EP0964694A1 - Immunomodulation induite par la mucine - Google Patents

Immunomodulation induite par la mucine

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Publication number
EP0964694A1
EP0964694A1 EP97946409A EP97946409A EP0964694A1 EP 0964694 A1 EP0964694 A1 EP 0964694A1 EP 97946409 A EP97946409 A EP 97946409A EP 97946409 A EP97946409 A EP 97946409A EP 0964694 A1 EP0964694 A1 EP 0964694A1
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EP
European Patent Office
Prior art keywords
mucin
molecule
muc1
animal
tandem repeat
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97946409A
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German (de)
English (en)
Other versions
EP0964694A4 (fr
Inventor
Jianlin Gong
Donald Kufe
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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Publication of EP0964694A1 publication Critical patent/EP0964694A1/fr
Publication of EP0964694A4 publication Critical patent/EP0964694A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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 .
  • cytotoxic T lymphocytes include, e . g. , decreasing, preventing, suppressing or augmenting the effects of cytotoxic T lymphocytes in an animal .
  • 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.
  • 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.
  • PEM episialin
  • 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.
  • 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.
  • 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.
  • G+C highly conserved
  • bp 60 base pair tandemly repeating units that confer allelic variation and expression of a polymorphic core of the polypeptide moiety.
  • 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 .
  • mouse homologue of MUC1 includes a tandem domain consisting of 16 repeated units of 20-21 amino acids.
  • Flanking the 30-90 DF3/MUC1 tandem repeats 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.
  • tandem repeats The functional role of the tandem repeats has been proposed to reside in the formation of a carbohydrate scaffold.
  • 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.
  • the structure containing the mucin tandem repeat segments is proteolytically cleaved from the transmembrane and cytoplasmic domains.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • the mucin after which the tandem repeat segment is patterned is DF3/MUC1.
  • tandem repeat segments patterned after repeats seen in other mucins 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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) .
  • 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.
  • the cell can be transfected with the nucleic acid of the invention, so that it expresses the tandem repeat-containing molecule.
  • 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 .
  • 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.
  • 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.
  • 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. ).
  • 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.
  • 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.
  • decreasing the deleterious effects is meant a lessening of the severity of a disease condition existing in an animal .
  • 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.
  • cytotoxic T lymphocytes By “deleterious effects of cytotoxic T lymphocytes” is meant a disorder caused directly or indirectly by the presence or activity of cytotoxic T lymphocytes .
  • tumor cell 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.
  • mucins are DF3/MUC1, MUC2 , MUC3 , MUC4 , the Xenopus integumentary mucin, and the porcine submaxillary mucin.
  • 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 .
  • 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 .
  • in vivo is meant within the body of an animal.
  • ex vivo is meant outside and separate from the body of an animal .
  • 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.
  • transduced cell 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.”
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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.
  • Example 1 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.
  • 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
  • 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
  • mice C57B1/6 mice were injected subcutaneously with 2 x 10 6 MC-38/Neo or MC-38/MUC1 cells treated with 200 Gy ⁇ -irradiation. After 14 d, the mice were immunized again with the irradiated cells. After another 14 d, 5 x 10 5 or 1 x 10 6 tumor cells in 100 ⁇ l phosphate buffered saline (PBS) were injected subcutaneously in the flank of the immunized mice. In certain experiments, tumor cells were first incubated with 25 ⁇ g/ml MAb DF3 , 5 ⁇ g/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 assays. Cytotoxic T lymphocyte assays. Cytotoxic T lymphocyte assays. Cytotoxic T lymphocyte assays
  • T lymphocyte activity was determined by the release of lactate dehydrogenase (LDH) .
  • LDH lactate dehydrogenase
  • 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.
  • reactivity with MAb DF3 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
  • 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) .
  • mice 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 10 6 irradiated MC-38/Neo cells. The mice were then challenged with 5 x 10 5 (FIG. 2A, left panel) or 1 x 10 6 (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.
  • 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.
  • MC-38/Neo FIG. 2B, left panel
  • MC- 38/MUC1 FIG. 2B, right panel
  • 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.
  • mice immunized with MC-38/MUC1 cells 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 10 6 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 ( ⁇ ) . 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.
  • 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.
  • 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) .
  • DF3/MUC1 molecules as both an inhibitor of cytotoxic T lymphocyte function and as a target for cytotoxic T lymphocyte activity.
  • 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 .
  • cytotoxic T lymphocytes induced against MC- 38/MUC1 are active against both MC-38/MUC1 and MC-38/Neo cells.
  • DF3/MUC1 inhibits the effector function of certain activated cytotoxic T lymphocytes, but not the process of activation itself.
  • 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.
  • 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-
  • 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 ⁇ MC-38/MUC1 tumor cells incubated with 25 ⁇ g/ml mouse IgG (O) , 25 ⁇ g/ml MAb DF3 (•) or 5 ⁇ g/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 ⁇ g/ml MAb DF3 and a 25% tumor incidence after treatment with 5 ⁇ /ml MAb DF3 (FIG. 4A, left) . By contrast, MC-38/MUC1 cells treated with 25 ⁇ g/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 ⁇ g/ml mouse IgG (o) , 25 ⁇ g/ml MAb DF3 ( ⁇ ) or no antibody (0) .
  • 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) .
  • 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 10 5 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) .
  • 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.
  • these findings indicate that the tandem repeats of DF3/MUC1 interact with certain activated cytotoxic T lymphocytes and abrogate their function by inducing apoptosis.
  • 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
  • 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 .
  • the mucin tandem repeat expressed in the tissue or organ be from the same species as the transplant recipient.
  • the tandem repeat segment can be derived from a human mucin, e.g. DF3/MUC1, MUC2 , MUC3 or MUC4.
  • 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
  • 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” ) .
  • primary or secondary human cells e.g., fibroblasts, epithelial cells including mammary and intestinal epithelial cells, endothelial
  • 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.
  • 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.
  • nucleic acid 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.
  • the nucleic acid can encode a non-mucin related polypeptide linked to the mucin-derived portion of the molecule.
  • 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.
  • 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.
  • tandem repeat-containing molecule e.g. a mucin or a fragment of a mucin, or any of the genetically-engineered hybrids referred to above
  • 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.
  • An in vivo therapy can provide a means of augmenting the immune response against a tumor.
  • 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.
  • a cell attachment ligand on the vector or the viral particle to accomplish preferential targeting of specific cells in accordance with standard methods .
  • 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, ⁇ -fetoprotein promoter for hepatocellular carcinoma (Huber et al . , 1991, Proc. Natl. Acad. Sci.
  • the molecule can be administered locally to the tumor site, or intravenously, orally, intraperitoneally, intrapulmonarily, or by another appropriate route.
  • 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.
  • a molecule e.g., polypeptide or glycoprotein
  • a nucleic acid encoding the polypeptide portion of that molecule.
  • 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.
  • the therapy of the invention is believed to induce apoptosis in activated cytotoxic T lymphocytes.
  • Ex vivo-based Human Therapies can use either a polypeptide- based molecule or a nucleic acid which expresses such a molecule .
  • 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.
  • 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.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO : 2 :

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Abstract

L'invention concerne un procédé destiné à réduire ou à prévenir les effets toxiques des lymphocytes T cytotoxiques, qui consiste à identifier un animal possédant une cellule non tumorale exposée à une attaque par les lymphocytes T cytotoxiques de l'animal, et à fournir ladite cellule une molécule présentant au moins une séquence répétée en tandem d'une mucine. L'invention concerne également un procédé destiné à augmenter la réponse immunitaire contre une cellule tumorale exprimant une mucine, à l'aide d'une composition qui se lie à la séquence répétée en tandem de la mucine et qui bloque les effets inhibiteurs des CTL de la mucine.
EP97946409A 1996-10-25 1997-10-24 Immunomodulation induite par la mucine Withdrawn EP0964694A4 (fr)

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US738262 1991-07-31
US73826296A 1996-10-25 1996-10-25
PCT/US1997/019784 WO1998017300A1 (fr) 1996-10-25 1997-10-24 Immunomodulation induite par la mucine

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AU741258B2 (en) * 1996-09-10 2001-11-29 Biomira Inc. MUC-1 as an immunosuppressive therapeutic agent for the treatment of inflammatory and autoimmune conditions
US20050169898A1 (en) 1997-04-15 2005-08-04 Jianlin Gong Cell fusions and methods of making and using the same
AU744042B2 (en) * 1997-04-15 2002-02-14 Dana-Farber Cancer Institute, Inc. Dendritic cell hybrids
GB9904695D0 (en) * 1999-03-01 1999-04-21 Imp Cancer Res Tech Peptide
US8031190B2 (en) 2004-05-03 2011-10-04 Microsoft Corporation Translating two-dimensional user input on three-dimensional scene

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WO1995003825A1 (fr) * 1993-07-30 1995-02-09 Finn Olivera J Peptides synthetiques a repetitions en tandem multiples, a base de mucine et d'analogues, et utilisations
WO1998010783A1 (fr) * 1996-09-10 1998-03-19 Biomira Inc. Le muc-1, agent therapeutique immunosuppresseur traitant les etats inflammatoires et auto-immunitaires

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WO1995003825A1 (fr) * 1993-07-30 1995-02-09 Finn Olivera J Peptides synthetiques a repetitions en tandem multiples, a base de mucine et d'analogues, et utilisations
WO1998010783A1 (fr) * 1996-09-10 1998-03-19 Biomira Inc. Le muc-1, agent therapeutique immunosuppresseur traitant les etats inflammatoires et auto-immunitaires

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DENTON G ET AL: "INDUCTION OF ANTIBODY RESPONSES TO BREAST CARCINOMA ASSOCIATED MUCINS USING SYNTHETIC PEPTIDE CONSTRUCTS AS IMMUNOGENS" CANCER LETTERS,US,NEW YORK, NY, vol. 70, no. 3, 16 July 1993 (1993-07-16), pages 143-150, XP002035983 ISSN: 0304-3835 *
GRAHAM R A ET AL: "The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine" CANCER IMMUNOLOGY AND IMMUNOTHERAPY,DE,BERLIN, vol. 2, no. 42, 1 January 1996 (1996-01-01), pages 71-80, XP002075466 ISSN: 0340-7004 *
HILKENS J ET AL: "Is episialin/MUC1 involved in breast cancer progression?" CANCER LETTERS, vol. 90, no. 1, 1995, pages 27-33, XP000915262 ISSN: 0304-3835 *
PEAT N ET AL: "TISSUE-SPECIFIC EXPRESSION OF A HUMAN POLYMORPHIC EPITHELIAL MUCIN MUC1 IN TRANSGENIC MICE" CANCER RESEARCH, vol. 52, no. 7, 1992, pages 1954-1960, XP000915268 ISSN: 0008-5472 *
REDDISH MARK A ET AL: "Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope sialyl-Tn-KLH cancer vaccine in active specific immunotherapy." CANCER IMMUNOLOGY IMMUNOTHERAPY, vol. 42, no. 5, 1996, pages 303-309, XP000914873 ISSN: 0340-7004 *
See also references of WO9817300A1 *
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WO1998017300A1 (fr) 1998-04-30

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