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Tolerance and Immunity to MUC1 in a Human MUC1 Transgenic Murine Model1

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Tolerance and Immunity to MUC1 in a Human MUC1 Transgenic Murine Model1 [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 mucin, MUd, is a large transmembrane glyco TR region between MUC1 and its murine homologue, Mud (1 1). protein 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 glycoprotein expressed on most simple epithelia. It has an ex dicted to be similar to that seen with endogenous self proteins; 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 immune system 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 carcinoma of the breast, lung, pancreas, colon, and endogenous expression of the MUCJ gene 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 vaccine 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 epitopes 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@glycosylation, 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 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. TOLERANCE IN MUC1 TRANSOENIC MICE 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 melanoma 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
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