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WO1997018822A1 - Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci - Google Patents

Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci Download PDF

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Publication number
WO1997018822A1
WO1997018822A1 PCT/US1996/018675 US9618675W WO9718822A1 WO 1997018822 A1 WO1997018822 A1 WO 1997018822A1 US 9618675 W US9618675 W US 9618675W WO 9718822 A1 WO9718822 A1 WO 9718822A1
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Prior art keywords
protein
deltex
nucleic acid
vertebrate
notch
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PCT/US1996/018675
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English (en)
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WO1997018822A9 (fr
Inventor
Spyridon Artanavis-Tsakonas
Kenji Matsuno
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Yale University
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Priority to EP96942785A priority Critical patent/EP0869802A4/fr
Priority to AU11614/97A priority patent/AU728798B2/en
Priority to JP9519885A priority patent/JP2000502246A/ja
Publication of WO1997018822A1 publication Critical patent/WO1997018822A1/fr
Publication of WO1997018822A9 publication Critical patent/WO1997018822A9/fr

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to vertebrate deltex genes and their encoded protein products, as well as derivatives and analogs thereof.
  • the invention further relates to production of vertebrate Deltex proteins, derivatives and antibodies.
  • Related therapeutic compositions and methods of therapy and diagnosis are also provided. 0
  • Notch locus plays a central role in regulative events influencing cell fate decisions in Drosophila in a very broad spectrum of developing tissues (reviewed in Artavams-Tsakonas 0 and Simpson, 1991 , Trends Genet. 7:403-408; and in Artavams-Tsakonas et al. , 1991 , Ann.
  • Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al., 1991, Cell 67:687-699; Coffrnan et al., 1990, Science 249: 1438-1441, Ellisen et al, 1991 , Cell 66-649-661 ; Weinmaster et al. , 1991 , Development 113- 199-205) Two human Notch
  • Notch encodes a large, structurally-complex transmembrane protein, rr consistent with an involvement in cell-cell communication (Wharton et al , 1985, Cell 43:567-581 ; Kidd et al., 1986, Mol. Cell. Biol. 3:194-3108).
  • Notch has an extracellular domain containing 36 tandem EGF-like repeats and 3 Notch/hnll repeats The intracellular domain bears several common structural motifs including 6 cdclO/SW16/ankyr ⁇ n repeats ("ANK" repeats) Lux et al., 1990, Nature 344:36-42; Breeden and Nasmyth, 1987, Nature
  • mice 35 homologs isolated from mice (Weinmaster et al., 1991, Development 1 13.199-205,
  • ANK repeats are crucial for Notch-mediated signaling events. Both EGF- ke repeats and ankyrtn motifs are found in a variety of proteins known to mteract with other protein molecules Indeed, evidence has shown a direct interaction between Notch and the products of the Delta and Serrate loci, which also encode transmembrane proteins containing EGF- 0 hke repeats (Fehon et al., 1990, Cell 61:523-534; Rebay et al., 1991, Cell 67:687-699)
  • Deltex has been demonstrated to play a critical role in development and other physiological processes, in particular, in the signaling pathway of Notch which is involved in cell fate (differentiation) determination
  • Notch which is involved in cell fate (differentiation) determination
  • Drosophila Deltex mediates the intracellular portion of the signal transduction cascade involved in Notch function (Diede ⁇ ch et al., 1994, Development 120.473-481)
  • the ANK repeat motif is shared by many proteins and has been implicated in protein-protein interactions (Lux et al., 1990, Nature 344:36-42, Thompson et al., 1991,
  • the Drosophila Su(H) gene encodes a protein of 594 amino acids and binds to the promoters of several viral and cellular genes and interacts directly with a viral transactivator protein termed Epstein-Barr virus nuclear antigen 2 (EBNA2), which enables a virus to subvert the normal program of B cell differentiation (Sch Stammguth, F. , et al, 1992, Cell 69: 1 199; Furukawa T. , et al.. 1991 , J. Biol.
  • EBNA2 Epstein-Barr virus nuclear antigen 2
  • the present invention relates to nucleotide sequences of vertebrate deltex
  • the mvennon further relates to fragments and other de ⁇ vatives, and analogs, of vertebrate Deltex proteins, 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 , bv recombinant methods, is provided. 15
  • the invention relates to human deltex nucleic acids and proteins
  • the invention relates to mammalian deltex nucleic acids and proteins
  • the invention relates to vertebrate Deltex protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a vertebrate Deltex protein, including but not limited to the SH3- bmding domains, ⁇ ng-H2-Zmc fingers, domains which mediate binding to Notch or to a _ ,.
  • the present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex proteins and nucleic acids
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic
  • a 3 Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state.
  • a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.
  • Therapeutics which antagonize, or inhibit, vertebrate are provided.
  • Antagonist Therapeutics are administered for therapeutic effect.
  • Therapeutics which promote vertebrate Notch and/or Deltex function are administered for therapeutic effect.
  • disorders of cell fate in particular hyperproliferative (e.g. , cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • hyperproliferative e.g. , cancer
  • hypoproliferative disorders involving aberrant or undesirable levels of expression or activity or localization of vertebrate Notch and/or Deltex protein can be diagnosed by detecting such levels, as described more fully infra.
  • a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex which mediates binding to a Notch protein or a fragment thereof.
  • the invention also provides methods of inactivating Notch function in a cell, methods of identifying a compound that inhibits or reduces the binding of a vertebrate
  • Deltex protein to a Notch protein, and methods of expanding non-terminally differentiated cells.
  • Figure 1A-F Nucleotide sequence (SEQ ID NO: l) and deduced amino acid sequence (SEQ ID NO:2) of Drosophila deltex cDNA.
  • FIG. 2A-C Composite nucleotide sequence (SEQ ID NO: 11) derived from the cDNA (nucleotide 1 to 2547), and deduced amino acid sequence (SEQ ID NO: 12) of the human deltex locus. The predicted amino acid sequence is depicted below the DNA sequence. The symbol: * designates the start of T05200 and $ the end of T05200. Core H and C residues in Rtng-H2-zinc finger are shown by underlining. PCR primers hdx-1 to 4
  • SEQ ID NO:26 (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), respectively, are indicated in bold.
  • X and N represent amino acid residues and nucleotides, respectively, not yet determined.
  • Figure 3 Aligned amino acid sequences of human Deltex (SEQ ID NO: 12) and Drosophila Deltex (SEQ ID NO:2) proteins. Those positions at which residues are identical are shaded. Sites in which amino acids are chemically similar are boxed.
  • FIG. 4A-B Amino acid sequence ol Drosophila Deltex (SEQ ID NO:2) 5 and designated fragments implicated in protein-protein interactions. Fragments A-D
  • the present invention relates to nucleotide sequences of vertebrate deltex genes, and amino acid sequences of their encoded Deltex proteins.
  • the invention further relates to fragments and other derivatives, and analogs, of vertebrate Deltex proteins. 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 0 methods, is provided.
  • the invention relates to a human deltex gene and protein.
  • the invention relates to a mammalian ? _ deltex gene and protein.
  • the invention also relates to vertebrate Deltex 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) vertebrate Deltex protein.
  • Such functional activities include but are not limited to antigenicity [ability to bind
  • the mvenuon further relates to fragments (and de ⁇ vauves and analogs thereof) of a vertebrate Deltex protein which comprise one or more domains of a vertebrate Deltex protem (see infra), including but not limited to the SH3-bind ⁇ ng domains, r ⁇ ng-H2- zmc fingers, domains which mediate bmdmg to Notch (or a derivauve thereof contaimng the
  • Notch ANK repeats or to a second Deltex molecule or fragment thereof, or any combinauon of the foregoing
  • Drosophila deltex as a probe were unsuccessful In contrast to such prior failures, the present invention is based on the successful cloning of human deltex As described by way of example below, we have used an innovative methodology to clone the transcription unit corresponding to human deltex As described therein (see Section 6), our results show a significant structural conservation of Deltex in humans, indicative of functional conservation Moreover, we demonstrate that human Deltex displays direct molecular interaction with both human and Drosophila Notch intracellular ANK repeats (see Section 7) Knowledge of the sequence of human deltex allows the identification of regions strongl) conserved between Drosophila and human deltex, and provides a method for readily isolating other vertebrate deltex genes by use of such strongly conserved regions (see Sections 5 6 and 8 infra).
  • the vertebrate deltex nucleic acid and ammo acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of vertebrate deltex mRNA and protem, to study expression thereof, to produce vertebrate Deltex protems, fragments and other derivatives, and analogs thereof, m the study, assay, and manipulation of differentiation and other physiological processes, and are of therapeutic and diagnostic use, as described infra
  • the agomsts and antagomsts of Deltex function can be used to alter the ability of a cell to differentiate
  • the vertebrate deltex nucleic acids and antibodies can also be used to clone vertebrate deltex homologs of other species, as described infra Such vertebrate deltex homologs are expected to exhibit significant homology to each other, and encode protems which exhibit the ability to bind to a Notch protein
  • the present invention also relates to therapeutic and diagnostic methods and compositions based on vertebrate Deltex protems and nucleic
  • Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state.
  • a Therapeutic of the invention is administered to treat 0 a nervous system disorder or to promote tissue regeneration and repair.
  • Therapeutics which antagonize, or inhibit, Notch and/or vertebrate Deltex function are administered for therapeutic effect.
  • Therapeutics which promote Notch and/or vertebrate Deltex function are administered for 5 therapeutic effect.
  • disorders of cell fate in particular hyperproliferative (e.g. , cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such 0 levels, as described more fully infra.
  • hyperproliferative e.g. , cancer
  • hypoproliferative disorders involving aberrant or undesirable levels of expression or activity or localization of Notch and/or vertebrate Deltex protein can be diagnosed by detecting such 0 levels, as described more fully infra.
  • a Therapeutic of the invention is a protem consisting of at least a fragment (termed herein "adhesive fragment") of vertebrate Deltex that mediates binding to a Notch protein, a second Deltex protein, or a fragment of Notch or Deltex.
  • the mvenuon is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of human deltex, and the identification of regions of human Deltex which are predicted to bind to the ANK repeats of Notch, or which are predicted to bind to regions of human Deltex.
  • human deltex nucleic acids comprise the cDNA sequence shown in Figure 2A-C (SEQ ID NO: 11), or the coding region thereof (nucleotide numbers 504-2363), or nucleic acids encoding a human Deltex protem (e.g. , having the sequence of
  • the mvention provides nucleic acids consisung of at least 8 nucleotides
  • the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides,
  • nucleic acids are provided which comp ⁇ se a sequence complementary to at least 10, 25, 50, 100, or 200 nucleoudes or the entire codmg region of a vertebrate deltex gene In a specific embodiment, the sequence is naturally occurring.
  • the mvention provides nucleic acids comprising at least 110, 150, or 200 continuous nucleotides of the sequence of SEQ ID NO 11 In other embodiments, the mvention provides a nucleic acid comp ⁇ smg the first 25, 50, 100, 150, 200, or 230 ammo acids of SEQ ID NO.12
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the ammo terminal 180 ammo acids (encoded, e.g., by nucleotide numbers 504-1044 of SEQ ID: 11) of human deltex, or fragments thereof
  • the vertebrate deltex nucleic acid has at least 50% identity over the correspondmg nucleotide sequence of an identically sized human deltex In another embodiment this identity is greater than 55 %
  • the nucleotide sequence identity of the vertebrate deltex is greater than 60% In a more preferred embodiment this identity is greater than 65 %
  • the nucleotide sequence identity of the vertebrate deltex is greater than 70% over that of the correspondmg nucleotide sequence of identically sized human deltex
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encodmg the central region ammo acids of human deltex (e.g. , nucleotide numbers 1045-1821 of SEQ ID NO 11) or fragments thereof
  • the nucleic acids encodmg the central ammo acids of the vertebrate Deltex protein has at least 50% nucleotide sequence identity with the correspondmg human deltex sequence of identical size In another embodiment this identity is greater than 55%. In a preferred embodiment, this nucleotide sequence identity is greater than 60% . In a more preferred embodiment this identity is greater than 65%.
  • the homology of the nucleic acids encoding the central region amino acids of the vertebrate deltex has a nucleotide sequence identity that is greater than
  • vertebrate deltex nucleic acids comprise those nucleic acids which are substantially homologous to the nucleic acids encoding the 180 carboxy terminal amino acids of human deltex (nucleotide numbers 1822-2366), or fragments thereof.
  • the nucleic acids encoding the carboxy terminal 0 region of the vertebrate Deltex protein has at least 50% nucleotide sequence identity over the corresponding human deltex sequence of identical size. In another embodiment this identity is greater than 55%. In a preferred embodiment, this identity is greater than 60%. In a more preferred embodiment this identity is greater than 65 % .
  • the identity of the nucleotides encoding the amino terminal amino acids of the vertebrate deltex is greater than 70% over that of the corresponding nucleotide sequence of identically sized human deltex.
  • a nucleic acid which is hybridizable to a vertebrate deltex nucleic acid e.g. , having sequence SEQ ID NO: 11
  • procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci.
  • Tris-HCl pH 7.4
  • 5 mM EDTA 5 mM EDTA
  • 0.1 % SDS Tris-HCl
  • the wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed
  • Filters are hybridized for 48 h at 65 °C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe. Washing of filters is done at
  • nucleic acids encoding derivatives (e.g. , fragments) of vertebrate Deltex proteins (see Section 5.6), and vertebrate deltex antisense nucleic acids (see Section 5.11) are additionally provided.
  • a "nucleic acid encoding a fragment or portion of a vertebrate Deltex protein” shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the vertebrate Deltex protein and not the other contiguous portions of the vertebrate Deltex protein as a continuous
  • an _ expression library is obtained or is constructed by methods known in the art.
  • mRNA e.g. , human
  • cDNA is made and ligated into an expression vector (e.g. , a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced.
  • an expression vector e.g. , a bacteriophage derivative
  • Various screening assays can then be used to select for the expressed vertebrate Deltex product.
  • anti-human Deltex antibodies e.g. , human
  • PCR is used to amplify the desired vertebrate deltex sequence in a genomic or cDNA library, prior to selection (see, by way of example Section 8, infra).
  • Oligonucleotide primers representing known vertebrate deltex sequences, 35 preferably regions known to be conserved between Drosophila and human, can be used as primers in PCR.
  • the synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest.
  • PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp").
  • the DNA being amplified can include human mRNA or cDNA or genomic DNA.
  • a segment of a vertebrate deltex gene homolog After successful amplification of a segment of a vertebrate deltex gene homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In a preferred aspect, human genes encoding Deltex proteins may be identified in this fashion. Alternatively to selection by hybridization, the PCR-amplified DNA can be inserted into an expression vector for expression cloning as described above.
  • the desired vertebrate deltex gene can be isolated as set forth in Example 8, by screening with a probe, or priming for PCR with an oligonucleotide, containing deltex sequences encoding regions highly conserved between human and Drosophila.
  • the human Deltex amino acid stretches 414-419 (SEQ ID NO:30), 475-480 (SEQ ID NO:31), 504-511 (SEQ ID NO: 32), 531-539 (SEQ ID NO:33) and 557-564 (SEQ ID NO:34) are conserved in Drosophila Deltex amino acid stretches 549-555 (SEQ ID NO: 35), 603-608 (SEQ ID NO:36), 632-639 (SEQ ID NO:37), 659-667 (SEQ ID NO:38) and 685- 692 (SEQ ID NO:39), respectively.
  • a pair of oligonucleotides comprising sequences separated by a length in the range from 50-500 nucleotides is used as primers in PCR.
  • the invention envisions the use of nucleic acids encoding conserved regions of the Deltex protein in combination to isolate the Deltex encoding nucleic acids of other organisms, by use in PCR to amplify the desired sequence or less preferably, without PCR, as a probe in selection by virtue of direct colony hybridization (e.g. , Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961).
  • the desired deltex gene can be isolated by a more gradual method of evolutionary walking via first isolating a deltex gene from a more closely related species, identifying the portions of deltex which are conserved cross-species, and then screening with a probe or priming for PCR with a nucleic acid containing the conserved sequence.
  • the evolutionary tree may first isolate a murine deltex gene using nucleic acids encoding human Deltex as a probe. A conserved portion of the murine deltex sequence is then used to screen or amplify deltex in an avian library; a conserved portion of the avian clone is used to screen an amphibian library, a conserved portion of the amphibian clone is used to screen a fish library, etc. If desired, the species to be selected in the next round of screening can be selected from among various species by hybridizing the deltex probe to a Southern blot containing genomic DNA from each species, and selecting a species to which hybridization occurs.
  • Any eukaryotic cell can potentially serve as the nucleic acid source for the molecular cloning of the vertebrate deltex 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 human cell (see, for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d. Ed., Cold Spring
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA 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 vertebrate deltex (of any species) gene or its specific RNA, or a fragment thereof e.g., the adhesive domain, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196, 180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 3961). Those DNA fragments with substantial homology to the probe will hybridize.
  • vertebrate Deltex has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, binding to Notch, or antigenic properties as known for vertebrate Deltex.
  • the vertebrate Deltex protein may be identified by binding of labeled antibody to the putatively vertebrate Deltex synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay )-type procedure.
  • the vertebrate deltex 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 vertebrate deltex DNA of another species (e.g., human).
  • Immunoprecipitation analysis or functional assays e.g., ability to bind Notch
  • mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against vertebrate Deltex protein.
  • a radiolabelled vertebrate deltex 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 vertebrate deltex DNA fragments from among other genomic DNA fragments.
  • RNA for cDNA cloning of the vertebrate deltex gene can be isolated from cells which express vertebrate Deltex. 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, pUC, or Blue script (Stratagene) 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. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, 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 vertebrate deltex gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced mto host cells via transformauon, transfecuon, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the desired gene may be identified and isolated after insernon mto a suitable cloning vector in a "shot gun" approach Enrichment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector.
  • transformation of host cells with recombinant DNA molecules that incorporate the isolated vertebrate deltex gene, cDNA, or synthesized DNA sequence enables generauon of muluple copies of the gene
  • the gene may be obtamed in large quanuties by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the vertebrate deltex sequences provided by the instant mvention include those nucleotide sequences encodmg substannally the same ammo acid sequences as found in native vertebrate Deltex protem, and those encoded amino acid sequences with functionally equivalent ammo acids, all as described in Section 5.6 infra for vertebrate Deltex derivatives
  • the nucleic acid coding for a vertebrate Deltex protem or a functionally active fragment or other derivative thereof can be inserted mto an appropriate expression vector, i e , a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence
  • an appropriate expression vector i e
  • the necessary transc ⁇ ptional and translational signals can also be supplied by the native vertebrate deltex gene and/or its flanking regions.
  • a variety of host-vector systems may be utilized to express the protein- coding sequence.
  • vectors include but are not limited to vertebrate cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc ), insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast contaimng yeast vectors, or bacteria transformed with bacte ⁇ ophage, DNA, plasmid DNA, or cosmid DNA
  • virus e.g., vaccinia virus, adenovirus, etc
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast contaimng yeast vectors, or bacteria transformed with bacte ⁇ ophage, DNA, plasmid DNA, or cosmid DNA
  • the expression elements of vectors vary m their strengths and specificities Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used
  • a molecule comprising a portion of a vertebrate deltex gene which encodes a protem that b ds to Notch or to a molecule compnsmg the Notch ANK repeats is expressed.
  • a molecule compnsmg a poruon of a vertebrate deltex gene which encodes a protem that binds to a fragment of a Deltex protem is expressed.
  • mammalian deltex gene is expressed, or a sequence encodmg a
  • the human deltex gene is expressed, or a sequence encodmg a functionally active portion of human
  • a chimeric protein comprising a Notch-binding domain of a vertebrate Deltex protem is expressed in other specific embodiments, a full-length
  • vertebrate deltex cDNA is expressed, or a sequence encodmg a functionally active portion of a vertebrate Deltex protem
  • a fragment of a vertebrate Deltex protem compnsmg a domain of the protem, or other de ⁇ vative, or analog of a vertebrate Deltex protem is expressed
  • any of the methods previously described for the insertion of DNA fragments mto a vector may be used to construct expression vectors containing a chimeric gene consisting of approp ⁇ ate transcnptional/translauonal control signals and the protem codmg sequences These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombinauon). Expression of a nucleic acid sequence
  • 2 encodmg a vertebrate Deltex protem or pepude fragment may be regulated by a second nucleic acid sequence so that the vertebrate Deltex protem or pepude is expressed in a host transformed with the recombinant DNA molecule
  • expression of a vertebrate Deltex protem may be controlled by any promoter/enhancer element known m the art Promoters which may be used to control vertebrate deltex gene expression mclude, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290 304-310), the promoter contained m 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.
  • plant expression vectors comprising the nopa ne synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); 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
  • Expression vectors containing vertebrate deltex 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 vertebrate deltex 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 vertebrate deltex 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 vertebrate deltex gene product in in vitro assay systems, e.g. , binding to
  • 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 vaccmia virus or adenovirus; msect viruses such as baculovirus, yeast vectors; bacte ⁇ ophage 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 engmeered vertebrate Deltex protein may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e g , phosphorylation, cleavage) of protems. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • the vertebrate Deltex protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protem, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of 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 methods commonly known in the art
  • such a chimeric product may be made by protem synthetic techniques, e g. , by use of a peptide synthesizer Both cDNA and genomic sequences can be cloned and expressed.
  • a vertebrate deltex cDNA sequence may be chromosomally integrated and expressed. Homologous recombination procedures known in the art may be used.
  • the mvention provides amino acid sequences of vertebrate Deltex, preferably human Deltex, and fragments and derivatives thereof which comprise an antigenic determinant (i.e., can be recognized by an antibody) or which are functionally acuve, as well as nucleic acid sequences encoding the foregoing.
  • "Functionally active" material as used herein refers to that material display mg one or more known functional activities associated with the full-length (wild-type) vertebrate Deltex protem product, e.g.
  • the mvention provides fragments of a vertebrate
  • Deltex protein consistmg of at least 6 ammo acids, 10 ammo acids, 50 amino acids, or of at least 75 ammo acids
  • the proteins comprise, or alternatively, consist essentially of; one or more of the SH3-b ⁇ nd ⁇ ng domams (e.g. , SEQ ID NOS: 17-21 of Table
  • one or more ⁇ ng-H2-z ⁇ nc finger domains e.g., SEQ ID NO:25
  • a portion which bmds to Notch e.g. , compnsmg the first approximately 230 ammo acids of vertebrate
  • Deltex or any combination of the foregoing, of a vertebrate Deltex protein. Fragments, or protems compnsmg fragments, lacking some or all of the foregoing regions of vertebrate
  • 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, lmmunoassay, etc. Chemically synthesized proteins, derivatives, and analogs can be similarly analyzed.
  • a vertebrate Deltex protein Once a vertebrate Deltex protein is identified, it 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 properues may be evaluated using any suitable assay (see Secuon 5.7).
  • the amino acid sequence of a vertebrate Deltex 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).
  • the structure of the vertebrate deltex genes and proteins can be analyzed by various methods known in the art.
  • the cloned DNA or cDNA corresponding to the vertebrate deltex gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975,
  • Southern hybridization can be used to determine the genetic linkage of vertebrate deltex.
  • Northern hybridization analysis can be used to determine the expression of the vertebrate deltex genes.
  • Various cell types, at various states of development or activity can be tested for vertebrate deltex gene expression.
  • 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 vertebrate deltex probe used.
  • Restriction endonuclease mapping can be used to roughly determine the genetic structure of the vertebrate deltex gene. Restriction maps derived by restriction 5 endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once d e nucleotide sequence is known.
  • DNA sequence analysis can be performed by any techniques known in the an, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol.
  • the amino acid sequence of a vertebrate Deltex protem can be de ⁇ ved by 20 deduction from the DNA sequence, or alternatively, by direct sequencing of the protem, e.g. , with an automated amino acid sequencer.
  • the ammo acid sequence of a representative vertebrate Deltex protem comprises the sequence substantially as depicted in Figure 2A-C (SEQ ID NO: 12), and detailed in Section 6, infra. .
  • the vertebrate Deltex protem sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.
  • a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a vertebrate Deltex protein and the corresponding regions of the gene sequence which encode such regions. Hydrophilic regions are predicted to be lmmunogenic.
  • Manipulation, translation, and secondary structure prediction, as well as open 3 readmg frame prediction and plotting, can also be accomplished using computer software programs available in the art. Other methods of structural analysis can also be employed These mclude but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modelmg (Flette ⁇ ck and Zoller (eds.), 1986, Computer Graphics and
  • a vertebrate Deltex protem may be used as an lmmunogen to generate antibodies which recognize such an immunogen
  • Such antibodies mclude but are not limited to polyclonal, monoclonal, chimeric, smgle chain, Fab fragments, and an Fab expression library
  • antibodies which specifically bmd to vertebrate Deltex 5 proteins are produced
  • an antibody which bmds to a vertebrate Deltex protem e g , mammalian, preferably human
  • bmd to (full length) Drosophila Deltex protem is produced
  • such an antibody is produced by us g as immunogen, regions least conserved between Drosophila o melanogaster and the vertebrate Deltex protem
  • antibodies to a particular domam of a vertebrate Deltex protem are produced.
  • an antibody is produced which bmds to a fragment of vertebrate Deltex which bmds to Notch; m another embodiment, an antibody bmds to a molecule compnsmg the first 230 amino-terminal ammo acids of 5 vertebrate Deltex In another embodiment the antibody binds to an ammo-terminal fragment of vertebrate Deltex containing not more than the first 200 ammo acids of vertebrate Deltex In yet another embodiment, an antibody bmds to a fragment of vertebrate Deltex which bmds to a second Deltex molecule.
  • rabbit polyclonal antibodies to an epitope of the vertebrate Deltex protem having a sequence depicted in Figure 2A-C or a subsequence thereof can be obtamed.
  • various host animals can be immunized by injection with a 5 native vertebrate Deltex protein, or a synthetic version, or derivative (e.g , fragment) thereof, mcludmg but not limited to rabbits, mice, rats, etc
  • Various adjuvants may be used to mcrease the lmmunological response, depending on the host species, and mcludmg but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithm, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dmitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gue ⁇ n) and corynebacte ⁇ um parvum.
  • polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a vertebrate Deltex 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 vertebrate Deltex 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 for example, the hyb ⁇ doma technique originally developed by Kohler and Milstein (1975, Nature 256 495-
  • monoclonal antibodies can be produced in germ-free animals (PCT Publication No. WO 89/12690 dated December 28, 1989)
  • human antibodies may be used and can be obtained by usmg human hyb ⁇ domas (Cote et al , 1983, Proc
  • Non-human antibodies can be humanized by the method of Winter (see
  • 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
  • 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
  • to select antibodies which recognize a specific domam of a vertebrate Deltex protem one may assay generated hyb ⁇ domas for a product which bmds to a vertebrate Deltex fragment contaming such domam.
  • a specific to human Deltex protein(s) one can select on the basis of positive bmdmg to a human Deltex protein and a lack of binding to Drosophila Deltex protem.
  • the invention further provides vertebrate Deltex protems, and derivatives
  • the vertebrate Deltex proteins are encoded by me vertebrate deltex nucleic acids described m Section 5.1 supra
  • the proteins, derivatives, or analogs are of mouse or rat; agricultural stock such as cow, sheep, horse, goat, pig and the like; pets such as cats, dogs; or other domesticated mammals, or primate Deltex proteins. 5
  • Deltex are within the scope of the present mvention.
  • the derivative or analog is functionally active, i e. , capable of exhibitmg one or more functional activities associated with a full-length, wild-type vertebrate Deltex protem.
  • vertebrate Deltex derivatives can be made by altering vertebrate deltex sequences by substitutions, additions or deletions that provide for functionally equivalent molecules Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same ammo acid sequence as a vertebrate deltex gene may be used m the practice of the present mvention These mclude but are not limited to nucleotide sequences compnsmg all or portions of vertebrate deltex genes which are altered by the substitution of different codons that encode a functionally equivalent ammo acid residue within the sequence, thus producmg a silent change. Likewise, the vertebrate
  • Deltex de ⁇ vatives of the mvention mclude, but are not limited to, those contaimng, as a 0 primary ammo acid sequence, all or part of the ammo acid sequence of a vertebrate Deltex protem mcludmg altered sequences in which functionally equivalent ammo acid residues are substituted for residues within the sequence resulting a silent change
  • one or more am o acid residues within the sequence can be substituted by another ammo acid _ j .
  • Substitutes for an ammo acid within the sequence may be selected from other members of the class to which the ammo acid belongs
  • the nonpolar (hydrophobic) ammo acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral ammo acids include glycine, se ⁇ ne, threomne, cysteine,
  • proteins consisting of or compnsmg a fragment of a vertebrate Deltex protein consisting of at least 10 (contmuous) amino acids of the vertebrate Deltex protein is provided in other embodiments, the fragment consists of at least 20 or 50 ammo acids of the vertebrate Deltex protem In specific embodiments, such fragments are not larger than 35, 100 or 200 ammo acids.
  • Derivatives or analogs of vertebrate Deltex m clude but are not limited to those peptides which are substantially homologous to human Deltex or fragments thereof
  • derivatives or analogs of vertebrate Deltex m include those peptides which are substantially homologous to the ammo terminal 180 ammo acids (1- 180) of human Deltex
  • the ammo terminal region of the vertebrate Deltex protem has at least 30% identity over the ammo terminal ammo acid sequence of identically sized human Deltex In another embodiment this identity is greater than 35%
  • the ammo terminal ammo acid identity of the vertebrate Deltex is greater than 45 % In a more preferred embodiment this identity is greater than 55 %
  • the homology of the ammo terminal ammo acids of the vertebrate Deltex is greater than 65 % over the correspondmg human Deltex ammo terminal ammo acid sequence of identical size
  • derivatives or analogs of vertebrate Deltex m include those peptides which are substantially homologous to the central region (ammo acids 181-441) of human Deltex, or fragments thereof
  • the central region of the vertebrate Deltex protem has at least 30% identity with the correspondmg human Deltex sequence of identical size In another embodiment this identity is greater than 35 %
  • the ammo acid identity of the central region of vertebrate Deltex and human Deltex is greater than 45 % In a more preferred embodiment this identm is greater than 55 %
  • the homology of the central amino acids of the vertebrate Deltex to corresponding human Deltex ammo acids of identical size is greater than 65%
  • derivatives or analogs of vertebrate Deltex m include but are not limited to those peptides which are substantially homologous to the carboxy terminal ammo acids of human Deltex or fragments thereof
  • the carboxy terminal region of the vertebrate Deltex protem (the carboxy terminal 180 ammo acids) has at least 45% identity over the ammo acid sequence of identical size In another embodiment this identity is greater than 50%
  • the amino terminal ammo acid identity of the vertebrate Deltex is greater than 55% In a more preferred embodiment this identity is greater than 60%.
  • the homology of the ammo terminal ammo acids of the vertebrate Deltex is greater than 65 %
  • derivatives or analogs of vertebrate Deltex comprise regions conserved between Drosophila and human Deltex (see Section 8).
  • the vertebrate Deltex protem derivatives and analogs of the mvention can be produced by various methods known in the art The manipulations which result m their production can occur at the gene or protem level.
  • the cloned vertebrate deltex gene sequence can be modified by any of numerous strategies known m the art (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Sprmg Harbor
  • the vertebrate Deltex-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, imtiation, and/or termination sequences, or to create variations 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 (Hutchmson et al., 1978, J. Biol. Chem 253:6551), use of TAB ® linkers (Pharmacia), etc.
  • Manipulations of the vertebrate deltex sequence may also be made at the protem level
  • vertebrate Deltex protein fragments or other derivatives or analogs which are differentially modified durmg or after translation, e.g. , by acetylation, phosphorylation, carboxylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified vertebrate Deltex protein or derivative thereof.
  • the phosphorylation state of the molecule may be important to its role in intracellular signal transduction of Notch function.
  • Phosphorylation can be carried out by reaction with an appropriate kinase (e.g. , possibly cdc2 or CK II).
  • Dephosphorylation can be carried out by reaction with an appropriate phosphatase.
  • Deltex protein which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, 0 nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the vertebrate Deltex protein sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid,
  • the vertebrate Deltex derivative is a chimeric, or fusion, protein comprising a vertebrate Deltex protein or fragment thereof (preferably consisting of at least a domain or motif of the vertebrate Deltex protein, or at least 10 amino 0 acids of the vertebrate Deltex 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
  • Such a chimeric product can be made by ligating the appropriate nucleic 5 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 methods 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 vertebrate Deltex protein which comprises a domain or motif of the vertebrate Deltex protein, e.g., a portion which binds to a Notch protein or to a second Deltex protein, an SH-3 binding domain, a ring-H2-zinc finger domain, etc.
  • a chimeric nucleic acid can be constructed, 5 encoding a fusion protein consisting of a vertebrate Deltex Notch-binding fragment joined to a non-Deltex protein.
  • a recombinant molecule can be constructed according to the invention, comprising coding portions of both a vertebrate deltex gene and another gene which is a member of the "Notch group.”
  • Another specific embodiment relates to a chimeric protein comprising a fragment of a vertebrate Deltex protein of at least six amino acids.
  • fusion proteins comprising human Deltex or various Notch fragments, are described in Section 7.
  • the invention provides vertebrate Deltex derivatives and analogs, in particular vertebrate Deltex fragments and derivatives of such fragments, that comprise or consist of one or more domains of the vertebrate Deltex protein, including but not limited to a region which binds to a Notch protein (or a molecule comprising the ANK repeats thereof), a region which binds to a second Deltex protein or portion thereof, an SH3-binding domain, or a ring-H2-zinc finger domain.
  • the vertebrate Deltex derivative may lack all or a portion of one or more of the foregoing domains.
  • the aforesaid domains consist of approximately the following amino-acid sequences (see
  • Ring-H2-zinc finger domain SEQ ID NO:25
  • binding fragments e.g., smaller than those set forth above, can be identified by routine methods, e.g. , by construction of nucleic acids encoding such fragments and assays for binding (e.g. , via the interaction trap method described in Section 7 infra.
  • fragments comprising specific domains of vertebrate Deltex are those comprising domains in the respective vertebrate Deltex protein most homologous to the specific domain of the human Deltex protein.
  • Deltex as well as Deltex-Notch interactions were detected.
  • Figure 5 summarizes the Deltex-Deltex interactions we have detected.
  • Fragment A interacts with Fragment A (homotypic interactions).
  • Fragment B interacts with Fragment B (homotypic interactions).
  • Fragment C interacts with Fragment C (homotypic interactions).
  • we detected interactions between fragments C and B we can only detect the fragment C-B interaction if fragment C is tested as the "bait" (i.e. , as the LexA fusion). If Fragment B is the bait, this interaction is not detected. All the other
  • Fragment A consists of amino acids 1-303.
  • Fragment B consists of amino acids 306-486.
  • Fragment C consists of amino acids 514-737.
  • Drosophila Notch ANK repeats as well as the ANK repeats of both human Notch proteins (encoded by TAN-1 and hN) were tested in this interaction assay and showed positive binding to fragment D.
  • the following fragments containing the ANK repeat region were used: Drosophila Notch amino acids: 1889-2076 (numbering per
  • vertebrate Deltex regions are provided that are most of the vertebrate Deltex regions.
  • inhibitors e.g. , peptide inhibitors
  • the nucleic acid sequences encodmg Notch or vertebrate Deltex proteins or fragments thereof, for use m such assays, can be isolated from porcine, bovme, equine, felme, canine, as well as primate sources and any other mammals in which homologs of known genes can be identified
  • me Notch protem or portion thereof compnsmg the ANK repeats which can be expressed and assayed for bmdmg to Deltex or a Deltex derivative can be derived from any of the Notch homologs human hN, human TAN-1, Xenopus, and Drosophila.
  • vertebrate Deltex proteins, fragments or derivatives thereof, of the mvention include, but are not limited to,
  • the derivatives, analogs, and peptides of the mvenuon can be produced by various methods known in the art
  • the manipulations which result in their production can occur at the gene or protem level
  • the nucleic acid sequence can be mutated in vitro or in vivo; and manipulations of the sequence may also be made at the protein level.
  • analogs and peptides can be chemically synthesized.
  • various 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 (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g.
  • 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 art for detecting binding in an immunoassay and are within the scope of the 5 present invention.
  • a method of identifying a molecule that inhibits or reduces the binding of a vertebrate Deltex protein to a Notch protein comprises the 5 steps of contacting a Notch protein and a vertebrate Delta protein such that binding between the Notch protein and the Deltex protein can occur, in the presence of one or more molecules which are desired to be tested for the ability to inhibit or reduce bindmg between the Notch protem and the Deltex protein, and identifying the molecules that mhibit or reduce the bindmg of the Deltex protem to the Notch protein.
  • Any of various binding assays known in the art can be used to carry out such a method, including but not limited to yeast interaction trap assays, cell culture in vitro aggregation assays, and soluble bmdmg assays usmg purified Notch and Deltex protems.
  • a specific embodiment is as follows: Cultured cells are cotransfected with plasmid expression constructs that place Notch and deltex under distinct mducible promoters. Notch expression in these cells is first induced to ensure proper cell surface localization; Deltex expression is then induced. These cells are then aggregated with cells expressing Delta, to produce mutual cappmg of Notch and Delta at the point of mutual contact (see Singer J., 1992, Science, 255.1671-1677; Fehon et al., 1990,
  • Deltex colocalizes with the capped Notch by virtue of its bmdmg to Notch
  • the cells are then incubated m the presence of one or more molecules (preferably, purified molecules) which are desired to be tested for the ability to mhibit bmdmg between Notch and Deltex.
  • Molecules which inhibit or reduce die bindmg of Deltex to Notch will result m an increased localization of Deltex throughout the cell cytoplasm. This mcreased localization can be determmed accordmg to methods known in the art (e.g. , immunofluorescent staining with antibody to Deltex). The method can also be carried out usmg derivatives of Notch and Deltex that mediate bindmg to Deltex and to Notch, respectively.
  • the invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the mvention.
  • therapeutic compounds include vertebrate Deltex proteins and analogs and derivatives (including fragments) thereof (e.g., as described hereinabove); antibodies thereto (as described hereinabove); nucleic acids encodmg the vertebrate Deltex proteins, analogs, or derivatives (e.g. , as described hereinabove); and vertebrate deltex antisense nucleic acids.
  • the Antagonist Therapeutics of the invention are those Therapeutics which antagonize, or inhibit, a vertebrate Deltex function and/or Notch function
  • Such Antagonist Therapeutics are most preferably identified by use of known convenient in vitro assays, e.g., based on their ability to inhibit bmdmg of vertebrate Deltex to another protein (e.g., a Notch protein), or inhibit any known Notch or vertebrate Deltex function as preferably assayed in vitro or in cell culture, although genetic assays (e.g., in
  • Drosophila or mouse may also be employed.
  • the Antagonist may also be employed.
  • Therapeutic is a protein or derivative thereof comprising a functionally active fragment such 5 as a fragment of vertebrate Deltex which mediates bindmg to Notch, or an antibody thereto.
  • such an Antagonist Therapeutic is a nucleic acid capable of expressing a molecule comprising a fragment of vertebrate Deltex which binds to Notch, or a vertebrate deltex antisense nucleic acid (see Section 5.11 herein). It should be noted that 0 preferably, suitable in vitro or in vivo assays, as described infra, should be utilized to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue, since the developmental history of the tissue may determine whether an Antagonist or Agonist Therapeutic is desired.
  • a nucleic acid containing a portion 5 of a vertebrate deltex gene is used, as an Antagonist Therapeutic, to promote vertebrate deltex inactivation by homologous recombination (Koller and Smithies, 1989, Proc. Natl.
  • Agonist Therapeutics of the invention promote ° vertebrate Deltex function.
  • Such Agonist Therapeutics mclude but are not limited to proteins and derivatives comprising the portions of Notch that mediate binding to vertebrate
  • Molecules which retain, or alternatively inhibit, a desired vertebrate Deltex property can be used therapeutically as inducers, or inhibitors, respectively, of such property and its physiological 0 co ⁇ elates.
  • a peptide e.g. , m the range of 6-50 or 15-25 amino acids; and particularly of about 10, 15, 20 or 25 amino acids
  • an Antagonist Therapeutic is used to treat or prevent human or 5 other malignancies associated with increased Notch expression (e.g.
  • Derivatives or analogs of vertebrate Deltex can be tested for the desired activity by procedures known in the art, mcludmg but not limited to the assays described m the examples infra.
  • molecules compnsmg Deltex fragments which bind to Notch ANK repeats can be obtamed and selected by expressing deletion mutants of human Deltex (or of a 5 nucleotide sequence of another species and assaymg for bmdmg of die expressed product to
  • peptide libraries can be screened to select a peptide with the desired activity; such screening can be earned out by assaymg,
  • the Agonist and Antagonist Therapeutics of the mvention have therapeutic utility for disorders of cell fate
  • the Agomst Therapeutics are administered therapeutically
  • Notch or vertebrate Deltex function for example, 15 m patients where Notch or vertebrate Deltex protem is lacking, genetically defective, biologically inactive or underactive, or underexpressed, and (2) m diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of vertebrate Deltex agomst administration The absence or decreased levels m Notch or vertebrate Deltex
  • vertebrate Deltex protem e g , Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc
  • hybridization assays to detect Notch or vertebrate Deltex expression by detecting and/or visualizing respectively Notch or vertebrate deltex mRNA (e.g.. Northern assays, dot blots, in situ hybridization, etc )
  • a sample of cells from such malignancy is plated out or grown m culture, and the cells are then exposed to a Therapeutic.
  • a Therapeutic which inhibits survival or growth of the malignant cells is selected for therapeutic use in vivo.
  • Many assays standard in the art can be used to assess such survival and/or growtii; for example, ceil proliferation can be assayed by measuring 3 H-thym ⁇ d ⁇ ne incorporation, by direct cell
  • the malignant cell cultures are separately exposed to (1) an Agonist
  • the result of the assay can mdicate which type of Therapeutic has therapeutic efficacy.
  • a Therapeutic is indicated for v a e which exhibits the desired effect, inhibition or promotion of cell growth, upon a patient cell sample from tissue havmg or suspected of havmg a hyper- or hypoproliferative disorder, respectivel)
  • hyper- or hypoproliferative disorders mclude but are not limited to those described in Sections 5.8.1 through 5 8.3 infra
  • a Therapeutic is indicated for use in treating nerve mjury or a nervous system degenerative disorder (see Section 5.8.2) which exhibits in
  • an Antagomst Therapeutic of the mvention is also indicated in diseases or disorders determmed or known to involve a Notch or Deltex dominant activated phenotype ("gam of function" mutations ) Administration of an Agonist
  • Therapeutic is indicated in diseases or disorders determmed or known to mvolve a Notch or Deltex dommant negative phenotype ("loss of function" mutations)
  • the functions of various structural domains of die Notch protein have been investigated in vivo, by ectopically expressmg a series of Drosophila Notch deletion mutants under the hsp70 heat-
  • shock promoter as well as eye-specific promoters (see Rebay et al., 1993, Cell
  • Deltex binds to the Notch ANK repeat region.
  • in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if a
  • cells of a patient tissue sample suspected of being pre-neoplastic are similarly plated out or grown in vitro, and exposed to a Therapeutic.
  • the Therapeutic which results in a cell phenotype diat is more normal (i.e., less representative of a pre-neoplastic state, neoplastic state, malignant state, or transformed phenotype) is selected for therapeutic use.
  • Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present.
  • characteristics associated with a transformed phenotype include a more rounded cell mo ⁇ hology, looser substratum attachment, loss of contact inhibition, loss of anchorage 0 dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc. (see Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York pp. 436-446).
  • the in vitro assays described supra can be carried out using a cell line, rather than a cell sample derived from the specific patient to be treated, in which the cell line is derived from or displays characteristic(s) associated with the malignant, neoplastic or pre-neoplastic disorder desired to be treated or prevented, or is derived from the neural or other cell type upon which an effect is desired, according to the
  • an antagonist of Notch and/or Deltex function that can be used as an Antagonist Therapeutic is a molecule comprising a Deltex protein or portion thereof that mediates binding to Notch, covalently bound to a protease or
  • protease preferably is able to cleave a Notch protein.
  • the molecule is preferably a fusion protein (i.e. , the covalent bond is a peptide bond).
  • the Deltex protein is preferably a vertebrate protein, most preferably human.
  • the invention provides a method of targeting or inactivating proteins to which
  • Deltex binds (e.g. , Notch) in a cell.
  • the molecule comprising the
  • Deltex protein or portion thereof and the protease sequences is produced through chemical 5 or via molecular biological techniques.
  • This molecule e.g. , fusion protein
  • This molecule is introduced into the cell by techniques known in the art (e.g., transfection of the cell with a nucleic acid encoding the molecule such that its expression occurs intracellularly). Inside the cell, the molecule can bind to Notch and/or ouier Deltex binding partners. Upon such binding, the
  • protease portion of the molecule cleaves the protein to which the molecule is bound, thus inactivating it.
  • a fusion protein contaming domain I of human Deltex and the protease thermolysin when introduced into the cell would bind to and cleave Notch, thereby inactivating the Notch signaling pathway.
  • Molecules which would inactivate protein function e.g. , by binding thereto, can be used as an alternative to proteases.
  • the Antagonist Therapeutics are administered therapeutically (including prophylactically): (1) in diseases or disorders involving increased (relative to normal, or desired) levels of Notch or vertebrate Deltex function, for example, where the Notch or vertebrate Deltex protein is overexpressed or overactive; and (2) in diseases or disorders
  • in vitro (or in vivo) assays indicate the utility of vertebrate Deltex antagonist administration.
  • the increased levels of Notch or vertebrate Deltex function can be readily detected by methods such as those described above, by quantifying protein and/or RNA.
  • In vitro assays with cells of patient tissue sample or the appropriate cell line or cell type, to criz determine therapeutic utility can be carried out as described above.
  • malignancy or dysproliferative changes are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.
  • malignancies of the colon and cervix can exhibit increased expression of human Notch relative to such non-malignant tissue (see PCT Publication WO 94/07474 published April 14, 1 94, incorporated by reference herein in its entirety).
  • malignancies of the colon or cervix are treated or prevented by administering an effective amount of an Antagonist Therapeutic of the invention.
  • the presence of increased Notch expression in colon, and cervical cancer suggests that many more cancerous and hyperproliferative conditions exhibit upregulated Notch.
  • various cancers e.g. , breast cancer, squamous adenocarcinoma, seminoma, melanoma, and lung cancer, as well as other hyperproliferative disorders, can be treated or prevented by
  • Nervous system disorders involving cell types which can be tested as 0 described supra for efficacy of intervention with Antagonist or Agonist Therapeutics, and which can be treated upon thus observing an indication of merapeutic utility, 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 demyelinauon.
  • Nervous system lesions which may be treated in a patient (including human and non-human vertebrate 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: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of 0 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
  • malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non- nervous system tissue; (iv) infectious lesions, in which a portion of the nervous system is
  • TM 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;
  • demyelinated lesions in which a portion of die 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 leukoencephalopafhy, 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 the survival or differentiation of neurons (see also Section 5.8).
  • Therapeutics which elicit any of die following effects may be useful according to the invention: (i) increased survival time of neurons in culture;
  • motor neuron dysfunction may be measured by assessmg the physical manifestation of motor neuron disorder, e.g. , weakness, motor neuron conduction velocity, or functional disability.
  • motor neuron disorders that may be treated accordmg to the invention mclude but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy mat 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 mcludmg but not lunited 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 me post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Ma ⁇ e-Tooth Disease).
  • disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy mat 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 mcludmg but
  • a Therapeutic of the mvention is used for promotion of tissue regeneration and repair, mcludmg but not lunited to treatment of benign dysprohferative disorders
  • Specific embodiments are directed to treatment of cirrhosis of the liver (a condition m which scarring has overtaken normal liver regeneration processes) , treatment of keloid (hypertrophic scar) formation (disfiguring of die skm in which die scarring process interferes with normal renewal), psoriasis (a common skm condition characterized by excessive proliferation of the skm and delay in proper cell fate determination), and baldness (a condition in which terminally differentiated hair follicles (a tissue rich in Notch) fail to function properly).
  • Deltex agonists and antagonists can also be used to manipulate the differentiation state of non-terminally differentiated (e.g , stem and progenitor) cells
  • a stem cell can be exposed to such an agonist to inhibit its differentiation and achieve expansion of the stem cell population by incubation in vitro under growth conditions.
  • Such stem cells have use in transplantation for in vivo repopulation of their progeny cells and tissue regeneration. (For mediods mat can be used in die foregoing, see
  • a method for d e expansion of a precursor cell comprises contacting die cell with an amount of a vertebrate (e.g. , human) Deltex
  • die precursor cell can be but is not limited to a hematopoietic precursor cell, epithelial precursor cell, kidney precursor cell, neural precursor cell, skin precursor cell, osetoblast precursor cell, chondrocyte precursor cell, liver precursor cell, and muscle cell.
  • the Therapeutics of the invention can be administered to prevent progression
  • Such administration is indicated where the Therapeutic is shown in assays, as described supra, to have utility for treatment or prevention of such disorder.
  • Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, 2 _,b _ 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. 68-79.) Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, witi out significant alteration in structure or function.
  • Metaplasia is a form of controlled cell growdi 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. 3 Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growdi, involving a loss in individual cell uniformity and in die architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism Dysplasia characteristically occurs where tiiere exists chronic irritation or inflammation, and is often found in die 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 die desirability of prophylactic/dierapeutic administration of a Therapeutic of die invention.
  • Such characteristics of a transformed phenotype include morphology 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 protem, 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
  • fibrocystic disease cystic hyperplasia, mammary dysplasia, particularly adenosis (bemgn epidielial hyperplasia)
  • adenosis bemgn epidielial hyperplasia
  • a patient which exhibits one or more of the following predisposing factors for malignancy is treated by administration of an effective amount of a
  • Therapeutic a chromosomal translocation associated wirh a malignancy e.g. , the
  • an Antagonist Therapeutic of die invention is administered to a human patient to prevent progression to breast, colon, or cervical cancer.
  • a Therapeutic of die invention can be administered to prevent a nervous system disorder described in Section 5.8.2, or other disorder (e.g. , liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.
  • a nervous system disorder described in Section 5.8.2 or other disorder (e.g. , liver cirrhosis, psoriasis, keloids, baldness) described in Section 5.8.3.
  • the Therapeutics of die invention can be tested in vivo for die desired dierapeutic 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 present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding vertebrate
  • Antisense refers to a nucleic acid capable of hybridizing to a portion of a vertebrate deltex RNA (preferably mRNA) by virtue of some sequence complementarity. Such antisense nucleic acids have utility as Antagonist
  • Therapeutics of die invention can be used in me treatment or prevention of disorders as described supra in Section 5.8 and its subsections.
  • the antisense nucleic acids of die invention can be oligonucleotides diat are double -stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be direcdy administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • me vertebrate deltex antisense nucleic acids provided by die instant invention can be used for the treatment of tumors or other disorders, the cells of which tumor type or disorder can be demonstrated (in vitro or in vivo) to express a vertebrate deltex gene or a Notch gene. Such demonstration can be by detection 5 of RNA or of protein.
  • the invention further provides pharmaceutical compositions comprising an effective amount of die vertebrate deltex antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra in Section 5.12.
  • a pharmaceutically acceptable carrier as described infra in Section 5.12.
  • Mediods for 0 treatment and prevention of disorders (such as tiiose described in Sections 5.8 and 5.9) comprising administering the pharmaceutical compositions of die invention are also provided.
  • die invention is directed to methods for inhibiting the expression of a vertebrate deltex nucleic acid sequence in a prokaryotic or eukaryotic cell comprising providing die cell with an effective amount of a composition compnsmg an antisense vertebrate deltex nucleic acid of the invention.
  • Vertebrate deltex antisense nucleic acids and tiieir uses are described in detail below.
  • the vertebrate deltex antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 oligonucleotides).
  • the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • 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 odier appending groups such as
  • a vertebrate deltex antisense oligonucleotide is provided, preferably of single-stranded DNA.
  • such an oligonucleotide comprises a sequence antisense to me sequence encoding an SH3- binding domain or a Notch-binding domain of vertebrate deltex or zinc finger domain, most preferably, of human deltex.
  • the oligonucleotide may be modified at any position on its structure witii substituents generally known in die art.
  • the vertebrate deltex antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracd, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
  • 2-thiouridine 5-carboxymed ⁇ ylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-med ⁇ ylguanine, l-methylinosine, 2,2-dimed ⁇ ylguanine,
  • 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 phosphoramidodiioate, a phosphoramidate, a phosphordiamidate, a me iylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • die oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual /3-units, the strands run parallel to each other (Ga ier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide may be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • Oligonucleotides of the invention may be synthesized by standard mediods known in die art, e.g., by use of an automated DNA syndiesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA syndiesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by die method of Stein et al. (1988, Nucl. Acids Res.
  • mediylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Nad. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • die vertebrate deltex 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).
  • die oligonucleotide is a 2'-0-methylribonucleotide (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).
  • the vertebrate deltex antisense nucleic acid of the invention is produced intracellular ly 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 die 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 vertebrate deltex 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 m the art.
  • Vectors can be plasmid, viral, or others known in die art, used for replication and expression in vertebrate cells.
  • Expression of the sequence encoding die vertebrate deltex antisense RNA can be by any promoter known in die art to act in vertebrate, preferably human, cells.
  • Such promoters can be inducible or constitutive.
  • Such promoters mclude 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.
  • the antisense nucleic acids of die invention comprise a sequence complementary to at least a portion of an RNA transcript of a vertebrate deltex gene, preferably a human deltex gene.
  • a sequence complementary to at least a portion of an RNA means a sequence having sufficient complementarity to be able to hybridize with die
  • RNA forming a stable duplex; in the case of double-stranded vertebrate deltex antisense nucleic acids, a single strand of the duplex DNA may tiius be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both die degree of complementarity and the lengtii of the antisense nucleic acid. Generally, die longer the hybridizing nucleic acid, die more base mismatches with a vertebrate deltex RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in die art can ascertain a tolerable degree of mismatch by use of standard procedures to determine die melting point of the hybridized complex.
  • the vertebrate deltex antisense nucleic acids can be used to treat (or prevent) malignancies or other disorders, of a cell type which has been shown to express vertebrate deltex or Notch.
  • die malignancy is cervical, breast, or colon cancer, or squamous adenocarcinoma.
  • Malignant, neoplastic, and pre-neoplastic cells which can be tested for such expression include but are not limited to ose described supra in
  • a single-stranded DNA antisense vertebrate deltex oligonucleotide is used.
  • Malignant (particularly, tumor) cell types which express vertebrate deltex or Notch RNA can be identified by various methods known in die art. Such methods include but are not limited to hybridization with a vertebrate deltex or ⁇ totc ⁇ -specific nucleic acid
  • RNA from the cell type can be translated in vitro into Notch or vertebrate Deltex, immunoassay, etc.
  • primary tumor tissue from a patient can be assayed for Notch or vertebrate Deltex expression prior to treatment, e.g. , by immunocytochemistry or in situ hybridization.
  • compositions of die invention comp ⁇ sing an effective amount of a vertebrate deltex antisense nucleic acid m a pharmaceutically acceptable carrier, can be administered to a patient havmg a malignancy which is of a type that expresses Notch or vertebrate deltex RNA or protem.
  • the amount of vertebrate deltex antisense nucleic acid which wdl 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 Where possible, it is desirable to determine die antisense cytotoxicity of the tumor type to be treated in vitro, and tiien m useful animal model systems prior to testing and use m humans.
  • compositions comprising vertebrate deltex antisense nucleic acids are administered via hposomes, microparticles, or microcapsules
  • it may be desirable to utilize hposomes targeted via antibodies to specific identifiable tumor antigens Leonetti et al , 1990, Proc. Nad Acad. Sci U.S. A
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the mvention
  • 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.
  • Therapeutic of the invention e.g. , encapsulation in hposomes, microparticles, microcapsules, expression by recombinant cells, receptor mediated endocytosis (see, e.g. ,
  • Methods of introduction include but are not limited to mtradermal, intramuscular, lntrape ⁇ toneal, intravenous, subcutaneous, intranasal, epidural, and oral routes
  • 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 witii other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer die pharmaceutical compositions of the invention locally to die 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 cadieter, 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.
  • die Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ibid.)
  • die Therapeutic can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and
  • a controlled release system can be placed in proximity of die therapeutic target, i.e. , the brain, ti us requiring only a fraction of the systemic dose (see, e.g. , Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • administration of a Therapeutic into a Notch- expressing cell is accomplished by linkage of the Therapeutic to a Delta (or other toporydimic) protein or portion thereof capable of mediating binding to Notch.
  • a Notch-expressing cell wi i die linked Therapeutic results in binding of die linked Therapeutic via its Delta portion to Notch on the surface of die cell, followed by uptake of the linked Therapeutic into the Notch-expressing cell.
  • die Therapeutic is delivered intracellularly (e.g., by expression from a nucleic acid vector, or by linkage to a Delta protein capable of binding to Notch followed by binding and internalization, or by receptor-mediated or diffusion mechanisms).
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so diat it becomes intracellular, e.g. , by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g. , a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g.
  • nucleic acid Therapeutic can be introduced intracellularly and incorporated witiiin host cell DNA for expression, by homologous recombination.
  • Alzheimer's disease Targeted intravenous; intradiecal
  • the present invention also provides pharmaceutical compositions. Such as
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of die Federal or a state government or listed in the U.S. Pharmacopeia or otiier generally recognized pharmacopeia for use in 0 animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which die dierapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mmeral oil, sesame oil and die Id e.
  • Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk,
  • composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pdls, capsules, powders, sustained-release formulations and the like.
  • the composition can be
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's
  • compositions will contain a therapeutically effective amount of ie Therapeutic, preferably in purified form, togetiier widi a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • ie Therapeutic preferably in purified form, togetiier widi a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • die composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • me composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to mammals.
  • compositions for intravenous administration are solutions in ster ⁇ e isotonic aqueous buffer.
  • the composition may also include a solubdizing agent and a local anesmetic 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 die quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating die quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • 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 die 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 those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ed ylamino eti anol, histidine, procaine, etc.
  • the amount of the Therapeutic of die invention which will be effective in the ° treatment of a particular disorder or condition wdl depend on die nature of the disorder or condition, and can be determined by standard clinical techniques.
  • 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 die route of administration, and 5 the seriousness of die disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • suitable dosage ranges for intravenous administration 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 mgredient in die 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 die ingredients of d e pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in e form prescribed by a governmental agency regulating die manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by die agency of manufacture, use or sale for human adniinistration.
  • Vertebrate Deltex proteins, analogues, derivatives, and subsequences diereof, vertebrate deltex nucleic acids (and sequences complementary diereto), anti-vertebrate Deltex antibodies have uses in diagnostics.
  • Such molecules can be used in assays, such as 0 immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting vertebrate Deltex expression, or monitor e treatment diereof.
  • an immunoassay is carried out by a method comprising contacting a sample derived from a patient with an anti-vertebrate Deltex antibody under conditions such diat immunospecific binding can occur, and detecting or measuring d e amount of any immunospecific binding by the antibody.
  • such binding of antibody, in tissue sections, preferably in conjunction with binding of anti-Notch can be used to detect aberrant Notch and/or vertebrate Deltex localization or aberrant levels of Notch-vertebrate Deltex colocalization in a disease state.
  • antibody to vertebrate " Deltex can be used to assay in a patient tissue or serum sample for the presence of vertebrate Deltex where an aberrant level of vertebrate Deltex is an indication of a diseased condition.
  • Aberrant levels of vertebrate Deltex binding abUity in an endogenous Notch protein, or aberrant levels of binding ability to Notch (or otiier vertebrate Deltex ligand) in 5 an endogenous vertebrate Deltex protein may be indicative of a disorder of cell fate (e.g. , cancer, etc.)
  • aberrant levels is meant increased or decreased levels relative to that present, or a standard level representing diat present, in an analogous sample from a subject not having the disorder.
  • the immunoassays which can be used include but are not lunited to competitive and non-competitive assay systems using techniques such as western blots. 5 radioi munoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitm reactions, lmmunodiffusion assays, agglutination assays, complement-fixation assays, lmmunoradiomet ⁇ c assays, fluorescent immunoassays, protein A immunoassays, to name 0 but a few.
  • Vertebrate deltex genes and related nucleic acid sequences and subsequences, mcludmg complementary sequences, and odier toporydi ⁇ uc gene sequences can also be used hybridization assays Vertebrate deltex nucleic acid sequences, or subsequences thereof compnsmg 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 aberrant changes in vertebrate Deltex expression and/or activity as described supra.
  • a hybridization assay is carried out by a mediod comprising contacting a sample containing nucleic acid with a nucleic acid
  • Human deltex was isolated dirough a combination of computer and biochemical screens. Initially, a human expressed sequence tag database was screened for
  • a series of two 5' primers (hdx-1 (SEQ ID NO:26) and hdx-2 (SEQ ID NO:27)) and two 3' primers (hdx-3 (SEQ ID NO:28) and hdx-4 (SEQ ID NO:29 )) were synthesized based on die DNA sequence of gnl I dbest I 24254 T05200.
  • PCR reactions were performed using die four different primer combinations and a human fetal brain cDNA library (Invitrogene) as the template The PCR product was sequenced and found to have the same DNA sequence as gnl I dbest I 24254 T05200.
  • the PCR product generated using die hdx-1 and hdx-4 primers was dien labeled and used to screen another human fetal brain cDNA library
  • the isolate was sequenced (SEQ ID NO: 11) and die predicted protein determined (SEQ ID NO.12) ( Figure 2A-C).
  • the predicted human Deltex product has 720 amino acids and an estimated molecular mass of approximately 80 kDa.
  • the 180 amino terminal residues of human Deltex have an approximate identity of 33% widi die co ⁇ esponding amino acid residues of Drosophila Deltex and the nucleic acids encoding tiiese amino acids have an approximate 52% identity.
  • the 180 carboxy terminal amino acids of human Deltex have an approximate 48 % identity widi die co ⁇ esponding amino acid residues of Drosophila Deltex and the nucleic acids encoding diese carboxy terminal amino acids have an approximate 49% identity.
  • a structural analysis of human Deltex protein revealed a conserved 0 structure among Deltex proteins (see Figure 3).
  • human Deltex Like Drosophila Deltex, human Deltex has both ring-H2-zinc finger (amino acids 411-471) (SEQ ID NO:25) and putative SH3-binding domains. Noticeably absent from the human Deltex are the two opa repeats that subdivide die primary structure of the Drosophila Deltex into
  • Domain I corresponds to the N-terminal 303 amino acids of
  • Domam II of Drosophila Deltex contains a putative SH3-bind ⁇ ng site
  • SH2 and SH3 domams are conserved protem modules so named based on tiieir homology to the oncogene Src (Src Homology) These motifs have been implicated in mediating protein-protein interactions in a number of signal transduction patiiways (reviewed in Cell 71:359-362;Science 252:668-674; Trends Cell Biol 3-8-13, FEBS 307:55-61) Recently, a complementary motif mat bmds to die SH3 domam has been identified and called simply an * SH3-b ⁇ nd ⁇ ng domam' (“SH3-BD”) (Science 259: 1157-1161) The core bmdmg region of SH3-BD is prolme-rich and approximately ten residues in length As shown m Table III, tins motif, as defined from
  • the EcoRI-cDNA insert was subcloned directiy bo orientations mto Bluesc ⁇ pt KS. Overlappmg deletions were produced on the insert usmg die
  • GenBank FASTA server 85:2444-2448 available by the GenBank FASTA server through BITNET.
  • one protem segment is fused to the DNA-bmdmg domam of the LexA protem, which in mrn bmds to me promoter of a LexAop-lacZ reporter construct without activating transcription. These constructs are referred as pEG.
  • a second foreign protem segment is fused to an acidic transcriptional activation domain that does not bmd DNA on its own. These constructs are referred to as pJG Coexpression of these two proteins in yeast cells results in the functional reconstruction of an active LexA "hybrid" transcription factor if the foreign proteins physically interact with one another.
  • die pEGhDeltex construct contains die entire coding region of human Deltex;
  • pJGhNotch-1 encodes die ankyrin repeats region of human Notch- 1 from amino acids 1826-2147;
  • pJGhNotch-2 encodes die ankyrin repeats region of human Notch- 2 from amino acids 1772-2084;
  • pJGhNotch encodes die ankyrin repeats of Drosophila Notch from amino acids 1827-2259;
  • JGfHairless contains the entire coding region of Drosophila Hairless.
  • Deltex protein demonstrates a conserved structure m tiiese two evolutionary distant species. Knowledge of die conserved regions of the protein allows one to design synthetic degenerate primers for use m hybridization and PCR reactions which enable the cloning of Deltex encodmg nucleic acids in otiier organisms.
  • a murme deltex gene is obtamed as follows: Standard techmques are utilized to synthesize a series of degenerate primers encoding amino acids 414-419 in Drosophila (SEQ ID NO 30) and 549-555 in human (SEQ ID NO: 35) m a 5' to 3' orientation A second series of degenerate primers correspondmg to die antisense strand of the nucleic acids encoding ammo acids 475-480 in Drosophila (SEQ ID NO:31) and 603-608 in human (SEQ ID NO.36) is also syntiiesized.
  • the two series of primers are added to a mixture contaming mouse embryomc cDNA as template for the PCR amplification PCR is carried out at a range of stringencies, accordmg to mediods commonly known, to allow for varymg degrees of nucleotide similarity between the known deltex sequences and die mouse nucleic acid homolog bemg isolated
  • mouse deltex gene is molecularly cloned and sequenced through techniques known in the art. This segment is used as a probe to isolate a complete cDNA and genomic clone The complete nucleotide sequence of the mouse deltex homolog is determined by sequence analysis. 9. DEPOSIT OF MICROORGANISMS
  • Plasmid pBS hdx containing a cDNA insert encoding a full-length human deltex as a EcoRI insert in Bluescript vector was deposited by S. Leslie Misrock, of Pennie & Edmonds, 1155 Avenue of the Americas, New York, New York 10036 on behalf of Yale University on November 17, 1995, with the American Type Culture Collection, 1201 Parklawn Drive, Rockville, Maryland 20852, under d e provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures, and assigned accession number 97341.
  • AAATGCTAGA AAAACCGTTT TTACCATCAA ACGTGAATTC TTAAGCTGCG CCTAAACGAA 60
  • AGC ATT CGG CGT ACC CAA CAG GCG CCG TAT CCC TTG GTG AAA CTA ACG 980
  • GGC ATC GTG TGG GAG TGG GAG AAC GAC GGC GGC GCA TGG ACG GCC TAC 866 Gly He Val Trp Glu Trp Glu Asn Asp Gly Gly Ala Trp Thr Ala Tyr 110 115 120

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Abstract

Séquences de nucléotides de gènes Deltex de vertébrés, et séquences d'acides aminés des protéines Deltex de vertébrés codées par ceux-ci. L'invention concerne également des fragments et d'autres dérivés et analogues des protéines Deltex de vertébrés, ainsi que les anticorps de ceux-ci. Les acides nucléiques codant pour ces fragments ou dérivés sont également compris dans l'invention. La production des protéines et dérivés cités, par exemple, par recombinaison, est décrite. Selon un mode de réalisation spécifique, l'invention porte sur les acides nucléiques et protéines Deltex chez l'homme. La présente invention porte également sur des procédés et des compositions thérapeutiques et diagnostiques à base de protéines, d'acides nucléiques et d'anticorps Deltex de vertébrés. L'invention concerne également des procédés d'inactivation d'une fonction Notch dans une cellule, des procédés d'identification d'un composé inhibant ou réduisant la liaison d'une protéine Deltex à une protéine Notch, et des procédés visant l'expansion de cellules à terminaisons non différenciées.
PCT/US1996/018675 1995-11-22 1996-11-22 Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci WO1997018822A1 (fr)

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EP96942785A EP0869802A4 (fr) 1995-11-22 1996-11-22 Proteines, acides nucleiques et anticorps deltex de vertebres, et procedes et compositions relatifs a ceux-ci
AU11614/97A AU728798B2 (en) 1995-11-22 1996-11-22 Vertebrate deltex proteins, nucleic acids, and antibodies, and related methods and compositions
JP9519885A JP2000502246A (ja) 1995-11-22 1996-11-22 脊椎動物Ｄｅｌｔｅｘタンパク質、核酸および抗体ならびに関連方法および組成物

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000012698A1 (fr) * 1998-08-31 2000-03-09 Astrazeneca Ab Gene zdx humain analogue au deltex
WO2000026364A1 (fr) * 1998-11-03 2000-05-11 European Molecular Biology Laboratory Regulateur de l'activite de signalisation notch
US6149902A (en) * 1995-09-29 2000-11-21 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
WO2000073329A3 (fr) * 1999-05-26 2001-02-22 Univ Manchester Utilisation de gènes du type notch
US6262025B1 (en) 1995-06-28 2001-07-17 Imperial Cancer Research Technology, Ltd. Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
WO2002012890A3 (fr) * 2000-08-04 2003-03-13 Lorantis Ltd Essai
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon

Citations (2)

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WO1992019734A1 (fr) * 1991-05-03 1992-11-12 Yale University DOMAINES DE LIAISON DANS DES PROTEINES NOTCH ET $i(DELTA)
WO1995019779A1 (fr) * 1994-01-21 1995-07-27 Yale University Proteines et acides nucleiques deltex, anticorps diriges contre ceux-ci, et procedes et compositions associes

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WO1992019734A1 (fr) * 1991-05-03 1992-11-12 Yale University DOMAINES DE LIAISON DANS DES PROTEINES NOTCH ET $i(DELTA)
WO1995019779A1 (fr) * 1994-01-21 1995-07-27 Yale University Proteines et acides nucleiques deltex, anticorps diriges contre ceux-ci, et procedes et compositions associes

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ARTAVANIS-TSAKONAS S, SIMPSON P: "CHOOSING A CELL FATE: A VIEW FROM THE NOTCH LOCUS", TRENDS IN GENETICS., ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM., NL, vol. 07, no. 11/12, 1 November 1991 (1991-11-01), NL, pages 403 - 408, XP001056502, ISSN: 0168-9525, DOI: 10.1016/0168-9525(91)90220-K *
BLANK V, KOURILSKY P, ISRAEL A: "NF-KAPPAB AND RELATED PROTEINS: REL/DORSAL HOMOLOGIES MEET ANKYRIN-LIKE REPEATS", TRENDS IN BIOCHEMICAL SCIENCES., ELSEVIER, HAYWARDS., GB, vol. 17, no. 04, 1 January 1992 (1992-01-01), GB, pages 135 - 140, XP001002610, ISSN: 0968-0004, DOI: 10.1016/0968-0004(92)90321-Y *
See also references of EP0869802A4 *
XU T, ARTAVANIS-TSAKONAS S: "DELTEX, A LOCUS INTERACTING WITH THE NEUROGENIC GENES, NOTCH, DELTA AND MASTERMIND IN DROSOPHILA MELANOGASTER", GENETICS, GENETICS SOCIETY OF AMERICA, AUSTIN, TX, US, vol. 126, 1 November 1990 (1990-11-01), US, pages 665 - 677, XP002907354, ISSN: 0016-6731 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6262025B1 (en) 1995-06-28 2001-07-17 Imperial Cancer Research Technology, Ltd. Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US8415303B2 (en) 1995-06-28 2013-04-09 Imperial Cancer Research Technology, Ltd. Method of treatment using a soluble human delta protein fragment
US6783956B2 (en) 1995-06-28 2004-08-31 Yale University Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US7118890B2 (en) 1995-06-28 2006-10-10 Yale University Antibodies to vertebrate delta proteins and fragments
US7928071B2 (en) 1995-06-28 2011-04-19 Yale Universtiy Nucleotide and protein sequences of vertebrate delta genes and methods based thereon
US6149902A (en) * 1995-09-29 2000-11-21 Yale University Manipulation of non-terminally differentiated cells using the notch pathway
US8222213B2 (en) 1997-07-23 2012-07-17 Yale University Activated amino- and carboxy-terminal forms of Notch
US6436650B1 (en) 1997-07-23 2002-08-20 Yale University Activated forms of notch and methods based thereon
US6692919B1 (en) 1997-07-23 2004-02-17 Yale University Activated forms of notch and methods based thereon
US7727732B2 (en) 1997-07-23 2010-06-01 Yale University Methods for identifying modulators of Notch activation
WO2000012698A1 (fr) * 1998-08-31 2000-03-09 Astrazeneca Ab Gene zdx humain analogue au deltex
WO2000026364A1 (fr) * 1998-11-03 2000-05-11 European Molecular Biology Laboratory Regulateur de l'activite de signalisation notch
WO2000073329A3 (fr) * 1999-05-26 2001-02-22 Univ Manchester Utilisation de gènes du type notch
WO2002012890A3 (fr) * 2000-08-04 2003-03-13 Lorantis Ltd Essai

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AU1161497A (en) 1997-06-11
CA2238404A1 (fr) 1997-05-29
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