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WO2000068269A1 - Recepteurs chimeriques constitutifs et leurs procedes d'utilisation - Google Patents

Recepteurs chimeriques constitutifs et leurs procedes d'utilisation Download PDF

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Publication number
WO2000068269A1
WO2000068269A1 PCT/US2000/012793 US0012793W WO0068269A1 WO 2000068269 A1 WO2000068269 A1 WO 2000068269A1 US 0012793 W US0012793 W US 0012793W WO 0068269 A1 WO0068269 A1 WO 0068269A1
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Prior art keywords
receptor
crf
ligand
chimeric
chimeric peptide
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PCT/US2000/012793
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English (en)
Inventor
Wylie W. Vale
Soren M. Nielsen
Lisa Z. Nielsen
Marilyn H. Perrin
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The Salk Institute For Biological Studies
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Priority to EP00932265A priority Critical patent/EP1177215A1/fr
Priority to CA002372807A priority patent/CA2372807A1/fr
Priority to AU50008/00A priority patent/AU5000800A/en
Publication of WO2000068269A1 publication Critical patent/WO2000068269A1/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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • 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
    • C07K14/575—Hormones
    • C07K14/57509—Corticotropin releasing factor [CRF] (Urotensin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to novel chimeric proteins for use in modulating signal transduction.
  • inducible receptors which are activated (or deactivated) by the binding of a ligand to the receptor.
  • the formation of the complex with the receptor results in a change in conformation with the receptor undergoing a change which results in a signal being transduced.
  • the conformational change in the receptor results in binding to other proteins, where the other proteins are activated and may carry out various functions.
  • the receptor is autophosphorylated or phosphorylated, resulting in a change in its activity.
  • Constitutively active receptors are of theoretical, practical and physiological importance. From a theoretical viewpoint, knowledge of residues in the receptor that account for constitutive activity will further our understanding of structural determinants that determine the mechanism of action of the receptor. From a practical point of view, constitutively active receptors would prove extremely useful in screening for libraries of compounds that inactivate the receptors or that act as inverse agonists. Finally, the roles of receptor mutations that result in constitutively active receptors in disease states are of increasing interest and importance. Therefore, there is a need in the art for methods to render inducible receptors constitutively active. The resulting receptor proteins will have broad and general applicability. BRIEF DESCRIPTION OF THE INVENTION
  • the present invention is directed to novel chimeric constitutively active receptor proteins and DNA sequences encoding these proteins.
  • the chimeric receptors comprise a receptor, or functional fragments thereof, operatively fused to a ligand therefor, or functional fragments thereof.
  • the receptor and ligand are not normally found associated together and only associate to induce activity of the receptor.
  • the receptor is rendered capable of constitutive transduction of a signal and activation of signaling pathway(s), whereby the cell may be turned on to carry out various functions relating to the signaling pathway.
  • a wide variety of receptors and ligands therefor may be employed in the practice of the present invention, wherein the receptors and ligands may be naturally occurring or synthetic.
  • nucleic acid constructs, vectors, cells, and transgenic animals which are capable of expressing the invention chimeric receptors.
  • methods for transforming an inducible receptor into a chimeric constitutively active receptor by fusing a ligand therefor to the receptor are also provided.
  • Figure 1 provides a schematic describing a chimeric construct according to the invention in which r/h CRF(1-41) replaces the N-terminal domain of hCRF-Rl.
  • Figure 2 presents a schematic describing a chimeric construct according to the present invention, wherein r/h CRF(l-43) is fused to hCRF-Rl via an engineered putative eighth transmembrane helix fused to the intracellular C-terminal domain of the receptor. In this chimera, the peptide portion has a free C-terminus.
  • Figure 3 is a schematic outline of Rl and related constructs. Based on alignment and structural modeling of the transmembrane region of secretin-like receptors, transmembrane segment 1 starts at position 124 in Rl.
  • Constructs in the lower left of the figure all have the N-terminal domain corresponding to residues 1-111 of the receptor replaced with the indicated portion of CRF and are expressed with the HA-signal peptide placed upstream of the peptide portion.
  • the construct in the lower right has residues 1-16 of CRF inserted into the N-terminal domain of Rl between residue 28 and 29.
  • the gray shaded dots in transmembrane segments 3 and 5, respectively, indicate residues important for binding of a Rl -specific non-peptide antagonist.
  • Figures 4A and 4B graphically show the constitutive activation and stimulation of Rl and peptide/Rl chimeras.
  • Figure 4A shows the level of cAMP produced by host cells in the absence (white bars) and in the presence (gray bars) of 10 ⁇ M antalarmin.
  • Figure 4B shows the level of c AMP produced by host cells in the absence (white bars) and in the presence (black bars) of 10 ⁇ M urocortin.
  • cAMP level is normalized in each experiment to that observed in the presence of 10 ⁇ M antalarmin. Data are presented as mean ⁇ S.E.M. from 4-8 independent experiments each performed in triplicate. The absolute level of cAMP in presence of 10 ⁇ M antalarmin is similar for all constructs.
  • Figures 5 A and 5B depict the dose response with urocortin and antalarmin on CRF(1-16)/R1 ⁇ N ( Figure 5A) and CRF(1-16[L8A])/R1 ⁇ N ( Figure 5B). Data are presented as mean ⁇ SD from triplicate determinations and are representative of 3 independent assays performed in parallel. For CRF(1-16)/R1 ⁇ N, the IC50 for inhibition of constitutive activation by antalarmin is 14 ⁇ 1 nM. For CRF(1-16[L8A])/R1 ⁇ N , the EC 50 for stimulation by urocortin is 140 ⁇ 20 nM. DETAILED DESCRIPTION OF THE INVENTION
  • chimeric proteins comprising receptors, or functional derivatives thereof, operatively fused to a ligand therefor, or functional derivatives thereof.
  • a receptor By operatively fusing a receptor to its respective ligand, preferably the agonist, the receptor is thereby locked into a constitutively active state, which is distinct from basal activity.
  • constitutively activated chimeric receptor(s) of the present invention can transduce signal indefinitely.
  • This kinetic trapping mechanism represents a novel, potent, long-lasting positive regulatory mechanism with potentially profound and wide-ranging physiological implications. Any receptor undergoing this type of constitutive activation would be expected to contribute dominantly to the overall basal tone in the body, thereby modifying tonicity caused by continuously released hormones and neuro transmitters.
  • inducible receptors may be employed in the practice of the present invnetion.
  • Receptors contemplated for use in the practice of the present invention are readily recognized by those skilled in the art, and include those associated with signal transduction, transcription, the uptake of nutrients, cell adhesion, cell aggregation, endocytosis, and the like.
  • receptors contemplated for use in the practice of the present invention can be cell surface associated receptors, membrane associated receptors, cytoplasmic receptors, nuclear receptors, combinations thereof, and the like.
  • Nuclear receptors include members of the steroid/thyroid hormone receptor superfamily, and the like.
  • Cell surface associated receptors are of particular interest including those which may be involved with one or more second messenger pathways, particularly pathways involved with a protein kinase.
  • Cell surface receptors include ion channel receptors, G-protein coupled receptors, receptors with single transmembrane segments or tyrosine kinase- containing receptor, and the like, as well as functional derivatives thereof.
  • Ion channel receptors contemplated for use in the practice of the present invention include nicotinic, NMDA and non-NMDA, GABA, 5-HT, and the like.
  • G protein-coupled receptors contemplated for use in the practice of the present invention include all subtypes of the opioid, muscarinic, dopamine, adrenergic, cAMP, opsins, angiotensin, serotonin, thyrotropin, gonadotropin, substance-K, substance-P and substance-R receptors, melanocortin, metabotropic glutamate, corticotropin-releasing factor receptors (CRF-R), or any other GPCR receptors known to couple via G proteins (e.g., the secretin-like family of GPCRs, also designated the class 2 or class B receptor family, including receptors for secretin, calcitonin, gastric inhibitory peptide, growth hormone-releasing hormone, glucagon, glucagon-like peptide I, parathyroid hormone
  • Receptors with single transmembrane segments contemplated for use in the practice of the present invention include growth factor receptors, insulin, cytokine, natriureticm, and the like.
  • Ligands contemplated for use in the practice of the present invention will depend on the specific receptor employed and can be readily identified by those of skill in the art.
  • Ligands contemplated include naturally-occurring, semi-synthetic and synthetic agonists and antagonists, and modifiers of the activity of agonists and antagonists which are capable of inducing their respective receptor to transduce signals within the cell.
  • ligands contemplated for use in the practice of the present invention include natural ligands (e.g., neurotransmitters, growth factors, hormones, steroids, autoacoids, chemotactic factors, exogenous stimulants such as odorants, cytokines, modifications thereof, and the like), recombinant peptides, peptidomimetics, antibodies or fragments thereof, synthetic molecules (e.g., drug, or any other agent which is capable of inducing a signal), and the like.
  • natural ligands e.g., neurotransmitters, growth factors, hormones, steroids, autoacoids, chemotactic factors, exogenous stimulants such as odorants, cytokines, modifications thereof, and the like
  • synthetic molecules e.g., drug, or any other agent which is capable of inducing a signal
  • the phrase "functional derivative" of either protein or nucleic acid is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the non-derivatized (i.e., parental) protein or nucleic acid sequence.
  • a functional derivative of a receptor will possess the ability to transduce signals, whereas a functional derivative of a ligand will be able to either inhibit or induce signal transduction upon association with the receptor.
  • a functional derivative of a protein can contain post-translational modifications such as covalently linked carbohydrate, or the like, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include mutants, fragments, segments, variants, analogs, or chemical derivatives of a molecule.
  • the term “functional derivative” also is intended to include chimeric combinations of one or more receptors, i.e., recombinant receptors comprising domains from different receptors (e.g., transmembrane domains from different receptors, DNA binding domain swaps, and the like).
  • the chimeric receptor will comprise only those portions of a cell surface receptor and ligand therefor necessary for signal transduction.
  • the chimeric receptor when the chimeric is derived from a cell surface receptor, the chimeric receptor will comprise at least one transmembrane domain and the cytoplasmic domain of a cell surface receptor operatively fused to a portion of an agonist therefor.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect(s) of the molecule, and the like. Procedures for coupling such moieties to a molecule are well known in the art.
  • the term "operatively fused" in the context of the present invention refers to any type of covalent or non-covalent binding, linkage, fusion or association, including chemical and biological fusions, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • Such fusion performed to join together components of desired functions to generate a desired combination of functions, i.e., constitutive activity, includes covalent bonding, hydrophobic/hydrophilic interaction, Van der Walls forces, ion pairing, ligand- receptor interaction, epitope-antibody binding site interaction, enzyme-substrate interaction, liposome-hydrophobic interaction, nucleotide base pairing, membrane- hydrophobic interaction, and the like.
  • the fusion does not contemplate the endogenous (i.e., normal) interaction of receptor and ligand, but instead contemplates a permanent interaction typically associated with pre- and post-translational fusions, such as recombinant constructs, and the like.
  • ligand in a presently preferred embodiment of the present invention, is fused to transmembrane or extracellular domain(s) of its cognate receptor.
  • ligand(s) can be introduced into receptor(s) in a variety of locations, e.g., within and/or attached to at least one intracellular domain (i.e., hinge domain, DNA binding domain, cytoplasmic domain, and the like), within and/or attached to at least one transmembrane domain of the receptor, within and/or attached to an exogenous transmembrane domain introduced to a chimeric receptor, within and/or attached to at least one extracellular domain at the amino terminus and/or carboxyl terminus of the domain, and/or inserted therebetween, and the like.
  • intracellular domain i.e., hinge domain, DNA binding domain, cytoplasmic domain, and the like
  • the term "introduced” refers to the addition, insertion, replacement, substitution, and the like, of the ligand, or derivatives thereof, or alternatively, the nucleic acid encoding the ligand, preferably the open reading frame, to the receptor, or functional derivatives thereof.
  • polynucleotide or nucleic acid refers to one which is not naturally occurring, or is made by the artificial combination of two otherwise non- contiguous segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • DNA sequences encoding the structural coding sequence of a gene product can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences may also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions.
  • a chimeric receptor can be further modified to facilitate signal transduction and/or protein orientation.
  • the signal sequence at the 5' terminus of the open reading frame (ORF) which directs the chimeric protein to the surface membrane will be the signal sequence of the extracellular domain.
  • ORF open reading frame
  • associated with the signal sequence will be a naturally occurring cleavage site, which will also typically be the naturally occurring cleavage site associated with the signal sequence or the extracellular domain.
  • Such modifications to facilitate signal transduction can be introduced either pre- or post-translational, and may be placed upstream or downstream of the ligand in order to insure insertion of the chimeric receptor in the plasma membrane of the cell (e.g., HA-signal peptide, and the like). Additional modifications which will facilitate chimeric receptor orientation, function, stability, and the like, are known to those skilled in the art.
  • the constructs may be designed so as to avoid their interaction with other surface membrane proteins native to the target host. Thus, for the most part, one will avoid the chimeric receptor binding to other proteins present in the surface membrane.
  • one may select a transmembrane domain which is known not to bind to other transmembrane domains, one may modify specific amino acids, e.g. substitute for a cysteine residue, or the like.
  • the present invention also provides DNA molecules encoding the chimeric proteins of the present invention.
  • Invention DNA molecules generally comprise a DNA sequence encoding the chimeric protein, and associated regulatory sequences such as transcription promoter, a transcription terminator, and the like.
  • prokaryotic expression vectors such as plasmid vectors containing replication and control sequences which are compatible with the host cells are used as cloning vectors for the DNA molecules of the present invention.
  • Other vectors such as ⁇ -phage, cosmids, or yeast artificial chromosomes may also be employed in the practice of the present invention.
  • the vector ordinarily carries a replication site, as well as sequences which encode proteins that are capable of providing phenotypic selection of transformed cells.
  • promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally into the chosen vector.
  • the promoters are operably linked to a nucleic acid sequence encoding the chimeric protein.
  • the promoters may be inducible or constitutive and provide a means to express the encoded chimeric protein in the host. Following expression, the polypeptide may be purified by standard methods such as described below.
  • a DNA sequence encoding the chimeric proteins of the present invention may be inserted into a suitable eukaryotic expression vector, which in turn is used to transfect eukaryotic cells.
  • a eukaryotic expression vector as used herein, is meant to indicate a DNA construct containing regulatory elements which direct the transcription and translation of DNA sequences encoding invention chimeric proteins. Such regulatory elements include promoters, enhancers, transcription terminators and polyadenylation signals. By virtue of the inclusion of these elements operably linked within the DNA constructs, the resulting eukaryotic expression vectors contain the information necessary for expression of the polypeptides of interest.
  • the vector comprises nucleic acid encoding a chimeric receptor operatively linked to an inducible promoter, thereby providing for inducible expression of the chimeric receptor in host cells.
  • Suitable vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art.
  • Any cell line can be used as a suitable "host" in the practice of the present invention.
  • Host cells contemplated for use in expressing recombinant chimeric proteins of interest include mammalian cells, avian cells, insect cells, fungal cells, and the like.
  • cells contemplated for use in the practice of the present invention include transformed cells, non-transformed cells, neoplastic cells, primary cultures of different cell types, and the like.
  • fungal cells contemplated for use in the practice of the present invention include species of yeast (a.g., Saccharomyces spp., Schizosaccharomyces spp.), filamentous fungi (e.g., Aspergillus spp., Neurospora spp.), and the like.
  • yeast a.g., Saccharomyces spp., Schizosaccharomyces spp.
  • filamentous fungi e.g., Aspergillus spp., Neurospora spp.
  • Cultured mammalian cells may be used as host cells in the practice of the present invention.
  • Cultured mammalian cells contemplated for use herein include human monocytoid, lymphocytoid, fibroblastoid cell lines, and the like.
  • a useful mammalian cell line is the HeLa-tat cells that are HeLa derived cells.
  • Mammalian expression vectors contemplated for use in carrying out the present invention include a promoter capable of directing the transcription of a cloned gene or cDNA.
  • Preferred promoters include viral promoters and cellular promoters. Other promoters will be readily recognized by those skilled in the art.
  • Such mammalian expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the polypeptide or protein of interest.
  • Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
  • Also contained in the expression vectors may be a polyadenylation signal located downstream of (i.e., 3' to) the coding sequence of interest.
  • Cloned DNA sequences may be introduced into cultured mammalian cells by methods well known in the art, including, for example, calcium phosphate-mediated transfection, electroporation, protoplast fusion, biolistics, using DNA-coated particles, transfection, infection (where the chimeric construct is introduced into an appropriate virus, particularly a non-replicative form of the virus), and the like.
  • a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
  • Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, methotrexate, and the like.
  • the selectable marker may be an amplifiable selectable marker such as the DHFR gene, or the like. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., which is incorporated herein by reference). The choice of selectable markers is well within the level of ordinary skill in the art.
  • chimeric receptors i.e., converting inducible receptors into constitutively active receptors.
  • Methods for making chimeric receptors include chemical synthesis, recombinant nucleic acid technology (including viral and non-viral vectors), purification from natural sources (and thereafter manipulation), or any other methods which may be used to make chimeric polypeptides.
  • Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J. D. et al., Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, Calif. (1987); Darnell, J. E.
  • a method for treating and diagnosing diseases and disorders associated with non-normal, i.e., constitutive or insufficient, signal transduction including neurological disorders such as Parkinsonism, Alzheimers, depression, and the like, diabetes, growth abnormalities, cancer therapy, surgery adjuvants, and the like.
  • neurological disorders such as Parkinsonism, Alzheimers, depression, and the like
  • therapeutic methods are provided wherein tissue or cells from a subject are transformed with nucleic acid capable of expressing at least one chimeric receptor.
  • Other means for modulating signal transduction employing chimeric receptors will be readily recognized by those skilled in the art.
  • chimeric receptors are produced by transformation of host cells from a given individual with retroviral vector constructs directing the synthesis of the chimeric construct. By transformation of such cells and reintroduction of the cells into the patient one may achieve autologous gene therapy applications.
  • screening assays for identifying modulators of inducible receptors.
  • use of screening assays permits the identification of agonists, neutral antagonists, negative antagonists, and receptor inhibitors capable of reducing the constitutively active state of invention chimeric receptors.
  • Such methods comprise determining the effect of test compounds on chimeric receptor activity upon exposure of chimeric receptors to a library of compounds. Thus, compounds which increase the level of signal transduction are identified as agonists and compounds which decrease the level of signal transduction are identified as antagonists.
  • ligands Two classes of ligands have been identified, particularly with respect to antagonists, i.e., neutral antagonists which block only agonist induced effects without changing basal activity, and inverse agonists, or negative antagonists, which also block basal receptor activity. Agonists which also increase the signal transduction activity irrespective of whether a ligand is pre-associated with the receptor can also be identified, and are contemplated as within the scope of the present invention. Any synthetic, semi-synthetic or naturally-occurring compound can be evaluated, including further evaluating known ligands for cell-surface receptors. Small molecules (e.g., Antalarmin, Astressin, and the like) are included among the compounds which can be evaluated and/or identified by employing invention receptors in assays described herein.
  • antagonists i.e., neutral antagonists which block only agonist induced effects without changing basal activity
  • inverse agonists, or negative antagonists which also block basal receptor activity.
  • Any reporter can be employed which will facilitate identification of the level of signal transduction, e.g., by monitoring the level of secondary messengers. Additional parameters which can be monitored will be readily recognized by those of skill in the art. In addition, other means for identifying ligands for receptors with constitutive activity will be readily apparent to those employing the present invention (see, e.g., Sadee et al., U.S. Patent No. 5,882,944, the entire contents which are hereby incorporated by reference).
  • the practice of the present invention is useful to determine or to screen for new pharmaceuticals useful for treating disease states mediated by receptors capable of displaying constitutive activity, to enhance the clinical utility of existing pharmaceuticals targeted to receptors, to devise therapeutic treatments from agents identified by the screening methods of the invention, and the like.
  • compounds identified according to the present invention are useful in the treatment of diseases and disorders associated with prolonged agonist exposure, such as narcotic addiction, drug resistance, and the like.
  • methods for studying the interaction between receptors and their respective ligands employing chimeric receptors of the present invention are provided.
  • Chimeric receptors of the present invention are suitable for such studies because of the binding stability associated with the ligand and the ligand binding domain.
  • the present invention is also directed to a transgenic non-human eukaryotic animal (preferably a rodent, such as, but not limited to, a mouse), the germ cells and/or somatic cells of which contain nucleic acid according to the present invention which codes for chimeric receptor.
  • the nucleic acid encoding chimeric receptor according to the present invention is introduced into the animal to be made transgenic, or an ancestor of the animal, at an embryonic stage, preferably at the one-cell, or fertilized oocyte stage, and generally not later than about the 8-cell stage.
  • transgene as used herein, means a gene which is incorporated into the genome of the animal and is expressed in the animal, resulting in the presence of protein in the transgenic animal. There are several means known to those of skill in the art by which such a gene can be introduced into the genome of the animal embryo so as to be chromosomally incorporated and expressed.
  • Chimeric non-human mammals in which fewer than all of the somatic and germ cells contain nucleic acid encoding chimeric receptor are also provided by the present invention. Such animals are produced when fewer than all of the cells of the morula are transfected in the process of producing the transgenic mammal.
  • Chimeric non-human mammals having human cells or tissue engrafted therein are also encompassed by the present invention. Such chimeras can be used for testing expression of chimeric receptors in human tissue and/or for testing the effectiveness of therapeutic and/or diagnostic agents associated with delivery vectors which preferentially bind to chimeric receptors of the present invention. Methods for providing chimeric non-human mammals will be readily apparent to those skilled in the art employing the present invention.
  • transgenic non-human mammals may be used for the production of transgenic non-human mammals of the present invention.
  • Animals carrying nucleic acid encoding invention chimeric receptor(s) can be used to test compounds or other treatment modalities which may prevent, suppress or cure a human disease associated with non-normal, i.e., constitutive or insufficient, signal transduction. Such animals can also serve as a model for testing of diagnostic methods for the same human pathologies or diseases. Such pathologies or diseases include cancer, neurological diseases, and the like. Transgenic or chimeric animals according to the present invention can also be used as a source of cells for cell culture.
  • a CRF receptor see, e.g., Perrin et al., U.S. Patent No.
  • 5,728,545) is made constitutively active by constructing, for example, a chimera in which all or a fragment of r/h CRF replaces the first extracellular domain (i.e., N- terminus) of the CRF receptor.
  • An optimized signal peptide e.g. the HA-signal peptide
  • This approach ensures a free N-terminus for the peptide (see Figure 1).
  • Rl human CRF-R 1
  • pCI vector Promega
  • Silent Mlul- and BspEI restriction sites were created at position 285 and position 450, respectively, and the endogenous BspEI site at position 1121 was removed.
  • the chimeras CRF(l-16)/Rl ⁇ N and CRF( 17-41)/R1 ⁇ N have residues 1-111 replaced by the HA-signal peptide followed by the indicated portions of rat/human CRF (Vaughan, J., Donaldson, C, Bittencourt, J., Perrin, M. H., Lewis, K., Sutton, S., Chan, R., Turnbull, A. V., Lovejoy, D., Rivier, C, Rivier, j., Sawchenko, P. E., and Vale, W. (1995) Nature 378, 287-292).
  • Chimera CRF(1-16)/R1 has the c-myc epitope and a glycine residue separating it from CRF(1- 16) (EQKLISEEDLGSEEPPISLDLTFHLLR) (SEQ ID NO: 8) inserted between residue 28 and 29 of Rl. All chimeras were identified by restriction enzyme digestion and verified by automatic sequencing.
  • Table 1 Summarized basal activity and potency of urocortin on Rl and peptide/Rl chimeras. Data are presented as mean ⁇ S.E.M from (n) independent experiments each performed in triplicate. The cAMP level is normalized in each experiment to that observed in the presence of 10 ⁇ M antalarmin. Because of the low level of urocortin stimulation of CRF(1 -16)/R1 ⁇ N, the EC50 could not be determined. Chimeras were designed between Rl and the amino-terminal residues (1 to 16) or the carboxy-terminal residues (17 to 41) of CRF, and the CRF peptide portion positioned in place of the N-terminal domain of the receptor (Fig 3).
  • these chimeras lack the signal peptide of the receptor, they are expressed using the HA- signal peptide derived from influenza hemaglutinin and introduced at the amino- termini of the chimeric receptors.
  • the introduction of the HA-signal peptide ensures proper membrane targeting of the expressed constructs.
  • the HA-signal peptide is cleaved by the expressing cells, leaving the CRF peptide with a free amino- terminus, and tethered at its carboxy end to the transmembrane region of the receptor.
  • CRF(1-16) The significance of the proximity between CRF(1-16) and the transmembrane region of the receptor was examined by inserting CRF(1-16) into the N-terminal domain of Rl between residues 28 and 29 within the intact receptor (Fig 3).
  • This chimera, CRF(1-16)/R1 does not display constitutive activity (Fig 4 A and Table 1), but shows a large response to urocortin with similar potency EC50 - 0.2 nM to that of
  • RASSL receptor activated solely by a synthetic ligand
  • This chimera does not respond to endogenous levels of agonist like a RASSL.
  • the CRF(1- 16)/R1 ⁇ N chimera will be constitutively activated upon expression.
  • the activity of this chimera can be pharmacologically inhibited by an orally active, Rl -specific, non- peptide antagonist like antalarmin.
  • This type of engineered chimera may be designated as a RISSL (receptor inhibited solely by a synthetic ligand).
  • a constitutively active CRF receptor is a chimera in which the r/h CRF (1-41) (or a fragment thereof) is positioned downstream of an engineered eighth transmembrane helix fused to the intracellular C-terminal domain of the receptor.
  • the peptide portion has a free C-terminus.
  • the rat/human CRF, human urocortin, and astressin were kindly provided by Dr. J. Rivier (Salk Institute for Biological Studies).
  • the non-peptide antagonist Antalarmin was kindly provided by Dr. G. Chrousos (NIH).
  • the cDNAs encoding the human CRF-R 1 and CRF-R2 ⁇ I were cloned by RT- PCR from human mRNA (Clontech) and inserted into the pCI vector as EcoRi/Xbal fragments. Both cDNAs contain a Kozak sequence upstream the initiating ATG codon.
  • PCR was used to generate the desired fragments using Pfu polymerase (Stratagene) in a slightly modified version of methods previously described (Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Gene 77(1), 61- 8).
  • Pfu polymerase (Stratagene)
  • the generated fragment was inserted into the pCI expression vector containing appropriate parts of the wildtype receptor. All fragments generated by PCR, were verified by fluorescent dideoxynucleotide sequencing (Perkin Elmer).
  • constructs with an engineered eighth transmembrane helix consisting of 24 leucines fused to the C-terminal domain of the receptor were designed using a BamHI site which was introduced at the end of the C-terminus of the wildtype receptor in place of the stop-codon. This site was subsequently used to insert a BamHI Xbal fragment encoding residues as described below.
  • N-terminally modified constructs were designed by introducing the described residues followed by residues 112-415 in the hCR-Rl. Thus, residues 1- 111 in the wildtype receptor are deleted in these constructs.
  • the purified plasmids (Qiagen maxi-prep) were transiently transfected into
  • COS-M6 cells using the DEAE-dextran method as previously described (Perrin et al., U.S. Patent No. 5,728,545).
  • the cells were grown in a humidified atmosphere with 5% CO 2 in, Dulbecco's modified Eagle's medium 1885, supplemented with 10% fetal calf serum, 2 mM glutamine and 0.2 mg/ml gentamicin.
  • H-89 was added again two hours before the cAMP assay which was performed as previously described (Perrin et al.), except that cells were pre-treated with IB MX (Sigma) for 10 minutes before test compounds were applied. Incubation was continued for another 10 min., and the cells were extracted with 0.1 N HCL/95% EtOH and assayed for cAMP using a kit (Biome. Tech.) for before incubation with ligands for 10 minutes.
  • Tables 2 A and 2B show cAMP ratios for N-terminal modified (Table 2A) or C-terminal modified (Table 2B) hCRFl receptor constructs. Values are calculated as mean (+/-) s.e.m. For each construct, the calculated cAMP level with 1 uM Antalarmin added is defined as 1 in each experiment and all other cAMP levels are shown as ratio of this level. The number of independent experiments is indicated by (n). Table 2A
  • invention receptors having ligand replacing the N terminal 111 amino acids of the receptor have greater constitutive activation than if ligand is fused to the C terminus of the receptor.
  • Each of the receptors remains responsive to natural ligands therefor (e.g., CRF and urocortin).
  • small molecule antagonists effectively inhibit the constitutive activity of the receptors, thereby demonstrating the utility of such receptors in assaying for antagonists of receptor function.

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Abstract

La présente invention concerne de nouvelles protéines de récepteur à activité constitutive chimériques et des séquences d'ADN codant ces protéines. Les récepteurs chimériques comprennent un récepteur ou des fragments fonctionnels de ce dernier, qui est/sont fusionné(s) de manière efficace à un ligand destiné au récepteur ou à des fragments fonctionnels de ce dernier. En général on ne trouve pas le récepteur et le ligand associés ensemble et ils ne s'associent que pour induire l'activité du récepteur. La fusion du ligand au récepteur permet cependant au récepteur d'assurer la transduction constitutive d'un signal et d'activer un mécanisme de signalisation dans la cellule, cette cellule pouvant ainsi être induite à effectuer diverses fonctions relatives au mécanisme de signalisation. Une grande diversité de récepteurs de surface cellulaire et de ligands associés peut être utilisée dans la présente invention, lesdits récepteurs et lesdits ligands pouvant être naturels ou synthétiques. On décrit également des constructions d'acide nucléique, des vecteurs, des cellules et des animaux transgéniques qui sont capables d'exprimer les récepteurs chimériques. On décrit également des procédés qui permettent de transformer un récepteur inductible en un récepteur chimérique constitutivement actif au moyen de la fusion d'un ligand approprié au récepteur ; et des procédés de production de ces mêmes récepteurs chimériques ainsi que des procédés d'utilisation de ces derniers, y compris des dosages permettant d'identifier d'autres ligands pour des récepteurs et des applications dans le domaine de la thérapie et du diagnostic.
PCT/US2000/012793 1999-05-10 2000-05-10 Recepteurs chimeriques constitutifs et leurs procedes d'utilisation WO2000068269A1 (fr)

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AU50008/00A AU5000800A (en) 1999-05-10 2000-05-10 Constitutive chimeric receptors, and methods of use thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009103965A1 (fr) * 2008-02-19 2009-08-27 Asterion Limited Liants modifiés
WO2010115207A1 (fr) * 2009-04-03 2010-10-07 The Regents Of The University Of California Chimères entre récepteurs gpc et partenaires de liaison associés
US20240024505A1 (en) * 2022-07-12 2024-01-25 Trustees Of Boston University Synthetic cellular signaling pathways and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: "Expression cloning of a human corticotropin-releasing-factor receptor", PROC. NATL. ACAD. SCI. USA,, vol. 90, October 1993 (1993-10-01), pages 8967 - 8971, XP002930926 *
MONTECLARO ET AL.: "The amino-terminal domain of CCR2 is both necessary and sufficient for high affinity of monocyte chemoattractant protein 1?", THE JOURNAL OF BIOLOGICAL CHEMISTRY,, vol. 272, no. 37, 12 September 1997 (1997-09-12), pages 23186 - 23190, XP002930925 *
VAUGHAN ET AL.: "Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor", NATURE,, vol. 378, 16 November 1995 (1995-11-16), pages 287 - 292, XP002980927 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009103965A1 (fr) * 2008-02-19 2009-08-27 Asterion Limited Liants modifiés
WO2010115207A1 (fr) * 2009-04-03 2010-10-07 The Regents Of The University Of California Chimères entre récepteurs gpc et partenaires de liaison associés
US20240024505A1 (en) * 2022-07-12 2024-01-25 Trustees Of Boston University Synthetic cellular signaling pathways and uses thereof
US12239718B2 (en) * 2022-07-12 2025-03-04 Trustees Of Boston University Synthetic cellular signaling pathways and uses thereof

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AU5000800A (en) 2000-11-21
CA2372807A1 (fr) 2000-11-16

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