WO1993011253A1 - Complexe recepteur/facteur neurotrophique ciliaire exempt de cellules - Google Patents

Complexe recepteur/facteur neurotrophique ciliaire exempt de cellules Download PDF

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WO1993011253A1
WO1993011253A1 PCT/US1992/010632 US9210632W WO9311253A1 WO 1993011253 A1 WO1993011253 A1 WO 1993011253A1 US 9210632 W US9210632 W US 9210632W WO 9311253 A1 WO9311253 A1 WO 9311253A1
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cntf
receptor
cell
cells
complex
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PCT/US1992/010632
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English (en)
Inventor
Nancy Ip
Nikos Panayotatos
Thomas H. Aldrich
Samuel Davis
Daniel Everdeen
Steven H. Nye
Stephen P. Squinto
Neil Stahl
Joanne Conover
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority claimed from US07/865,878 external-priority patent/US5332672A/en
Application filed by Regeneron Pharmaceuticals, Inc. filed Critical Regeneron Pharmaceuticals, Inc.
Publication of WO1993011253A1 publication Critical patent/WO1993011253A1/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
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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

Definitions

  • the present invention provides for substantially purified cell-free CNTF/receptor complex and to related hybrid or mutant proteins or peptides that may, in alternative embodiments of this invention, be used to promote or antagonize cell proliferation and/or differentiation, and that may be used in methods for diagnosing
  • the invention further provides for the interaction of such complexes with receptor components that are shared by IL- 6, LIF and CNTF signal transduction pathways.
  • CNTF Ciliary neurotrophic factor
  • CNTF is believed to induce the - differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain [Hughes et al., Nature 335:70-73 (1988)]. Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons [Skaper and Varan, Brain Res. 2££:39-46 (1986)].
  • CNTFR or CNTFR ⁇ The CNTF receptor (CNTFR or CNTFR ⁇ ) has been cloned and expressed in eukaryotic cells, as described in U.S. Patent Application Serial No. 07/700,677, entitled "The Ciliary Neurotrophic Factor
  • CNTFR glycophosphatidyl inositol linkage
  • CNTFR is related to a number of receptors, referred to herein as the CNTF/IL-6/LIF receptor family, including IL-6, LIF, G-CSF and oncostatin M (OSM) [Bazan, Neuron 7:197-208 (1991); Rose and Bruce, Proc. Natl. Acad. Sci. 8_8_: 8641-8645, (1991 )], but appears to be most closely related to the sequence of the receptor for IL-6.
  • IL-6 has not been shown to be a GPI-linked protein [e.g.. Taga, et al. dislike Cell 5_8_:573-581 (1989); Hibi, et al., , Cell 85.: 1 149-
  • LIF, G-CSF and OSM are all broadly acting factors that, despite having unique growth-regulating activities, share several common actions with IL-6 during hemopoiesis as well as in other processes. For example, all can inhibit the proliferation and induce the differentiation of the murine myeloid leukemia cell line, M1 [Rose and Bruce, Proc. Natl. Acad. Sci. ££: 8641-8645 (1991 )].
  • M1 murine myeloid leukemia cell line
  • the use of related receptor systems may provide a basis for the similar biological actions of these hemopoietic cytokines - G-CSF, IL-6, OSM and LIF all have receptor components that are structurally homologous to gp130 [Fukunaga et al., EMBO J.
  • hemopoietic factors that share receptor components with CNTF would ' enable the utilization of CNTF and its specific receptor components for activation of targeted cells that are normally responsive to such hemopoietic factors.
  • the present invention relates to a cell-free CNTF/receptor complex. It is based, in part, on the discovery that cell-free CNTF/receptor complex is biologically active on a broader spectrum of cell types than those that express the CNTF receptor.
  • the CNTF/receptor acts as a differentiation factor in cell types that express receptors belonging to the CNTF/IL-6/LIF receptor family.
  • the present invention is further based on the ability of
  • CNTF/receptor complex under normal physiological buffer conditions.
  • equimolar amounts e.g., 80 mM
  • normal physiological buffer conditions 100 mM Tris- 1 0 Hcl, 50 mM NaCI, pH 8.0
  • This CNTF/receptor complex may be purified via gel filtration and utilized in assays described infra.
  • the invention further provides for hybrid or mutant proteins related to the CNTF/receptor complex which function as 1 5 either agonists or antagonists of cellular differentiation factors.
  • a hybrid or mutant CNTFR may be unable to bind CNTF but be capable of signal transduction.
  • This hybrid or mutant may be utilized to promote or enhance the differentiation, proliferation, growth or survival of cells that are 20 responsive to the CNTF/receptor complex, including cells that express receptors that are members of the CNTF/IL-6/LIF receptor family independent of CNTF levels.
  • a mutant receptor may exhibit an increased binding affinity for CNTF, but be unable to effectively induce signal 25 transduction. Such a mutant may be useful in binding to and neutralizing CNTF without eliciting secondary effects on cell differentiation.
  • the invention also provides for in vitro or in vivo diagnostic methods and for assay systems for use in testing target cells for sensitivity to a particular treatment involving the
  • the invention further provides for therapeutic methods for treating not only CNTF-related disorders but also disorders of differentiation and/or proliferation related to any target cell which is responsive to cell-free CNTF/receptor complex or related compounds.
  • the present invention also provides for a method of producing substantially purified, biologically active CNTFR or related molecules in- bacteria.
  • the present invention is also based on the discovery that
  • CNTF and LIF act on neuronal cells via the IL-6 transducing receptor component gp130 and a gp130-like second receptor comp ⁇ nent(referred to as LIF ⁇ ), together which, when bound to CNTF and CNTFR initiate signal transduction using a signalling pathway comparable to IL-6.
  • LIF ⁇ IL-6 transducing receptor component
  • the invention provides for the utilization of CNTF and CNTFR to induce a response in cells that are normally responsive to LIF or IL-6 (presumably because they express gp130 and LIFR ⁇ ).
  • the present invention also provides for a method of targeting cells with CNTFR(CNTFR ⁇ ) so that CNTF can be used to selectively initiate signal transduction in such cells.
  • the invention further provides for the use of CNTF in place of LIF to prevent differentiation of cultured embryonic stem cells.
  • FIGURE 1 Nucleic acid sequence (SEQ ID NO:1) of human CNTF and the deduced amino acid sequence (SEQ ID NO:2).
  • FIGURE 2 Nucleic acid sequence (SEQ ID NO: 3) of
  • FIGURE 3 DNA sequences of the PCR primers used in the construction of pRPN151. Small characters indicate positions at which the DNA sequence was modified in order to optimize expression without modification of the protein sequence. Sense:(SEQ ID NO:5) Anti Sense:(SEQ ID NO:6).
  • FIGURE 4 Physical and restriction map of pRPN151. The length of the plasmid in base pairs (bp), the positions of a few unique restriction sites, as well as the physical location of the huCNTRFI (dotted bar) and the beta lactamase (solid bar) genes are shown.
  • FIGURE 5 Isolation of active receptor by gel filtration.
  • (A) Elution profile of an S100-HR column, monitored by absorbance at 280 nm. Nucleic acids contribute approximately 50% to the absorbance of the major peak but less than 10% to the smaller one.
  • (B) Proteins eluting in fractions 9-14 (20 ⁇ l per lane) and 16-21 (200 ⁇ l per lane), were analyzed by SDS-PAGE. Total protein extract applied to the column (lane E) is also shown, along with size markers of 14, 21, 31 , 45, 66 and 90 kD (lane M). R-, indicates the position of the receptor band at 40 kD.
  • FIGURE 6 Receptor ligand complex formation by native PAGE. A constant amount of receptor (1 ug) was mixed with the indicated amounts (in ug) of rat CNTF and analyzed by native PAGE. The positions of the bands corresponding to CNTFR, CNTF, and the CNTF/receptor complex are indicated.
  • FIGURE 7 Growth of MAH cells following treatment with
  • CNTF, LIF and FGF CNTF, LIF and FGF.
  • CNTF affects neuronal differentiation.
  • MAH cells were treated with CNTF (10 ng/ml) for 24hr.
  • Total RNA was prepared and subjected to northern analysis using a CNTFR probe and a GAPDH probe.
  • the transcript sizes for CNTFR and GAPDH were 2kb.
  • FIGURE 9 Dose-dependent tyrosine phosphorylation of proteins in response to CNTF, LIF and FGF.
  • Total cell lysates were prepared from MAH cells (A) or EW-1 cells (B) following a 5 mi ⁇ treatment with various concentrations (0.1 -100 ng/ml) of CNTF, LIF or FGF. Lysates were immunoprecipitated with anti-phosphotyrosine antibody, electrophoresed and immunoblotted with anti- phosphotyrosine antibody as described in Experimental procedures.
  • C SK-N-LO cells were treated with 50 ng/ml of CNTF for 5 or 15 min prior to anti-phosphotyrosine immunoprecipitation and blotting as described above.
  • FIGURE 10 Unique protein tyrosine phosphorylation patterns of CNTF, or LIF treated cells compared to FGF or NGF treated cells.
  • EW-1 cells following treatment with 50 ng/ml of CNTF, LIF or FGF were immunoprecipitated using anti-phosphotyrosine antibody.
  • ERK1 and ERK2 were precipitated from PC12 cell lysates using ERK-specific antibody. Immunoblotting was performed with ERK antibody.
  • C Total cell lysates were prepared from EW-1 cells treated with CNTF (50 ng/ml) or LIF (50 ng/ml) and PC12 cells treated with NGF (50 ng/ml). Lysates were electrophoresed and immunoblotted with anti-phosphotyrosine antibody.
  • FIGURE 11 Time course comparison of protein tyrosine phosphorylation changes to tis11 induction in response to CNTF and
  • MAH cells were treated with 50 ng/ml of CNTF or LIF for 5-60 min. Total cell lysates were immunoprecipitated and immunoblotted with anti-phosphotyrosine antibody as described in Fig. 3.
  • B. MAH cells were similarly treated with CNTF or LIF for 15- 120 min. Total RNA were prepared, fractionated by formaldehyde agarose gel electrophoresis and hybridized to tis11 and c-fos DNA probes as described in Experimental procedures.
  • C MAH or EW-1 cells were treated with CNTF (50 ng/ml), LIF (50 ng/ml) or FGF for 30 min. Total RNA were prepared, and expression of tis1 and c-fos were analyzed as above. The transcript sizes for tis11 and c-fos were 2.3 and 2kb, respectively.
  • FIGURE 12 Comparison of tyrosine phosphorylation changes in response to CNTF, LIF or IL-6 in EW-1 and M1 cells.
  • EW-1 or M1 cells were treated with CNTF (50 ng/ml), LIF (50 ng/ml) or mlL-6 (100 ng/ml) for 5 minutes.
  • Total cell lysates were immunoprecipitated and immunoblotted with antiphosphotyrosine antibody as described supra.
  • FIGURE 13 Effects of protein kinase inhibitors on CNTF- and LIF-induced protein tyrosine phosphorylation and tis1 1 gene expression MAH (A) or EW-1 (B) cells were treated with protein kinase inhibitors H-7 (40 ug/ml) or staurosporine (100 ng/ml) for 15 minutes prior to addition of CNTF (50 ng/ml), LIF (50 ng/ml) or mlL-6 (100 ng/ml). Total cell lysates were immunoprecipitated and immunoblotted with anti-phosphotyrosine antibody as described supra.
  • MAH(C) or M1 (D) cells were treated with protein kinase inhibitors H-7 (40 ug/ml) for 30 min prior to addition of CNTF (50 ng/ml), LIF (50 ng/ml) or mlL-6 (100 ng/ml).
  • Total RNA were prepared and subjected to northern analysis using a tis11 probe.
  • FIGURE 14 The CLIPs are co-regulated by CNTF and LIF (A), are on the cell surface (B), and one of the CLIPs (CLIP2) is gp130.
  • A. CLIP1 and CLIP2 are co-regulated by CNTF and LIF. EW-1 cells were treated for five or 60 minutes with either CNTF (50 ng/ml) or LIF (50 ng/ml), as indicated; after the 60 minute timepoints either additional CNTF (lanes 3 and 7) or LIF (lanes 4 and 6) were added to the cells for five additional minutes. Total cell lysates were then immunoprecipitated and immunoblotted with anti- phosphotyrosine antibody. B.
  • Biotinylation assay reveals that CLIP1 and CLIP2 are on the cell surface.
  • EW-1 cells were surface biotinylated as described herein.
  • the figure shows the anti- phosphotyrosine immunoblot for control (C) or CNTF stimulated (CNTF) cells that were subsequently biotinylated or left non- biotinylated before separation into unbound (UB) or bound (B) fractions on streptavidin-agarose.
  • C. CLIP2 is gp130.
  • the figure shows the anti-phosphotyrosine immunoblot of lysates from control (C) or CNTF/LIF stimulated EW-1 cells that were immunoprecipitated with the anti-phosphotyrosine antibody ( ⁇ pTyr) or the gp130-specific antibody ( ⁇ gp130).
  • the immunoprecipitating antibodies were either used individually or in sequential manner, as indicated.
  • FIGURE 15 Ubiquitous distribution of gp130 mRNA contrasts with restricted neuronal distribution of CNTFR ⁇ mRNA.
  • RNA was prepared from the indicated lines and subjected to northern analysis using either a human gp130 cDNA probe (top panels), or a rat CNTFR cDNA probe (bottom panels); the weaker hybridization to the rodent lines with the gp130 probe is due to poor cross-species hybridization.
  • SH-SY5Y neuroblastoma
  • EW-1 ewing's sarcoma
  • SK-N-LO neuroepithelioma
  • MAH sympathoadrenal progenitor
  • M1 myeloid progenitor
  • B9 IL-6 dependent B cell hybridoma that does not respond to CNTF.
  • FIGURE 16 Ubiquitous distribution of gp130 mRNA in tissues. Total RNA and northern analysis was conducted as in FIGURE 15.
  • FIGURE 17 gp130 blocking antibodies prevent CNTF/LIF induced tyrosine phosphorylations and gene inductions. Antibodies were examined for their ability to block tyrosine phosphorylations induced by CNTF and LIF in EW-1 cells. Tyrosine phosphorylations of CLIP 1 and CLIP2 (panel A), as well as tis 11 gene expression induced by CNTF or LIF (panel B) were both completely blocked by anti-gp130.
  • FIGURE 18 Effect of LIF and CNTF on ES cells.
  • ES cells maintained in the absence of feeder ceils, but in the presence of LIF (10-20ng/ml) remained as undifferentiated, compact colonies of small cells.
  • Lower concentrations of LIF (less than 10 ng/ml) resulted in the differentiation of the ES cells over a period of 2-7 days, as evidenced by the presence of endoderm-like cells and large, flat cells, with some cell death occurring (Panel 18A).
  • FIGURE 19 Expression of CNTFR in ES cells. Northern analysis of RNA from ES cells and rat brain indicating expression of CNTFR.
  • FIGURE 20 Induction of tisll by CNTF and LIF in ES cells.
  • ES cells were plated and maintained in an indifferentiated state in the presence of either CNTF (20ng/mi) or LIF (20ng/ml). Total cellular RNA was prepared, electrophoresed on a formaldehyde agarose gel, transferred to nylon membrane and hybridized to 32p. ⁇ labelled tis11 probe. In ES cells, CNTF and LIF both produced similar inductions in tis11 gene expression.
  • FIGURE 21 Schematic models of G-CSF, IL-6, CNTF and LIF receptor complexes.
  • A Model depicting known components of indicated cytokine receptor complexes.
  • B Revised "unified" models of CNTF and LIF receptor complexes assuming that CLIP1 is LIFR ⁇ .
  • CNTFR ⁇ is all that is required to convert a functional LIF receptor complex into a functional CNTF receptor complex.
  • Factors represented as squares; ⁇ subunits are known to exist for the IL-6 and CNTF receptor complexes, and are thus depicted with solid lines (asterisk adjacent to CNTFR ⁇ /membrane junction indicates GPI-linkage), while potential LIFR ⁇ component is indicated by a dashed line.
  • the present invention relates to a cell free CNTF/receptor complex and related compounds and their use in promoting the survival, differentiation, proliferation and/or growth of cells which may or may not express CNTFR.
  • the detailed description of the invention is divided into the following subsections: (i) the CNTF/Receptor Complex.
  • the present invention is based on the further discoveries that CNTF and LIF act on neuronal cells via a shared signalling pathways that involves the IL-6 signal transducing receptor component gp130 and that CNTF and LIF require a second, CLIPI/LIFR ⁇ component to initiate signal transduction.
  • CNTF and LIF act on neuronal cells via a shared signalling pathways that involves the IL-6 signal transducing receptor component gp130 and that CNTF and LIF require a second, CLIPI/LIFR ⁇ component to initiate signal transduction.
  • the present invention relates to the formation of a stable, biologically active cell-free CNTF/receptor complex. It is based, in part, on the production and purification of useful amounts of CNTF and CNTFR and their ability to form a stable, biologically active complex under normal physiological conditions.
  • CNTF and CNTFR may be prepared by first cloning and sequencing a gene encoding each respective protein. Each cloned gene may then be expressed in a prokaryotic or eukaryotic expression system. Any of a number of protocols available to one skilled in the art may be utilized to clone and sequence CNTF and CNTFR. For example, but not by way of limitation, CNTF may be cloned and sequenced preceding expression in a bacterial expression system, as described in U.S. Patent
  • CNTFR may, by way of example and not of limitation, be cloned and sequenced, as described in U.S. Patent Application No. 07/700,677, entitled “The Ciliary Neurotrophic Factor,” filed May 15, 1991 by Davis, et al., and International Application No. PCT/US91/03896 by Davis et al., filed June 3, 1991.
  • CNTFR having a sequence substantially as set forth in Figure 1 and CNTFR having a sequence substantially as set forth in Figure 2 may be used.
  • the recombinant CNTFR gene may be expressed and purified utilizing any number of methods.
  • CNTFR may be prepared from bacterial cells that express recombinant CNTFR as follows.
  • the gene encoding human CNTFR may be subcloned into a bacterial expression vector, such as for example, but not by way of limitation, pCP110.
  • the resulting plasmid, pRPN151 encodes a recombinant, mature form of human CNTFR (huCNTFR), consisting of 327 amino acids of the mature huCNTFR coding region and three additional amino acids, Met Ser Thr, at the NH terminus. Additional manipulations at the beginning of the coding region, as described in Example Section 6, further optimize huCNTFR expression without modifying the protein sequence.
  • This recombinant plasmid may then be transformed into a suitable strain of bacteria, such as E_. coli strain RFJ26 and grown under culture conditions known in the art to induce synthesis of recombinant protein, so as to obtain useful amounts of recombinant huCNTFR.
  • the recombinant huCNTFR may be purified by any technique which allows for the subsequent formation of a stable, biologically active CNTF/receptor complex.
  • huCNTFR may be recovered from RFJ26/pRPN151 cells as inclusion bodies, followed by quantitative extraction in 8M guanadinium chloride and dialysis as in Section 7, infra.
  • the present invention provides for a method of further purifying CNTFR comprising gel filtration.
  • proteins other than CNTFR that are expressed at low levels may also be purified by this method.
  • the CNTF/receptor complex may be formed subsequent to the purification of CNTF and CNTFR. Any ratios of CNTF and CNTFR which produce a stable CNTF/receptor complex may be used, including but not limited to, 1 :1 , 2:1 , 3:1 , etc.
  • equimolar amounts e.g., 80 nM
  • a physiological buffer solution e.g., 100 mM Tris HCI, 50 mM NaCI, pH 8.0
  • the mixture may then be applied to a gel filtration column and the peak corresponding to the CNTF/receptor complex may be recovered for use in numerous assays described infra.
  • the present invention provides for complexes in which CNTF and CNTFR are covalently or, preferably, non-covalently linked.
  • the present invention further relates to any complex or molecule which may be used to either promote or, alternatively to antagonize cell differentiation.
  • the CNTF/receptor complex imparting such an effect may be encoded by a hydrid or chimeric nucleic acid sequence.
  • This hybrid or chimeric nucleic acid sequence may be constructed by any of the numerous recombinant DNA methods known in the art such that sequences encoding functional portions of both CNTF and CNTFR are translationally linked; subcloned into either a prokaryotic or eukaryotic expression plasmid such that expression of the hybrid or chimeric nucleic acid sequence is controlled by any of a number of promoter elements compatible with the prokaryotic or eukaryotic host system as well as the orientation of the hybrid gene within the expression plasmid.
  • the nucleic acid sequences encoding functional portions of CNTF and CNTFR are subcloned in the same orientation and under control of the same regulatory sequences, resulting in a "dicistronic" construction.
  • the nucleic acid region spanning the fusion junction of the CNTF and CNTFR gene will either possess a sequence promoting splicing of the initial transcript prior to translation or, alternatively, this region will encode a peptide sequence known in the art to promote post- translational proteolytic processing by a protease active in the host cell.
  • the construction of such a hybrid or chimeric molecule promotes the expression of equimolar amounts of both functional components of the CNTF/receptor complex and allows for purification of the CNTF/receptor complex directly from either the prokaryotic or eukaryotic host cell.
  • the present invention also relates to nucleic acid sequences that encode a mutant CNTFR.
  • a given CNTFR gene can be mutated in vitro or in vivo, to create site- specific changes, additions or deletions in the coding region. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site directed mutagenesis [Hutchinson, et al.,, J. Biol. Chem.
  • hybrid or mutant molecules or complexes prepared as above may possess a number of characteristics that differ from those of the native CNTF/receptor complex.
  • such a hybrid or mutant may be able to promote signal transduction in the absence of CNTF (i.e., without the formation of the CNTF/receptor complex).
  • a hybrid or mutant may be capable of binding CNTF without resulting in signal transduction. These CNTFR blocking mutants or hybrids may then be assayed for their ability to act as antagonists of signal transduction in the presence of CNTF in any of the assay systems described infra. In preferred embodiments, such CNTFR blocking mutants or hybrids may bind CNTF with a higher affinity than that of native CNTFR for CNTF. In yet another embodiment of the invention, a CNTF mutant may be produced that binds to the CNTFR such that the resulting complex is incapable of signal transduction.
  • the present invention relates- to a stable CNTF/receptor complex.
  • a biologically active CNTF/receptor complex in a particular embodiment of the invention as described in Section 5.1 and Example Section 6, a biologically active CNTF/receptor complex.
  • CNTF/receptor complex is formed by adding equimolar amounts of CNTF and CNTFR in a physiological buffer solution at room temperature.
  • the CNTF/receptor complex of this particular embodiment possesses a different mobility in native polyacryiamide gels than either purified fractions of CNTF or CNTFR.
  • the CNTF/receptor complex may also be characterized according to its biological activity. In CNTF responsive cells, the activity of the CNTF/receptor complex corresponds to that of CNTF.
  • CNTF promotes cell differentiation as well as the survival of primary neurons.
  • Target cells for CNTF that express CNTFR include, but are not limited to, cells of the ciliary ganglion, dorsal root ganglion, hippocampus, and motor neurons.
  • CNTF mediates growth arrest and differentiation of MAH cell lines. Exposure of a MAH cell line to CNTF rapidly induces a pattern of tyrosine phosphorylation of three distinct CLIP proteins. In addition, phosphorylation of these CLIP genes immediately precedes induction of a characteristic immediate early gene, tis1 1.
  • the CNTF/receptor complex mediates similar effects as described supra on target cells which do not express the CNTF receptor (see, for example, Section 8, infra), provided such cells express a second component referred to herein as a "signal transducing component;" i.e.. a second component that interacts with receptor molecules to induce signal transduction, e.g., gp130 associated with the IL 6 receptor system or the beta chain of the receptor for Leukemia
  • a target cell for the CNTF/receptor complex may be any cell conducive to identification through a signal transduction assay in vitro (e.g., as discussed supra, such as a target cell which demonstrates a phenotypic differentiation, the expression of immediate early genes or the phosphorylation of CLIP proteins) in response to treatment with the CNTF/receptor complex, or a hybrid or mutant thereof that either mimics or alters the normal " physiological effect of the CNTF/receptor complex.
  • CNTF/receptor complex or a related hybrid or mutant compound
  • target cells can be assayed by any of a number of phenotypic and/or biochemical responses which are characteristic of the specific cell type. If the target cells are responsive to CNTF, the activity of the complex may be measured as a function of CNTF-related biological effects, such as the survival of ciliary ganglion neurons, dorsal root ganglion neurons, or motomeurons r etc.
  • the target cells are not responsive to CNTF, but are CNTF/receptor complex responsive, they may be assayed for other markers of differentiation.
  • an M1 cell line may be utilized to test the ability of the CNTF/receptor complex, or a hybrid or mutant protein thereof, to promote differentiation in a manner similar to the IL-6 or LIF- receptor pathway. Undifferentiated M1 cells are round and phase bright and do not adhere to the subtrate. Upon differentiation, as promoted through the CNTF/IL-6/LIF receptor family, the M1 cells become more differentiated and adhere to the substrate. Therefore, M1 cultures may easily be scored for this differentiation phenotype.
  • Cell specific markers may also be utilized, as was described supra for MAH cells, such as an alteration in patterns of CLIP proteins or other patterns of phosphorylation and the activation of immediate early genes (e.g., tia 11 ; See Section 9, infra) .
  • CNTF/receptor complex The ability of the CNTF/receptor complex to promote phenotypic differentiation in M1 cells (e.g., belonging to the IL-6/LIF receptor family) indicates that any other target cell responding to this complex is a target cell for the CNTF/receptor.
  • myeloid leukemia cells such the M1 cell line
  • other potential target cells for the CNTF/receptor, or hybrids and mutants thereof include leukemia cells, hematopoietic stem cells, megakaryocytes and their progenitors, DA1 cells, osteoclasts, osteobiasts, hepatocytes, adipocytes, kidney epithelial cells, embryonic stem cells, renal mesangial cells, T cells, B cells, etc.
  • a target cell for the CNTF/receptor complex may be any cell conducive to identification through a signal transduction assay in vitro (e.g., as discussed supra, such as a target cell which demonstrates a phenotypic differentiation, the expression of immediate early genes or the phosphorylation of CLIP proteins) in response to treatment with the CNTF/receptor complex, or a hybrid or mutant thereof that either mimics or alters the normal physiological effect of the CNTF/receptor complex. 5.2.1. DIRECT 1251-hCNTF BINDING ASSAY As discussed supra, another embodiment of the invention relates to the isolation of CNTFR mutants that are altered in their binding capacity for CNTF.
  • mutagenesis of pCMX-hCNTFR(l2) is followed by a direct i25
  • 10 ⁇ g hCNTF (560 ⁇ g/ml in 10 mM NaP0 4 , pH7.4) may be iodinated with 1 mCi i25
  • reaction may be quenched with an equal volume of buffer containing 0.1 M Nal, 0.1% BSA and 0.1% cytochrome C, 0.3% HOAc, 0.05% phenol red and 0.02% NaN 3 . Aliquots may be removed for determination of TCA precipitatable counts. The remainder may be loaded onto a BioRad PD - 10 Biogel column equilibrated with 0.05 M NaP0 4 , 0.1 M NaCI, 0.5 mg/ml protamine sulfate and 1 mg/ml BSA. Fractions may be collected and TCA precipitable counts determined. Next, COS cells may be transfected with mutagenized plasmid DNA.
  • the media may be removed and replaced with 0.25 ml of binding buffer (RPM1 1640 with 10% FBS and 0.1% NaN 3 ) containing 125
  • -hCNTF may be for 60 minutes at room temperature. After incubations are complete, the i25
  • Non-specific binding may be determined by the addition of at least 100 fold excess unlabelled hCNTF. After the last wash the plates may be 5 autoradiographed.
  • CNTFR mutants exhibiting either high or little or no CNTF binding may be selected for further analysis.
  • Supernatants from transfected cell lines of interest may be utilized in any number of differentiation assays to determine the ability of each mutant to 1 0 promote signal transduction.
  • the M1 cell assay system is useful for measuring the signal transducing ability of members of the 1 5 CNTF/IL-6/LIF receptor family.
  • This assay system is also useful to practice further embodiments of the invention, namely to identify mutant CNTF receptors that transduce signals without binding CNTF, and mutant CNTF receptors that bind CNTF but do not induce signal transduction.
  • CNTF mutant CNTF receptors that transduce signals without binding CNTF
  • mutant CNTF receptors that bind CNTF but do not induce signal transduction 20
  • a CNTFR mutant gives a weak or non existent signal in the 1251-hCNTF direct binding assay
  • this mutant receptor is scored in the M1 assay for the ability to promote phenotypic differentiation.
  • a CNTFR mutant showing increased binding may also be assayed in the M1 system.
  • the mutant CNTF 25 receptor may be mixed with varying amounts of unlabeled CNTF and
  • the mutant CNTFR acts as an antagonist to signal transduction pathway
  • different target cells of the CNTF/IL-6/LIF family may be identified using the same type of assay as described for M1 cells.
  • target cells may be identified in an assay system that demonstrates signal transduction, such as phosphorylation of CLIP proteins or immediate early gene induction, as described in Section 9, infra.
  • signal transduction such as phosphorylation of CLIP proteins or immediate early gene induction, as described in Section 9, infra.
  • a culture of putative target cells may be exposed to an effective concentration of
  • CNTF/receptor complex and then evaluated for phosphorylation of CLIP proteins, induction of tis11 immediate early gene expression, etc., in which such evidence of signal transduction indicates that the cells are indeed targets for CNTF/receptor complex.
  • the CNTF/receptor complex may be used to promote differentiation, proliferation or survival in vitro or in vivo of cells that are responsive to the CNTF/receptor complex including cells that express receptors of the CNTF/IL-6/LIF receptor family, or any cells that express the appropriate signal transducing component as evidenced by the characteristics (e.g., phosphorylation of CLIP proteins and/or immediate early gene induction) set forth in section 5.2, supra. Mutants or hybrids may alternatively antagonize cell differentiation or survival. 5.3.1.
  • the present invention may be utilized to identify new target cells for potential therapeutic or diagnostic applications of the CNTF/receptor complex.
  • assays identifying changes in morphology e.g.. • progression from rounded to flat cells and the extension of cellular processes and the transition from free-floating to attached cells
  • biochemical markers e.g.. the expression of cell specific markers, the activation of cellular genes, such as the immediate early gene, tis 11 , or the alteration in phosphorylation patterns, such as the CLIP proteins
  • cell growth or proliferation may be utilized to identify such novel target cells.
  • cells responsive to the CNTF/receptor complex may be used to identify CNTF/receptor complex related hybrid or mutant compounds.
  • such cells may be exposed to varying concentrations of CNTF/receptor complex related hybrid or mutant compound, and then the presence or absence and magnitude of physiological effects, such as cell proliferation, cellular morphology, phosphorylation of CLIP proteins, immediate early gene induction, etc. may be determined.
  • physiological effects such as cell proliferation, cellular morphology, phosphorylation of CLIP proteins, immediate early gene induction, etc. may be determined.
  • physiological changes should be similar to those produced by CNTF/receptor complex.
  • the hybrid or mutant acts as an antagonist of CNTF/receptor complex action
  • the physiological changes associated with CNTF/receptor complex action should be diminished or eliminat ed.
  • Any such target cell identified as a cell responsive to the CNTF/receptor complex through alterations of morphological or biochemical patterns is a candidate for use in diagnostic assays.
  • Such assays involve testing cells biopsied from patients for sensitivity to a particular treatment protocol involving the CNTF/receptor complex, or a hybrid or mutant thereof which promotes or antagonizes the signal transduction within this fa ' mily of receptors.
  • the present invention may be utilized to diagnose diseases and disorders of the nervous system which may be associated with alterations in the pattern of CNTF or CNTFR expression, and hence, formation of the CNTF/receptor complex.
  • diseases and disorders of the nervous system which may be associated with alterations in the pattern of CNTF or CNTFR expression, and hence, formation of the CNTF/receptor complex.
  • an abnormal response to CNTF/receptor complex in cells taken from a patient may support a diagnosis of a neurological disorder in the patient.
  • Such cell biopsies can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease .
  • states associated with changes in CNTF expression including in particular, conditions resulting in damage and degeneration of neurons known to respond to CNTF, such as parasympathetic neurons, cholinergic neurons, spinal cord neurons, neuroblastoma cells and cells of the adrenal medulla, such diseases and conditions include but are not limited to central nervous system trauma, infarction, infection, degenerative nerve disease, malignancy, or post-operative changes including but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Chorea, and amyotrophic lateral sclerosis.
  • the present invention has utility regarding any target cell identified through a bioassay system as described supra.
  • Any such target cell is a candidate for use in an in vitro system to detect, prognose, diagnose, or monitor the condition of the differentiation disorder or disease including, but not limited to malignant or neoplastic conditions, and in particular diseases or disorders involving the following cells: leukemia cells, hematopoietic stem cells, megakaryocytes and their progenitors, DA1 cells, osteoclasts, osteoblasts, hepatocytes, adipocytes, kidney epithelial cells, embryonic stem cells, renal mesangial cells, T cells, B cells, etc.
  • the present invention may be used to treat disorders of any cell responsive to the CNTF/receptor complex, including cells that are responsive to CNTF, as well as cells that are not.
  • disorders of cells that express members of the CNTF/IL-6/LIF family may be treated according to these methods. Examples of such disorders include but are not limited to those involving the following cells: leukemia cells, hematopoietic stem cells, megakaryocytes and their progenitors, DA1 cells, osteoclasts, osteoblasts, hepatocytes, adipocytes, kidney epithelial cells, embryonic stem cells, renal mesangial cells, T cells, B cells, etc.
  • the present invention provides for methods in which a patient suffering from a CNTF-related neurological or differentiation disorder or disease is treated with an effective amount of the CNTF/receptor complex, or a hybrid or mutant thereof.
  • the CNTF/receptor complex or appropriate hybrids or mutants thereof may be utilized to treat disorders or diseases as described for CNTF in International Application PCT/US90/05241 by Sendtner, et al. and for CNTFR in U.S. Patent Application Serial No. 07/700,677, entitled "The Ciliary Neurological Receptor,” filed May 15, 1991 by Davis et al.
  • Therapeutic methods comprising administering the CNTF/receptor complex, a CNTFR mutant inducing signal transduction without binding CNTF or a CNTF/receptor complex antagonist (e.g.. a CNTFR mutant with a high CNTF binding affinity that does not induce signal transduction), are within the scope of the invention.
  • the present invention also provides for pharmaceutical compositions comprising the CNTF/receptor complex, hybrid or mutant thereof in a suitable pharmacologic carrier.
  • the CNTF/receptor complex, hybrid or mutant thereof may be administered systemically or locally. Any appropriate mode of administration known in the art may be used, including, but not limited to, intravenous, intrathecal, intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or by local injection or surgical Implant. Sustained release formulations are also provided for. 5.3.2.1. FORMATION OF THE ACTIVE INGREDIENT
  • the active ingredient which may comprise the stable CNTF/receptor complex, or a hybrid or mutant thereof, should be formulated in a suitable pharmaceutical carrier for administration in vivo by any appropriate route including, but not limited to injection (e.g...
  • the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
  • a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
  • the concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used.
  • the dose used should be sufficient to achieve circulating plasma concentrations of active ingredient that are efficacious.
  • a circulating serum concentration level ranging from about 50 picomolar to 100 nanomolar may be used.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • CNTF/receptor complex preparations are provided, in which more than one CNTF/receptor complex are linked together either directly or through another member, such as a bead.
  • gp130 In the case of IL-6, a complex between IL-6 and its receptor component binds gp130, which then somehow activates the signal transduction process [Taga et al., Cell 5 [: 573-581 (1989); Hibi et al., Cell 85_: 1149-1157 (1990)].
  • the ability of gp130 to transduce functional signals correlates with its ability to be phosphorylated on tyrosine [Murakami et al., Proc. Natl. Acad- Sci. USA ££: 11349-11353 (1991 )].
  • tyrosine Murakami et al., Proc. Natl. Acad- Sci. USA ££: 11349-11353 (1991 )].
  • CNTF responsive neuronal cell lines examined displayed indistinguishable phenotypic and biochemical responses to LIF; in contrast, LIF responsive hemopoietic cells did not respond to CNTF.
  • CNTF and LIF responses in neuronal cells appear to initiate with the tyrosine phosphorylation of the three CLIPs, at least two of which (CLIPi and CLIP2) are cell surface proteins that can interact to form a stable, immunoprecipitable complex.
  • the CNTF receptor complex apparently also includes another cell surface protein, CLIP1 , that is tyrosine phosphorylated in response to CNTF and can directly interact with gp130 (Figure 21 A).
  • CLIP1 another cell surface protein
  • LIF is known to bind a recently cloned gp130-related receptor component with a molecular weight of approximately 190 kD (hereon LIFR ), and the existence of a LIF-receptor ⁇ (hereon LIFR ) has also been proposed [Gearing et al, Cell ££: 9-10 (1991)].
  • Our data indicates that the LIF receptor complex also includes CLIP1 and gp130 ( Figure 21 A).
  • the receptor complex for IL-6/CNTF/LIF-related G-CSF is apparently a homodimer of the gp130-reiated G-CSF receptor [Fukunaga et al, EMBO J. 1H-2855-2865 (1991)] ( Figure 21 A).
  • ⁇ - subunit dimerization and/or activation leads to activation of the signalling process, as proposed for receptor tyrosine kinases and some cytokine receptors [Aaronson et al., Science 254:1 146-1 153 (1991); De Vos et al., Science 255. 306-312 (1992)].
  • the ⁇ receptor components would ' act to modulate the binding of the factors to the ⁇ components, and thus be responsible for conferring ligand specificity upon the shared transducing machinery.
  • Cross-linking data [Godard et al., J. Biol. Chem. 267 (in press)] might suggest that for LIF, as with G-CSF, such components may not be required.
  • the CNTFR component would be all that is required to convert a functional LIF receptor into a functional CNTF receptor.
  • complexes containing these factors together with soluble forms of their receptors may act as heterodimeric factors for cells that are not capable of responding to the factor alone (because they do not express the appropriate receptor), but which do express the appropriate transducing components.
  • heterodimeric complexes actually operate as soluble factors in vivo is supported by the homology between the receptor components and one of the two subunits of natural killer cell stimulatory factor, a normally occurring heterodimeric factor [Gearing and Cosman, Cell £6_:9-10 (1991)].
  • G-protein coupled receptors similarly interact with a small number of signal transducing heterotrimeric G-proteins, allowing a vast array of different signals (eg. neurotransmitters, polypeptide hormones and odorants) to converge on a relatively modest number of signalling pathways [Gilman, Ann. Rev. Biochem. 56: 615-647 (1987)]. More directly relevant to the gp130-coupled receptor systems are those of IL-3, IL-5 and GM-CSF.
  • the subunits are themselves tyrosine phosphorylated, but do not appear to have inherent kinase activity.
  • the multi-component IL2 receptor also utilizes a subunit (IL2R ) that is responsible for high affinity binding and signal transduction, and this chain is tyrosine phosphorylated by a src-like tyrosine kinase (lck) with which it physically associates (reviewed in Miyajima et al., in press).
  • CNTF appears to be quite unusual compared to its distant cytokine relatives. Most importantly, CNTF has a very restricted receptor component distribution and, thus far, it is primarily cells of the nervous system that appear responsive to CNTF [Davis et al., Science 253: 59- 63 (1991)]. This restriction contrasts with the broad actions of the cytokines related to CNTF; the CNTF example suggests that additional related cytokines displaying a very restricted range of actions may exist. Identification of the MAH cell line provides a neuronal precursor cell line which displays physiologically relevant responses to CNTF and LIF as well as to factors using unrelated receptor systems, such as FGF and NGF.
  • MAH cell line should contribute to the understanding of how different factors, utilizing distinct signalling pathways, can interact to effect the growth and differentiation of neuronal progenitor cells. Contrasting the responses of MAH cells and hemopoietic cell lines to the cytokines should also provide insight into the mechanisms by which distinct cellular contexts alter the perception and interpretation of a very similar initial signal.
  • CNTF and LIF share components of a receptor complex/signal transduction pathway. Accordingly, CNTF alone, or in combination with CNTFR(CNTF ⁇ ), may prove to be useful as a means of initiating a response in a cell normally responsive to LIF. Depending on the receptor components a cell has, it may respond to CNTF or to a combination of CNTF and CNTFR. If a cell has gp130,
  • LIFR ⁇ and CNTF ⁇ (as appears to be the case with ES cells), CNTF alone will have the same effect at LIF. If the cell has only gp130 and LIFR ⁇ , CNTF and CNTFR in combination would mimic the effect of LIF.
  • CNTF can be utilized in place of LIF to prevent the differentiation of ES cell.
  • Embryonic stem (ES) cells totipotent cells isolated from pre-implantation- stage mouse embryos, can be cultured and manipulated in vitro and then reincorporated into a host embryo where they can develop normally and contribute to all cell lineages including the germ line.
  • ES cells thereby, provide an ideal vector system for the introduction of a specific mutation into mice.
  • Maintenance of the totipotent ES cells in culture requires either the presence of a feeder layer of fibroblasts (eg., STO cells) or the soluble factor leukemic inhibitory factor (LIF) Smith, et al. Nature 336. 688-690 (1988); Williams, et al. Nature 3365. 684-687 (1988).
  • the use of feeder cells is very reliable, but the preparation of the feeder layers is time-consuming. STO cells must be treated for 2-3 hrs. with mitomycin C to arrest their growth, then after several washes with PBS the STO cells can be plated onto gelatin-coated plates.
  • the plates can be used the following day, but are only good for 1 week after plating (Robertson, Nature 323. 445-448 (1987). To circumvent the problem of feeder layers, Williams et al., Nature (1988) supra and Pease and Williams, Exp. Cell Res. 190. 209-211., (1990) found that ES cells maintained in the absence of feeder cells retained their potential to form germ- line chimeras, provided LIF was included in the culture media.
  • ES cells cultured in the presence of CNTF, but in the absence of feeder cells and LIF, will retain the characteristic stem- cell morphology of compact colonies of small cells. This is the first example of a factor other than LIF which will prevent the differentiation of ES cells.
  • LIF feeder cells and LIF
  • CNTF/CNTFR soluble CNTFR
  • sCNTFR soluble CNTFR
  • the present invention contemplates the targeting of cells by complexing the CNTFR to the surface of a target cell and subsequently using CNTF to activate such a cell.
  • the CNTF/CNTFR complex is modified in such a way as to make it specific for a particular target cell.
  • a linking molecule a molecule that is capable of binding to both the CNTFR as well as the target cell.
  • a linking molecule would allow flexible binding to a naturally occurring receptor on a target cell.
  • attachment of a linking molecule with a terminal galactose to CNTFR would allow for attachment of such a complex to asialoglycoprotein receptors in the liver.
  • Another example might include attachment of an Fc containing linking molecule to CNTFR that would allow attachment of CNTFR to target cells containing Fc receptors.
  • antibodies preferably monoclonal antibodies
  • antibodies that recognize a particular cell surface receptor could be linked to the CNTFR.
  • an epitope could be attached to CNTFR, wherein such an epitope is recognized by a linking antibody that binds both the CTNFR-epitope complex and an epitope on a target cell. If an antibody directed against a target cell binds to an unknown epitope, it could be identified by panning random peptide expression libraries [see, for example PNAS 8JLL 1865 (1992)]. If CNTFR is attached to the surface of a target cell, the cell can then be activated by the addition of CNTF.
  • CNTFR attached to a linking molecule can be combined with CNTF.
  • the active agent would be the CNTF/CNTFR/Iinking molecule complex.
  • HepG2 cells which respond to LIF and IL6 in the "acute phase response" involving the transcriptional upregulation of a variety of genes including fibrinogen are used to provide an assay system for CNTF agonists or antagonists. It has been demonstrated that reporter constructs consisting of ChAT and the responsive gene's upstream sequences (i.e. the fibrinogen promoter) will accurately report functional signalling by IL-6 by upregulating ChAT activity.
  • a reported construct linked to a secreted or cell surface enzyme such as alkaline phosphatase
  • HepG-2 cells transfected with CNTFR would provide a valuable assay system for screening for CNTF activators and/or inhibitors.
  • Plasmid pRPN151 was generated by replacing the DNA between the unique Sail and EagI restriction sites in pCP1 10 with a
  • Human CNTFR1 consists of the 327 amino acids of the mature huCNTFR sequence and three additional amino acids of the sequence Met-Ser-Thr at the NH 2 - terminus. The three additional amino acids were included in order to signal translation initiation at the desired amino acid position and in order to simplify subsequent genetic engineering manipulations.
  • the DNA sequence was further modified at the beginning of the coding region in order to optimize expression without modification of the protein sequence. This was accomplished by incorporating the desired changes into the sense PCR primer ( Figure 3). Plasmid pRPN151 was then transfected into the £. coli strain, RFJ26. Under appropriate induction conditions of RFJ26/pRPN151 cells, huCNTFRI reached levels representing 10-
  • recombinant CNTF receptor forms a stable complex with CNTF.
  • M1 cells a myeloid leukemia-derived cell line, were 1 5 cultured in Dulbecco's Modified Eagle's Medium and 10% horse serum.
  • Undifferentiated M1 cells are round and phase bright and do not adhere to the substrate. These are scored (-). As the cells become more differentiated, they adhere to the substrate, become 25 less phase bright, assume irregular to spindle shaped morphology, and extend processes. Cultures are scored (+) to (++++) depending on the extent to which they have these features. Under optimal conditions, IL-6 treatment gives a score of (++) and LIF gives a score of (+++). Extremely high concentrations of CNTF and CNTFR gives the strongest expression of these characteristics and were scored (++++).
  • Protein Kinase inhibitors used include H-7 [1 -(5-lsoquinolinesulfonyl)-2-methylpiperaine dihydrochloride, Seikagaku Kogyo Co.) and staurosporine (Kamiya Biomed. Co.).
  • Antiphosphotyrosine monoclonal antibodies conjugated to agarose beads was from Upstate Biotech., Inc. (NY).
  • MAH cells were maintained in culture as previously described [Birren et. al., Neuron 4:189-201 (1990)]. Briefly, cells were plated onto dishes precoated with poly-D-lysine (100 ug/ml) and lam ⁇ nin (10 ug/ml), at a density of 6 K/6 mm well, or 40 K 16 mm well. Medium used was modified L15-C02 medium supplemented with 10% FBS and dexamethasone (5 uM).
  • IARC-EW-1 Ewing sarcoma cells
  • SK-N-LO neuroepithelioma cells
  • PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 6% horse serum, 6% calf serum, 2mM L-glutamine and 100units/ml penicillin and streptomycin.
  • RNA ISOLATION AND ANALYSIS Cells were plated at a density of 5x10 6 cells on 100 mm dishes, and treated with factors for various periods of time. Total RNA was prepared by guanidinium thiocyanate method as described previously [Chomczynski et al., Anal. Biochem. 162;156-159 (1987)]. Ten ug RNA was electrophoresed on a formaldehyde agarose gel, transferred to a nylon membrane (MSI), and hybridized to 32p-probes labelled by random oligo-priming (Stragene).
  • the probes used included tis11 (2.3 kb EcoRI fragment), c-fos (1kb Pst fragment), rat CNTF receptor (rCNTFR, 0.4kb Pst fragment), and GAPDH (1.25kb Pst fragment).
  • Proteins were eluted from the agarose beads wit 200ul of 1 X protein loading dye and boiled for 3 min. Fifty microliter of either the total protein samples or the imunoprecipitate was electrophoresed on 10% SDS- polyacryiamide gels, immunoblotted with anti-phosphotyrosine antibodies as previously described [Glass et al., Cell 6j6:405-4l 3] and specific proteins detected with 1251-labeled goat anti-mouse polyclonal antibody (1 ul of 4.91 uCi/ug per 1 ml buffer, DuPont).
  • the cell lysate was immunoprecipitated with an ERK-specific antibody (Zymed, Inc.), followed by a Goat anti-mouse IgG antibody conjugated to agarose. The precipitate was electrophoresed as above and immunoblotted with the same ERK antibody.
  • Lysates were precipitated with immobilized anti-phosphotyrosine antibody as described, then bound phosphoproteins were removed from the beads by boiling for 5 minutes in 50 mM tris pH 8.2 containing 1% SDS.
  • Biotinylated proteins were precipitated from this solution by incubation for 1 hour with 20L streptavidi ⁇ -agarose (Pierce). The supernatant containing the non-biotinylated proteins was subjected to SDS PAGE after the addition of sample buffer.
  • the beads containing biotinylated proteins were washed once in the binding buffer, then the bound biotinylated proteins were eluted from the beads by boiling for 5 minutes in 2X SDS PAGE sample buffer containing 10% ⁇ -mercaptoethanol. Anti-phosphotyrosine immunoblotting on these samples was performed as described above.
  • the MAH cell line utilized in this example was derived by immortalizing sympathoadrenal progenitors with the v-myc oncogene [Birren et al., Neuron 4 189-201 (1990)].
  • Treatment of MAH cells with CNTF dramatically blocked the increase in cell number that normally occurs upon the culture of these cells.
  • LIF which has effects on mature sympathetic neurons similar to those of CNTF, also blocked the normally occurring increase in MAH cell number of cells in CNTF- or LIF-treated cultures remained essentially constant over a 4 day period, while the control cultures continued to accumulate at an exponential rate (Figure 7B). The effects of both
  • CNTF and LIF displayed a very similar dose-dependency, with EC 50 values of approximately 50pg/ml (or 2 pM) (Figure 7C); this dose- dependency is similar to that observed for the survival effect of CNTF on ciliary neurons [Masiakowski et al., J. Neurochem £7:1003- 1012 (1991 )].
  • basic fibroblast growth factor (FGF) acted as a potent mitogenic agent for these cells
  • MAH cells were arrested in the G1 phase of the cell cycle reminiscent of many factors that induce a transition between a proliferative state and cell differentiation.
  • CNTF has been shown to induce cholinergic differentiation of sympathetic progenitors, and both CNTF. and LIF induce cholinergic differentiation of mature sympathetic neurons.
  • Choline acetyltransferase ChAT
  • CNTF acts as a growth arrest/differentiative factor for sympathoadrenal progenitor cells. These actions appear quite distinct from those of FGF.
  • FGF In addition to acting as a mitogenic agent for MAH cells, FGF induces neurite outgrowth and initiates neuronal differentiation (but not cholinergic differentiation) of these cells; FGF-induced differentiation may yield an NGF-dependent cell.
  • multiple factors may normally be capable of effecting the differentiation of neuronal progenitors.
  • MAH cells appear to be a very useful model system in which to dissect the roles of various factors in mediating various aspects of neuronal differentiation.
  • cytokines do not utilize receptors which contain intrinsic tyrosine kinase activity, tyrosine phosphorylation is rapidly induced by a variety of different cytokines.
  • CNTF induces tyrosine phosphorylation in responsive cells, " and to compare these phosphorylations with those induced by its distant structural relatives, we first examined CNTF and LIF responses in MAH cells as well as in neuroepitheliomas and Ewing's sarcoma.
  • CNTF- receptor positive cell lines with different phenotypic responses to CNTF displayed indistinguishable phosphorylation patterns in response to CNTF.
  • the observed tyrosine phosphorylation responses were dose-dependent, with maximal induction obtained at 10 ng/ml for both CNTF and LIF.
  • the CLIPs are not phosphorylated in response to NGF in the NGF-responsive pheochromocytoma cell line, PC12 ( Figure 10C).
  • ERKs extracellular signal-regulated kinases
  • ERKs also known as MAP or MBP kinases
  • tyrosine phosphorylation [Boulton et al., Cell 65:663-675 (1991)] and rapidly occurs following the binding of receptor tyrosine kinases to their cognate ligands (e.g., for NGF, see Figure 10C).
  • the ERKs are also activated in response to a diverse set of both mitogenic and differentiative agents that do not utilize receptor tyrosine kinases.
  • CNTF, LIF and FGF in EW-1 cells.
  • CNTF or LIF did not induce the rapid tyrosine phosphorylation of a 40 kD protein ( Figure 10A) that could be identified as ERK2
  • NGF do not result in CLIP phosphorylation.
  • stimulation of tyrosine kinase receptors results in rapid activation of the ERKs, which are not rapidly phosphorylated by either CNTF or LIF.
  • CNTF and LIF both produced an induction in tisl l gene expression which followed the induction of CLIP phosphorylation. Maximal activation occurred at 45 minutes and returned to control levels by 120 minutes (Figure 1 1 B). Similar gene activation kinetics for /s11 were observed in EW-1 cells. No induction of c-fos expression was observed in MAH cells with either CNTF or LIF ( Figure 11 B). However, bFGF, unlike CNTF and LIF, induced c-fos gene expression in the absence of f/s1 1 gene induction in MAH cells ( Figure 11 C). CNTF and LIF induced both tisl 1 and c-fos ( Figure 11 C) in EW-1 cells.
  • LIF induces the tyrosine phosphorylation of proteins identical in size to CLIP1 , CLIP2 and CLIP3 in the M1 myeloid progenitor cell line, • whereas IL-6 induces the tyrosine phosphorylation of only two of these proteins, corresponding to CLIP2 (presumably p160) and CLIP3;
  • the f s11 induction appears to be characteristic of responses to several of these distantly related cytokines, and the mechanism of induction by these different cytokines displays a similar sensitivity to protein kinase inhibitors.
  • the M1 cells did not express CNTF receptors and did not respond to CNTF, while the MAH, Ewing's sarcoma and neuroepithelioma cell lines examined did not respond to IL-6.
  • the finding that cell lines can be found which segregate responsiveness to CNTF, LIF or IL-6 suggests that no two of these factors utilize an identical receptor.
  • IL-6, CNTF and LIF share gp130 was investigated by using a monoclonal antibody (AM64) specific for human gp130 [Hibi et al., Cell f>3_: 1149-1157 (1990)] in concert with a human cell line responsive to both CNTF and LIF.
  • This antibody does not bind any gp130-related proteins, nor does it bind gp130 from rodent species.
  • Immunoprecipitation of gp130 revealed that it was strongly tyrosine phosphorylated in response to either CNTF or LIF in EW-1 cells, and that this phosphorylated gp130 co-migrated with CLIP2 (compare lanes 3 and 7 with lanes 2 and 6 in Figure 14C).
  • the anti-gp130 antibody could be used to completely deplete CLIP2 from extracts of CNTF/LIF-induced EW-1 cells
  • EW-1 cells were starved for 1 hour in defined medium in the presence or absence of a cocktail of anti-gp130 antibodies (2ug/ml). The cells were treated for 5 minutes or 45 minutes with various factors prior to tyrosine phosphorylation assays and RNA analysis, respectively.
  • Anti-gp130 antibodies which have been shown to inhibit IL-6 responses in hepatoma cell lines, were examined for their ability fo block tyrosine phosphorylations induced by CNTF and LIF in
  • gp130 might function as a transducer for factors other than IL-6 based on the finding that gp130 transcripts were much more widely distributed than those for IL-6R [Hibi et al., Cell 6 ; 1149-1157 (1990)].
  • ES cells were grown on a feeder layer of STO cells (growth-arrested with mitomycin C, Sigma Chemical Co.) and maintained in Dulbecco's modified Eagle media (DMEM, Irvine Scientific) supplemented with 10% FBS (Lot #11 11 1020, Hyclone,), 0.1 mM ⁇ -mercaptoethanol (Sigma Chemical Co.), 292 mg/ml of L-glutamine, 100 U/ml penicillin G, and 100 mcg/ml streptomycin sulfate (100X stock of L-glutamine, penicillin and streptomycin sulfate, Irvine Scientific).
  • DMEM Dulbecco's modified Eagle media
  • LIF synthetic human LIF, Amgen Biologicals
  • ES cells were passaged every 3-4 days onto newly-made feeder cell plates as described previously ((Robertson et al. Nature 323. 445-448 (1986).
  • ES cells were passaged 2 times, in the presence of LIF (20 ng/ml), onto gelatin- coated plates (0.1% gelatin from porcine skin, Sigma Chemical Co.), few STO cells remained after the second passage.
  • the ES cells were grown for 1 day in the presence of LIF (20 ng/ml), washed free of LIF, and then cultured in the presence of either
  • Total RNA was prepared from ES cells, grown in the absence of STO feeder cells and maintained in the presence of LIF
  • RNA was electrophoresed on a formaldehyde agarose gel, transferred to a nylon membrane (MSI), and hybridized to 32p labelled CNTF receptor cDNA probe (800 bp, Pstl fragment) labelled by random oligo-priming (Stratagene).
  • Recombinant rat CNTF was iodinated using the Bolton- Hunter method (Bolton and Hunter, Biochem J. 133: 529-539. (1973).
  • ES cells were plated at a density of 2.5 x 106 cells / 35 mm well on gelatin plates and grown in the presence of 20 ng/ml LIF 4 days prior to binding. Media was removed from the wells and the cells were washed once with assay buffer (PBS, pH 7.4, containing BSA (1 mg/ml), 0.1 mM bacitracin, 1 - mM PMSF and leupeptin (1 ⁇ g/ml)). The cells were incubated with i25
  • ES cells maintained in the absence of feeder cells, but in the presence of LIF (10-20 ng/ml) remained as undifferentiated, compact colonies of small cells.
  • LIF 10-20 ng/ml
  • lower concentrations of LIF resulted in the differentiation of the ES cells over a period of 2-7 days, as evidenced by the presence of endoderm-like cells and large, flat cells.
  • Some cell death also occurred (FIGURE 18, Panel A).
  • ES cells were grown on gelatin plates with varying concentrations of CNTF. Low concentrations of CNTF, 5 pg/ml to 10 ng/ml CNTF, resulted in differentiation and some cell death.
  • concentrations of greater than 10ng/ml up to 50 ng/ml CNTF maintained ES cells as small compact colonies of cells (FIGURE 18, Panel B).
  • ES cells maintained in the absence of either LIF or CNTF appeared endoderm- like or large and flat over a period of 2-7 days (FIGURE 18, Panel C).
  • RNA from ES cells indicated that CNTF receptor mRNA is present, albeit at low levels compared to adult rat brain (FIGURE 19).
  • CNTF (20ng/ml) or LIF (20ng/ml).
  • the cells were washed twice in defined medium, starved for 2 hours in defined medium prior to the addition of CNTF (50ng/ml) or LIF (50ng/ml) for 45 minutes.
  • RNA was prepared, electrophoresed on a formaldehyde agarose gel, transferred to a nylon membrane (MSI), and hybridized to 32p-iabelled f/ ' s11 probe ( Figure 20).
  • MSI formaldehyde agarose gel
  • Figure 20 32p-iabelled f/ ' s11 probe
  • CNTF and LIF both produced similar inductions in f/s1 1 gene expression, indicating responsiveness of ES cells to both of these cytokines.
  • ATC ACT CTG GCC CTG GCT GCC GCT GCC GCC ACT GCC AGC AGT CTC TTG 1401 He Thr Leu Ala Leu Ala Ala Ala Ala Ala Thr Ala Ser Ser Leu Leu 360 365 370

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Abstract

Complexe stable, biologiquement actif de récepteur/facteur neurotrophique ciliaire (CNTF) et ses hybrides ou mutants. L'invention est fondée en partie sur la découverte que le complexe de récepteur CNTF facilite la différentiation par un acheminement conducteur de signaux sur des cellules cibles qui n'expriment pas le récepteur de CNTF. Un mutant de récepteur/CNTF facilite la transduction de signaux sans lier le CNTF. L'invention concerne en outre un mutant de blocage du récepteur/CNTF mais n'ayant aucune fonction de transduction de signaux. L'invention identifie également des composants partagés par les acheminements de transduction de signaux de l'IL-6, du CNTF, du LIF et de l'OSM, et le démarrage de la transduction de signaux sur la base de la présence de ces composants. L'invention concerne en outre des applications thérapeutiques et diagnostiques en fonction de la capacité du complexe de récepteur/CNTF, de l'hybride ou du mutant de provoquer une réponse physiologique sur la celllule cible appropriée.
PCT/US1992/010632 1991-12-02 1992-12-01 Complexe recepteur/facteur neurotrophique ciliaire exempt de cellules WO1993011253A1 (fr)

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US80156291A 1991-12-02 1991-12-02
US801,562 1991-12-02
US07/865,878 US5332672A (en) 1991-12-02 1992-04-09 Prevention of ES cell differentiation by ciliary neurotrophic factor
US865,878 1992-04-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6146886A (en) * 1994-08-19 2000-11-14 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997929A (en) * 1989-01-05 1991-03-05 Synergen, Inc. Purified ciliary neurotrophic factor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4997929A (en) * 1989-01-05 1991-03-05 Synergen, Inc. Purified ciliary neurotrophic factor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 58, issued 11 August 1989, T. TAGA et al., "Interleukin-6 Triggers the Association of its Receptor with a Possible Signal Transducer, gp130", pages 573-581. *
MOLECULAR CELL BIOLOGY, Volume 11, No. 9, issued 1991, K.A. LORD et al., "Leukemia Inhibitory Factor and Interleukin-6 Trigger the Same Immediate Early Response, Including Tyrosine Phosphorylation, upon Induction of Myeloid Leukemia Differentiation", pages 4371-4379. *
NATURE, Volume 342, Number 6252, issued 21 December 1989, K.A. STOCKLI et al., "Molecular Cloning, Expression and Regional Distribution of Rat Ciliary Neurotrophic Factor", pages 920-923. *
SCIENCE, Volume 253, issued 05 July 1991, S. DAVIS et al., "The Receptor for Ciliary Neurotrophic Factor", pages 59-63. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6146886A (en) * 1994-08-19 2000-11-14 Ribozyme Pharmaceuticals, Inc. RNA polymerase III-based expression of therapeutic RNAs
US6852535B1 (en) 1994-08-19 2005-02-08 Sirna Therapeutics, Inc. Polymerase III-based expression of therapeutic RNAS

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