EP1108018A1 - Gene zdx humain analogue au deltex - Google Patents

Gene zdx humain analogue au deltex

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Publication number
EP1108018A1
EP1108018A1 EP99941752A EP99941752A EP1108018A1 EP 1108018 A1 EP1108018 A1 EP 1108018A1 EP 99941752 A EP99941752 A EP 99941752A EP 99941752 A EP99941752 A EP 99941752A EP 1108018 A1 EP1108018 A1 EP 1108018A1
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European Patent Office
Prior art keywords
seq
positions
deltex
sequence
polypeptide
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EP99941752A
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German (de)
English (en)
Inventor
Anastasia Mikhael Khoury-Christianson
Joanne Susan Kays
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AstraZeneca AB
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AstraZeneca AB
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Publication of EP1108018A1 publication Critical patent/EP1108018A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to nucleic acid and amino acid sequences pertaining to a Deltex (hZDX) Notch signaling biomolecule which is integral to the cellular process of differentiation and proliferation.
  • the intracellular signaling protein binds the ankyrin repeat region of Notch and functions as a positive regulator of the Notch signaling pathway.
  • Molecular sequences are provided for the design and synthesis of entities that modulate biological and/or pharmacological activity of the native biomolecule.
  • the sequences are also provided for employment to identify small molecule compounds that modulate biological and/or pharmacological activity of the native biomolecule.
  • Biologically-effective antisense molecules are provided, as well as dominant negative mutant versions of hZDX which are suitable for therapeutic use.
  • the invention is also drawn toward the study, prevention, diagnosis, and treatment of pathophysiological disorders related to the Notch signaling pathway. Background of the Invention
  • Multicellular development is governed by the combinatorial and sequential activity of genes that gradually restrict the developmental potential of cell lineages during differentiation.
  • a fundamental cell-fate control mechanism regulating multicellular development is defined by the Notch-signaling pathway.
  • Notch-signaling pathway Developmental and genetic studies of wild type and activated Notch-receptor expression in diverse organisms suggest that Notch plays a general role in development by governing the ability of undifferentiated precursor cells to respond to specific signals. Notch signaling has been conserved throughout evolution and controls the differentiation of a broad spectrum of cell types.
  • the Notch receptor exists at the plasma membrane as a heterodimeric molecule. Logeat, F., et al., PNAS, 95(14):8108.
  • the Notch receptor family mediates the specification of numerous cell fates during development. Studies on the expression, mutant phenotypes and developmental consequences of unregulated receptor activation have implicated Notch signaling proteins in a general mechanism of local cell signaling, which includes interactions between equivalent cells and between different cell types. Genetic approaches have identified components of the signaling cascade, including a conserved family of extracellular ligands as . well as factors that associate with the Notch intracellular domain. Several vertebrate Notch receptors have been discovered and play important roles in normal development and tumorigenesis.
  • Notch proteins are detected at elevated levels relative to the surrounding normal tissue, for example, in metaplastic cervical tissues as well as in cancerous lesions.
  • the available data support the notion that Notch signaling activity is correlated with the differentiation state of these human tissues.
  • Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp tissue boundaries, i.e., discrete differentiation.
  • the tight localization of Notch activity in vertebrate system contrasts with the broad distribution of Notch ligands. Neumann, C.J., et al, Science, 281:409 (1998).
  • the present invention is directed to an isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a polypeptide comprising a sequence having at least about 80% homology to a member selected from: (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:3 positions 3-91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336).
  • Isolated and purified polynucleotides of the present invention include but are not limited to sequences which comprise SEQ ID NO: 1 and/or SEQ ID NO:2.
  • the current invention is directed to a purified polypeptide comprising the amino acid sequence as depicted in SEQ ID NO:3 or a variant thereof as defined herein.
  • a preferred embodiment of the invention is an isolated and purified biologically effective antisense polynucleotide molecule comprising an oligomer in the range from about 12 to about 25 nucleotides in length which is complementary to a region within SEQ ID NO:l selected from positions 469- 556, 650-791, and 1110-1189.
  • the present invention is also directed to an isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a biologically effective dominant negative mutant variant of SEQ ID NO:3 which has the ability to modulate a biological and/or pharmacological activity of a Deltex regulator of a Notch pathway.
  • a further preferred embodiment of the invention is a biologically effective dominant negative mutant polypeptide variant of SEQ ID NO:3.
  • the instant invention is further directed to methods of identifying compounds that modulate a biological and/or pharmacological activity of a protein which regulates a Notch pathway, comprising: (a) combining a candidate compound modulator with a Deltex polypeptide derived from SEQ ID NO:3, and (b) measuring an effect of the candidate compound modulator on the biological and/or pharmacological activity of the polypeptide.
  • Biological activity as used herein in reference to a Deltex polypeptide refers to the ability of the Deltex to interact with and/or bind to with a Notch protein, particularly the intracellular ankyrin repeat region of Notch.
  • Pharmacological activity as used herein in reference to a Deltex biomolecule, particularly a hZDX molecule of the present invention refers to the ability of the biomolecule to mediate cell proliferation and/or differentiation, transformation, and other processes related to abnormal growth and differentiation including but not limited to angiogenesis; and/or the ability to bind or interact with a signal transduction molecule, activator, cofactor, terminal kinase or ligand and/or the direct or indirect transcriptional activation of one or more genes.
  • Dominant negative mutant refers to a polypeptide or a nucleic acid coding region sequence which has been changed with regard to at least one position in the sequence, relative to the corresponding wild type native version at a position which changes an amino acid residue position at an active site required for biological and/or pharmacological activity of the native peptide.
  • dominant negative mutants of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 as well as variants thereof contemplated herein include, but are not limited to, polypeptide species which manifest any change (substitution and/or deletion) with regard to at least one amino acid which corresponds to the following SEQ ID NO:3 positions: E23, W24, W31, Y34, F41, R53, D69, Y75, W82, R93, H103, E113, W114, W121, Y124, Y131, D144, Y150, H159, T184, Y214, R256, R281, S303, C412, C415, C445, C453, C469, C372, H447, and/or H450.
  • Dominant negative mutant embodiments of the invention are therefore nucleic acids which encode peptides, as well as the peptides themselves.
  • Biologically effective as used herein in reference to antisense nucleic acid molecules as well as dominant negative mutants refers to the ability of these molecules to modulate the biological and/or pharmacological activity of a Deltex regulator of the Notch signaling pathway of the present invention, including direct or indirect modulation of transcriptional activation of one or more genes, and/or transcription/translation of nucleic acid coding regions of the protein of the present invention.
  • Biologically effective antisense molecules as well as nucleic acids which encode biologically effective dominant negative mutant versions of SEQ ID NO:3, or derivatives thereof, are preferred embodiments of the present invention.
  • variants refers to sequences substantiality as shown having changes, e.g., a polypeptide sequence comprising a sequence which differs from the sequence referred to by at least one amino acid substitution, addition, or deletion, preferrably a conservative amino acid substitution, that demonstrate or perform substantially the same biological and/or pharmacological activity in substantially the same way, as well as truncated versions of these variants.
  • modulation is used herein to refer to the capacity to either enhance or inhibit a function of a biological molecule including, but not limited to, a biological and/or pharmacological activity of a Deltex molecule, or to the capacity to either enhance or inhibit a functional property of a nucleic acid regulatory or coding region.
  • Modulate physiology refers to the biophysiological regulation of cells and/or tissue and the treatment of pathophysiological disorders related thereto.
  • Direct administration refers to the direct administration of nucleic acid molecules, peptides, or compounds as well as contemplated derivatives/variants of the present invention.
  • Direct administration includes but is not limited to ex vivo as well as in vivo gene therapy techniques.
  • Purified as used herein refers to molecules, either nucleic acid or amino acid sequences, that are removed from their natural environment and isolated or separated from at least one other component with which they are naturally associated.
  • Expression vector refers to nucleic acid vector constructions to direct the transcription of nucleic acid regions in host cells.
  • Expression vectors include but are not limited to plasmids, retro viral vectors, viral and synthetic vectors.
  • Transformed host cells as used herein refer to cells which harbor one or more nucleic acids of the present invention.
  • the Drosophila Notch gene encodes a 300kD transmembrane protein with an extracellular domain containing 36 epidermal growth factor (EGF)-like repeats, 3 lin 12 repeat, and an intracellular domain bearing 6 tandem cdcl0/SW16/ankyrin repeats (ankyrin repeats), and a PEST region. Matsuno, K., et al, Development, 121:2633 (1995). Human Notch
  • Human Notch and Notch receptor molecules are of significant importance in the assays for modulation of the biological activity of the novel human cytoplasmic Notch signaling protein, Deltex (hZDX), described herein as well as high throughput screening methods for agents which modulate the biological and/or pharmacological activity of hZDX (the novel intracellular Deltex Notch signaling protein, e.g., SEQ ID NO:3, binds the ankyrin repeat region of Notch and functions as a positive regulator of the Notch signaling pathway).
  • Deltex novel intracellular Deltex Notch signaling protein, e.g., SEQ ID NO:3
  • Notch 3 Anchored Intracellular Region nucleotides are represented at positions 4942-7041; Notch 3 Ankyrin Repeat nucleotides are represented at positions 5425- 6069.
  • Notch 3 receptor EMBL U97669 nucleotides 79-7041).
  • Intracellular Region amino acids are represented by positions 1622-2321; Notch 3 Ankyrin Repeats amino acids are represented by positions 1783-1997.
  • the EMBL entry for human Notch 4 is U95299.
  • the human Notch receptor coding sequence is represented by positions 91 through 6102.
  • Ankyrin Repeats are located at positions 1581-1792 of the translated sequence.
  • Drosophila Deltex The Drosophila Deltex gene encodes a 737-amino acid Deltex (dDX) protein. Two opa repeats subdivide the primary structure into three domains, designated as domains I, II and III. Drosophila Deltex contains a SH3-binding domain in domain II and a ring H 2 -zinc finger in domain III.
  • N-terminal amino acids 1-303 correspond to domain I.
  • Results indicate that, while each of the three domains of dDX is capable of homotypic interactions, only domain I mediates associations with the ankyrin repeats of Notch.
  • Overexpression studies of dDX and an 'activated' Notch receptor suggest that dDx acts as a positive regulator in the Notch pathway.
  • the fact that overexpression of dDx and activated Notch give similar phenotypes is consistent with the idea that dDx acts as a positive regulator of the pathway.
  • the involvement of the dDx protein in Notch signaling is mediated by the ankyrin binding Dx domain I. Matsuno, K., et al, Development, 121 :2633 (1995).
  • SEQ ID NO: 3 is the reference amino acid sequence used herein which comprises three other ORF resuting polypeptides: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
  • Region I of the novel human cytoplasmic Notch signaling protein (SEQ ID NO:3), Deltex (hZDX) that interacts with the Notch receptor ankyrin repeat region corresponds to 0 residue positions 1-178.
  • Regions in SEQ ID NO:3 which are expected to confer biological and/or pharmacological activity include: positions 1-383, 1-336, 1-211, 92-383, 92-336, and 218-383.
  • preferred polypeptides which are expected to confer biological and/or pharmacological activity comprise, for example, SEQ ID NO:3 positions 3-91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336.
  • Proline-rich Regions otherwise known as potential binding sites for SH3-domains and/or E3 ubiquitin ligases, corresponds to SEQ ID NO:3 residue positions 178 -181, 222- 231, 289-294, 356-362, and 387-393.
  • PEST Region otherwise known as a potential site for targeting protein for degradation corresponds to SEQ ID NO:3 residue positions 271-320.
  • RLNG-H2 Zinc Finger or C3H Zinc Finger corresponds to SEQ ID NO:3 residue positions
  • Region III a highly inter-species conserved region appears in SEQ ID NO:3 residue positions 481-622 Residues which are believed to be important for biological and/or pharmacological activity of hZDX exist at SEQ ID NO:3 positions E23, W24, W31, Y34, F41, R53, D69, Y75, W82, R93, H103, El 13, W114, W121, Y124, Y131, D144, Y150, H159, T184, Y214, R256, R281, and S303.
  • cysteine residues which are believed to be important for 5 biological and/or pharmacological activity of hZDX exist at positions 412, 415, 445, 453, 469, and 372 of SEQ ID NO:3. Furthermore important histidines exist at positions 447, and 450. These residue positions particularly pointed out, are each (as well as combinations thereof) example embodiments, set forth herein, for substitution or deletion to create dominant negative mutant versions of the novel human cytoplasmic Notch signaling proteins.
  • a second open reading frame is provided that runs from position 485-2347 of SEQ ID NO:
  • SEQ ID NO:4 represents an alternate embodiment of the novel human Deltex Notch signaling protein. GRAIL2 analysis predicts SEQ ID NO:4 to be the best translation. Uberbacher, E.C., et al, PNAS, 88:11261 (1991); Xu, Y., et al, Computer Applications in the Biosciences, 10:613 (1994).
  • a third open reading frame is provided that runs from position 752-2347 of SEQ ID
  • SEQ ID NO:l (a Kozak sequence spans positions 747-752).
  • SEQ ID NO:5 represents another embodiment of the human Deltex Notch signaling protein disclosed herein.
  • a fourth open reading frame is provided that runs from position 1130-2347 of SEQ ID NO:l.
  • SEQ ID NO:6 represents yet another embodiment of the novel
  • T lymphoblastic lymphomas Aberrant Notch signaling has been associated with various different types of cancers including T lymphoblastic lymphomas, leukemias, adenocarcinomas, lung carcinomas, cervical neoplasias as well as various other conditions. See, e.g., Ellisen, L.W., et al, Cell,
  • hZDX plays a role in lung carcinoma, colon adenocarcinoma, and myelogenous leukemias in view of the fact that hZDX message is upregulated in these cancer cell lines compared to normal cells from similar tissue.
  • the most striking hZDX mRNA differential expression pattern noted herein is between normal lung message and lung carcinoma A549 cell line message.
  • the human cytoplasmic Notch signaling protein, Deltex (hZDX) message for in normal lung tissue is very weak; whereas, the message is extremely strong, for example, in A549 cells.
  • differential message levels are clearly notable between normal colon mucosal lining and colorectal adenocarcinoma SW480.
  • differential message levels are clearly demonstrated between normal peripheral blood leukocyte and chronic myelogenous leukemia K-562.
  • the assay for cellular transformation demonstrates positive control cells exhibited foci or films of cells as do cells transfected with SEQ ID NO:2. Negative control cells, however, show no foci or film formations. The results indicate that SEQ ID NO:2 transform cells so that they lose cell-to-cell-contact inhibiton of growth and gain the ability to proliferate in a semi-solid medium. These two characteristics distinguish malignant cells from their normal counterparts.
  • the ability to effectively decrease the biologically effective level of hZDX, or the mRNA level of hZDX, for example through an antisense approach or the administration of a dominant negative mutant version, or the administration of a compound which modulates a biological and/or pharmacological activity of hZDX, is expected to effect cell proliferation and/or differentiation.
  • the present invention relates to molecules capable of modulating Notch signal transduction in order to regulate, modulate and/or inhibit disease conditions including but not limited to abnormal cell differentiation and/or proliferation.
  • molecules of the present invention may be included in methods for treating diseases comprising roliferation or metabolic disorders, for example cancer, fibrosis, and other disorders related to abnormal growh and differentiation including but not limited to vasculogenesis and/or angiogenesis.
  • diseases comprising roliferation or metabolic disorders, for example cancer, fibrosis, and other disorders related to abnormal growh and differentiation including but not limited to vasculogenesis and/or angiogenesis.
  • the identification of effective small compounds which specifically modulate Notch signal transduction by modulating the activity of the human Deltex function, i.e., biological and/or phrmacological activity is therefore an object of the invention.
  • Bioly effective antisense molecules a purified polynucleotide comprising an oligomer in the range from about 12 to about 25 nucleotides in length which is complementary to a region within SEQ ID NO:l
  • nucleic acids which encode biologically effective dominant negative mutant versions of SEQ ID NO:3, or derivatives thereof, as well as dominant negative mutant versions of SEQ ID NO:3, and derivatives thereof, examples of each of which are described infra, are preferred embodiments of the present invention and are intended to fall within the scope of the claims appended hereto.
  • the present invention also provides a method of treatment for a patient in need of such treatment, videlicet for a patient who suffers a pathological condition mediated by the Notch signaling pathway or a biomolecule of the present invention, comprising administering an effective amount of a biologically effective antisense nucleic acid molecule derived from SEQ ID NO:l; or administering an effective amount of a nucleic acid which encodes a biologically effective dominant negative mutant version of hZDX; or administering a compound that modulates the biological and/or pharmacological activity of hZDX which was identified by a method described herein.
  • the present invention relates to variants of nucleic acid sequences sequences (e.g., SEQ ID NO:l and SEQ ID NO:2) and amino acid sequences (e.g., SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO:6) substantially as shown, which have changes, e.g., a polypeptide sequence comprising a sequence which differs from the sequence referred to by at least one amino acid substitution, preferrably a conservative amino acid substitution (or a nucleic acid sequence which encodes therefor), that demonstrate or perform substantially the same biological and/or pharmacological activity in substantially the same way, as well as molecules which comprise truncated versions.
  • changes e.g., a polypeptide sequence comprising a sequence which differs from the sequence referred to by at least one amino acid substitution, preferrably a conservative amino acid substitution (or a nucleic acid sequence which encodes therefor), that demonstrate or perform substantially the same biological and/or pharmacological activity in substantially the same
  • variant as used herein is intended to encompass all contemplated biologically effective dominant negative mutants, several species of which are set forth herein.
  • a preferred variant of the molecule as depicted in SEQ ID NO:3 is one comprising an amino acid sequence having at least 80% amino acid sequence homology (identity) to SEQ ID NO:3 or a biologically and/or pharmacologically active substantial fragment thereof (e.g., SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:3 positions 3-91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336); a more preferred variant is one having at least 90% amino acid sequence homology; and a most preferred variant is one having at least 95% amino acid sequence homology to the amino acid sequence as depicted in SEQ ID NO:3 or a biologically and/or pharmacologically active substantial fragment thereof.
  • Variants within the scope of this invention also include biologically-effective dominant negative mutant
  • a variant of the Deltex regulator of the Notch signaling pathway, hZDX, e.g., SEQ ID NO:3, of the present invention may have an amino acid sequence that is different by one or more amino acid substitutions. Embodiments which comprise amino acid deletions and/or additions are also contemplated.
  • the variant may have conservative changes (amino acid similarity), wherein a substituted amino acid has similar structural or chemical properties, for example, the replacement of leucine with isoleucine.
  • a variant may have nonconservative changes, e.g., replacement of a glycine with a tryptophan.
  • Amino acid substitutions may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as a biological and/or pharmacological activity of the native molecule is retained.
  • amino acid substitutions are important to construct contemplated biologically effective dominant negative mutants, several species of which are set forth herein.
  • Negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine phenylalanine, and tyrosine.
  • At least one amino acid residue position at an active site required for biological and/or pharmacological activity in the native peptide is changed to produce an agent or entity having reduced activity or which is devoid of detectable activity.
  • Suitable substitutions of amino acids include the use of a chemically derivatized residue in place of a non-derivatized residue. D-isomers as well as other known derivatives may also be substituted for the naturally occurring amino acids. See, e.g., U.S. Patent No. 5,652,369, Amino Acid Derivatives, issued July 29, 1997. Example substitutions are set forth in TABLE 1 as follows:
  • “Homology” as used herein is a measure of the identity of nucleotide sequences or amino acid sequences. In order to characterize the homology, subject sequences are aligned so that the highest order homology (match) is obtained. "Identity" per se has an art- recognized meaning and can be calculated using published techniques. Computer program methods to determine identity between two sequences, for example, include DNAStar software (DNAStar Inc., Madison, Wl); the GCG program package (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387); BLASTP, BLASTN, FASTA (Atschul, S. F. et al, J Molec Biol . (1990) 215:403).
  • Homology as defined herein is determined conventionally using the well known computer program, BESTFIT (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis., 53711).
  • BESTFIT Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis., 53711.
  • Eighty percent of homology is therefore determined, for example, using the BESTFIT program with parameters set such that the percentage of identity is calculated over the full length of the reference sequence, e.g., SEQ ID NO:3, and gaps of up to 20% of the total number of amino acids in the reference sequence are allowed, and wherein up to 20% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 20% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • the BESTFIT program with parameters set such that the percentage of identity is calculated over the full length of the reference sequence, e.g., SEQ ID NO:3, and gaps of up to 20% of the total number of amino acids in the reference sequence are allowed, and wherein up to 20% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 20% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • Percent homologies are likewise determined, for example, to identify preferred species, within the scope of the claims appended hereto, which reside within the range of about 80 percent to 100 percent homology to SEQ ID NO:3 as well as biologically and/or pharmacologically active derivatives thereof and biologically effective dominant negative mutants contemplated herein.
  • Percentage similarity (conservative substitutions) between two polypeptides may also be scored by comparing the amino acid sequences of the two polypeptides by using programs well known in the art, including the BESTFIT program, by employing default settings for determining similarity.
  • the present invention relates, in part, to the inclusion of the polynucleotide encoding hZDX in an expression vector which can be used to transform host cells or organisms. Such transgenic hosts are useful for the production of the regulator of the Notch signaling pathway as well as valuable variations thereof contemplated herein.
  • the nucleic acid sequence also provides for the design of antisense molecules, example embodiments of which are provided herein, which are useful in downregulating, diminishing, or eliminating expression, e.g., transcription and/or translation of sequences which comprise SEQ ID NO:2 in cells.
  • the regulator molecule of the Notch signaling pathway of the present invention is used in screening assays to identify antagonists or inhibitors which bind to, or interact with hZDX, emulate its substrate, or otherwise inactivate the biomolecule or compete biologically, e.g., competitive interaction or competitive binding inhibition, with SEQ ID NO:3.
  • the regulator of the Notch signaling pathway, as well as derivatives contemplated herein are used in screening assays to identify agonists which agonize or mimic the biological and/or pharmacological activity, induce the production of or prolong the biological halflife of the molecule in vivo or in vitro.
  • the invention also relates to pharmaceutical compositions which comprise molecules as depicted in SEQ ID NO:2 or SEQ ID NO:3 or variants of these molecules as defined herein, including antisense molecules, or a compound identified by means of a described method, for the treatment of pathological disorders related to or mediated by hZDX.
  • a purified polynucleotide which comprises a nucleic acid sequence which encodes a polypeptide comprising the sequence as depicted in SEQ ID NO: 3 or a variant of SEQ ID NO:3, including but not limited to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:3 positions 3-91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336.
  • the SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 coding regions, derived from SEQ ID NO:l or SEQ ID NO:2, can be obtained from existing cDNA (e.g., ORIGENE (Rockville, MD) or other suitable cDNA source, for example, by PCR amplification using primers, e.g., SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, as described below.
  • PCR primers are disclosed herein to amplify hZDX coding regions from each of three start sites through the common stop codon. 5'ORFl
  • Each of the 5' end PCR primers incorporate a Kozak sequence prior to the ATG.
  • PCR reactions for example, employ materials from Perkin-Elmer, Foster City, CA
  • the insert is unidirectionally ligated into pCMVSport2 (Gibco BRL Lifesciences, Gaithersburg, MD) and pcDNA3.1(+) (Invitrogen, San Diego, CA, vectors for mammalian cell transfections; as well as into pFastBacl (Gibco BRL Lifesciences,
  • Bac-to-BacTM Baculovirus Expression System.
  • EcoRI-Xbal nucleic acid coding region fragments of hZDX are each ligated unidirectionally into the pFastBacTMHT vector (Gibco BRL Lifesciences, Gaithersburg, MD to generate hZDX protein having 6x histidine affinity tags for standard affinity protein purification from insect cells. See, also, Example II.
  • Purified protein may then be used, for example: 1) to isolate and purify proteins from cell lysates that bind the Notch signaling protein hZDX; 2) in binding experiments to determine if anti-hZDX antibodies prevent hZDX biological interactions; and 3) in screening assays to identify small molecules that modulate hZDX biological and/or pharmacological interaction with the intracellular portion of Notch protein, e.g., ankyrin repeats, thereby modulating the Notch signaling pathway.
  • the interruption of the Notch signaling pathway potentially has a role as a therapeutic method in certain types of cancers.
  • Small molecule modulators including but not limited to antagonists, to hZDX-Notch interaction are expected to be developed as therapeutic agents for treatment of pathophysiological disorders related to the Notch signaling pathway.
  • a purified polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising a sequence having at least about 80% homology to a member selected from: (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:3 positions 3-91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336).
  • a polynucleotide comprising a nucleic acid sequence which encodes a polypeptide comprising a sequence selected from: (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:3 positions 3- 91, SEQ ID NO:3 positions 1-178, SEQ ID NO:3 positions 92-211, and SEQ ID NO:3 positions 218-336), which encodes a polypeptide comprising a sequence wherein one or more of the following positions corresponding to SEQ ID NO:3 are substituted or deleted: (E23, W24, W31, Y34, F41, R53, D69, Y75, W82, R93, H103, El 13, W114, W121, Y124, Y131, D144, Y150, H159, T184, Y214, R256, R281, S303, C412, C415, C445, C453, C469, C372, H447, and H450).
  • Antisense Molecules Various nucleic acid sequences complementary to SEQ ID NO:l and/or SEQ ID NO:2 are used in another embodiment of the invention to modulate the Notch signaling pathway by affecting gene expression, e.g., transcription and/or translation of the subject sequences, in cells.
  • Pharmacological activity of an endogenous gene may be modulated by affecting the transcription and/or translation, for example, of the endogenous gene by use or administration of anti-sense constructs to produce anti-sense transcripts or by direct delivery of anti-sense oligomers.
  • Antisense constructs and oligomers may each be used as embodiments of the present invention and each are related to therapeutic method embodiments practiced via direct administration as defined herein.
  • oligos for example, are demonstrated to be effective in reducing proliferation of A549 cells after 24 hrs incubation at 1 and 2.5 micromolar concentrations: complement to SEQ ID NO:l positions 479-495, 666-683, and 753-770.
  • Antisense molecules which comprise oligomers in the range from about 12 to about 25 nucleotides which are complementary the regions of SEQ ID NO:l and/or ID NO:2 are preferred embodiments of the invention.
  • Antisense molecules comprising oligomers from about 12 to about 25 nucleotides in length which are complementary to a region within SEQ ID NO:l selected from positions 469- 556, 650-791, and 1110-1189 are particularly preferred embodiments.
  • Oligonucleotides which comprise sequences complementary to the following positions of SEQ ID NO:l are example embodiments of the invention: SEQ ID NO:l positions 469-480, 470- 481, 471-482, 472-483, 473-484, 474-485, 475-486, 476-487, 477-488, 478-489, 479- 490, 480-491, 481-492, 482-493, 483-494, 484-495, 485-496, 486-497, 487-498, 488-499, 489-500, 490-501, 491-502, 492-503, 493-504, 494-505, 495-506, 496-507, 497-508, 498-
  • Oligonucleotides which comprise sequences complementary to the following positions of SEQ ID NO:l are further example embodiments of the invention: SEQ ID NO:l positions 650-661, 651-662, 652-663, 653-664, 654-665, 655-666, 656-667, 657-668, 658-669, 659- 670, 660-671, 661-672, 662-673, 663-674, 664-675, 665-676, 666-677, 667-678, 668-679,
  • Oligonucleotides which comprise sequences complementary to the following positions of SEQ ID NO:l are further example embodiments of the invention: SEQ ID NO:l positions 1110-1121, 1111-1122, 1112-1123, 1113-1124, 1114-1125, 1115-1126, 1116-1127, 1117- 1128, 1118-1129, 1119-1130, 1120-1131, 1121-1132, 1122-1133, 1123-1134, 1124-1135, 5 1125-1136, 1126-1137, 1127-1138, 1128-1139, 1129-1140, 1130-1141, 1131-1142, 1132- 1143, 1133-1144, 1134-1145, 1135-1146, 1136-1147, 1137-1148, 1138-1149, 1139-1150, 1140-1151, 1141-1152, 1142-1153, 1143-1154, 1144-1155, 1145-1156, 1146-1157, 1147- 1158, 1148-1159, 1149-1160, 1150-1161, 1151-1162, 11
  • Oligonucleotides which comprise sequences complementary to and hybridizable to the recited area of hZDX mRNA are contemplated for therapeutic use.
  • Nucleotide sequences that are complementary to the hZDX-encoding nucleic acid sequence can be synthesized for antisense therapy. These antisense molecules may be DNA,
  • Antisense molecules described herein may be introduced into cells by microinjection or by expression from vectors harboring the antisense sequence. Antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce the biological and/or pharmacological activity of the regulator of the Notch signaling pathway, hZDX.
  • Embodiments of biological molecules which modulate the Notch signaling pathway described herein i.e., nucleic acids or dominant negative mutant versions thereof as well as antisense embodiments may be administered to a subject via gene therapy to boost or attenuate the corresponding biological and/or pharmacological activity or gene expression of an endogenous Deltex Notch signaling protein, e.g., hZDX.
  • an endogenous Deltex Notch signaling protein e.g., hZDX.
  • Nucleic acid sequences of the present invention may be delivered ex vivo or in vivo to the cells of target organs in a tissue-specific manner.
  • the hZDX coding region as well as variants thereof contemplated herein can be ligated into viral vectors which mediate transfer of the hZDX nucleic acid coding regions by infection of recipient host cells.
  • Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. See, e.g., U.S. Patent No. 5,624,820, Episomal Expression Vector for Human Gene Therapy, issued April 29, 1997.
  • GENOVO Corporation for instance, Sharon Hill, PA, at the date of this application, have a readily commercially available expression vector portfolio which comprise an assortment of vectors complete with well-established methods which consistently demonstrate tissue-specific expression and inducible tissue- specific expression.
  • the GENOVO Corporation is an example source for vectors and methods to practice gene- therapy methods of the present invention.
  • Nucleic acid coding regions of the present invention are incorporated into effective expression vectors, which are directly administered or introduced into somatic cells for gene therapy (a nucleic acid fragment comprising a coding region, preferably mRNA transcripts, may also be administered directly or introduced into somatic cells). See, e.g., U.S. Patent No. 5,589,466, issued Dec. 31, 1996.
  • nucleic acids and vectors may remain episomal or may be incorporated into the host chromosomal DNA as a pro virus or portion thereof that includes the gene fusion and appropriate eukaryotic transcription and translation signals, i.e, an effectively positioned RNA polymerase promoter 5' to the transcriptional start site and ATG translation initiation codon of the gene fusion as well as termination codon(s) and transcript polyadenylation signals effectively positioned 3' to the coding region.
  • DNA derived from SEQ ID NO:l can be transferred into cells for gene therapy by non- viral techniques including direct microinjection, receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, or lipofection membrane fusion. These procedures and variations thereof are suitable for ex vivo, as well as in vivo gene therapy according to established methods in this art.
  • Generally Acceptable Vectors include direct microinjection, receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, or lipofection membrane fusion.
  • polynucleotide sequences which encode hZDX, a corresponding polypeptide, dominant negative mutant versions, fusion proteins, or antisense molecules may be used in recombinant DNA molecules that direct the expression of the respective molecule in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express the mediator of the Notch signaling pathway, as well as variations thereto and dominant negative mutants thereof. As will be understood by those of skill in the art, it may be advantageous to produce nucleotide sequences possessing non-naturally occurring codons.
  • SEQ ID NO:2 may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce the native biomolecule. Techniques for such manipulations are fully described in Sambrook, J., et al, Molecular Cloning Second Edition, Cold Spring Harbor Press (1990), and are well known in the art.
  • Expression vectors are described herein as nucleic acid sequences for the transcription of embodiments of the present invention. Such vectors can be used to express nucleic acid sequences in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells, human, and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast, or bacteria-animal cells, or bacteria- fungal cells, or bacteria-invertebrate cells.
  • mammalian expression vectors may be used to express the hZDX molecule as well as variants and derivativescontemplated herein.
  • Commercially available mammalian expression vectors which are suitable for recombinant expression, include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565), pLXIN and pSIR (CLONTECH), pIRES-EGFP (CLONTECH).
  • INVITROGEN corporation provides a wide variety of commercially available mammalian expression vector/systems which can be effectively used with the present invention.
  • INVITROGEN Carlsbad, CA. See, also, PHARMLNGEN products, vectors and systems, San Diego, CA.
  • Baculoviral expression systems may also be used with the present invention to produce high yields of biologically acive hZDX.
  • Vectors such as the CLONETECH, BacPakTM Baculovirus expression system and protocols are preferred which are commercially available. CLONTECH, Palo Alto, CA. Miller, L.K., et al, Curr. Op. Genet. Dev. 3:97 (1993); O'Reilly, D.R., et al, Baculovirus Expression Vectors: A Laboratory Manual, 111.
  • Vectors such as the INVITROGEN, MaxBacTM Baculovirus expression system, insect cells, and protocols are also preferred which are commercially available. INVITROGEN, Carlsbad, CA.
  • Host cells transformed with a nucleotide sequence which encodes the activator of the Notch signaling pathway of the present invention may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • Embodiments of the present invention are host cells transformed with a purified polynucleotide comprising a nucleic acid sequence to encode the polypeptide having the sequence as depicted in SEQ ID NO:3 or a contemplated variant thereof.
  • Cells of this type or preparations made from them may be used to screen for modulators of the biological and/or pharmacological activity of the native molecules, e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, as well as alternate versions described.
  • modulators of the biological and/or pharmacological activity of the native molecules e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, as well as alternate versions described.
  • Eukaryotic recombinant host cells are especially preferred. Examples include but are not limited to yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • L cells L- M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • the expression vector may be introduced into host cells to express the activator of the Notch signaling pathway, hZDX, and derivatives thereof via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation.
  • Commercially available kits applicable for use with the present invention for hererologous expression including well-characterized vectors, transfection reagents and conditions, and cell culture materials are well-established and readily available. CLONTECH, Palo Alto, CA; INVITROGEN, Carlsbad, CA; PHARMINGEN, San Diego, CA; STRATAGENE, LaJolla, CA.
  • the expression vector-containing cells are clonally propagated and individually analyzed to determine the level of hZDX production.
  • Identification of host cell clones which express hZDX may be performed by several means, including but not limited to immunological reactivity with antibodies described herein, and/or the presence of host cell-associated specific biological activity, and/or the ability to covalently cross-link specific substrate to hZDX with the bifunctional cross-linking reagent disuccinimidyl suberate or similar cross-linking reagents.
  • the Deltex molecules of the present invention may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath, J., Protein Exp. Purif. 3:263 (1992)), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • the inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and the coding region is useful to facilitate purification.
  • CLONTECH Palo Alto, CA.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a nascent form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3, HEK293 etc., have specific cellular machinery and characteristic mechanisms for such post- translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express SEQ ID NO:2/SEQ ID NO:3, for example may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • the human Deltex can be produced in the yeast S.cerevisiae following the insertion of the optimal cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of the heterologous protein.
  • vectors such as EmBLyex4 or the like are ligated to the beta subunit cistron. See, e.g., Rinas, U., et al, Biotechnology, 8:543 (1990); Horowitz, B., et al, J. Biol. Chem., 265:4189 (1989).
  • the Deltex cistron is ligated into yeast expression vectors which may employ any of a series of well-characterized secretion signals.
  • the levels of expressed hZDX are determined by the assays described herein.
  • a variety of protocols for detecting and measuring the expression of the novel molecule as well as functional derivatives thereof, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes may be employed.
  • Well known competitive binding techniques may also be employed. See, e.g., Hampton, R., et al.
  • Screening Assays Methods are provided to screen compounds individually, or libraries of compounds, for the identification of compounds which have the ability to modulate a biological and/or pharmacological activity of a Deltex Notch signaling protein, particularly hZDX described herein.
  • the present invention is also directed to methods of screening for compounds which modulate the expression (transcription and/or translation) of DNA or RNA encoding hZDX.
  • Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non- proteinaceous organic molecules (e.g., small molecule compounds).
  • Example assays described herein are used to identify agents which modulate hZDX biological interaction with Notch, as well as the pharmacological activity of hZDX.
  • modulating agents are contemplated for therapeutic purposes, i.e., administration for the treatment of pathophysiological disorders related to the biological and/or pharmacological activity of the novel human cytoplasmic Notch signaling protein, Deltex (hZDX).
  • Compounds may modulate an ultimate biological and/or pharmacological activity by increasing or attenuating the expression of DNA or RNA encoding hZDX or a function of the native Deltex Notch signaling protein.
  • Compounds that modulate the expression of DNA or RNA encoding hZDX or the function of the polypeptide may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or activity.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • the human Deltex described herein as well as contemplated variants can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment or entity employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the abolition or modulation of activity or the formation of binding complexes, between an hZDX molecule and the agent being tested, may be measured, for example, by means provided (see, Examples appended hereto).
  • the present invention provides a method for screening a plurality of compounds for specific binding affinity with the native polypeptide SEQ ID NO: 3 or a variant thereof contemplated herein, comprising providing a plurality of compounds; combining an embodiment of hZDX of the present invention with each of a plurality of compounds for a time sufficient to allow binding under suitable conditions; and detecting binding of an embodiment of the activator of the Notch signaling pathway, to each of the plurality of compounds, thereby identifying the compounds which specifically bind the hZDX polypeptide.
  • Disruption of hZDX binding to Notch for example, is readily measurable by means of competitive binding assays well known in the art.
  • Methods of identifying compounds that modulate a biological and/or pharmacological activity of a Deltex Notch signaling protein are generally preferred, which comprise combining a candidate compound modulator with a purified polypeptide comprising the amino acid sequence as depicted in SEQ ID NO: 3 or a variant of SEQ ID NO: 3 having at least about 80% homology to a member selected from (SEQ ID NO:3, SEQ ID NO:4, SEQ .
  • Preferred assays for modulators of hZDX fall into two general categories: 1) direct measurement of a biological activity, e.g., binding to Notch, and 2) measurement of downstream events in the signaling cascade including cell/tissue/organism physiological manifestations. See Examples appended hereto.
  • a nucleic acid sequence which encodes a Deltex Notch signaling protein as depicted in SEQ ID NO:3 may be ligated to a heterologous sequence to encode a fusion protein for use in a yeast 2-hybrid system.
  • a heterologous sequence to encode a fusion protein for use in a yeast 2-hybrid system.
  • Chimeric constructs are also used to express a 'bait', according to methods well known using a yeast two-hybrid system, using accessory native peptides that are expected to be associated with hZDX, e.g., Notch (described supra).
  • the two-hybrid system uses the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA-binding site that regulates the expression of an adjacent reporter gene.
  • An especially preferred embodiment of the present invention is a method for treatment of a patient in need of such treatment for a dysfunctional- Notch signaling pathway related condition which is mediated by a human activator of the Notch signaling pathway, e.g., SEQ ID NO:3, comprising administration of a therapeutically effective amount of a modulating compound identified using sequences comprising sequences as depicted in SEQ ID NO:l and/or SEQ ID NO:3 or a contemplated variant thereof as a pharmacological target in methods contemplated herein.
  • a human activator of the Notch signaling pathway e.g., SEQ ID NO:3
  • a method of modulating a biological and/or pharmacological activity of a protein which mediates the Notch signaling pathway in a cell, tissue, or organism comprises administering an effective amount of a polynucleotide comprising a nucleic acid sequence derived from SEQ ID NO:l contemplated herein including but not limited to nucleic acid sequences which encode biologically effective fragments, dominant negative mutant versions, and antisense molecules.
  • a polynucleotide comprising a nucleic acid sequence derived from SEQ ID NO:l contemplated herein including but not limited to nucleic acid sequences which encode biologically effective fragments, dominant negative mutant versions, and antisense molecules.
  • Peptide regions are selected to be used for antibody production against Region I (Region I is described supra) of the human cytoplasmic Notch signaling protein, Deltex (hZDX) SEQ ID NO:3.
  • the antibodies are used to identify, isolate, and characterize hZDX.
  • Antibodies are, moreover, developed as a diagnostic tools to determine if biopsy cells from suspected tumors contain more or less hZDX than their normal counterparts, thereby indicating the suspect cells are cancerous or abnormal.
  • the antibodies are expected to act as neutralizing agents.
  • Peptide sequences are demonstrated for antibody production against the human cytoplasmic Notch signaling protein, Deltex SEQ ID NO:3: • amino acids 46-54 of SEQ ID NO:3 plus extra Cys for conjugation of peptide to carrier protein KLH
  • a variety of protocols for measuring the human Deltex polypeptide, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the human Deltex polypeptide is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, D.E. et al, J. Exp. Med.
  • antibodies may be utilized to produce antibodies specific for hZDX polypeptide fragments, or the full- length nascent polypeptide, e.g., SEQ ID NO:3. Specifically, it is readily apparent to those skilled in the art that antibodies may be generated which are specific for the fully functional protein or fragments thereof.
  • Antibody affinity columns are made by adding the antibodies to Aff ⁇ gel-10 (Biorad), a gel support which is activated with N hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) with appropriate detergent and the cell culture supernatants or cell extracts containing hZDX using appropriate membrane solubilizing detergents are slowly passed through the column.
  • the column is then washed with phosphate buffered saline/detergent until the optical density falls to background, then the protein is eluted with 0.23M glycine-HCl (pH 2.6)/detergent.
  • Purified hZDX polypeptide is then dialyzed against phosphate buffered saline/detergent.
  • hZDX molecules can be separated from other cellular proteins by use of an immunoaffmity column made with monoclonal or polyclonal antibodies specific for full length nascent hZDX, or polypeptide fragments of the the Notch signaling pathway activator . molecule.
  • hZDX polypeptides described herein may be used to affinity purify biological effectors from native biological materials, e.g. disease tissue. Affinity chromatography techniques are well known to those skilled in the art.
  • a hZDX peptide described herein or an effective fragment thereof, is fixed to a solid matrix, e.g.
  • CNBr activated Sepharose according to the protocol of the supplier (e.g., Pharmacia, Piscataway, NJ), and a homogenized/buffered cellular solution containing a potential molecule of interest is passed through the column. After washing, the column retains only the biological effector which is subsequently eluted, e.g., using 0.5M acetic acid or a NaCl gradient. Diagnostic Assays
  • SEQ ID NO:l oligonucleotides or antisense molecules described herein may be used in diagnostic assays of body fluids or biopsied tissues to detect the expression level of the novel human Deltex molecule described herein.
  • sequences derived from SEQ ID NO:l by means of hybridization assays, even for the detection of single nucleotide polymorphisms, well-known in the art, can be used to detect the presence of the mRNA transcripts in a patient or to monitor the modulation of transcripts during treatment.
  • Drmanac S et al, Accurate Sequencing by Hybridization for DNA Diagnostics and Individual Genomics, Nature Biotechnology 16:54(1998); Drmanac S, et al, Gene-Representing cDNA Clusters Defined by Hybridization of Clones from Infant Brain Libraries with Short Oligonucleotide Probes, Genomics, 37: 29 (1996) .
  • PCR can also be applied to detect sequences of the invention in suspected samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence, e.g., SEQ ID NO:l, set forth herein.
  • the primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nucleotides or more (usually not more than 2000 nucleotides). See, e.g., Perkin Elmer, PCR Bibliography, Roche Molecular Systems, Branchburg, New Jersey; CLONTECH products, Palo Alto, CA; U.S. Patent No. 5,629,158, Solid Phase Diagnosis of Medical Conditions, issued May 13, 1997.
  • compositions which comprise at least one nucleic acid oligomer derived from SEQ ID NO:l are contemplated as commercially significant aspects of the current invention.
  • compositions which comprise a derivative nucleic acid of SEQ ID NO:l, a dominant negative mutant coding region, an antisense sequence, a polypeptide as depicted in SEQ ID NO:3 or a variation thereof contemplated herein, or a compound identified by means encompassed by the claims appended hereto that modulates the biological and/or pharmacological activity a protein which mediates the Notch signaling pathway, e.g., SEQ ID NO:3, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, PA). To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of a polypeptide, nucleic acid, or compound modulator.
  • compositions of the invention are administered to an individual or used in amounts sufficient to treat or diagnose the Notch signaling pathway- related disorders.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, eg, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of compound, peptide, or nucleic acid which ameliorate or prevent a dysfunctional apoptotic condition. The exact dosage is chosen by the individual physician in view of the patient to be treated.
  • compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular. Administration of pharmaceutical compositions is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tissue), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of a protein which mediates the Notch signaling pathway can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • an effective but non-toxic amount of the compound, nucleic acid, or peptide desired can be employed as an the Notch signaling pathway modulating agent.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult human/per day.
  • the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day.
  • the range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. Even more particularly, the range varies from about 0.05 to about 1 mg/kg.
  • the dosage level will vary depending upon the potency of the particular compound. Certain compounds will be more potent than others.
  • the dosage level will vary depending upon the bioavailability of the compound. The more bioavailable and potent the compound, the less compound will need to be administered through any delivery route, including but not limited to oral delivery.
  • the dosages of hZDX modulators are adjusted when combined to achieve desired effects.
  • dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
  • Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells and conditions.
  • a human cDNA fragment (EMBLEST AC# R06577) was identified which encodes a peptide with similarity to the C3H zinc finger region of mouse FXl-Tl, but not to that of Drosophila Deltex. The clone was of interest because of this apparent close relationship to the mammalian homolog of deltex.
  • ElectroMAX DH10BTM cells Gibco BRL Life Technologies, Gaithersburg, MD, #18290-015
  • the cells are plated on LB-ampicillin agar and the plates are incubated overnight. Resulting colonies are PCR-screened with primers specific for the desired sequence.
  • PCR primers are shown which are employed to amplify the hZDX coding region from the start site for ORF1 (position 479 through the stop codon at nucleotide 2347 of SEQ ID NO: 1).
  • the template for these reactions can be a human spleen cDNA library, human spleen polyA RNA, or cDNA isolated via the magnetic capture method.
  • PCR reactions use materials from the Perkin-Elmer (Foster City, CA) GeneAmp® XL PCR Kit.
  • Antisense oligonucleotides complementary to a portion of the human cytoplasmic Notch signaling protein, Deltex (hZDX) cDNA may be employed, for example, to transfect a cancer cell line that overexpresss hZDX such as the A549 lung carcinoma cell line (American Type Culture Collection (ATCC # CCL-185, Manassas, VA).
  • the antisense oligo is added to cell culture medium at 10 uM every other day for 2-14 days prior to harvest.
  • the antisense sequence to the entire SEQ ID NO:2, for example, is first cloned into expression plasmid pcDNA3.1+ (Invitrogen, Carlsbad, CA).
  • the resulting construct is transfected into A549 cells using LipoTAXITM Transfection Reagent (Stratagene, La JoUa, CA). Cell proliferation is assayed by measuring 3 H thymidine incorporation via the well-known method described by Caltgirone S., et al, Am. J. Respir. Cell Mol. Biol., 17(1):51 (1997). Relative levels of ⁇ - thymidine in cells transfected with the antisense oligo or antisense expression construct are compared to that of vector-only- tranfected (control) cells as well as untransfected cells. A decrease in 3 H-thymidine incorporation in the cells transfected with the antisense oligo/construct vs. the control and untransfected cells indicate that blocking/reducing the expression of hZDX in A549 cells results in a reduction of cell proliferation in this lung carcinoma cell line.
  • Example IV Induce Cell Proliferation
  • an in vivo activity assay is a cell proliferation assay based on hZDX overexpression.
  • Cells for example, IMR90 (ATCC #CCL- 186), LL-86 (ATCC # CCL-190), 293 (ATCC # CRL-1573), or NIH3T3 (ATCC # CRL-1658), are transfected using LipoTAXITM Transfection Reagent (Stratagene, La Jolla, CA) or CalPhosTM Mammalian Transfection Kit (Clontech, Palo Alto, CA) with a sequence which comprises SEQ ID NO:2, for example, or an effective fragment thereof, cloned into a standard expression vector, e.g., pcDNA3.1+.
  • a standard expression vector e.g., pcDNA3.1+.
  • a reporter construct may be used, for example, by means of the mammalian HES-1 promoter (Sasai Y., et al, Genes Dev., 6: 2620 (1992); Takebayashi, K., et al, J. Biol. Chem., 269(7):5150 (1994)) or the HES-2 promoter (Nishimura, et al, Genomics, 49(1):69 (1998)) fused to any of a number of reporter sequences commercially available.
  • HES-1 promoter Sasai Y., et al, Genes Dev., 6: 2620 (1992); Takebayashi, K., et al, J. Biol. Chem., 269(7):5150 (1994)
  • HES-2 promoter Neishimura, et al, Genomics, 49(1):69 (1998) fused to any of a number of reporter sequences commercially available.
  • Reporter genes commercially available for use in this example include but not limited to the likes of luciferase (expressed in pGL3 Basic vector, Promega, Madison, Wl, or pGL3 Enhancer vector (Promega, Madison, Wl); assayed via Promega Luciferase Assay System (# E1500); b-galactosidase (expressed in pBgal -Basic vector, Clontech, Palo Alto, CA; or pB gal-Enhancer vector, Clontech, Palo Alto, CA (assayed via Clontech Luminescent B-gal Genetic Reporter System II); Green Fluorescent Protein (GFP) (expressed in Clontech vector pEGFP-1 (assayed via measurement of fluorescence from cells at 525 nm); secreted alkaline phosphatase (SEAP) (expressed in Clontech pSEAP2-Basic vector #6049-1 or pSEAP2-Enhancer vector #6051
  • HeLa S3 (ATCC # CCL-2.2), 293 cells (ATCC # CRL-1573), or NIH3T3 (ATCC # CRL-1658) cells are transfected with the following constructs (or combination of constructs) and separately with control vectors: a) control reporter plasmid (Promega pGL3-Control # E1741 or Clontech pBgal-
  • HES-reporter and hZDX e.g., SEQ ID NO:2
  • expression vector e.g., pcDNA3.1+
  • Notch3 AIC in expression vector, e.g. pcDNA3.1+.
  • a further example of a reporter assay employs enhancer of split [E(spl)] m gamma promoter for HES-1 or -2 in the above experiments as described in Eastman, D., et al, Mol. Cell. Biol., 17(9):5620 (1997).
  • Example VI High Throughput Screening - Scintillation Proximity Assay (SPA)
  • SPA High Throughput Screening - Scintillation Proximity Assay
  • hZDX and Notch e.g., Notch3, AIC separately expressed in either insect cells using the baculovirus expression system, or other mammalian cell lines such as CHO, Cos, or MEL. See, e.g., Needham M, et al, Protein Expr.
  • HZDX Baculovirus Expression System.
  • HZDX can be tagged via expression as a fusion protein from commercially available expression vectors containing sequences, for example, FLAG, or c- myc/6-His to aid in purification and detection as is well-known in the art.
  • Materials, purification and detection methods for FLAG-tag are available, for example, from Kodak, New Haven CT.
  • Vectors, for use in the assay include pFLAG.MAC for N-terminal expression of FLAG or pFLAG.CTC for C-terminal expression of FLAG (Kodak, New Haven CT).
  • Purification method uses anti-FLAG M2 Affinity Gel. Detection is performed using monoclonal antibody Anti-FLAG M2 Monoclonal Antibody (# IB 13010) (Kodak, New Haven CT). C-myc/6-His-tag vectors, methods and detection reagents are obtained from Invitrogen Carlsbad CA: vector pcDNA3.1(-)/Myc-His XpressTM; and, purification: XpressTM Purification System; and, detection: Anti-myc Antibody (# R950-25) or Anti-His(C-term) Antibody (# R930-25). After expression and purification of the tagged hZDX the purified protein is fluorescently labeled through conjugation to BODIPY FL (Molecular Probes Eugene, OR) per the protocol provided by the manufacturer.
  • BODIPY FL Molecular Probes Eugene, OR
  • Membranes of cells expressing Notch AIC are dissociated to release the Notch AIC protein by resuspending the membranes in 0.1 - 1% Triton X-100. Excess lipids are pelleted via centrifugation. Luo H., et al, Biochim Biophys Acta, 1052:119 (1990); Reyl-Desmars F., et al., J.B.C., 264:18789 (1989). Solubilized Notch3 AIC is bound to polylysine coated SPA beads (Amersham, Arlington Heights, IL) per the manufacturer's protocol.
  • the results demonstrated with stable cell lines from 2 different transfections were as follows: At 1 x 10 3 cells per plate, the positive control PDGF cells exhibited foci or films of cells as did the cells transfected with pcDNA3.1(+) with the SEQ ID NO:2 insert. The negative control pcDNA3.1(+) cells, however, seeded at 1 x 10 3 cells showed no foci or film formations. Only positive control cells showed foci at 1 x 10 5 and 1 x 10 4 cells per plate and none of the plates had any visible foci or films of cells when plated at 1 x 10 2 cells.
  • SEQ ID NO:2 transformed cells so that they lost their cell- to-cell-contact inhibiton of growth and had the ability to proliferate in a semi-solid medium. These two characteristics are among several that distinguish malignant cells from their normal counterparts. These results suggest that hZDX described herein may play a role in transforming normal cells to malignant cells.
  • Example VIII Efficacy (Pharmacological Activity) Screen To further evaluate the ability of a compound, polynucleotide, or peptide to inhibit human tumor growth, for example, human tumor cells are injected into SCID mice (severe combined immunodeficiency) to form palpable tumor masses.
  • an agent in inhibiting tumor growth can be determined as follows: approximately 1 x 10 7 cells of the CCL 221 cell line (ATCC, Rockville, Md.), a human ras- dependent colon adenocarcinoma cell line, for example, is suspended in 100 ⁇ l DMEM and injected subcutaneously into SCID mice such that two tumors per mouse are formed. SCID mice receive cancer cells and the tumors are grown for 7 days without treatment; on the 7th day (Day 0) tumor maximal diameters and animal weights are recorded and the mean tumor size for the mice is determined. On Day 1 (eight days following tumor cell injection), treatment of the mice with the candidate compound or vehicle alone is begun.
  • mice One group of the mice (controls) are injected intraperitoneally with 0.2 ml of vehicle and a second group of mice receives compound by intraperitoneal injection.
  • Various doses of the agent can be tested in separate groups of mice. On Day 7 and Day 14, animal weight and maximal tumor diameter is measured. Average maximal tumor size for each group on Day 0, Day 7, and Day 14 are compared Day 14, one high dose animal is followed for an additional to determine whether the agent produces a dose-dependent inhibition of tumor growth. Toxicity effects can be examined by tracking weight and by harvesting lungs, livers, and spleens of the animals for histological staining.

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Abstract

L'invention porte sur la biomolécule signal Notch de Deltex humaine (hZDX) isolée et purifiée, sur une séquence d'ADNc codant pour la Deltex native, sur la zone structurelle de codage, et sur la séquence des résidus d'acides aminés, ainsi que sur des procédés utilisant ces séquences pour identifier des composés modulant l'activité biologique ou pharmacologique desdites biomolécules et exerçant de ce fait une action régulatrice sur la physiologie des cellules et des tissus.
EP99941752A 1998-08-31 1999-08-25 Gene zdx humain analogue au deltex Withdrawn EP1108018A1 (fr)

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