+

WO1999032621A2 - Sequences d'acide nucleique codant des proteines de co-activateur de recepteur nucleaire et utilisation de ces sequences - Google Patents

Sequences d'acide nucleique codant des proteines de co-activateur de recepteur nucleaire et utilisation de ces sequences Download PDF

Info

Publication number
WO1999032621A2
WO1999032621A2 PCT/US1998/025478 US9825478W WO9932621A2 WO 1999032621 A2 WO1999032621 A2 WO 1999032621A2 US 9825478 W US9825478 W US 9825478W WO 9932621 A2 WO9932621 A2 WO 9932621A2
Authority
WO
WIPO (PCT)
Prior art keywords
src
receptor
transcription
steroid
cells
Prior art date
Application number
PCT/US1998/025478
Other languages
English (en)
Other versions
WO1999032621A3 (fr
Inventor
Chen-Shian Suen
Donald Edward Frail
Cecil Richard Lyttle
Original Assignee
American Home Products Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by American Home Products Corporation filed Critical American Home Products Corporation
Priority to AU18010/99A priority Critical patent/AU1801099A/en
Publication of WO1999032621A2 publication Critical patent/WO1999032621A2/fr
Publication of WO1999032621A3 publication Critical patent/WO1999032621A3/fr

Links

Classifications

    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the technical field of this invention concerns nucleic acid sequences encoding nuclear receptor associated proteins which are involved in gene transcription.
  • this invention concerns nucleic acid sequences encoding steroid nuclear receptor coactivators (SRC-3), proteins encoded by said nucleic acid sequences and uses thereof.
  • SRC-3 steroid nuclear receptor coactivators
  • Nuclear hormone receptors are a superfamily of transcription factors which as a class are involved in ligand-dependent transcriptional control of gene expression.
  • Steroid hormone receptors are a distinct class of the nuclear receptor superfamily, characterized in that the ligands are steroid hormones.
  • the receptors for glucocorticoids (GR), mineralcorticoids (MR), progestins (PR), androgens (AR) and estrogens (ER) are examples of classical steroid receptors.
  • GR glucocorticoids
  • MR mineralcorticoids
  • PR progestins
  • AR androgens
  • ER estrogens
  • this superfamily consists of receptors for non-steroid hormones such as vitamin D, thyroid hormones and retinoids.
  • Nuclear hormone receptors are characterized by a modular structure in comprising six distinct structural and functional domains, A to F: a variable N-terminal region (domain A/B), followed by a centrally located, highly conserved DNA-binding domain (hereinafter referred to as DBD; domain C), a variable hinge region (domain D), a conserved ligand-binding domain (hereinafter referred to as LBD; domain E) and a variable C-terminal region (domain F).
  • DBD DNA-binding domain
  • LBD conserved ligand-binding domain
  • domain F variable C-terminal region
  • the DBD consists of approximately 66 to 70 amino acids and is responsible for DNA-binding activity: it targets the receptor to specific DNA sequences called hormone responsive elements (hereinafter referred to as HRE) within the transcription control unit of specific target genes on the chromatin.
  • HRE hormone responsive elements
  • Steroid receptors such as GR, MR, PR and AR recognize similar HRE DNA sequences, while the ER recognizes a different DNA HER sequence. After binding to DNA, the steroid receptor is thought to interact with components of the basal transcriptional machinery and with sequence- specific transcription factors, thus modulating the expression of specific target genes.
  • a hormone ligand for a nuclear receptor When a hormone ligand for a nuclear receptor enters the cell and is recognized by the LBD, it will bind to the specific receptor protein, thereby initiating an allosteric alteration of the receptor protein (Cell, supra) . As a result of this alteration the ligand/receptor complex switches to a transcriptionally active state and as such is able to bind through the presence of the DBD with high affinity to the corresponding HRE on the chromatin DNA. In this way the ligand/receptor complex modulates expression of the specific target genes. The diversity achieved by this family of receptors results from their ability to respond to different ligands. Steroid hormone receptors are involved in embryonic development, adult homeostasis and organ physiology.
  • Estrogen E 2
  • E 2 exerts numerous biological effects in different tissues through an interaction with the ER (1 , 2).
  • Amino acid sequence analyses, transient transfection studies, and mutational dissections of ER indicate that ER has the classical modular structure described above (3).
  • the N-terminal A/B domain of the ER contains a transactivation function, referred to as transcriptional activation function 1 (TAF-1 ).
  • the DNA binding domain DBD contains two zinc fingers and is responsible for DNA recognition.
  • the LBD and a second transactivation function, referred to as TAF-2 is located at the C-terminal of ER. Upon binding to hormone, the ER undergoes an activation and transformation step.
  • the activated ER interacts with specific estrogen response elements (EREs) that are located in the promoter region of estrogen-regulated genes and influence its target gene transcription.
  • ERP estrogen response elements
  • ER ⁇ a new estrogen receptor
  • ER ⁇ appears to be distinct from the more commonly known estrogen receptor referred to as ER ⁇ .
  • the DBD of ER ⁇ is 90% identical to that of ER ⁇ .
  • the overall homology between the ligand binding domain (LBD) of ER ⁇ and ER ⁇ is less than 55 %.
  • LBD ligand binding domain
  • ER ⁇ can stimulate transcription from an ERE in a ligand-dependent manner.
  • the biological significance of the existence of two ER subtypes is not clear.
  • the potential functional differences and differential localization between ER ⁇ and ER ⁇ (7) may contribute to the selective actions of E 2 in different target tissues.
  • NRs nuclear hormone receptors
  • the ligand-activated NRs may stabilize and/or promote formation of the preinitiation complex (PIC) of the basal transcriptional apparatus and facilitate transcription by RNA polymerase II. These effects may be transmitted in part by direct interactions between NRs and basal transcriptional factors (8, 9, 10, 1 1 , 1 2).
  • NR- induced transcription of different target genes may be transmitted through indirect interactions, mediated by intermediary proteins called transcriptional coactivators.
  • intermediary proteins called transcriptional coactivators.
  • NR-associated proteins that interact with steroid/thyroid receptors have been reported (reviewed in 1 3, 14).
  • NR-associated proteins include unliganded thyroid hormone receptor (TR) and retinoic acid receptor (RAR)
  • TR thyroid hormone receptor
  • RAR retinoic acid receptor
  • proteins that interact with NRs in a ligand-dependent manner and augment transcription have also been identified (1 2).
  • TAF-1 steroid receptor coactivator-1
  • TAF-2 transcription intermediate factor-2
  • ARA70 androgen receptor-associated protein
  • CBP CREB binding protein
  • TAF n 1 35 2
  • NR-stimulated gene transcription was markedly enhanced by coexpression of these coactivators with NRs.
  • TAF-1 , TIF2 and CBP have been shown to augment ER ⁇ -stimulated gene transcription and to date, only TAF-1 has been shown to modulate ER ⁇ -mediated gene transcription (23).
  • ER- ⁇ or ER- ⁇ selectively interact with specific nuclear receptor coactivators and corepressors.
  • the present invention provides for novel NR coactivators, having ligand mediated activity which selectively interact with certain NRs.
  • NR coactivators are specifically steroid receptor coactivators, and in particular, are novel estrogen or progesterone receptor coactivators , which are able to regulate the expression of nuclear receptor-responsive genes, and which interestingly, demonstrate specific selectivity for ER ⁇ over ER ⁇ .
  • the present invention provides for nucleic acid sequences encoding a novel steroid receptor coactivator, SRC-3, that selectively enhances expression of ER- ⁇ , and PR-responsive genes. Additionally, SRC-3 augments ER ⁇ over ER ⁇ -mediated reporter gene transcription.
  • the present invention provides a novel nucleotide sequence encoding a SRC-3 having a putative amino acid sequence of SEQ ID No. 2.
  • the nucleotide sequence of human SRC-3 is shown in SEQ ID No. 1 .
  • Methods of using SRC-3 and SRC-3 encoding nucleic acids are also provided.
  • Recombinant SRC-3 and anti-SRC-3 antibodies find use in drug screening, diagnostics, and therapeutics.
  • the SRC-3s provide valuable reagents in developing specific biochemical assays for screening compounds that agonize or antagonize selected transcription factor receptors involved in regulating gene expression associated with human pathology, such as steroid-dependent cancers.
  • FIG. 3 Interactions between clone 31 and ER a.
  • Clone 31 the portion of nucleic acid sequence encoding SRC-3 corresponding to amino acids 620-1423 of SEQ ID no. 2, interacts with the LBD (TAF-2), but not the N-terminal (TAF-1 ), of hER a in yeast.
  • 31 /pACT2 plasmid was transformed with either TAF1 /pAS2 or TAF2/pAS2 plasmid into yeast strain CG-1 945 and plated on SD/-His/-Trp/-Leu in the presence or absence of 0.1 uM 1 7b-estradiol (E 2 ) to select for transformants in which the two proteins interact.
  • Clone 31 protein was immobilized on the surface of a CM 5 sensor chip (certified) and either ER a or ER b were injected across the surface in the presence of estradiol. The refractory units, a measure of the amount of estrogen receptor bound, is recorded over the period of time indicated.
  • E The interaction of clone 31 protein with ER a is influenced by ligand, as determined by SPR. A basal level of interaction is observed between clone 31 protein and ER a in the absence of ligand. Estradiol promoted the interaction of clone 31 protein with ER a and ICI 182,780 destabilized this interaction.
  • SRC-3 contains a transcription activation function when tethered to GAL4 DNA binding domain.
  • SRC-3 SRC-3/pM
  • clone 31 31 /pM
  • pG5CAT GAL4 response reporter
  • Modulating transcription means altering transcription, and includes changing the rate of transcription initiation, the level of transcription, or the responsiveness of transcription/transcription initiation to regulatory controls.
  • Neoplastic state of cells means any new growth of cells which may be benign or malignant.
  • substantially pure or isolated mean that the SRC-3 protein, SRC-3 protein fragment, or nucleic acid encoding a SRC-3 or SRC- 3 fragment are unaccompanied by at least some of the material with which it is normally associated in its natural state.
  • a composition of a substantially pure SRC-3 or portion thereof may contain excipients and additives useful in diagnostic, therapeutic and investigative reagents.
  • “Substantial sequence identity” means that a portion of the protein or nucleic acid presents at least about 70%, more preferably at least about 80%, and most preferably at least about 90% sequence identity with a SRC-3 sequence portion.
  • the differences are preferably conservative, i.e., an acidic for an acidic amino acid substitution or a nucleotide change providing a redundant codon.
  • Dissimilar sequences are typically aggregated within regions rather than being distributed evenly over the polymer.
  • a substantially identical sequence hybridizes to a complementary SRC-3- encoding sequence under low stringency conditions, for example, at 42 °C and 6x SSC (0.9M saline/0.09M sodium citrate) and that remains bound when subject to washing at 50°C with 1 x SSC.
  • a biologically active SRC-3 or SRC-3 -3 fragment retains one or more of the SRC-3's native functions such as the ability to specifically bind ER- ⁇ over ER- ⁇ or anti-SRC-3 antibodies, or to modulate or facilitate transcription or transcription initiation of selective NR. Exemplary assays for biological activity are described below and in the working exemplification.
  • a preferred embodiment of the present invention is a cDNA obtained from a human lymphoma library having a long open reading frame of about 4269 base pairs which encode a putative protein of about 1 423 amino acids with a predicted molecular weight of about 1 55 kilodaltons.
  • An additional preferred embodiment is a murine genomic nucleotide sequence corresponding to amino acids 1 9-631 of p/CIP.
  • Specific binding is empirically determined by contacting, for example a SRC-3, with a mixture of components and identifying those components that preferentially bind the SRC-3. Specific binding may be conveniently shown by a number of methods, including but not limited to yeast and mammalian hybrid systems and competitive binding studies. For example, a human cDNA library was screened using the yeast two-hybrid assay to identify potential SRC-3 coactivators. In yeast and mammalian cells, SRC- 3 interacted with the LBD of ER ⁇ , and this interaction was dependent on the presence of ligand. Furthermore, SRC-3 augmented ligand-induced transcriptional activity of ER a and progesterone receptor (PR).
  • SRC-3 Surface Plasmon resonance (SPR) analysis using a BIAcore 2000 system was used as described (26).
  • SRC-3 can be used in similar strategies to assess the interaction between SRC-3 and other known transcription factors, including other nuclear receptors, and to identify novel SRC-3 interacting proteins.
  • the potential for an interaction between SRC-3 and the transcription factor CBP was assessed using a transfection assay in mammalian cells.
  • SRC-3 enhanced the activity of the ER ⁇ and this stimulation was further enhanced in the presence of CBP, indicating the potential for an interaction between SRC-3 and CBP.
  • yeast two-hybrid system the mammalian two-hybrid system, surface plasmon resonance assays, immunoprecipitation assays, or other assays could be used to demonstrate a direct interaction between SRC-3 and CBP.
  • these assays systems could be used to broadly screen for known and novel proteins that interact with SRC-3.
  • the invention provides recombinantly produced SRC-3 proteins,
  • SRC-3 analogs and fragments thereof are readily modified through physical, chemical, and molecular techniques disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • fragments of the SRC-3-encoding sequences are spliced with heterologous sequences to produce fusion proteins.
  • Such fusion proteins find particular use in modulating gene transcription in vitro and in vivo.
  • a substantially pure or isolated SRC-3 or SRC-3 portion encoding nucleic acid is generally at least about 1 % nucleic acid weight of said SRC-3 encoding nucleic acid; preferably at least about 10%; more preferably at least about 50%; and most preferably at least 90% .
  • Nucleic acid weight percentages are determined by dividing the weight of the SRC-3 or SRC-3 portion encoding nucleic acid, including alternative forms and analogs such as alternatively spliced or partially transcribed forms, by the total nucleic acid weight present.
  • modified SRC-3 encoding sequences or related sequences encoding proteins with SRC-3-like functions there will generally be substantial sequence identity between at least a portion thereof and a portion of a SRC-3, preferably at least about 40%, more preferably at least 80%, most preferably at least 90%, particularly conservative substitutions, particularly within the leucine charged domains and regions encoding protein domains involved in protein-protein interactions, particularly SRC-3- transcription factor interactions.
  • SRC-3 encoding nucleic acids can be subject to alternative purification, synthesis, modification or use by methods disclosed herein or otherwise known in the art.
  • the nucleic acids can be modified to alter stability, solubility, binding affinity and specificity, methylation, etc.
  • the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
  • Exemplary labels include radioisotopes, fluorescers, biotinylation, etc.
  • Nucleic acids encoding at least a portion of a SRC-3 are used to identify nuclear factors which interact with that SRC-3 using expression screening in yeast or mammalian cells as described in Current Protocols in Molecular Biology.
  • a yeast cDNA library containing fusion genes of cDNA joined with DNA encoding the activation domain of a transcription factor are transfected with fusion genes encoding a portion of a SRC-3 and the DNA binding domain of a transcription factor.
  • Clones encoding SRC-3 binding proteins provide for the complementation of the transcription factor and are identified through transcription of a reporter gene. See, e.g. Fields and Song (1 989) Nature 340, 245-246 and Chien et al.
  • the invention also provides vectors comprising nucleic acids encoding a SRC-3 or portion or analog thereof.
  • vectors comprising nucleic acids encoding a SRC-3 or portion or analog thereof.
  • a large number of vectors, including plasmid and viral vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts.
  • Vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • the inserted SRC-3 coding sequences may be synthesized, isolated form natural sources, prepared as hybrids, etc. Ligation of the coding sequences to the transcriptional regulatory sequences may be achieved by known methods.
  • vectors may also include a promoter operably linked to the SRC-3 encoding portion.
  • Suitable host cells may be transformed/transfected/infected by any suitable method including electroporation, CaCI 2 mediated DNA uptake, viral infection, microinjection, microprojectile, or other established methods.
  • nucleic acids encoding one or more SRC-3s may be introduced into cells by recombination events. For example, a sequence can be microinjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene encoding a SRC-3, an analog or pseudogene thereof, or a sequence with substantial identity to a SRC-3-encoding gene.
  • recombination based methods such as non- homologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, etc.
  • Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. subtiiis, Saccharomyces cerevisiae, A549 cells and CHO, COS, HeLa cells and immortalized mammalian myeloid and lymphoid cell lines.
  • Preferred host cell lines include but are not limited to human cancer cell lines such as MCF-7 and T47D cells.
  • such expression systems utilize inducible expression strategies like the TET ON/OFF system that is commercially available.
  • Such cell lines are useful to define the role of SRC-3 in tumorigenesis and to assess the effect of potential compounds that modulate SRC-3 interactions and activity on tumorigenesis.
  • a large number of transcription initiation and termination regulatory elements/regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art.
  • the particular choice of vector/host cell is not critical to the invention.
  • SRC-3 encoding oligonucleotides can also be used to identify other SRC-3s or transcription factor coactivators. For example, 32 P-labeled SRC- 3 encoding nucleic acids are used to screen cDNA libraries at low stringency to identify similar cDNAs that encode proteins with SRC-3 related domains. Additionally, SRC-3 related proteins are isolated by PCR amplification with degenerate oligonucleotide probes using the sequences disclosed herein. Other experimental methods for cloning SRC-3 are also set out in the working exemplification below. Other useful cloning, expression, and genetic manipulation techniques for practicing the inventions disclosed herein are known to those skilled in the art.
  • compositions and methods disclosed herein may be used to effect gene therapy. See, e.g., Gutierrez et al. (1 992) Lancet 339, 71 5- 721 .
  • cells are transfected with SRC-3 sequences operably linked to gene regulatory sequences capable of effecting altered SRC-3 expression or regulation.
  • cells may be transfected with SRC-3 complementary antisense polynucleotide.
  • Antisense modulation may employ SRC-3 antisense sequences operably linked to gene regulatory sequences.
  • Cells are transfected with a vector comprising a SRC-3 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to SRC-3 encoding mRNA.
  • Transcription may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration.
  • single-stranded antisense nucleic acid sequences that bind to genomic DNA or mRNA encoding at least a portion of SRC-3 may be administered to the target cell at a concentration that results in a substantial reduction in SRC-3 expression.
  • the invention provides methods and compositions for identifying agents useful in modulating gene transcription.
  • agents find use in the diagnosis or treatment of broad range of disease including, but not limited to, cancer, cardiovascular diseases, microbial and fungal interactions and particularly immune disease, bone protection, etc.
  • the ability to develop rapid and convenient high-throughput biochemical assays for screening compounds that interfere with the process of transcription in human cells opens a new avenue for drug development. An overview of this therapeutic approach is presented in Peterson & Baichwal ( 1 993), Trends in Biotechnology.
  • prospective agents are screened from large libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds, see, e.g. Lam et al. ( 1 991 ) Nature 354, 82- 86. Alternatively, libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. Examples of such modifications are disclosed herein. Useful agents are identified with a range of assays employing SRC-
  • SRC-3 encoding nucleic acids.
  • protein binding assays include assaying labeled nuclear receptor binding to immobilized SRC-3, labeled SRC-3 or SRC-3 peptide binding to immobilized nuclear receptors, etc.
  • Many appropriate assays are amenable to scaled- up, high throughput usage suitable for volume drug screening. Such screening will typically require the screening of at least about 10, preferably at least about 100, and more preferably at least about 1000 prospective agents per week. The particular assay used will be determined by the particular nature of the SRC-3 interactions.
  • Assays may employ single SRC-3s, SRC-3 fragments, SRC-3 fusion products, partial SRC-3 /complexes, or the complete basal transcription complex comprising an SRC-3 nucleic acid, depending on the associational requirements of the subject transcription factor.
  • Useful agents are typically those that bind to or modify the association of transcription associated coactivators, especially SRC-3s.
  • Preferred agents include those capable of modulating the expression of Pol II genes, particularly oncogenes and genes transcribed by members of the nuclear receptor superfamily. Preferred agents modify transcription complexes comprising SRC-3.
  • Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like.
  • Structural identification of an agent may be used to identify, generate, or screen additional agents.
  • peptide agents may be modified in a variety of ways to enhance their stability, such a using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxyl terminus, e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.
  • Other methods of stabilization may include encapsulation, for example, in liposomes, etc.
  • Agents may be prepared in a variety of ways known to those skilled in the art. For example, peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers. Alternatively, peptide (and protein and nucleic acid agents) are readily produced by known recombinant technologies.
  • Such assay systems could also be used to screen for potential therapeutics, including peptide and chemical ligands.
  • potential therapeutics, activators or inhibitors of SRC-3 function could act by either modulating an interaction between SRC-3 and an interacting protein ("class I") or by modulating an activity of SRC-3, including an enzymatic activity (“class II”).
  • Interaction assays including but not limited to two-hybrid assays, immunoprecipitation assays, and surface plasmon resonance (SPR) assays, could be used to identify "class I" therapeutics.
  • chemical compounds or peptides can be screened for their ability to modulate the interaction between SRC-3 and ER ⁇ by SPR.
  • Activity assays including but not limited to mammalian transfection assays in which SRC-3 enhancement of an nuclear receptor response is observed, could be used to identify "class M" compounds.
  • compounds could be screened for their ability to modulate the enhancement of nuclear receptor activity by SRC-3.
  • Such compounds may modulate the interactions of SRC-3 with interacting proteins or they may modulate a known or unknown activity of SRC-3, including an enzymatic activity.
  • compositions and selected agents disclosed herein may be administered by any convenient way that will depend upon the nature of the compound.
  • oral administration is preferred and enteric coatings may be indicated where the compound is not expected to retain after exposure to the stomach environment.
  • the amount administered will be empirically determined, typically in the range of about 1 to 1000 ⁇ g/kg of recipient.
  • Large proteins are preferably administered parenterally, conveniently in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like.
  • a physiologically acceptable carrier e.g., phosphate buffered saline, saline, deionized water, or the like.
  • such compositions are added to a retained physiological fluid such as blood or synovial fluid.
  • the amount administered will be empirically determined, typically in the range of about 10 to 1000 ⁇ g/kg of the recipient.
  • Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts.
  • SRC-3s may also be modified with a label capable of providing a detectable signal either directly or indirectly.
  • exemplary labels include radioisotopes, fluorescers, etc.
  • a SRC-3 may be expressed in the presence of a labeled amino acid such as 35 S-methionine.
  • labeled SRC-3s and analogs thereof find use, for example, as probes in expression screening assays for proteins that interact with SRC-3s, or, for example, SRC-3 binding to other transcription factors in drug screening assays.
  • Anti-SRC-3 antibodies and fragments (Fab, etc.) thereof find use in modulating SRC-3 involvements in transcription complexes, screening SRC- 3 expression libraries, etc. In addition, these antibodies can be used to identify, isolate, and purify structural analogs of SRC-3s. Anti-SRC-3 antibodies also find use for subcellular localization of SRC-3s under various conditions such as infection, during various cell cycle phases, induction with cytokines, protein kinases such as C and A, etc.
  • compositions are also provided for therapeutic intervention in disease, for example, by modifying SRC-3s or SRC-3 encoding nucleic acids.
  • Oiigopeptides can be synthesized in pure form and can find many uses in diagnosis and therapy. These oiigopeptides can be used, for example, to modulate native SRC-3 interaction with other SRC-3s or other transcription factors or DNA.
  • the oiigopeptides will generally be more than six and fewer than about 60 amino acids, more usually fewer than about 30 amino acids, although large oiigopeptides may be employed.
  • a SRC-3 or a portion thereof may be used in purified form, generally greater than about 50%, usually greater than about 90% pure. Methods for purifying such peptides to such purities include various forms of chromatographic, chemical, and electrophoretic separations disclosed herein or otherwise known to those skilled in the art.
  • This invention is based on the molecular cloning, structural analysis and characterization of nucleic acid sequences encoding SRC-3.
  • the present invention demonstrates that a nucleic acid sequence encoding a portion of human SRC-3, i.e., clone 31 , preferentially interacted with ER ⁇ over ER ⁇ (Figs. 3C, 3D), and the transcriptional activity of ER a but not ER b was augmented by SRC-3 (Figs. 4B, 4C).
  • the transcriptional enhancement was not exclusively limited to ER a, since SRC-3 also enhanced PR-stimulated transcriptional activity.
  • smaller amounts of SRC-3 were required to observe the transcriptional augmentation of PR than for ER a (Figs.
  • the present invention provides that SRC-3 enhances transcriptional activity of both the TAF-2-defective ER a (ER a-TAF-1 ) and the TAF-1 deleted ER a (ER a-TAF-2) (Fig. 5B).
  • SRC-3 had no effect on the transcriptional activity of a construct in which both the TAF-1 is deleted and the TAF-2 is mutated (ER a-null), suggesting that SRC-3 activity required the presence of an intact TAF domain.
  • a transcriptional coactivator enhances gene transcription by bridging transcription factors with the components of the basal transcriptional machinery. Therefore, a transcriptional coactivator would be expected to have a transcriptional activation function that activates target gene expression through either the disruption of the nucleosome structure or the stabilization of the pre-initiation complex (39, 40, 41 ), resulting in an increased rate of transcription initiation.
  • a transcriptional coactivator would be expected to have a transcriptional activation function that activates target gene expression through either the disruption of the nucleosome structure or the stabilization of the pre-initiation complex (39, 40, 41 ), resulting in an increased rate of transcription initiation.
  • TAF-1 , TIF2, and CBP both clone 31 and SRC-3 activated heterologous gene transcription when fused to the DNA binding domain of GAL4, thus demonstrating the presence of a transcriptional activation function.
  • LXXLL consensus core motifs
  • AIB1 the gene encoding SRC-3, referred as AIB1 , was identified as a segment of chromosome 20 that is amplified in some primary breast tumors and the expression of AIB1 mRNA was increased in over half of these tumors (35).
  • SRC3/AIB1 may contribute to the development of cancers in tissues in which it is expressed, particularly steroid-dependent cancers (35).
  • Plasmid pM is a commercially available mammalian expression vector that expresses an insert as a fusion protein with GAL4 DNA binding domain.
  • a Northern analysis using 5 mg of poly A + mRNA from various human tissues was performed to determine the size of the message and the tissue distribution of SRC-3.
  • Poly A + mRNAs (5 mg) of various human tissues were separated in a formaldehyde gel and transferred to Hybond-N membrane (Amersham, Arlington Heights, IL).
  • Prehybridization was carried out for 4 hours in 50% formamide, 5X SSPE, 5X Denhardt's solution, 1 % glycine and 100 ug/ml denatured salmon sperm DNA at 42° C.
  • Hybridization was conducted overnight under the same conditions with 2 X 10 6 cpm/ml denatured probe (0.8-kilobase pair fragment of SRC-3).
  • the membrane was washed in 0.1 X SSC, 0.1 % SDS at 65° C for 30 minutes and exposed to X-OMAT AR film (Kodak, Rochester, NY) overnight at -80° C with intensifying screens.
  • Fig. 2A A major mRNA transcript with an approximate size of 8.5 to 9.0 kb was detected (Fig. 2A).
  • the transcript was abundant in uterus, mammary gland, pituitary, testis, heart, and skeletal muscle, it is relatively low in bone marrow, and it is barely detectable in liver, lung, brain, kidney, stomach, and adrenal gland (Fig. 2A and data not shown) indicating that SRC-3 is differentially expressed among tissues.
  • a minor species " 5.5 kb
  • the transcript of SRC-3 was expressed abundantly in Burkitt's lymphoma cells, colorectal SW480 cells (Fig. 2B) and MCF-7 breast cancer cells (data not shown).
  • Human cancer cells lines including MCF-7 human breast cancer cells, A549 human lung cancer cells, HeLa human cervical carcinoma cells, and Ishikawa endometrial adenocarcinoma cells, were grown in 1 50 mm cell culture plates with Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), penicillin ( 100 units/ml), and streptomycin 100 (mg/ml). When cells reached confluence, nuclear extract and cytosolic proteins were prepared as previously described (25).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin 100 units/ml
  • streptomycin 100 streptomycin 100
  • SRC-3 protein was detected using a rabbit anti-peptide antibody specific to an internal 1 5 amino acid sequence (91 2 to 926) that shows minimal sequence similarity to both TAF-1 and TIF-2. The membrane was then incubated with horseradish peroxidase-conjugated second antibody. The immunoreactive SRC-3 was visualized using the ECL detection system (Amersham, Arlington Heights, IL) following the procedures recommended by the supplier.
  • Expression plasmids containing SRC-3, or fragments of SRC-3 are created to make cell lines to monitor the role of SRC-3 in tumorigenesis.
  • a vector expressing SRC-3 antisense sequences, or fragments of SRC-3 that possess dominant negative phenotypes, are expressed in monitored MCF-7 cells which express high levels of SRC-3 as shown.
  • the tumorigenicity of the resulting cell lines are correlated with the reduced levels of SRC-3 expression.
  • a vector containing SRC-3 sequences are expressed in T47D cells, a breast cancer cell line expressing low levels of SRC-3, or other cancer cell lines expressing low levels of SRC-3, and the tumorigenicity of the resulting cell lines are correlated with the increased levels of SRC-3 expression.
  • Antibodies that recognize two different peptide sequences of SRC-3 were generated in rabbits to examine the expression of SRC-3 protein.
  • a single band with a molecular weight of approximately 1 60 kD was detected predominantly in the nuclear fraction of MCF-7 breast cancer cells, suggesting SRC-3 is present in the nucleus (Fig. 2C).
  • Western blot analysis of nuclear extracts prepared from several human cancer cell lines clearly showed that SRC-3 was highly expressed and most abundant in human breast cancer MCF-7 cells (Fig. 2D).
  • SRC-3 protein was detected in whole cell lysates of a number of different human tumor cell lines, including breast, ovary, prostate, colon, lung. The quantity of protein differed among the different tumor cell lines (Fig 2E and F).
  • a sandwich ELISA assay was used also used to determine the quantity of SRC-3 in extracts.
  • one antibody to SRC-3 was immobilized on a surface (capture antibody), plates were washed, nuclear extracts were incubated with a different SRC-3 antibody that was biotinylated (detection antibody), plates were washed, and the amount of SRC-3 was quantified using a reaction that detects the amount of detection antibody bound, and therefore the amount of SRC-3 in the sample.
  • an increasing amount of nuclear extract is used, an increasing amount of signal was obtained, and this signal was linear in the range of 0.1 to 0.3 OD units.
  • the amount of SRC-3 detected in MCF-7 cells was greater than that observed in the A549 cells.
  • SRC-3 protein was also be detected in MCF-7 and A549 cells by immunofluorescence using SRC-3 antibodies.
  • Cells were grown on chamber slides, fixed in paraformaldehyde, incubated with SRC-3 antibodies, washed, and incubated with a fluorescent labeled second antibody.
  • yeast two-hybrid protein/protein interaction assays both the N-terminal (TAF-1 ) and the ligand binding domain (TAF-2) of hER a were ligated into the pAS2 vector, and clone 31 , the original clone identified in yeast that contains amino acids 621 -1423 of SRC-3, was subcloned into the pACT2 plasmid.
  • plasmids Following cotransformation of plasmids into yeast strain CG 1 945, colonies were selected on SD/-His/-Trp/-Leu plates.
  • the ligand binding domain (LBD) of hER a and hER b were ligated into the pM vector (ER a/pM, and ER b/PM), and clone 31 was ligated into the pVP1 6 vector (31 /VP1 6).
  • the reporter plasmid expressing the chloramphenicol acetyltransferase (CAT) gene was under control of the GAL4 response element (pG5CAT).
  • Human lung A549 cells were routinely maintained in MEM containing 5% fetal bovine serum (FBS). Cells were seeded into 6-well plates (Falcon, Franklin Lakes, NJ), and plasmids were transiently introduced by using the calcium phosphate co-precipitation method protocol (Promega, Madison, Wl). Transfections were done in the presence or absence of 1 7 ⁇ -estradiol (10 nM) using plasmids pSVGal (0.25 ⁇ g) as an internal control, reporter plasmid pG5CAT (2 //g), ER/pM (0.8 ⁇ g), 31 /VP1 6 (0.8 ⁇ g), and pGEM-4Z as a carrier DNA (Promega, Madison, Wl). After 48 hours, cells were harvested and extracts were assayed for CAT and b-galactosidase activities, b-galactosidase activity was used to correct for differences in transfection efficiency.
  • FBS fetal bovine serum
  • clone 31 In yeast, clone 31 , a nucleic acid sequence encoding the last 801 amino acids of SRC-3, interacted with the LBD (TAF-2), but not the N- terminal (TAF-1 ) of hER ⁇ (Fig. 3A). Furthermore, this interaction was dependent upon the presence of ligand (Fig. 3A) . To test whether this interaction between the hER ⁇ and clone 31 occurs in mammalian cells, a mammalian two-hybrid protein-protein interaction assay was conducted.
  • SRC-3 transcriptional coactivator activity was measured by a luciferase assay in which CHO cells were maintained in DMEM/F-1 2 tissue culture medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin ( 100 ug/ml).
  • FBS fetal bovine serum
  • penicillin 100 units/ml
  • streptomycin 100 ug/ml
  • CHO cells 1 .5 X 10 5 cells/well
  • phenol red-free DMEM/F-1 2 supplemented with 5% charcoal dextran-treated FBS.
  • cells were transfected with 0.05 mg receptor expression vector, 2 mg reporter plasmid, and 0 to 3 mg of SRC-3 expression vectors.
  • RSV-bgal served as an internal control and pGEM4Z plasmid used as a carrier DNA. After 4 hours, cells were treated with 10% glycerol and incubated in the presence or absence of 10 nM E 2 and/or 1 mM of the anti-estrogen ICI
  • Luciferase assays were performed according to the supplier's protocol (Promega, Madison, Wl), and light emission was detected using a microlumat LB96P luminometer (Wallac Inc., Gaithersburg, MD). In the absence of clone 31 (31 /VP1 6), E 2 ( 10 nM) stimulated CAT activity approximately 8-fold when cells were transfected with the LBD of ER ⁇ (ER ⁇ /PM), consistent with the presence of a known transcription activation function (TAF-2) in this region (Fig. 3B).
  • TAF-2 transcription activation function
  • E 2 -stimulated CAT activity was augmented by an additional 10 to 1 5 fold.
  • hER human ER
  • ICI 1 82,780 inhibited this interaction.
  • CAT activity was stimulated by E 2 when cells were transfected with the LBD of ER b (ER b/pM), suggesting the presence of a transcription activation function within the LBD of ER b.
  • E 2 -stimulated CAT activity was only marginally enhanced (Fig. 3C).
  • clone 31 preferentially interacts with the LBD of ER ⁇ .
  • SPR assays using a BIAcore 2000 system (Pharmacia Biosensor, Uppsala, Sweden) were used as described previously (26). Briefly, clone 31 , ER a, and ER b were expressed in prokaryotic expression vectors, pET-28 (Novagen, Madison, Wl) and pFLAGMAC (Kodak-IBI, Rochester, NY), and purified proteins (approximately 80% pure) were obtained using the Ni + 2 affinity, M2-agarose affinity, Superdex 200, and Mono Q columns.
  • One of the interacting components, clone 31 was immobilized on a dextran layer bound to a gold surface (sensor chip), while the other component, either ER ⁇ or ER b, was provided by constant flow.
  • the SPR detector records changes in the refractive index of the medium close to the dextran layer, which is in turn directly proportional to the mass of macromolecules bound to the surface. The response is converted to arbitrary resonance units and plotted as a function of time.
  • a surface with 347 RU of the immobilized clone 31 was titrated with increasing amounts of purified ER a and ER b and saturable interactions were detected for both proteins (data not shown).
  • the transcriptional activity of ER a in the presence or absence of SRC-3 was assessed to determine if SRC-3 is a transcriptional coactivator that enhances E 2 -stimulated gene transcription.
  • SRC-3 1 7 ⁇ -estradiol (E 2 ) stimulated ER a-mediated reporter gene transcription approximately 4 to 5 fold over that of the control (Fig. 4A).
  • E 2 1 7 ⁇ -estradiol
  • the E 2 -stimulated transcriptional activity was enhanced up to 22 fold in the presence of increasing amounts of SRC-3.
  • Anti-estrogen ICI 1 82,780 completely blocked this transcriptional augmentation.
  • SRC-3 did not affect basal level transcription of ER a under the assay conditions used (data not shown).
  • Example 4 The protein/protein interaction assay described in Example 4 above demonstrated a preferential interaction of clone 31 with the LBD of ER a over ER b (Fig. 3C). Therefore, we investigated whether ER b-stimulated reporter gene transcription could also be augmented by SRC-3. As in the case of hER a, E 2 stimulated ER b-mediated reporter gene transcription (Fig. 4C). However, this ER b-stimulated transcriptional activity was not augmented with increasing amounts of SRC-3 (Fig. 4C). In some experiments, a slight augmentation was observed, but never more than two fold.
  • a radioligand binding assay indicated that similar amounts of ER a and ER b were expressed in the assay, a result consistent with the similar amounts of transcriptional activity observed in the absence of SRC-3 (data not shown).
  • This preferential stimulation of ER a activity by SRC-3 is consistent with our previous finding that clone 31 , a C-terminal derivative of SRC-3, interacted preferentially with the LBD of the ER a.
  • TAF transactivation function
  • SRC-3 contains an autonomous transcriptional activation function
  • both full length SRC-3 and a fragment thereof, namely, clone 31 were fused to the DNA binding domain (DBD) of GAL (1 -147) and transfected into the CHO cells with a GAL4 reporter plasmid.
  • the reporter activity was stimulated by both SRC-3 (SRC-3/pM) and clone 31 (31 /pM) (Fig. 6).
  • SRC-3 SRC-3/pM
  • clone 31 31 /pM
  • a fragment of SRC-3 was isolated from the mouse genome by PCR. This fragment was sequenced (SEQ ID NO 3) and encodes amino acids 1 9- 631 of the mouse protein. These amino acids are contained within three short exons separated by two large introns, suggesting that the genomic structure of SRC-3 is quite complex. This fragment can now be used as a probe to isolate larger genomic fragments encoding the entire SRC-3 mouse gene from libraries containing 100 kb inserts or more. Fragment SRC-3/1 9- 631 is used to create a vector containing a selection marker embedded within 5' and 3' genomic sequences and the vector used to obtain homologous recombinants in embryonic stem cells to functional "knockout" sequences 1 9-631 , or any part thereof.
  • Tremblay GB Tremblay A
  • Copeland NG Gilbert D
  • Jenkins NA Labrie F
  • Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein. Mol Cell Biol 1 5: 1 554-1 563.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Steroid Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne un nouveau co-activateur 3 de récepteur stéroïde (SRC-3), ainsi qu'un co-activateur de transcription de récepteur nucléaire stéroïde qui a été cloné et caractérisé. Le SRC-3 représente un nouveau membre qui appartient à une famille de co-activateurs de récepteur stéroïde et qui comprend TAF-1 et TIF-2. Le SRC-3 a fait preuve d'une transcription de gène stimulée par récepteur nucléaire et sélective dans des conditions de dépendance envers les ligands. Cette invention concerne également des dosages d'interaction protéine-protéine qui comprennent des analyses BIACORE et qui ont prouvé que l'interaction entre SRC-3 et ER était bien plus importante que celle observée entre SRC-3 et ER b. Une fonction de transactivation intrinsèque a été observée dans la moitié de la terminaison C du SRC-3. Le SRC-3 a été exprimé de manière différentielle dans divers tissus et, parmi plusieurs cellules tumorales examinées, était plus abondant dans la fraction nucléaire de cellules du cancer du sein MCF-7. Le SRC-3 représente ainsi un troisième membre de la famille des co-activateurs de récepteur stéroïde, et possède une répartition tissulaire distincte ainsi qu'une sélectivité intrinsèque entre ER et ER b.
PCT/US1998/025478 1997-12-22 1998-12-01 Sequences d'acide nucleique codant des proteines de co-activateur de recepteur nucleaire et utilisation de ces sequences WO1999032621A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18010/99A AU1801099A (en) 1997-12-22 1998-12-01 Nucleic acid sequences encoding nuclear receptor coactivator proteins and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6851197P 1997-12-22 1997-12-22
US60/068,511 1997-12-22

Publications (2)

Publication Number Publication Date
WO1999032621A2 true WO1999032621A2 (fr) 1999-07-01
WO1999032621A3 WO1999032621A3 (fr) 1999-09-16

Family

ID=22083052

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/025478 WO1999032621A2 (fr) 1997-12-22 1998-12-01 Sequences d'acide nucleique codant des proteines de co-activateur de recepteur nucleaire et utilisation de ces sequences

Country Status (2)

Country Link
AU (1) AU1801099A (fr)
WO (1) WO1999032621A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11497772B2 (en) 2020-10-28 2022-11-15 Baylor College Of Medicine Targeting of SRC-3 in immune cells as an immunomodulatory therapeutic for the treatment of cancer

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0871729A1 (fr) * 1995-09-15 1998-10-21 Baylor College Of Medicine Compositions coactivatrices de recepteurs de steroides et leurs procedes d'utilisation
ES2281105T3 (es) * 1996-07-12 2007-09-16 Institut National De La Sante Et De La Recherche Medicale (Inserm) Factor intermediario de transcripcion-2 tif2.
CA2295332C (fr) * 1997-06-17 2011-02-15 Paul Meltzer Nouveau co-activateur du recepteur steroidien,le aib1

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11497772B2 (en) 2020-10-28 2022-11-15 Baylor College Of Medicine Targeting of SRC-3 in immune cells as an immunomodulatory therapeutic for the treatment of cancer
US11633428B2 (en) 2020-10-28 2023-04-25 Baylor College Of Medicine Targeting of SRC-3 in immune cells as an immunomodulatory therapeutic for the treatment of cancer
US11633429B2 (en) 2020-10-28 2023-04-25 Baylor College Of Medicine Targeting of SRC-3 in immune cells as an immunomodulatory therapeutic for the treatment of cancer

Also Published As

Publication number Publication date
AU1801099A (en) 1999-07-12
WO1999032621A3 (fr) 1999-09-16

Similar Documents

Publication Publication Date Title
Boersma et al. Estrogen Receptors alpha and beeta Two Receptors of a Kind
Suen et al. A transcriptional coactivator, steroid receptor coactivator-3, selectively augments steroid receptor transcriptional activity
US5639616A (en) Isolated nucleic acid encoding a ubiquitous nuclear receptor
Vadlamudi et al. Functional and biological properties of the nuclear receptor coregulator PELP1/MNAR
Castillo et al. A divergent role of COOH-terminal domains in Nurr1 and Nur77 transactivation
US6476206B1 (en) p40 protein acts as an oncogene
JP2006068010A (ja) レチノイドｘ受容体相互作用性ポリペプチドならびに関連する分子および方法
US7232890B2 (en) AIB1, a novel steroid receptor co-activator
AU771564B2 (en) Androgen receptor coactivators
JP2008156360A (ja) 新規な核内受容体の非ゲノム活性のモジュレーター（ｍｎａｒ）およびその使用
Obendorf et al. FoxG1, a member of the forkhead family, is a corepressor of the androgen receptor
WO1999032621A2 (fr) Sequences d'acide nucleique codant des proteines de co-activateur de recepteur nucleaire et utilisation de ces sequences
Elsholtz Molecular biology of prolactin: cell-specific and endocrine regulators of the prolactin gene
WO1989012687A1 (fr) Recepteur d'acide retinoique
Meng et al. ERR-10: a new repressor in transcriptional signaling activation of estrogen receptor-α
AU2002212142B2 (en) Coaster, a human coactivator of steroid receptors
WO2002077229A2 (fr) Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
WO2002083726A2 (fr) Nouvelle proteine a doigt de zinc et ses utilisations
AU2002212142A1 (en) Coaster, a human coactivator of steroid receptors
Guo Human androgen receptor-associated hereditary prostate cancer factor and its function.
US20020197670A1 (en) Membrane associated progesterone receptor
EP1489172A1 (fr) Recepteur nucleaire err gamma 3
NIKODEM A Divergent Role of COOH-Terminal Domains in Nurrl and Nur77 Transactivation
Steketee Molecular mechanisms of androgen receptor functions
Nawaz Role of the PY Motif Containing Protein, WBP-2 in ER, PR Signaling and Breast Tumorigenesis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载