WO1994011401A1 - Homologue humain du gene de la cadherine e et procedes d'utilisation - Google Patents

Homologue humain du gene de la cadherine e et procedes d'utilisation Download PDF

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WO1994011401A1
WO1994011401A1 PCT/US1993/011097 US9311097W WO9411401A1 WO 1994011401 A1 WO1994011401 A1 WO 1994011401A1 US 9311097 W US9311097 W US 9311097W WO 9411401 A1 WO9411401 A1 WO 9411401A1
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protein
cadherin
human
nucleic acid
subject
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PCT/US1993/011097
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English (en)
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David L. Rimm
Jon S. Morrow
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Yale University
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Priority to AU56694/94A priority Critical patent/AU5669494A/en
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to the human epithelial-cadherin (E-cadherin) gene and its encoded protein product, as well as derivatives and analogs of the human E-cadherin protein. Production of human E-cadherin proteins, derivatives and antibodies is also provided.
  • the invention further relates to therapeutic and diagnostic methods and compositions.
  • CAMs Cell adhesion molecules
  • CAMs are cell surface glycoproteins which mediate specific cell-cell adhesions involved in embryonic development and maintaining tissue form and function.
  • E-cadherin is a cell adhesion molecule that is also known as uvomorulin, L-CAM and Cell CAM 120/80. E-cadherin localizes to the lateral surfaces and is concentrated in the adherens junctions of intestinal epithelial cells. It is present in epithelial cells from all organs examined, and related "cadherin family" molecules have been identified in brain, muscle, placenta, and other organs. This molecule has been attributed to play a role in initiation of the formation of the cortical cytoskeleton, establishment of polarity (Nelson, 1991, Sem. Cell. Bio. 2:375-385; Nelson and Hammerton, 1989, J. Cell Biol.
  • E-cadherin is a 120 kilodalton (kD) membrane spanning protein with a large, glycosylated, amino-terminal extracellular domain and a 150 amino acid
  • the extracellular domain is cleavable with trypsin in the presence of Ca ++ , resulting in an 80 kD peptide that contains three putative repeat structures (possibly involved in Ca + + binding) and a highly conserved amino-terminal 113 amino acid region. Within this region is a HAV
  • E-cadherin The murine and avian homologs of E-cadherin have been cloned and sequenced (see, e.g. , Ringwald et al., 1987, EMBO J. 6:3647-3653;
  • E-cadherin have not been available prior to the present invention. A knowledge of such sequences is of primary importance since proteins having human sequences and antibodies thereto are greatly preferred over those of other species for human therapeutic and diagnostic purposes. Knowledge of the complete
  • E-cadherin sequences is also important for deriving appropriate strategies in the generation of derivatives and fragments, for example, in choosing an appropriate restriction enzyme for cleavage in the isolation of portions of the coding sequence.
  • the present invention relates to nucleotide sequences of the human E-cadherin gene, and the amino acid sequences of the encoded E-cadherin protein.
  • the invention further relates to fragments and other derivatives, and analogs, of the human E-cadherin protein, as well as antibodies thereto. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g. , by recombinant methods, is provided.
  • the invention relates to human E-cadherin protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a human E-cadherin protein, including but not limited to the amino-terminal processed region, the HAV homotypic binding domain, one or more of the three repeat domains, the conserved cysteine domain, the transmembrane region, the extracellular region, the cytoplasmic domain, and any combination of the foregoing.
  • the present invention further relates to therapeutic and diagnostic methods and compositions based on E-cadherin proteins and nucleic acids.
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention.
  • therapeutic compounds include: E-cadherin proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the E-cadherin proteins, analogs, or derivatives; and E-cadherin antisense nucleic acids.
  • a Therapeutic of the invention is administered to treat or prevent a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state, or to inhibit or ameliorate metastatic tumor development.
  • Methods of promoting nerve or tissue regeneration, of promoting wound healing, of treating an inflammatory disorder, and of treating or preventing gestational disease or fetal wastage are also provided.
  • the invention provides the complete nucleotide sequence of human cDNA from liver and colon coding for E-cadherin, and the sequence of the encoded human E-cadherin protein. Also described are specific anti-human E-cadherin antibodies, and multiple human cloned fragments of both liver and colon
  • E-cadherin cDNAs some of which have been expressed in both eukaryotic and prokaryotic expression systems.
  • FIG. 1 A schematic diagram of E-cadherin showing structural features including the amino (N)-terminal processed region, the HAV homotypic binding sequence, the three repeat domains, and the highly conserved carboxy (C)-terminal cytoplasmic domain. Also shown are some of the proteins thought to interact, either directly or indirectly, with the cytoplasmic domain.
  • FIG. 1 A restriction map of the liver E-cadherin clone. The translation start is shown preceding the mature N terminus of the protein. Three proposed repeat domains are shown and the homotypic adhesion sequence (HAV) is located in the first repeat. The hashed portion of the sequence denotes the region originally published by Mansouri et al. (1988, Differentiation 38:67-71). The regions labeled e250 and cyto 20 are regions that have been produced as fusion proteins for in vitro binding studies and antibody production.
  • HAV homotypic adhesion sequence
  • FIG. 1 The complete nucleotide (SEQ ID NO: 1) and protein (SEQ ID NO:2) sequences of human liver E-cadherin.
  • Figure 4 Series of E-cadherin clones shown by location and source. Leftmost column lists the clone name. Numbers beneath lines are the starting and ending nucleotide of each clone. Clones with hashmarks on one end are either clones whose ends are not yet sequenced or fusion clones with artefactual sequence on the end as shown. Part A: liver clones. Part B: colon clones. Solid bar: characterized E-cadherin sequence; cross-hatched area:
  • FIG. 1 Schematic diagram of plasmid pCMV-NeoPoly 1.
  • pCMV-NeoPoly 1 is a 6.7 kb plasmid that was constructed and kindly provided by Dr. Eric R. Fearon.
  • Known unique restriction sites Xhol, EcoRV, BamHI, StuI, Nhel, Hindlll, and Sstl.
  • the present invention relates to nucleotide sequences of the human E-cadherin gene, and the amino acid sequence of the encoded E-cadherin protein.
  • the invention further relates to fragments and other derivatives, and analogs, of the human E-cadherin protein. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g. , by recombinant methods, is provided.
  • the invention also relates to human E-cadherin protein derivatives and analogs of the invention which are functionally active, i.e. , they are capable of displaying one or more known functional activities associated with a full-length (wild-type) E-cadherin protein.
  • Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a E-cadherin protein for binding) to an anti-E-cadherin protein antibody], immunogenicity (ability to generate antibody which binds to a E-cadherin protein), ability to bind (or compete with a E-cadherin protein for binding) to a receptor or ligand for a E-cadherin protein, suppression of cell invasiveness, therapeutic activity, etc.
  • the invention further relates to fragments (and derivatives and analogs thereof) of a human E-cadherin protein which comprise one or more domains of a human E-cadherin protein (see Section 6), including but not limited to the amino-terminal processed region, the HAV homotypic binding domain, one or more of the three repeat regions, the conserved cysteine domain, the extracellular region, transmembrane region, cytoplasmic domain, and any combination of the foregoing.
  • Antibodies to the human E-cadherin protein and its derivatives and analogs are additionally provided.
  • the present invention further relates to therapeutic and diagnostic methods and compositions based on E-cadherin proteins and nucleic acids.
  • the invention provides for treatment by administration of a therapeutic compound of the invention.
  • therapeutic compounds include: human E-cadherin proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the human
  • a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non- malignant state (e.g. , metaplastic condition) into a neoplastic or a malignant state, or to inhibit or ameliorate metastatic tumor development.
  • a nucleic acid encoding a human E-cadherin protein or fragment thereof is used in gene therapy. Methods of promoting nerve or tissue regeneration, of promoting wound healing, of treating an inflammatory disorder, and of treating or preventing gestational disease or fetal wastage are also provided.
  • E-cadherin plays a role in developmental and other physiological processes.
  • the nucleic acid and amino acid sequences and antibodies thereto of the invention can also be used for the detection and quantitation of human E-cadherin mRNA, to study expression thereof, to produce human E-cadherin proteins, fragments and other derivatives, and analogs thereof, in the study and manipulation of differentiation and other physiological processes.
  • the invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of human E-cadherin cDNAs from liver and colon, and the construction and recombinant expression of human E-cadherin chimeric/fusion derivatives and production of antibodies thereto.
  • the invention relates to the nucleotide sequences of human
  • the human E-cadherin nucleic acid comprises the nucleotide sequence (SEQ ID NO: 1 ) shown in
  • Figure 3 in particular, from nucleotides numbers 116-2749, or 566-2749, or 1037-2748, or fragments thereof.
  • a nucleic acid is provided which comprises the nucleotide sequence depicted in Figure 3 (SEQ ID NO: 1) from nucleotide numbers 1-1053, 510-2686, 1332-3000, 540-1500, 348-906, 890-1648, 384-1208, 641-2046, 685-1336, 880-1661, 1199-1742, 1373-1742, 1705-2204, or 2458-2775 (see Fig. 4).
  • the nucleotide sequence encodes all or a portion of the amino acid sequence (SEQ ID NO:2) shown in Figure 3.
  • the invention provides nucleic acids consisting of at least 8 nucleotides (i.e. , a hybridizable portion) of a human E-cadherin sequence; in other embodiments, the nucleic acids consist of at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a human E-cadherin sequence.
  • the invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences.
  • nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 30, 50, 100, or 200 nucleotides or the entire coding region of a human E-cadherin gene.
  • the longest stretch of identity among the human E-cadherin sequence of Figure 3 and the published mouse and chicken E-cadherin sequences is 29 nucleotides.
  • Nucleic acids comprising a portion of the noncoding sequence shown in Figure 3 are also provided, as are nucleic acids complementary thereto.
  • the nucleic acids of the invention do not consist of the nucleotide sequence shown in Figure 3
  • nucleic acids from nucleotide numbers 617-1036; preferably, such nucleic acids also do not consist of a portion of such nucleotide sequence.
  • E-cadherin proteins (see Section 5.6) are additionally provided.
  • a human E-cadherin DNA can be cloned and sequenced by the method described in Section 6, infra.
  • Any human cell potentially can serve as the nucleic acid source for the molecular cloning of the E-cadherin gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g. , a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. (See, for example. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press. Ltd., Oxford, U.K. Vol.
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will lack introns and will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
  • identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a E-cadherin (of any species) gene or its specific RNA, or a fragment thereof, e.g. , an extracellular, or cytoplasmic region (see Section 5.6), is available and can be purified, or synthesized, and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).
  • DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map, either available or deduced from a known nucleotide sequence. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g. , has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, binding activity, or antigenic properties as known for a
  • E-cadherin protein By use of an antibody to a E-cadherin protein, the
  • E-cadherin protein may be identified by binding of labeled antibody to the putatively E-cadherin protein synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.
  • ELISA enzyme-linked immunosorbent assay
  • the E-cadherin gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified E-cadherin DNA of human or of another species (e.g. , mouse, chicken). Immunoprecipitation analysis or functional assays (e.g. , binding to a receptor or ligand; see infra) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences.
  • specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against a E-cadherin protein.
  • a radiolabelled E-cadherin cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to identify the E-cadherin DNA fragments from among other genomic DNA fragments.
  • Alternatives to isolating the human E-cadherin genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes a human E-cadherin protein.
  • E-cadherin gene can be isolated from human cells (e.g. , epithelial cells) which express a E-cadherin protein. Other methods are possible and within the scope of the invention.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and E-cadherin gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, microinjection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.
  • transformation of host cells with recombinant DNA molecules that incorporate the isolated E-cadherin gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained in large quantities by growing
  • transformants isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the nucleotide sequence coding for a human E-cadherin protein or a functionally active fragment or other derivative thereof can be inserted into an appropriate expression vector, i. e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence.
  • the necessary transcriptional and translational signals can also be supplied by the native E-cadherin gene and/or its flanking regions.
  • host-vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g.
  • vaccinia virus adenovirus
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities.
  • any one of a number of suitable transcription and translation elements may be used.
  • a chimeric protein comprising the extracellular domain or repeat region or other domain of a human E-cadherin protein is expressed.
  • a full-length human E-cadherin cDNA is expressed, or a sequence encoding a functionally active portion of a human E-cadherin protein.
  • a fragment of a human E-cadherin protein comprising a domain of the protein, or other derivative, or analog of a human E-cadherin protein is expressed.
  • any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a human E-cadherin protein or peptide fragment may be regulated by a second nucleic acid sequence so that the E-cadherin protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a E-cadherin protein may be controlled by any promoter/enhancer element known in the art.
  • Promoters which may be used to control E-cadherin gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. , 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981 , Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), an adenovirus promoter,
  • CMV cytomegalovirus
  • prokaryotic promoters such as the ⁇ - lactamase (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727- 3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
  • ⁇ P L ⁇ P L
  • trc promoters see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981 , Nucl. Acids Res.
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al. , 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • a pGEX vector (Pharmacia) is used for expression in bacteria.
  • a nucleotide sequence encoding a human E-cadherin or fragment or derivative thereof is operatively linked to a promoter, wherein the promoter is not a human E-cadherin gene promoter.
  • Expression vectors containing human E-cadherin gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
  • first approach the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted E-cadherin gene.
  • second approach the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.
  • recombinants containing the E-cadherin insert can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the E-cadherin gene product in in vitro assay systems, e.g. , binding to a ligand or receptor, binding with antibody.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered E-cadherin protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification (e.g. , glycosylation, cleavage) of proteins.
  • mammalian, yeast, and baculovirus host cells can glycosylate proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • cDNA and genomic sequences can be cloned and expressed.
  • the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay, etc.
  • a human E-cadherin protein may be isolated and purified by standard methods including chromatography (e.g. , ion exchange, affinity, and sizing column chromatography). centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the functional properties may be evaluated using any suitable assay (see Section 5-7).
  • the amino acid sequence of a human E-cadherin protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. Once the amino acid sequence is thus known, the protein can be synthesized by standard chemical methods known in the art (e.g. , see Hunkapiller et al., 1984, Nature 310:105-111).
  • a human E-cadherin protein includes but is not limited to one containing, as a primary amino acid sequence, all or part of the amino acid sequence substantially as depicted in Figure 3 (SEQ ID NO:2), as well as fragments and other derivatives, and analogs thereof.
  • the invention relates to mature human E-cadherin proteins, e.g.. those having an amino acid sequence substantially as depicted in Figure 3 from amino acid numbers 151-878.
  • a protein comprises the amino acid sequence as depicted in Figure 3 from amino acid numbers 153-878.
  • the invention relates to an E-cadherin protein having an amino acid sequence substantially as depicted in Figure 3 from amino acid numbers 1-878.
  • Purified proteins comprising the foregoing sequences are also provided.
  • the invention provides purified human E-cadherin proteins and fragments thereof that are free of detergents, substantially non-denatured, and/or free of other human cell membrane components.
  • the human E-cadherin protein or fragment thereof e.g. , comprising the extracellular domain
  • is glycosylated e.g. , as obtained by expression in mammalian cells.
  • the human E-cadherin protein or fragment thereof is nonglycosylated (e.g.. as obtained by expression in bacteria). Nonglycosylated mature E-cadherin proteins are believed to be capable of homotypic binding.
  • the cloned DNA or cDNA corresponding to the E-cadherin gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g. , Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis (see infra). Polymerase chain reaction (PCR; U.S. Patent Nos.
  • the stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific E-cadherin probe used, whether it be human or other species.
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of the human E-cadherin gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once the nucleotide sequence is known.
  • DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al. , 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699; Sequenase, U.S. Biochemical Corp.), or Taq polymerase, or use of an automated DNA sequenator (e.g. , Applied Biosystems, Foster City, CA).
  • the cDNA sequence of a human E-cadherin gene comprises me sequence substantially as depicted in Figure 3 (SEQ ID NO: 1), and described in Section 6, infra.
  • the amino acid sequence of a human E-cadherin protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g. , with an automated amino acid sequencer.
  • the amino acid sequence of a representative human E-cadherin protein comprises the amino acid sequence substantially as depicted in Figure 3 (SEQ ID NO:2).
  • the sequence of the mature E-cadherin protein is substantially as depicted in Figure 3 from amino acid numbers 151-878.
  • the E-cadherin protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a E-cadherin protein and the corresponding regions of the gene sequence which encode such regions.
  • Biochemistry 13:222 can also be done, to identify regions of a E-cadherin protein mat assume specific secondary structures.
  • Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.
  • a human E-cadherin protein may be used as an immunogen to generate antibodies which recognize such an immunogen.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • antibodies which specifically bind to human E-cadherin proteins are produced.
  • such an antibody recognizes the human E-cadherin protein having the sequence shown in Figure 3 (SEQ ID NO:2), or a portion thereof.
  • such an antibody specifically binds to human, but not mouse or chicken, E-cadherin.
  • antibodies to a particular domain e.g. , the cytoplasmic domain of a human E-cadherin protein are produced.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to a human E-cadherin protein or derivative or analog.
  • various host animals can be immunized by injection with a native human E-cadherin protein, or a synthetic version, or derivative (e.g. , fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a human E-cadherin peptide (e.g. , identified by the procedure of Hopp and Woods (1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824)).
  • a hydrophilic portion of a human E-cadherin peptide e.g. , identified by the procedure of Hopp and Woods (1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824).
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies can be used.
  • monoclonal antibodies can be produced in germ-free animals (PCT Publication No.
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • E-cadherin of another species e.g. , mouse, chicken
  • antibodies can be used in methods known in the art relating to the localization and activity of the protein sequences of the invention (e.g. , see Section 5.7, infra), e.g. , for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc., diagnostically, and
  • merapeutically e.g. , for inhibiting E-cadherin function.
  • the invention further relates to derivatives (including but not limited to fragments) and analogs of human E-cadherin proteins.
  • the derivative or analog is functionally active, i.e. , capable of exhibiting one or more functional activities associated with a full-length, wild- type human E-cadherin protein.
  • such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization, for promotion or inhibition of E-cadherin protein activity, etc.
  • Such molecules which retain, or alternatively inhibit, a desired human E-cadherin protein property e.g.
  • binding to a receptor or ligand can be used as inducers, or inhibitors, respectively, of such property and its physiological correlates.
  • a receptor or ligand such as possibly Notch protein
  • Derivatives or analogs of E-cadherin proteins can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in Section 5.7.
  • E-cadherin derivatives can be made by altering E-cadherin sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • nucleotide coding sequences Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a human E-cadherin gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of human E-cadherin genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the E-cadherin derivatives of the invention include, but are not limited to, diose containing, as a primary amino acid sequence, all or part of the amino acid sequence of a human E-cadherin protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • proteins consisting of or comprising a fragment of a human E-cadherin protein consisting of at least 30 amino acids of the E-cadherin protein is provided.
  • the fragment consists of at least 6, 10, 50, 75, or 100 amino acids of the E-cadherin protein.
  • a protein comprising a fragment of the amino acid sequence shown in Figure 3 (SEQ ID NO:2) from amino acid numbers 728-878, which can be bound by anti-E-cadherin antibody.
  • a purified protein is provided which comprises a derivative or fragment of a human E-cadherin protein, with the proviso that said purified protein is not a mature human E-cadherin protein comprising amino acids 151-878 as depicted in Figure 3 (SEQ ID NO:2).
  • the human E-cadherin protein derivatives and analogs of the invention can be produced by various mediods known in the art.
  • E-cadherin gene sequence can be modified by any of numerous strategies known in the art (Maniatis, 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring
  • E-cadherin gene uninterrupted by translational stop signals, in the gene region where the desired E-cadherin protein activity is encoded.
  • the E-cadherin-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or term.:.ation sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551).
  • Manipulations of the human E-cadherin sequence may also be made at the protein level.
  • human E-cadherin protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g. , by acetylation, glycosylation or deglycosylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, etc.
  • analogs and derivatives of human E-cadherin proteins can be chemically synthesized.
  • a peptide corresponding to a portion of a E-cadherin protein which comprises the desired domain can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the human E-cadherin protein sequence.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids.
  • the human E-cadherin derivative is a chimeric, or fusion, protein comprising a human E-cadherin protein or fragment thereof (preferably consisting of at least a domain or region of the E-cadherin protein, or at least 30 amino acids of the E-cadherin protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a human
  • E-cadher in-coding sequence joined in-frame to a coding sequence for a different protein).
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by mediods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g. , by use of a peptide synthesizer.
  • a specific embodiment relates to a chimeric protein comprising a fragment of a E-cadherin protein which comprises a domain or motif of the E-cadherin protein, e.g.
  • the extracellular domain the extracellular domain, transmembrane region, cytoplasmic domain, amino-terminal processed region, homotypic binding domain, conserved cysteine domain, HAV sequence, repeat region, repeat #1, repeat #2, repeat #3, or any combination of the foregoing (see Section 7).
  • Another specific embodiment relates to a chimeric protein comprising a fragment of a human E-cadherin protein of at least six amino acids.
  • the fusion protein comprises or consists of the extracellular portion of human E-cadherin or a functional fragment thereof joined via a peptide bond to a transmembrane domain joined via a peptide bond to the cytoplasmic (signalling) domain or functional fragment thereof of another receptor or adhesion molecule (e.g. , DCC (Deleted in Colorectal Cancer), P-cadherin, N-cadherin; N-CAM (neural cell adhesion molecule); receptor tyrosine kinases such as growth factor receptors like epidermal growth factor receptor, fibroblast growth factor receptor, neu, etc.).
  • DCC deleted in Colorectal Cancer
  • P-cadherin, N-cadherin; N-CAM neural cell adhesion molecule
  • receptor tyrosine kinases such as growth factor receptors like epidermal growth factor receptor, fibroblast growth factor receptor, neu, etc.
  • human E-cadherin fusion proteins consisting of a human E-cadherin fragment capable of generating anti-E-cadherin antibody fused to the carboxyl-terminus of glutathione-S-transferase, are described in Section 8 hereof.
  • Another specific embodiment relates to a protein comprising portions of the human E-cadherin sequence which appear in different order or are missing amino acid sequence relative to native E-cadherin.
  • a protein comprising a portion of me E-cadherin amino acid sequence shown in Figure 3 (SEQ ID NO:2) is provided, with the proviso mat the protein does not contain amino acids numbers 153-307.
  • the invention relates to human
  • E-cadherin protein derivatives and analogs in particular human E-cadherin fragments and derivatives of such fragments, mat comprise one or more domains of a human E-cadherin protein, including but not limited to the extracellular domain, transmembrane region, cytoplasmic domain, amino-terminal processed region, homotypic binding domain, HAV sequence, conserved cysteine domain, repeat domain, repeat #1, repeat #2, and repeat #3.
  • the amino acid sequences representing the foregoing domains for the protein having the sequence shown in Figure 3 are described in Section 7 infra.
  • a protein comprises one or more of human E-cadherin repeats #1 , #2, and #3.
  • a protein comprises the human E-cadherin transmembrane region and cytoplasmic domain.
  • the invention relates to a derivative or analog of human E-cadherin mat lacks one or more domains of a human E-cadherin protein.
  • the invention also provides human E-cadherin fragments, and analogs or derivatives of such fragments, which mediate binding to other proteins, and nucleic acid sequences encoding the foregoing.
  • fragments, analogs, and derivatives which bind to alpha, beta or gamma catenin, actin, spectrin/fodrin, and/or ankyrin are envisioned.
  • E-cadherin proteins, derivatives and analogs can be assayed by various methods known in the art.
  • immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labelled. Many means are known in the ert for detecting binding in an immunoassay and are within the scope of the present invention.
  • the ability to bind to another protein can be demonstrated by in vitro binding assays, noncompetitive or competitive, by methods known in the art.
  • a second E-cadherin protein alpha, beta, or gamma catenin; actin, spectrin/fodrin, ankyrin, 220 kD undercoat protein (Itoh et al., 1991 , J. Cell Biol. 115(5): 1449-1462), or ouierwise
  • the ability of an E-cadherin derivative or analog to bind to an identical molecule (“homotypic binding") or to native E-cadherin can be assayed by methods known in the art.
  • physiological correlates of E-cadherin introduction into cells can be assayed.
  • me ability to suppress cell invasion can be assayed by known methods (see, e.g. , Vleminckx et al., 1991 , Cell 66:107-119).
  • the human E-cadherin proteins, derivatives (including fragments) and analogs thereof; antibodies thereto; nucleic acids encoding the human E-cadherin proteins, derivatives, and analogs, and E-cadherin antisense nucleic acids have therapeutic utility in the modulation of functions mediated by human E-cadherin.
  • Such therapeutical ly useful molecules provided by the invention are termed herein "Therapeutics.
  • the Therapeutics have therapeutic value for various diseases and disorders.
  • One specific embodiment of the invention relates to Therapeutics which antagonize, or inhibit, a E-cadherin protein function.
  • Such Therapeutics are most preferably identified by use of known convenient in vitro assays, e.g. , based on their ability to inhibit binding of E-cadherin to other proteins, or inhibit any known E-cadherin function as assayed in vitro, although in vivo assays may also be employed.
  • a Therapeutic is a protein or derivative thereof comprising a functionally active fragment such as a fragment of a human E-cadherin protein which binds to another protein; such a Therapeutic can be used, e.g. , in soluble form, to competitively inhibit the function mediated by such fragment.
  • a Therapeutic is a protein comprising the homotypic binding domain, or an analog/competitive inhibitor of a E-cadherin signal-transducing function, a nucleic acid capable of expressing one of the foregoing proteins, a human E-cadherin antisense nucleic acid (see infra), or an anti-human E-cadherin antibody which neutralizes a functional activity of E-cadherin.
  • a nucleic acid containing a portion of a dysfunctional E-cadherin gene is used, to promote E-cadherin inactivation by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al. , 1989, Nature 342:435-438).
  • Therapeutics can be used to promote E-cadherin function.
  • Such Therapeutics include but are not limited to human E-cadherin proteins and derivatives and analogs of the invention which are functionally active, i.e. , they are capable of displaying one or more known functional activities associated with a full-length (wild-type) E-cadherin protein.
  • functional activity is the ability to suppress cell invasion or metastasis.
  • such a Therapeutic comprises one or more domains of the human E-cadherin protein, preferably the homotypic binding domain.
  • the Therapeutic is a chimeric/fusion protein comprising (a) an extracellular domain or functional derivative thereof that is of human
  • E-cadherin (b) a transmembrane domain, and (c) a cytoplasmic signalling domain or functional derivative thereof of a protein normally expressed by a cell type representative of the cell to which the Therapeutic is delivered.
  • me cytoplasmic domain can be of N-CAM or N-cadherin.
  • a recombinant nucleic acid encoding and capable of expressing such a chimeric molecule is introduced into a host cell.
  • E-cadherin function is administered to promote cell invasion.
  • a Therapeutic which promotes E-cadherin function is administered to inhibit cell invasion and metastasis.
  • introduction into cell of a nucleic acid encoding a human E-cadherin or fragment thereof which mediates homotypic binding is therapeutically useful for promotion of adhesion of such cells and prevention of their invasion and metastasis.
  • tumor cells direct delivery of the nucleic acid to such tumor cells in vivo is envisaged.
  • such cells to be used for introduction of the E-cadherin nucleic acid can be any cells to be administered in vivo for therapeutic effect; introduction into such cells of the E-cadherin-encoding sequences prior to administration of the cell to a patient can prevent the cell from subsequently becoming or behaving like an invasive tumor cell (see also Section 5.9).
  • a Therapeutic which exhibits E-cadherin homotypic binding ability is administered to treat or prevent malignancy, or metastasis of a malignancy. This is described further in Sections 5.8.1 and 5.8.2.
  • a Therapeutic which promotes E-cadherin function e.g. , comprising the extracellular domain or homotypic binding domain, and dius capable of mediating homotypic binding and resultant adhesion of cells attached to or expressing the Therapeutic
  • a Therapeutic which promotes E-cadherin function is used for treatment of benign dysproliferative disorders.
  • Specific embodiments are directed to treatment of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), and baldness (a condition in which terminally differentiated hair follicles fail to function properly).
  • a Therapeutic which promotes E-cadherin function (e.g. , comprising the extracellular domain or homotypic binding domain, and dius capable of mediating homotypic binding) is used to promote wound healing, including the treatment of burns, and to promote me re-epithelialization of the skin, mucosal surfaces, or cornea.
  • fibroblasts obtained from a patient are transfected in vitro with a nucleic acid encoding human E-cadherin or a derivative thereof capable of homotypic binding, in order to form a synmetic skin graft that, when applied to the site of a patient's wound, provides a protective autologous barrier to promote wound healing.
  • Incorporation of human E-cadherin, or cells expressing the same, in synthetic organs is also envisioned.
  • E-cadherin function is used to treat or prevent gestational disease, or fetal wastage, for example, spontaneous abortions, and developmental abnormalities of the fetus or neonate.
  • the Therapeutic is administered into the amniotic sac or intrauterinely.
  • E-cadherin is normally expressed in human placenta.
  • E-cadherin function (in particular, its function in establishing an impermeability barrier in epithelial cells) can be used for the treatment or prevention of inflammatory disorders, e.g. , Crohn's disease or sclerosing cholangitis. Crohn's disease and sclerosing cholangitis are associated with decreased permeability of epithelial cells. It has been reported that E-cadherin is required to establish the impermeability barrier in epithelial cells.
  • a Therapeutic preferably one which promotes adhesiveness mediated by E-cadherin and dius exhibits E-cadherin homotypic binding ability, include but are not limited to diose described below in this and the subsequent subsection.
  • Such malignancies and related disorders include but are not limited to those listed in Table 1 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co. , Philadelphia):
  • gestational proliferative disease e.g. , molar
  • the Therapeutics of the invention which exhibit adhesiveness to cells, or homotypic binding, can be administered to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 1.
  • Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
  • Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic of the invention.
  • characteristics of a transformed phenotype include mo ⁇ hology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc. (see also id., at pp. 84-90 for characteristics associated with a transformed or malignant phenotype).
  • leukoplakia a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ, are pre-neoplastic lesions indicative of the desirability of prophylactic intervention.
  • fibrocystic disease cystic hyperplasia, mammary dysplasia, particularly adenosis (benign epithelial hyperplasia), or atypical papillomatosis
  • adenosis benign epithelial hyperplasia
  • atypical papillomatosis is indicative of the desirability of prophylactic
  • a patient with a strong family history of breast cancer is treated with a Therapeutic, for prevention of breast cancer.
  • a patient which exhibits one or more of me following predisposing factors for malignancy is treated by administration of an effective amount of a Therapeutic: a chromosomal translocation associated with a malignancy (e.g. , the Philadelphia chromosome for chronic myelogenous leukemia, t(14;18) for follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possible forerunners of colon cancer), benign monoclonal gammopathy (a possible forerunner of multiple myeloma), and a first degree kinship with persons having a cancer or precancerous disease showing a Mendel ian (genetic) inheritance pattern (e.g.
  • Fanconi's aplastic anemia, and Bloom's syndrome see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113); or one of the foregoing precancerous conditions, or neurofibromatosis (e.g. , tuberous sclerosis, von Hippel-Lindau disease, multiple exostoses), genodermatosis (e.g. , polydysplastic epidermolysis bullosa), immune deficiency syndrome (e.g. , Wiskott-Aldrich syndrome, X-linked agammaglobulinemia), chromosome breakage or polyploidy (e.g.
  • Therapeutics which antagonize E-cadherin function, and thus promote cell invasiveness can be used therapeutically, e.g. , to promote tissue repair and regeneration.
  • a particular embodiment is directed to the promotion of nerve regeneration.
  • Therapeutics which antagonize E-cadherin function include but are not limited to human E-cadherin antisense nucleic acids, anti-human E-cadherin monoclonal antibodies (e.g. , directed against the homotypic binding region, repeat region), and human E-cadherin peptide fragments or analogs thereof (e.g. of the
  • E-cadherin extracellular domain which when administered preferably in soluble form will competitively inhibit homotypic binding or adhesiveness of E-cadherin).
  • Nervous system disorders which are dius envisioned for treatment include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
  • Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:
  • traumatic lesions including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
  • ischemic lesions in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
  • malignant lesions in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue;
  • infectious lesions in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
  • degenerative lesions in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease,
  • Huntington's chorea or amyotrophic lateral sclerosis; (vi) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to. vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
  • diabetes diabetes neuropathy
  • lesions caused by toxic substances including alcohol, lead, or particular neurotoxins and (ix) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
  • Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting neurite extension or survival or differentiation of neurons. For example, and not by way of limitation, Therapeutics which elicit any of the following effects may be useful according to the invention:
  • a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.
  • Such effects may be measured by any method known in the art.
  • increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981 , Ann. Rev. Neurosci.
  • neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g. , weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, or degenerative disease that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary
  • Nucleic acids encoding human E-cadherin or functional derivatives thereof, and expression vectors comprising the same can be introduced into cells such that the E-cadherin nucleic acid sequences are stably incorporated in the cell and capable of expression by the cell and/or its progeny cells. Such introduction can occur in vivo, by or following in vivo administration of the E-cadherin encoding nucleic acid; or in vitro, after which the recombinant cell can be introduced into an animal, most preferably a human, for pu ⁇ oses of gene therapy (i.e., therapeutic benefit via expression of the protein encoded by the introduced, heterologous gene sequence) (for reviews relating to gene therapy, see, e.g. , Karson et al., 1992, J. Reprod. Med. 37(6):508-514; Thompson. 1992, Science 258:744-746; Cline, 1985, Pharmac. Ther. 29:69-92).
  • gene therapy i.e., therapeutic benefit via expression of
  • a nucleic acid encoding the complete human E-cadherin protein or a functional derivative thereof capable of homotypic binding is introduced into a cell of an animal, either in vitro followed by introduction of the transformed cell or in vivo (e.g. , by direct injection into the animal) to prevent progression to malignancy, to prevent cell invasion, or to prevent metastasis.
  • the nucleic acid is directly injected into or otherwise delivered to a tumor cell, to prevent metastasis.
  • tumor cells include but are not limited to the solid tumors listed in Table 1, supra.
  • recombinant cells engineered to secrete an E-cadherin protein or derivative can be used to provide soluble E-cadherin to competitively inhibit E-cadherin adhesive function on cells, thus promoting cell invasion.
  • Cells into which an E-cadherin-encoding nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as
  • T lymphocytes B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g. , as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the E-cadherin-encoding nucleic acid if not directly introduced into the tumor ceil in vivo, is preferably introduced into a cell of the same cell type as the tumor cell.
  • the cell used for gene therapy is autologous to the patient.
  • stem cells are preferred for use. Any stem cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
  • stem cells include but are not limited to hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, and embryonic heart muscle cells. Epithelial stem cells are preferred for use.
  • Epithelial stem cells or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures
  • ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture (Rheinwald, 1980, Meth. Cell Bio. 21A:229; Pittelkow and Scott, 1986, Mayo Clinic Proc. 61 :771). If the ESCs are provided by a donor, a mediod for suppression of host versus graft reactivity (e.g. , irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.
  • HSC hematopoietic stem cells
  • any technique which provides for the isolation, propagation, and maintenance in vitro of HSC can be used in this embodiment of the invention.
  • Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic.
  • Non-autologous HSC are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient.
  • human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g.
  • the HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long- term culturing, and can be done by any techniques known in the art. Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified Dexter cell culture techniques (Dexter et al. , 1977, J. Cell Physiol. 91:335) or Witlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612).
  • the nucleic acid encoding E-cadherin or a derivative thereof is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the E-cadherin sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g. , Cline, 1985, Pharmac. Ther.
  • the technique should provide for the stable transfer of the heterologous gene sequence to the cell, so that the heterologous gene sequence is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • recombinant cells can be introduced by various methods known in the art (see Section 5.12 infra).
  • epithelial cells are injected, e.g. , subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g. , hematopoietic stem or progenitor cells
  • Recombinant blood cells are preferably administered intravenously.
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc. , and can be determined by one skilled in the art.
  • E-cadherin or a derivative thereof is directly administered in vivo for therapeutic effect, whereby it is expressed to produce E-cadherin or a derivative thereof.
  • This can be accomplished by any of numerous methods known in the art, e.g. , by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g. , by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
  • nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • the present invention also provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding a human E-cadherin protein or a portion thereof.
  • Antisense refers to a nucleic acid capable of hybridizing to a portion of a human E-cadherin RNA (preferably mRNA) by virtue of some sequence
  • antisense nucleic acids have utility as antagonists of E-cadherin function, and can be used where cell invasion is desired (e.g. , to promote nerve or other tissue regeneration).
  • the antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • the invention further provides pharmaceutical compositions comprising an effective amount of the E-cadherin antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described in Section 5.12.
  • the invention is directed to mediods for inhibiting the expression of a human E-cadherin nucleic acid sequence in a prokaryotic or eukaryotic cell, comprising providing the cell with an effective amount of a composition comprising an antisense E-cadherin nucleic acid of the invention.
  • the human E-cadherin antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides). In specific aspects, the oligonucleotide is at least 10
  • the oligonucleotide has a sequence that is not identical or 100% complementary to any same-size portion of the mouse or chicken E-cadherin nucleotide coding sequences or flanking sequences.
  • the antisense oligonucleotide is not 100% complementary to a same size nucleic acid having a sequence depicted in Figure 3 from nucleotide numbers 617-1036 or a portion thereof.
  • the oligonucleotide is complementary to the nucleotide sequence encoding a domain or portion thereof of the human
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
  • the antisense nucleic acid is antisense to a sequence encoding one or more domains of the E-cadherin protein.
  • a human E-cadherin antisense oligonucleotide is provided, preferably of single-stranded DNA.
  • such an oligonucleotide comprises a sequence antisense to me sequence encoding the extracellular domain of a human E-cadherin protein, or the repeat domain thereof.
  • the oligonucleotide may be modified at any position on its structure with substituents generally known in the art.
  • the E-cadherin antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
  • hypoxanthine xantine
  • 4-acetylcytosine 5-(carboxyhydroxylmedhyl) uracil
  • 5-carboxymethylaminomethyl-2-d ⁇ iouridine 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine.
  • the oligonucleotide comprises at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
  • the oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al. , 1987, Nucl. Acids Res.
  • the oligonucleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent,
  • Oligonucleotides of the invention may be synthesized by standard memods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligos may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports (Sarin et al. , 1988. Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451 ), etc.
  • the E-cadherin antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g. , PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al. , 1990, Science 247: 1222- 1225).
  • the oligonucleotide is a 2'-0-med ⁇ ylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • me E-cadherin antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the E-cadherin antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology mediods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of me sequence encoding the E-cadherin antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells.
  • Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a human E-cadherin gene.
  • absolute complementarity almough preferred, is not required.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • the longer the hybridizing nucleic acid the more base mismatches with a E-cadherin RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. In another embodiment, 100% complementary sequences are envisioned.
  • E-cadherin antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • E-cadherin antisense nucleic acids can be administered by methods as described supra in Section 5.9. 5.1 1. DEMONSTRATION OF THERAPEUTIC
  • the Therapeutics of the invention can be tested in vivo for the desired therapeutic or prophylactic activity.
  • such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used.
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • a Therapeutic of the invention e.g. , encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis (see, e.g. , Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular,
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the
  • compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection;
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • a reservoir such as an Ommaya reservoir.
  • liposomes targeted via antibodies to specific identifiable tumor antigens Leonetti et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265: 16337-16342).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by methods as described supra in Section 5.9.
  • administer the Therapeutics of the invention may be desirable to administer the Therapeutics of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g. , in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the present invention also provides pharmaceutical compositions.
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile
  • composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the Therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and diose formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triemylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration of a protein Therapeutic are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • E-cadherin expression has diagnostic and prognostic utility.
  • the loss of expression or improper localization of expressed E-cadherin correlates with severity and degree of differentiation in some cancers (see Shimoyama and Hirohashi, 1991 , Cancer Res. 51 :2185-2192).
  • decreased expression or change in localization of E-cadherin in human tumor cells relative to the level of expression or localization, respectively, in non- malignant cells (preferably of the same cell type) indicates a poor prognosis and the presence of an invasive malignancy.
  • an anti-human E-cadherin antibody is used diagnostically in conventional immunoperoxidase staining of a surgical specimen to predict metastatic potential of an epithelial cell cancer such as breast, prostate, ovarian, gastric, or squamous cell cancer.
  • disorders of cell fate in particular precancerous conditions such as metaplasia and dysplasia, and hyperproliferative (e.g. , cancer) or
  • hypoproliferative disorders involving aberrant or undesirable levels of expression or activity of a E-cadherin protein can be diagnosed by detecting such levels.
  • human E-cadherin proteins, analogues, derivatives, and subsequences thereof, E-cadherin nucleic acids (and sequences complementary thereto), anti- human-E-cadherin protein antibodies, and other proteins and derivatives and analogs thereof which interact with human E-cadherin proteins, and inhibitors of such E-cadherin-protein interactions have uses in diagnostics.
  • Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting E-cadherin expression, or monitor the treatment thereof.
  • an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-human E-cadherin protein antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • antibody to E-cadherin can be used to assay in a patient tissue or serum sample for the presence of E-cadherin where an aberrant level of E-cadherin is an indication of a diseased condition.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent
  • Human E-cadherin genes and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays.
  • Human E-cadherin nucleic acid sequences, or subsequences thereof comprising about at least 8 nucleotides can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in human E-cadherin expression and/or activity as described supra.
  • such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to human E-cadherin DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
  • E-cadherin nucleotide sequence are used in diagnostic PCR.
  • a pair of purified oligonucleotide primers is provided: a first oligonucleotide having a sequence which is the same as a first portion of the nucleotide sequence depicted in Figure 3 (SEQ ID NO: 1); and a second oligonucleotide having a sequence which is complementary to a second portion of the nucleotide sequence shown in Figure 3 (SEQ ID NO: 1) and thus able to prime DNA synthesis off the opposite DNA strand from the first oligonucleotide: in which the second portion is situated 3' to the first portion in the sequence shown in Figure 3.
  • oligonucleotides are then used as primers in PCR to amplify DNA fragments spanning from the first portion to the second portion in the E-cadherin gene, thus allowing the detection of full-length E-cadherin nucleic acids or portions thereof (depending on the identity of the primers and dius their position within the E-cadherin gene) in a sample from a patient.
  • the characteristics (e.g. , size) of the amplified fragment, or the lack of any amplification, where differing from mat achieved with a wild- type human E-cadherin gene, can indicate the presence of an abnormality associated with a change in human E-cadherin gene sequence.
  • primers from the noncoding regions, flanking the coding sequence are employed.
  • the oligonucleotide primers are at least 15, and are preferably 18 or 24 nucleotides.
  • the primers consist of nucleotide sequences that are not identical or complementary to any same-size fragment of the chicken or mouse E-cadherin cDNAs or the coding regions or flanking regions thereof. 6. ISOLATION OF HUMAN E-CADHERIN
  • the amino-terminal processed region (leader sequence) is cleaved post-translationally to produce the mature protein.
  • the SHAVS sequence shown to be necessary for homotypic interaction is identical in sequence and location to that seen in mouse and chicken.
  • Human E-cadherin shows the same three internal repeat (putative Ca + + binding) structures seen in mouse with 79-86% similarity to the mouse sequence and 22-36% similarity between repeats.
  • transmembrane binding domain are identically conserved as are the three consensus sequences for N-glycosylation in that region in spite of the fact that this region is less similar overall (69% homology).
  • the other N-glycosylation sequence in the middle of the second repeat is conserved but not identical.
  • the region with the highest similarity is the carboxy terminal region of the molecule with the 24 transmembrane amino acids showing 100% identity and the cytoplasmic domain showing 95% similarity.
  • the human E-cadherin protein and domains thereof can be characterized as containing the following amino acids: Amino acids of Figure 3
  • E-cadherin protein or portion thereof SEQ ID NO:2
  • FLEC coding sequence clone
  • the FLEC -encoded protein dius is missing amino acids 1 to about 145 of the sequence shown in Figure 3.
  • the FLEC clone thus encodes the entire mature E-cadherin protein sequence (amino acids 151-878 of Fig. 3), but differs in me protein sequence corresponding to the amino-terminal processed region of human E-cadherin.
  • a full-length human E-cadherin coding sequence can be generated from bsFLEC and bsL5.1 (both deposited with the ATCC; see Section 10).
  • Both bsFLEC and bsL5. 1 represent E-cadherin sequences cloned in "bluescript phagemid II" from Stratgene Inc.
  • L5.1 represents the 5 ' end of the E-cadherin clone (see Fig. 4) and is cloned into the EcoR1 site of the plasmid.
  • bsFLEC is cloned between the EcoR1 and Xhol sites.
  • Clone e250 contains nucleotide numbers 589-832 of me human E-cadherin sequence (Fig. 2; SEQ ID NO: 1 ). The e250-encoded protein thus contains the HAV binding domain, and is expected to competitively inhibit homotypic binding of E-cadherin. Clone e250 was made by subcloning a
  • Clone cyto 20 contains nucleotide numbers 2297-2750 of the human E-cadherin sequence (Fig. 2; SEQ ID NO: 1). Clone cyto 20 was made by performing PCR with 16-mer primers just 5' and just 3' of nucleotide numbers 2297-2749 in Figure 3. The primers were designed so as to incorporate an in-frame BamHI site at nucleotide number 2296. The PCR-amplified product was then cleaved with BamHI and subcloned into pGEX-3X. Expression in E. coli HB101 yielded a 42 kd fusion protein as detected by SDS-PAGE.
  • Fusion proteins were produced and purified by affinity chromatography according to standard procedures (Smith and Johnson, 1988, Gene 67:31-40) and utilized for immunization of rabbits (for polyclonal antibody production) and of mice (for monoclonal antibody production).
  • the polyclonal antibodies thus obtained were tested against native human E-cadherin in colonic extracts and in extracts from a number of cell lines and were shown to bind with high affinity.
  • lymph nodes or spleen were taken for fusion with myeloma cells after only one immunization and one boost.
  • the initial injection of fusion protein into mice was in Freund's complete adjuvant; the boost was given 13 days later in Freund's incomplete adjuvant.
  • the lymph nodes or spleen were taken, and fusions with myeloma cells were performed, generating hybridomas for screening.
  • the hybridomas were screened for reactivity with the fusion protein used as immunogen, and ten candidate hybridomas were identified as reactive with the e250-encoded protein, and are subject to verification.
  • accession numbers accession numbers 1201 Parklawn Drive, Rockville, Maryland 20852, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, and assigned the indicated accession numbers.
  • E. coli HB101 bsFLEC (containing FLEC) 69123
  • E. coli HB101 bsL5.1 (containing L5.1) 69122
  • GGC AGA GTG AAT TTT GAA GAT TGC ACC GGT CGA CAA AGG ACA GCT ATT 310 Gly Arg Val Asn Phe Glu Asp Cys Thr Gly Arg Gln Arg Thr Ala lle

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Abstract

L'invention concerne des séquences de nucléotides du gène humain de la cadhérine E et des séquences d'acides aminés de la protéine codée de la cadhérine E. L'invention concerne aussi des fragments et d'autres dérivés ainsi que des analogues de la protéine de la cadhérine E humaine, ainsi que des anticorps les concernant. Les acides nucléiques codant de tels fragments ou dérivés font également partie de l'invention, de même que des acides nucléiques antisens de la cadhérine E humaine. On décrit la production de ces protéines et dérivés, par exemple par génie génétique. Dans des variantes spécifiques, l'invention concerne des dérivés et analogues de l'invention liés à la protéine de la cadhérine E humaine qui se révèlent fonctionnellement actifs ou comprennent un ou plusieurs domaines d'une protéine de la cadhérine E humaine, englobant notamment la région traitée du terminal amino, le domaine de liaison homotypique HAV (His-Ala-Val), un ou plusieurs des trois domaines de répétition, la région transmembranaire, la région extracellulaire, le domaine cytoplasmique et l'une ou l'autre de leurs combinaisons. L'invention concerne enfin des procédés thérapeutiques et diagnostiques et des compositions à base de protéines et d'acides nucléiques de cadhérine E.
PCT/US1993/011097 1992-11-17 1993-11-16 Homologue humain du gene de la cadherine e et procedes d'utilisation WO1994011401A1 (fr)

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EP0821060A2 (fr) * 1996-07-24 1998-01-28 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Diagnostic et thérapie des tumeurs malignes humaines basés sur des mutations d'E-cadherin
WO1999016791A2 (fr) * 1997-09-29 1999-04-08 Adherex Inc. Composes et procedes de regulation d'adherence cellulaire
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WO2000002917A2 (fr) * 1998-07-10 2000-01-20 Adherex Technologies, Inc. Composes permettant de moduler des fonctions induites par la cadherine et techniques afferentes
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US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
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