EP0964694A1 - Mucin-mediated immunomodulation - Google Patents

Abstract

Disclosed is a method of decreasing or preventing the deleterious effects of cytotoxic T lymphocytes that involves identifying an animal having a non-tumor cell at risk of attack by the animal's cytotoxic T lymphocytes, and providing to the cell a molecule having at least one tandem repeat segment of a mucin. Also disclosed is a method of augmenting the immune response against a tumor cell expressing a mucin, using a composition that binds to the tandem repeat segment of the mucin and blocks the CTL-inhibiting effects of the mucin.

Description

MUCIN-MEDIATED IMMUNOMODULATION Background of the Invention This invention relates to the modulation of the immune response of an animal, and in particular to modulating the effects of cytotoxic T lymphocytes of an animal including, e . g. , decreasing, preventing, suppressing or augmenting the effects of cytotoxic T lymphocytes in an animal . The references cited throughout this document are incorporated herein by reference in their entirety.

The mucins are glycoprotein molecules present on the surface of many cell types, including glandular epithelial cells. The polypeptide portion of a mucin is frequently characterized by a tandemly repeated unit of amino acids. The mucins are also characterized by their variable, high molecular masses and high content of 0- linked carbohydrate chain moieties.

Human cells express at least four mucin families, known as MUC1, MUC2 , MUC3 and MUC4. DF3/ UC1 is one member of the MUC1 family of carcinoma-associated antigens (also known as episialin, PEM) , with core proteins ranging from 160 to 230 kDa. (Abe, M. & Kufe, D. ., J. Immunol . 139, 257-261 (1987); Abe, M. & Kufe, D. ., Cancer Res . 49, 2834-2839 (1989))

DF3/MUC1 glycoproteins are normally expressed on the apical borders of secretory epithelial cells (Kufe, D.W., et al . Hybrido a 3, 223-232 (1984)) such as in breast tissue. However, the polarity of expression of DF3/MUC1 glycoproteins along apical borders is lost to overexpression throughout the entire cell membrane of transformed cells in carcinomas of the breast and other epithelial tissues. (Kufe, et al . (1984); Friedman, E.L., et al . Cancer Res . 46, 5189-5194 (1986); Arklie, J., et al . Int . J. Cancer 28, 23-29 (1981); Hilkens, J. , et al . Int . J. Cancer 34, 197-206 (1984) .

The DF3/MUC1 gene has a variable number of highly conserved (G+C) -rich 60 base pair (bp) tandemly repeating units that confer allelic variation and expression of a polymorphic core of the polypeptide moiety. (Gendler, S., et al . J. A . J. Biol . Chem. 263, 12820-12823 (1988); Siddiqui, J., et al . Proc . Na tl . Acad . Sci . USA 85, 2320- 2323 (1988)) The 60 base pair repeat unit of DF3/MUC1 is translated to a 20 amino acid unit.

The Xenopus integumentary mucin (Hoffman, J. J^". Biol . Chem. 263, 7686-7690 (1988)), the porcine submaxillary mucin (Timpte, C, et al . J. Biol . Chem . 263, 1081-1088 (1988)), the human intestinal MUC2 and UC3 (Gum. J.R., et al . J. Biol . Chem. 264, 6480-6487 (1989) ) and the human tracheobrondial MUC4 mucin (Porchet, N. , et al . Biochem . Biophys . Res . Commun . 175, 414-422 (1991)) also contain, like human DF3/MUC1, tandemly repeated domains. Moreover, the mouse homologue of MUC1 includes a tandem domain consisting of 16 repeated units of 20-21 amino acids. (Spicer, A. P., et al . Biol . Chem . 266, 15099-15109 (1991))

Flanking the 30-90 DF3/MUC1 tandem repeats (van de iel-van Kemenade et al . J. Immunology 151, 767-776 (1993)) are an N-terminal region that contains hydrophobic signal sequences and a C-terminal region that includes a transmembrane domain, followed by a 72 amino acid cytoplasmic tail. (Abe, et al . (1989); Lan, M.S., et al . J. Biol . Chem. 265, 15294-15299 (1990); Ligtenberg, M.J.L., et al . J^". Biol . Chem. 265, 5573-5578 (1990); reschner, D.H., et al . Eur. J. Biochem. 189, 463-473 (1990))

The functional role of the tandem repeats has been proposed to reside in the formation of a carbohydrate scaffold. As a consequence of the proline residues and the O-glycosidic linkages, the extracellular domain of DF3/MUC1 and other mucins is predicted to have an extended and rigid structure. (Jentoft, N. Trends Biochem . Sci . 15, 291-294 (1990); van de Wiel-van Kemenade et al . (1993))

Under certain circumstances, the structure containing the mucin tandem repeat segments is proteolytically cleaved from the transmembrane and cytoplasmic domains. (Ligtenberg, M.J., et al . J. Biol . Chem. 267, 6171-6177 (1992a)) Such cleavage may contribute to release of the mucin from malignant cells and the high circulating levels of DF3/MUC1 in patients with cancers of the breast and other tissues. (Hayes, D., et al . J. Clin . Oncol . 4, 1542-1550 (1986)) The DF3/MUC1 genetic locus resides on chromosome lq21-24 and includes seven exons that span 4-7 kilobases (kb) depending on the number of tandem repeat segments. (Lan et al . (1990); Swallow, D.M., et al . Ann . Human Genet . 51, 289-294 (1987); Merlo, G.R., et al . Cancer Res . 49, 6966-6971 (1989))

Certain mucins function as ligands for the selectin family of carbohydrate-binding proteins and thereby play a role in cell adhesion. (Shimizu, Y. & Shaw, S. Nature 366, 630-631 (1993)) However, the precise function of DF3/MUC1 has remained unclear.

Studies have reported that cell-cell interactions are reduced in cells transfected with a DF3/MUC1 cDΝA (Ligtenberg, M.J.L., et al . J. Cancer Res . 52, 2318-2324 (1992b); van de Wiel-van Kemenade et al . (1993)) and that DF3/MUC1 inhibits adhesion of eosinophils to antibody- coated targets. (Hayes, D.F., et al. J. Immunol . 145, 962-970 (1990)) More recent findings support involvement of DF3/MUC1 in intracellular signaling. (Zrihan-Licht , S., et al . FEBS Lett . 356, 130-137 (1994); Pandey, P., et al . Cancer Res . 55, 4000-4003 (1995)) Reportedly, tyrosine phosphorylation of the cytoplasmic tail is associated with binding to the SH2 - and SH3 -containing adaptor protein Grb2 , and the DF3/Grb2 complex associates with the Ras activator protein Sos . (Pandey et al . (1995)) These results suggest that of DF3/MUC1 glycoprotein may function as a transmembrane receptor.

Summary of the Invention It has now been found that mucins in general, and DF3/MUC1 in particular, play a significant role in the modulation of immune effector cell function, by inhibiting the cytotoxic action of cytotoxic T lymphocytes. DF3/MUC1 has been shown to inhibit cytotoxic T lymphocyte killing in vivo and in vi tro . The tandem repeat structure of the mucin is responsible for this effect.

The invention based upon this discovery involves two principle embodiments: (1) down regulating undesirable cytotoxic T lymphocyte response by the use of molecules containing one or more tandem repeat segments derived from a mucin, and (2) up regulating a useful cytotoxic T lymphocyte response against cells which bear mucin molecules on their surface, by the use of compounds which bind to the tandem repeat region of the mucin.

Thus, one aspect of this invention is an in vivo method of decreasing or preventing the deleterious effects of cytotoxic T lymphocytes in an animal. The method includes identifying an animal that harbors a non- tumor cell(s) that is at risk of attack by cytotoxic T lymphocytes, and providing to that cell a molecule that includes at least one tandem repeat segment of a mucin. The molecule is provided in an amount effective to decrease or prevent the deleterious effects of an attack by cytotoxic T lymphocyte on the non-tumor cell(s) . In preferred embodiments of the method, the mucin after which the tandem repeat segment is patterned is DF3/MUC1. However, tandem repeat segments patterned after repeats seen in other mucins, such as MUC2 , MUC3 , MUC4 , or a mucin of a non-human mammal, are also useful. The molecule preferably contains at least two tandem repeat segments, and more preferably at least three or four. Most preferred are those containing at least five tandem repeats (e.g., at least seven) . It is not necessary that the full natural complement of 30-90 tandem repeat segments be used in order to observe the immunosuppressant effect. In fact, for reasons of economy and ease of handling, it is preferred that the molecule used contains fewer than 30 tandem repeat segments, and preferably fewer than 25 or even 20. Where the molecule is a protein expressed from a nucleic acid such as a plasmid, possible instability of the plasmid favors keeping the number of tandem repeats to 15 or fewer, and more preferably 10 or fewer, for example eight, nine, or ten.

In addition to the mucin-derived tandem repeat segments, the molecule can include other amino acid sequences, such as mucin-derived sequences that represent some or all of the amino acid sequences of a mucin outside of the tandem repeat domain. For example, the molecule may have the amino acid sequence of a DF3/MUC1 mucin, or other mucin, except for the transmembrane domain and/or the cytoplasmic domain of the mucin. It may thus include less than all or none of the mucin amino acid sequence outside of the tandem repeat domain.

Alternatively, the molecule can include a transmembrane domain from a non-mucin molecule, or another heterologous sequence which, for example, permits the molecule to bind to the surface of a cell, or confers some other advantageous characteristic. The molecule can be a glycoprotein or lipoprotein.

The method of the invention can be used to decrease or prevent the deleterious effects of cytotoxic T lymphocytes associated with autoimmune disease or transplant rejection, e.g. where an allogeneic or xenogeneic target cell is transplanted into the animal. In the latter case the target cell (s) can constitute essentially all or only a part of a transplanted organ or tissue.

The method of the invention can be carried out by introducing into the target cell a nucleic acid which is expressed within the target cell, i.e., an expression vector. The nucleic acid encodes a polypeptide that includes at least one mucin tandem repeat segment .

Alternatively, the molecule is provided to the target cell by administering to the patient a therapeutic composition containing the molecule, and thereby contacting the target cell(s) directly with the exogenously produced molecule.

Conditions treatable by the methods of the invention include those disorders attributable at least in part to activation of cytotoxic T lymphocytes, such as autoimmune diseases (e.g., diabetes, lupus, and multiple sclerosis) and conditions characterized by cytotoxic T lymphocyte induced granuloma formation, e . g. , tuberculosis, sarcoidosis, leprosy, Crohn's disease, hypersensitivity pneumonitis, and primary biliary cirrhosis. The methods of the invention can also be used to treat malignant disorders of cytotoxic T lymphocytes, e . g. , activated T cell leukemia/lymphoma (ATLL) .

Typically the method of the invention involves first identifying an animal (e.g., a human patient) in need of treatment to decrease or prevent a cytotoxic T lymphocyte response, and then introducing into the animal a molecule containing a tandem repeat segment of a mucin. The introducing step may be accomplished by administering to the animal a therapeutic composition including the molecule, or by introducing into a cell of the animal an expression vector encoding the polypeptide portion of the molecule, where the expression vector has expression control sequences that permit expression in the cell.

The molecules and tandem repeat-encoding nucleic acids of the invention can be used to treat a cell ex vivo, prior to implantation into an animal. The cell, preferably a non-tumor cell, can be from the first animal or from a second, donor animal, and is characterized as being at risk of attack by the first animal's cytotoxic T lymphocytes. The cell can be supplied in the form of separate, individual cells (e.g., cultured cells), or a tissue or organ. The cell is contacted with a molecule that includes a tandem repeat segment of a mucin, so that the tandem repeat segment is at the surface of the cell. Alternatively, the cell can be transfected with the nucleic acid of the invention, so that it expresses the tandem repeat-containing molecule. Thereafter the cell is implanted into the first animal. The invention includes such transfected, non-tumor-derived cells, e.g., mammalian cells, particularly those which express an isolated DNA encoding a protein or glycoprotein having at least one DF3/MUC1 tandem repeat. The transfected mammalian cell can be part of a tissue or organ that has been removed from a mammal .

Also within the invention is a transgenic, non- human mammal containing in its nucleated cells a transgene encoding a polypeptide that includes at least one tandem repeat segment of a human mucin. This mammal can be used as a source of transplantable cells, tissues or organs, wherein a plurality of cells of the tissue or organ express the polypeptide. The second aspect of the invention involves a method of augmenting the immune response to a tumor cell in an animal . The method includes the step of identifying an animal suspected of harboring a tumor cell bearing a mucin on its surface, wherein the mucin' s presence on the tumor cell inhibits the animal's immune response to the tumor cell. A therapeutic composition containing a molecule that includes (1) an antibody which binds to an epitope within the tandem repeat domain of the mucin, (2) an antigen-binding fragment of such an antibody, or (3) another compound which is capable of binding to the tandem repeat domain and thereby preventing the latter' s interaction with cytotoxic T lymphocytes, is administered to the animal. Preferably, the therapeutic composition is one that is not suitable for use as a diagnostic: e.g., it contains no detectable label such as a radioisotope. It also preferably contains no toxic moieties attached to the antibody, e.g. ricin or other toxins typically used in preparing immunotoxins . The method can include an additional step, prior to the introducing step, of determining whether a tumor cell of the animal bears the mucin on its surface.

In a preferred embodiment of this aspect of the invention, the mucin is DF3/MUC1 and the epitope bound by the antibody includes any three-residue portion of the sequence Trp-Arg-Pro-Ala-Pro-Gly-Ser (SEQ ID NO: 2) , which represents the epitope bound by MAb DF3 (ATCC accession no. ). Preferably, the epitope includes a four or six-residue portion of that sequence, and most preferably the epitope consists of all seven residues. The antibody can be, for example, MAb DF3 , a fragment of MAb DF3 , or a humanized form of MAb DF3.

Definitions Brief definitions of certain technical terms are presented below in order to aid the understanding of the instant invention.

The descriptions and definitions presented herein sometimes refer to "molecule," "cell," "antibody,"

"nucleic acid," and other like terms in the singular. It will be understood by workers in the art that many terms used to describe molecules may be used in the singular and referred to either a single molecule or to a plurality of such molecules. For example, in a preparation of "an antibody, " there are in fact many individual molecules present . In such instances terms are to be understood in context. Such terms are not to be limited to meaning either singular or multiple unless specifically stated to be so limited.

By "decreasing the deleterious effects" is meant a lessening of the severity of a disease condition existing in an animal .

By "preventing the deleterious effects" is meant serving to avert at least in part the occurrence of a disease condition which would otherwise be expected to develop in the subject animal.

By "deleterious effects of cytotoxic T lymphocytes" is meant a disorder caused directly or indirectly by the presence or activity of cytotoxic T lymphocytes .

By "tumor cell" is meant an animal cell which is transformed from its normal differentiated state to a state of continued growth, either in vivo or in vi tro . A non-tumor cell is any animal cell other than a tumor cell.

"Mucin" is used herein to mean a glycoprotein identical to a naturally-occurring glycoprotein which can be found expressed on the surface of many animal cells and which is characterized by an extracellular, tandemly repeated peptide sequence, high content of O-linked carbohydrate chain moieties, and high but variable molecular weight. Examples of mucins are DF3/MUC1, MUC2 , MUC3 , MUC4 , the Xenopus integumentary mucin, and the porcine submaxillary mucin.

By "organ" or "tissue" is meant an organized and differentiated mass of cells exercising a specific function in an animal. Blood cells are considered a tissue . By "expression vector" is meant an isolated nucleic acid which can be introduced into an animal cell, and which contains (1) a coding sequence and (2) regulatory sequences operably linked to the coding sequence, the regulatory sequences making possible the expression of the coding sequence in the cell. It includes plasmids and viral vectors, but does not include very large, multi-gene DNAs such as mammalian chromosomes or cosmids .

By " in vivo" is meant within the body of an animal. By " ex vivo" is meant outside and separate from the body of an animal .

By a "transfected cell" is meant a cell into which has been incorporated an exogenous nucleic acid, or a descendent of such a cell which contains a copy of the exogenous nucleic acid. The nucleic acid may or may not be expressed at any given time.

By a "transduced cell" is meant a transfected cell that has the nucleic acid incorporated into the genome of the cell. The transduced cell can replicate the nucleic acid and pass the replicated copies to descendent cells. A "transgenic non-human mammal" is an animal the nucleated cells of which contain a "transgene." By "transgene" is meant a gene or coding sequence which is introduced into germ line cells of an animal. Transgenic non-human mammals are taught in Leder et. al . , U.S. Patent No. 4,736,866, which is incorporated herein by reference .

By "antibody" is meant either a double chain or single chain antibody, and either a monoclonal or polyclonal antibody. A "fragment" of an antibody is a portion of an antibody, less than the whole, which is capable of binding to the antibody's antigen: for example an Fab' fragment.

As the term is used herein, a "DF3/MUC1 tandem repeat segment" is any contiguous 20 amino acid segment of SEQ ID N0:1, SEQ ID NO : 1 being 2 tandemly repeated units of DF3/MUC1: 40 amino acids in total.

Correspondingly, a "tandem repeat segment" of any given mucin is a single peptide unit that is tandemly repeated in that mucin, which unit can begin at any residue within the tandem repeat. One or more glycosyl moieties can be present attached to an amino acid in a tandem repeat segment .

In the descriptions presented below, particular vectors, expression systems, regulatory nucleic acid sequences, libraries, cells, cell lines, animals, etc. are used. However, those skilled in the art will realize that comparable vectors, cells, etc. can be substituted.

The teaching of the Examples is exemplary of the invention disclosed herein. The Examples do not limit the invention which is described by the claims appended hereto.

Brief Description of the Drawings

FIG. 1 is a set of graphs illustrating cell surface expression of DF3/MUC1 and MHC class molecules in MC-38 cell transfectants , as analyzed by flow cytometry. The control tracing almost completely overlaps the shaded area in panel MC-38/Neo-MAb DF3. FIG. 2A and FIG. 2B are graphs depicting DF3/MUC1 mediated inhibition of the cytotoxic T lymphocyte effector response generated by immunization with MC- 38/Neo cells. FIGS. 3A, 3B and 3C are graphs illustrating the induction of CD8+ cytotoxic T lymphocytes in mice immunized with irradiated MC-38/MUC1 cells.

FIG. 4A and FIG. 4B are graphs showing the inhibition of activated cytotoxic T lymphocyte effectors by the tandem repeat structure of DF3/MUC1.

FIG. 5A and FIG. 5B are graphs illustrating that deletion of the entire DF3/MUC1 tandem repeat structure abrogates the inhibition of activated cytotoxic T lymphocyte effectors.

Detailed Description

The findings presented in Example 1 below demonstrate that the cytotoxic T lymphocyte response generated against MC-38 adenocarcinoma cells in syngeneic mice is abrogated by expression of a molecule with a DF3/MUC1 tandem repeat, but that cytotoxic T lymphocytes induced against DF3/MUC1 positive MC-38 cells are unaffected by DF3/MUC1 expression. The findings indicate that DF3/MUC1 inhibits the effector function of certain activated cytotoxic T lymphocytes, but not the process of activation itself. Example 2 demonstrates that blocking the tandem repeat structure of DF3/MUC1 with antibody prevents abrogation of cytotoxic T lymphocyte function. Moreover, expression of DF3/MUC1 deleted of tandem repeat segments has no effect on activated cytotoxic T lymphocytes. These findings demonstrate that the tandem repeat structure is responsible for the observed inhibition of cytotoxic T lymphocytes, and that blocking the tandem repeat with an antibody reverses the inhibitory effect. Example 3 concerns transgenic non- human mammals, useful for generation of cells, tissues and organs for transplantation, e.g., xenotransplantation. Example 4 discusses methods for genetically engineering cells to express the polypeptides and glycoproteins of the invention. Example 5 outlines a number of ways in which the molecules, constructs and methods of the invention can be applied to human therapy.

EXAMPLE 1: DF3 /MUC1 tandem repeat inhibits CT1 Cytotoxic T lymphocyte function General Methods

MC-38 cell transfectants . The human DF3/MUC1 cDNA was isolated from an MCF-7 cell cDNA library (Siddiqui et al . (1988); Merlo, et al . (1989)) and cloned into the pLNSX retroviral vector with expression driven by the SV40 promoter. The plasmid was transfected into the

PA317 packaging cell line. G418 resistant colonies were pooled and supernatants used to retrofect murine (C57B1/6) MC-38 adenocarcinoma cells (Fox, B.A. et al . J. Biol Response Mod. 9, 499-511 (1990)). Cells transduced with the empty pLNSX vector have been designated MC- 38/Neo and those expressing a DF3/MUC1 molecule as MC- 38/MUC1. In addition, a MUC1 cDNA (designated MUC1-D) devoid of DF3/MUC1 tandem repeats was confirmed by sequencing and cloned into PLNSX. Transfectants expressing the devoid form have been designated MC-

38/MUC1-D. Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L- glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Indirect immunofluorescence of cells transfected with a nucleic acid that encodes a DF3/MUC1 molecule was performed using MAb DF3. (Kufe, et al . (1984) ) MC-38/MUC1-D cells were analyzed by Northern blotting of total cellular RNA using the MUC1 cDNA (Merlo, et al . (1989)) and by immunoblotting with a rabbit polyclonal antibody prepared against the MUC1 cytoplasmic domain. Other antibodies used for cell analysis were Ml/42/3.9.8 anti-MHC Class I, 2.43 anti- CD8, GK1.5 anti-CD4, PK136 anti-NK cells (TIB126, 210, 207, HB-191, respectively; ATCC; Rockville, MD) , mouse IgG and rat IgG (F-8765 and R9255; Sigma Chemical Co., St. Louis, MO), anti-mouse IgG FITC and anti-rat IgG FITC (Boehringer Mannheim, IN) .

Tumoriqenicity . C57B1/6 mice were injected subcutaneously with 2 x 10⁶ MC-38/Neo or MC-38/MUC1 cells treated with 200 Gy γ-irradiation. After 14 d, the mice were immunized again with the irradiated cells. After another 14 d, 5 x 10⁵ or 1 x 10⁶ tumor cells in 100 μl phosphate buffered saline (PBS) were injected subcutaneously in the flank of the immunized mice. In certain experiments, tumor cells were first incubated with 25 μg/ml MAb DF3 , 5 μg/ml MAb DF3 or mouse IgG for 1 h at 4°C. The cells were washed with PBS prior to subcutaneous injection. Tumor development was assessed by measurement with calipers. Cytotoxic T lymphocyte assays. Cytotoxic

T lymphocyte activity was determined by the release of lactate dehydrogenase (LDH) . (CytoTox, Promega, Madison, WI) (Franks, et al . J. Immunol . Methods 171, 259-262 (1994), (Brander, C, et al . Eur. J. Immunol . 23, 3217- 3223 (1993)) . Splenocytes isolated from mice were subjected to Ficoll density gradient centrifugation to remove dead cells. The splenocytes were incubated with MC-38 target cells at varying E:T (effectors: target) ratios in V-bottom microtitration plates (Nunc, Roskilde, Denmark) , centrifuged for 3 min at lOOOg and incubated for 4 h at 37°C. At the end of co-culture, 50 μl supernatant/well were transferred to an assay plate and incubated with 50 μl of substrate mixture for 30 min at room temperature. The reaction was terminated by addition of stop solution. Absorbance values were determined at 490-492 nm using a BIO-RAD microplate reader (model 3550, BIO-RAD Laboratories, CA) . Specific target cell killing by effector cells was determined by the formula: Cytotoxicity (%) = 100 x (Experiment release - Spontaneous release) / (Maximum release - Spontaneous release) .

Results

Murine MC38 adenocarcinoma cells were stably transfected with a retroviral vector that expresses a nucleic acid that encodes a DF3/MUC1 molecule with fewer than 10 DF3/MUC1 tandem repeat segments. In the experiment illustrated in Fig. 1, reactivity with MAb DF3 (shaded areas, upper panels) and an anti-MHC Class I antibody (MAb Ml/42/3.9.8 ; shaded areas, lower panels) was determined by flow cytometry. Mouse IgG (solid line) was used as a control. MC-38 cells transfected with the empty vector (MC-38/Neo) exhibited no detectable reactivity with MAb DF3 , while MC-38/MUC1 cells demonstrated cell surface expression of the DF3/MUC1 molecule (FIG. 1) . The two cell lines expressed similar levels of MHC Class I (FIG. 1) .

To determine the effects of DF3/MUC1 expression on tumorigenicity of MC-38 cells, syngeneic C57B1/6 mice were immunized twice at two week intervals with 2 x 10⁶ irradiated MC-38/Neo cells. The mice were then challenged with 5 x 10⁵ (FIG. 2A, left panel) or 1 x 10⁶ (FIG. 2A, right panel) MC-38/Neo (D) or MC-38/MUC1 (■) cells. As a control, mice were mock immunized with PBS and challenged with MC-38/Neo (O) cells. Tumors 3 mm or greater in diameter were scored as positive. Tumor incidence was calculated from groups of eight mice. No tumors developed with innocula of 5 x 10⁵ and 1 x 10⁶ MC- 38/Neo cells. By contrast, the immune response generated against irradiated MC-38/Neo cells failed to prevent the growth of MC-38/MUC1 tumors.

Splenocytes were isolated from the mice immunized with irradiated MC-38/Neo cells and were challenged with 5 x 10^s MC-38/Neo (FIG. 2B, D) or MC-38/MUC1 (FIG. 2B ■) cells. Using MC-38/Neo (FIG. 2B, left panel) or MC- 38/MUC1 (FIG. 2B, right panel) cells as targets, cytotoxic T lymphocyte activity was determined by the 4h LDH release assay. Cytotoxic T lymphocytes from the immunized mice induced lysis of MC-38/Neo, but not MC- 38/MUC1, targets. The same results were obtained with cytotoxic T lymphocytes from immunized mice bearing MC- 38/MUC1 tumors. These findings demonstrate that DF3/MUC1 can play a role in rendering targets resistant to the killing effects of cytotoxic T lymphocytes.

Similar studies were performed to assess induction of CD8+ cytotoxic T lymphocytes in mice immunized with MC-38/MUC1 cells. Mice were immunized twice at two week intervals with 2 x 10⁶ irradiated MC-38/MUC1 cells and then challenged with MC-38/Neo (•) or MC-38/MUC1 (o) cells (FIG. 3A) . As a control, mice were mock immunized with PBS and challenged with MC-38/MUC1 cells (Δ) . Tumors 3 mm or greater in diameter were scored as positive. Tumor incidence was calculated from groups of eight mice. In the MC-38/MUC1 - immunized animals, challenge with MC-38/Neo or MC-38/MUC1 cells failed to result in tumor growth.

FIG. 3B shows that cytotoxic T lymphocytes from the MC-38/MUC1- immunized mice induced lysis of both MC- 38/Neo and MC-38/MUC1 cells in vi tro . Splenocytes isolated from mice immunized with irradiated MC-38/MUC1 cells and challenged with MC-38/MUC1 cells were incubated with MC-38/Neo (FIG. 3B left panel) or MC-38/MUC1 cells (FIG. 3B, right panel) as targets. Cytotoxic T lymphocyte activity was determined by the LDH release assay.

To define the effector cells responsible for antitumor activity, mice immunized with MC-38/MUC1 cells were injected intraperitoneally on days -4 and 4 with rat IgG, anti-NK MAb PK136, anti-CD4 MAb GK1.5 or anti-CD8 Mab 2.43. On day 0, 5 x 10⁵ (FIG. 3C, solid bar) or 1 x 10⁶ (FIG. 3C, hatched bar) MC-38/MUC1 cells were injected subcutaneously. Tumor incidence was determined from groups of five mice. Depletion of the respective cell population by 80-90% was confirmed by flow cytometric analysis of splenocytes. The finding that injection of the anti-CD8 antibody selectively increases tumor incidence indicated that CD8+ T cells constitute the antitumor activity of mice immunized with MC-38/MUC1 (FIG. 3C) .

These results support a role for DF3/MUC1 molecules as both an inhibitor of cytotoxic T lymphocyte function and as a target for cytotoxic T lymphocyte activity. In this context, cytotoxic T lymphocytes induced against MC-38/Neo cells are shown to be inhibited by expression of DF3/MUC1 molecules in vivo and in vi tro . However, cytotoxic T lymphocytes induced against MC- 38/MUC1 are active against both MC-38/MUC1 and MC-38/Neo cells. These findings support a mechanism in which DF3/MUC1 inhibits the effector function of certain activated cytotoxic T lymphocytes, but not the process of activation itself. Moreover, induction of CD8+ cytotoxic T lymphocyte activity against DF3/MUC1 is not subject to the inhibition observed for cytotoxic T lymphocytes directed against other MC-38 antigens.

These findings thus provide the first evidence for abrogation of an antitumor immune response by a tumor- associated antigen. Given the high levels of DF3/MUC1 expression on human carcinomas and the loss of polarity observed in normal secretory epithelium (Kufe, et al .

(1984) ) , DF3/MUC1 appears to play a role in modulating the effector function of cytotoxic T lymphocytes, if any, generated against these tumors. Example 2: Blocking DF3/MUC1 tandem repeat reverses inhibition of cytotoxic T lymphocyte function

MAb DF3 reacts with the Trp-Arg-Pro-Ala-Pro-Gly-

Ser (SEQ ID NO:2) epitope within the 20 amino acid

DF3/MUC1 tandem repeat segment (Perey, L. , et al . Cancer Res . 52, 2563-3568 (1992)). Mice were immunized twice with irradiated MC-38/Neo cells (FIG. 4A, left panel) or mock immunized with PBS (FIG. 4A, right panel) . The mice were challenged with 1 x 10^δ MC-38/MUC1 tumor cells incubated with 25 μg/ml mouse IgG (O) , 25 μg/ml MAb DF3 (•) or 5 μg/ml MAb DF3 (o) for 1 h at 4°C and then washed twice with PBS before injection. Tumors 3 mm or greater in diameter were scored as positive. Tumor incidence was calculated from groups of eight mice. There was no detectable tumor growth after treatment with 25 μg/ml MAb DF3 and a 25% tumor incidence after treatment with 5 μ/ml MAb DF3 (FIG. 4A, left) . By contrast, MC-38/MUC1 cells treated with 25 μg/ml mouse IgG grew as tumors in 87.5% of challenged animals (FIG. 4A, left) . Moreover, as an additional control, treatment with MAb DF3 had no effect on growth of MC-38/MUC1 tumors in mice mock immunized with PBS (FIG. 4A, right) .

Splenocytes from mice immunized with irradiated MC-38/Neo cells were incubated with MC-38/MUC1 cells treated with 25 μg/ml mouse IgG (o) , 25 μg/ml MAb DF3 (■) or no antibody (0) . As a control MC-38/Neo cells served as the target cells (D) (FIG. 4B) . Cytotoxic T lymphocytes from the MC-38/Neo- immunized mice induced lysis of MC-38/Neo cells, but not MC-38/MUC1 cells (FIG. 4B) . While treatment of the MC-38/MUC1 cells with IgG had no detectable effect, treatment with MAb DF3 increased lysis of these targets (FIG. 4B) . These results indicate that the tandem repeat structure of DF3/MUC1 is responsible for inhibition of cytotoxic T lymphocyte effectors activated against MC-38/Neo cells.

To further address the role of the tandem repeats in blocking cytotoxic T lymphocyte effectors, MC-38 cells that stably express a MUC1 cDNA devoid of the tandem repeat sequences (MUC1-D) were generated. Expression of MUCl-1 was confirmed by Northern blot. Immunoblotting with rabbit polyclonal antibody prepared against the DF3/MUC1 cytoplasmic domain confirmed antigen expression, while there was no detectable reactivity of the transfectants with MAb DF3.

Mice were immunized with irradiated MC-38/Neo cells and then challenged with 5 x 10⁵ MC-38/Neo (D) , MC- 38/MUC1 (■) or MC-38/MUC1-D (•) cells (FIG. 5A) . Tumor incidence (≥ 3 mm diameter) was determined for groups of eight mice. Mice immunized with irradiated MC-38/Neo cells failed to develop tumors when challenged with MC- 38/Neo cells or MC-38 cells expressing the tandem repeat deleted form of DF3/MUC1 (MC-38/MUC1-D) (FIG. 5A) . Splenocytes from the mice immunized with irradiated MC-38/Neo cells were incubated with MC-38/Neo (D) , MC-38/MUC1 (■) or MC-38/MUC1-D (•) cells as targets. Cytotoxic T lymphocytes from the mice immunized with irradiated MC-38/Neo cells induced lysis of MC-38/Neo and MC-38/MUC1-D cells, but not the MC-38/MUC1 targets (FIG. 5B) .

These results provide evidence that the mucin tandem repeat structure of DF3/MUC1 is involved in abrogation of cytotoxic T lymphocyte function. An extended and rigid DF3/MUC1 glycoprotein could sterically block interaction of a cytotoxic T lymphocyte with antigen presented in the context of an MHC molecule. However the finding that binding of MAb DF3 to the DF3/MUC1 tandem repeat segment reverses the inhibitory effect in vivo and in vi tro supports a mechanism other than stearic interference.

Without limiting the invention to any particular theory, one potential explanation for the results is that the tandem repeats directly interact with certain activated cytotoxic T lymphocytes and thereby abrogate their effector function. The importance of the tandem repeat structure in abrogating cytotoxic T lymphocyte effector function is also supported by experiments with a DF3/MUC1 devoid of the tandem repeats (FIGS. 5A and 5B) . The MC-38/MUC1-D cells are effective targets for cytotoxic T lymphocytes generated against MC-38/Neo cells. Taken together, these findings indicate that the tandem repeats of DF3/MUC1 interact with certain activated cytotoxic T lymphocytes and abrogate their function by inducing apoptosis.

Example 3 Transgenic non-human animals

Transgenic animals (e.g. mammals such as mice and other rodents, pigs, cows, dogs, cats, goats, sheep, horses, and non-human primates such as chimpanzees and baboons) expressing a molecule containing a mucin tandem repeat segment are useful for providing cells, tissues or organs that display a cell -surface molecule having at least one mucin tandem repeat segment. Cells, tissues and organs so produced can be used as source materials for transplantation into a recipient animal. Such transplants will be less subject to attack by the cytotoxic T lymphocytes of the transplant recipient . In these methods it is preferable, but not required, that the mucin tandem repeat expressed in the tissue or organ be from the same species as the transplant recipient. For a human transplant recipient, the tandem repeat segment can be derived from a human mucin, e.g. DF3/MUC1, MUC2 , MUC3 or MUC4. However, mucin molecules from other animals may also be effective when used in humans .

Transgenic animals that express a nucleic acid encoding a molecule having a mucin tandem repeat segment can be created by methods known in the art . A method is taught by Leder et al . , U.S. Patent No. 4,736,866, which is incorporated herein in its entirety. Transplantable cells, tissues or organs can be harvested from the donor animal by standard methods.

Example 4 Transfected cells expressing a polypeptide of the invention

When cells are to be genetically modified for the purposes of carrying out a method within the scope a this invention by, e . g. , gene therapy, a nucleic acid molecule that contains a mucin cDNA or a nucleic acid sequence encoding a polypeptide having at least one mucin tandem repeat segment may be contained within an expression vector and introduced into primary or secondary human cells (e.g., fibroblasts, epithelial cells including mammary and intestinal epithelial cells, endothelial cells, nucleated elements of the blood including lymphocytes and bone marrow cells, glial cells, hepatocytes, keratinocytes, muscle cells, neural cells, or the precursors of these cell types) by standard methods of transfection including, but not limited to, liposome-, polybrene-, or DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection, or velocity driven microprojectiles ( "biolistics" ) . Alternatively, one could use a system that delivers nucleic acid by viral vector. Viruses known to be useful for gene transfer include adenoviruses, adeno associated virus, herpes virus, mumps virus, poliovirus, retroviruses , Sindbis virus, and vaccinia virus such as canary pox virus. (see generally, Wolff, J.A. Ed. Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser, Boston, MA, 1994, and other publications available to those of skill in the art) . The promoter and other expression control sequences needed to drive expression of the polypeptide would be selected as appropriate for the particular expression desired. For example, if expression in chondrocytes is desired, e.g., in order to reduce CTL- induced destruction of joints, then chondrocyte-specific promoters and/or enhancers would be utilized.

Example 5: Human Therapy

The mucin tandem repeat functions to prevent the cytolytic action of cytotoxic T lymphocytes against a cell displaying the mucin tandem repeat on its surface. Therapies can be designed to exploit this inhibitory action, or to overcome it, depending upon the therapeutic result desired. Examples of both types of therapies are presented below.

In some therapies a nucleic acid is employed while in others a molecule such as a polypeptide or glycoprotein is employed. A nucleic acid useful in this invention is one that encodes some or all of the polypeptide portion of a mucin, including at least one tandem repeat segment. The polypeptide can contain one or more mucin tandem repeat segments and, optionally N- terminal and/or C-terminal flanking regions of a mucin polypeptide. As an alternative to one or both of the flanking regions, or in addition thereto, the nucleic acid can encode a non-mucin related polypeptide linked to the mucin-derived portion of the molecule. For example, a nucleic acid can be designed to encode a polypeptide including one or more mucin tandem repeat segments placed between a signal sequence and a transmembrane domain from a non-mucin molecule. Alternatively, the tandem repeat segment (s) can be joined to a distinctive epitope (for identification purposes) , or to a ligand, such as a single-stranded antibody, which binds to a receptor on the cells for which protection from cytotoxic T lymphocyte desired. Nucleic acids can be delivered to cells as described above.

As an alternative to genetic therapy, one can deliver the tandem repeat-containing molecule (e.g. a mucin or a fragment of a mucin, or any of the genetically-engineered hybrids referred to above) itself. The molecule can be harvested from natural sources or from recombinant expression systems, or can be prepared by chemical synthesis. It is preferably a protein or glycoprotein . Molecules having a mucin tandem repeat segment, or nucleic acids encoding them, can be used for decreasing or preventing the deleterious effects of cytotoxic T lymphocytes in autoimmune disease, graft or transplant rejection and conditions characterized by the formation of granulomas . In vivo or ex vivo based therapeutic approaches can be used.

In vivo-based Human Therapies An in vivo therapy can provide a means of augmenting the immune response against a tumor. For example, after surgical removal of a primary tumor that overexpresses a mucin, residual cells may be targeted by treating the vicinity of the tumor with a composition containing a retroviral vector encoding a single chain antibody or fragment thereof that binds a tandem repeat segment of the mucin. The antibody or fragment is expressed within the residual tumor cells, and binds to the mucin molecule within or on the surface of the tumor cells. This method prevents the mucin from blocking the action of cytotoxic T lymphocytes against the tumor cells. The method can, of course, be used instead of surgery in appropriate situations, e.g. where a tumor is not accessible by surgery.

Malignant cells distal to the primary tumor site may be reached by delivering the vector intravenously. Targeting of tumor cells can be accomplished by the use of a retrovirus, which targets proliferating cells. Alternatively, one could utilize a cell attachment ligand on the vector or the viral particle to accomplish preferential targeting of specific cells in accordance with standard methods .

An example of a non-viral vector system is a molecular conjugate composed of a plasmid attached to poly-L-lysine by electrostatic forces. Poly-L-lysine covalently binds to a ligand that can bind to a receptor on tumor cells (Cristiano et al . , 1995, J. Mol. Med 73:479-486). A promoter inducing relatively tumor- specific expression can be used to achieve a further level of targeting: for example, α-fetoprotein promoter for hepatocellular carcinoma (Huber et al . , 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043) or the tyrosinase promoter for melanoma (Hart et al . , 1995, Br . Med. Bull. 51 (3 ): 647-655) . Alternatively, a constitutively active promoter could be used, e . g. , the SV40 promoter or CMV promoter.

As an alternative to genetic therapy, one can administer directly the antibody or other molecule that binds to a tandem repeat segment of a mucin. The molecule can be administered locally to the tumor site, or intravenously, orally, intraperitoneally, intrapulmonarily, or by another appropriate route.

Conversely, one can inhibit the effector function of cytotoxic T Lymphocytes in vivo by treating with either a molecule (e.g., polypeptide or glycoprotein) having at least one mucin tandem repeat segment, or with a nucleic acid encoding the polypeptide portion of that molecule. For example, one could intravenously inject into the patient a soluble form of DF3/MUC1, e.g., a glycoprotein lacking the transmembrane and cytoplasmic domain of DF3/MUC1. This mode of therapy is useful in disorders such as autoimmune disease, transplant rejection, and conditions characterized by granuloma formation. Granuloma formation, which is nucleated by cytotoxic T lymphocytes which recruit white cells, is involved in a number of conditions, including tuberculosis, sarcoidosis, leprosy, Crohn's disease, hypersensitivity pneumonitis and biliary cirrhosis. Other disorders which can be treated with the methods of the invention are multiple sclerosis and malignancies of cytotoxic T lymphocytes such as lymphomas and leukemia. Without intending to be bound by a particular theory, the therapy of the invention is believed to induce apoptosis in activated cytotoxic T lymphocytes.

Ex vivo-based Human Therapies Ex vivo therapies can use either a polypeptide- based molecule or a nucleic acid which expresses such a molecule .

In an ex vivo method, one would harvest cells from a first or a second animal as a cell, tissue or organ sample for transplantation into the first animal. The sample is treated, ex vivo, with an exogenously provided molecule having at least one mucin tandem repeat segment. The treatment is designed to incorporate the molecule into the membranes of the cells of the sample, or otherwise to leave the molecule in association with the cell surface (e.g., by binding to a receptor). Incorporation into the cellular membrane would be accomplished, e.g., by using a molecule that includes a transmembrane domain or a lipid moiety, and preferably by delivering the molecule in or attached to liposomes. The cells, organ, or tissue is then implanted into the first animal. By this method, the severity of transplant rejection is lessened. Alternatively, one can use a nucleic acid described above in an ex vivo therapy. This approach could entail harvesting the sample from a first animal and transducing or transfecting the cells of the sample with a nucleic acid designed as taught above. The transduction or transfection step could be accomplished by any standard means used for ex vivo gene therapy, including calcium phosphate precipitation, lipofection, electroporation, viral infection, naked DNA techniques, and biolistic gene transfer. Cultured cells that have been successfully transduced can optionally be selected, for example via a drug resistance gene. The cells can alternatively be simply screened for the expression of a marker such as green fluorescent protein. The sample may then be injected or implanted into the patient. It should be noted that whether a polypeptide or nucleic acid is used, it is not necessary to treat 100% of the cells in a sample to be transplanted or implanted in an animal. However, as the proportion of cells that ' are treated is increased, one will expect to see a greater decrease in cytotoxic T lymphocytes attack against the transplanted cells.

Other embodiments are within the claims below.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Dana-Farber Cancer Institute, Inc.

(ii) TITLE OF THE INVENTION: MUCIN-MEDIATED IMMUNOMODULATION

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson P.C.

(B) STREET: 225 Franklin Street (C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 2.0

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/738,262 (B) FILING DATE: 25-OCT-96

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Fraser, Janis K.

(B) REGISTRATION NUMBER: 34,819

(C) REFERENCE/DOCKET NUMBER: 00530/082001 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-542-5070

(B) TELEFAX: 617-542-8906

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not applicable

(D) TOPOLOGY: not applicable (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro

1 5 10 15

Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly 20 25 30

Ser Thr Ala Pro Pro Ala His Gly 35 40

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not applicable

(D) TOPOLOGY: not applicable

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Trp Arg Pro Ala Pro Gly Ser 1 5

What is claimed is :

Claims

1. An in vivo method of decreasing or preventing the deleterious effects of cytotoxic T lymphocytes in an animal, comprising identifying an animal harboring a target cell at risk of attack by the animal's cytotoxic T lymphocytes, wherein said target cell is not a tumor cell; and providing to the target cell, in vivo, a molecule comprising at least one tandem repeat segment of a mucin, said molecule being provided in an amount effective to decrease or prevent the deleterious effects of cytotoxic T lymphocyte attack on said target cell.

2. The method of claim 1, wherein the mucin is DF3/MUC1.

3. The method of claim 1, wherein the mucin is selected from the group consisting of MUC2 , MUC3 , MUC4 , or a mucin of a non-human mammal.

4. The method of claim 2, wherein the molecule comprises at least two DF3/MUC1 tandem repeat segments.

5. The method of claim 2, wherein the molecule comprises at least five DF3/MUC1 tandem repeat segments.

6. The method of claim 2, wherein the molecule comprises less than all or none of the amino acid sequence of DF3/MUC1 outside of the terminal repeat domain.

7. The method of claim 1, wherein the deleterious effects are associated with autoimmune disease.

8. The method of claim 1, wherein the target cell is an allogeneic or xenogeneic target cell transplanted into the animal, and the deleterious effects are associated with transplant rejection.

9. The method of claim 8, wherein the target cell is within a transplanted organ or tissue.

10. The method of claim 1, wherein the providing step comprises introducing into the target cell a nucleic acid which is expressed within the target cell, said nucleic acid encoding a polypeptide comprising said tandem repeat segment .

11. The method of claim 1, wherein the providing step comprises contacting the target cell with the molecule, said molecule being exogenously produced.

12. The method of claim 11, wherein the mucin is DF3/MUC1 and the molecule lacks the transmembrane domain of DF3/MUC1.

13. The method of claim 12, wherein the molecule also lacks the cytoplasmic domain of DF3/MUC1.

14. The method of claim 1, wherein the molecule is a glycoprotein.

15. A method of decreasing or preventing the cytotoxic T lymphocyte response in an animal, comprising identifying an animal in need of said decreasing or preventing, and introducing into the animal a molecule comprising a tandem repeat segment of a mucin.

16. The method of claim 15, wherein the mucin is DF3/MUC1.

17. The method of claim 16, wherein the molecule comprises less than all, or none, of the DF3/MUC1 sequence outside of the tandem repeat domain.

18. The method of claim 17, wherein the molecule lacks at least the transmembrane domain of DF3/MUC1.

19. The method of claim 15, wherein the introducing step is accomplished by administering a therapeutic composition comprising said molecule to the animal .

20. The method of claim 15, wherein the introducing step is accomplished by introducing into a cell of the animal an expression vector encoding the polypeptide portion of the molecule.

21. A method of decreasing or preventing the deleterious effects of cytotoxic T lymphocytes in an animal , comprising providing a non-tumor cell obtained from a first animal or a second animal, wherein said cell, when implanted into the first animal, is at risk of attack by the first animal's cytotoxic T lymphocytes; providing to the cell, ex vivo, a molecule comprising a tandem repeat segment of a mucin, so that the tandem repeat segment is at the surface of the cell; and implanting the cell into the first animal.

22. The method of claim 21, wherein the mucin is DF3/MUC1.

23. The method of claim 21, wherein the mucin is selected from the group consisting of MUC2 , MUC3 , MUC4 , or a mucin of a non-human mammal .

24. The method of claim 22, wherein the molecule comprises at least two DF3/MUC1 tandem repeat segments.

25. The method of claim 22, wherein the molecule comprises at least five DF3/MUC1 tandem repeat segments.

26. The method of claim 22, wherein the molecule comprises less than all or none of the amino acid sequence of DF3/MUC1 outside of the terminal repeat domain.

27. The method of claim 21, wherein the deleterious effects are associated with autoimmune disease .

28. The method of claim 21, wherein the providing step comprises introducing into the target cell a nucleic acid which is expressed within the target cell, said nucleic acid encoding a polypeptide comprising said tandem repeat segment .

29. The method of claim 21, wherein the providing step comprises contacting the target cell with the molecule, said molecule being exogenously produced.

30. The method of claim 21, wherein the molecule is a glycoprotein.

31. A transfected mammalian cell containing an isolated nucleic acid encoding a polypeptide comprising a mucin tandem repeat segment, wherein the cell is not a tumor cell .

32. The transfected mammalian cell of claim 31, wherein the cell is not within an animal.

33. The transfected mammalian cell of claim 31, wherein the transfected mammalian cell is part of a tissue or organ that has been removed from a mammal.

34. The transfected mammalian cell of claim 31 wherein said nucleic acid encodes a polypeptide comprising a DF3/MUC1 tandem repeat segment.

35. A transgenic non-human mammal containing a transgene encoding a polypeptide comprising a tandem repeat segment of a human mucin.

36. A transplantable tissue or organ of the transgenic non-human mammal of claim 35, wherein a plurality of cells of the tissue or organ express the polypeptide .

37. A method of augmenting the immune response to a tumor cell in an animal, comprising identifying an animal suspected of harboring a tumor cell bearing a mucin on its surface, wherein the mucin' s presence on the tumor cell inhibits the animal's immune response to the tumor cell, and introducing into the animal a therapeutic composition comprising a molecule comprising (1) an antibody which binds to an epitope within a mucin tandem repeat segment, or (2) an antigen-binding fragment of said antibody, wherein the therapeutic composition comprises no detectable label.

38. The method of claim 37, comprising the additional step of, prior to the introducing step, determining whether a tumor cell of the animal bears the mucin on its surface.

39. The method of claim 37, wherein the mucin is DF3/MUC1.

40. The method of claim 39, wherein the epitope comprises a three-residue portion of the sequence Trp-

Arg-Pro-Ala-Pro-Gly-Ser (SEQ ID N0:2) .

41. The method of claim 40, wherein the epitope comprises a six-residue portion of the sequence Trp-Arg- Pro-Ala-Pro-Gly-Ser (SEQ ID NO: 2) .

42. The method of claim 41, wherein the antibody is MAb DF3 (ATCC accession no. ) , or a humanized form of Mab DF3.

43. An in vivo method of decreasing or preventing the deleterious effects of cytotoxic T lymphocytes in an animal, comprising identifying an animal suspected of having a disorder attributable at least in part to activation of cytotoxic T lymphocytes, and introducing into the animal a molecule comprising a mucin tandem repeat segment in an amount effective to decrease or prevent the deleterious effects of activated cytotoxic T lymphocytes associated with the disorder.

44. The method of Claim 43, wherein the disorder is characterized by cytotoxic T lymphocyte induced granuloma formation.

45. The method of Claim 44, wherein the condition is selected from the group consisting of tuberculosis, sarcoidosis, leprosy, Crohn' s disease, hypersensitivity pneumonitis, primary biliary cirrhosis and multiple sclerosis .

46. The method of Claim 43, wherein the disorder is a malignant disorder of cytotoxic T lymphocytes.

47. The method of Claim 46, wherein the disorder is T cell Leukemia/Lymphoma.

48. The method of Claim 1, wherein the molecule comprises 20 or fewer tandem repeat segments of the mucin.

49. The method of Claim 10, wherein the molecule comprises 15 or fewer tandem repeat segments of the mucin.

50. The method of Claim 15, wherein the molecule comprises 20 or fewer tandem repeat segments of the mucin.

51. The method of Claim 20, wherein the molecule comprises 15 or fewer tandem repeat segments of the mucin.

52. The method of Claim 21, wherein the molecule comprises 20 or fewer tandem repeat segments of the mucin.

53. The method of Claim 28, wherein the molecule comprises 15 or fewer tandem repeat segments of the mucin.

54. The method of Claim 31, wherein the molecule comprises 15 or fewer tandem repeat segments of the mucin.