[go: up one dir, main page]

WO1996004389A1 - Recepteurs chimeres de rtk/cytokine - Google Patents

Recepteurs chimeres de rtk/cytokine Download PDF

Info

Publication number
WO1996004389A1
WO1996004389A1 PCT/US1995/009952 US9509952W WO9604389A1 WO 1996004389 A1 WO1996004389 A1 WO 1996004389A1 US 9509952 W US9509952 W US 9509952W WO 9604389 A1 WO9604389 A1 WO 9604389A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
cell
cells
tyrosine
lifrβ
Prior art date
Application number
PCT/US1995/009952
Other languages
English (en)
Inventor
Neil Stahl
Thomas Farruggella
George D. Yancopoulos
Original Assignee
Regeneron Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals, Inc. filed Critical Regeneron Pharmaceuticals, Inc.
Priority to AU32393/95A priority Critical patent/AU3239395A/en
Publication of WO1996004389A1 publication Critical patent/WO1996004389A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • RTK's protein receptor tyrosine kinases
  • the extracellular portion ' of each receptor protein tyrosine kinase is generally the most distinctive portion of the molecule, as it provides the protein with its ligand-recognizing characteristic. Binding of a ligand to the extracellular domain results in signal transduction via an intracellular tyrosine kinase catalytic domain which transmits a biological signal to intracellular target proteins.
  • TrkB binds and mediates the functional responses to brain derived neurotrophic factor (BDNF), neurotrophin 4/5 (NT-4/5), and, to a lesser extent, neurotrophin-3 (NT-3) [Ip et al. (1992), Proc Natl Acad Sci USA. 89: 3060-3064; Klein et al. (1992), Neuron. 8: 947-956; Squinto et al. (1991 ), Cell. 65: 1-20].
  • BDNF brain derived neurotrophic factor
  • NT-4/5 neurotrophin 4/5
  • NT-3 neurotrophin-3
  • Trk gene family has now been expanded to include the trkC locus, with NT-3 having been identified as the preferred ligand for trkC [Lamballe et al. (1991), Cell. 66: 967-979]
  • RTK families identified to date include the EGF, FGF, Eph, Tyro-3, Ror, PDGF, MET/sea/ron, IgF, Nep/Tyro10, and Ret family. All known growth factor RTKs appear to undergo dimerization following ligand binding ([Schlessinger and Ullrich. (1992), Neuron. 9: 383-391 ; Ullrich and Schlessinger. (1990), Cell. 61 : 203-211 ]; molecular interactions between dimerizing cytoplasmic domains lead to activation of kinase function. In some instances, such as the growth factor platelet derived growth factor (PDGF), the ligand is a dimer that binds two receptor molecules [Hart et al.
  • PDGF growth factor platelet derived growth factor
  • the tissue distribution of a particular tyrosine kinase receptor within higher organisms provides relevant data as to the biological function of the receptor.
  • the tyrosine kinase receptors for some growth and differentiation factors, such as fibroblast growth factor (FGF) are widely expressed and therefore appear to play some general role in tissue growth and maintenance.
  • FGF fibroblast growth factor
  • members of the Trk RTK family [Glass and Yancopoulos. (1993), Trends In Cell Biology.
  • receptors are more generally limited to cells of the nervous system, and the Nerve Growth Factor family consisting of NGF, BDNF, NT-3 and NT-4/5 (known as the neurotrophins) which bind these receptors promote the differentiation of diverse groups of neurons in the brain and periphery [Lindsay. (1993), Neurotrophic Factors. 257-284.].
  • the Nerve Growth Factor family consisting of NGF, BDNF, NT-3 and NT-4/5 (known as the neurotrophins) which bind these receptors promote the differentiation of diverse groups of neurons in the brain and periphery [Lindsay. (1993), Neurotrophic Factors. 257-284.].
  • the cellular environment in which an RTK is expressed may influence the biological response exhibited upon binding of a ligand to the receptor.
  • a neuronal cell expressing a Trk receptor is exposed to a neurotrophin which binds that receptor, neuronal survival and differentiation results.
  • the same receptor is expressed by a fibroblast, exposure to the neurotrophin results in proliferation of the fibroblast [Glass et al. (1991 ), Cell. 66: 405-413].
  • the extracellular domain provides the determining factor as to the ligand specificity, and once signal transduction is initiated the cellular environment will determine the phenotypic outcome of that signal transduction.
  • Trks are somewhat unusual with regard to RTK's in that the receptors as well as at least some of their cognate ligands have been identified. Regions of homology in the Trk's as well as other RTK's, in combination with the use of PCR technology, has rapidly enabled the cloning of an abundant number of novel protein tyrosine kinases, wherein the cognate ligand has yet to be discovered (hence, such receptors are termed "orphan" receptors). For such orphan receptors, despite the lack of cognate ligands, knowledge of the tissues in which such receptors are expressed provides insight into the regulation of the growth, proliferation and regeneration of cells in target tissues.
  • cytokines include erythropoietin, as well as many of the interleukins, and growth hormone, utilize receptors that, at least initially, have an entirely different intracellular signalling pathway.
  • CNTF, IL-6, LIF, OSM, and IL-11 form a subfamily of cytokines (referred to herein as the "CNTF family") that mediate their biological actions through activation of the related signal transducing receptor ⁇ components gp130 and LIFR ⁇ [Stahl and Yancopoulos. (1993), Cell. 74: 587-590].
  • Activation of the receptor requires either homodimerization of gp130 for IL-6, or heterodimerization of gp130 with LIFR ⁇ for CNTF, LIF, and OSM [Davis et al. (1993), Science. 260: 1805-1808; Murakami et al.
  • Activation of cells that express gp130 and LIFR ⁇ occurs upon exposure to LIF, whereas CNTF also requires interaction with an ⁇ -receptor component (which may be in soluble form) to activate gp130/LIFR ⁇ expressing cells. Similarly, IL-6 activates gp130-expressing cells only in the presence of its ⁇ -receptor component.
  • the CNTF family receptor ⁇ components preassociate
  • Jaks intracellular tyrosine kinases of the Jak/Tyk family (hereon referred to as the Jaks), which then become activated upon ⁇ component dimerization [Lutticken. (1994), Science. 263: 89-92; Stahl et al. (1994), Science. 263: 92-95].
  • Other members of the cytokine receptor superfamily also bind to and activate specific Jaks: receptor components for erythropoietin , growth hormone , GM-CSF, GCSF, and IL-3 use Jak2 [Argetsinger et al. (1993), Cell.
  • IFN-a requires Jak1 and Tyk2 [Muller et al. (1993), Nature. 366: 129-135; Velazquez et al. (1992), Cell. 70: 313-322], IFN-g requires Jak1 and Jak2 [Watling et al. (1993), Nature. 366: 166-170], while IL2 and IL4 use Jak3 [Johnston et al. (1994), Nature.
  • Both gp130 and LIFR ⁇ appear to be unique in that they can utilize distinct combinations of Jak1 , Jak2, or Tyk2 in a cell-specific manner [Stahl et al. (1994), Science. 263: 92-95].
  • the cytokines play a vital role in the hemopoiesis, inflammation and immunity as well as response to infection, their pleiotropic character results in their cross reactivity on many types of cells. Depending upon the cell types with which they react, their potential benefits are often outweighed by their lack of specificity, thus limiting their therapeutic usefulness due to undesirable side effects.
  • LIF is a pleiotropic cytokine that is toxic when administered to animals [Metcalf and Gerating. (1989), Proc Natl Acad Sci. 86: 5948-5952].
  • LIF's activities are highly desirable and would be therapeutically worthwhile if they could be specifically activated in a particular cell type.
  • chimeric receptors whereby the extracellular domain is one utilized by a growth factor, such as a neurotrophin, that recognizes a narrow range of cell populations, and the cytoplasmic domain is one which is capable of initiating signal transduction via a cytokine intracellular signal transduction pathway.
  • a growth factor such as a neurotrophin
  • An object of the invention is to target specific cell populations using chimeric receptors.
  • Another object of the invention is to stimulate the survival, growth, differentiation and/or proliferation of cytokine responsive cell populations using non-cytokine related growth factors.
  • An object of the present invention is to provide chimeric receptors consisting of extracellular domains of receptor tyrosine kinases and intracellular domain of the CNTF cytokine family receptor components gp130 and LIFR ⁇ .
  • Another object of the invention is to provide assay systems for agonists and antagonists of ctyokine related signal transduction pathways.
  • Fig. 1 Phosphorylation of STAT3 and PTP1 D upon activation of truncated chimeric receptors containing the cytoplasmic domains of gp130 and LIFR ⁇ .
  • a series of truncated chimeric receptors were constructed containing extracellular and transmembrane portions of TrkC fused to the cytoplasmic domains of gp130 (called TG, A) or the extracellular portion of the human EGF receptor fused to the transmembrane and cytoplasmic domains of LIFR ⁇ (called EL, B); each receptor also contained a C-terminal repeat of three 10 amino acid sequence from c-myc that is recognized by the monoclonal antibody 9E10 (indicated by triangle in schematic [Evan et al. (1985), Mol Cell Biol.
  • Proteins immunoprecipitated with anti-phosphotyrosine were first immunoblotted with anti-STAT3 [Zhong et al. (1994), Science. 264: 95-98]; the blots were then stripped and probed with anti-PTP1 D (Signal Transduction Labs). All immunoblots were visualized with enhanced chemiluminescence [Boulton et al. (1994), J Biol Chem. 269: 1 1648-1 1655].
  • tyrosine motifs added to cytokine receptor cytoplasmic domains can specify STAT3 phosphorylation.
  • a series of truncated receptor molecules were constructed (TGt series) each containing a single tyrosine phosphorylation site in which the gp130 Y1 motif was replaced with each of the indicated tyrosine motifs derived from elsewhere on gp130.
  • B Chimeric receptors with TrkC extracellular and transmembrane domain fused to the cytoplasmic domain of murine EPOR were constructed that contain the triple myc epitope tag.
  • Two versions also have sequences encoding 5 additional amino acids as indicated in the schematic inserted between the end of the EPOR sequences, but 5 * of the triple myc tag.
  • vectors encoding these constructs were transfected into COS 7 cells and analyzed as described in Fig. 1. DETAILED DESCRIPTION OF THE INVENTION
  • chimeric receptors are created containing extracellular and transmembrane portions of a receptor tyrosine kinase (RTK) and the cytoplasmic domains of gp130 or LIFR ⁇ , or, in the alternative, extracellular portions of an RTK, and the transmembrane and cytoplasmic domains of gp130 or LIFR ⁇ .
  • RTK receptor tyrosine kinase
  • receptor tyrosine kinase refers to any member of the superfamily of transmembrane tyrosine kinases that serve as receptors for a variety of protein factors that promote cellular proliferation, differentiation and survival.
  • Receptor tyrosine kinases include, but are not limited to, the Trks, which bind members of the neurotrophin family, including Nerve Growth Factor (NGF), which preferentially binds TrkA, as well as Brain Derived
  • cytoplasmic domain refers to the full length cytoplasmic domain of gp130 or LIFR ⁇ , or truncated versions that lack distal portions of the cytoplasmic domain, yet retain the membrane proximal Jak binding domain. For example, as shown in Figure 1, both gp130 and LIFR ⁇ contain 5 tyrosine motifs.
  • TrkC which binds the TrkC receptor, induces tyrosine phosphorylation of two downstream targets of the CNTF family of cytokines-the transcriptional activator STAT3 [Akira et al. (1994), Cell. 77: 63-71 ; Stahl, et al. (1995), Science, 276: 1359-1353; Velazquez et al. (1992), Cell. 70: 313-322] and the tyrosine phosphatase PTP1 D [Boulton et al. (1994), J Biol Chem. 269: 1 1648-1 1655].
  • chimeric receptors made according to the invention are used to enable the RTK dependent survival, growth, proliferation and/or differentiation of target cells, the growth, survival, proliferation and/or differentiation of which are normally influenced or controlled by cytokines.
  • a target cell for expression of an RTK/gp130 or LIFR ⁇ chimera may be any cell which demonstrates a phenotypic response (such as differentiation, the expression of immediate early genes or the phosphorylation of CLIP or JAK proteins) in response to treatment with the CNTF/receptor complex, the IL-6/receptor complex, LIF or OSM, or a hybrid or mutant thereof that either mimics or alters the normal physiological effect of the CNTF/receptor complex.
  • Examples of cell types which respond to these cytokines include myeloid leukemia cells such the M1 cell line, other leukemia cells, hematopoietic stem cells, megakaryocytes and their progenitors, DA1 cells, osteoclasts, osteoblasts, hepatocytes, adipocytes, kidney epithelial cells, stem cells, renal mesangial cells, T cells, B cells, etc.
  • myeloid leukemia cells such the M1 cell line, other leukemia cells, hematopoietic stem cells, megakaryocytes and their progenitors, DA1 cells, osteoclasts, osteoblasts, hepatocytes, adipocytes, kidney epithelial cells, stem cells, renal mesangial cells, T cells, B cells, etc.
  • the present invention includes the nucleotide sequence that encodes the chimeric receptors described herein, as well as host cells and microorganisms and vectors that carry the recombinant nucleic acid molecules.
  • Cells that express receptor protein may be genetically engineered to produce receptor, as described supra, by transfection, transduction, electroporation, microinjection, via a transgenic animal, etc. of nucleic acid encoding chimeric receptor in a suitable expression vector.
  • the host cell carrying the recombinant nucleic acid is an animal cell, such as COS.
  • the host cell is a bacterium, preferably Escherichia coli.
  • any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding the chimeric receptors described herein. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (genetic recombination). Expression of nucleic acid sequence encoding the receptor protein or peptide fragment may be regulated by a second nucleic acid sequence so that the receptor protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of receptor may be controlled by any promoter/enhancer element known in the art.
  • Promoters which can be used to control receptor expression include, but are not limited to the long terminal repeat as described in Squinto et al., (1991 , Cell 65:1-20); the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the CMV promoter, the M-MuLV 5 * terminal repeat the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981 , Proc. Natl. Acad. Sci. U.S.A.
  • alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel. 1 :161 -171 ), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science
  • Expression vectors containing receptor-encoding gene inserts can be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
  • first approach the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA hybridization using probes comprising sequences that are homologous to an inserted gene.
  • second approach the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g.. thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • marker e.g. thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombinants containing the gene insert can be identified by the absence of the marker gene function.
  • recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant vector. Such assays can be based, for example, on the physical or functional properties of the receptor-encoding gene product, for example, by binding of the receptor to neurotrophic factor or to an antibody 5 which directly recognizes the receptor.
  • Cells of the present invention may transiently or, preferably, constitutively and permanently express receptors or portions thereof.
  • the present invention provides for cells that express receptors described herein or o portions thereof and that also contain recombinant nucleic acid comprising an immediate early gene promoter [e.g. the fos or jun promoters (Gilman et al., 1986, Mol. Cell. Biol. 6:4305-4316)].
  • an immediate early gene promoter e.g. the fos or jun promoters (Gilman et al., 1986, Mol. Cell. Biol. 6:4305-4316)
  • the binding secondarily induces transcription off the immediate 5 early promoter.
  • Such a cell may be used to detect receptor/ligand binding by measuring the transcriptional activity of the immediate early gene promoter, for example, by nuclear run-off analysis, Northern blot analysis, or by measuring levels of a gene controlled by the promoter.
  • the immediate early promoter may be o used to control the expression of f _s_ or iun or any detectable gene product, including, but not limited to, any of the known reporter genes, such as a gene that confers hygromycin resistance (Murphy and Efstratiadis, 1987, Proc. Natl. Acad. Sci. U.S.A. 84:8277-8281) chloramphenicol acetyltransferase (CAT), 5 neomycin phosphotransferase (neo), beta-gaiactosidase beta-glucuronidase, beta-galactosidase, etc. of detecting or measuring neurotrophin activity.
  • CAT chloramphenicol acetyltransferase
  • neo 5 neomycin phosphotransferase
  • beta-gaiactosidase beta-glucuronidase beta-galactosidase, etc.
  • cells engineered to express a chimeric protein described herein are treated using the ligand specific for the extracellular domain of the chimeric receptor.
  • Such cells are utilized in assay systems to identify cognate ligands of the orphan receptors by measuring a predetermined phenotypic change in the chimeric receptor-expressing cells in the presence of a potential ligand.
  • the chimeric receptors made as described herein are used in assay systems that may be used to identify agents that act as agonists or antagonists of CNTF or other cytokine members of the CNTF receptor family that utilize signal transduction pathways in common with the CNTF receptor family.
  • Cells that express TRK/gp130 or TRK/LIFR ⁇ receptor, or mutants thereof lacking specific tyrosine motifs may be exposed to a test agent and the tyrosine phosphorylation of either the ⁇ -component(s) or the signal transduction component(s) are compared to the tyrosine phosphorylation of the same component(s) in the absence of the test agent.
  • Such assay systems are useful to identify small molecules that are capable of initiating or blocking signal transduction induced by specific cytokines.
  • cells expressing the ⁇ -component of a specific receptor and one or more signal transducing components may exhibit tyrosine phosphorylation of the ⁇ -component in the absence of any transduction inducing ligand.
  • Such cells are useful to determine the ability of a test agent to inhibit this step in the signal transduction pathway by contacting the cells with test agents and measuring the effect of the test agents on the tyrosine phosphorylation.
  • the cytokine domain is "engineered" to contain altered or additional motifs that endow novel activity to the receptor, thereby enabling modulation of downstream signaling pathways activated by the receptor.
  • a short tyrosine motif added to the cytoplasmic domain of the EPO receptor can enable that receptor to mediate tyrosine phosphorylation of STAT3.
  • This motif alone (as expressed by a cell or in a cell-free system) may be useful for selecting agonists or antagonists of cytokines, such as the CNTF family, that specifically interact with the STAT3 related segment of the signal transduction pathway.
  • chimeric receptor expressing cells engineered in vivo or engineered in vitro or ex vivo may be implanted and subsequently treated with a ligand recognizing the extracellular domain.
  • Ligands capable of recognizing chimeric receptors expressed by a targeted cell population may then be used to treat diseases or disorders which involve the targeted cells, or alternatively, to enhance the growth and/or survival or other function of targeted cells.
  • the chimeric receptors may be supplied to the system using appropriate vectors, or they may be produced within the system via, for example, appropriately engineered precursor cells.
  • a chimeric receptor responsive to, for example, a neurotrophin would enable use of the neurotrophin to elicit a response in cell or tissue cultures, in whole animals, in particular cells or tissues within whole animals or tissue culture systems, or over specified time intervals (including during embryogenesis) in embodiments in which the chimera expression is controlled by an inducible or developmentally regulated promoter.
  • the CMV promoter may be used to control expression of a TRK/cytokine chimeric receptor in transgenic animals.
  • transgenic animals refers to non-human transgenic animals, including transgenic mosaics, which carry a transgene in some or all of their cells, which include any non-human species, and which are produced by any method known in the art, including, but not limited to microinjection, cell fusion, transfection, electroporation, etc.
  • the animals may be produced by a microinjection of zygotes by a method such as that set forth in "Brinster et al. [1989, Proc. Natl. Acad. Sci. U.S.A. 82:4438-4442].
  • Techniques for gene therapy or implantation of genetically engineered cells are known in the art. For example, U.S. Patent No.
  • RTK:gp130 or RTK ⁇ IFR ⁇ chimeras are expressed by cells such that neurotrophins may be utilized in place of LIF to activate the cells. This is of particular use in view of the fact that although LIF has desirable therapeutic properties, it is toxic when administered to animals. [Metcalf and Gearing. (1989), Proc Natl Acad Sci. 86: 5948-5952].
  • the use of the chimeric RTK:gp130 or RTK ⁇ IFR ⁇ is a novel way to specifically activate the LIF/IL-6 signal transduction pathway in a certain subset of all LIF responsive cells.
  • LIF has a profound effect on bone morphology, resulting in an increase in both the number of osteoblastic cells as well as an increase in the amount of bone deposition [Evans et al. (1994), Biochem Biohpys Res Commun. 199: 220-226; Metcalf and Gerating.
  • a chimeric RTK:cytokine receptor such as TrkC:gp130 in the osteoblastic cell lineage would enable control of the level of osteoblastic activity and thus the rate of bone deposition through controlling the NT3 levels in the cells environment.
  • NT-3 would be desirable in this regard as the stimulating factor since there are few natural NT-3 targets outside of the nervous system; thus the chimeric TrkC:gp130 receptors would be a rather specific target of NT-3 in the periphery.
  • transgenic mice in which the TrkC:gp130 cDNA is controlled by a promoter that is only expressed in cells of the osteoblastic lineage may be created.
  • One such promoter is that controlling osteocalcin, which is only expressed by mature osteoblasts .
  • the cDNA encoding the chimeric receptor may be introduced via a precursor cell from which osteoblastic cells arise.
  • a precursor cell that is capable of differentiating into an osteoblastic cell in culture has recently been purified from mice by fluorescence activated cell sorting (FACS) [Van Vlasselaer et al. (1994), Blood. 84: 753-763].
  • FACS fluorescence activated cell sorting
  • Osteoprogenitor cell cultures may be transfected in vitro with DNA encoding a chimeric RTK:cytokine receptor such as TrkC:gp130.
  • the cDNA for TrkC:gp130 is placed downstream of an osteoblastic-specific promoter such as the osteocalcin promoter described above, or using a more general promoter that would be expressed in all of the cells of the lineage such as that for alkaline phosphatase or ⁇ -actin. Having developed a stable osteoprogenitor population that expresses the TrkC:gp130 chimeric receptor, these cells are reintroduced into bone marrow. Systemic administration of NT-3 would then be used to mimic the effect of LIF and induce net bone deposition.
  • an osteoblastic-specific promoter such as the osteocalcin promoter described above
  • a more general promoter that would be expressed in all of the cells of the lineage such as that for alkaline phosphatase or ⁇ -actin.
  • chimeric receptors as described herein are introduced into, for example, stem cells, simultaneously with the introduction of genes used for correction of human diseases via gene therapy.
  • stem cells engineered to express human adenosine deaminase (ADA) [Luskey, B., et al. (1990) Ann. N.Y.Acad.Sci. 612:398-406] may be simultaneously engineered to express chimeric receptors according to the present invention, thereby enabling use of, for example, neurotrophins, to support the transplanted stem cells in vivo.
  • ADA human adenosine deaminase
  • gp130 and LIFR ⁇ we constructed chimeric receptors to facilitate mutational analysis of their cytoplasmic domains. Since activation of gp130 by IL-6 involves homodimerization of gp130 [Davis et al. (1993), Science. 260: 1805-1808; Murakami et al. (1993), Science. 260: 1808-1810], we employed extracellular domains from receptor tyrosine kinases such as TrkC and EGF receptor that dimerize upon binding neurotrophin 3 (NT-3) or EGF respectively.
  • receptor tyrosine kinases such as TrkC and EGF receptor that dimerize upon binding neurotrophin 3 (NT-3) or EGF respectively.
  • EL ⁇ Y 5 comprises amino acids 1-646 of the EGF receptor and amino acids 834-1027 of LIFR ⁇ .
  • EL ⁇ Y 4 - 5 comprises amino acids 1 -646 of the EGF receptor and amino acids 834-1000 of LIFR ⁇ .
  • EL ⁇ Y 3 . 5 comprises amino acids 1-646 of the EGF receptor and amino acids 834-980 of LIFR ⁇ .
  • EL ⁇ Y 1 - 5 comprises amino acids 1-646 of the EGF receptor and amino acids 834-948 of LIFR ⁇ .
  • TrkC/gp130 chimeras were constructed by fusing the
  • TG ⁇ Y 4 - 5 comprises the same extracellular segment derived from TrkC and amino acids 644 to 904 of gp130.
  • TG ⁇ Y 3 - 5 comprises the same extracellular segment derived from TrkC and amino acids 644 to 813 of gp130.
  • TG ⁇ Y 2 - 5 comprises the same extracellular segment derived from TrkC and amino acids 644 to 765 of gp130.
  • TG ⁇ Y 1 - 5 comprises the same extracellular segment derived from TrkC and amino acids 644 to 757 of gp130.
  • EXAMPLE 1 As predicted, fusion proteins (called TG for TrkC:gp130 or EG for EGFR:gp130) containing these extracellular domains linked to the cytoplasmic domain of gp130 expressed in COS cells gave factor-induced tyrosine phosphorylation of the chimeric receptor and the associated Jak (Fig 1A and data not show). Although there is no known cytokine that induces homodimerization of LIFR ⁇ , a fusion protein (EL for EGFR:LIFRb) containing the EGFR extracellular domain and the transmembrane and cytoplasmic domains of LIFR ⁇ gave robust tyrosine phosphorylation of the chimeric receptor and the associated Jak (Fig 2B).
  • TG for TrkC:gp130 or EG for EGFR:gp130 containing these extracellular domains linked to the cytoplasmic domain of gp130 expressed in COS cells gave factor-induced tyrosine phosphorylation of the chimeric receptor and the
  • Y ⁇ SYCPP
  • Y 2 IYIDV
  • Y 3 MYQPQ
  • Y 4 GYKPQ
  • Y 5 GYRPQ.
  • Each of these chimeric receptors were then expressed in COS cells, and analyzed for their ability to induce tyrosine phosphorylation of two downstream targets of the CNTF family of cytokines-the transcriptional activator STAT3 [Akira et al. (1994), Cell. 77: 63-71 ; Boulton et al. (1994), Submitted. ; Zhong et al. (1994), Science.
  • STAT3 is preferentially activated by the CNTF cytokine family relative to other cytokines such as IFN-g, GM-CSF, or IL-3, and the receptor sequences that might be required for its activation are unknown [Boulton et al. (1994), Submitted. ].
  • Fig. 1C One interpretation of this result is that there are multiple sites in the gp130 cytoplasmic domain that are capable of mediating STAT3 phosphorylation, the last of which is removed in the truncation mutant TGDY2-5.
  • Both of the tyrosine motifs in the cytoplasmic regions of gp130 and LIFRb that were implicated in STAT3 phosphorylation have the sequence YXXQ, a motif which is indeed present in multiple copies in both gp130 and LIFR ⁇ .
  • YXXQ could be considered candidates for mediating STAT3 activation, although additional sequences may also be required.
  • Previous analysis of other cytokines showed that GCSF could mediate STAT3 activation, while GMCSF could not [Boulton et al. (1994), Submitted. ].
  • the murine GCSF receptor contains the sequence AYVLQ, while neither the GMCSF receptor nor the EPO receptor have sequences matching this motif.
  • the present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur des récepteurs chimères consistant en domaines extracellulaire de tyrosine kinases réceptrices et en domaines cytoplasmiques de cytokines, sur leurs méthodes de production, et sur leur utilisation pour le ciblage de cellules répondant à la cytokine.
PCT/US1995/009952 1994-08-04 1995-08-04 Recepteurs chimeres de rtk/cytokine WO1996004389A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU32393/95A AU3239395A (en) 1994-08-04 1995-08-04 Rtk/cytokine receptor chimeras

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28599094A 1994-08-04 1994-08-04
US08/285,990 1994-08-04

Publications (1)

Publication Number Publication Date
WO1996004389A1 true WO1996004389A1 (fr) 1996-02-15

Family

ID=23096562

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/009952 WO1996004389A1 (fr) 1994-08-04 1995-08-04 Recepteurs chimeres de rtk/cytokine

Country Status (4)

Country Link
AU (1) AU3239395A (fr)
IL (1) IL114825A0 (fr)
WO (1) WO1996004389A1 (fr)
ZA (1) ZA956545B (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998002558A3 (fr) * 1996-07-17 1998-05-07 Univ New Jersey Med Recepteurs chimeres pour transduction de signaux jak-stat
WO1998039418A1 (fr) * 1997-03-07 1998-09-11 Ariad Gene Therapeutics, Inc. Nouvelles applications de la technique de la therapie genique
WO2000018932A3 (fr) * 1998-09-25 2000-11-02 Regeneron Pharma Antagonistes a base de recepteurs, modes d'elaboration et d'utilisation
WO2011138423A1 (fr) * 2010-05-05 2011-11-10 Addex Pharma Sa Récepteurs chimériques et procédés d'identification d'agents présentant une activité sur des récepteurs à un passage transmembranaire de type 1
US11786553B2 (en) 2019-03-01 2023-10-17 Allogene Therapeuctics, Inc. Chimeric cytokine receptors bearing a PD-1 ectodomain
US12043655B2 (en) 2019-03-01 2024-07-23 Allogene Therapeutics, Inc. Constitutively active chimeric cytokine receptors
US12163169B2 (en) 2018-03-02 2024-12-10 Allogene Therapeutics, Inc. Inducible chimeric cytokine receptors

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010151A1 (fr) * 1991-11-22 1993-05-27 Immunex Corporation Recepteur de l'oncostatine m et facteur d'inhibition de la leucemie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010151A1 (fr) * 1991-11-22 1993-05-27 Immunex Corporation Recepteur de l'oncostatine m et facteur d'inhibition de la leucemie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIDEYA OHASHI ET AL.: "Ligand-induced activation of chimeric receptors between the erythropoieitin receptor and receptor tyrosine kinases", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, no. 1, 4 January 1994 (1994-01-04), WASHINGTON US, pages 158 - 162 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998002558A3 (fr) * 1996-07-17 1998-05-07 Univ New Jersey Med Recepteurs chimeres pour transduction de signaux jak-stat
WO1998039418A1 (fr) * 1997-03-07 1998-09-11 Ariad Gene Therapeutics, Inc. Nouvelles applications de la technique de la therapie genique
WO2000018932A3 (fr) * 1998-09-25 2000-11-02 Regeneron Pharma Antagonistes a base de recepteurs, modes d'elaboration et d'utilisation
WO2011138423A1 (fr) * 2010-05-05 2011-11-10 Addex Pharma Sa Récepteurs chimériques et procédés d'identification d'agents présentant une activité sur des récepteurs à un passage transmembranaire de type 1
WO2011138414A1 (fr) * 2010-05-05 2011-11-10 Addex Pharma Sa Méthodes et outils pour le criblage d'agents présentant une activité sur des récepteurs de la superfamille des récepteurs des facteurs de nécrose tumorale
US12163169B2 (en) 2018-03-02 2024-12-10 Allogene Therapeutics, Inc. Inducible chimeric cytokine receptors
US11786553B2 (en) 2019-03-01 2023-10-17 Allogene Therapeuctics, Inc. Chimeric cytokine receptors bearing a PD-1 ectodomain
US12043655B2 (en) 2019-03-01 2024-07-23 Allogene Therapeutics, Inc. Constitutively active chimeric cytokine receptors

Also Published As

Publication number Publication date
ZA956545B (en) 1996-03-29
AU3239395A (en) 1996-03-04
IL114825A0 (en) 1995-12-08

Similar Documents

Publication Publication Date Title
EP0758381B1 (fr) Ligands de la famille des eph a activite biologique
EP1001024B1 (fr) Protéine codée par le proto-oncogène trkB, utilisée comme système d'analyse permettant de détecter l'activité de la neurotrophine
US5648334A (en) Methods of treatment using ciliary neurotrophic factor
WO1996004389A1 (fr) Recepteurs chimeres de rtk/cytokine
US5426177A (en) Ciliary neurotrophic factor receptor
AU656732B2 (en) The ciliary neurotrophic factor receptor
US5747033A (en) Method of enhancing the biological activity of Eph family ligands
US5622862A (en) Assay systems for trkB neurotrophin activity
US5656473A (en) Human Dmk receptor
US5849897A (en) Nucleic acid molecule encoding ciliary neurotrophic factor receptor
AU710728B2 (en) Denervated muscle kinase (dmk), a receptor of the tyrosine kinase super family
US6602683B1 (en) Biologically active Eph family ligands
Zhang The expression of neurotrophin receptors during embryonic development
JP2004201693A (ja) 除神経筋キナーゼ（ｄｍｋ）、チロシンキナーゼスーパーファミリーのレセプター
Kenny Molecular cloning and early embryonic expression of twoeph-related receptor tyrosine kinases
HK1024935B (en) Protein encoded by the trkb proto-oncogene, used in assay systems for neurothophin activity

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase