EP1054969A2 - Complexes proteiques de retinoblastome et proteines interagissant avec le retinoblastome - Google Patents

Complexes proteiques de retinoblastome et proteines interagissant avec le retinoblastome

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Publication number
EP1054969A2
EP1054969A2 EP99932509A EP99932509A EP1054969A2 EP 1054969 A2 EP1054969 A2 EP 1054969A2 EP 99932509 A EP99932509 A EP 99932509A EP 99932509 A EP99932509 A EP 99932509A EP 1054969 A2 EP1054969 A2 EP 1054969A2
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Prior art keywords
protein
complex
glutl
derivative
activity
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German (de)
English (en)
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Meijia Yang
Krishnan Nandabalan
Vincent Peter Schulz
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CuraGen Corp
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CuraGen Corp
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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4736—Retinoblastoma protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention discloses complexes of the retinoblastoma protein (RB) with other proteins, in particular, complexes of RB with RN-tre, RB with the cellular apoptosis susceptibility protein (CAS), RB with the gamma-interferon inducible protein (IP-30), RB with the ribosomal protein L6, RB with the cytokine IK factor, RB with lactate dehydrogenase B (LDH-B), RB with Nlkl(Nek 2), RB with cyclophilin A, RB with Zap 3, RB with the oxidoreductase 17-beta-hydroxysteroid (17 beta-HSD6), RB with CD24*, RB with Set*, RB with the glutamate transporter GluTl*, and RB with clone 115392*.
  • CAS cellular apoptosis susceptibility protein
  • IP-30 gamma-interferon inducible protein
  • the present invention also discloses antibodies to RB complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy.
  • the present invention further discloses CD24*, Set*, GluTl*, and 115392* genes and proteins, as well as derivatives, fragments and analogs, thereof.
  • RB Human Retinoblastoma Protein complexes with one or more of the following proteins: RN-tre, cellular apoptosis susceptibility protein CAS, gamma-interferon inducible protein IP-30, ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl(Nek 2), cyclophilin A, Zap 3, oxidoreductase 17-beta-hydroxysteroid (17 beta-HSD6), CD24*, Set*, glutamate transporter GluTl* or clone 115392*, have not been previously described prior to the disclosure of the present invention.
  • RN-tre cellular apoptosis susceptibility protein CAS
  • gamma-interferon inducible protein IP-30 ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl(Nek 2), cyclophil
  • RB Human Retinoblastoma Protein
  • GenBank Ace. No. M28419, U.S. Patent 4,942123; Lee, et al, 1987. Nature 329: 642-645 is considered a classical tumor suppressor protein.
  • the RB protein belongs to the retinoblastoma "tumor suppressor family" that includes RB, pRb2/pl30, and pi 07.
  • retinoblastoma Members of the retinoblastoma family are involved in implementing and controlling three major aspects of cellular life: (/) proliferative growth, (a) differentiation, and (Hi) apoptosis (see e.g., Stiegler, 1998. J. Cell. Biochem. Suppl. 30-31:30-36).
  • the activities of these proteins are highly regulated, enabling them to precisely establish control. Proteins that interact with RB have potentially wide ranging effects on both normal physiological processes and important pathogenic processes.
  • the phosphoprotein is the critical control protein for cellular passage through the GI restriction point of the cell cycle (see e.g., Weinberg, 1995. Cell 81:323-330).
  • RB needs to be phosphorylated by cyclin-dependent kinases before mammalian cells can continue the cell cycle.
  • RB Growth suppression by RB is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif (e.g., insulin, papilloma virus E6 and E7 proteins), and the C pocket binds the nuclear c-Abl tyrosine kinase (see e.g., Whitaker, et al, 1998. Mol. Cell. Biol. 18:4032-4042).
  • LXCXE peptide motif e.g., insulin, papilloma virus E6 and E7 proteins
  • C pocket binds the nuclear c-Abl tyrosine kinase (see e.g., Whitaker, et al, 1998. Mol. Cell. Biol. 18:4032-4042).
  • Retinoblastoma protein binds to several proteins involved in transcription, including but not limited to the (i) E2F, (ii) the transcription factor UBF (see e.g., Cavanaugh, et al, 1995. Nature 374:177-180); (iii) the transcription regulator Brm protein (see e.g., Singh, et al, 1995. Nature 374:562-565); (iv) the transcriptional regulator RIZ (see e.g., Buyse, et al, 1995. Proc. Natl. Acad. Sci. USA 92:4467- 4471); (v) the transcriptional activation protein IE2 (see e.g., Choi, et al, 1995. Virology
  • RB is implicated in the pathogenesis of a number of cancers, including, but not limited to cervical cancer, breast cancer, retinoblastomas, pituitary tumors, lymphoma, small cell carcinoma of the lung, esophageal cancer, glioblastoma, familial melanoma, pancreatic cancer, sarcoma and bladder cancer.
  • the mechanism of RB-mediated carcinogenesis has been elucidated for a number of cancers due to its central role in the control of cell cycle progression.
  • the human papilloma virus E7 oncoprotein has been demonstrated to bind to and inactivate RB. See e.g., Dyson, et al, 1989.
  • RB has been demonstrated to bind to, and is concomitantly down-regulated by, the cellular oncoprotein MDM2 in human sarcomas, gliomas and squamous cell carcinomas. See e.g., Xiao, et al, 1995. Nature 375:694-698.
  • RB has been demonstrated to prevent the interaction of cellular and viral oncoproteins (such as El A and c-Myc) with a transcription factor. See e.g., Hateboer, et al, 1993. Proc. Natl. Acad. Sci. USA 90:8489-8493.
  • RB In addition to the function as tumor suppressor, RB displays an independent role in the differentiation of some cell types. For example, the nerve growth factor-mediated accumulation of hypophosphorylated RB is an important signal for differentiation of cells. See e.g., Li, et al, 1996. J. Neurochem. 66:2287-2294. Thus, in summary, RB has been centrally implicated in numerous physiological processes, including, but not limited to: (i) proliferative growth, (ii) differentiation, and (Hi) apoptosis.
  • RB has been strongly implicated in play a role in numerous pathophysio logical conditions, including, but not limited to: (i) hyperproliferative states (e.g., tumorigenesis and metastatic tumor dissemination); (ii) degenerative states (e.g., neurodegeneration) and (Hi) response to viral infection.
  • hyperproliferative states e.g., tumorigenesis and metastatic tumor dissemination
  • degenerative states e.g., neurodegeneration
  • Hi response to viral infection.
  • the protein designated "Related to the -N-terminus of tre” (hereinafter designated RN-tre; submitted as GenBank Ace. Nos. D13644; Nomura, et al, 1994. DNA Res. 1:27-35) is a 97-100 kD protein which appears to have been derived from a fusion of an amino-terminal tre-like region to a carboxy-terminal region encoding a deubiquinating enzyme. See e.g., Matoskova, et al,
  • the RN-tre gene maps to chromosomal location 10pl3, a region known to be a site of translocations in various leukemias and a monosomy syndrome characterized by developmental anomalies.
  • the tre domain within the N-terminus is conserved among several proteins in species from yeast to humans and has demonstrable protein-binding properties.
  • the best known substrate for RN-tre is eps8, a protein involved in mitogenic signaling. See e.g., Biesova, et al, 1997. Oncogene 14:233-241
  • the human chromosome segregation gene homolog (hereinafter designated CAS; GenBank Ace. No. U33286; Brinkmann, et al, 1995. Proc. Natl. Acad. Sci. USA 92:10427-
  • CAS is a cellular apoptosis susceptibility gene.
  • CAS plays a critical role in toxin- and tumor necrosis factor alpha 1 -mediated cell death. See e.g., Brinkmann, et al, 1996. Biochemistry 35:6891-6899.
  • the protein is found to be highly expressed in human leukemia, colon cancer, and breast cancer cell lines, as well as human testis and fetal liver, all of which contain actively dividing cells, supporting a role for the protein in either cell proliferation, or in a homeostatic response to cell proliferation.
  • the CAS gene has been mapped to chromosome 20ql3.2, a region that harbors mutations associated with aggressive breast tumors.
  • the human gamma-interferon-inducible protein IP-30 (GenBank Ace. No. J03909; Luster, et al, 1988. J. Biol. Chem. 263:12036-12043) is induced by interferon gamma which is believed to orchestrate cellular response to various pathogens. See e.g., Boehm, et al, 1997. Ann. Rev. Immunol. j_5: 749-795. Immunohistochemical studies indicate a possible lysosomal localization for IP-30, supporting a role in protein breakdown. The exact function of IP-30 is not yet known, but it may play a role in gamma-interferon mediated immune reactions.
  • V Human Ribosomal Protein (L6)
  • the human ribosomal protein L6 (GenBank Ace. No. X69391; Zaman, 1993. Nucl. Acids Res. 21:1673) is the human homolog of the rat L6 ribosomal protein, which is a structural component of the large ribosomal subunit, and may serve to regulate protein translation (see e.g., Chan, et al, 1996. Biochem. Mol. Biol. Int. 39:431-438).
  • the human IK factor (GenBank Ace. No. S74221; Krief, et al, 1994. Oncogene 9:3449- 3456; U.S. Patent No. 5,612,195; PCT Publication WO 93/11232; GenBank Ace. No. A25270) is a cytokine which has been shown to be secreted by a number of cancer cell lines. IK inhibits gamma-interferon induction in normal as well as leukemic and other cancer cell lines. The factor is also believed to play a role in the escape of cancer cells from immunorecognition.
  • LDH-B Human lactate dehydrogenase B
  • GenBank Ace. No. Y00711 GenBank Ace. No. Y00711 ; Sakai, et al, 1987. Biochem. J. 248:933-936
  • LDH-B GenBank Ace. No. Y00711 ; Sakai, et al, 1987. Biochem. J. 248:933-936
  • LDH-B Human lactate dehydrogenase B
  • Nlkl (also known as Nek 2; GenBank Ace. No. Ul 1050) is the human homolog of the mitotic regulator NIMA of Aspergillus nidulans.
  • the serine/threonine specific kinase Nlkl (hereinafter designated Nlkl (Nek 2)) has been shown to exhibit a 47% identity to NIMA within its catalytic domain. See e.g., Fry, et al, 1995. J. Biol. Chem. 270:12899-12905.
  • Nlkl(Nek 2) appears to function similarly to NIMA. See e.g., Fry and Nigg, 1995. Curr. Biol.
  • Nlkl(Nek 2) differs from NIMA in that Nlkl(Nek 2) activity peaks in the S/G2 phase of the cell cycle and not in mitosis, and Nlkl (Nek 2) lacks CDC2 phosphorylation sites and C-terminal PEST sequences found in NIMA. Accordingly, the exact function of Nlkl (Nek 2) remains unknown, although, as with other members of the Nlkl (Nek) family, there is strong evidence that it functions in cell cycle control. (IX) Cyclophilin A
  • Cyclophilin A (GenBank Ace. No. Y00052; Haendler, et al, 1987. EMBO J. 6:947-950), is a widely expressed, small, soluble protein that is the target of cyclosporin A binding. Cyclophilin A functions as a chemo-attractant for macrophages and eosinophils which promote the inflammatory response. Cyclophilin A has also been associated with systemic lupus erythematosus, Lyme disease and rheumatoid arthritis.
  • cyclophilin A when bound by cyclosporin, is inhibition of calcineurin phosphatase activity, thus affecting a number of cell activities, such as dephosphorylation of transcription factors to prevent their import into the nucleus. See e.g., LeHir, et al, 1995. Lab Invest. 73:727-733.
  • cyclophilin A itself is targeted to the nucleus, where it can act as a DNase involved in cell apoptosis. See e.g., Montague, et al, 1997. J. Biol. Chem. 272:6677-6684.
  • cyclophilin A has also been demonstrated to specifically associate with the HIV-1 gag polyprotein p55gag, but not other primate immunodeficiency virus coat proteins. This association appears to be necessary for replication of the virus. See e.g., Franke, et al, 1994. Nature 372:359-362; Thali, et al, 1994. Nature 372:363-365.
  • the human Zap 3 gene (GenBank Ace. No. L40403; Sherrington, et al, 1995. Nature 375:754-760) is located in the early-onset Alzheimer's disease-associated AD3 locus at 14q24.3. While it is pro line-rich, similar to many secreted proteins, the function of the protein sequence has yet to be quantitatively elucidated.
  • Human oxidoreductase (GenBank Ace. No. AFO 16509; Biswas and Russell, 1997. J. Biol. Chem. 272:15959-15966) is an isoform of the oxidative 17 beta-hydroxysteroid (17 beta- HSD6), and a human relative of rat retinol dehydrogenase (see e.g., Chai, et al, 1996. Gene 169:219-222).
  • 17 beta-HSD6 is implicated in a wide range of disorders associated with retinol (i.e., vitamin A) imbalance. Furthermore, 17 beta-HSD6 is implicated in disorders associated with androsterone deficiency. (XII) Set* . GluTl* . CD24 and 115393
  • the present invention also discloses novel splice variants of Set and GluTl (the proteins which are encoded are hereinafter designated as Set* and GluTl*, respectively), as well as novel open reading frames with the genes for CD24 and 115392 (the proteins which are encoded are hereinafter designated as CD24* and 115392*, respectively), which interact with RB, as disclosed in this invention.
  • CD24 The hybrid signal transduction protein CD24 (GenBank Ace. No. L33930; Kay, et al,
  • CD24 functions to inhibit differentiation and activation of antibody-producing cells; it is also implicated in the differentiation of granulocytes. It is also believed to transduce extracellular signals by release of second messengers, including calcium. See e.g., Lund- Johansen, et al, 1993. Eur. J. Immunol. 23:2782-2791.
  • CD24 has been shown to interact with the members of the protein tyrosine kinase family (c-fgr and lyn products) and, thus, it may function to mediate signaling via phosphorylation cascades.
  • CD24 is also highly expressed on the surface of small cell lung carcinomas, thus tending to implicate this protein in tumorigenesis. See e.g., Jackson, et ⁇ l., 1992. Cancer Res. 52:5264-5270.
  • the human Set gene (GenBank Ace. No. M93651; von Lindern, et al, 1992. Mol. Cell. Biol. 12:3346-3355) encodes a putative oncogene-stimulating protein, Set.
  • the Set gene has been localized to chromosomal region 9q34, which is associated with with various human leukemias . See e.g., Adachi, et al, 1994. J. Biol. Chem. 269:2258-2262.
  • SET and a smaller fusion product (SET-CAN) is located to the nucleus, in specific association with the nuclear pore complex. SET-CAN is believed to regulate nucleocytoplasmic transport and cell cycle progression. See e.g., Fornerod, et al, 1996. Oncogene 13:1801-1808.
  • the human glutamate transporter protein (hereinafter designated GluTl; GenBank Ace. No. D26443; Nomura, et al, 1994. DNA Res. 1:223-229), is an astroglial specific glutamate transporter. Alterations in astroglial glutamate transport have been implicated in the pathogenesis of hepatic encephalopathy and, possibly, in Alzheimer's Disease.
  • the human mRNA clone 115392 (GenBank Accession No. L43578; Tirnms, et al, 1996. Genome Res. 5:71-78) is an expressed sequence associated with Hunter Syndrome (mucopolysaccharidosis type II), a fatal X-linked recessive disorder.
  • Hunter Syndrome molecular genetic disorder
  • the translation product for this mRNA has not been currently defined, and, thus, no function has yet been assigned.
  • RB retinoblastoma protein
  • the present invention discloses herein compositions and methodologies for the production of protein complexes comprised of RB with proteins which interact with (i.e., bind to) said RB protein.
  • the proteins which have been demonstrated to form complexes with the RB protein will be designated hereinafter as "RB-IP” (for an RB-interacting protein) whereas a complex of the RB protein and an RB-IP will be hereinafter be designated as "RB-RB-IP”.
  • the present invention relates to complexes of RB, and derivatives, fragments and analogs thereof, with the following proteins: RN-tre, cellular apoptosis susceptibility protein CAS, gamma-interferon inducible protein IP-30, ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl(Nek 2), cyclophilin A, Zap 3, oxidoreductase 17-beta-hydroxysteroid (17 beta-HSD6), CD24*, Set*, glutamate transporter GluTl* or clone 115392*, and their derivatives, analogs and fragments.
  • RN-tre cellular apoptosis susceptibility protein CAS
  • gamma-interferon inducible protein IP-30 ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl(Nek 2),
  • the present invention discloses further the identification of the novel proteins, Set* and GluTl*, which are encoded by mRNA splice variants of the Set and G/w77 genes, respectively. These aforementioned splice variants generated by the mRNAs encoding Set* and GluTl* are produced by the RNA splicing at splice sites other than the processing sites used to process the mRNAs encoding Set and GluTl, respectively. Additionally, the present invention discloses a novel protein encoded by an open reading frame (ORF) in the CD24 mRNA which is not part of, and does not overlap with, the nucleotide sequence coding for the known CD24 protein.
  • ORF open reading frame
  • the present invention further discloses the nucleotide and amino acid sequences of Set* and GluTl* (the human Set* and GluTl* and homologs of other species), as well as derivatives (e.g., fragments) and analogs thereof.
  • the present invention also discloses the amino acid sequence of CD24* and 115392*, and derivatives (including fragments and analogs thereof).
  • Nucleic acids able to hybridize to or complementary to the foregoing nucleotide sequences, such as the inverse complement (i.e., a nucleic acid that has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand) of the foregoing sequences, are also provided.
  • nucleic acids which are hybridizable to or complementary to (e.g., being the inverse complement) the portions of the nucleotide sequences encoding Set* and GluTl* which span the alternate splice junctions of the Set* and GluTl* mRNAs, respectively (i.e., the point in the Set* or GluTl * nucleotide sequence at which the 5' and 3' splice sites were joined in processing the Set* and GluTl * mRNAs).
  • the present invention also discloses CD24*, Set*, GluTl*, and 115392* derivatives and analogs which possess functional biological activity (i.e., they are capable of displaying one or more known functional biological activities of a wild-type CD24*, Set*, GluTl*, or 115392* protein).
  • Such functional biological activities include, but are not limited to: ( ) the ability to bind with, or compete for, interaction with RB; (ii) antigenicity (i.e., the ability to bind or compete with CD24*, Set*, GluTl*, or 115392* for binding to an anti-CD24*, anti-Set*, anti- GluTl* or anti-115392* antibody, respectively) and (Hi) immunogenicity (i.e., the ability to generate an antibody that binds CD24*, Set*, GluTl*, or 115392*, respectively).
  • antigenicity i.e., the ability to bind or compete with CD24*, Set*, GluTl*, or 115392* for binding to an anti-CD24*, anti-Set*, anti- GluTl* or anti-115392* antibody, respectively
  • immunogenicity i.e., the ability to generate an antibody that binds CD24*, Set*, GluTl*, or 115392*,
  • the present invention will further provide methodologies for the modulation (i.e., inhibiting or enhancing) the activity of RB*RB-IP complexes, and CD24*, Set*, GluTl*, and 115392* proteins.
  • the protein components of these aforementioned complexes have been implicated in various cellular functions, including, but not limited to: (i) physiological processes (e.g., cell cycle control, cellular differentiation and apoptosis); (ii) response to viral infection; (Hi) intracellular signal transduction; (iv) transcriptional control; and (v) pathophysiological processes (e.g., hyperproliferative disorders including tumorigenesis and tumor spread, degenerative disorders including neurodegenerative diseases, virus infection).
  • the present invention discloses methodologies for the screening RB «RB-IP complexes, and CD24*, Set*, GluTl*, and 115392* proteins, as well as derivatives and analogs of the RB «RB-IP complexes, CD24*, Set*, Glutl*, and 115392* mRNAs, and CD24*, Set*, GluTl*, and 115392* proteins for their ability to alter cell functions, particularly those cell functions in which RB and/or an RB-IP has been implicated, as non-exclusively listed supra.
  • the present invention also discloses therapeutic and prophylactic, as well as diagnostic, prognostic, and screening methodologies and compositions which are based upon RB*RB-LP complexes (as well as the nucleic acids encoding the individual proteins which participate in these aforementioned RB-RB-IP complexes) as well as CD24*, Set*, GluTl*, and 115392* proteins and nucleic acids.
  • Therapeutic compounds of the present invention include, but are not limited to: (i) RB «RB-IP complexes and complexes where one or both members of the complex is a derivative or analog of RB or an RB-IP; CD24*, Set*, GluTl*, or 115392* proteins and derivatives, fragments and analogs thereof; (ii) antibodies to and nucleic acids encoding the foregoing; and (iii) antisense nucleic acids to the nucleotide sequences encoding the complex components and CD24*, Set*, GluTl *, or 115392* antisense nucleic acids. Diagnostic, prognostic and screening kits will also be provided.
  • Figure 1 illustrates the CD24 nucleotide acid sequence (GenBank Accession No. L33930; [SEQ ID NO : 1 ] ) .
  • the initiation methionine codon ATG of CD24 is boxed at the start of the
  • CD24 coding sequence which is underlined and denoted by callout "A".
  • the translational stop codon TAA used for CD24 is boxed at the end of the underlined sequence.
  • the prey sequence identified in the assay described in the Specific Examples Section, infra begins at nucleotide 689, denoted by the arrow and callout "B".
  • potential open reading frames (ORFs) included in the identified prey sequence are denoted by callouts "C", "D", and "E”. It should be noted, however, that only the open reading frame (ORF) of callouts "A” and “C” represent potential in vivo protein products, as determined in the Specific Examples Section, infra.
  • FIG. 1 illustrates the CD24 nucleotide and amino acid sequences (Frame A; [SEQ ID NOS:2 and 3, respectively), and the CD24* nucleotide and amino acid sequences (Frame B;
  • the known Set protein-encoding sequence is underlined, flanked by the initiation codon ATG (denoted by callout "A") and the termination codon TAA (denoted by callout "C”).
  • the prey sequence identified in the Specific Examples Section infra begins at nucleotide 2076, donated by the white arrow and callout "F", and falls outside of the known coding sequence for Set. Nucleotides which are identical to the known consensus sequence for 5'- splice sites are shown in bold, and the last base of the exon (exon 1) is denoted by an arrow and callout "B".
  • a 3'- splice site with nucleotides identical to the known consensus sequence for 3'- splice sites, is shown in bold, and the first base of the exon (exon 2) is denoted by an arrow and callout "E”.
  • the stop codon TAA for Set* is denoted by callout "G”.
  • Figure 4 illustrates the Set* nucleotide sequence [SEQ ID NO:7] and associated amino acid sequence [SEQ ID NO:8].
  • the amino acid residue at which the sequence deviates from that of Set because of alternate splicing is denoted by arrow "A”.
  • the prey sequence identified in the assay described in the Specific Examples Section, infra begins at nucleotide 2076 of the Set sequence ( Figure 3), and is denoted by arrow "B".
  • Figure 5 illustrates the nucleotide sequence of GluTl (GenBank Accession No. D26443;
  • a 5'- splice site with nucleotides identical to the known consensus sequence for 5'- splice sites shown in bold, and the last nucleotide of the exon (exon 1), is denoted by an arrow and callout "B".
  • a compatible 3'- splice site with nucleotides identical to the known consensus sequence for 3'- splice sites shown in bold, and the first base of the exon (exon 2) is denoted by an arrow and callout "E”.
  • the stop codon TAA for GluTl * is denoted by callout "G”.
  • a branch point consensus sequence for this exon with bases matching the consensus bases shown in bold, and is denoted by callout "D”.
  • Figure 6 illustrates the GluTl* nucleotide sequence [SEQ ID NO: 10] and associated amino acid sequence [SEQ ID NO:l 1].
  • the amino acid residue at which the sequence deviates from that of GluTl because of alternate splicing is denoted by arrow "A”.
  • the prey sequence identified in the assay described in the Specific Examples Section, infra begins at nucleotide 1789 of the original GluTl sequence ( Figure 5), denoted by arrow "B".
  • Figure 7 illustrates the 115392* nucleotide sequence (GenBank Accession No. L43578;
  • Figure 8 illustrates the specificity of RB interactions. Shown is the matrix of results of the yeast two hybrid system assays. The results of assays using the bait proteins RB and Bl are indicated to the left of the rows, and the prey proteins RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, 115392*, RbAP46 and PI are indicated above the columns. A positive interaction between the indicated bait and prey proteins is indicated as "+" in the box forming the intersection between the particular bait and prey proteins; a lack of interaction is designated by an empty box.
  • Boxes labeled A through P indicate the results of matings and growth of yeast expressing RB and RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, 115392*, respectively.
  • the box labeled Q indicates the mating and growth of yeast expressing bait protein RB and protein Rb AP46, recapitulating a previously known interaction. See e.g., Hwang, et al, 1991. Nature 350:160-162.
  • the present invention is based upon the identification of proteins which possess the ability to interact with RB (hereinafter referred to as "RB-IPs") using an improved, modified form of the yeast two hybrid system.
  • RB-IPs proteins which possess the ability to interact with RB
  • RN-tre cellular apoptosis susceptibility protein CAS, gamma-interferon inducible protein IP-30, ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl(Nek 2), cyclophilin A, Zap 3, oxidoreductase 17-beta-hydroxysteroid (17 beta-HSD6), CD24*, Set*, glutamate transporter GluTl* or clone 115392* (wherein "*” indicates splice variants of Set and GluTl, and novel open reading frames (ORFs) of CD24 and 115392* as determined in the Specific Examples Section, infra, respectively)
  • RB RB- IP complexes.
  • These RB»RB-IP complexes are implicated in the modulation of various functional activities of RB, and of its binding partners. These functional activities include, but are not limited to: (i) physiological processes (e.g., cell cycle control, cellular differentiation and apoptosis); (ii) response to viral infection; (Hi) intracellular signal transduction; (iv) transcriptional control; and (v) pathophysiological processes (e.g., hyperproliferative disorders including tumorigenesis and tumor spread, degenerative disorders including neurodegenerative diseases, virus infection).
  • physiological processes e.g., cell cycle control, cellular differentiation and apoptosis
  • response to viral infection e.g., viral infection
  • Hi intracellular signal transduction
  • iv transcriptional control
  • pathophysiological processes e.g., hyperproliferative disorders including tumorigenesis and tumor spread, degenerative disorders including neurodegenerative diseases, virus infection.
  • the present invention discloses methodologies for the screening of proteins which interact with (e.g., bind to) RB.
  • the present invention further relates to RB complexes (RB*RB- IPs), in particular RB complexed with one of the following proteins: RN-tre, cellular apoptosis susceptibility protein CAS, gamma-interferon inducible protein IP-30, ribosomal protein L6, cytokine IK factor, lactate dehydrogenase B (LDH-B), Nlkl (Nek 2), cyclophilin A, Zap 3, oxidoreductase 17-beta-hydroxysteroid (17 beta-HSD6), CD24*, Set*, glutamate transporter GluTl * or clone 115392*.
  • RN-tre cellular apoptosis susceptibility protein CAS
  • gamma-interferon inducible protein IP-30 ribosomal protein L6, cytokine IK factor, lactate de
  • the present invention further discloses complexes of RB or derivatives, analogs and fragments of RB, with RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*, or derivatives, analogs and fragments thereof.
  • complexes bind an anti-RB»RB-IP complex antibody.
  • complexes of human RB with human proteins are disclosed.
  • the present invention also discloses methodologies for the production and/or isolation of RB»RB-IP complexes.
  • the present invention provides methods of using recombinant DNA techniques to express both RB and its binding partner (RB-IP), or fragments, derivatives or homologs of one or both members of the complex, either where both binding partners are under the control of one heterologous promoter (i. e. , a promoter not naturally associated with the native gene encoding the particular complex component) or where each is under the control of a separate heterologous promoter.
  • the present invention discloses novel proteins encoded by novel splice variants of the Set and GluTl genes, which encode proteins designated Set* and GluTl*, that also possess the ability to interact with RB.
  • the nucleotide and associated amino acid sequences of Set* and GluTl* are depicted in Figures 3-4 and 5-6, respectively [SEQ ID NOS:6-8 and 9-11, respectively].
  • the present invention also discloses a protein which interacts with RB, and is encoded by an ORF found within the CD24 gene which does not overlap with the nucleotide sequence encoding the CD24 protein.
  • the nucleotide and associated amino acid sequences of CD24* are depicted in Figure 2 [SEQ ID NOS:4 and 5, respectively]. Additionally, the present invention discloses a protein encoded by a nucleotide sequence within the EST sequence 115392 (the protein is hereinafter designated 115392*) which interacts with RB. The nucleotide sequence of EST sequence 115393* and its associated amino acid sequence are depicted in Figure 6 [SEQ ID NOS : 12 and 13 , respectively] .
  • the present invention discloses the nucleotide sequences of Set* and GluTl * and their encoded proteins, as well as the mRNA nucleotide sequence (including the DNA sequence corresponding to the mRNA sequence) and associated amino acid sequence of CD24*.
  • the present invention further discloses CD24*, Set*, GluTl*, and 115392* proteins, and derivatives (including but not limited to fragments) and homologs and paralogs thereof, as well as nucleic acids encoding the CD24*, Set*, GluTl*, and 115392* proteins, derivatives, fragments and homologs.
  • the present invention also provides CD24* protein, and Set*, GluTl*, and 115392* proteins and genes encoding these proteins, of many different species, preferably vertebrates and more preferably mammals.
  • the CD24* protein, and the Set*, GluTl*, and 115392* proteins, and genes are of human origin.
  • the production of the aforementioned proteins and derivatives are also disclosed (e.g., by recombinant methodologies).
  • the present invention further relates to CD24*, Set*, GluTl*, and 115392* derivatives and analogs that possess functional biological activity (i.e., are capable of displaying one or more known functional activities associated with a full-length, wild-type CD24*, Set*, GluTl*, or 115392*)
  • Such functional biological include, but are not limited to: (i) the ability to form a complex with RB; (ii) antigenicity (i.e., the ability to bind, or compete with CD24*, Set*, GluTl*, or 115392* for binding, to an anti-CD24*, anti-Set*, anti-GluTl*, or anti-115392* antibody, respectively); (Hi) immunogenicity (i.e., the ability to generate an antibody that binds to CD24*, Set*, GluTl*, or 115392*, respectively) and the like.
  • the present invention provides methods of treating or preventing diseases or disorders associated with aberrant levels of RE RB-IP complexes or CD24*, Set*, GluTl*, or 115392*, or aberrant levels of the activity of one or more of the components of the complex by administration of the RB*RB-IP complexes, CD24*, Set*, GluTl*, or 115392*, or modulators of RB*RB-IP complex formation or activity, mutants of RB or the RB-IP which increase or decrease binding affinity, small molecule inhibitors/enhancers of complex formation, antibodies that either stabilize or neutralize the complex, and the like.
  • RB «IP Complexes and CD24*. Set*. GluTl*. and 115392* Proteins. Derivatives and Analogs
  • the present invention discloses RB»RB-IP complexes and, in particular aspects, complexes of RB and RN-tre, RB and CAS, RB and IP-30, RB and L6, RB and IK, RB and LDH-B, RB and Nlkl (Nek 2), RB and cyclophilin A, RB and Zap 3, RB and 17 beta-HSD6, RB and CD24*, RB and Set*, RB and GluTl*, and RB and 115392*.
  • the RB*RB-IP complexes are complexes of human proteins.
  • the present invention also discloses complexes of derivatives (including fragments) and analogs of RB with an RB-IP, complexes of RB with derivatives (including fragments) and analogs of an RB-IP, and complexes of derivatives (including fragments) and analogs of RB and derivatives (including fragments) and analogs of an RB-IP.
  • fragments, derivatives or analogs of an RB «RB-IP complex include complexes where one or both members of the complex are fragments, derivatives or analogs of the wild-type RB or RB-IP protein.
  • the RB «RB- IP complexes in which one or both members of the complex are a fragment, derivative or analog of the wild type protein are functional biologically active RB*RB-IP complexes.
  • the native proteins, derivatives or analogs of RB and/or the RB-IP are derived from animals or plant sources.
  • physiological processes e.g., cell cycle control, cellular differentiation and a
  • the present invention provides methods for the screening of RB*RB-IP complexes, particularly complexes of RB with RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392*, and the CD24*, Set*, GluTl*, and 115392* proteins, as well as derivatives and analogs of the RB»RB- IP complexes, and CD24*, Set*, GluTl*, and 115392* proteins, for the ability to alter cell functions, particularly those cell functions in which RB and or an RB-IP has been implicated, such as but not limited to: (i) physiological processes (e.g., cell cycle control, cellular differentiation and apoptosis); (ii) response to viral infection; (Hi) intracellular signal transduction; (iv) transcriptional control; and (v) pathophysiological processes (
  • such derivatives or analogs which possess the desired immunogenicity or antigenicity may be used in immunoassays, for immunization, for inhibition of RB»RB-IP complex activity, and the like.
  • Derivatives or analogs which retain or enhance, or alternatively lack or inhibit, a specific property of interest e.g., participation in an RB*RB-IP complex
  • An other specific embodiment of the present invention relates to an RB*RB-IP complex of a fragment of an RB and/or a fragment of RB-IP which may be bound by an anti-RB and/or anti -RB-IP antibody or antibody specific for an RB*RB-IP complex when such a fragment is included within a given RB-RB-IP complex. Fragments and other derivatives or analogs of RB*RB-IP complexes can be tested for the desired activity by procedures well-known within the art, including, but not limited to those assays described in Section (VI), infra.
  • the present invention provides RB»RB-IP complexes comprising fragments of one or both members of the complex.
  • these fragments consist of, but are not limited to, fragments of RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392*, identified as interacting with RB in the improved, modified yeast two hybrid assay.
  • fragments include, but are not limited to: amino acid residues 758-827 of RN-tre; amino acid residues 714-971 of CAS; amino acid residues 142-303 and 146-303 of IP-30; amino acid residues 64-288 of L6; amino acid residues 1-162 of IK; amino acid residues 265-334 of LDH-B; amino acid residues 319-445 of Nlkl(Nek 2); amino acid residues 1-165 of cyclophilin A; amino acid residues 296-331 of Zap 3; amino acid residues 1-317 of 17 beta-HSD6; amino acids 1-64 of CD24* as depicted in Figure 2 [SEQ ID NO:6]; amino acid residues 105-116 of Set* as depicted in Figure 4 [SEQ ID NO:8]; amino acid residues 297-316 of GluTl* as depicted in Figure 6 [SEQ ID NO:l 1] and amino acid residues 33-158 of 115392* as depicted in Figure 7 [SEQ ID NO
  • fragments, or proteins comprising fragments, which lack some or all of the aforementioned amino acid residues of either member of the RB»RB-IP complex assay, are also provided.
  • nucleic acids which encode the foregoing are also disclosed herein.
  • the present invention further relates to CD24*, Set*, GluTl *, and 115392* proteins as well as derivatives and homologs and paralogs of CD24*, Set*, GluTl*, and 115392* proteins.
  • human CD24*, Set*, GluTl*, and 115392* genes and proteins are disclosed.
  • the native proteins, fragments, derivatives or analogs of CD24*, Set*, GluTl*, or 115392* are derived from animals (e.g., mouse, rat, pig, cow, dog, monkey, human, fly, or frog) or from plants.
  • the fragment, derivative or analog is possesses functional biological activity (i.e., is capable of exhibiting one or more functional activities associated with full-length, wild-type CD24*, Set*, GluTl*, or 115392*, for example, the ability to bind RB, immunogenicity or antigenicity).
  • nucleotide sequences encoding human RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24, Set, GluTl, and clone 115392 are known (GenBank Accession No. M28419; GenBank Accession No. D13644; GenBank Accession No. U33286; GenBank Accession No. J03909; GenBank Accession No. X69391 ; GenBank Accession No. S74221; GenBank Accession No. Y00711; GenBank Accession No. Ul 1050; GenBank Accession No. Y00052; GenBank Accession No.
  • nucleotide sequences encoding CD24, Set, GluTl, and clone 115392 are provided in Figures 1, 3, 5, and 7 (SEQ ID NOS: 1, 6, 9, and 12), respectively.
  • nucleic acids encoding RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta- HSD6, CD24*, Set*, GluTl*, or clone 115392* may be readily obtained by any method known within the art (e.g., by PCR amplification using synthetic primers hybridizable to the 3'- and 5'- termini of the sequence and/or by cloning from a cDNA or genomic library using an oligonucleotide specific for the gene sequence. These aforementioned methodologies are fully described in Section (II), infra. Homologs (i.e., those nucleic acids encoding RB, RN-tre, CAS, IP-30, L6, IK, LDH-B,
  • Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392* of species other than human) or other related sequences (e.g., paralogs) may be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well-known within the art for nucleic acid hybridization and cloning (e.g., as described in Section (II) for CD24*, Set*, GluTl *, and 115392* sequences).
  • the RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392* proteins either alone or in a complex may be obtained by methods well-known within the art for protein purification and recombinant protein expression.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein may be inserted into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence).
  • an appropriate expression vector i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence.
  • the necessary transcriptional and translational signals may also be supplied by the native promoter for RB or any RB-IP genes, and/or their flanking regions.
  • host- vector systems may be utilized to express the protein coding sequence.
  • host vector systems include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • the expression elements of vectors vary in their overall strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be utilized.
  • the RB «RB-IP complexes are obtained by expressing the entire RB sequence and an RB-IP coding sequence in the same cell, either under the control of the same promoter or two separate promoters.
  • a derivative, fragment or homolog of RB and/or a derivative, fragment or homolog of an RB-IP are recombinantly expressed.
  • the derivative, fragment or homolog of RB and/or the RB-IP protein forms a complex with a binding partner identified by a binding assay, such as the modified yeast two hybrid system, and more preferably forms a complex that binds to an anti-RB «RB-IP complex antibody.
  • any of the methods described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleotide sequences encoding RB and an RB-IP, or derivatives, fragments or homologs thereof, may also be regulated by a second nucleotide sequence so that the gene(s) or fragment(s) thereof are expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins may be controlled by any promoter/enhancer known within the art.
  • the promoter which is utilized is not native (i.e., is not the wild-type promoter) to the genes for RB or the specific RB-IP.
  • Promoters which may be utilized include, but are not limited to: (i) the SV40 early promoter (see e.g., Bernoist & Chambon, 1981. Nature 290:304-310): (//) the promoter contained in the 3'- long terminal repeat of Rous sarcoma virus (see e.g., Yamamoto, et al, 1980. Cell 22:787-797); (Hi) the Herpesvirus thymidine kinase promoter (see e.g., Wagner, et al, 1981. Proc. Natl.
  • transcriptional control regions which exhibit tissue specificity and have been utilized in transgenic animals may also be utilized. These transcriptional control regions include, but are not limited to: ( ) the elastase I gene control region which is active in pancreatic acinar cells (see e.g., Swift, et al, 1984. Cell 38:639-646; (ii) the insulin gene control region which is active in pancreatic ⁇ -cells (see e.g., Hanahan, et al, 1985. Nature 315:115-122): (Hi) the immunoglobulin gene control region which is active in lymphoid cells (see e.g., Alexander, et al, 1987. Mol. Cell Biol.
  • mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (see e.g., Leder, et al, 1986. Cell 45:485-495);
  • ⁇ -fetoprotein gene control region which is active in liver (see e.g., Krumlauf, et al, 1985. Mol. Cell. Biol. 5:1639-1648);
  • ⁇ -globin gene control region which is active in myeloid cells (see e.g., Kollias, et al, 1986.
  • a vector which comprises:
  • a promoter operably-linked to nucleotide sequences encoding RB and/or an RB-IP e.g., RN- tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl *, or 115392*
  • a vector is utilized which comprises a promoter operably-linked to nucleotide sequences encoding both RB and an RB-IP, one or more origins of replication, and optionally, one or more selectable markers.
  • an expression vector containing the coding sequences, or portions thereof, of RB and an RB-IP (e.g., RN-tre, CAS, IP-30, L6, EK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392*), either together or separately, is constructed, by way of example but not of limitation, by initially sub-cloning the gene sequences into the EcoRI restriction site of each of the three pGEX vectors (glutathione S-transferase expression vectors; Promega Corp.; Madison, WI.; see also Johnson, 1988. Gene 7:31-40).
  • This aforementioned cloning system allows for the expression of selected gene products in the correct reading frame.
  • Expression vectors containing the sequences of interest may be identified by three general methodologies: (i) by nucleic acid hybridization; (ii) by the presence or absence of "marker” gene function and/or (c) by the expression of the inserted sequences.
  • RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*, or other RB-IP sequences may be detected by nucleic acid hybridization to probes comprising sequences homologous and complementary to the inserted sequences.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" functions (e.g., binding to an anti-RB, anti-RB-IP, or anti-RB*RB-IP complex antibody, resistance to antibiotics, occlusion body formation in baculovirus, and the like) caused by insertion of the sequences of interest in the vector.
  • certain "marker” functions e.g., binding to an anti-RB, anti-RB-IP, or anti-RB*RB-IP complex antibody, resistance to antibiotics, occlusion body formation in baculovirus, and the like
  • certain "marker” functions e.g., binding to an anti-RB, anti-RB-IP, or anti-RB*RB-IP complex antibody, resistance to antibiotics, occlusion body formation in baculovirus, and the like
  • recombinant expression vectors can be identified by assaying for RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*, or other RB-IP products expressed by the recombinant vector.
  • assays can be based, for example, on the physical or functional properties of the interacting species in in vitro assay systems (e.g., formation of a RB»RB-IP complex or immunoreactivity to antibodies specific for the protein).
  • Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*, or other RB-IP molecules are identified and the complexes or individual proteins are isolated, various methods well-known within the art may be used to propagate them. Moreover, when a suitable host system and growth conditions have been established, recombinant expression vectors may then be propagated and amplified in quantity.
  • the expression vectors or derivatives which can be used include, but are not limited to: human or animal viruses; insect viruses; yeast vectors; bacteriophage vectors and plasmid and cosmid vectors.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies or processes the expressed proteins in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically-engineered RB and/or RB-IP may be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification (e.g., glycosylation, phosphorylation, etc.) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein is achieved.
  • expression in a bacterial system can be used to produce an non-glycosylated core protein, while expression in mammalian cells ensures "native" glycosylation of a heterologous protein.
  • different vector/host expression systems may effect processing reactions to different extents.
  • the RB and/or RB-IPs, or fragments, homologs or derivatives thereof may be expressed as fusion or chimeric protein products comprising the protein, fragment, homolog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein.
  • Such chimeric products can be made by ligating the appropriate nucleic acids encoding the desired amino acids to each other in the proper coding frame by methods known in the art, and expressing the chimeric products in a suitable host by methods commonly known in the art.
  • a chimeric product can be made by protein synthetic techniques (e.g., by use of a peptide synthesizer).
  • Chimeric genes comprising portions of RB and/or an RB-IP, or CD24*, Set*, GluTl *, or 115392*, fused to any heterologous protein- encoding sequences may be constructed.
  • a specific embodiment of the present invention discloses a chimeric protein comprising a fragment of RB and/or an RB-IP, or CD24*, Set*, GluTl*, or 115392*, of at least six amino acid residues in length.
  • fusion proteins which possess the interacting domains of the RB protein and an RB-IP (e.g., RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*) and/or, optionally, a hetero-functional reagent (e.g., a peptide linker between the two domains) where such a reagent promotes the interaction of the respective RB and RB-IP binding domains.
  • a hetero-functional reagent e.g., a peptide linker between the two domains
  • fusion proteins may be particularly useful where the stability of the interaction is desirable due to the formation of the complex as an intra-molecular reaction (e.g., in the production of antibodies specific to the RB «RB-IP complex). More specifically, RB and/or derivatives of an RB-IP, or CD24*, Set*, GluTl *, or
  • 115392* may be generated by altering their respective sequences by substitutions, additions and/or deletions which provide for functionally-equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as an RB or RB-IP gene can be used in the practice of the present invention.
  • nucleotide sequences comprising all or portions of RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl *, and 115392* genes that are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a "silent" alteration.
  • RB and RB-IP derivatives of the present invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of RB or an RB-IP, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • nucleic acids encoding proteins, and proteins consisting of, or comprising a fragment of RB or an RB-IP comprised of at least 6 (contiguous) amino acid residues of RB or an RB-IP are disclosed.
  • the aforementioned amino acid fragments are comprised of at least 10, 20, 30, 40, 50, 100 or 200 amino acids of RB or an RB-IP.
  • Derivatives or analogs of RB and RB-IPs include, but are not limited to, molecules comprising regions that are substantially homologous to RB or RB-IPs, in various embodiments, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known within the art, or whose encoding nucleic acid is capable of hybridizing to the complement (e.g., the inverse complement) of a sequence encoding RB or an RB-IP under stringent, moderately stringent, or non-stringent conditions, as described supra, Section (I).
  • complement e.g., the inverse complement
  • the RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392* derivatives and analogs of the present invention may be produced by various methods known within the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned RB and RB-IP gene sequences can be modified by any of numerous strategies known in the art. See e.g., Sambrook, et al, 1989. Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  • sequences of interest may be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. It should be noted, however, that in the production of the gene encoding a derivative or analog of RB or an RB-IP, care should be taken to ensure that the modified gene retains the original translational reading frame, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.
  • the RB and/or RB-IP-encoding nucleotide sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis and in vitro site-directed mutagenesis (see e.g., Hutchinson, et al, 1978. J. Biol. Chem. 253:6551-6558), the use of TABTM linkers (Pharmacia, Upsala, Sweden), and the like.
  • the individual gene product or complex can be isolated and analyzed. This is achieved by assays based on the physical and/or functional properties of the protein or complex, including, but not limited to, radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, cross-linking to marker- labeled product, and the like.
  • the RB-RB-IP complexes, and CD24*, Set*, GluTl*, and 115392* proteins may be isolated and purified by standard methodologies known within the art (either from natural sources or recombinant host cells expressing the complexes or proteins) including, but not limited to: ( ) column chromatography (e.g., ion exchange, affinity, gel exclusion, reversed-phase high pressure, fast protein liquid, etc.); (ii) differential centrifugation; (Hi) differential solubility or (iv) by any other standard technique used for the purification of proteins. Functional properties may be evaluated using any suitable assay known in the art.
  • the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene from which it was encoded.
  • the protein (or its derivative) may be synthesized by standard chemical methods known within the art. See, e.g., Hunkapiller, et al, 1984. Nature 310:105-111.
  • such RB»RB-IP complexes, as well as CD24*, Set*, GluTl*, and 115392* proteins include, but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequences, as well as fragments and other analogs and derivatives thereof, including proteins homologous thereto.
  • RB and/or RB-IP sequences may be made at the protein level. Included within the scope of the present invention are derivatives or complexes of RB or RB-IP fragments, derivatives or analogs of CD24*, Set*, GluTl*, and 115392*, and RB, RB-IP, CD24*, Set*, GluTl*, and 115392* fragments, derivatives and analogs that are differentially modified during or after translation (e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like).
  • any of numerous chemical modifications may also be performed by well-known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 , acetylation, formylation, oxidation, reduction, synthesis in the presence of tunicamycin, and the like.
  • the RB and/or RB-IP sequences are modified to include a fluorescent label.
  • the RB and/or the RB- IP are modified so as to possess a heterofunctional reagent which may be utilized to cross-link the protein to other members of the complex or to other RP-IPs.
  • analogs and derivatives of RB and/or an RB-IP, or analogs and derivatives of CD24*, Set*, GluTl*, or 115392* can be chemically synthesized by use of a peptide synthesizer.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the RB and/or an RB-IP.
  • the amino acid sequence of RB, or an RB-IP isolated from the natural source, as well as those expressed in vitro, or from synthesized expression vectors in vivo or in vitro can be determined from analysis of the DNA sequence, or alternatively, by direct sequencing of the isolated protein. Such analysis may be performed by manual sequencing or through use of an automated amino acid sequenator.
  • the RB»RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* proteins, may also be analyzed by hydrophilicity analysis. See e.g., Hopp and Woods, 1981. Proc. Natl. Acad. Sci. USA 78: 3824-3828.
  • Secondary structural analysis may also be performed to facilitate the identification of specific regions of the RB and or an RB-IP that assume specific structural motifs. See e.g., Chou and Fasman, 1974. Biochemistry 1_3: 222-23.
  • Computer software programs may also be utilized in various analyses including, but not limited to: manipulation, translation, secondary structure prediction, hydrophilicity and hydrophobicity profiles, open reading frame prediction and plotting, and determination of sequence homologies.
  • Other methods of structural analysis including, but not limited to: X-ray crystallography (see e.g., Engstrom, 1974. Biochem. Exp. Biol. H:7-13); mass spectroscopy and gas chromatography (see e.g., Methods in Protein Science, 1997. J.
  • CD24*, Set*, GluTl*, AND 115392* discloses the nucleotide sequences encoding CD24*, Set*, GluTl*, and 115392*.
  • the CD24*, Set*, GluTl *, and 115392* nucleic acids comprise the sequences set forth in SEQ ID NOS:4, 7, 10, and 12, respectively, or portions thereof, or nucleotide sequences encoding, in whole or in part, a CD24*, Set*, GluTl*, or 115392* protein (e.g., a protein comprising the amino acid sequence of SEQ ID NOS:5, 8, 11 or 13, respectively, or portions thereof).
  • the present invention also discloses purified nucleic acids comprised of at least six (6) nucleotides (i.e., a hybridizable portion) of a CD24*, Set*, GluTl *, or 115392* nucleic acid sequence.
  • the nucleic acids consisting of at least six (6) nucleotides of a Set* or GluTl * nucleotide sequence are comprised of those sequences which span the splice junctions specific to Set* and GluTl*, respectively (i.e., the portion of the Set* or GlutTl * nucleotide sequence containing the sequence at which the 5'- and 3'-splice sites used in processing Set* and GluTl * mRNA but not the Set mRNA or GluTl mRNA were joined).
  • nucleic acid sequence consisting of nucleotides both 5'- and 3'-of the splice site at nucleotide numbers 212- 213 of the Set* nucleotide sequence as depicted in Figure 4 [SEQ ID NO: 7] or nucleotide numbers 860-861 of the GluTl * nucleotide sequence as depicted in Figure 6 [SEQ ID NO: 10].
  • the nucleic acids consist of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a CD24*, Set*, GluTl*, or 115392* sequence, or a full-length CD24*, Set*, GluTl *, or 115392* coding sequence.
  • the nucleic acid sequences are smaller than 35, 200 or 500 nucleotides in length. The nucleic acid sequences may be either single or double-stranded.
  • the invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences, in particular the invention provides the inverse complement to nucleic acids hybridizable to the foregoing sequences (i.e., the inverse complement of a nucleic acid strand has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand; thus, for example, where the coding strand is hybridizable to a nucleic acid with no mismatches between the coding strand and the hybridizable strand, then the inverse complement of the hybridizable strand is identical to the coding strand).
  • nucleic acids which comprise a sequence complementary to (specifically are the inverse complement of) at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a CD24*, Set*, GluTl*, or 115392* gene.
  • a nucleic acid which is hybridizable to a CD24*, Set*, GluTl *, or 115392* nucleic acid e.g., having sequence SEQ ID NOS:4, 7, 10, and 12 respectively
  • a nucleic acid encoding a CD24*, Set*, GluTl *, or 115392* derivative, (or the complement of the foregoing) under conditions of low stringency is provided.
  • procedures using such conditions of low stringency are as follows. See e.g., Shilo and Weinberg, 1981. Proc. Natl. Acad. Sci. USA 78:6789-6792.
  • Filters containing DNA are pre-hybridized for 6 hours at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 x 10 6 cpm 32 P-labeled probe.
  • Filters are incubated in hybridization mixture for 18-20 hours at 40°C, and then washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1 % SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60°C. Filters are blotted dry and exposed to X-ray film for autoradiography. If necessary, filters are washed for a third time at 65-68°C and re-exposed to X-ray film. Other conditions of low stringency which may be used are well known in the art.
  • a nucleic acid which is hybridizable to a CD24*, Set*,
  • GluTl *, or 115392* nucleic acid or a nucleic acid encoding a CD24*, Set*, GluTl* or 115392* derivative (or a complement of the foregoing) under conditions of moderate stringency is provided.
  • procedures using such conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 hours at 55°C in a solution containing 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with 5-20 x 10 6 cpm 32 P-labeled probe.
  • Filters are incubated in hybridization mixture for 18-20 hours at 55°C, and then washed twice for 30 minutes at 60°C in a solution containing IX SSC and 0.1% SDS. Filters are blotted dry and exposed to X-ray film for autoradiography. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.1% SDS. Other conditions of moderate stringency which may be used are well-known in the art.
  • nucleic acid which is hybridizable to a CD24*, Set*, GluTl * or 115392* nucleic acid (or a complement of the foregoing) or to a nucleic acid encoding a CD24*, Set* GluTl* or 115392* derivative under conditions of high stringency is provided.
  • pre-hybridization of filters containing DNA is carried out for 8 hours to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C in pre-hybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 x 10 6 cpm of 32 P-labeled probe.
  • nucleic acid encoding a fragment or portion of CD24*, Set*, GluTl* or 115392* shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of CD24*, Set*, GluTl*, or 115392* protein, and not the other contiguous portions of the CD24*, Set*, GluTl*, or 115392* as a continuous sequence.
  • Sequences within the 3 '-non-translated regions of the CD24, Set, and GluTl mRNAs were identified as encoding a protein or proteins that interact with RB using an improved, modified version of the yeast two hybrid system.
  • the present inventors have identified interacting proteins encoded by nucleotide sequences identical to the portion of the nucleotide sequence of CD24 that is 3'- to base 689, of the nucleotide sequence of Set that is 3'- to base 2076, and of the nucleotide sequence of GluTl that is 3' to base 3408.
  • potential open reading frames can be identified using the N.C.B.I. BLAST program "ORF Finder" available to the public. Because all known protein translation products are at least 60 amino acids or longer (see e.g., Creighton, 1992. Proteins, 2 nd Ed., W.H. Freeman and Co., New York), only those ORFs potentially encoding a protein of 60 amino acids or more are considered. Furthermore, if an initiation methionine codon (ATG) and a translational stop codon (TGA, TAA, or TGA) are identified, then the boundaries of the protein are defined. Other potential proteins include any open reading frames that extend to the 5 '-terminus of the nucleotide sequence, in which case the open reading frame predicts the
  • nucleotide sequence encoding the portions of the CD24*, Set* and GluTl* identified as interacting with RB are 3' to the translational stop codon of the nucleotide sequence encoding CD24, Set and GluTl, respectively, indicates that CD24*, Set* and GluTl* could be encoded by mRNAs resulting from splicing of unprocessed CD24, Set and GluTl gene transcripts at splice sites other than the splice sites used in processing the known CD24, Set and GluTl mRNAs.
  • the Set* and GluTl * sequences were determined by identifying alternate 5' and 3'-splice sites in the Set and GluTl sequence. Alternate splice sites within the CD24 sequence were not detected.
  • Determination of 5' and 3' splice points for protein splice variants can be performed by any method known within the art.
  • the 5' and 3' splice points may be ascertained by the following methodology:
  • Potential 5'-splice sites can be identified in the coding sequence of the known protein (e.g., Set and GluTl).
  • the sequence of 5'-splice sites has an invariant GT sequence at the start of the intron, and the remaining bases are not invariant, but the preferred consensus sequence is AG:GTAAGT, with the colon indicating the splice point (see Padgett, et ⁇ l., 1984. Ann. Rev. Biochem. 55:1119-1150).
  • Potential splice sites can be identified in order of the number of residues matching this consensus sequence, requiring at a minimum, the invariant GT and 4/6 matches in the other consensus bases.
  • the nucleotides which are 5 to 14 nucleotides 5'- of the last intronic G base can contain at most two non-T, non-C bases.
  • the sequence between potential 5'-splice sites and the start of the nucleotide sequence encoding the detected interacting protein region can be scanned for the invariant AG: sequence, where the base preceding the invariant region must be a C or T. Potential sites are then examined to determine if they meet the requirement of at least 8 of 10 C or T bases in positions -5 to -14 from the splice site.
  • a splice variant is named after the known protein, followed by a *, and if more than one splice variant for a given gene is identified, the name is followed by a * and number.
  • New splice variant proteins must encode at least 60 amino acid residues to constitute a viable in vivo product. Further, the 3 '-terminus of slice variants must, by definition, extend into the identified interacting sequence.
  • a 5 '-splice site was identified at nucleotides 214-221 of the Set nucleotide sequence (indicated by "B” on the Set nucleotide sequence in Figure 3, with the last base of the first exon being nucleotide number 215, as indicated by the arrow in callout "B” in Figure 3, and a 3'-splice site was identified at nucleotides 2061 to 2074 of the Set nucleotide sequence (indicated by callout "E” on the Set nucleotide sequence in Figure 3, with the first base of the second exon being nucleotide number 2074, as indicated by the arrow in Figure 3, callout at "E”).
  • the translation stop codon of Set* was identified as nucleotides 2211 to 2213 of the Set nucleotide sequence indicated as callout "E" in Figure 3.
  • the branch point consensus region for Set* splicing was identified at nucleotides 2053 to 2060 of the Set nucleotide sequence as depicted in Figure 3
  • GluTl * a 5'-splice site was identified at nucleotides 1037-1044 of the GluTl sequence, as depicted in Figure 5 [SEQ ID NO:9] and as indicated by callout "B", with the last base of the first exon being nucleotide number 1038, as indicated by the arrow in Figure 5
  • PCR polymerase chain reaction
  • Oligonucleotide primers that hybridize to sequences at the 3'- and 5 '-termini of the identified sequences can be used as primers to amplify by PCR sequences from a nucleic acid sample (cDNA or DNA), preferably a cDNA library, from an appropriate source (e.g., the sample from which the initial cDNA library for the improved, modified yeast two hybrid assay fusion population was derived).
  • a nucleic acid sample preferably a cDNA library
  • an appropriate source e.g., the sample from which the initial cDNA library for the improved, modified yeast two hybrid assay fusion population was derived.
  • PCR can be carried out, for example, by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp TM ).
  • the DNA being amplified can include genomic DNA or cDNA sequences from any eukaryotic species.
  • nucleic acid homologs e.g., to obtain CD24*, Set*, GluTl*, or 115392* sequences from species other than humans, or to obtain human sequences with homology to CD24*, Set*, GluTl*, or 115392*
  • amplify nucleic acid homologs e.g., to obtain CD24*, Set*, GluTl*, or 115392* sequences from species other than humans, or to obtain human sequences with homology to CD24*, Set*, GluTl*, or 115392*
  • low stringency conditions are preferred.
  • moderately stringent conditions are preferred.
  • nucleic acid containing all or a portion of the CD24*, Set*, GluTl*, or 115392* sequence that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone.
  • This 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.
  • nucleotide sequences of the entire CD24*, Set*, GluTl * or 115392* genes, as well as additional genes encoding CD24*, Set*, GluTl*, or 115392* proteins and analogs may be identified.
  • Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the CD24*, Set*, GluTl*, or 115392* gene.
  • the nucleic acids can be isolated from vertebrates, including: mammalian, human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, plants, and the like.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see e.g., Sambrook, et al, 1989. Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985. DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • the clones derived from such genomic DNA may contain regulatory and intronic 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. Alternatively, one may use DNase in the presence of manganese to fragment the DNA, or the DNA can be physically sheared (e.g., by sonication).
  • the linear DNA fragments can then be separated according to size by standard techniques including, but not limited to, agarose and/or polyacrylamide gel electrophoresis, and column chromatography. Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways.
  • a portion of the CD24*, Set*, GluTl *, or 115392* (of any species) gene e.g., a PCR amplification product obtained as described above, or an oligonucleotide having a sequence of a portion of the known nucleotide sequence
  • its specific RNA, or a fragment thereof may be purified and labeled, and the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (see e.g., Benton and Davis, 1977. Science 196:180-182; Grunstein and Hogness, 1975. Proc. Natl. Acad. Sci. U.S.A. 72:3961-3964).
  • DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map if such is available, or by DNA sequence analysis and comparison to the known nucleotide sequence of CD24*, Set*, GluTl *, or 115392*. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immuno logical properties of its expressed product.
  • cDNA clones or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, for example, possesses similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, or antigenic properties or ability to bind RB, as is known for CD24*, Set*, GluTl*, or 115392*.
  • the protein may be identified by binding of labeled antibody to the putatively CD24*, Set*, GluTl*, or 115392* synthesizing clones, in an enzyme-linked immunosorbent assay (ELISA)-type procedure.
  • ELISA enzyme-linked immunosorbent assay
  • An alternative to isolating the CD24*, Set*, GluTl * or 115392* cDNA includes, but is not limited to, chemically synthesizing the gene sequence itself from a known sequence. Other methods are possible and within the scope of the present invention.
  • the identified and isolated nucleic acids can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the pBlueScript vector (Stratagene, La Jolla, CA). Insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified to ensure compatibility.
  • any site desired may be produced by ligating nucleotide sequences (e.g., linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • the cleaved vector and the CD24*, Set*, GluTl*, or 115392* gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.
  • transformation of host cells with recombinant DNA molecules that incorporate the isolated CD24*, Set*, GluTl*, or 115392* gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene.
  • the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.
  • the CD24*, Set*, GluTl*, or 115392* sequences provided by the instant invention include those nucleotide sequences encoding substantially the same amino acid sequences as found in native CD24*, Set*, GluTl*, or 115392* proteins, and those encoded amino acid sequences with functionally equivalent amino acids, as well as those encoding other CD24*, Set*, GluTl*, or 115392* derivatives or analogs, as described in Section (I), supra, for CD24*, Set*, GluTl*, or 115392* derivatives and analogs
  • the RB»RB-IP complexes e.g., RB complexed with RN-tre, CAS, IP-30, L6, IK, cyclophilin A, Zap 3, Nlkl (Nek 2), 17 beta-HSD6, CD24*, Set*, GluTl*, or 115392*), or fragments, derivatives or homologs thereof, or CD24*, Set*, GluTl*, or 115392* proteins, and fragments, homologs and derivatives thereof, may be utilized as immunogens to generate antibodies which immunospecifically bind such immunogens.
  • Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, and single chain antibodies, F ab fragments, and F ab expression libraries.
  • antibodies to complexes of human RB and a human RB-IP are produced.
  • complexes formed from fragments of an RB and an RB-IP, where the fragments contain the protein domain that interacts with the other member of the complex are used as immunogens for antibody production.
  • CD24*, Set*, GluTl*, or 115392* proteins or fragments, derivatives, or homologs thereof are used as immunogens.
  • RB RB-IP complex
  • CD24* Set*, GluTl*, or 115392* protein
  • derivative, fragment or analog thereof various host animals can be immunized by injection with the native RB*RB-IP complex, or CD24*, Set*, GluTl*, or 115392* protein, or a synthetic version, or a derivative of the foregoing, such as a cross-linked RB»RB-IP.
  • host animals include, but are not limited to, rabbits, mice, rats, etc.
  • adjuvants can be used to increase the immunological response, depending on the host species, and include, but are not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin (BCG) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • Culture techniques include, but are not limited to: ( ) the hybridoma technique (see Kohler & Milstein, 1975. Nature 256:495-497); ( ) the trioma technique see Rosen, et al, 1977. Cell 11:139-147); (Hi) the human B-cell hybridoma technique (see Kozbor, et al, 1983.
  • monoclonal antibodies can be produced in germ-free animals utilizing a recently developed technology (see e.g., PCT Patent Publication U590/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (see e.g., Cole, et al, 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human B cells with EBV virus in vitro (see e.g., Cole, et al, 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • human hybridomas see e.g., Cole, et al, 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030
  • transforming human B cells with EBV virus in vitro see e.g., Cole, et al, 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96.
  • techniques developed for the production of "chimeric antibodies” are developed for the production of "chimeric antibodies"
  • techniques described for the production of single-chain antibodies may be adapted to produce RB»RB-IP complex-specific and CD24*-, Set*-, GluTl*-, and 115392*-specific single-chain antibodies.
  • An additional embodiment of the invention utilizes the techniques described for the construction of F ab expression libraries (see e.g., Huse, et al, 1989. Science 246:1275-1281) to allow rapid and easy identification of monoclonal F ab fragments with the desired specificity for the RB*RB-IP complexes, or individual CD24*, Set*, GluTl*, or 115392* proteins, derivatives or analogs thereof.
  • Non-human antibodies can be "humanized" by known methods (see e.g., U.S. Patent No. 5,225,539). Antibody fragments that contain the idiotypes of RB «RB-IP complexes or of CD24*,
  • Set*, GluTl*, or 115392* proteins can be generated by techniques known in the art.
  • fragments include, but are not limited to: the F (ab , )2 fragment which can be produced by pepsin digestion of the antibody molecule; the F ab , fragments that can be generated by reducing the disulfide bridges of the F (ab , )2 fragment; the F ab fragments that can be generated by treating the antibody molecular with papain and a reducing agent; and F v fragments.
  • screening for the desired antibody can be accomplished by various techniques known within the art (enzyme-linked immunosorbent assay (ELISA)).
  • ELISA enzyme-linked immunosorbent assay
  • To select antibodies specific to a particular domain of the RB»RB-IP complex, or CD24*, Set*, GluTl*, or 115392* protein one may assay generated hybridomas for a product that binds to the fragment of the RB*RB-IP complex, or CD24*, Set*, GluTl*, or 115392* protein, that contains such a domain.
  • antibodies are generated that immunospecifically recognize the portion of Set* or GluTl* having an amino acid sequence that differs from the Set or GluTl amino acid sequence and that do not immunospecifically recognize Set or GluTl.
  • antibodies specific to a domain of the RB*RB-IP complex are also disclosed, as are antibodies to specific domains of CD24*, Set*, GluTl*, and 115392* proteins.
  • anti-RB*RB-IP complex antibodies and fragments thereof, or anti-CD24*, anti-Set*, anti-GluTl*, and anti-115392*, or fragments thereof, containing the binding domain are utilized as Therapeutic moieties (see infra).
  • RB RB-IP complexes (particularly RB complexed with RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*), or CD24*, Set*, GluTl*, and 115392* proteins, may be markers of normal: (i) physiological processes (e.g., cell cycle control, cellular differentiation and apoptosis);
  • physiological processes e.g., cell cycle control, cellular differentiation and apoptosis
  • Detecting levels of RB*RB-IP complexes may be used in prognosis, to follow the course of disease states, to follow therapeutic response, and the like.
  • RB RB-IP complexes and the individual components of the RB «RB-IP complexes (e.g., RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, and 115392*), and derivatives, analogs and subsequences thereof, RB and/or RB-IP, and CD24*, Set*, GluTl*, and 115392* nucleic acids (and sequences complementary thereto), and anti-RB «RB-IP complex antibodies and antibodies directed against the individual components that can form RB»RB-IP complexes, and anti-CD24*, anti-Set*, anti- GluTl *, and anti-115392* antibodies, have uses in diagnostics.
  • RB RB-IP complexes and the individual components of the RB «RB-IP complexes
  • RB-IP complexes e
  • Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders, and treatment thereof, characterized by aberrant levels of RB «RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* proteins.
  • such an immunoassay is performed by use of a method comprising contacting a sample derived from a patient with an anti-RB «RB-IP complex antibody, or anti-CD24*, anti-Set*, anti-GluTl*, or anti-115392* antibody under conditions such that immunospecific binding can occur, and detecting or measuring the amount of any immunospecific binding by the antibody.
  • such binding of antibody, in tissue sections can be used to detect aberrant RB*RB-IP complex formation, or aberrant CD24*, Set*, GluTl*, or 115392* protein localization, or aberrant (e.g., high, low or absent) levels of RB*RB-IP complex or complexes, or CD24*, Set*, GluTl*, and 115392* proteins.
  • an antibody against an RB»RB-IP complex can be used to assay a patient tissue or serum sample for the presence of the RB*RB-IP complex, where an aberrant level of said complex is an indication of a diseased condition.
  • an antibody against CD24*, Set*, GluTl*, or 115392* can be used to assay a patient tissue or serum sample for the presence of CD24*, Set*, GluTl *, or 115392* where an aberrant level of CD24*, Set*, GluTl*, and 115392* is an indication of a diseased condition.
  • aberrant levels as utilized herein, is defined as meaning increased or decreased levels relative to that present, or a standard level representing that present, in an analogous sample from a portion of the body, or from a subject not having the specific disorder.
  • the immunoassays which may be utilized include, but are not limited to: competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, and other similar methodologies.
  • competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradio
  • Nucleic acids encoding the various protein constituents of the RB»RB-IP complexes and encoding the CD24*, Set*, GluTl*, and 115392* proteins, and related nucleotide sequences and subsequences, including complementary sequences, can also be used in hybridization assays.
  • the RB and/or RB-IP nucleotide sequences, or subsequences thereof, comprising about at least six (6) nucleotides, may be used as hybridization probes.
  • Hybridization assays may then be utilize to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant levels of the mRNAs encoding the constituent proteins of an RB «RB-IP complex, or CD24*, Set*, GluTl*, and 115392* protein, as described supra.
  • a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing to RB or an RB-IP DNA or RNA, under conditions such that hybridization can occur, and detecting or measuring any resulting hybridization.
  • the hybridization assay is carried out using nucleic acid probes capable of hybridizing to RB and to a binding partner (i.e., IP) of to measure concurrently the expression of both members of an RB»RB-IP complex.
  • a binding partner i.e., IP
  • the expression of mRNAs encoding CD24*, Set*, GluTl*, or 115392* is quantitatively ascertained.
  • diseases and disorders involving or characterized by aberrant levels of RB*RB-IP complexes may be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting aberrant levels of RB»RB-IP complexes, or non-complexed RB and/or an RB-IP proteins or nucleic acids or functional activity including, but not restricted to: (i) binding to an interacting partner or (ii) detecting mutations in RB and/or an RB-IP RNA, DNA or protein (e.g., translocations, truncations, changes in nucleotide or amino acid sequence relative to wild- type RB and/or RB-IP) that cause increased or decreased expression or activity of a RB •RB-IP complex and/or RB and/or protein that binds to RB.
  • Such diseases and disorders include, but are not limited to, those described in Section (V).
  • levels of RB»RB-IP complexes and the individual components of RB»RB-IP complexes may be detected by immunoassay; levels of RB and/or RB-IP RNA may be detected by hybridization assays (e.g., Northern blots, dot blots); binding of RB to an RB-IP may be measured by binding assays commonly known in the art, translocations and point mutations in RB and/or in genes encoding RB-IPs may be detected by Southern blotting, RFLP analysis, PCR using primers that preferably generate a fragment spanning at least most of the RB and/or RB-IP gene, sequencing of the RB and/or RB-IP genomic DNA or cDNA obtained from the patient, and the like.
  • Assays which are well-known within the art (e.g., immunoassays, nucleic acid hybridization assays, activity assays, etc.) may be used to determine whether one or more particular RB*RB-IP complexes are present at either increased or decreased levels, or are absent, in samples from patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder as compared to the levels in samples from subjects not having such a disease or disorder.
  • immunoassays e.g., immunoassays, nucleic acid hybridization assays, activity assays, etc.
  • these aforementioned assays may also be used to determine whether the ratio of the RB-RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, to the non-complexed components of the RB»RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, (i.e., RB and/or the specific RB-IP in the complex of interest, is increased or decreased in samples from patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, as compared to the ratio in samples from subjects not having such a disease or disorder).
  • levels of one or more particular RB»RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* are determined to be increased in patients suffering from a particular disease or disorder, or having a predisposition to develop such a disease or disorder, then the particular disease or disorder, or predisposition for a disease or disorder, can be diagnosed, have prognosis defined for, be screened for, or be monitored by detecting increased levels of the one or more RB «RB-IP complexes, , or CD24*, Set*, GluTl*, or 115392*, the mRNA that encodes the members of the one or more particular RB «RB-IP complexes, or CD24*, Set*, GluTl*, or 115392*, or RB-RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, functional activity.
  • diseases and disorders involving increased levels of one or more RB*RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* may be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting increased levels of the one or more RB-RB-IP complexes, , or CD24*, Set*, GluTl*, or 115392*, the mRNA encoding both members of the complex, or complex functional activity, or by detecting mutations in RB or the RB-IP (e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to the native, wild-type RB or RB-IP) which function to stabilize or increase RB*RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, formation.
  • mutations in RB or the RB-IP e.g., translocation
  • levels of one or more particular RB*RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* are determined to be decreased in patients suffering from a particular disease or disorder or having a predisposition to develop such a disease or disorder, then the particular disease or disorder or predisposition for a disease or disorder can be diagnosed, have its prognosis determined, be screened for, or be monitored by detecting decreased levels of the one or more RB-RB-IP complexes, or CD24*, Set*, GluTl*, or 115392*, the mRNA that encodes the members of the particular one or more RB*RB-IP complexes, or CD24*, Set*, GluTl*, or 115392*, or RB»RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, functional activity.
  • diseases and disorders involving decreased levels of one or more RB «RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be detected, by detecting decreased levels of the one or more RB»RB-IP complexes, , or CD24*, Set*, GluTl*, or 115392*,the mRNA encoding the members of the one or more complexes, or complex functional activity, or by detecting mutations in RB or the RB-IP (e.g., translocations in nucleic acids, truncations in the gene or protein, changes in nucleotide or amino acid sequence relative to wild-type RB or the RB-IP) which function to inhibit or reduce RB»RB-IP complex formation.
  • mutations in RB or the RB-IP e.g., translocations in nucleic acids, truncations in the gene or protein,
  • Also disclosed by the present invention are methodologies for the detection of an RB «RB-IP complex, or CD24*, Set*, GluTl *, or 115392* protein, in cell culture models that express particular RB «RB-IP complexes or CD24*, Set*, GluTl*, or 115392* proteins, or derivatives thereof, for the purpose of characterizing or preparing RB»RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* proteins for harvest.
  • This aforementioned embodiment includes, but is not limited to, cell sorting of prokaryotes (see e.g., Davey and Kell, 1996. Microbiol. Rev.
  • tissue specimens from eukaryotes, including mammalian species such as human (see e.g., Steele, et al, 1996. Clin. Obstet. Gynecol. 39:801- 813) and continuous cell cultures (see e.g., Orfao and Ruiz-Arguelles, 1996. Clin. Biochem. 29:5- 9).
  • tissue specimens from eukaryotes, including mammalian species such as human (see e.g., Steele, et al, 1996. Clin. Obstet. Gynecol. 39:801- 813) and continuous cell cultures (see e.g., Orfao and Ruiz-Arguelles, 1996. Clin. Biochem. 29:5- 9).
  • isolation methodologies may also be used as methods of diagnosis, as described supra.
  • Kits for diagnostic use are also disclosed herein, and are comprised, within one or more containers, of an anti-RB «RB-IP complex antibody or an anti-CD24*, anti-Set*, anti-GluTl*, or anti-115392* antibody, and, optionally, a labeled binding partner to the antibody.
  • the anti-RB «RB-IP complex antibody, or anti-CD24*, anti-Set*, anti-GluTl*, or anti-115392* antibody may be labeled with a detectable marker (e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety)
  • a detectable marker e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety
  • a kit is disclosed which comprises, in one or more containers, a nucleic acid probe capable of hybridizing to RB and/or an RB-IP mRNA.
  • a kit can comprise, in one or more containers, a pair of primers (e.g., each in the size range of 6-30 nucleotides) that are capable of serving as primers in an amplification-based reaction, including, but not limited to: (/) polymerase chain reaction (PCR; see e.g., Innis, et al, 1990. PCR Protocols, Academic Press, Inc., San Diego, CA); (ii) ligase chain reaction; (Hi) use of Q ⁇ replicase; (iv) cyclic probe reaction or various other methods known within the art, under such appropriate reaction conditions that at least a portion of an RB and/or an RB-IP, nucleic acid undergoes amplification.
  • PCR polymerase chain reaction
  • ligase chain reaction e.g., ligase chain reaction
  • Hi use of Q ⁇ replicase
  • cyclic probe reaction or various other methods known within the art, under such appropriate reaction conditions that at least a portion of an RB and/
  • a kit may, optionally, further comprise (in one or more containers) a predetermined amount of a purified RB*RB-IP complex, RB and/or an RB-IP, or nucleic acids thereof , for use as, for example, a standard or control.
  • Such Therapeutics may include, but are not limited to: (i) RB*RB-IP complexes, (ii) RB; (Hi) the individual RB-IP proteins and analogs and derivatives (including fragments) of the foregoing; (iv) antibodies specific thereto; (v) nucleic acids encoding RB and/or an RB-IP, and analogs or derivatives thereof; (vi) RB and/or RB-IP antisense nucleic acids and (vii) RB «RB-IP complex and CD24*, Set*, GluTl*, and 115392* modulators (e.g., inhibitors, agonists and antagonists).
  • IPs binding partners
  • a wide-range of diseases affected by intracellular signal transduction and transcriptional regulation may be treated or prevented by administration of a Therapeutic which modulates (i.e., inhibits, antagonizes, enhances or promotes) RE RB-IP complex activity, or
  • CD24*, Set*, GluTl*, or 115392* activity Both RB and cyclophilin A have been implicated in the cellular response to viral infection.. In accord, all of these aforementioned disorders may thus be treated or prevented by administration of a Therapeutic that modulates (i.e., inhibits, antagonizes, enhances or promotes) RB»RB-IP complex activity, or CD24*, Set*, GluTl*, or 115392* activity.
  • Diseases or disorders associated with aberrant levels of RB»RB-IP complex levels or activity, or aberrant levels of CD24*, Set*, GluTl*, or 115392* may be treated by administration of a Therapeutic that modulates RB «RB-IP complex formation or activity, or CD24*, Set*, GluTl*, or 115392* activity.
  • the activity or level of RB is modulated by administration of an RB-IP.
  • the activity or level of an RB-IP is modulated by administration of RB itself.
  • RB RB «RB-IP levels or activity, or increased CD24*, Set*, GluTl*, or 115392* levels or activity
  • Therapeutics that antagonize (i.e., reduce or inhibit) RB-RB-IP complex formation or activity, or CD24*, Set*, GluTl*, and 115392* levels or activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) RB-RB-IP complex formation or activity, or CD24*, Set*, GluTl*, and 115392* levels or activity.
  • Therapeutics which may be used include, but are not limited to: ( ) RB or an RB-IP, or analogs, derivatives or fragments thereof; (ii) anti-RB»RB-IP complex antibodies, and anti- CD24*, anti-Set*, anti-GluTl*, and anti-115392* antibodies, fragments and derivatives thereof containing the binding region thereof; (Hi) nucleic acids encoding RB or an RB-IP; (iv) concurrent administration of RB and RB-IP antisense nucleic acids, or CD24*, Set*, GluTl *, or 115392* antisense nucleic acids; and/or (v) RB and/or RB-IP, or CD24*, Set*, GluTl*, or 115392* nucleic acids that are dysfunctional (e.g., due to a heterologous (non-RB and/or non- RB-IP, or non-CD24*, non-Set*, non-GluTl*, or non-115392
  • a nucleic acid containing a portion of an RB and or an RB-IP gene in which the RB and/or RB-IP sequences flank (i.e., are both 5-' and 3'- to) a different gene sequence is used, as an RB and/or an RB-IP antagonist, so as to promote RB and/or RB-IP inactivation by homologous recombination.
  • an RB and/or an RB-IP antagonist so as to promote RB and/or RB-IP inactivation by homologous recombination.
  • mutants or derivatives of a first RB-IP protein that possess greater affinity for RB than that possessed by a second RB-IP may be administered to compete with said second RB-IP protein for RB binding, thereby reducing the levels of RB complexes containing the second RB- IP.
  • Other Therapeutics which function to inhibit RB-RB-IP complex or CD24*, Set*, GluTl*, or 115392* function may be identified by use of various in vitro assays well-known within the art (e.g., based upon their ability to inhibit RB*RB-IP binding).
  • Therapeutics which function as antagonists in RB»RB-IP complex formation or activity, or CD24*, Set*, GluTl*, or 115392* activity are administered therapeutically or prophylactically in the following situations: (i) in diseases or disorders involving an increased (relative to normal or desired) level of RB «RB-IP complex, or CD24*, Set*, GluTl*, and 115392* proteins, for example, in patients where RB»RB-IP complexes or CD24*, Set*, GluTl*, or 115392* proteins are overactive or over- expressed; or (ii) in diseases or disorders wherein in vitro and/or in vivo assays indicate the utility of RB «RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, antagonist administration.
  • Increased levels of RB*RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* protein may be readily detected by quantifying protein and/or RNA (e.g., obtained from a patient tissue sample from biopsy tissue or the like) and performing an in vitro assay for RNA or protein levels, structure and/or activity of the expressed RB»RB-IP complex (or the RB and RB-IP mRNA), or CD24*, Set*, GluTl*, or 115392* protein or mRNA.
  • protein and/or RNA e.g., obtained from a patient tissue sample from biopsy tissue or the like
  • CD24*, Set*, GluTl*, or 115392* protein or mRNA may be readily detected by quantifying protein and/or RNA (e.g., obtained from a patient tissue sample from biopsy tissue or the like) and performing an in vitro assay for RNA or protein levels, structure and/or activity of the expressed RB»RB-IP complex (or the
  • immunoassays to detect and/or visualize RB «RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* protein (e.g., Western blot, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect concurrent expression of RB and an RB-IP, or individual CD24*, Set* GluTl *, and 115392* mRNA.
  • SDS sodium dodecyl sulfate
  • a specific embodiment of the present invention discloses methodologies of reducing RB*RB-IP complex expression (i.e., the expression of the two components of the RB*RB-IP complex and/or formation of the complex), or CD24*, Set*, GluTl*, or 115392* expression, by targeting mRNAs that express the protein moieties.
  • RNA-based therapeutics currently fall within three classes: ( ) antisense species; (ii) ribozymes or (Hi) RNA aptamers. See e.g., Good, et ⁇ l, 1997. Gene Therapy 4:45-54. Antisense oligonucleotides have been the most widely utilized methodology.
  • Ribozyme therapy involves the administration, induced expression, etc., of small RNA molecules which possess enzymatic activity to cleave, bind, or otherwise inactivate specific RNA species so as to reduce or eliminate expression of particular proteins. See e.g., Grassi and Marini, 1996. Annals of Medicine 28:499-510; Gibson, 1996. Cancer and Metastasis Rev. 15:287-299.
  • the design of "hairpin” and "hammerhead" RNA ribozymes is necessary to specifically target a particular mRNA, such as the mRNA encoding RB.
  • RNA aptamers are specific RNA ligands for proteins (e.g., such as for Tat and Rev RNA) which can specifically inhibit their translation. See e.g., Good, et al, 1997. Gene Therapy 4:45-54. Aptamers specific for RB or an RB-IP may be identified by many methods well-known within the art (e.g., protein-protein interaction assay).
  • the activity or level of RB is reduced by the administration of an RB-IP, or a nucleic acid that encodes an RB-IP, or antibody that immunospecifically-binds to an RB-IP, or a fragment or a derivative of the antibody containing the binding domain thereof.
  • the level or activity of an RB-IP may be reduced by administration of an RB or an RB nucleic acid, or an antibody that immunospecifically-binds RB, or a fragment or derivative of the antibody containing the binding domain thereof.
  • diseases or disorders associated with increased levels of RB or a particular RB-IP may be treated or prevented by administration of a Therapeutic that increases RB «RB-IP complex formation, if the complex formation acts to reduce or inactivate RB or the particular RB-IP through the RB*RB-IP complex formation.
  • Such diseases or disorders can be treated or prevented by administration of one member of the RB «RB-IP complex, including mutants of a member of the complex that possess increased affinity for the other member of the RB»RB-IP complex (so as to cause increased complex formation), administration of antibodies or other molecules that stabilize the RB*RB-IP complex, and the like.
  • RB*RB-IP complex or RB, or a particular RB-IP
  • a Therapeutic that promotes (i.e., increases or supplies) RB»RB-IP complexes or function. Examples of such a
  • Therapeutic include, but are not limited to: (i) RB»RB-IP complexes and derivatives, analogs and fragments thereof that are functionally active (e.g., active to form RB «RB-IP complexes); (ii) non-complexed RB and RB-IP proteins, and derivatives, analogs, and fragments thereof and/or (Hi) nucleic acids encoding the members of an RB*RB-IP complex, or functionally active derivatives or fragments thereof (e.g., for use in gene therapy).
  • derivatives, homologs or fragments of RB and/or an RB-IP that increase and or stabilize RB*RB-IP complex formation.
  • Therapeutics which promote RB»RB-IP complex function, or CD24*, Set*, GluTl*, and 115392* function are administered therapeutically (including prophylactically) in the following clinical situations: (/) in diseases or disorders involving an absence or decreased (relative to normal or desired) level of RB»RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, proteins, for example, in patients where RB*RB-IP complexes (or the individual components necessary to form the complexes), or CD24*, Set*, GluTl*, or 115392* is lacking, genetically defective, biologically inactive or underactive, or under-expressed or (ii) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of RB»RB-IP complex, or CD24*, Set*, GluTl*, or 115392* agonist administration.
  • RB-RB-IP complex or CD24*, Set*, GluTl*, or 115392* protein or function
  • a patient tissue sample e.g., from biopsy tissue
  • the activity or level of RB is increased by administration of an RB-IP, or derivative or analog thereof, a nucleic acid encoding an RB-IP, or an antibody that immunospecifically-binds an RB-IP, or a fragment or derivative of the antibody contains the binding domain thereof.
  • the activity or levels of an RB-IP are increased by administration of RB, or derivative or analog thereof, a nucleic acid encoding RB, or an antibody that immunospecifically-binds RB, or a fragment or derivative of the antibody contains the binding domain thereof.
  • suitable in vitro or in vivo assays are utilized to determine the effect of a specific Therapeutic, and whether its administration is indicated for treatment of the affected tissue.
  • in vitro assays may be carried out with representative cells or cell types involved in a patient's disorder to determine if a Therapeutic has a desired effect upon such cell types.
  • Therapeutic compounds Prior to utilization in humans, Therapeutic compounds may be tested in suitable in vivo animal model systems.
  • Therapeutics of the present invention may be useful in treating or preventing diseases or disorders associated with cell hyperproliferation or loss of control of cell proliferation, particularly cancers, malignancies and tumors.
  • the Therapeutics of the present invention may be assayed by any method known within the art for efficacy in treating or preventing malignancies and related disorders.
  • Such assays include in vitro assays using transformed cells or cells derived from the tumor of a patient, or in vivo assays using animal models of cancer or malignancies, or the like.
  • potentially effective Therapeutics may function to inhibit proliferation of tumors or transformed cells in culture, cause regression of tumors in animal models in comparison to controls, or other similar physiological response.
  • a malignancy or cancer has been shown to be amenable to treatment by modulating (i.e., inhibiting, antagonizing, enhancing or agonizing) RE RB-IP complex activity, or CD24*, Set*, GluTl*, and 115392* activity
  • that cancer or malignancy can be treated or prevented by administration of a Therapeutic that modulates RB*RB-IP complex formation and function, or CD24*, Set*, GluTl*, and 115393* function, including supplying RB*RB-TP complexes and the individual binding partners of an RB»RB-IP complex.
  • malignancy or dysproliferative changes are treated or prevented in, for example, the bladder, breast, colon, lung, prostate, pancreas, or uterus.
  • the Therapeutics of the present invention which are effective in the therapeutic or prophylactic treatment of cancer or malignancies may also be administered for the treatment of pre-malignant conditions and/or to prevent the progression of a pre-malignancy to a neoplastic or malignant state.
  • Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia or, most particularly, dysplasia has occurred.
  • non-neoplastic cell growth consisting of hyperplasia, metaplasia or, most particularly, dysplasia has occurred.
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in its structure or function. For example, it has been demonstrated that endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of mature or fully differentiated cell substitutes for another type of mature cell. Metaplasia may occur in epithelial or connective tissue cells. Dysplasia is generally considered a precursor of cancer, and is found mainly in the epithelia. Dysplasia is the most disorderly form of non-neoplastic cell growth, and involves a loss in individual cell uniformity and in the architectural orientation of cells. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the presence of one or more characteristics of a transformed or malignant phenotype displayed either in vivo or in vitro within a cell sample derived from a patient is indicative of the desirability of prophylactic/therapeutic administration of a Therapeutic of the present invention which possesses the ability to modulate RBVRBIP complex activity.
  • Characteristics of a transformed phenotype include, but are not limited to: (i) morphological changes; (ii) looser substratum attachment; (Hi) loss of cell-to-cell contact inhibition; (iv) loss of anchorage dependence; (v) protease release; (vi) increased sugar transport; (VII) decreased serum requirement; (viii) expression of fetal antigens, (ix) disappearance of the 250 Kdal cell-surface protein, and the like. See e.g., Richards, et al, 1986. Molecular Pathology (W.B. Saunders Co., Philadelphia, PA).
  • 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: (i) a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome (bcr/ abl) for chronic myelogenous leukemia and t(14;18) for follicular lymphoma, etc.); (ii) familial polyposis or Gardner's syndrome (possible forerunners of colon cancer); (Hi) monoclonal gammopathy of undetermined significance (a possible precursor of multiple myeloma) and (iv) a first degree kinship with persons having a cancer or pre-cancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amy
  • a Therapeutic of the present invention is administered to a human patient to prevent the progression to breast, colon, lung, pancreatic, uterine cancer, melanoma or sarcoma.
  • a Therapeutic is administered to treat or prevent hyperproliferative or benign dysproliferative disorders.
  • Therapeutics of the present invention can be assayed by any method known in the art for efficacy in treating or preventing hyperproliferative diseases or disorders, such assays include in vitro cell proliferation assays, in vitro or in vivo assays using animal models of hyperproliferative diseases or disorders, or the like.
  • Potentially effective Therapeutics include, but are not limited to, Therapeutics that reduce cell proliferation in culture or inhibit growth or cell proliferation in animal models in comparison to controls.
  • a hyperproliferative disorder has been shown to be amenable to treatment by modulation of RB*RB-IP complex activity, or CD24*, Set*, GluTl*, or 115392* activity
  • that hyperproliferative disease or disorder can be treated or prevented by administration of a Therapeutic that modulates RB*RB-IP complex formation, or CD24*, Set*, GluTl* or 115392* activity (including supplying RB «RB-IP complexes and/or the individual binding partners of said complex).
  • Specific embodiments of the present invention are directed to treatment or prevention of cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid/hypertrophic scar formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia).
  • cirrhosis of the liver a condition in which scarring has overtaken normal liver regeneration processes
  • treatment of keloid/hypertrophic scar formation configured of the skin in which the scarring process interferes with normal renewal
  • psoriasis a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination
  • benign tumors e.g., prostatic hyperplasia
  • tissue hypertrophy e.g., prostatic hyperplasia
  • RB and certain binding partners of RB have been implicated in the deregulation of cellular maturation and apoptosis, which are characteristic of neurodegenerative disease (see e.g., Isslebacher, et al, 1997. Principals of Internal Medicine, 13 th Ed., McGraw Hill, New York, NY). Additionally, cyclophilin A is also implicated in mitochondrial dysfunction, again considered an underlying cause of neurodegenerative diseases and disorders.
  • Therapeutics of the present invention particularly, but not limited to those that modulate (or supply) RB»Zap 3, RB «GluTl*, RB»cyclophilin A and RB»Nlkl(Nek2) complexes maybe effective in treating or preventing neurodegenerative disease.
  • Therapeutics of the invention which modulate RB*RB-IP complexes involved in neurodegenerative disorders can be assayed by any method known in the art for efficacy in treating or preventing such neurodegenerative diseases and disorders.
  • Such assays include in vitro assays for regulated cell maturation or inhibition of apoptosis or in vivo assays using animal models of neurodegenerative diseases or disorders, or the like.
  • Potentially effective Therapeutics for example but not by way of limitation, promote regulated cell maturation and prevent cell apoptosis in culture, or reduce neurodegeneration in animal models in comparison to controls.
  • a neurodegenerative disease or disorder has been shown to be amenable to treatment by modulation of RB*RB-IP complex activity
  • the disease or disorder may be treated or prevented by administration of a Therapeutic that modulates RB •RB-IP complex formation.
  • RB and cyclophilin A are strongly implicated in various viral infection mechanisms, including those for HIV-l .
  • Viral diseases of a wide array of tissues including the respiratory tract, the CNS, the skin, the genitourinary tract, the eyes and ears, the immune system, the gastrointestinal tract, and the musculoskeletal system, affect a vast number of humans, of all ages.
  • Therapeutics of the invention particularly those that modulate (or supply) RB»RB-IP complex (particularly RB «cyclophilin A complex) activity may be effective in treating or preventing viral diseases or disorders.
  • Therapeutics of the invention can be assayed by any method known in the art for efficacy in treating or preventing such viral diseases and disorders.
  • Such assays include widely used in vitro assays using cell culture models, and in vivo assays using animal models of viral diseases or disorders. See e.g., McGeoch, et al, 1986. J. Gen. Virol. 67:813-825.
  • Potentially effective Therapeutics by way of example but not of limitation, reduce viral responses in animal models in comparison to controls.
  • a viral disease or disorder has been shown to be amenable to treatment by modulation of cyclophilin A complex activity
  • that viral disease or disorder can be treated or prevented by administration of a Therapeutic which functions to modulate RB «cyclophilin A complex formation (including supplying RB •cyclophilin A complexes).
  • nucleic acids comprising a sequence encoding RB and/or an RB-IP, or CD24*, Set*, GluTl*, or 115392*, or functional derivatives thereof, are administered to modulate RB»RB-IP complexes, or CD24*, Set*, GluTl*, or 115392* function, by way of gene therapy.
  • a nucleic acid or nucleic acids encoding both RB and an RB-IP e.g.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
  • the nucleic acid produces its encoded protein(s) that mediates a therapeutic effect by modulating the RB «RB-IP complex, or CD24*, Set*, GluTl*, or 115392*, function.
  • the Therapeutic comprises a RB and a RB-IP nucleic acid, or CD24*, Set*, GluTl*, or 115392* nucleic acid, that is part of an expression vector that expresses the RB and RB-IP proteins, or expresses CD24*, Set*, GluTl*, or 115392*, or fragments or chimeric proteins thereof, in a suitable host.
  • such a nucleic acid has promoter(s) operably linked to the RB and the RB-IP coding region(s), or linked to the CD24*, Set*, GluTl*, or 115392* coding region, said promoter(s) being inducible or constitutive, and, optionally, being tissue-specific.
  • a nucleic acid molecule is used in which the RB and RB-IP coding sequences, or CD24*, Set*, GluTl *, or 115392* coding sequences, and any other desired sequences, are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intra-chromosomal expression of the RB and the RB-IP nucleic acids.
  • the RB and RB-IP coding sequences or CD24*, Set*, GluTl *, or 115392* coding sequences, and any other desired sequences.
  • Delivery of the Therapeutic nucleic acid into a patient may be either direct (i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect (i.e., cells are first transformed with the nucleic acid in vitro, then transplanted into the patient). These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product.
  • a nucleic acid-ligand complex may be produced in which the ligand comprises a fusogenic viral peptide designed so as to disrupt endosomes, thus allowing the nucleic acid to avoid subsequent lysosomal degradation.
  • the nucleic acid may be targeted in vivo for cell-specific endocytosis and expression, by targeting a specific receptor. See e.g., PCT Publications WO 92/06180; WO93/14188 and WO 93/20221.
  • the nucleic acid may be introduced intracellularly and incorporated within host cell genome for expression by homologous recombination.
  • a viral vector that contains the RB and/or the RB-IP nucleic acids, or CD24*, Set*, GluTl*, or 115392* nucleic acid is used.
  • retroviral vectors may be employed (see e.g., Miller, et al, 1993. Meth. Enzymol 217:581-599) which have been modified to delete those retroviral-specific sequences which are not required for packaging of the viral genome and its subsequent integration into host cell DNA.
  • RB and/or RB-IP preferably both RB and RB-IP nucleic acids, or CD24*, Set*, GluTl *, or 115392* nucleic acids, to be used in gene therapy is/are cloned into the vector, which facilitates delivery of the gene into a patient. See e.g., Boesen, et al, 1994. Biotherapy 6:291-302; Kiem, et al, 1994. Blood 83:1467-1473. Additionally, adenovirus is an especially efficacious "vehicle" for the delivery of genes to the respiratory epithelia. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle.
  • Adenoviruses also possess the advantageous ability to infect non-dividing cells.
  • Adenovirus-associated virus has also been proposed for use in gene therapy. See e.g., Walsh, et al, 1993. Proc. Soc. Exp. Biol. Med. 204:289-300.
  • An additional approach to gene therapy in the practice of the present invention involves transferring a gene into cells in in vitro tissue culture by such methods as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection.
  • the methodology of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection pressure (e.g., antibiotic resistance) so as facilitate the isolation of those cells which have taken up, and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to the in vivo administration of the resulting recombinant cell by any method known within the art including, but not limited to: transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences of interest, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and similar methodologies which ensure that the necessary developmental and physiological functions of the recipient cells are not disrupted by the transfer. See e.g., Loeffler & Behr, 1993. Meth. Enzymol 217: 599-618.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • the resulting recombinant cells may be delivered to a patient by various methods known within the art including, but not limited to: injection of epithelial cells (e.g., subcutaneously); the application of recombinant skin cells as a skin graft onto the patient and the intravenous injection of recombinant blood cells (e.g., hematopoetic stem or progenitor cells).
  • recombinant blood cells e.g., hematopoetic stem or progenitor cells.
  • the total amount of cells which are envisioned for use depend upon the desired effect, patient state, etc., and may be determined by one skilled within the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include, but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes and blood cells (e.g., T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes and hematopoetic stem or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.).
  • the cell utilized for gene therapy may be autologous to the patient.
  • stem or progenitor cells which can be isolated and maintained in vitro, may be utilized.
  • stem cells include, but are not limited to, hematopoetic stem cells (HSC), stem cells of epithelial tissues (e.g., skin, lining of the gut, embryonic heart muscle cells, liver stem cells) and neural stem cells (see e.g., Stemple & Anderson, 1992. Cell 71:973-985).
  • HSC hematopoetic stem cells
  • epithelial tissues e.g., skin, lining of the gut, embryonic heart muscle cells, liver stem cells
  • neural stem cells see e.g., Stemple & Anderson, 1992. Cell 71:973-985.
  • HSC hematopoetic stem cells
  • any technique which provides for the isolation, propagation, and maintenance in vitro of HSC may be used in this specific embodiment of the invention.
  • HSCs utilized for gene therapy are, preferably, autologous to the patient.
  • non-autologous HSCs are, preferably, utilized in conjunction with a method of suppressing transplantation immune reactions of the future host/patient.
  • HSCs may be highly enriched (or produced in a substantially-pure form), by any techniques known within the art, prior to administration to the patient. See e.g., Witlock & Witte, 1982. Proc. Natl. Acad. Sci. USA 79:3608-3612.
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • Additional methods may be adapted for use to deliver a nucleic acid encoding the RB and/or RB-IP proteins, or functional derivatives thereof, and are within the scope of the present invention.
  • RB*RB-IP complex or CD24*, Set*, GluTl*, or 115392* protein formation and function may be inhibited by the use of anti-sense nucleic acids for the RB protein and/or RB-IPS, and is preferably comprised of both the RB protein and RB-IP.
  • the present invention discloses the therapeutic or prophylactic use of nucleic acids (of at least six nucleotides in length) which are anti-sense to a genomic sequence (gene) or cDNA encoding the RB protein and/or RB-IPS, or portions thereof.
  • Such anti-sense nucleic acids have utility as Therapeutics which inhibit RE RB-IP complex or CD24*, Set*, GluTl*, or 115392* protein formation or activity, and may be utilized in a therapeutic or prophylactic manner.
  • Another specific embodiment of the present invention discloses methodologies for the inhibition of the expression of the RB protein and RB-IP nucleic acid sequences, within a prokaryotic or eukaryotic cell, which is comprised of providing the cell with an therapeutically- effective amount of an anti-sense nucleic acid of the RB protein and RB-IP or derivatives thereof.
  • the anti-sense nucleic acids of the present invention may be oligonucleotides which may either be directly administered to a cell or which may be produced in vivo by transcription of the exogenous, introduced sequences.
  • the anti-sense nucleic acid may be complementary to either a coding (i.e., exonic) and/or non-coding (i.e., intronic) region of the RB protein or RB- IP mRNAs.
  • the RB protein and RB-IP anti-sense nucleic acids are, at least, six nucleotides in length and are, preferably, oligonucleotides ranging from 6-200 nucleotides in length.
  • the anti-sense oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • the anti-sense oligonucleotides may be DNA or RNA (or chimeric mixtures, derivatives or modified versions thereof), may be either single- stranded or double-stranded and may be modified at a base, sugar or phosphate backbone moiety.
  • the anti-sense oligonucleotide of the present invention may include other associated functional groups, such as peptides, moieties which facilitate the transport of the oligonucleotide across the cell membrane, a hybridization-triggered cross-linking agent, a hybridization-triggered cleavage-agent, and the like. See e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; PCT Publication No. WO 88/09810.
  • the RB protein and RB-IP antisense oligonucleotides comprise catalytic RNAs or ribozymes. See, e.g., Sarver, et al, 1990. Science 247:1222-1225.
  • the anti-sense oligonucleotides of the present invention may be synthesized by standard methodologies known within the art including, but not limited to: (/) automated phosphorothioate-mediated oligonucleotide synthesis (see e.g., Stein, et al, 1988. Nuc. Acids Res. 16:3209) or (II) methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (see e.g., Sarin, et al, 1988. Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451).
  • the RB protein and RB-IP antisense nucleic acids are produced intracellularly by transcription of an exogenous sequence.
  • a vector may be produced which (upon being exocytosed by the cell) is transcribed in vivo, thus producing an antisense nucleic acid (RNA) species.
  • RNA antisense nucleic acid
  • the aforementioned vector may either remain episomal or become chromosomally-integrated, so long as it can be transcribed to produce the desired antisense RNA.
  • the vectors utilized in the practice of the present invention may be derived from bacterial, viral, yeast or other sources known within the art, which are utilized for replication and expression in mammalian cells.
  • RNAs may be facilitated by any promoter known within the art to function in mammalian, preferably, human cells.
  • promoters may be inducible or constitutive and include, but are not limited to: (i) the SV40 early promoter region; (II) the promoter contained in the 3'-terminus long terminal repeat of Rous sarcoma virus (RSV); (in) the Herpesvirus thymidine kinase promoter and (tv) the regulatory sequences of the metallothionein gene.
  • the RB protein and RB-IP antisense nucleic acids may be utilized prophylactically or therapeutically in the treatment or prevention of disorders of a cell type which expresses (or preferably over-expresses) the RB protein»RB-IP complex.
  • Cell types which express or over- express the RB protein and RB-IP RNA may be identified by various methods known within the art including, but are not limited to, hybridization with RB protein- and RB-IP-specific nucleic acids (e.g., by Northern hybridization, dot blot hybridization, in situ hybridization) or by observing the ability of RNA from the specific cell type to be translated in vitro into the RB protein and the RB-IP by immunohistochemistry.
  • primary tissue from a patient may be assayed for the RB protein and or RB-IP expression prior to actual treatment by, for example, immunocytochemistry or in situ hybridization.
  • compositions of the present invention comprising an effective amount of a RB protein and RB-IP antisense nucleic acid contained within a pharmaceutically-acceptable carrier may be administered to a patient having a disease or disorder which is of a type that expresses or over-expresses RB protein»RB-IP complex RNA or protein.
  • the amount of RB protein and/or RB-IP antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will be dependant upon the nature of the disorder or condition, and may be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity in vitro, and then in useful animal model systems prior to testing and use in humans.
  • compositions comprising RB protein and RB-IP antisense nucleic acids may be administered via liposomes, microparticles, or microcapsules. See e.g., Leonetti, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451.
  • RB «RB-IP complexes, and CD24*, Set*, GluTl* and 115392* proteins, and derivatives, fragments and analogs thereof can be assayed by various methods.
  • Potential modulators e.g., inhibitors, agonists and antagonists
  • RB «RB-IP complex activity, or CD24*, Set*, GluTl*, or 115392* activity e.g., anti-RB «RB-IP, anti-CD24*, anti-Set*, anti- GluTl*, and anti-115392* antibodies, and RB and RB-IP antisense nucleic acids, can be assayed for the ability to modulate RB*RB-IP complex formation and/or activity, and CD24*, Set*, GluTl*, or 115392* activity.
  • immunoassays include, but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme linked immunosorbent assay(ELISA), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immuno fluorescence assays, protein A assays, immunoelectrophoresis assays, and the like.
  • RIA radioimmunoassays
  • ELISA enzyme linked immunosorbent assay
  • sandwich immunoassays immunoradiometric assays
  • gel diffusion precipitin reactions immunodiffusion assays
  • in situ immunoassays Western blots
  • precipitation reactions agglutination assays
  • complement fixation assays immuno fluorescence assays
  • protein A assays
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are all intended to be included within the scope of the present invention.
  • RB and RB-IP genes both endogenous genes and those expressed from cloned DNA containing these genes
  • RB and RB-IP genes can be detected using techniques known within the art, including, but not limited to Southern hybridization; Northern hybridization; restriction endonuclease mapping; DNA sequence analysis; polymerase chain reaction amplification (PCR; see e.g., U.S. Patent Nos. 4,683,202, 4,683,195, and 4,889,818; Gyllenstein, et al, 1988. Proc. Natl. Acad. Sci. USA 85:7652-7657; Loh, et al, 1989. Science 243:217-220) or RNase protection with probes specific for RB or RB-IP genes, in various cell types.
  • Southern hybridization can be used to detect genetic linkage of RB or RB-IP gene mutations to physiological or pathological states.
  • Various cell types at various stages of development, can be characterized for their expression of RB and a RB-IP (particularly expression of RB and an RB-IP at the same time and in the same cells), or CD24*, Set*, GluTl*, or 115392* expression.
  • the stringency of the hybridization conditions for northern or Southern blot analysis can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific probes used. Modifications to these methods and other methods commonly known in the art can be used.
  • VIII Binding Assays
  • Derivatives e.g., fragments
  • analogs of RB-IPs can be assayed for binding to RB by any method known in the art, for example (i) the modified, improved yeast two hybrid assay system (described infra), (ii) immunoprecipitation with an antibody that binds to RB in a complex followed by analysis by size fractionation of the immunoprecipitated proteins (e.g., by denaturing or non-denaturing polyacrylamide gel electrophoresis), (iii) Western analysis, (iv) non-denaturing gel electrophoresis, and the like.
  • the modified, improved yeast two hybrid assay system described infra
  • immunoprecipitation with an antibody that binds to RB in a complex followed by analysis by size fractionation of the immunoprecipitated proteins e.g., by denaturing or non-denaturing polyacrylamide gel electrophoresis
  • Western analysis e.g., by denaturing or non-denaturing
  • a specific embodiment of the present invention provides a methodology for the screening of a derivative or analog of RB for biological activity comprising contacting said derivative or analog of RB with a RB-IP, and detecting the formation of a complex between said derivative or analog of RB and said protein; wherein detecting formation of said complex indicates that said derivative or analog of RB has biological (e.g., binding) activity.
  • another embodiment of the invention relates to a method for screening a derivative or analog of a RB-IP protein for biological activity comprising contacting said derivative or analog of said protein with RB; and detecting the formation of a complex between said derivative or analog of said protein and RB; wherein detecting the formation of said complex indicates that said derivative or analog of said protein has biological activity.
  • the present invention discloses methodologies relating to the modulation of the activity of a protein that possesses the ability to participate in a RB»RB-IP by administration of a binding partner of that protein, or derivative or analog thereof.
  • RB, and derivatives and analogs thereof can be assayed for the ability to modulate the activity or level of an RB-IP by contacting a cell or administering an animal expressing an RB-IP gene with an RB protein, or a nucleic acid encoding an RB protein, or an antibody that immunospecifically-binds the RB protein, or a fragment or derivative of said antibody containing the binding domain thereof, and measuring a change in RB-IP levels or activity, wherein a change in RB-IP levels or activity indicates that RB can modulate RB-IP levels or activity.
  • an RB-IP can be assayed for the ability to modulate the activity or levels of an RB protein by contacting a cell or administering an animal expressing a gene encoding said protein with RB, or a nucleic acid encoding RB, or an antibody that immunospecifically-binds RB, or a fragment or derivative of said antibody containing the binding domain thereof, wherein a change in RB levels or activity indicates that the RB-IP can modulate RB levels or activity.
  • RB and several of the identified binding partners of RB (RB-IPs), e.g., RN-tre, CAS, IK, Nlkl (Nek 2), LDH-B, CD24*, and Set*, play roles in the control of cell proliferation and, therefore, cell-transformation and tumorigenesis.
  • RB-IPs e.g., RN-tre, CAS, IK, Nlkl (Nek 2), LDH-B, CD24*, and Set*
  • the present invention discloses methodologies for screening RB «RB-IP complex and and CD24* and Set* proteins, (and derivatives, fragments, analogs and homologs, thereof) for the ability to alter cell proliferation, cell transformation and/or tumorigenesis in vitro and in vivo.
  • cell proliferation may be assayed by measuring 3 H-thymidine incorporation, by direct cell count, by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g., c-fos, c-myc) cell-cycle markers, and the like.
  • proto-oncogenes e.g., c-fos, c-myc
  • the RB»RB-IP complex and and CD24* and Set* proteins may also be screened for activity in inducing or inhibiting cell transformation (or the progression to malignant phenotype) in vitro.
  • the proteins and protein complex of the present invention may be screened by contacting either cells with a normal phenotype (for assaying for cell transformation) or a transformed cell phenotype (for assaying for inhibition of cell transformation) with the protein or protein complex of the present invention and examining the cells for acquisition or loss of characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo) including, but not limited to: colony formation in soft agar, a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250 Kdal cell-surface protein, and the like. See e.g., Luria, et al, 1978. General Virology, 3rd ed. (John Wiley & Sons, New York, NY).
  • the RB «RB-IP complex and and CD24* and Set* proteins, (and derivatives, fragments, analogs and homologs, thereof) may also be screened for activity to promote or inhibit tumor formation in vivo in non-human test animal.
  • a vast number of animal models of hyperproliferative disorders e.g., tumorigenesis and metastatic spread
  • the proteins and protein complex may be administered to a non-human test animal (preferably a test animal predisposed to develop a type of tumor) and the non-human test ariimals is subsequently examined for an increased incidence of tumor formation in comparison with controls animals which were not administered the proteins or protein complex of the present invention.
  • the proteins and protein complex may be administered to non-human test animals possessing tumors (e.g., animals in which tumors have been induced by introduction of malignant, neoplastic, or transformed cells or by administration of a carcinogen) and subsequently examining the tumors within the test animals for tumor regression in comparison to controls. Accordingly, once a hyperproliferative disease or disorder has been shown to be amenable to treatment by modulation of RB*RB-IP complex activity that disease or disorder may be treated or prevented by administration of a Therapeutic which modulates RB «RB-IP complex formation.
  • RB As well as the complexes and proteins previously described supra, which are implicated in the control of cell cycle progression, RB, and the RB-IPs cyclophilin A, Zap 3, 17 beta-HSD6, and GluTl*, specifically have been implicated in cellular apoptosis. In addition, in the case of Zap 3, expression of this protein is genetically-linked to neurodegenerative disease.
  • the RB»RB-IP complexes (particularly the RB «cyclophilin A, RB «Zap 3, RB*17 beta-HSD6, and RB»GluTl* complexes) and derivatives, analogs and fragments thereof, nucleic acids encoding the RB and RB-IP genes, anti-RB «RB-IP antibodies, and other modulators of RB*RB-IP complex activity, may be tested for activity in treating or preventing neurodegenerative disease in in vitro and in vivo assays.
  • a Therapeutic may be assayed for activity in treating or preventing neurodegenerative disease by contacting cultured cells which exhibit an indicator of a neurodegenerative disease.
  • an assay of the level of over-expression of the ⁇ -A4 peptide, in vitro may be used to ascertain the efficacy of a specific Therapeutic by comparing the level of said indicator within the cells contacted with the Therapeutic with said level of said indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the Therapeutic has activity in treating or preventing neurodegenerative disease.
  • cell culture models for neurodegenerative disease include, but are not limited to: (i) cultured rat endothelial cells from affected and non- affected individuals (see e.g., Maneiro, et al, 1997. Methods Find. Exp. Clin. Pharmacol. 19:5- 12); ( t) P19 murine embryonal carcinoma cells (see e.g., Hung, et al, 1992. Proc. Natl. Acad. Sci. USA 89:9439-9443) and (in) dissociated cell cultures of cholinergic neurons from the nucleus basalis of Meynert (see e.g., Nakajima, et al, 1985. Proc. Natl. Acad. Sci. USA 82:6325- 6329).
  • a Therapeutic may be assayed for activity in treating or preventing neurodegenerative disease by administering the Therapeutic to a test animal which exhibits symptoms of a neurodegenerative disease (e.g., premature development of cognitive deficiencies in transgenic animals expressing ⁇ -APP) or that is predisposed to develop symptoms of a neurodegenerative disease; and measuring the change in said symptoms of the neurodegenerative disease following the administration of said Therapeutic; wherein a reduction in the severity of the symptoms of the neurodegenerative disease, or prevention of the symptoms of the neurodegenerative disease, indicates that the
  • Such a test animal may be any one of a number of animal models known within the art for neurodegenerative disease. These aforementioned animal models (including those for Alzheimer's Disease and trisomy 21), accurately mimic natural human neurodegenerative diseases. See e.g., Campbell, et al, 1997. Mol. Psychiatry 2:125-129; Schultz, et al, 1997. Mol. Cell. Biochem. 174:193-197. Examples of specific neurodegenerative animals models include, but are not limited to: ( ) the partial trisomy 16 mouse (see e.g., Holtzman, et al, 1996. Proc. Natl. Acad. Sci.
  • interactants RB and cyclophilin A are strongly implicated in viral infection mechanisms, including that for HIV-1.
  • numerous human diseases directly result from virulent and opportunistic viral infection. See e.g., Belshe, 1984.
  • RB «RB-IP complexes (particularly RB •cyclophilin A complexes), and derivatives, analogs, and fragments thereof, nucleic acids containing the RB and RB-IP genes (in particular the cyclophilin A gene); anti-RB*RB-IP antibodies (in particular anti-RB «cyclophilin A antibodies) and various other modulators of the RB «RB-IP complex activity, may be tested for activity in treating or preventing viral diseases in both in vitro and in vivo assays.
  • a Therapeutic may be assayed for activity in treating or preventing viral disease by contacting cultured cells that exhibit an indicator of a viral reaction (e.g., formation of inclusion bodies) in vitro with the Therapeutic, and comparing the level of said indicator in the cells contacted with the Therapeutic with said level of said indicator in cells not so contacted, wherein a lower level in said contacted cells indicates that the
  • Therapeutic has activity in treating or preventing viral disease.
  • Cell models which may be used for such assays include, but are not limited to: (i) viral infection of T lymphocytes (see e.g., Selin, et al, 1996. J. Exp. Med. 183:2489-2499); (ii) hepatitis B infection of de-differentiated hepatoma cells (see e.g., Raney, et al, 1997. J. Virol. 71:1058-1071); (Hi) viral infection of cultured salivary gland epithelial cells (see e.g., Clark, et al, 1994.
  • a Therapeutic of the present invention my be assayed for activity in treating or preventing viral disease by administering said Therapeutic to a test animal having symptoms of a viral infection (e.g., characteristic respiratory symptoms in animal models of virally-induced asthma), or which test animal does not exhibit a viral reaction and is subsequently challenged with an agent that elicits an viral reaction, and measuring the change in the viral reaction after the administration of said Therapeutic; wherein a reduction in said viral reaction or a prevention of said viral reaction indicates that the Therapeutic has activity in treating or preventing viral disease.
  • Animal models which may be utilized for such assays include, but are not limited to: (i) respiratory viral infections in guinea pigs (see e.g., Kudlacz and Knippenberg, 1995. Inflamm.
  • mice (ii) influenza virus infection of mice (see e.g., Dobbs, et al, 1996. J. Immunol. 157:1870-1877); (Hi) neurotrophic virus infection of mice (see e.g., Barna, et al, 1996. Virology 223:331-343); (tv) measles infection in hamsters (see e.g., Fukuda, et al, 1994. Ada Otolaryngol. Suppl (Stockh.) 514:111-116); (v) encephalomyocarditis infection of mice (see e.g., Hirasawa, et al, 1997. J. Virol. 71:4024-4031) and (vi) cytomegalovirus (CMV) infection of mice (see e.g., Orange and Biron, 1996. J. Immunol. 156:1138-1142).
  • CMV cytomegalovirus
  • the present invention discloses methodologies for assaying and screening derivatives, fragments, analogs and homologs of RB-IP for binding to RB.
  • the derivatives, fragments, analogs and homologs of the RB-IP which interact with RB may be identified by means of a yeast two hybrid assay system (see e.g., Fields & Song, 1989. Nature 340:245-246) or; preferably, a modification and improvement thereof, as described in U.S. Patent Applications Serial Nos. 08/663,824 (filed June 14, 1996) and 08/874,825 (filed June 13, 1997), both of which are entitled "Identification and Comparison of Protein-Protein Interactions that Occur in
  • the identification of interacting proteins by the improved yeast two hybrid system is based upon the detection of the expression of a reporter gene (hereinafter "Reporter Gene"), the transcription of which is dependent upon the reconstitution of a transcriptional regulator by the interaction of two proteins, each fused to one half of the transcriptional regulator.
  • reporter Gene a reporter gene
  • the bait RB (or derivative, fragment, analog or homolog) and prey protein (proteins to be tested for ability to interact with the bait protein) are expressed as fusion proteins to a DNA-binding domain, and to a transcriptional regulatory domain, respectively, or vice versa.
  • the prey population may be one or more nucleic acids encoding mutants of RB-IP (e.g., as generated by site-directed mutagenesis or another method of producing mutations in a nucleotide sequence).
  • the prey populations are proteins encoded by DNA (e.g., cDNA, genomic DNA or synthetically generated DNA).
  • the populations may be expressed from chimeric genes comprising cDNA sequences derived from a non-characterized sample of a population of cDNA from mammalian RNA.
  • recombinant biological libraries expressing random peptides may be used as the source of prey nucleic acids.
  • the present invention discloses methods for the screening for inhibitors of RB-IP.
  • the protein-protein interaction assay may be performed as previously described herein, with the exception that it is performed in the presence of one or more candidate molecules.
  • inhibition of the protein interaction is necessary for the yeast cells to survive, for example, where a non-attenuated protein interaction causes the activation of the URA3 gene, causing yeast to die in medium containing the chemical 5-fluoroorotic acid. See e.g., Rothstein, 1983. Meth. Enzymol 101 :167-180.
  • the proteins comprising the bait and prey populations are provided as fusion (chimeric) proteins, preferably by recombinant expression of a chimeric coding sequence containing each protein contiguous to a pre-selected sequence.
  • the pre- selected sequence is a DNA-binding domain that may be any DNA-binding domain, so long as it specifically recognizes a DNA sequence within a promoter (e.g., a transcriptional activator or inhibitor).
  • the pre-selected sequence is an activator or inhibitor domain of a transcriptional activator or inhibitor, respectively.
  • the regulatory domain alone (not as a fusion to a protein sequence) and the DNA-binding domain alone (not as a fusion to a protein sequence) preferably, do not detectably interact, so as to avoid false-positives in the assay.
  • the assay system further includes a reporter gene operably linked to a promoter that contains a binding site for the DNA-binding domain of the transcriptional activator (or inhibitor). Accordingly, in the practice of the present invention, the binding of the RB fusion protein to a prey fusion protein leads to reconstitution of a transcriptional activator (or inhibitor), which concomitantly activates (or inhibits) expression of the Reporter Gene.
  • the present invention discloses a methodology for detecting one or more protein-protein interactions comprising the following steps: (i) recombinantly- expressing the RB (or a derivative, fragment, analog or homolog thereof) in a first population of yeast cells of a first mating type and possessing a first fusion protein containing the RB sequence and a DNA-binding domain; wherein said first population of yeast cells contains a first nucleotide sequence operably-linked to a promoter which is "driven” by one or more DNA- binding sites recognized by said DNA-binding domain such that an interaction of said first fusion protein with a second fusion protein (comprising a transcriptional activation domain) results in increased transcription of said first nucleotide sequence; (ii) negatively selecting to eliminate those yeast cells in said first population in which said increased transcription of said first nucleotide sequence occurs in the absence of said second fusion protein; (ii ' i) recombinantly expressing in a second population of yeast cells of
  • the bait (a RB sequence) and the prey (a library of chimeric genes) are combined by mating the two yeast strains on solid media for a period of approximately 6-8 hours. In a less preferred embodiment, the mating is performed in liquid media.
  • the resulting diploids contain both types of chimeric genes (i.e., the DNA-binding domain fusion and the activation domain fusion).
  • the DNA sequences encoding the pairs of interactive proteins are isolated by a method wherein either the DNA- binding domain hybrids or the activation domain hybrids are amplified, in separate reactions.
  • the amplification is carried out by polymerase chain reaction (PCR; see e.g., Innis, et al, 1990.
  • PCR Protocols (Academic Press, Inc., San Diego, CA) utilizing pairs of oligonucleotide primers specific for either the DNA-binding domain hybrids or the activation domain hybrids.
  • the PCR amplification reaction may also be performed on pooled cells expressing interacting protein pairs, preferably pooled arrays of interactants.
  • Other amplification methods known within the art may also be used including, but not limited to, ligase chain reaction; Q ⁇ -replicase or the like. See e.g., Kricka, et al, 1995. Molecular' Probing, Blotting, and Sequencing (Academic Press, New York, NY).
  • the plasmids encoding the DNA- binding domain hybrid and the activation domain hybrid proteins may also be isolated and cloned by any of the methods well-known within the art.
  • a shuttle yeast to E. coli
  • the genes may be subsequently recovered by transforming the yeast DNA into E. coli and recovering the plasmids from the bacteria. See e.g., Hoffman ,et al, 1987. Gene 57:267-272.
  • compositions and Therapeutic/Prophylactic Administration discloses methods of treatment and prophylaxis by the administration to a subject of an pharmaceutically-effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified and the subject is a mammal, and most preferably, human.
  • Formulations and methods of administration that can be employed when the Therapeutic comprises a nucleic acid are described in Sections 6(i) and 6(z ' ⁇ ), supra.
  • a Therapeutic of the present invention including, but not limited to: (i) encapsulation in liposomes, microparticles, microcapsules; (ii) recombinant cells capable of expressing the Therapeutic; (ii ' i) receptor-mediated endocytosis (see, e.g., Wu & Wu, 1987. J. Biol. Chem. 262:4429-4432); (iv) construction of a Therapeutic nucleic acid as part of a retroviral or other vector, and the like.
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the Therapeutics of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically-active agents. Administration can be systemic or local.
  • Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the Therapeutic locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant. In a specific embodiment, administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the Therapeutic may be delivered in a vesicle, in particular a liposome. See e.g.. Langer. 1990. Science 249:1527-1533.
  • the Therapeutic can be delivered in a controlled release system including, but not limited to: a delivery pump (see e.g., Saudek, et al, 1989. New Engl J. Med. 321:574 and a semi-permeable polymeric material (see e.g., Howard, et al, 1989. J. Neurosurg. 71:105).
  • the controlled release system can be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release 1984. (CRC Press, Bocca Raton, FL).
  • the Therapeutic nucleic acid may 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 that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by 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., Joliot, et al, 1991. Proc. Natl. Acad. Sci. USA 88:1864-1868), and the like.
  • a nucleic acid Therapeutic can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions comprise a therapeutically-effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration of the Therapeutics of the present invention are generally about 20-500 micrograms ( ⁇ g) of active compound per kilogram (Kg) body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the present invention also provides a pharmaceutical pack or kit, comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions and Therapeutics of the present invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions and Therapeutics of the present invention.
  • Optionally associated with such container(s) may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • yeast two hybrid system was used to identify protein interactions.
  • Yeast is a eukaryote, and therefore, intermolecular protein interactions detected in this type of system would be expected to demonstrate protein interactions that occur under physiological conditions. See e.g., Chien, et al, 1991. Proc. Natl. Acad. Sci. USA 88:9578-9581.
  • Expression vectors were constructed to encode two hybrid proteins. For the "forward screen", one hybrid consisted of the DNA binding domain of the yeast transcriptional activator Gal4 fused to a portion of RB; whereas the other hybrid consisted of the Gal4 activator domain fused to "prey" protein sequences encoded by a mammalian cDNA library.
  • Each of the vectors was then inserted into complementary (a and alpha) mating types of yeast using methods known within the art. See e.g., Chien, et al, 1991. Proc. Natl. Acad. Sci. USA 88:9578-9581. Mating of the yeast strains was carried out to express both vector constructs within the same yeast cells, thus allowing interaction to occur. Interaction between the bait and prey domains led to transcriptional activation of Reporter Genes containing cw-binding elements for Gal4.
  • the Reporter Genes encoding the indicator protein beta-galactosidase, and metabolic markers for uracil and histidine auxotrophy, were included in specific fashion in one or the other of the yeast strains used in the mating.
  • yeast were selected for successful mating, expression of both fusion constructs, and expression of RB-IPs.
  • Yeast clones which contained interacting regions were picked and grown in individual wells of microtiter plates. The plasmids containing the RB-IPs were then isolated and characterized.
  • the prey cDNAs were obtained from a commercial human fetal brain cDNA library of 3.5 x 10 6 independent isolates (Cat. #HL4029AH; Clontech, Palo Alto, CA).
  • the library was synthesized from Xho l-dT 15 primed fetal brain mRNA (from a mixture of five male and female 19-22 week fetuses) that was directionally cloned into pACT2, a yeast Gal4 activation domain cloning vector including the LEU2 gene for selection in yeast deficient in leucine biosynthesis.
  • the RB binding domain clone was constructed by inserting a BamHl/Sall fragment of RB from amino acid residues 301 to 928 of RB (GenBank Accession Number M28419; nucleotides 1040 to 2925 between the BamHl and Sail sites of pAS-1 (Clontech).
  • This vector is a yeast DNA-binding domain cloning vector that contains the TRP1 gene for selection in yeast strains deficient in tryptophan biosynthesis.
  • the bait sequence was confirmed by nucleic acid sequencing to confirm that PCR amplification reproduced an accurate copy of the RB sequence. This test determined that as predicted, the bait sequence encoded an interacting domain identical to human RB amino acid residues 301 to 928.
  • the bait was expressed by lithium acetate/polyethylene glycol transformation (see e.g., Ito, et al, 1983. J. Bacteriol 153:163-168) into the yeast strain YULH (mating type a, ura3, his3, lys2, Ade2, trpl, leu2, gal4, gal80, GAL1-URA3, GALl-lacZ), while the prey sequences were expressed by transformation into the yeast strain N106 r (mating type ⁇ , ura3, hi 3, ade2, trpl, leu2, gal4, gal80, cyK, Lys2::GALl UAS -HIS3 T ⁇ TA -HIS3, ura 3::GALl UAS -GAL TATA -lacZ).
  • the two transformed populations were then mated using standard methods within the art.
  • the cells were grown until mid-to-late log phase on media that selected for the presence of the appropriate plasmids.
  • the two mating strains, alpha and a were then, diluted in YAPD media (see e.g., Sherman, et al, 1991. Getting Started with Yeast, Vol. 194, Academic Press, New York, NY), filtered onto nitrocellulose membranes, and incubated at 30°C for 6-8 hours.
  • the cells were then transferred to medium selective for the desired diploids (i.e., yeast harboring reporter genes for ⁇ -galactosidase, uracil auxotrophy, and histidine auxotrophy, and expression of the vectors encoding the bait and prey).
  • the mating products were then plated on SC (synthetic complete) medium (see e.g., Kaiser, 1994. Methods in Yeast Genetics, 1994 Ed., Cold Spring Harbor Laboratory Press, New York, NY) lacking adenine and lysine (to select for successful mating), leucine and tryptophan (to select for expression of genes encoded by both the bait and prey plasmids), and uracil and histidine (to select for protein interactions).
  • SC synthetic complete
  • SCS medium for SC Selective medium.
  • the selected clones were tested for expression of ⁇ -galactosidase to confirm the formation of a RB*RB-IP interaction.
  • Filter-lift ⁇ -galactosidase assays were then performed as a modified version of the protocol of Breeden and Nasmyth (1985. Cold Spring Harbor Quant. Biol. 50:643-650). Colonies were patched onto SCS plates, grown overnight, and replica plated onto Whatman No. 1 filters. The replica filters were subsequently assayed for ⁇ -galactosidase activity (i.e., positive colonies turned a visible blue).
  • the cells contained within colonies which were positive for protein interaction contained a mixture of DNA-binding and activation-domain plasmids. These cells were individually plated, and regrown as single isolates in individual wells of 96-well plates. Ten microliters of each isolate were lysed, the inserts within the pACT2 and pASS-1 plasmids were amplified by polymerase chain reaction using primers specific for the flanking sequences of each vector, and approximately 200 amino-terminal bases of each insert were determined using an ABI Model 377 sequenator. Comparison to known sequences was made using the "BLAST" program publicly available through the National Center for Biotechnology Information.
  • the determined nucleic acid sequences and corresponding amino acid sequences of the splice variants Set* and GluTl *, and the open reading frames encoding CD24* and 115392*, are shown in Figures 2, 4, 6, and 7, respectively. Determination of the splice variants sequences for Set*, and GluTl*, and the open reading frames (ORFs) encoding proteins CD24* and 115392*, is described in the Specific Examples Section, infra.
  • yeast cells were created that express the individual plasmids encoding RB, RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl (Nek 2), cyclophilin A, Zap 3, CD24*, Set*, GluTl*, and 115392*. These yeast cells were plated on SCS plates, grown overnight, and examined for growth. No growth was found for RB and all RB-IP proteins, confirming that they were not "self-activating" proteins, that is, these proteins require interaction with a second protein domain for a functional activation complex.
  • plasmids containing RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, CD24*, Set*, GluTl*, and 115392* inserts were transformed into strain N106 r (mating type alpha) and mated with yeast strain YULH (mating type a) expressing proteins other than RB.
  • Promiscuous binders that is, RB-IP able to bind with many other proteins in a non-specific fashion, would interact non-specifically with non-RB domains, and would be discarded as non-specific interactants. None of the interactants showed binding to proteins other than those described in the following paragraph.
  • the isolated bait plasmid for RB and separately the plasmid encoding human bait protein 1 (Bl) were used to transform yeast strain YULH (mating type a).
  • RB complexed specifically with RN-tre (Box A), CAS (Box B), IP-30 (Box C), L6 (Box D), IK (Box E), LDH-B (Box F), Nlkl (Nek 2) (Box G), cyclophilin A (Box I), Zap 3 (Box J), P2 (Box K), CD24* (Box L), Set* (Box M), GluTl* (Box N), and 115392* (Box O), as well as the known interactant RbAP46 (Box P). RB did not react non- specifically with the prey PI.
  • the intersection of the RB row (top) with the RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlkl(Nek 2), cyclophilin A, Zap 3, 17 beta-HSD6, CD24*, Set*, GluTl*, 115392*, and RbAP26 columns indicates growth (i.e., a positive interaction), but the intersection of the RB row with the column for PI indicates no growth (i.e., no protein interaction).
  • Regions within the 3' untranslated regions of the known protein cDNAs for CD24, Set, and GluTl were identified as encoding a protein or proteins that interact with RB using the improved, modified yeast two hybrid system.
  • the present invention discloses interacting nucleotide sequences identical to the nucleotide sequence of CD24 3' of nucleotide 689 (as depicted in Figure 1; SEQ ID NO:l); the nucleotide sequence of Set 3' of nucleotide 2175 ( Figure 3; SEQ ID No:6) and the nucleotide sequence of GluTl 3' of nucleotide 3408 (as depicted in Figure 5; SEQ ID No:9).
  • the human expressed clone 115392 was detected as an interacting prey, 3' of nucleotide 343 of clone 115392 sequence. This represented the first evidence that the 115392 sequence encodes a functional protein or protein domain.
  • an analysis was performed in order to ascertain whether there was any open reading frames (ORFs) within each of the three forward translation frames.
  • ORFs open reading frames
  • the interacting protein domains were encoded by sequences of known orientation within the prey sequence vector, and thus only forward translations could explain the detected interacting species.
  • Open reading frames (ORFs) were defined by performing a "BLAST" analysis with "ORF Finder". For CD24, only two open reading frames were detected.
  • the second open reading frame included the detected interacting region for the CD24 sequence.
  • This novel protein, called CD24* is depicted in Figure 2, Frame B [SEQ ID NO:4 for the nucleic acid sequence and SEQ ID NO:5 for the amino acid sequence]. It should be noted that no splice variant containing the interacting domain and an upstream 5'- coding sequence could be detected, using the methods described immediately above.
  • Determination of 5'- and 3 '-splice points for protein splice variants was performed as follows. First, potential 5 '-splice sites were identified in the coding sequence of the known protein. The sequence must contain an invariant GT sequence at the start of the intron. The remaining nucleotides were not invariant, but the preferred consensus sequence was AG:GTAAGT, with the colon indicating the splice point. See e.g., Padgett, et al, 1984. Ann. Rev. Biochem. 55:1119-1150. Potential splice sites were identified in order of their matching to this consensus, with a minimum of 4/6 matches beyond the invariant nucleotides.

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Abstract

La présente invention concerne des complexes de la protéine du rétinoblastome (RB) avec des protéines identifiées comme interagissant avec RB par un système amélioré de criblage à deux hybrides de levures modifiés. Les protéines identifiées comme devant interagir avec RB sont RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlk1(Nek 2), cyclophiline A, Zap 3, CD24*, Set*, GluT1* et 115392*. Ainsi, l'invention présente concerne des complexes de RB et de RN-tre, CAS, IP-30, L6, IK, LDH-B, Nlk1(Nek 2), la cyclophiline A, Zap 3, CD24*, Set*, GluT1* et 115392* ainsi que des dérivés, fragments et analogues desdits composés. L'invention concerne également les gènes et protéines CD24*, Set*, GluT1* et 115392* ainsi que des dérivés, fragments et analogues de ces composés. L'invention a également trait à des techniques de criblage de complexes visant à améliorer l'efficacité du traitement et/ou de la prévention de certaines maladies et troubles, notamment de pathologies hyperprolifératives dont le cancer, les maladies neurodégénératives et les maladies virales.
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CA2669520C (fr) 2006-11-13 2016-07-26 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Procedes de traitement d'un cancer a l'aide de molecules d'arnic dirigees contre cd24
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