5, and B16.9. to facilitate study of the parameters that regulate immunological Equal quantities of cell lysates from parental cells and MUC1F transfectants were separated on 6% denaturing polyacrylamide gels, transferred to polyvinylidene difluoride tolerance to the tumor-associated antigen MUC1. This experimental membranes, and tested for reactivity with MUC1F (M2 antibody) or an isotype control model will enable us to study the effect of endogenous expression of antibody. The transfectants expressed large quantities of MUCIF. as demonstrated by M2 the MUCJ gene on the ability of mice to produce protective immune reactivity with an antigen of greater than Mr 200,000. Specificity was confirmed by the lack of reactivity in isotype control lanes. responses to tumors, and it represents an improved model system for evaluating the efficacy of anti-MUC1 vaccine formulations in vivo within the context of existing tolerance mechanisms. were injected s.c. with 2 X iO@MUC1F-expressing Bl6.9 cells (Fig. Immunohistochemical analysis demonstrated that the 10-kb SacII 3A). MUC1.Tg mice developed progressively growing tumors with an genomic DNA fragment resulted in reliable expression of the MUCJ average volume of 1.31 ±1.05 cm3 at day 26 postinjection. Con gene in tissues that normally express MUC 1; no expression was seen versely, tumor growth in wt mice was significantly delayed in ectopic sites. This finding is consistent with previous results using (P < 0.05); only one of seven wt mice had a tumor at day 26 this construct in outbred mice, in which the correct temporal and postinjection. By day 34 postinjection, five of seven wt mice devel spatial pattern of expression were conferred by the transgene (23). oped tumors with an average volume of 0.1 1 ±0.06 cm3. Similar results were obtained with Bl6.2 cells (data not shown). Groups of 7 MUC1.Tg and wt mice were injected s.c. with control B16 transfec A tants, and both strains developed tumors at approximately the same rate (Fig. 3B). Similar results were observed when wt and MUC1.Tg mice were injected with 4 X iO@cells per mouse. Expression of MUC1 in tumors from MUCJF-transfected Bl6 cells was compared in wt and MUC 1.Tg mice. Tissue and tumor samples flimor volume were obtained for immunohistochemical analysis 4.5—6weeks after (cmi) injection. Tumors from MUC1.Tg mice (n = 10) were heteroge neously positive for MUC1 expression by immunohistochemistry (Fig. 4A). In contrast, tumors from wt mice (n = 12) were consistently MUC1-negative as determined by immunohistochemical analysis (Fig. 4C). These results suggested that MUC1-expressing tumor cells 30* 34* were eliminated in the wt mice and that MUC1 negative variants were not eliminated and eventually progressed in wt mice. Days Humoral Immunity in MUC1.Tg Mice. Sera from wt and trans genic mice challenged with MUC1-expressing tumors were tested for B antibodies that bind to a 40-residue synthetic peptide, corresponding to two copies of the TR (Fig. 5). Five of seven wt mice developed strong MUC1-specific 1gM and IgG1 responses, whereas none of the MUC1.Tg mice developed an 1gM response, and only one of the MUC1.Tg mice developed an IgGl antibody response. Similarly, Thmor volume three of seven wt mice produced a strong IgG3 antibody response, (cin@) whereas zero of seven MUC1 .Tg produced this response. MUC1- reactive IgG2a or IgG2b antibodies were not detected in any mice (data not shown). Antibody responses to control antigens (Keyhole Limpet Hemocyanin and other peptides) were similar among the control and MUC1.Tg animals (data not shown). To investigate the immune response of MUC1 .Tg mice to self peptides derived from the TR region of the MUC1 protein, wt and MUC1 .Tg mice were immunized with peptides corresponding to 1.2 Days copies (24 amino acids) or 5.25 copies ( 105 amino acids) of the Fig. 3. Rates of tumor growth in transgenic and wt mice. wt (EJ) and MUCI.Tg (0) MUC1 TR. Immunization of mice with a synthetic peptide corre C57BI/6 mice were injected with 2 X l0@MUC1-expressing Bl6 murine melanoma cells (A) and 2 X l0@°Bl6control transfectants (B). a, statistically significant difference in sponding to 1.2 copies of the MUC1 TR (TAPPA) resulted in a weak tumor volume (P < 0.05) as determined by the two-sample t test. The results are 1gM and no IgG antibody response in both wt and Tg mice (data not representative of six independent experiments. Bars, SEM. 318

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Fig. 4. Immunohistochemical staining of MUCI-transfected Bl6 melanoma tumors from wt and MVCI.Tg mice. Serial sections of tumor tissue were stained for expression of MUCIF with HMFG2 antibody, reactive with the MUC1 TR, or with an isotype control antibody as a negative controL Tumors from MUC1.Tg mice(A; n = 10) were heterogeneously positive for MUC1F expression by immunohistochemistry. In contrast, tumors from wt mice (C; n = 12) were consistently MUCIF negative as determined by immunohistochemical analysis. Specificity of the antibody staining was confirmed by the lack of staining in tumors from either group when stained with an isotype control antibody (B and D).

It was hypothesized that the endogenous expression of MUC1 Evidence that MUC1.Tg mice are tolerant to MUC1 is provided by would result in immunological tolerance to MUC1. MUC1.Tg mice the finding that sera from MUC1.Tg mice challenged with MUC1- challenged with MUC1-expressing B16 melanoma cells developed expressing tumors (with one exception) contained neither 1gM nor progressively growing MUC1-positive tumors. In contrast, wt mice IgG MUC1-reactive antibodies. In contrast, wt mice challenged with challenged with MUC1-expressing B16 cells failed to produce MUC1-expressing tumors produced approximately 10-30 p@g/mlof MUC1-positive tumor cells; only MUC1-negative variants were de MUC1 TR-specific 1gM and IgO antibodies. Further evidence of tected. One interpretation of these results is that the wt mice devel immune tolerance is provided by the results of immunizing the o_ a MUC1-specific immune response that effectively eliminated MUC1.Tg animals with MUCI synthetic peptides. wt mice immu the MUC1-positive tumor cells. This interpretation is consistent with nized with the 105-mer developed strong 1gM and IgG immune previous studies that demonstrated immunological rejection of responses. In contrast, MUC1.Tg mice developed strong 1gM anti MUC1-transfected tumor cells in wt mice (12—14).The development body responses but only weak IgG antibody responses. These results of MUC1-negative tumor variants observed in the wt mice may be indicate that in MUC1.Tg mice, the B-cell compartment does not explained by the presence of 10—18%of cells that were MUC1- undergo robust antibody class switching when challenged with the negative; these tumor cells were not eliminated by the anti-MUC1 MUC1 TR 105-mer, which suggests that the T-cell compartment is response. The MUC1-negative variants probably appeared in the affected by the endogenous expression of MUC1. Immunological MUC1-transfected B16 cell lines (although they were cloned follow tolerance to transgene products has been shown in several other ing transfection) as result of down-regulation or inactivation of cx transgenic model systems (31, 32). For example, transgenic mice pression of the MUC1 transgene or promoter. The outgrowth of expressing the soluble antigen hen egg lysozyme (HEL) showed MUC1-negative tumor variants in the wt mice underscores a funda immunological tolerance at the level of T cells; low-level expression mental problem in the development of -specific vac of HEL resulted in T-cell tolerance, whereas the induction of B-cell cines. Tumor populations display antigemc heterogeneity and retain tolerance required high levels of HEL expression (31). Studies that the capacity to alter antigen expression by different mechanisms. The evaluate the status of the MUC1.Tg T-cell compartment are in pro observed loss of MUC1 expression reinforces the widely held belief gross. that effective tumor will need to include multiple antigenic An alternateexplanation for a lack of detectable circulating anti components to completely eradicate the entire population of tumor bodies in the MUC1.Tg mice is that MUC1-specific antibodies may cells. complex with endogenously expressed MUC1. The likelihood of this 319

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is decreased by the fact that MUC1 is expressed at the lumenal or apical membrane of epithelial cells, a site of limited antibody access. MUCI.Tg Within the limits of immunoperoxidase detection, the MUC1.Tg 14000@ LI “@‘@Type animals showed no evidence of antibody deposition in the kidneys or other organ sites. Moreover, no inflammatory cell infiltrates were 12000• observed in any organs, except at sites of tumor growth. Recent I- studies suggest that the inability of B cells to undergo class switching can be overcome by immunizing mice with the TAPPA peptide 10000@ I conjugated to a very strong carrier protein (33). Using this immuno 111 0 gen, both wt and MUC1.Tg mice could be shown to produce strong :@ 8000 1gM and IgG antibody responses to the TAPPA portionof the mol I- ecule (33). The observation that sera obtained from the MUC1.Tg 6000• mice immunized with TR-KLH produce detectable 1gM and IgG MUC1-specific antibodies provides evidence that circulating MUC1- 4000@ reactive antibodies in MUC1.Tg mice can be detected by conventional solid-phase ELISA techniques. 200O@ I A 100 - gM ‘ IgGi ‘ lgG2a ‘ lgG2b ‘ lgG3 75 Fig. 6. Humoral anti-MUC1 immune responses in MUCI.Tg and wt mice immunized 25 with a 105-residue MUC1 TR peptide. One week following the third injection with MUCI-l05-merpeptide,seraofwt mice(n= 5)exhibitedhigh1gMandIgGanti-MUCI 20 A 105-mar titers. In contrast, MUC1.Tg mice (n = 5) developed high 1gM titers but only IgGi U weak IgG titers 1 week following the third injection of MUC1-lO5-mer. IgG responses (@ig/mlofsore) 15 were significantly different between wt and MUC1.Tg mice (P < 0.05) as detennined by the two-sample : test. Titer is expressed as the reciprocal of the dilution of sera that @ 10 resulted in A@5 readings that were 2.1 times greater than those observed in nonim munized mice. *, statistically significant difference in antibody titer (P < 0.05) as 5 determined by the two-sample : test.

WT MUC1 Tg In summary, we have produced MUC1.Tg mice that express the B human MUCJ transgene in a pattern consistent with that observed in 25 humans. The findings that MUC1.Tg mice are unable to reject tumors that express the MUC1 protein and that B lymphocytes from these 20 mice fail to class switch in response to immunization with MUC1 peptides provide indirect evidence that MUC1.Tg mice are tolerant to @ IgG3 stimulation by MUC1 at the level of the . Thus, the MUC1.Tg (j.tgfml of sore) mice provide a useful model for investigating immunity and mecha U 10 nisms of central and peripheral tolerance to the tumor antigen MUC1. This model also provides opportunities to evaluate anti-MUC1 vac 5, cine formulations on the background of existing tolerogenic mecha 0' nisms and to evaluate the autoimmune consequences of producing WT MUC1 Tg anti-MUC1 immune responses.

ACKNOWLEDGMENTS C We acknowledgethe expertiseof SureshSavarirayanin embryo injection and implantation, Antia Jennings for expert tissue processing and sectioning, and Amy Weaver for statistical assistance. We thank Dr. Olia Finn for the 1gM generous gift of the MUC1—l05-merpeptide.We acknowledge the graphical (pg/mI of sore) expertise of Julie Jenson and Marvin Ruona.

U REFERENCES 1. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell. J., Pemberton, L., Lalani, E. N., and Wilson, D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. 3. Biol. Chem., 265: 15286—15293,1990. 2. Gendler, S. 3., Taylor-Papadimitriou, 3., Duhig, T., ROthbard, J., and Burchdll, 3. A Fig. 5. Humoral immune responses of MUC1.Tg and wt mice injected with MUC1- highlyimmunogenicregionof a humanpolymorphicepithelialmucinexpressedby expressing Bl6 melanoma cells. Sees from MUC1.Tg and wt mice primed with 2 X 10@ is made up of tandem repeats. J. Biol. Chem., 263: 12820—12823,1988. MUC1-expressing Bl6 murine melanoma cells were analyzed at 26-34 days postinjection 3. Lan, M. S., Batra, S. K., Qi, W-N., Metzgar, R. S., and Hollingswonh, M. A. Cloning forlevelsof MUC1reactiveantibodiesbyELISA.wimicegeneratedstronganti-MUCI, and sequencing of a human pancreatic apomucin cDNA. J. Biol. Chem., 265: IgGI (5 of 7; A), Ig03 (3 of 7; B), and 1gM (5 of 7; C) antibody responses. In contrast, 15294—15299,1990. MUC1.Tgmicefailedtodemonstrate1gMorIgG3antibodyresponsestoMUC1,andonly 4. Gendler, S. J., and Spicer, A. P. Epithelial mucin . Annu. Rev. Physiol., 57: one mousedemonstratedan lgOl response.The resultswere representativeoffour 607—634,1995. independentexperiments. 5. Barnd, D. L., Lan, M. S., Metzgar, R. S.. and Finn, 0. J. Specific, major histocom 320

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. TOLERANCE IN MUC1 TRANSOENIC MICE

patibility complex-unrestricted recognition of turner-associated by human of self-reactiveBlymphocytesintransgenicmice.Nature(Lond.),334:676—682, cytotoxic T cells. Proc. Nail. Aced. Sci. USA, 86: 7159—7163, 1989. 1988. 6. Jerome,K.R.,Domenech,N.,andFinn,0. J. Tumor-specificcytotoxicTcellclones 21. Nemazee, D. A., and Burki, K. Clonal deletion of B lymphocytes in a transgenic frompatientswithbreastandpancreaticadenocarcinomarecognizeEBV-immortal mouse bearing anti-MHC class I antibody genes. Nature (Loud.), 337: 562-566, ized B cells transfected with polymorphic epithelial mucin complementary DNA. J. 1989. Immunol., 151: 1654—1662,1993. 22. Yule, 1. D., Basten. A., and Allen, P. M. Hen egg-white lysozyme-specific T cells 7. Kotera, Y., Fontenot, J. D., Pecher, G., Metzgar, V., and Finn, 0. J. Humoral elicited in hen egg-white lysozyme-transgenic mice retain an imprint of self-toler immunity against a tandem repeat epitope of human MUC1 in sera from breast, ance. J. Immunol., 151: 3057—3069,1993. pancreas and colon cancer patients. Cancer Res., 54: 2856—2860,1994. 23. Peat, N., Gendler, S. i., Lalani, E-N., Duhig, T., and Taylor-Papadimitriou, J. 8. Taylor-Papadimitriou, J. Report on the first international workshop on carcinoma Tissue-specificexpressionof a humanpolymorphicepithelialmucin(MUC1)in associated mucins. hit. 3. Cancer, 49: 1-5, 1991. transgenic mice. Cancer Res., 52: 1954-1960. 1992. 9. Xing, P. X., Prenzoska,J., Quelch, K., and McKenzie, I. F. Second generation 24. Talmadge,J.E.,Talmadge,C.B..Thar,B.,McEwen,R.,Meeker,A.K.,andTribble, anti-MUC1 peptide monoclonal antibodies. Cancer Res., 52: 2310-2317, 1992. H. in vivo immunologicselection of class I majorhistocompatibilitycomplex gene 10. Thang,S., Graeber,L A., Helling, V., Ragupathi,V.. AdIUri,S.. Lloyd, K. 0., and deletion variants from the B16-BL6 melanoma. J. NatI. Cancer Inst., 78: 1215—1221, Livingston, P. 0. Augmenting the immunogenicity of synthetic MUCI peptide 1987. vaccines in mice. Cancer Rca., 56: 3315—3319,1996. 25. Gunning, P., Leavitt, J., Muscat, G., Ng, S-Y., and Kedes, L. A human @-actin 11. Spicer, A. P., Parry, G., Patton, S., and Gendler, S. J. Molecular cloning and analysis expressionvectorsystemdirectshigh-levelaccumulationofantisensetranacripis. ofthemousehomologueofthetumor-associatedmucin,MUCI,revealsconservation Proc. NatI. Acad. Sci. USA, 84: 4831-4835, 1987. of potentialO-glycosylationsites,transmembrane,andcytoplasmicdomainsanda 26. Burdick, M. D., Harris, A., Reid, C. 3., Iwamura, T., and Hollingsworth, M. A. loss of minisatellite-like polymorphism. 3. Biol. Chem., 266: 15099-15109, 1991. expressed on MUC1 produced by pancreatic and colon tumor cell 12. Apostolopoulos,V.,Xing,P.X.,andMcKenzie,I.F.C. Murineimmuneresponseto lines. 3. Biol. Chem., 272: 24198-24202, 1997. cells transfected with human MUCI: immunization with cellular and synthetic 27. Pemberton, L, Taylor-Papadimitriou, J., and Gendler, S. J. Antibodies to the cyto antigens. Cancer Rca., 54: 5186-5193, 1994. plasmic domain of the MUC1 mucin show conservation throughout mammals. 13. Graham,R.A.,Burchell,J.M.,Beverley,P.,andTaylor-Papadimitriou,J.Intramus Biochem.Biophys.Res.Commun.,185:167—175,1992. cular immunizationwithMUC1eDNA can protectC57 mice challengedwith 28. Fontenot, J. D., Tjandra, N., Bu, D., Ho, C., Montelaro, R. C., and Finn, 0. J. MUCI-expressing syngeneic mouse tumour cells. hit. J. Cancer, 65: 664-670, 1996. Biophysical characterization ofone-, two-, and three-tandem repeats ofhuman mucin 14. Lalani, E. N., Berdichevsky,F., Boshell, V., Shearer,M., Wilson, D., Status, H., (MUC1) protein core. Cancer Res., 53: 5386—5394,1993. Gendler, S. I., and Taylor-Papadimitriou, 3. Expression of the gene coding for a 29. Fontenot,J.D.,Finn,0. J.,Dales,N.,Andrews,P.C.,andMontelaro,R.C.Synthesis human mucin in mouse mammaiy tumor cells can affect their tumorigenicity. 3. Biol. of largemulti-determinateimmunogensusingthe poly-proline13-turnhelixmotif. Chem., 266: 15420—15426.1991. Pept. Rca., 6: 330—336, 1993. 15. Disis,M.L.,andCheever,M.A.Oncogenicproteinsastumorantigens.Curr.Opin. 30. Spicer, A. P., Rowse, G. J., Lidner, T. K., and Gendler, S. J. Delayed mammazytumor Immunol., 8: 637—642,1996. progression in Mud null mice. J. Biol. Chem., 270: 30093—30101, 1995. 16. Robbins,P. F., andKawakami,Y. Humantumorantigensrecognizedby Tcells. Curr. 31. Adelstein,S., Pritchard-Btiscoe,H.,Anderson,T. A., Crosbie,3., Gammon,G. Ophs. Immunol., 8: 628—636,1996. Loblay, R. H., Basten, A., and Goodnow, C. C. Induction of self-tolerance in T cells 17. Jones, L A., Chin, L T., Longo, D. L, and KrUisbeek, A. D. Peripheral clonal butnotB cellsof transgenicmiceexpressinglittleselfantigen.Science(Washington elimination of functional T cells. Science (Washington DC), 250: 1726—1729,1990. DC), 251: 1223—1225,1991. 18. Kappler,J. W., Roehm,N., andMarrack,P.1 cell toleranceby clonal eliminationin 32. Milich, D. R., Jones, J. E., Hughes, J. L., Maruyama, T., Price, J., Melhado, I., and thethymus.Cell,49: 273—280,1987. Jirik, F. Extrathymic expression ofthe intracellular hepatitis B core antigen results in 19. Mondino,A.,Khonits,A.,andJenkins,M.K.TheanatomyofT-cellactivationand T celltoleranceintransgenicmice.J. Immunol.,152:455—466,1994. tolerance. Proc. NatI. Aced. Sci. USA, 93: 2245—2252,1996. 33. Rowse, G. J., Tempero, R., Hollingsworth, M. A., and Gendler, S. J. MUCI peptide 20. Goodnow,C.C., Crosbie,J., Adelstein,S.,Lavoie,T. B., Smith-Gill,S.J., Brink, immunogens produce different antibody responses in native and MUCI transgemc R. A., Pritchard-Btiscoe,H..Wotherspoon,J. S., Loblay, R. H., Raphael,K.. Trent, mice. in: J. Taylor Papadimitriou (ed), 4th International Workshop on Carcinoma R. J., and Basten, A. Altered immunoglobulinexpression and functionalsilencing Associated Mucins, p. 83. Cambridge, UK: imperial Cancer Research Fund, 1996.

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Gerald J. Rowse, Richard M. Tempero, Michelle L. VanLith, et al.

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