EP0812354A2 - RECEPTEUR $g(g) DE RETINOIDE X HUMAIN (hRXR-GAMMA) - Google Patents

RECEPTEUR $g(g) DE RETINOIDE X HUMAIN (hRXR-GAMMA)

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Publication number
EP0812354A2
EP0812354A2 EP96903616A EP96903616A EP0812354A2 EP 0812354 A2 EP0812354 A2 EP 0812354A2 EP 96903616 A EP96903616 A EP 96903616A EP 96903616 A EP96903616 A EP 96903616A EP 0812354 A2 EP0812354 A2 EP 0812354A2
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EP
European Patent Office
Prior art keywords
hrxr
nucleic acid
polypeptide
sequence
cells
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Legal status (The legal status 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 status listed.)
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EP96903616A
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German (de)
English (en)
Inventor
William W. Lamph
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Ligand Pharmaceuticals Inc
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Ligand Pharmaceuticals Inc
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Publication of EP0812354A2 publication Critical patent/EP0812354A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors

Definitions

  • This invention relates to the cloning and uses of a human retinoid X receptor subtype.
  • Retinoic acid is a vitamin A metabolite which has been recognized as inducing a broad spectrum of biological effects.
  • a variety of structural analogues of retinoic acid have been synthesized that also have been found to be bioactive. Some, such as Retin-A ® (registered trademark of Johnson & Johnson) and Accutane ® (registered trademark of Hoffmann- aRoche) , have found utility as therapeutic agents for the treatment of various pathological condi ⁇ tions.
  • Metabolites of vitamin A and their synthetic analogues are collectively herein called "retinoids”. Synthetic retinoids have been found to mimic many of the pharmacological actions of retinoic acid.
  • retinoic acid is not reproduced in full by all bioactive synthetic retinoids.
  • Medical professionals are interested in the medicinal applications of retinoids. Among their uses approved by the FDA is the treatment of severe forms of acne and psoriasis. Evidence also exists that these compounds can be used to arrest and, to an extent, reverse the effects of skin damage arising from prolonged exposure to the sun. Other evidence indicates that these compounds may be useful in the treatments of a variety of cancers including melanoma, cervical cancer, some forms of leukemia, and basal and squamous cell carcinomas. Retinoids have also been shown to be efficacious in treating premalignant cell lesions, such as oral leukoplakia, and to prevent the occurrence of malignancy.
  • Retinoids are able to cross passively biological membranes and control cell functions by using specific intracellular receptors as signal transducers. These intracellular receptors, located in the nucleus in the presence of their retinoid ligands, function as ligand- activated transcription factors that modulate gene expression through binding to specific DNA sequences located in the regulatory regions of target genes.
  • Retinoids regulate the activity of two distinct intracellular receptor subfamilies; the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) .
  • the RAR and RXR subfamilies are divided into six subtypes, based upon their primary sequence homology, their ability to bind to various retinoid analogues, and by their promoter recognition sequence specificity (Mangelsdorf DJ, Umesono K, and Evans RM 1994 Retinoid receptors. In: Sporn MB, Roberts AB, and Goodman DS (eds) The Retinoids: Biology. Chemistry, and Medicine.
  • the RARs have three subtypes denoted a , ⁇ , and ⁇ .
  • the RXRs also have three known subtypes, a, ⁇ , and ⁇ .
  • RARs and RXRs share common structure and functional domains with other members of the steroid hormone receptor superfamily, comprising an amino-terminal region of variable length, a DNA-binding domain located in the central region, and a ligand-binding domain encompass ⁇ ing most of the carboxy-terminal end of the proteins.
  • RARs and RXRs differ in several aspects.
  • the RARs and RXRs are divergent in primary struc- ture, e.g., the ligand-binding domains of RARO and RXR ⁇ have only approximately 27% amino acid identity (i.e., "homology").
  • RARs bind to both 9-cis retinoic acid (9cRA) and all- trans retinoic acid (tRA) with equally high affinity, displaying K ⁇ values of 0.2 - 0.8 nM (Allenby G, et al. , 1993, "Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.” Proc Natl Acad Sci USA 90:30-34; Allegretto EA, et al. , 1993, "Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast: correlation with hormone binding and effects of metabolism.” J. Biol. Chem. 268:26625-26633) .
  • RXRs bind with high affinity and speci ⁇ ficity to 9cRA (Levin AA, et al. , 1992, 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor
  • RXR ⁇ mRNA has been shown to be most abundant in the liver, kidney, lung, muscle and intestine.
  • RARs and RXRs have different target gene specificity.
  • response elements in cellular retinol binding protein type II (CRBPII) and apolipopro- tein Al genes confer responsiveness to RXR, but not to RAR.
  • RAR has also been shown to repress RXR-mediated activation through the CRBPII RXR response element (Mangelsdorf et al., Cell. 66:555-61 (1991)) .
  • the RXR class of retinoid receptors not only function as effector molecules for 9-cis RA but also function as heterodimeric partners for other members of the intra ⁇ cellular receptor superfamily including RARs, the thyroid hormone receptor, the peroxisome proliferator-activator receptor (PPAR) , the vitamin D receptor, and a number of other intracellular receptors whose ligands have not yet been identified (orphan receptors) (Leid,M., Kastner,P.,
  • RXR-j ⁇ was first identi ⁇ fied in either human or rat cells biochemically by a number of laboratories using functional assays to charac- terize protein molecules that increased the DNA binding properties of VDR, RAR, TR, and H2BPII (Hamada,K., Gleason,S.L. , Levi,B.Z., Hirschfeld,S. , Appella,E. and Ozato,K. (1989) Proc. Natl. Acad. Sci. U. S. A., 86, 8289-8293) . Upon isolation and cloning of these molecules it became evident that these molecules were the human counterparts of mouse RXR-S.
  • RXR family of receptors Some members of the RXR family of receptors have been described in humans, rat, chicken (Rowe,A. , Eager,N.S. and Brickell,P.M. (1991) Development, 111, 771-778) and xenopus (Blumberg,B. , Mangelsdorf,D.J. , Dyck,J.A., Bittner,D.A. , Evans,R.M. and De Robertis,E.M. (1992) Proc. Natl. Acad. Sci. U. S. A., 89, 232.1-2325) . To date only two subtypes of RXR receptors, and ⁇ , have been characterized from humans (Mangelsdorf,D.J.
  • the present invention relates to hRXR- ⁇ polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such polypeptides and nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides and nucleic acids, and methods relating to all of the foregoing.
  • the hRXR- ⁇ polypeptides, nucleic acids, and antibodies are useful for establishing the tissue specific expression pattern of hRXR- ⁇ gene. For example, a Northern blot can be used to reveal tissue specific expression of the gene.
  • the present invention is based upon the identifica ⁇ tion and isolation of a novel human retinoid X receptor subtype termed hRXR- ⁇ that is activated by binding of 9-cis retinoic acid or LG100069, i.e., (E) -4- [2- (5,6,7, 8- Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-propenyl] benzoic acid.
  • hRXR- ⁇ has 463 amino acids and a predicted molecular weight of 55 kD.
  • the invention features an isolated, purified, enriched or recombinant nucleic acid encoding a hRXR- ⁇ polypeptide.
  • isolated in reference to nucleic acid is meant a polymer of 2 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized.
  • the isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
  • nucleic acid enriched in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2 - 5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the DNA from other sources may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19.
  • This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleic acid does not require absolute purity (such as a homogeneous prepara ⁇ tion) ; instead, it represents an indication that the sequence .is relatively purer than in the natural environ ⁇ ment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity.
  • the claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA) .
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure indi ⁇ vidual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • cDNA synthetic substance
  • the process which includes the construe- tion of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • a hRXR- ⁇ polypeptide is meant two or more contiguous amino acids set forth in the full length amino acid sequence of SEQ ID NO:2, wherein said contiguous amino acids have a sequence different from those of mouse RXR- ⁇ polypeptides.
  • the hRXR- ⁇ polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained.
  • the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length nucleic acid sequence SEQ ID NO:l or at least 27, 30, 35, 40 or 50 contiguous nucleotides thereof and the hRXR- ⁇ polypeptide comprises, consists essentially of, or consists of at least 9, 10, 15, 20, or 30 contiguous amino acids of a hRXR- ⁇ polypeptide.
  • “comprising” is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.
  • compositions and probes of the present invention may contain human nucleic acid encoding a hRXR- ⁇ polypeptide but are substantially free of nucleic acid not encoding a human hRXR- ⁇ polypeptide.
  • the human nucleic acid encoding a hRXR- ⁇ polypeptide is at least 18 contiguous bases of the nucleotide sequence set forth in SEQ. ID NO. 1 and will selectively hybridize to human genomic DNA encoding a hRXR- ⁇ polypeptide, or is complementary to such a * sequence.
  • the nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be blood, semen, and tissue of humans; and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
  • the nucleic acid is a unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, and obtaining antibodies to polypeptide regions.
  • unique nucleic acid region is meant a sequence present in a full length nucleic acid coding for a hRXR- ⁇ polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 12 or 20 contiguous nucleotides present in the full length nucleic acid encoding a hRXR- ⁇ polypeptide.
  • the invention also features a nucleic acid probe for the detection of a hRXR- ⁇ polypeptide or nucleic acid encoding a hRXR- ⁇ polypeptide in a sample.
  • the nucleic acid probe contains nucleic acid that will hybridize to a sequence set forth in SEQ ID N0:1, but not to a mouse RXR- ⁇ nucleic acid sequence under high stringency hybridization conditions.
  • the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 27, 30, 35, 40 or 50 contiguous amino acids of the full-length sequence set forth in SEQ ID N0:2.
  • high stringency hybridization conditions those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinyl- pyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml) , 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS. Under stringent hybridization conditions
  • Methods for using the probes include detecting the presence or amount hRXR- ⁇ RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to hRXR- ⁇ RNA.
  • the nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a hRXR- ⁇ polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al . , in Nonisotopic DNA Probe Techniques. p. 275 Academic Press, San Diego (Kricka, ed. , 1992) hereby incorporated by reference herein in its entirety, including any drawings) .
  • Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
  • the invention features recombinant nucleic acid comprising a contiguous nucleic acid sequence encoding a hRXR- ⁇ polypeptide, preferably in a cell or an organism.
  • the recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1 and a vector or a promoter effective to initiate transcription in a host cell.
  • the recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence compli- mentary to an RNA sequence encoding a hRXR- ⁇ polypeptide and a transcriptional termination region functional in a cell.
  • the invention features an isolated, enriched, purified or recombinant hRXR- ⁇ polypeptide.
  • isolated in reference to a polypeptide is meant a polymer of 2 (preferably 7, more preferably 13, most preferably 25) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized.
  • the isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term "isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
  • sequence is the only amino acid chain present, but that it is the predominate sequence present (at least 10 - 20% more than any other sequence) and is essentially free (about 90 - 95% pure at least) of non-amino acid material naturally associated with it.
  • enriched in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2 - 5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term "significantly” here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more prefer ⁇ ably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no amino acid from other sources.
  • the amino acid from other sources may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid.
  • purified in reference to a polypeptide does not require absolute purity (such as a homogeneous prepara ⁇ tion) ; instead, it represents an indication that the sequence is relatively purer than in the natural environ ⁇ ment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml) . Purification of at least one order of magnitude, prefer ⁇ ably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The sub ⁇ stance is preferably free of contamination at a function- ally significant level, for example 90%, 95%, or 99% pure.
  • recombinant hRXR- ⁇ polypeptide is meant a hRXR- ⁇ polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g. , present in a different cell or tissue than found in nature) , purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • This invention features recombinant hRXR- ⁇ polypeptides obtainable using techniques known to those skilled in the art, including those described in McDonnell et al . , PCT Publication No. WO 94/23068 published October 13, 1994, Evans et al . , U.S. Patent 5,071,773, and PCT application, PCT/US91/00399 filed January 22, 1991 (International Publication No. WO 91/12258) , incorporated by reference herein.
  • vector pBacPAK ⁇ (Clontech) or vector pBacPAK9 (Clontech) is used to express recombinant hRXR- ⁇ polypeptide in insect cells.
  • vector pYES2 (Invitrogen) is used to express recombinant hRXR- ⁇ polypeptide in yeast cells.
  • pBKCMV (Stratagene) is used to express recombinant hRXR- ⁇ poly ⁇ peptide in mammalian cells.
  • the hRXR- ⁇ polypeptide contains at least 9, 10, 15, 20, or 30 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2.
  • the invention features a puri ⁇ fied antibody (e.g.. a monoclonal or polyclonal antibody) having specific binding affinity to a hRXR- ⁇ polypeptide.
  • the antibody contains a sequence of amino acids that is able to specifically bind to a hRXR- ⁇ polypeptide.
  • An anti-peptide antibody may be prepared with techniques known to those skilled in the art, including, but not limited to, those disclosed in Niman, PCT application PCT/US88/03921 (International Publication No. WO 89/04489) , incorporated by reference herein.
  • Antibodies having specific binding affinity to a hRXR- ⁇ polypeptide may be used in methods for detecting the presence and/or amount of a hRXR- ⁇ polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the hRXR- ⁇ polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container means containing the antibody and a second container means having a conjugate of a binding partner of the antibody and a label.
  • the invention features a hybridoma which produces an antibody having specific binding affinity to a hRXR- ⁇ polypeptide.
  • hybrida an immortalized cell line which is capable of secreting an antibody, for example a hRXR- ⁇ antibody.
  • the hRXR- ⁇ antibody comprises a sequence of amino acids that is able to specifically bind a hRXR- ⁇ polypeptide.
  • the invention provides transgenic, nonhuman mammals containing a transgene encoding a hRXR- ⁇ polypeptide or a gene effecting the expression of a hRXR- ⁇ polypeptide.
  • Such transgenic nonhuman mammals are partic ⁇ ularly useful as an in vivo test system for studying the effects of introducing a hRXR- ⁇ polypeptide, regulating the expression of a hRXR- ⁇ polypeptide (i.e.. through the introduction of additional genes, antisense nucleic acids, or ribozymes) .
  • transgenic animal is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • the transgenic DNA may encode for a hRXR- ⁇ polypeptide. Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor.
  • the invention describes a recombinant cell or tissue containing a purified nucleic acid coding for a hRXR- ⁇ polypeptide.
  • the nucleic acid may be under the control of its genomic regu- latory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • exogenous it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the hRXR- ⁇ polypeptide.
  • Another aspect of the invention features a method of detecting the presence or amount of a compound capable of binding to a hRXR- ⁇ polypeptide.
  • the method involves incubating the compound with a hRXR- ⁇ polypeptide and detecting the presence or amount of the compound bound to the hRXR- ⁇ polypeptide.
  • the present invention also features novel compounds capable of binding hRXR- ⁇ poly- peptide that are identified by methods described above.
  • a cell or an in vi tro system is transformed with a vector expressing hRXR- ⁇ polypeptide and a reporter gene which becomes activated when a ligand binds to hRXR- ⁇ polypeptide. Then said cell or in vi tro system is brought into contact with a test compound. An increase in the activity of the reporter gene would indicate that the test compound is capable of binding hRXR- ⁇ polypeptide.
  • the DNA-binding domain of hRXR- ⁇ is replaced with the DNA-binding domain of a well character ⁇ ized nuclear receptor, such as the glucocorticoid or estrogen receptor, to create a chimeric receptor able to activate a glucocorticoid- or estrogen-responsive reporter gene in the presence of the hRXR- ⁇ -specific ligand (Giguere, V. and Evans, RM 1990, "Identification of receptors for retinoids as members of the steroid and thyroid hormone receptor family", In : Packer L (ed) Retinoids. Part A: Molecular and Metabolic Aspects. Methods in Enzvmology.
  • the cell is transformed with the chimeric receptor.
  • the cell is also transformed with a reporter vector which comprises a segment encoding a reporter polypeptide under the control of a promoter and a segment of hormone response element (such as a glucocorticoid- or estrogen-responsive element) .
  • a suitable hormone or ligand is provided to the cell, a hormone receptor - hormone complex is formed and delivered to an appropriate DNA-binding region to thereby activate the hormone response element and cause expression of the reporter gene.
  • Activation of the reporter gene is detected by standard procedures used for detecting the product of the reporter gene.
  • the compounds identified by the method of this invention are particularly useful in the treatment of skin-related diseases, including, without limitation, actinic keratoses, axenic keratoses, inflammatory and non ⁇ inflammatory acne, psoriasis, ichthyoses and other keratinization and hyperproliferative disorders of the skin, eczema, atopic dermatitis, Darriers disease, lichen planus, prevention and reversal of glucocorticoid damage
  • the compounds are also useful for the prevention and treatment of cancerous and pre-cancerous conditions, including, premalignant and malignant hyperproliferative diseases such as cancers of the breast, skin, prostate, cervix, uterus, colon, bladder, esphoagus, stomach, lung, larynx, oral cavity, blood and lymphatic system, metaplasias, dysplasias, neoplasias, leukoplakias and papillomas of the mucous membranes and in the treatment of Kaposis sarcoma.
  • premalignant and malignant hyperproliferative diseases such as cancers of the breast, skin, prostate, cervix, uterus, colon, bladder, esphoagus, stomach, lung, larynx, oral cavity, blood and lymphatic system, metaplasias, dysplasias, neoplasias, leukoplakias and papillomas of the mucous membranes and in the treatment of Kaposi
  • the present compounds can be used as agents to treat diseases of the eye, including, without limitation, proliferative vitreoretinopathy (PVR) , retinal detachment, dry eye and other corneopathies, as well as in the treat ⁇ ment and prevention of various cardiovascular dyslipid- emias, prevention of restenosis and as an agent to increase the level of circulating tissue plasminogen activator (TPA) .
  • PVR proliferative vitreoretinopathy
  • TPA tissue plasminogen activator
  • Other uses for the compounds of the present invention include the prevention and treatment of conditions and diseases associated with Human papilloma virus (HPV) , including warts and genital warts, various inflammatory diseases such as pulmonary fibrosis, ileitis, colitis and Krohn's disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Lou Gehrigs disease, improper pituitary function, including insufficient production of growth hormone, modulation of apoptosis, including both the induction of apoptosis and inhibition of T-Cell activated apoptosis, restoration of hair growth, including combination therapies with the present compounds and other agents such as Minosidil, diseases associated with the immune system, including use of the present compounds as immunosuppressants and immuno- stimulants, modulation of organ transplant rejection and facilitation of wound healing, including modulation of chelosis.
  • the compounds identified herein may be used in combination with radiation therapy, chemotherapy and other biologicals such as interferons and interleukins.
  • the present invention also includes pharmaceutically acceptable compositions prepared for storage and subse ⁇ quent administration which include a pharmaceutically effective amount of an above-described product in a pharmaceutically acceptable carrier or diluent.
  • therapeutically effective amount is meant an amount of a pharmaceutical composition having a thera ⁇ Pontically relevant effect.
  • a therapeutically relevant effect relieves to some extent one or more symptoms of the disease or condition in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition.
  • the present invention relates to hRXR- ⁇ polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing.
  • nucleic acid molecules include the functional equivalents of the herein-described isolated nucleic acid molecules.
  • the degeneracy of the genetic code permits substitution of certain codons by other codons which specify the same amino acid and hence would give rise to the same protein.
  • the nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon.
  • portions or all of the hRXR- ⁇ gene could be synthesized to give a nucleic acid sequence significantly different from that shown in SEQ ID NO: 1. The encoded amino acid sequence thereof would, however, be preserved.
  • nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5' -end and/or the 3'-end of the nucleic acid formula shown in SEQ ID NO: 1 or a derivative thereof.
  • Any nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of SEQ ID NO:2 which is encoded by the nucleotide sequence.
  • the present inven- tion is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5' -end of the inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3'-end of the inventive nucleo- tide sequence or its derivative.
  • the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its 5'-end and/or 3'-end.
  • Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto.
  • All variations of the nucleotide sequence of the hRXR- ⁇ genes and fragments thereof permitted by the genetic code are, therefore, included in this invention.
  • the two polypeptides are functionally equivalent, as are the two nucleic acid molecules which give rise to their production, even though the differences between the nucleic acid molecules are not related to degeneracy of the genetic code.
  • a nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain another nucleic acid molecule of the present invention.
  • a chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989) .
  • nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest.
  • the synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, A Guide to Methods and Applications, edited by Michael et al., Academic Press, 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
  • PCR polymerase chain reaction
  • One skilled in the art can readily design such probes based on the sequence disclosed herein using methods of computer alignment and sequence analysis known in the art (cf.
  • hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemi- luminescence, and the like. After hybridization, the probes may be visualized using known methods.
  • the nucleic acid probes of the present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art.
  • the nucleic acid probe may be immobilized on a solid support.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
  • test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized. III. Probe Based Method And Kit For Detecting hRXR- ⁇ .
  • One method of detecting the presence of hRXR- ⁇ in a sample comprises a) contacting said sample with the above- described nucleic acid probe, under conditions such that hybridization occurs, and b) detecting the presence of said probe bound to said nucleic acid molecule.
  • a) contacting said sample with the above- described nucleic acid probe, under conditions such that hybridization occurs and b) detecting the presence of said probe bound to said nucleic acid molecule.
  • One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
  • a kit for detecting the presence of hRXR- ⁇ in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe.
  • the kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase) , and affinity labeled probes (biotin, avidin, or steptavidin) .
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compart ⁇ ment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents
  • nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • the present invention also relates to a recombinant DNA molecule comprising, 5' to 3' , a promoter effective to initiate transcription in a host cell and the above- described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecules.
  • the present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said cell.
  • the above- described molecules may be isolated and/or purified DNA molecules.
  • the present invention also relates to a cell or organism that contains an above-described nucleic acid molecule.
  • the peptide may be purified from cells which have been altered to express the peptide.
  • a cell is said to be "altered to express a desired peptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • the precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
  • Such regions will normally include those 5' -non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the sequence encoding an hRXR- ⁇ gene may be obtained by the above- described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3' -region naturally contiguous to the DNA sequence encod ⁇ ing an hRXR- ⁇ gene, the transcriptional termination signals may be provided. Where the transcriptional termi ⁇ nation signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences (such as a promoter region sequence and a hRXR- ⁇ sequence) . are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a hRXR- ⁇ gene sequence, or (3) interfere with the ability of a hRXR- ⁇ gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • transcriptional and translational signals recognized by an appropriate host are necessary.
  • the present invention encompasses the expression of the hRXR- ⁇ gene (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for the hRXR- ⁇ gene.
  • Prokaryotes most frequently are represented by various strains of E. coli . However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUCll ⁇ , pUC119 and the like; suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtll and the like; and suit ⁇ able virus vectors may include pMAM-neo, pKRC and the like.
  • the selected vector of the present invention has the capacity to replicate in the selected host cell.
  • prokaryotic hosts include bacteria such as E. coil, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the peptide will not be glycosylated.
  • the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • hRXR- ⁇ (or a functional derivative thereof) in a prokaryotic cell
  • a functional prokaryotic promoter may be either constitutive or, more preferably, regulatable (i.e., inducible or derepres- sible) .
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the j ⁇ -lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ) , the trp, recA, lacZ, lad, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et at., J. Bacteriol. 162:176-182(1985)) and the ⁇ -28-specific promoters of B.
  • subtilis (Gilman et at., Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468-478(1986)) .
  • Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiot. 1:277-282(1987)); Cenatiempo (Biochimie 68:505-516(1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).
  • progeny Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene sequence-encoding sequence.
  • ribosome binding sites are disclosed, for example, by Gold et at. (Ann. Rev. Microbiol. 35:365-404(1981)).
  • the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • “cell”, “cell line”, and “cell culture” may be used interchange ⁇ ably and all such designations include progeny.
  • the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent muta ⁇ tions. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
  • Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the hRXR- ⁇ polypeptide of interest.
  • Suitable hosts may often include eukaryotic cells.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CH0-K1, or cells of lymphoid origin and their derivatives.
  • Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 which may provide better capacities for correct post-translational processing.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459(1988).
  • baculovirus vectors can be engineered to express large amounts of hRXR- ⁇ in insects cells (Jasny, Science 238:1653 (1987); Miller et al. , In: Genetic Engineering (1986), Setlow, J.K., et al., eds. , Plenum, Vol. 8, pp. 277-297) .
  • yeast gene sequence expression systems can be utilized which incorporate promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides) .
  • transcriptional and translational regulatory signals may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • regula ⁇ tory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288(1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature (London) 290:304-
  • eukaryotic mRNA Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes hRXR- ⁇ (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG) . The presence of such codons results either in a formation of a fusion protein
  • AUG codon is not in the same reading frame as the hRXR- ⁇ coding sequence) .
  • a hRXR- ⁇ nucleic acid molecule and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
  • a vector may be employed which is capable of inte ⁇ grating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA.
  • cDNA expression vectors incorporating such elements include those described by Okayama, Molec. Cell. Biol. 3:280(1983) .
  • the introduced nucleic acid molecule can be incor ⁇ porated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coil
  • Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacitli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al. , Miami Wntr. Symp. 19:265-274(1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et at., J. Ctin. Hematol. Oncol.
  • the DNA construct (s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., trans- formation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • hRXR- ⁇ or fragments thereof results in the production of hRXR- ⁇ or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administra- tion of bromodeoxyuracil to neuroblastoma cells or the like) .
  • a variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions.
  • the peptide may be purified from tissues or cells which naturally produce the peptide.
  • the above- described isolated nucleic acid fragments could be used to expressed the hRXR- ⁇ protein in any organism.
  • the samples of the present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the. assay format, the detection method and the nature of the tissues, cells or extracts used as the sample.
  • source organism refers to the original organism from which the amino acid sequence of the subunit is derived, regardless of the organism the subunit is expressed in and ultimately isolated from.
  • the present invention relates to an antibody having binding affinity to a hRXR- ⁇ polypeptide.
  • the polypeptide may have the amino acid sequence set forth in SEQ ID NO:2, or mutant or species variation thereof, or at least 9 contiguous amino acids thereof (preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof).
  • the present invention also relates to an antibody having specific binding affinity to an hRXR- ⁇ polypeptide.
  • an antibody may be isolated by comparing its binding affinity to a hRXR- ⁇ polypeptide with its binding affinity to another polypeptide. Those which bind selectively to hRXR- ⁇ would be chosen for use in methods requiring a distinction between hRXR- ⁇ and other polypeptides.
  • the hRXR- ⁇ proteins of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
  • the hRXR- ⁇ peptide of the present invention can be used to produce antibodies or hybridomas.
  • the antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma which produces the above-described monoclonal antibody, or binding fragment thereof.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the anti- genicity of the polypeptide and the site of injection.
  • The.polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or 3-galactosidase) or through the inclusion of an adjuvant during immunization.
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Agl4 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al. , Exp. Cell Res. 175:109- 124(1988)).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, supra (1984) ) .
  • antibody containing anti- sera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specific ⁇ ity using one of the above-described procedures.
  • the above-described antibodies may be detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like) , enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluor ⁇ escent labels (such as FITC or rhodamine, and the like) , paramagnetic atoms, and the like.
  • the above-described antibodies may also be immobil ⁇ ized on a solid support.
  • solid supports include plastics such as polycarbonate, complex carbo ⁇ hydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10(1986); Jacoby et al. , Meth. Enzym. 34 Academic Press, N.Y. (1974)).
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immuno- chromotography.
  • Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the hRXR- ⁇ peptide sequence with acidic residues, while maintaining hydro- phobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
  • the present invention encompasses a method of detect ⁇ ing a hRXR- ⁇ polypeptide in a sample, comprising: a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and b) detecting the presence of said antibody bound to the polypeptide.
  • the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample.
  • Incubation conditions vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay.
  • immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays
  • Examples of such assays can be found in Chard, "An Introduction to Radioimmunoassay and Related Techniques" Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al.
  • the immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
  • the test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.
  • kits contains all the necessary reagents to carry out the previously described methods of detection.
  • the kit may comprise: i) a first container means containing an above-described antibody, and ii) second container means containing a conjugate comprising a binding partner of the antibody and a label.
  • the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alter ⁇ native, if the primary antibody is labeled, the chromo- phoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the com- partmentalized kit may be as described above for nucleic acid probe kits.
  • the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • the present invention also relates to a method of detecting a compound capable of binding to a hRXR- ⁇ polypeptide comprising incubating the compound with hRXR- ⁇ and detecting the presence of the compound bound to hRXR- ⁇ .
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
  • the present invention also relates to a method of detecting an agonist or antagonist of hRXR- ⁇ activity comprising incubating cells that produce hRXR- ⁇ in the presence of a compound and detecting changes in the level of hRXR- ⁇ activity.
  • the compounds thus identified would produce a change in activity indicative of the presence of the compound.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art.
  • the present invention also encompasses a method of agonizing (stimulating) or antagonizing hRXR- ⁇ associated activity in a mammal comprising administering to said mammal an agonist or antagonist to hRXR- ⁇ in an amount sufficient to effect said agonism or antagonism.
  • DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g.. the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al.. Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985) ) .
  • Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA) , Taconic (Germantown, NY) , Harlan Sprague Dawley (Indianapolis, IN) , etc.
  • transgenic mouse female mice are induced to superovulate. Females are placed with males, and the mated females are sacri- ficed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice. See Hammer et. al. , Cell 63:1099-1112 (1990) .
  • ES cells embryonic stem cells
  • methods for the culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipita ⁇ tion and direct injection also are well known to those of ordinary skill in the art. See, for example, Terato- carcinomas and Embryonic Stem Cells. A Practical Approach. E.J. Robertson, ed., IRL Press (1987) .
  • a clone containing the sequence(s) of the invention is co- transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra) .
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination.
  • Capecchi Science 244 : 1288- 1292 (1989) .
  • Methods for positive selection of the recombination event (i.e. , neo resistance) and dual positive-negative selection (i.e.. neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et. a ⁇ . , Nature 338: 153-156 (1989) , the teachings of which are incorporated herein.
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Procedures for the production of non-rodent mammals and other animals have been discussed by others. See Houdebine and Chourrout, supra; Purse1 e_t al. , Science 244 :1281-1288 (1989); and Simms et. al. , Bio/Technology fL:179-183 (1988) .
  • an expression vector containing the hRXR ⁇ coding sequence is inserted into cells, the cells are grown in vi tro and then infused in large numbers into patients.
  • a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous hRXR ⁇ in such a manner that the promoter segment enhances expression of the endogenous hRXR ⁇ gene (for example, the promoter segment is •transferred to the cell such that it becomes directly linked to the endogenous hRXR ⁇ gene) .
  • the gene therapy may involve the use of an adenovirus containing hRXR ⁇ cDNA targeted to a tumor, systemic hRXR ⁇ increase by implantation of engineered cells, injection with hRXR ⁇ virus, or injection of naked hRXR ⁇ DNA into appropriate tissues.
  • Target cell populations e.g.. hematopoietic or nerve cells
  • Target cell populations may be modified by introducing altered forms of hRXR ⁇ in order to modulate the activity of such cells.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g.. cDNA) encoding recombinant hRXR ⁇ protein into the targeted cell population (e.g.. tumor cells) .
  • nucleotide sequences e.g.. cDNA
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors contain- ing coding sequences. See, for example, the techniques described in Maniatis et al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, N.Y.
  • nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g.. lipo ⁇ somes or other lipid systems for delivery to target cells (See e.g.. Feigner et al. , Nature 337:387-8, 1989) .
  • gene transfer can be performed by simply injecting minute amounts of D ⁇ A into the nucleus of a cell, through a process of microinjection.
  • Capecchi MR Cell 22:479-88 (1980) .
  • Other methods have also been attempted for introducing D ⁇ A into larger numbers of cells. These methods include: transfection, wherein D ⁇ A is precipitated with CaP0 4 and taken into cells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol.
  • Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins. It has also been shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene.
  • DNA transfer means the process of introducing a foreign nucleic acid molecule into a cell.
  • Gene transfer is commonly performed to enable the expres ⁇ sion of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA.
  • Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cyto ⁇ plasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
  • gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
  • a vector having nucleic acid sequences encoding hRXR ⁇ is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expres ⁇ sion as set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
  • the nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
  • Gene replacement means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • compositions where it is mixed with suitable carriers or excipient(s) .
  • a therapeutically effective amount of a agent or agents such as these is administered.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determin ⁇ ing the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated ini ⁇ tially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disruption of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex component) .
  • IC 50 as determined in cell culture
  • levels in plasma may be measured, for example, by HPLC.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al. , in The Pharmacological Basis of Therapeutics. 1975, Ch. 1 p. 1) . It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dys ⁇ functions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) . The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods.
  • the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • a program comparable to that discussed above may be used in veteri ⁇ nary medicine.
  • such agents may be formulated and administered systemic- ally or locally. Techniques for formulation and adminis ⁇ tration may be found in Remington's Pharmaceutical Sciences. 18th ed. , Mack Publishing Co., Easton, PA (1990) .
  • Suitable routes may include oral, rectal, trans- dermal, vaginal, transmucosal, or intestinal administra ⁇ tion; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intra- thecal, direct intraventricular, intravenous, intraperi ⁇ toneal, intranasal, or intraocular injections, just to name a few.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physio- logically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formula ⁇ tion.
  • penetrants are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic admin ⁇ istration is within the scope of the invention.
  • the compositions of the present invention in particular, those formulated as solutions, may be adminis ⁇ tered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tab ⁇ lets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior.
  • the liposomal contents are both protected from the external microenvi- ronment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mix ⁇ ing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical formulations for parenteral adminis ⁇ tration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or syn ⁇ thetic fatty acid esters, such as ethyl oleate or triglyc- erides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl- methyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pig ⁇ ments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, option- ally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • a hRXR ⁇ nucleic acid sequence may be administered utilizing an ex vivo approach whereby cells are removed from an animal, transduced with the hRXR ⁇ nucleic acid sequence and reimplanted into the animal.
  • the liver can be accessed by an ex vivo approach by removing hepatocytes from an animal, transducing the hepatocytes in vi tro with the hRXR ⁇ nucleic acid sequence and reimplanting them into the animal ( e . g. , as described for rabbits by Chowdhury et al, Science 254: 1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3: 179-222, 1992) incorporated herein by reference.
  • hRXR ⁇ nucleic acid sequence into a cell can be used, including direct naked DNA uptake (e . g. , Wolff et al., Science 247: 1465-1468, 1990), receptor-mediated DNA uptake, e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu et al. , j Biol. Chem. 266: 14338-14342, 1991), and liposome-mediated delivery (e.g., Kaneda et al., Expt. Cell Res.
  • direct naked DNA uptake e . g. , Wolff et al., Science 247: 1465-1468, 1990
  • receptor-mediated DNA uptake e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor
  • the hRXR ⁇ or nucleic acid encoding hRXR ⁇ may also be administered via an implanted device that provides a support for growing " cells.
  • the cells may remain in the implanted device and still provide the useful and therapeutic agents of the present invention.
  • a human heart cDNA library Human Heart 5' -STRETCH in ⁇ -gtlO, was purchased from Clontech Laboratories Inc., Palo Alto, California.
  • a 1.7kb fragment isolated from a mouse RXR- ⁇ cDNA clone (pSKmRXR- ⁇ , (Mangelsdorf,D.J. , Borgmeyer,U. , Heyman,R.A., Zhou,J.Y., Ong,E.S., Oro,A.E., Kakizuka,A. and Evans,R.M.
  • PCR amplification of the 23 phage clones showed that 15 phages contained fragments greater than 500bp. These fragments fell into 9 different size groups ranging from 600bp to 1600bp. To ensure the fragments amplified by PCR were the phage clone fragments that hybridized to mouse RXR- ⁇ , Southern blot analysis was performed on the amplified inserts; all amplified fragments continued to hybridize with the mouse RXR- ⁇ probe.
  • Nucleotide sequence alignments showed that two of the clones (clone 10 and clone 27) were related to mouse RXR- ⁇ ; whereas three clones were human RXR- ⁇ and one clone was human RXR-S.
  • phage DNA was prepared and the inserts were subcloned from the phage DNA into pGEM-4Z. The size of each insert was 900bp for clone 10 and 1600bp for clone 27.
  • the nucleotide sequence of the complete human RXR- ⁇ cDNA was determined by sequencing fragments isolated by restriction digestion and subcloning into pGEM-4Z; both strands of the clone were sequenced. All nucleotide sequencing was performed with the Automated Laser Fluor ⁇ escence (A.L.F.) DNA Sequencer (Pharmacia Biotechnology, Piscataway, NJ) . The entire nucleotide sequence was compiled utilizing the Genetics Computer Group Sequence Analysis Software Package (Devereux,J. , Haeberli,P. and Smithies,O. (1984) Nucleic. Acids. Res., 12, 387-395).
  • sequences 5' to the PstI site in clone 10 were replaced with the corresponding sequences from clone 27.
  • This process generated a clone containing 1594 nucleotides. Sequence analysis indicates that the clone begins 28bp upstream of the initiator codon ATG and contains an in frame stop codon 12bp upstream of the ATG.
  • the clone contains an open reading frame of 1389nt, a short 3' untranslated region containing a poly A addition site (AATAAA) followed 14nt later by a string of 22 adenine residues.
  • AATAAA poly A addition site
  • the deduced amino acid sequence of human RXR- ⁇ predicts a protein of 463 amino acids.
  • a comparison of the amino acid sequences between human and mouse show 98% amino acid sequence identity (i.e., "homology").
  • In vi tro transcription followed by in vi tro translation yields a protein product that has an apparent molecular mass of 55Kd when analyzed by SDS-polyacrylamide gel electrophoresis.
  • a human multiple tissue Northern blot (Clontech Laboratories Inc.) containing lO ⁇ g of poly-A plus mRNA isolated from several human tissues was hybridized with the full length human RXR- ⁇ cDNA that was random prime labeled with [ 32 P]-dCTP. The hybridization and all washes were conducted under high-stringency.
  • a message of approximately 2.2kb that specifically hybridizes with the human RXR- ⁇ probe is found in high amounts in skeletal muscle and in lower amounts in heart, brain, lung and liver.
  • a smaller message of 1.24kb is also seen in human liver.
  • mouse tissues contain predominately two RXR- ⁇ transcripts of 2.5kb and 2.0kb (Mangelsdorf,D.J. , Borgmeyer,U. , Heyman,R.A., Zhou,J.Y.., Ong,E.S., Oro,A.E., Kakizuka,A. and Evans,R.M. (1992) Genes Dev. , 6, 329-344); whereas human tissues contain three RXR- ⁇ transcripts of 6.5kb, 2.2kb and 1.24kb.
  • the different expression patterns seen in mouse between the 2.5kb and the 2.Okb fragment are not seen in human tissues.
  • the 2.5kb transcript is seen in brain and lung, the 2.Okb transcript is present in kidney and liver and both transcripts are expressed in mouse heart and muscle. There is no evidence that a similar expression pattern exists in human tissues.
  • the predominate transcript detected in human tissues is
  • RNA-protection probe for the human RXR- ⁇ isoform was constructed by PCR amplification from the full length hRXR- ⁇ cDNA clone; a 159bp fragment corresponding to nucleotides 1395-1553 of human RXR- ⁇ was amplified. The resulting fragment was cloned into pGEM4Z, and its identity was confirmed by nucleotide sequencing. The specificity of this probe for human sequences was verified by testing the probe against both mouse, rat and human
  • RNAs known to contain the corresponding mRNA The cRNA probe was produced by linearizing the plasmid with EcoRI and followed by in vitro transcription in the presence of
  • [ 3 P] -UTP utilizing T7 polymerase A probe corresponding to human GAPDH was purchased from Ambion Inc and is util ⁇ ized in all assays to control for RNA content in the sample.
  • RNA-protection analysis was performed as described (Zinn et. al., 1984; Melton et. al., 1984). Hybridization of cRNA probes was carried out at 45°C overnight, followed by the addition of 300 ⁇ l of RNase digestion buffer containing 40 ⁇ g/ml of RNase A and 700 U/ml of RNase Tl. RNase digestion was performed at 25°C for 1 hour. The RNase resistant fragments were resolved by electrophoresis on 6% urea-polyacrylamide sequencing gels. To control for RNA loading, a GAPDH probe was included in all samples.
  • hRXR- ⁇ is not expressed in two hematopoietic cell lines, HL-60 and RPMI 8226, in a cervical carcinoma cell line, ME-180, in a breast cancer cell line, MCF-7, or in a prostate carcinoma cell line, LNCaP.
  • hRXR- ⁇ cDNA clone produced a protein that was transcriptionally competent
  • applicant used a transient co-transfection assay with a reporter construct specific for the RXR class of retinoid receptors (CRBP (2) -tk-LUC) (Mangelsdorf,D.J. , Umesono,K., Kliewer,S.A. , Borgmeyer,U. , Ong,E.S. and Evans,R.M. (1991) Cell, 66, 555-561) .
  • a vector expressing human RXR- ⁇ is transfected into CV-1 cells along with a reporter for monitoring RXR-activity and the CV-1 cells are exposed to retinoid specific compounds.
  • CV-1 cells were transiently transfected with a mixture of plasmids including the hRXR- ⁇ expression vector, a reporter plasmid containing two copies of the RXR response element from the cellular retinoic acid binding protein (CRBP, 2
  • CRBP cellular retinoic acid binding protein
  • -tk-LUC cellular retinoic acid binding protein
  • MOLECULE TYPE DNA (genomic)
  • AGCGTATTCC CCACTTCTCT GACCTCACCT TGGAGGACCA 920 GGTCATTTTG CTTCGGGCAG GGTGGAATGA ATTGCTGATT 960
  • Lys Asp Leu lie Thr Thr Cys Arg Asp Asn Lys Asp Cys Leu 170 175 180
  • Thr Asn lie Cys His Ala Ala Asp Lys Gin Leu Phe Thr Leu 270 275 280
  • Val Glu Trp Ala Lys Arg lie Pro His Phe Ser Asp Leu Thr
  • Leu Arg Leu Pro Ala Leu Arg Ser lie Gly Leu Lys Cys Leu 425 430

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Abstract

L'invention concerne un nouveau récepteur de rétinoïde, le récepteur η de rétinoïde X humain. Le hRXRη module la transcription de certains gènes en présence de certains composés de rétinoïdes. Le hRXRη diffère des récepteurs de rétinoïde connus par sa séquence nucléotidique, sa séquence d'acides aminés et son modèle d'expression dans des tissus. L'invention concerne l'acide nucléique isolé, purifié ou enrichi codant des polypeptides de hRXRη ainsi que des vecteurs les contenant, des cellules transformées à l'aide desdits vecteurs et des procédés de criblage de composés capables de lier des polypeptides de hRXRη. En outre, l'invention concerne des polypeptides de hRXRη isolés, purifiés, enrichis ou recombinés, des anticorps présentant une affinité de liaison spécifique vis-à-vis des polypeptides de hRXRη, ainsi que des hybridomes produisant ces anticorps.
EP96903616A 1995-01-23 1996-01-19 RECEPTEUR $g(g) DE RETINOIDE X HUMAIN (hRXR-GAMMA) Withdrawn EP0812354A2 (fr)

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PCT/US1996/000847 WO1996023070A2 (fr) 1995-01-23 1996-01-19 RECEPTEUR η DE RETINOIDE X HUMAIN (hRXR-GAMMA)
US377423 2003-02-28

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CN110592095A (zh) * 2019-09-26 2019-12-20 浙江海洋大学 曼氏无针乌贼维甲酸x受体基因的克隆和表达

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GB9907461D0 (en) * 1999-03-31 1999-05-26 King S College London Neurite regeneration
JP2003533184A (ja) * 2000-03-30 2003-11-11 オックスフォード バイオメディカ（ユーケイ）リミテッド レチノイン酸レセプターβ２、そのアンタゴニスト、および神経障害治療用の遺伝子治療ベクター
WO2005101008A1 (fr) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Diagnostics et therapeutiques pour traiter des maladies associees au recepteur gamma de retinoide x (rxrg)

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JPH05504479A (ja) * 1990-02-09 1993-07-15 ザ　ソーク　インスティテュート　フォア　バイオロジカル　スタディーズ レチノイドレセプター組成物および方法
DE10199015I2 (de) * 1991-12-18 2004-01-29 Salk Inst For Biological Studi Mitteln zur Modulierung von Verfahren durch Retinoid Rezeptoren und dafür nützliche Verbindungen.
CA2106382A1 (fr) * 1992-01-24 1993-07-25 Mark Leid Recepteurs nucleaires proteiques heterodimeriques, genes codant pour ces recepteurs et leur utilisation

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CN110592095A (zh) * 2019-09-26 2019-12-20 浙江海洋大学 曼氏无针乌贼维甲酸x受体基因的克隆和表达

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