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WO1989009223A1 - Proteines de liaison d'adn comprenant un recepteur d'androgene - Google Patents

Proteines de liaison d'adn comprenant un recepteur d'androgene Download PDF

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Publication number
WO1989009223A1
WO1989009223A1 PCT/US1989/001238 US8901238W WO8909223A1 WO 1989009223 A1 WO1989009223 A1 WO 1989009223A1 US 8901238 W US8901238 W US 8901238W WO 8909223 A1 WO8909223 A1 WO 8909223A1
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dna sequence
dna
androgen receptor
cdna
androgen
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PCT/US1989/001238
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English (en)
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Shutsung Liao
Chawnshang Chang
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Arch Development Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to DNA binding regulatory proteins and more particularly to DNA sequences encoding androgen receptor protein and novel DNA binding proteins designated TR2, to the polypeptide products of recombinant expression of these DNA sequences / to peptides whose sequences are based on amino acid sequences deduced from these DNA sequences/ to antibodies specific for such proteins and peptides, and to procedures for detection and quantification of such proteins and nucleic acids related thereto.
  • steroid hor ⁇ mones There are five major classes of steroid hor ⁇ mones: progestins, glucocorticoids, mineralocorticoids, androgens / and estrogens.
  • Receptor proteins/ each specific for a steroid hormone/ are distributed in a tissue specific fashion and in target cells, steroid hormones can form specific complexes with corresponding intracellular receptors.
  • Androgens such as testosterone
  • Androgens are respons- ible for the development of male secondary sex charac ⁇ teristics and are synthesized primarily in testis.
  • Cloning of a cDNA for androgen receptor (AR) has been difficult because, until recently, monospecific antibodies against AR have not been available for screening cDNA libraries.
  • Anti-AR autoantibodies were identified in the sera of prostate cancer patients, as described in Liao, S., et al., Proc. Nat'l. Acad. Sci. (USA), 82:8345 (1984) (one of the co-inventors herein), and were characterized with respect to their titer, affinity, and specificity. Subsequently, lymphocytes from the blood of those patients having high antibody titers were isolated, transformed with Epstein-Barr Virus (EBV), and cloned for anti-AR monoclonal antibody production. These monoclonal antibodies were found to interact with androgen receptors from rat prostate. An attempt to scale-up antibody production resulted in a decline of antibody secretion.
  • EBV Epstein-Barr Virus
  • novel DNA sequences comprise cDNA sequences encoding human and rat androgen receptor and human TR2 protein.
  • Alternate DNA forms such as genomic DNA, and DNA prepared by partial or total chemical synthesis from nucleotides as well as DNA with deletions or mutations, is also within the contemplation of the invention.
  • DNA sequences provided by the invention with homologous or heterologous species expression control DNA sequences, such as promoters, operators, regulators and the like, allows for iri vivo and ⁇ n vitro transcription to form messenger RNA which, in turn, is susceptible to translation to provide androgen receptor and TR2 proteins, and related poly- and oligo-peptides in large quantities.
  • AR and TR2 encoding DNA is operatively associated with a viral (T7) regulatory (promoter) DNA sequence allowing for in vitro transcription and translation in a cell free system to provide, e.g., a 79 kD and 98 kD human androgen receptor (hAR) protein, 79 kD and 98 kD rat androgen receptor (rAR) protein and smaller forms of these proteins, as well as TR2 protein, including 20 kD and 52 kD species.
  • T7 regulatory (promoter) DNA sequence allowing for in vitro transcription and translation in a cell free system to provide, e.g., a 79 kD and 98 kD human androgen receptor (hAR) protein, 79 kD and 98 kD rat androgen receptor (rAR) protein and smaller forms of these proteins, as well as TR2 protein, including 20 kD and 52 kD species.
  • Systems provided by the invention included transformed E. coli DH5 ⁇ cells, deposited January 25, 1989, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 in accordance with the U.S. Patent and Trademark Office's requirements for microorganism deposits, and designated EC-hAR3600 under A.T.C.C. Accession No. 67879; EC-rAR 2830, A.T.C.C. No. 67878; EC-TR2-5, A.T.C.C. No. 67877; and EC TR2-7, A.T.C.C. No. 67876.
  • Novel protein products of the invention include polypeptides having the primary structural con ⁇ formation (i.e., amino acid sequence) of AR and TR2 proteins as well as peptide fragments thereof and syn ⁇ thetic peptides assembled to be duplicative of amino acid sequences thereof.
  • Proteins, protein fragments, and synthetic peptides of the invention are projected to have numerous uses including therapeutic, diagnostic and prognostic uses and will provide the basis for prepara ⁇ tion of monoclonal and polyclonal antibodies specifi- cally immunoreactive with AR and TR2 proteins.
  • Preferred protein fragments and synthetic peptides include those duplicating regions of AR and TR2 proteins which are not involved in DNA binding functions and the most preferred are those which share at least one anti ⁇ genic epitope with AR and TR2 proteins.
  • polyclonal and monoclonal antibodies characterized by their ability to bind with high immunospecif city to AR and TR2 proteins and to their fragments and peptides, recognizing unique epitopes which are not common to other proteins especially DNA binding proteins.
  • AN1-6, AN1-7, AN1-15 monoclonal antibodies, designated AN1-6, AN1-7, AN1-15; and produced by hybridoma cell lines designated H-AN1-6, H-AN1-7, H-AN1-15; deposited January 25, 1989, under Accession Nos. HB 10,000;
  • antibodies are characterized by (a) capacity to bind androgen receptors from rat ventral prostate and synthetic peptides having sequences predicted from the structure of hAR-cDNA and rAR-cDNA; (b) specific immunological reactivity with, and capacity to reversibly immunobind to, naturally occurring and recombinant androgen receptors, in native and denatured conformations; and (c) specific immunological reactivity with, and capacity to reversibly immunobind to, proteinaceous materials including all or a substantially, immunologically significant, part of an amino acid sequence duplicative of that extant at residues 331 through 577 of hAR and corresponding amino acid sequences in rAR.
  • the monoclonal antibodies of the invention can be used for affinity purification of AR from human or rat prostate, and other sources such as AR-rich organs and cultured cells. Also provided by the present invention are novel procedures for the detection and/or quantification of normal, abnormal, or mutated forms of AR and TR2, as well as nucleic acids (e.g., DNA and mRNA) associated therewith.
  • antibodies of the invention may be employed in known immunological procedures for quantitative detection of AR and TR2 proteins in fluid and tissue samples, of DNA sequences of the invention (particularly those having sequences encoding DNA binding proteins) that may be suitably labelled and employed for quantitative detection of mRNA encoding these proteins.
  • Preferred polypeptide products of the inven ⁇ tion include the approximately 79 kD (starting from the second ATG/Met) and 98 kD (starting from the first ATG/Met) hAR polypeptides having the deduced amino acid sequence of 734 and 918 residues, respectively, as set out in Figure 3. Also preferred are the 79 kD and 98 kD - 9 -
  • rAR species polypeptides having the deduced sequence of 733 and 902 residues set out in Figure 3 and the 20 kD and 52 kD species human TR2 polypeptides having the same deduced amino acid sequence of 184 and 483 residues set out in Figure 4.
  • the preferred 79 kD and 98 kD hAR and rAR polypeptides may be produced ii vitro and are characterized by a capacity to specifically bind androgens with high specificity and by their im unopre- cipitatability by human auto-immune anti-androgen receptor antibodies.
  • Figure 1 illustrates the strategy employed in construction of a human androgen receptor cDNA vector
  • Figure 2 illustrates the strategy employed in construction of rat androgen receptor cDNA vectors
  • Figure 3 provides a 3715 base pair nucleotide sequence for a human androgen receptor (hAR) DNA clone and the deduced sequence of 734 and 918 amino acid residues for hAR proteins; and in addition provides a 3218 base pair nucleotide sequence for a rat androgen receptor (rAR) DNA clone and the deduced sequences of 733 and 902 amino acids for two rAR species;
  • hAR human androgen receptor
  • rAR rat androgen receptor
  • Figure 4 provides a 2029 base pair nucleotide sequence for a human TR2 DNA clone ⁇ and a deduced sequence of 483 amino acids for a "TR2-5" species and a deduced sequence of 184 amino acids for a "TR2-7" species;
  • Figure 5 provides an amino acid sequence alignment of the cysteine-rich DNA binding domain of human androgen receptor, glucocorticoid receptor, ineralocorticoid receptor, progesterone receptor, estrogen receptor, TR2, rat AR, chick vitamin D receptor (c-VDR), and the v-erb A oncogene product of avian erythroblastosis virus.
  • Figures 6, 7, and 8 illustrate, respectively, the in-frame fusion of three different parts of the AR gene (the N-terminal, the DNA-binding domain and the androgen-binding domain) to the N-terminal half of the trpE gene using pATH expression vectors.
  • Example 1 relates to the isolation, preparation, and partial structural analysis of cDNA for human and rat androgen receptors.
  • Example 2 relates to confirmation of the presence on the human X-chromosome of an AR-type cDNA sequence.
  • Example 3 relates to the preparation of human and rat cDNAs containing AR-type cDNA from different clones and ligation into the pGEM-3Z plasmid.
  • Example 4 relates to transcription and trans- lation of the AR-type cDNA plasmid DNA.
  • Example 5 relates to steroid binding activity of the expression product of Example 4.
  • Example 6 relates to the binding activity of the expression product of Example 4 to human auto-antibodies.
  • Example 7 relates to the characteriza- tion of TR2-CDNA.
  • Example 8 relates to the Ln vitro transcription and translation of TR2-CDNA.
  • Example 9 relates to the binding activity of TR2-CDNA expression product.
  • Example 10 relate to the androgen regulation of TR2 mRNA levels in the rat ventral prostate.
  • Example 11 relates to recombinant expression systems of the invention.
  • Example 12 relates to the production of fusion proteins and their use in producing polyclonal and monoclonal antibodies according to the invention.
  • Example 13 relates to use of DNA probes of the inventions.
  • Example 14 relates to development of transgenic animals by means of DNA sequences of the invention. These examples are for illustrative purposes only and are not intended in any way to limit the scope of the invention.
  • ⁇ GTll cDNA libraries The isolation of cDNA for human androgen receptor (hAR) and rat androgen receptor (rAR) was accomplished using ⁇ GTll cDNA libraries.
  • the human testis and prostate ⁇ GTll libraries were obtained from Clontech Co., Palo Alto, California and a rat ventral prostate ⁇ GTll library in E.coli Y1090 was constructed as described in Chang, et al., J. Biol. Chem., 262:11901 (1987).
  • clones were differentiated using oligonucleotide probes specific for various steroid receptors.
  • the cDNA libraries were initially screened with a set of 41-bp oligonucleotide probes designed for homology to nucleotide sequences in the DNA-binding domain of glucocorticoid receptors (GR) , estrogen recep ⁇ tors (ER), progesterone receptors (PR), mineralocorti- coid receptors (MR), and the v-erb A oncogene product of avian erythroblastosis virus.
  • the set of probes had the following sequence: TGTGGAAGCTGT/CAAAGTC/ATTCTTTAAAAGG/ AGCAA/GTGGAAGG.
  • the plaques were replicated on a nitrocellu ⁇ lose filter and screened with a 5'-end 32 P-labeled 41-bp oligonucleotide probes.
  • the conditions of hybridization were 25% formamide, 5X Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.1% SDS, 5X SSC (IX SSC is 150 mM NaCl, 15 mM sodium citrate), 100 yg/ml denatured salmon sperm DNA, and 1 ⁇ g/ml poly(A) at 30 ⁇ C.
  • the GR-cDNA clones were eliminated by screening with two GR-specific 24-bp probes that had nucleotide sequences identical to nucleotide segments immediately f next to the 5'-end or the 3'-end of the DNA binding- region of hGR-cDNA , i.e., TGTAAGCTCTCCTCCATCCAGCTC and CAGCAGGCCACTACAGGAGTCTCA. 244 and 14 clones, respec ? tively, were eliminated as hGR- and rGR-cDNA clones.
  • TR2- type cDNA and AR-type cDNA of Example 1 were probed with the TR2- type cDNA and AR-type cDNA of Example 1. With TR2-type cDNA fragments, no positive clones were detected, while 3 positive clones were obtained with a 1.9 kb fragment of AR-type cDNA from a human testis (clone AR 132), thereby confirming the presence of an AR-type cDNA sequence on the human X-chromosome. Because the X- chromosome has been implicated as the chromosome which contains an AR gene [Lyon, et al.. Nature (London), 2_27_:1217- (1970); Meyer, et al., Proc. Nat'l. Acad. Sci.
  • AR-type cDNA but probably not the "TR2-type” cDNA, contained the DNA sequence that could encode for androgen receptor.
  • Two human clones containing DNA inserts that overlapped to form a 2.7 kb cDNA were designated AR 132 and AR 5.
  • Two rat clones containing DNA inserts that overlapped to form a 2.8 kb cDNA were designated rAR 1 and rAR 4. After restriction enzyme digestion, the DNA segments from these AR-type clones were ligated, selected and amplified using pBR322 and pGEM-3Z vectors as described in Example 3 below.
  • Figure 1 relates to the strategy employed in the construction of a full length hAR-cDNA clone.
  • cDNA of clone AR 132 was digested with Eco RI to obtain a 1.9 kb fragment which was then digested with Kpn I to pro ⁇ vide a 1 kb Eco RI-Kpn I fragment.
  • This 1 kb fragment was ligated to a 3 kb fragment obtained by digestion of clone AR 5 with Kpn I and Pvu I.
  • the resulting 4 kb fragment was inserted into Eco RI and Pvu I-digested pBR322 vector and used to infect E. coli DH5 ⁇ .
  • the transformed clones were selected by tetracycline-resis- tance.
  • the plasmid with the DNA insert was digested with Cla I and Nde I to obtain a 2.6 kb fragment.
  • the fragment was blunt-ended with the Klenow fragment of E ⁇ coli DNA polymerase I and ligated to the cloning vector pGEM-3Z' plasmid DNA (Promega Biotec, Madison WI.) which was previously blunt-ended by digestion with Sma I.
  • E. coli DH5 ⁇ cells were transformed with the plasmid so formed (designated plasmid PhAR3600) and colonies containing the plasmid were selected by ampicillin resistance and amplified.
  • E. coli DH5 ⁇ cells, transformed with plasmid PhAR3600 were designated EC-hAR3600 and were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville,
  • the plasmid DNA was isolated and its structure analyzed by restriction enzyme mapping and sequencing.
  • the 2.0 kb hAR fragment obtained by NruI-BamHI digestion of a 2.6 kb hAR in pGEM3Z was then ligated to another 1.6 kb ECORI-NruI fragment of hHR to obtain the full length 3715 bp hAR.
  • the open reading frame is about 2.8 kb which is sufficient to code for a protein with " more than 900 amino acids. Near the middle of the protein is a cysteine-rich region with a 72 amino acid sequence highly homologous to regions in other steroid receptors considered to be the DNA binding domain.
  • the 2.4 kb Eco RI-Eco RI cDNA insert of clone rAR 1 was digested with Xmn I to obtain a 2.3 k b fragment.
  • This 2.3 kb Xmn I-EcoR I fragment was ligated to a 400 bp fragment that was obtained by digestion of another cDNA clone insert (Eco RI-Eco RI insert of rAR 4) with Pst I.
  • the ligated 2.7 kb fragment was inserted into Sma I and Pst I-digested pGEM-3Z vector and used to infect E. coli DH5o.
  • E_ ⁇ coli DH5 ⁇ cells were transformed with the plasmid and colonies containing the plasmid were selected by ampicillin resistance and amplified. These cells were designated EC-rAR 2830 and were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on January 25, 1989 under Accession No. 67878. As noted in Figure 2, this construction allowed for a transcription product translated beginning with the second of two in-frame methionine-specifying codons (designated ATG 2 ).
  • the 2.4 kb Eco RI-Eco RI cDNA insert of rAR 1 was digested with Hind III to obtain a 1.68 kb frag ⁇ ment.
  • the 1.68 kb Eco RI-Hind III fragment was ligated to a 1.15 kb DNA fragment obtained by digestion of another cDNA clone insert (rAR 6) with Hind III and Pst I.
  • the ligated 2.83 kb fragment was inserted into Eco RI and Pst I-digested pGEM 3Z vector and used to infect E. coli DH5 ⁇ .
  • E. coli (DH5 ⁇ ) cells were transformed with the plasmid and colonies containing the plasmid were selected by ampicillin resistance and amplified. As noted in Figure 2, this construction allowed for a transcription product translated beginning at the first of two in-frame methionine-specifying codons (designated ATG ⁇ ) .
  • Figure 3 provides the nucleotide sequence of the DNA sequence of the longer "full length” rat and human AR clones and includes the deduced amino acid sequences.
  • the first and second methionine-specifying codons are designated at amino acid positions 1 and 170 of rAR and positions 1 and 185 of hAR.
  • the linearized plasmid was transcribed in a reaction mixture containing 40 mM Tris-HCl, pH 7.5, 6 mM MgCl 2 , 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 500 ⁇ M each of ATP, GTP, CTP, and UTP, 160 units ribonuclease inhibitor, 5 ⁇ g plasmid, 30 units T7 RNA polymerase (Promega Biotec, Madison, WI) and diethylpyrocarbonate (DEPC)- treated water to a final volume of 100 ul.
  • T7 RNA polymerase was used in the transcription of the plasmid DNA, because a T7 promotor, rather than the SP6 promotor, was found ahead of the 5'-end of the ligated AR-cDNA.
  • RNA trans ⁇ cribed was isolated and then translated in a rabbit reticulocyte lysate system.
  • RNA was carried out in a micro- coccal nuclease-treated rabbit reticulocyte lysate
  • the reticulocyte lysate of Example 4 containing the newly synthesized protein was incubated with 17 ⁇ [ 3 H]-methyl-17s-hydroxy- estra-4,9,ll-trien-3-one ([ 3 H] R1881), a potent syn ⁇ thetic androgen that binds AR with high affinity [Liao, et al., J. Biol. Chem., 248_:6154 (1973)].
  • RNA transcribed from the cloned cDNA, as described in Example 4 was translated in a rabbit reticulocyte lysate system and aliquots of the lysate were then incubated with 5 nM [ 3 H] R1881 (87 Ci/mmol) in the absence or presence of 25 nM, 50 nM, or 250 nM of non-radioactive steroid.
  • the final incubation volume was 100 ⁇ l.
  • the radioactive androgen binding was measured by the hydroxylapatite-filter method as des- - 19 -
  • Non-radioactive [ 3 H] R1881-bound (% of control) steroid added 25 nM 50 nM 250 nM
  • the active natural andro ⁇ gen, 17s-hydroxy-5 ⁇ -androstan-3-one(5 ⁇ -dihydro-testos- terone) competed well with [ 3 H] R1881 binding, but the inactive 58-isomer did not compete well with [ 3 H] R1881 suggesting that it does not bind tightly to AR.
  • the binding activity was steroid specific; dexamethasone, hydrocortisone, progesterone, and 17s-estradiol did not compete well with the radioactive androgen for binding to the 79 kD protein.
  • Reticulocyte lysate containing translated AR was incubated with [ 3 H] R1881, as described in Example 4, and then incubated again in either the presence of or absence of 5 ⁇ l of human male serum containing anti ⁇ bodies to AR (anti-AR serum) for 4 hrs. at 4°C.
  • Rabbit serum containing anti-human immuglobulins (Anti-IgG) was then added as the second antibody. After 18 hrs. of incubation at 4°C, the mixture was centrifuged and the radioactivity associated with the precipitate was esti ⁇ mated. Human female serum, not containing anti-AR anti- body, was also used for comparison.
  • Anti-human immunoglobulin-dependent precipitation of hAR made by the translation of RNA transcribed from cloned cDNA
  • TR2-5 Of the more than 40 TR2-type human cDNA clones obtained, including the 30 described in Example 1, the clone designated TR2-5 was found to be 2029 base pairs
  • the open reading frame between the first ATG and terminator TAA can encode 483 amino acids with a calculated molecular weight of 52 kD.
  • the putative DNA binding region is underscored.
  • the putative initiator ATG matched closely with Kozak's concensus sequence for active start codons. [See, Kozak, M. , Nature, 308:241 (1984).] Two triplets upstream of this ATG codon is an in-frame terminator (TAA) further supporting initiator function for the ATG.
  • TAA in-frame terminator
  • TR-2 with open reading frames at the putative ligand-binding domains have been obtained. Some of these may code for receptors for new hormones or cellular effectors. It is anticipated that the knowledge of TR2-CDNA sequences will be utilized in isolation and structural analysis of other cellular receptors, their genes, and ligands (endogenous or therapeutic agents) that can regulate cellular growth and functions in both normal and diseased organs.
  • Figure 5 depicts an amino acid sequence align- ment of the cysteine-rich DNA binding domain of human androgen receptor, glucocorticoid receptor, mineralocor- ticoid receptor, progesterone receptor, estrogen recep ⁇ tor, human TR2 protein, rat AR, chick vitamin D receptor (c-VDR), and the v-erb A oncogene product of avian erythroblastosis virus.
  • the numbers in the left margin represent the positions of amino acid residues in the individual receptors. Common residues are boxed with solid lines. The residues in dotted boxed represent those not in common with those in the solid boxes.
  • V-erb A has two more amino acids at the starred posi ⁇ tion.
  • the human and rat cDNAs for AR have identical amino acid sequences, although for some amino acids different codons are employed. Also in this region, the homology between human AR or rat AR and other receptors is as follows: glucocorticoid receptor (GR), 76.4%; mineralo-corticoid receptors (MR), 76.4%; progesterone receptors (PR), 79.2%; estrogen receptors (ER), 55.6%; TR2, 45.8%; chick vitamin D receptor (c- VDR), 40,3%; and the v-erb A oncogene product of avian erythroblastosis virus, 40.3%.
  • GR glucocorticoid receptor
  • MR mineralo-corticoid receptors
  • PR progesterone receptors
  • ER estrogen receptors
  • TR2 chick vitamin D receptor
  • c- VDR chick vitamin D receptor
  • v-erb A oncogene product of avian erythroblastosis virus, 40
  • the homology between human AR or rat AR and hGR, hMR, or hPR is about 45-55%, whereas the homology between human AR and rat AR and hER is less than 20%.
  • human and rat AR appear to be more closely related to GR, MR, and PR, than to v-erb A or to receptors for estrogen, vitamin D, and thyroid hormones.
  • TR2 (amino acids 111 to 183) has a high homology with the steroid receptor super-family as follows: retinoic acid receptor (RAR), [Giguere, et al..
  • VD 3 R [McDonnell, et al. , Science, £35:1214 (1987)], 53%; hERRl and hEER2, [Giguere, V., et al.. Nature, 331:91 (1988)], 51% estrogen receptor (ER), [Hollenberg, et al.. Nature, 318:635 (1985)], 51%; glucocorticoid receptor (GR) [Hollenberg, et al.. Nature, 318:635
  • TR2-7 were isolated and ligated to an EcoRI digested pGEM-3Z vector for i i vitro transcription essentialy as described in Example 3.
  • E * coli DH5 ⁇ cells, transformed with these plasmids were designated EC TR2-5 and EC TR2-7 and were deposited with the
  • SDS- polyacrylamide gel electrophoresis PAGE
  • the translation lysate was passed over a DNA cellulose column.
  • the bound product was then eluted, concentrated and applied to SDS-PAGE.
  • the results indi ⁇ cated that the translated proteins were indeed DNA- binding proteins.
  • TR2-5 expression product steroid binding activity may involve some post-translation modifications missing in the rabbit reticulocyte lysate system.
  • the TR2-5 translated protein may be steroid independent or may bind to an unidentified ligand present in the human testis or rat ventral prostate.
  • TR2 mRNA The size of TR2 mRNA was determined by Northern blot analysis with TR2-5 cDNA insert as a probe. One 2.5 kb band was detected which should include enough sequence information to code for a 52 kD protein.
  • TR2 mRNA tissue distribution was also analyzed by dot hybridization. The hybridization was visualized by densitometric scanning of the autoradio- graphs, individual dots were cut and radioactivity measured by liquid scintillation counting [See, Chang, et al., J. Biol. Chem., 26_2:2826 (1987)].
  • TR2 mRNA was most abundant in the rat ventral prostate with the relative amounts in other tissues being: prostate.100%, seminal vesicle 92%; testis, 42%; submaxillary gland, 18%; liver, 13%; kidney, ⁇ 1%; and uterus, ⁇ 1%.
  • rat ventral prostate is an androgen- sensitive organ and contains the greatest amount of AR and TR2 mRNA
  • RNA dot hybridization and Northern blot analysis Total RNA was extracted from the ventral prostate of normal rats, rats castrated and rats previously castrated and treated with
  • TR2 mRNA levels were then measured by dot hybridization as described above. The results show that flutamide injection, like castration, increased TR2 mRNA levels.
  • the change in the AR or TR2 protein levels could be due to a change in mRNA stab ⁇ ility and utilization or a change in the regulation of gene transcription.
  • the activation or inactivation by androgen of specific genes to different degrees in the same organ may suggest that androgen is involved in the structuring of the pattern of gene expression in the target cell.
  • AR-cDNA and TR2- cDNA may readily be used for large scale production of gene products.
  • the most efficient transcription units can be constructed using viral, as well as non-viral, vectors with regulatory signals that can function in a variety of host cells.
  • SV40, pSV2, adenoviruses, and bovine papilloma virus DNA have been used successfully for introduction of many eukaryotic genes into eukaryotic cells and permit their expression in a controlled genetic environment. These and similar systems are expected to be appropriate for the expression of AR- and TR2-genes.
  • the two most widely used methods the "calcium phosphate precipitation" and the "DEAE-dextran technique" can be used.
  • Genes can be introduced into cells either transiently, where they continue to express for up to 3 days, or, more permanently to form stably transformed cell-lines.
  • the expressed proteins can be detected by androgen binding or antibody assays.
  • the expression of cloned AR-genes was achieved as follows in a eukaryotic system. NIH 3T3 cells, contact-inhibited cells established from NIH Swiss mouse embryo, were co-transfected with hAR cDNA inserted into pBPVMTH vectors as described by Gorman, "DNA Cloning", 2:143-190 D. M. Glover, ed.; (Oxford, Washington, D.C. 1985). Transfected cells were cloned and grown in multiple-well cell culture plates. About 100 individual cell lines were isolated. Of these, 6 demonstrated [ 3 H] R1881-binding activity at least 4-fold the activity of cells transfected with pSV2 vector alone, i.e., without the hAR cDNA sequence.
  • hAR and rAR cDNAs were inserted into a number of expression vectors including pUR, ⁇ GTll, pKK223-3, pKK233-2, pLEX, pATHl, pATH2, pATHIO, and pATHll.
  • Vectors with AR cDNA inserts were used to infect E. coli strains (JM109, DH5o, Y1089, JM105, and RR1). According to polyacrylamide gel electrophoresis analysis, the infected bacteria can synthesize AR fragments coded for by the AR cDNA inserts. Some of these AR polypeptides are degraded in culture. Amino terminal, DNA-binding, and androgen binding domains were used, as described in Example 12, to construct fusion proteins representing these domains.
  • oligopeptides representing sequences unique to AR, (i.e., PYGDMRLETARDHVLP; CPYGDMRLETARDHVLP; and
  • SIRRNLVYSCRGSKDCIINK were bound to BSA or KLH carrier proteins and were used to immunize mice. Spleen cells from these mice were fused to myeloma cells to produce hybrid antibody producing cells. Analysis by ELISA (enzyme-linked immunoassay) of the supernatants of 4 hybrid cultures appeared to indicate the presence of immunoglobulin that interacts with AR of rat ventral prostate. It is anticipated that these cells which produce monoclonal antibodies can be injected intraperitoneally into BALB/c mice previously treated with pristane. Ascites fluids can then be harvested and antibodies precipitated with ammonium sulfate.
  • the induced fusion proteins i.e., those proteins not present in the control pATH vector (no AR gene insert) were sliced from the gels and then used for immunization.
  • Fusion proteins other than the three specifically exemplified, can also be constructed using these means.
  • Anti-AR crude serum was then affinity-purified by differential suspension of immune serum containing TrpE protein(s) (both those TrpE proteins having and those TrpE proteins not having inserted AR sequences) expressed by pATH vectors.
  • TrpE protein(s) both those TrpE proteins having and those TrpE proteins not having inserted AR sequences
  • the bound antibodies can be removed from the suspension because TrpE protein is insoluble.
  • Antibodies specific against only the trpE protein were removed; antibodies specific for AR were isolated and again confirmed by both ELISA and double antibody precipitation.
  • the immunized rats were judged ready to be sacrificed for a fusion when their serum tested positive anti-AR antibodies by ELISA. Spleens were removed and grinded to release the cells into DMEM (Dulbeco's
  • DMEM with IX H-T, IX Methotrexate, 20% FCS, and IX PBS were distributed in 96-well plates. Plates were supplemented after 6 days with DMEM and 20% FCS.
  • Hybridomas were identified and assayed, using the ELISA assay of Engrail, et al ⁇ . , Bio. Chem. et Biophys. ACTA,
  • hybridomas that caused a positive reaction with the AR fusion protein were "limit diluted” to a concentration of 10 cells/ml and were then distributed among half of a 96-well plate. The remaining cells from the original well were transferred to a 24-well plate. Each of these plates had a thymocyte feeder layer.
  • the tymocyte feeder layer was made up of thymus cells isolated from an un-injected rat, purified through centrifugation, irradiated with 1200 to 1400 RADS, and diluted to 1 x 10 7 cells/ml of DMEM with 20% FCS. Positives from these thymocyte 96-well plates were again tested by ELISA.
  • the monoclonal antibodies were designated AN1-6, AN1-7, and AN1-15 and the three cell lines were designated HAN1-6, HAN1-7, and HAN1-15; Accession Nos. 10,000; 9,999; and 10,001; respectively, deposited on January 25, 1989 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852.
  • Sucrose gradient centrifugation was used to characterize the specificity of the three monoclonal anti-AR antibodies and their ability to react with non- denatured [ 3 H]AR.
  • Cytosol was prepared from the ventral prostates of castrated rates as follows. Rats were castrated by the scrotal route while under anesthesia. They were killed 18 hrs. laters by cervical dislocation and their ventral prostates were removed, minced with scissors, washed in Buffer A (50 mM sodium phosphate, pH 7.5, 1 mM EDTA, 2 mM DTT, 10 mM sodium molybdate, 10% (v/v) glycerol and 10j mM sodium floride) and homogenized in 2x the tissue volume of Buffer A + 0.1 mM bacitracin, 1 mM PMSF, and aprotinin (lTIU/ml).
  • Buffer A 50 mM sodium phosphate, pH 7.5, 1 mM EDTA, 2 mM DTT, 10 mM sodium molybdate, 10% (v/v) glycerol and 10j mM sodium floride
  • the homogenate was centrifuged at 5,000 * g for 10 mins., adjusted to 10 nM 3 H-androgen, spun at 225,000 x g for 45 mins. and treated with dextran-coated charcoal.
  • the [ H]AR and other steroid receptor complexes had a sedimendation coefficient of about 4-5S in the sucrose gradient media containing 0.4M KC1.
  • Anti-AR antibodies do not alter the sedimentation coefficient of 4-5S for [ 3 H]glucocorticoid receptors complexes of rat liver, estrogen receptor complexes of MCF-7 cells, and progesterone receptor complexes of T47D cells, but do shift the sedimentation coefficient of [ 3 H]A-AR complexes of rat ventral prostate from 4S to 9- 12S or to heavier units.
  • SDS-polyacrylamide gel electrophoresis analysis it was also found that all major ⁇ n vitro transcription/translation products of human and rat AR cDNAs were immunoprecipitatable by the anti-AR antibodies.
  • AR cDNA, TR2 cDNA, or their partial segments can be used as specific probes in these studies.
  • high molecular weight genomic DNA isolated from target organs, tumors, and cultured cells can be used in identifying and characterizing AR genes.
  • Different restriction endonucleases can be used to cleave DNA.
  • the fragments can be analyzed by Southern analysis (agarose. electrophoresis, transfer to nitrocellulose and hybridization with AR cDNA probes). After identification, selected fragments can be cloned and sequenced. It is also possible to use appropriate oligonucleotide fragments of AR or TR2 cDNA as primers to amplify genomic DNA isolated from normal and abnormal organs or cells by specific DNA polymerases.
  • the amplified genomic DNA can then be analyzed to identify sequence abnormality using the polymerase chain reaction (PCR) assay.
  • PCR polymerase chain reaction
  • Saiki, et al. Science, 230, 1350 (1985). See also, Mullis, K.B., U.S. Patent No. 4,683,202; July 28, 1987; and Mullis, K.B., U.S. Patent No. 4,683,195; July 28, 1987.
  • dot hybridization and Northern hybridization analysis could be used to characterize mRNA and AR or receptor-like molecules quantitatively and qualitatively. From these studies valuable information about the number of different forms of AR genes and their expression in androgen insensitive and sensitive tumor cells can be obtained.
  • DNAs and RNAs obtained from androgen sensitive and insensitive tumors and from cell lines from rats and humans with testicular feminization syndromes have been analyzed by the above methods. Preliminary studies indicated that abnormality in androgen responses may be due to sequence deletion/mutation in genes for ARs.
  • Transgenic techniques have been employed for expression of exogenous DNA. It may therefore be poss ⁇ ible to confer androgen sensitivity to animals with androgen receptor defects.
  • androgen insen ⁇ sitive animals such as testicular feminized mice or rats, are known to have defective AR genes or defective AR itself. If DNA containing a normal AR gene is injected into fertilized mouse embryos, the transgenic mice may carry and express the gene and produce a functional AR necessary for androgen responses. For micro-injection, it is necessary to use AR genes containing DNA that can be expressed in the insensitive animals.
  • Clones containing AR sequences will be characterized by endonuclease mapping, by Southern hybridization and by Sl-nuclease mapping. The 5' and 3' untranslated regions thus identified will aid in determining the minimal size of the DNA that would be required for tissue specific expression of the AR coding region.
  • Partial sequence analysis of the 5' and 3 ' regions would locate the minimal region that represents the promoter and the polyadenylation region. Approxi ⁇ mately 2 to 5 kb of upstream un-translated region and 0.5 to 1 kb of sequences downstream from the poly(A) site may be fused to the cDNA clone (minimal-gene) and injected into embryos of mice. Transgenic mice would be identified by analysis of their tail DNA using mini-gene specific probe(s). Normally only some of the transgenic mouse lines can express their transgenes.
  • Transgenes may be inactive because of the presence of inhibitory sequences, integration of the exogenous gene into a transcriptionally inactive chromosomal location, or the juxtaposition of the transgene and an endogenous enhancer.
  • androgen insensitivity may be due to various other factors and not due to abnormality in the AR gene or its expression.
  • the foregoing illustrative examples relate to the isolation of human and rat cDNAs encoding DNA bind ⁇ ing proteins including androgen receptor and TR-2 and more particularly describe the transcription of the corresponding cDNAs and translation of the corresponding mRNAs in cell-free systems.

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Abstract

On a mis au point des séquences d'ADN codant des polypeptides de liaison d'ADN comprenant un récepteur d'androgène (AR) ainsi que des polypeptides TR2. A titre d'illustration, l'ADNc d'AR humain et de rat ont des produits d'expression des polypeptides 79 kD et 98 kD pouvant être précipités immunologiquement par des anticorps de récepteur anti-androgène auto-immuns humains et sont capables de lier des androgènes spécifiquement et avec une grande affinité. On a également mis au point des anticorps ainsi que des procédés et des matières immunologiques permettant la détection du récepteur d'androgène et des polypeptides TR2, ainsi que des procédés et des matières d'hybridation permettant la détection de l'AR et d'acides nucléiques se rapportant au TR2.
PCT/US1989/001238 1988-03-30 1989-03-24 Proteines de liaison d'adn comprenant un recepteur d'androgene WO1989009223A1 (fr)

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WO1991007423A1 (fr) * 1989-11-17 1991-05-30 Arch Development Corporation Proteines de liaison d'adn comprenant un recepteur d'androgene
EP0365657A4 (en) * 1988-04-15 1991-08-21 University Of North Carolina At Chapel Hill Dna encoding androgen receptor protein
US5614620A (en) * 1988-03-30 1997-03-25 Arch Development Corporation DNA binding proteins including androgen receptor
WO1999019354A1 (fr) * 1997-10-14 1999-04-22 Pharmacia & Upjohn Ab Nouveaux polypeptides apparentes au recepteur de vitamine d, sequence d'acide nucleique codant ces polypeptides et utilisation de ces derniers
WO2000013699A1 (fr) * 1998-09-04 2000-03-16 Ludwig Institute For Cancer Research Peptide antigenique code par un autre cadre de lecture ouvert de facteur stimulant la proliferation des macrophages
EP1375519A3 (fr) * 1992-04-07 2004-06-30 Immunomedics, Inc. Methode pour influencer les fonctions cellulaires en utilisant des anticorps
US7118885B2 (en) 1997-10-14 2006-10-10 Pfizer Inc. Nucleic acid encoding vitamin D receptor related polypeptide

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US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

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US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences

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Title
Biochemical and Biophysical Research Communications, Academic Press (Orlando, USA, Volume 153, issued May 1988, TRAPMAN Et al., "Cloning, Structure and Expression of a cDNA Encoding the Human Androgen Receptor", pages 241-248, see the entire document. *
Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor Laboratory (New York, USA), Volume LI, published 1986, MULLIS et al., " Specific Enzymatic Amplification of DNA in Vitro: The Polymerase Chain Reaction", pages 263-273, see the entire document. *
Journal of Endocrinological Investigation (Milan, Italy), Volume 10, Supplement 2, published 1987, GOVINDAN et al., "Cloning of the Human Androgen Receptor cDNA", page 63, see the entire Abstract. *
Nature (London, UK), Volume 324, issued November 1986, SAIKI et al., "Analysis of Enzymatically Amplified B-Globin and HLA-DQ DNA with Allele-Specific Oligonucleotide Probes", pages 163-166, see the entire document. *
Proceedings of the National Academy of Sciences, USA (Washington, USA), Volume 85, issued October 1988, CHANG et al., "Structural Analysis of Complementary DNA and Amino Acid Sequences of Human and Rat Androgen Receptors", pages 7211-7215, see the entire document. *
Progress in Cancer Research and Therapy, Raven Press (New York, USA), Volume 35, issued July 1988, GOVINDAN et al., "Cloning of the Human Androgen Receptor cDNA", pages 49-54, see the entire document. *
Science (Washington, USA), Volume 240, issued April 1988, CHANG et al., "Molecular Cloning of Human and Rat Complementary DNA Encoding Androgen Receptor", pages 324-326, see the entire document *
Science (Washington, USA), Volume 240, issued April 1988, LUBAHN et al., "Cloning of Human Androgen Receptor Complementary DNA and Localization to the X Chromosome", pages 327-330, see the entire document. *
See also references of EP0407462A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5614620A (en) * 1988-03-30 1997-03-25 Arch Development Corporation DNA binding proteins including androgen receptor
EP0365657A4 (en) * 1988-04-15 1991-08-21 University Of North Carolina At Chapel Hill Dna encoding androgen receptor protein
US6307030B1 (en) 1988-04-15 2001-10-23 The University Of North Carolina At Chapel Hill Androgen receptor proteins, recombinant DNA molecules coding for such, and use of such compositions
US6821767B1 (en) 1988-04-15 2004-11-23 The University Of North Carolina At Chapel Hill Androgen receptor proteins, recombinant DNA molecules coding for such, and use of such compositions
US7129078B2 (en) 1988-04-15 2006-10-31 University Of North Carolina At Chapel Hill DNA encoding androgen receptor fragment
WO1991007423A1 (fr) * 1989-11-17 1991-05-30 Arch Development Corporation Proteines de liaison d'adn comprenant un recepteur d'androgene
EP1375519A3 (fr) * 1992-04-07 2004-06-30 Immunomedics, Inc. Methode pour influencer les fonctions cellulaires en utilisant des anticorps
WO1999019354A1 (fr) * 1997-10-14 1999-04-22 Pharmacia & Upjohn Ab Nouveaux polypeptides apparentes au recepteur de vitamine d, sequence d'acide nucleique codant ces polypeptides et utilisation de ces derniers
US7118885B2 (en) 1997-10-14 2006-10-10 Pfizer Inc. Nucleic acid encoding vitamin D receptor related polypeptide
WO2000013699A1 (fr) * 1998-09-04 2000-03-16 Ludwig Institute For Cancer Research Peptide antigenique code par un autre cadre de lecture ouvert de facteur stimulant la proliferation des macrophages

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