IE83306B1 - The ciliary neurotrophic factor receptor - Google Patents

Description

THE CILIARY NEUROTROPHIC FACTOR RECEPTOR . INTRODUCTION The present invention relates to the ciliary jxneurptrophic factor-receptor (CNTER), and.provides‘for.guTrp ,‘ receptor encoding nucleic acid and amino acid sequences.

It also relates to (i) assay systems for detecting CNTF activity; (ii) experimental model systems for studying the physiological roleyof_CNTF; (ii) diagnostic techniques for identifying CNTF—related neurologic conditions} (ivj therapeutic techniques-for the treatment of CNTF-related neurologic conditions, and (v) methods for identifying ' nolecules homologous to CNTFR.

. BACKGROUND OF THE INVENTION 2.1. CILIARY NEUROTROPHIC FACTOR Ciliary neurotrophic factor (CNTF) is a protein that is specifically required for the survival of embryonic The ciliary ganglion is In_the chick ciliary ganglion, half of the -2... ) observed that removal of an eye prior to the period of‘ cell death results in the complete loss of ciliary ganglion neurons in the ipsilateral ganglion. Conversely, Narayanan. and Narayanan (1978, J. Embryol. Ex. Morphol. 44253470) ohserved that, by implanting an additional eye primordium and thereby"increasing the amount of available target tissue, ciliary ganglion neuronal cell death may he. decreased. These results are consistent with the _existence offa target derived neurotrophic factor which . acts upon ciliaryfiganglion neurons. ' In culture, ciliary ganglion (CG) neurons have been found to require a factor or factors for survival. Ciliary‘ ,.neurotrophic factor(s) (CNTF) activity has been identified ‘diseussien, see also Adler et a1., 1979, Science 204:1434- Out of 8000 trephie units (TU) present in a twe1ve~day 9' _embryo, 2500 T0 were_£ound present in eye tissue; activity appeared to be localized inia fraction containing the ciliary body and choroid coat. ' Subsequently, Barbin et al. (1984, J. Neurochem. _ :1468~1478) reported a procedure for enriching CNTE ffOWg_.. (83330 as»: ..3_ be associated with non-CG tissues, including rat sciatic nerve (Williams et al., 1984, Int. J. Develop. Neurosci 218:460-470). Manthorpe et al. (1986, Brain Res. 367:282~ 286) reported partial purification of mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating CNTF activity from chick eye. (In addition, Watters and Hendry (1987, J. Neurochem. 49:705-713) described a method for enriching CNTF activity approximately 20,b08~fold from bovine cardiac tissue under _non—denaturing conditions using heparin—affinity chromatography.. CNTF activity has also been identified in .damaged-brain tissue (Manthorpe et al., 1983, Brain Res. :47—56; Nieto-Sampedro et al., 1983, J. Neurosci. 3:22l9-2229) .

Carnow et al. (1985, J. Neurosci. 5: 1965-1971) and Rudge et al., (1987, Develop. Brain Res. 32:103—110) describe methods for identifying CNTF—like activity from Western blots of tissue extracts and then identifying protein bands containing CNTF activity by inoculating the nitrocellulose strips in a culture dish with CG neurons and identifying areas of cell survival using vital dyes. Using this method, Carnow et al. (1985, J. Neurosci. 5:1965-1971) observed that adult rat sciatic nerve and brain-derived CNTF activities appear to exhibit a different size (24kD) than chick CNTF (20.4 kD).

Recently, CNTF has been cloned and synthesized in bacterial expression systems, as described in W0 91/04315~ Using recombinant probes, CNTF-mRNA in tissues of adult rat appeared to be about 1.2 kb in size. Rat brain CNTF was cloned and found to be encoded by a mRNA having a short 5' untranslated region of 77 bp and an open reading frame of -4... bp, predicting a protein of about 200 amino acids (Stockli et al., 1989, Nature 342:920—923), Human CNTF was also cloned and sequenced (W0 91/04315): its coding sequences were substantially conserved relative to rat sequences, whereas noncoding sequences were less Conserved. .2. FUNCTH-)NAl. PRQPERTIESVOF CILIARY NEUROTROPHIC FACTOR A number of biological effects have been ascribed to CNTF. As discussed above, CNTF was originally described as an activity which supported the survival of neurons of the‘ ‘E8 chick ciliary ganglion, a component of the ' parasympathetic nervous system. A description of other biological properties of preparations known to contain CNTF activity follows: Saadat et al. (1989, J. Cell Biol. 108:1807-1816) observed that their most highly purified preparation of rat sciatic nerve CNTF induced cholinergic differentiation of rat sympathetic neurons in culture. Also, Hoffman (1988, J. Neurochem. 51:109-113) found that CNTF activity derived from chick eye increased the level of choline-O- acetyltransferase activity in retinal monolayer cultures.

Hughes et al. (1988, Nature 335:10—73) studied a population of bipotential glial progenitor cells-in cultures derived from the perinatal rat optic nerve and brain; these progenitor cells have been shown to give rise to, first, oligodendrocytes and then, to type 2 astrocytes.

Under the culture conditions used, oliqodendrocyte differentiation appeared to occur directly from an oligodendrocyte-type 2—astrocyte (0-2A) progenitor cell, whereas type 2 astrocyte differentiation appears to require the presence of an inducing protein similar or identical to CNTF (see also Anderson, 1989, Trends Neurosci. 12:83-85)- -5...

Heymanns and Unsicker (1979, Proc. Natl. Acad. sci, U.S.A. 4:7758—7762) observed that high-speed supernatants of neuroblastoma cell extracts produced effects similar to those associated with CNTF activity from chick eye or rat sciatic nerve; the presence of a protein similar but not identical to CNTF (by molecular weight) was indicated.

Ebendal (1987, J. Neurosci. Res. 17:19-24) looked for CNTF-like activity in a variety of rat and chicken tissues.

He observed CNTF—like activity among a fairly wide range of rat, but not in chicken tissues: rat liver, spleen T cells, and submandibular gland cells were found to be associated with low levels of CG survival promoting activity, whereas heart, brain, and skeletal muscle tissues were associated ‘with higher survival promoting activity. Among tissues tested the highest CNTF—like activity was observed to be associated with rat kidney.

While the above studies have shown that many tissue and cell extracts contain activities which support the survival of neuronal populations which are also responsive to CNTF, (i.e. they support the survival of E8 chick ciliary ganglion neurons in a tissue culture bioassay), it cannot be assumed that a single or identical protein is responsible for these activities. As shown for the family of fibroblast growth factors (FGFS) (Dionne et al. , 1990, EMBO J; g:2685-2692), for example, a number of distinct polypeptides or proteins possess identical biological activity in a single bioassay. , The neuronal specificity of chick eye and rat sciatic nerve CNTF were initially found to have some overlap with neuronal populations responsive to NGF. Although CNTF was observed to have.some overlapping neuronal specificity with NGF, distinguishing characteristics between them became most apparent in studies of the roles of CNTF and NGF in populations of developing neurons (Skaper and Varon, 1986.

Brain Res. 389:39-46). In addition to their differing ;: i ..6._ appeared to prevent cell death of mdtorneurons in lesioned ‘ .facial.nerve(VlIth cranial nerve) of newborn rat (Sendtner:5 :activities. .3. GROWTH FACTOR RECEPTORS A number of receptors which mediate binding and response to protein factors have been characterized and molecularly cloned over the last few years, including receptors for insulin, for platelet derived'growth factor, for epidermal growth factor and its relatives, for the fibroblast growth factors, and for various interleukins and hematopoietic growth factors. Recent data reveal that certain receptors can bind to multiple (related) growth factors, while in other cases the same factor can act on multiple (related) receptors (e.g. Lupu et al., 1990, which span the membrane, and intracellular domains that are intracellular portions of a given receptor often share The extracellular and common structural motifs with the corresponding regions of other receptors, suggesting evolutionary and functional ,relationships between different receptors. These relationships can often be quite distant and may simply reflect the repeated use of certain general domain structures. For example, a variety of different receptors that bind unrelated factors make use of "immunoglobulin" domains in their extracellular portions, while other receptors utilize "cytokine receptor" domains in their factor—binding regions (e.g. Akira.et al., 1990, The FASEB J. 4:2860-2867). extracellular binding domains (which thus bind different A large number of receptors with distinct factors) contain related intracytoplasmic domains encoding tyrosine-specific protein kinases that are activated in The mechanisms by which factor— mysterious.

. SUMMARY OF THE INVENTION The present invention relates to CNTF receptor (CNTFR) genes and proteins. It is based, in part, on the cloning and characterisation of the human CNTFR—gene and its expression in transfected COS cells.

The present invention provides for nucleic acid sequences which encode the CNTFR.

Thus, the invention provides an isolated nucleic acid molecule selected from: (a) a nucleic acid molecule having the sequence of Figure 2 and encoding a polypeptide capable of binding ciliary neurotrophic factor (CNTF); (b) a nucleic acid molecule whose sequence differs from that of Figure 2 by one or more substitutions, additions or deletions and encodes a polypeptide capable of binding to CNTF; (c) a nucleic acid molecule whose sequence differs from that of Figure 2 but, due to the degeneracy of the genetic code, encodes the amino acid sequence of Figure 2; (d) a nucleic acid molecule having a sequence complementary to that of (a), (b) or (c).

The invention also provides a nucleic acid vector comprising such a nucleic acid molecule.

The invention also provides a cell transformed, transfected or infected with such a nucleic acid molecule or vector.

The invention also provides an immortalised cell line comprising such cells.

It also provides CNTFR proteins/polypeptides. In particular, the invention provides CNTFR polypeptides encoded by nucleic acid molecules as just described. In a further aspect of the invention, CNTFR probes,including nucleic acid as well as antibody probes, may be -W9 -: used to identify CNTFR~related molecules- For example, the present invention provides for such molecules which form a complex with CNTFR and thereby participate in CNTFR function. As another example, the present .invention provides for receptor molecules which are homologous or cross—reactive antigenically, but not identical to CNTER. These particular embodiments are based on the discovery that the CNTFR bears homology to ti other biologically relevant molecules, including, most particularly, the IL-6 receptor, but also the PDGF receptor, the CSF—1 receptor, the prolactin receptor, the IL-2 and IL-4 receptors, the GM-CSF granulocyte macrophage colony stimulation factor receptor, pregnancy- specific alpha 1~beta glycoprotein, and carcinoembryonic antigen, a tumor marker.

The present invention also provides for assay systems for detecting CNTF activity, comprising cells which express high levels of CNTFR, and which are itherefore extremely sensitive to even very low concentrations of CNTF or CNTF—like molecules.l In yet further embodiments of the invention, CNTFR probes may be used to identify cells and tissues which are responsive to CNTF in normal or diseased states. For example, a patient suffering from a CNTF—related disorder may exhibit an aberrancy of CNTR expression.

In addition, the CNTFR genes and proteins of the invention may be used therapeutically. For example, and not by way of limitation, a circulating CNTFR may be used to deplete CNTF levels in areas of trauma to the central nervous system. Alternatively, a recombinant CNTFR gene may be inserted in tissues which would benefit from increased sensitivity to CNTF, such as motorneurons in patients suffering from any amyotrophic lateral sclerosis.

The invention also provides a method for identifying a cell which binds to CNTF, which method comprises detecting the expression of CNTFR by the cell by detecting the presence of CNTFR RNA having a sequence corresponding to that of the DNA sequence of Fig 2 or CNTFR protein, having the amino acid sequence of Fig 2.

The invention also provides an assay system for the screening of pharmaceutical compounds for CNTF activity comprising cells according to the invention which express recombinant CNTFR or a functionally active portion thereof, and a means for detecting the interaction between CNTF, or a molecule having an activity similar to CNTF, and CNTFR by measuring the physiological response of the cells to CNTF or a molecule having an activity similar thereto.

The invention also provides an experimental model system for studying the physiology of CNTF comprising cells according to the invention which express, on their surface, an increased number of CNTF receptors relative to the same type of cells which do not contain recombinant nucleic acid encoding a CNTF receptor.

The invention also provides a method of diagnosing a CNTF—related disorder comprising detecting a difference in .... xxxx ".4. A4: {"\‘l"|'V1'.‘D r\v' 2 f-'r:nmnn+- -I-hnrnnf in (‘P114 01‘ '— 11-'~ tissue from the patient relative to the amount of CNTFR in comparable cells or tissue from a healthy person, wherein the amount of CNTF receptor or fragment thereofi is measured‘ by: (i) exposing the cells or tissue to an anti~CNTFR antibody according to the invention under ' conditions which allow imnunospecific binding to CNTFR tn occur: and (ii) measuring the amount of antibody binding.

The invention also.provides a pharmaceutical’ composition comprising a polypeptide according to the invention and Pharmaceutically acceptable carrier.

The invention also provides polypeptides and nucleic acids of the invention for use in a method of treatment of neurologcial, neuromuscular or muscle disorders of the human or animal_body by therapy.

The invention also provides use of a polypeptide of the invention in the manufacture of a medicament for use in a method of treatment as described herein; The invention also provides a method of identifying molecules related to the CNTF receptor which are members" of the family of molecules which include the IL-6 ireceptor comprising: _ 7.. (1) . using an oligonucleotidc probe derived either from protein sequence data or from-the nucleic acid sequence set forth in Figure 2 (SEQ ID N01 1) to screen a DNA library for clones which hybridise to portions of CNTF receptor—encodin9 nucleic acid as defined in claim 1(3) ‘ (C); (11) isolating and propogating clones which hybridize to CNTF receptor encoding nucleic acid; _*:.-*i (iii) (iv) Figure 1- A. _ 12 _ determining the nucleic acid sequence of the clones isolated and propogated in step (ii); and comparing the nucleic acid sequences determined in step (iii) with the nucleic acid sequences of known members of the family.

. DESCRIPTION or THE FIGURES Schematic diagram of expression cloning using tagged ligand binding strategy. "B. Secondary iodinated antibody assay showed that in contrast to CO5 cells transfected with the original CDNA library, many COS cells transfected with DNA obtained after one round of panning expressed CNTF-binding sites (radioautograph done on 60 mm plate of transfected COS cells; each black dot represents a single transfected COS cell expressing a CNTF-binding site). C. The same assay as described in (B), but where COS cells had been transfected with a non-CNTFR encoding plasmid (negative clone) or a CNTFR encoding plasmid (positive clone).

Only small sections of each plate are shown- IL Resdlts of fluorescence activated cell sorting (FACS) analysis of COS cells transfected with the negative clone or the positive clone of (C).

Figure 2. Nucleic acid sequence of CNTFR—encoding CDNA and deduced amino acid sequence.

Figure 3. Alignment of the human CNTFR showing homologies in the immunoglobulin—like domain and the cytokine receptor- like domain. Numbers on the left indicate the amino acid number starting from the first methionine. Identical residues and conserved substitutions are marked by solid boxes. Gaps are introduced to maximise homology.

IL-6 = interleukin; CEA = carcinoembryonic antigen; PDGF = platelet derived growth factor; CSF-1 = colony stimulating factor-1; alpha 1-6 GP = alpha 1 B glycoprotein, PRL = prolactin, EPO = erythropoietin; IL-2 = interleukin 2; IL~4 = interleukin 4; GM—CSF = granulocyte macrophage colony stimulating factor.

Figure 4. Structural relationships between the CNTFR and other related receptors. The human IL-6 receptor and CNTFR have an immunoglobulin domain fused to the N-terminus of the proposed factor binding domain. A short acidic tether (zig zag line) connects the globular immunoglobulin and proposed factor binding domain. A proposed protein similar to gpl30 is shown in association with the CNTFR, as discussed in the text.

HuGRHR - human growth hormone receptor; RbPRLR — rabbit prolactin receptor; MOEPOR — mouse erythropoietin receptor; HuIL2RB - human interleukin-2 ' l _]_4 ._. receptor fi—chain; HuIL6R — human interleukin 6 receptor; HuCNTFR — human ciliary derived neurotrdphiC_- factor receptor; C — cysteine; X- unknown aming acid: W) — tryptophan; S >vserine; F ~ phenylalanine.

Figure 5. ‘Tissue localization of CNTFR message. RNA'was; prepared from the indicated tissues of_rat as described in section'8»l5{_DNA‘fragments of CNTFR were derived i from expreséion constructs containing these genes in- 'pCMX as described in section 8.1. Tissues: cerebellum (CB); hindbrain~(HB}3 midbrain (MB); thalamus (TH/HYP)A striatum (STRI):,hi@pocampus B (HIP B); hippocampus A‘ (HIP A);.cortex (CORT); olfactory bulb-(OLF): adult brain (AD BR); skin (SK); heart (HRT); muscle (MUS), lung (LUNG); intestine (INT); kidney (KID); liver 7 (LIV); spleen (SPL): thymus (THY): E1 liver (E17 LIV).

‘Yu- .‘,5 as-: 'Figure 6. pCMX with hCNTF-R gene insert. Construction of pCMX in copending application- Figure 7. Northern blot analysis of CNTF receptor expression in skeletal muscle. 10 pg of total RNA was run in each lane. Lane 1, mouse myoblast cell line i C2Cl2 mb; lane 2, mouse myotube cell line C2C12 mt; lane 3, rat myotube cell line H9C2 mt; lane 4, rat myotube cell line L6 mt; lane 5, rat soleus muscle; lane 6, rat extensor digitorum longus (EDL) muscle; lane 7, denervated skeletal muscle; lane 8, purified human myotubes: lane 9, skeletal muscle (this RNA sample was degraded): lane 10, adult rat cerebellum; lane 11, sham—operated soleus muscle; lane 12, 72 hour denervated rat soleus muscle: lane 13, sham-operated EDL muscle; lane 14, 72 hour denervated EDL muscle.

Figure 8. Anatomical diagram of the right hindlimb subjected to denervation surgery.

Figure 9. UNOP = unoperated soleus_muscles from animal group 1 (Table IV): solid bar represents right side and stippled bar represents left side; NONE = lesioned '(denervated, right side) and control (sham-operated, left side) soleus muscles without any injection, from animal group 2; ALB = lesioned (right) and control (left) soleus muscles treated with PBS/BSA (SC) from animal group 6: CNTF=lesioned (right) and control (left) soleus muscles treated with CNTF/BSA (sc) from I3!‘ animal group 5. Solid bars: lesioned; stippled bars: control . DETAILED DESCRIPTION OF THE INVENTION For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the invention will be divided into the following subsections: (i) cloning of the CNTF receptor; (ii) nucleic acid encoding the CNTF receptor; (iii) CNTFR peptides; (iv) expression of CNTF.receptor; (v) identification of molecules related to the CNTF receptor; and (vi) utility of the invention. .1. CLONING OF THE CILIARY NEUROTROPHIC FACTOR RECEPTOR The present invention enables the cloning of the CNTF receptor (CNTFR) by providing a method for selecting target cells which express CNTFR. By providing a means of enriching for CNTFR encoding sequences, the present invention enables the purification of CNTFR protein and the direct cloning of CNTFR-encoding DNA.

For example, CNTFR-bearing target cells may be selected, and CNTFR protein may be purified using methods known to one skilled in the art for the purification of a receptor molecule. For example, and not by way of limitation, CNTF or CNTF attached to a detectable molecule, as described in section 5.6.3, infra, in which the tag may be, for example, a radiolabel, antigenic determinant, or antibody, to name a few, (CNTF/tag) may be reversibly crosslinked to target cells, and membrane associated proteins from said target cells may be subjected to purification methods. Such purification methods may include SDS-PAGE, followed by detection of the position of _ 17 _ CNTF or CNTF/tag in the gel; for example, radiolabeled CNTF could be used, and, crosslinked to its receptor, may be visualized in the gel by autoradiography. Alternatively, anti~CNTF or anti—tag antibody could be used in the Western blot technique to identify the position of the CNTF/receptor complex in such gels.: Preparative gel electrophoresis could be used to isolate sufficient amounts of protein to enable amino acid sequencing of peptide fragments of_the receptor, or to enable production of anti~CNTFR antibody*which could be used to purify CNTFR molecules from target cell extracts. Amino acid sequence obtained from purified CNTFR may be used to design degenerate oligonucleotide probes which may be used to ‘identify CNTFR encoding cloned nucleic acid in a genomic DNA library or, preferably, in a CDNA library constructed from CNTFR producing target cells.

Alternatively, the CNTFR may be cloned by subtractive hybridization methods, in which mRNA may be prepared from target cells which express CNTFR, and then non—CNTFR encoding sequences may be subtracted by hybridizing the mRNA (or CDNA-produced therefrom) with mRNA or CDNA derived from cells such as neuronal cells which do ngt express the CNTFR . likely to be enriched in CNTFR-encoding sequences.

The nucleic acid remaining after subtraction is Nucleic acid prepared, preferably, from target cells enriched in CNTFR encoding sequences due to endogenous expression of ChTFR and/or due to subtraction techniques discussed supra, may also be used in expression cloning techniques to directly clone the CNTFR. For example, and not by way of limitation, total genomic DNA from target cells which express CNTFR may be prepared and then transfected into a cell line which does not express CNTFR and which is preferably derived from a different species from the target cell species (for example, DNA from a human CNTFR-encoding cell may be transfected into a mouse cell, _ 13 _ such as an L cell). Although a relatively small number of transfected cells may express CNTFR, such cells may be identified by rosetting techniques or immunofluorescence techniques as described in section 5.6.3, infra and may be isolated, for example, by fluorescence-activated cell sorting or using antibody-coupled magnetic beads or "panning" techniques, known to one skilled in the art. The CNTFR encoding DNA may be cloned from receptor—producing transfectants by producing a genomic library from the transfectants andithen isolating and propagating clones that contain either sequences conforming to CNTFR amino acid sequence or sequences homologous to species specific .genetic elements: for example, human DNA may be identified via Alu repeated sequences, which are distributed at high frequency throughout the human genome. For example, and not by way of limitation, cultured non-human cells comprising transfected human DNA encoding the CNTFR and which express human CNTFR may be selected, propagated, and then genomic DNA prepared from these cells may be used to transfect cultured non—human cells, and CNTFR expressing cells may be selected. This process may be repeated; its purpose is to decrease, by each transfection step, the amount of human DNA present in CNTFR encoding cells.

Accordingly, when the genomic DNA of transfected, human CNTFR expressing cells is cloned to generate a library, clones which include human DNA (and are identified, for example, by screening for distinctly human sequence elements) are more likely to comprise CNTFR-encoding sequences when repeated transfections have been performed.

RNA from a CNTFR expressing cell line or tissue source, or a cDNA expression library obtained from such a source may be introduced in pools into Xenopus oocytes by direct injection: oocytes injected with pools encoding the CNTFR may be identified by assaying for functional responses (e.g. ion fluxes) that may be induced by exposing _ 19 _ such oocytes to CNTF, or alternatively by detecting the presence of CNTF-binding sites on the surface of such injected oocytes. Repetitively dividing positive pools into smaller and smaller pools may lead to the identification of individual clones encoding the CNTFR.

Alternatively, a CDNA expression library may be derived from CNTFR bearing target cells and then utilized in transient expression assays. In a preferred embodiment of the invention, said expression library may incorporate the SV4O origin of replication and transient expression assays may be performed using COS cells. CNTFR-expressing transfectants may be identified as set forth above, and CNTFR encoding DNA may be retrieved using standard methods.

AThe nucleic acid sequence encoding the CNTFR may then be propagated and/or utilized in expression systems using methods substantially as set forth for nucleic acid encoding CNTF, as described in W0 91/04316- (1) instead of incubating the transfected cells with anti—receptor antibodies, the cells may be incubated first with tagged CNTF (for example.

CNTF myc) on ice for about 30 minutes, ~ 20 — * z centrifuged through phosphate buffered saline (PBS)/2% Ficoll to remove excess ligand, and then incubated with anti-tag antibody (for example, the anti—myc antibody 9El0) for about 30 minutes on ice. (ii) the cells may then be spun through PBS/2% Ficoll and then "panned" on plates coated with antibody that recognizes the anti~tag antibody (for example, if the anti-tag antibody is 9E10, anti- mouse antibody.

Then, after washing nonadherent cells from the plates, Hirt supernatants may be prepared from the adherent cells, and plasmid DNA may be precipitated in the presence of about 10-20pq of tRNA. The resulting plasmid DNA may then be introduced into suitable bacteria (for example DH10 B bacteria) by standard techniques, including, but not limited to, electroporation. The cultures grown from transformed bacteria may then be used to prepare plasmid DNA for another round of eukaryotic transfection and panning. After this second transfection, panning and plasmid DNA preparation and transformation, the bacterial transformants may be plated out on selective media, individual colonies may be picked and used for the preparation of plasmid DNA, and DNA prepared from a number of such clones may be used individually for COS cell transfection. Alternatively, more rounds of enrichment may be necessary before individual colonies are tested.

Resulting COS cells expressing CNTF binding sites may be identified by a number of techniques, including, but not limited to, indirect binding assays using radioactively labeled or fluorescently labeled indicator antibodies. An example of a CNTFR—encoding nucleic acid is comprised in pCMX-hCNTFR (I2), Figure 6, which has been deposited with the NRRL and assigned accession number B—18789. molecules may be enzymatically modified. _ 21 _ __ Clones identified in this manner may then be analyzed by restriction fragment mapping and nucleic acid sequencing using standard techniques. Fragments of the CNTFR-encoding CDNA may then be used to identify genomic DNA sequences which comprise the CNTFR gene, for example, from a genomic DNA library using standard hybridization techniques. ' Once obtained, a CNTFR gene may be cloned or subcloned using any method Known in the art. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, cosmids, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322, pUC, or Bluescript° (Stratagene) plasmid derivatives. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc.

The CNTFR gene may be inserted into a cloning vector which can be used to transform, transfect, or infect appropriate host cells so that many copies of the gene sequences are generated. This can be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. ,However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA It may prove advantageous to incorporate restriction endonuclease cleavage sites into the oligonucleotide primers used in polymerase chain reaction to facilitate insertion into vectors. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini: these ligated linkers may comprise specific chemically synthesised oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and CNTFR gene maybe modified by homopolymeric tailing.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate an isolated CNTFR gene, CDNA, or synthesised DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. .2 NUCLEIC ACID ENCODING CILIARY NEUROTROPHIC FACTOR RECEPTOR Using the methods detailed sgpra and in Example Section 6, infra, the following nucleic acid sequence was determined, and the corresponding amino acid sequence deduced. The sequence of the human CNTFR is depicted in Figure 2. Additionally, the invention relates to CNTFR genes isolated from porcine, ovine, bovine, feline, avian, equine, or canine, as well as primate sources and any other species in which CNTF activity exists.

Subsequences comprising hybridisable portions of the CNTFR sequence have use, e.g., in nucleic acid hybridisation assays; Southern and Northern blot analyses, etc.

As set out in 3 above, the invention provides an isolated nucleic acid molecule selected from; (a) a nucleic acid molecule having the sequence of Figure 2 and encoding a polypeptide capable of binding ciliary neurotrophic factor (CNTF); (b) a nucleic acid molecule whose sequence differs from .1 I r u-.* vs 1, L 1_'.|..1..'._..... -.4-J4-IA—u'n.-an or deletions and encodes a polypeptide capable of binding to CNTF; (c) a nucleic acid molecule whose sequence differs from that of Figure 2 but, due to the degeneracy of the genetic code,g encodes the amino acid sequence of Figure 2; (d) a nucleic acid molecule having a sequence complementary to that of (a), (b) or (c).

For example, the nucleic acid sequence depicted in Figure 2 can be altered by mutations such as substitutions, additions or deletions that provide for sequences encoding functionally equivalent molecules. According to the present invention, a molecule is functionally equivalent or active compared with a molecule having the sequence depicted in Figure 2 if it has the ability to bind CNTF, but it does not necessarily bind CNTF with an affinity comparable to that of natural CNTFR. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as depicted in Figure 2 may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of the CNTFR gene depicted in Figure 2 which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

In addition, the recombinant CNTFR—encoding nucleic acid sequences of the invention may be engineered so as to modify processing or expression of CNTFR. For example, and not by way of limitation, the CNTFR gene may be combined with a promoter sequence and/or a ribosome binding site, or a signal sequence may be inserted upstream of CNTFR encoding sequences to permit secretion of CNTFR and thereby facilitate harvesting or bioavailability.

Additionally, a given CNTFR can be mutated in yitgg or in yiyg to create and/or destroy translation,initiation, and/or termination sequences, or to create variations in coding regions, and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site—directed mutagenesis (Hutchinson, et al.,1978, J Biol.

Chem. 253:6551), use of TAB® linkers (Pharmacia) etc. .3 CILIARY NEUROTROPHIC FACTOR RECEPTOR-PEPTIDES The invention also provides CNTFR polypeptides encoded by nucleic acid molecules of the invention. The CNTFR protein having the amino acid sequence depicted in Figure 2 has a molecular weight of approximately 42 kd.

CNTFR polypeptides of the invention include those containing, as a primary amino acid sequence, an amino acid sequence as depicted in Figure 2, as well as altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.

Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine.

The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the invention are CNTFR polypeptides as described above but -25.. techniques. .4. EXPRESSION OF CILIARY NEUROTROPHIC FACTOR RECEPTOR A _ - In order to express recombinant CNTFR of the invention, the nucleotide sequence gf the inyention coding for a CNTFR1qrotein, pcan be inserted into an appropriate expression vector, i;g;, a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence. The necessary transcription and translation signals can also be supplied by the native CNTFR7gene and/or its flanking regions. A variety of host—vector systems may be utilized to express the protein—coding sequence. These include but are not limited to mammalian cell systems infected with virus (g;g;, vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus): microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expression elements of these vectors vary in their strengths and specificities. Depending on the host- Vlvector system utilized, any one of a number of suitable transcription and translation elements may be used.

In a preferred specific embodiment of the invention, the CNTFR gene may be comprised in the pCMX expression _ 27 _ vector, as deposited with the NRRL and assigned accession no. B~18790.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in yitrg recombinant DNA and synthetic techniques and in yigg recombinations (genetic ’ recombination). Expression of nucleic acid sequence encoding CNTFR protein or peptide fragment may be regulated by a second nucleic acid sequence so that CNTFR protein or _peptide is expressed in a host transformed with the ‘recombinant DNA molecule. For example, expression of CNTFR may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control CNTFR expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature ggg:304—310), the CMV promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus regions, which exhibit tissue specificity and have been _ 23 _ testicular, breast, lymphoid and mast cells (Leder et al., is active in liver (Pinkert et al., 1987, Genes and Devel. l:268—276), alpha—fetoprotein gene control region which is ’ active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. be identified by three general approaches: (a) DNA-DNA hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by DNA-DNA _.29._ hybridization using probes comprising sequences that are homologous to an inserted CNTFR gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (g;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.

For example,-if the CNTFR gene is inserted within the marker gene sequence of the vector, recombinants containing the CNTFR insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the CNTFR gene product, for example, by binding of the receptor to CNTF or to an antibody which directly recognizes the CNTFR.

In an additional embodiment, cells which do not normally express CNTFR may be transfected with recombinant-CNTFR encoding nucleic acid and then tested for the expression of functional CNTFR by exposing the transfectants to CNTF and then testing for an increase in CAMP levels.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus: insect viruses such as baculovirus; yeast vectors; bacteriophage _ 30 _ vectors (g;g;, lambda), and plasmid and cosmid DNA Vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be .elevated in the presence of certain inducers; thus, expression of the genetically engineered CNTFR protein may be controlled. Furthermore, different host cells have characteristic andgspecific mechanisms for the translational and post-translational processing and modification (g;g;, glycosylation, cleavage) of proteins.

Appropriate cell lines or host systems can be chosen to ‘ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast may be used to produce a glycosylated product. Expression in mammalian cells can be used to ensure "native" glycosylation of the heterologous CNTFR protein. Furthermore, different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.

Once a recombinant which expresses the CNTFR gene is identified, the gene product should be analyzed. This can be achieved by assays based on the physical or functional properties of the product; Once the CNTFR protein is identified, it may be isolated and purified by standard methods including chromatography (g;g;, ion exchange, affinity, and sizing column chromatography}, centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In particular, CNTFR protein may be isolated by binding to an affinity column comprising CNTF bound to a stationary support. . _ 31 _ Nucleic acid sequences complementary to DNA or RNA sequences encoding CNTFR or a functionally active portion thereof are also provided. In a particular aspect, antisense oligonucleotides can be synthesized, which are complementary to at least a portion of CNTFR mRNA. .5. IDENTIFICATION OF MOLECULES RELATED TO THE CILIARY NEUROTROPHIC FACTOR RECEPTOR Multiple receptor—factor systems have been defined in- which the same fagtor can bind to multiple receptors (see supra). As this may be the case for CNTF, the present invention allows for the identification of any additional CNTF receptors by the identical scheme used to obtain the 'CNTFR described here, except for the source of RNA used to prepare the CDNA expression library. A source may be chosen that would be likely to be expressing a distinct CNTF receptor; sources may be evaluated for the presence of CNTF—binding not attributable to the CNTFR (genetic probes and antibody reagents generated from the CNTFR sequence may be used to compare the protein responsible for CNTF binding in cell lines or tissue sources with the CNTF described here). In addition, because receptors are known which bind to more than one related factor (see sgpra), identification of the CNTFR should allow identification of any additional native ligands which bind this receptor.

In a further aspect of the invention, the CNTFR sequence of the invention as described above, may be used in the ' identification of CNTFR—re1ated molecules. T _ The CNTFR contains motifs which are shared with a variety of other receptors. The extracellular portion of the CNTFR contains both an "immunoglobulin" domain at its N—terminus, as well as a "cytokine receptor" domain which is separated from the "immunoglobulin" domain by a short hinge region. Although many receptors have homology to either the "immunoglobulin" or "cytokine receptor" domains, only one receptor — the IL-6 receptor — shares the same According to the present invention, by screening a DNA library (comprising genomic DNA or, preferably, CDNA) with oligonucleotides corresponding to CNTFR sequence derived either from protein sequence data or from the nucleic acid sequence set forth in Figure 2, clones may be identified which encode new members of the family described above. By decreasing the stringency of hybridisation, the -33.. duplicate filters carrying DNA from clones - generated using PCR, as outlined above. It may be observed that various clones may hybridise to some probes, but not others. New family members may also be identified by increasing the stringency of the hybridisation conditions, wherein new members not identical to probes derived from known members would hybridise less strongly at higher stringency.

‘ Alternatively, new family members may be identified by restriction mapping or sequencing analysis using standard techniques to reveal differences in restriction maps or sequences relative to known family members. techniques. _ 35 _ .6. UTILITY OF THE INVENTION .6.1. ASSAY SYSTEMS The present invention provides for assay systems in which CNTF activity or activities similar to CNTF activity resulting from exposure to a peptide or non—peptide compound may be detected by measuring a physiological response to CNTF in a cell or cell line responsive to CNTF which expresses the CNTFR molecules of-the invention. A , physiological response may comprise any of the biological effects of CNTF, including but not limited to, those described in Section 2.2, ggpra, as well as the transcriptional activation of certain nucleic acid -sequences (e.g. promoter/enhancer elements as well as structural genes), CNTF-related processing, translation, or phosphorylation, the induction of secondary processes in response to processes directly or indirectly induced by CNTF, and morphological changes, such as neurite sprouting, or the ability to support the survival of cells such as ciliary ganglion cells, motorneurons, Purkinje cells, or hippocampal neurons, to name but a few. have been produced that contain recombinant CNTFR—encoding — 36 - nucleic acid or selected by virtue of binding to CNTF, it may be desirable to ensure that the target cells respond characteristically to CNTF or compounds with CNTF—like activity. In the context of the present invention, the term CNTF—like activity is construed to mean biological activity which is similar but may or may not be identical to that of CNTF: such activities would include but are not limited_to those described in Section 2.2, supra or the activation of particular immediate early promoters such as the fgg or jug prbmoters.

The present invention provides for the development of novel assay systems which may be utilized in the screening ‘of compounds for CNTF— or CNTF-like activity. Target cells which bind to CNTF may be produced by transfection with CNTFR-encoding nucleic acid or may be identified and segregated by, for example, fluorescent-activated cell sorting, sedimentation of rosettes, or limiting dilution as described in Section 5.6.3, infra. of detecting low levels of CNTF activity. _ 37 i In particular, using recombinant DNA techniques, the present invention provides for CNTF target cells which are engineered to be highly sensitive to CNTF. For example, the CNTF-receptor gene, cloned according to the methods set forth in Section 5.1, may be inserted into cells which are naturally CNTF responsive such that the recombinant CNTFR gene is expressed at high levels and the resulting engineered target cells express a high number of CNTFRs on. their cell surface. i Alternatively, or additionally, the target cells may be engineered to comprise a recombinant gene which is expressed at high levels in response to CNTF/receptor _binding. Such a recombinant gene may preferably be associated with a readily detectable product. For example, and not by way of limitation, transcriptional control regions (i.e. promoter/enhancer regions) from an immediate early gene may be used to control the expression of a reporter gene in a construct which may be introduced into target cells. The immediate early gene/reporter gene construct, when expressed at high levels in target cells by virtue of a strong promoter/enhancer or high copy number, may be used to produce an amplified response to CNTFR binding. For example, and not by way of limitation, a CNTF~responsive promoter (such as the c—fos or c-jun promoter) may be used to control the expression of detectable reporter genes including fi—ga1actosidase, growth hormone, chloramphenicol acetyl transferase, neomycin phosphotransferase, luciferase, or B-glucuronidase.

‘Detection of the products of these reporter genes, well known to one skilled in the art, may serve as a sensitive indicator for CNTF or CNTF—like activity of pharmaceutical compounds.

The CNTFR—encoding or reporter gene constructs discussed above may be inserted into target cells using anY method known in the art, including but not limited to -38.. transfection, electroporation, calcium phosphate/DEAR dextran methods, and cell gun, as well as the production of transgenic animals bearing the above-mentioned constructs as transgenes, and from which CNTF target cells may be selected using the methods discussed supra. A Assay systems.of the present invention enable the efficient screening of pharmaceutical compounds for utility in the treatment of CNTF-associated diseases. For example, and not by way of limitation, it may be desirable to screen a pharmaceutical agent for CNTF activity and therapeutic efficacy in cerebellar degeneration. In a specific embodiment of the invention, Purkinje cells responsive to ' CNTF may be identified and isolated, and then cultured in is essential to have access to a reliable and sensitive j— 39 — screening system such as the methods the present invention provide. For another example, if a particular disease is found to be associated with a defective CNTF response in a particular tissue, a rational treatment for the disease would be supplying the patient with exogenous CNTF.

However, it may be desirable to develop molecules which have a longer half-life than endogenous CNTF, or which act as CNTF agonists, or which are targeted to a particular tissue. Accordingly, the methods of the invention can be used to produce efficient and sensitive screening systems which can be used_to identify molecules with the desired properties‘ identify CNTF antagonists. .6.2.

Similar assay systems could be used to EXPERIMENTAL MODEL SYSTEMS The present invention also provides for experimental «model svstems for studying the physiological,rg1e of CNTF.

In these model systems, CNTFR polypeptides of the invention may be either supplied to the system or produced within the system. Such model systems could be " used to study_the effects of CNTF excess or CNTF depletion.

The experimental model systems may be used to study the effects of increased or decreased response to CNTF in cell or tissue cultures '"~"5.6.2.1. MODELS FOR INCREASED CNTF ACTIVITY For example, and not by way of limitation, an experimental model system may be created which may be used to study the effects of excess CNTF activity. In such a system, the response to CNTF may be increased by engineering an increased number of CNTFRs on cells of the model system relative to cells which have not been so engineered. It may be preferable to provide an increased number of CNTFRS selectively on cells which normally express CNTFRS. A Cells may be engineered to produce increased numbers of CNTFR by infection with a virus which carries a CNTFR gene of the invention. Alternatively, the CNTFR gene maY be provided to the cells by transfection. response to endogenous CNTF may be increased. _ 40 _ By increasing the number of cellular CNTFRS, the If the model system contains little or no CNTF, CNTF may be added to the system. It may also be desirable to add additional CNTF to the model system in order to evaluate the effects of excess CNTF activity. Over expressing CNTF (or secreted CNTF) may be the preferable method for studying the effects of elevated levels of CNTF on cells already expressing CNTFR.

More preferably would be to express CNTFR in all cells (general expression) and determine which cells are then endowed with functional responsiveness to CNTF, thus allowing the potential identification of a second receptor component, if one exists. .6.2.2. MODELS FOR DECREASED CNTF ACTIVITY Alternative1y,«as an example, and not by way of limitation, an experimental model system may be created which may be used to study the effects of diminished CNTF activity. This system may permit identification of processes or neurons which require CNTF, and which may represent potential therapeutic targets. In such a system, the response to CNTF may be decreased by providing recombinant CNTFRS which are not associated with a cell y— 41- surface or which are engineered so as to be ineffective in transducing a response to CNTF. _ V For example’ CNTFR protein of the invention may be supplied to the system such that the supplied receptor may compete with endogenous CNTFR for CNTF binding, thereby diminishing the response to CNTF. The CNTFR may be a cell free receptor which is either added to the system or produced by the system. For example, a CNTFR protein which lacks the transmembrane domain may be produced by cells within the system: such as an anchorless CNTFR that may be secreted from the producing cell. Alternatively, CNTFR protein. may be added to an ‘extracellular space within the system.

In additional embodiments of the invention, a recombinant CNTFR gene may be used to inactivate or "knock out" the endogenous gene by homologous recombination, and thereby create a CNTFR deficient cell or tissue.

For example, and not by way of limitation, a recombinant CNTFR gene may be engineered to contain an insertional mutation, for example the neg gene, which inactivates CNTFR. Such a construct, under the control of a suitable promoter, may be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, transduction, injection, etc. Cells containing the construct may then be selected by G418 resistance. Cells which lack an intact CNTFR gene may then be identified, e.g. by Southern blotting or Northern blotting or assay of expression. _ 42 _ Alternatively, a recombinant CNTFR protein, peptide, or derivative which competes with endogenous receptor for CNTF may be erpressed on the surface of cells within the system, but may be engineered so as to fail to transduce a response to QNTF binding.

-The recombinant CNTFR proteins of the invention ~- . -may bind to CNTF with an ’ affinity that is similar to or different from the affinity‘ of endogenous’CNTFR to CNTF. ’To more effectively diminish the response to.CNTF,'the CNTFR Pr0t9iH: I may desirably_bind to CNTF with a greater affinity than that exhibited hyfthe native receptpr.'.W M I_f_._’_the CNTFR proteain-«is _P_3?0duced within ‘the’-model "system; nucleic acid encoding_the CNTFR:prQtein;maY be supplied to the system by infection, transduction, transfection, etc- I or as a transgene. As discussed supra, the CNTFR gene may be placed under the control of a suitable promoter, which may he, for example, a tissue-specific promoter or an inducible promoter or developmentally regulated promoter.

In a specific embodiment of the invention the endogenous CNTFR gene of a cell may be replaced by a mutant ‘CNTFR gene by homologous recombination.

In a further embodiment of the invention, CNTFR expression may be reduced by providing CNTFR expressing cells with an amount of CNTFR anti-sense RNA or DNA ;effective to reduce expression of CNTFR protein; _ 43d- . 6 . 3 . DIAGNOSTIC APPLICATIONS According to the present invention, CNTFR probes may be used to identify cells and tissues which are responsive to CNTF in normal or diseased states. The present invention provides for methods for identifying cells which are responsive to CNTF comprising detecting CNTFR expression in such cells. CNTFR expression may be evidenced by transcription of CNTFR mRNA or production of CNTFR protein. CRTFR expression may be detected using probes which identify CNTFR nucleic acid or protein.

One variety of probe which may be used to detect CNTFR expression is a nucleic acid probe, which may be used to detect CNTFR—encoding RNA by any method known in the art, including, but not limited to, ip sitg hybridization, Northern blot analysis. or PCR related techniques.

In order to measure the level of CNTFR, one may usetagged CNTF.According to the present invention, the term "tagged" CNTF should be construed to mean a CNTF molecule which is attached to a second detectable compound (the "tag"). The detectable compound may comprise radioisotope, a fluorescent moiety, or a ligand capable of binding to a receptor, or a substance which may be detected colorimetrically or which has catalytic activity.

The tag may comprise an antigenic determinant such that antibody is capable of binding to the tag. In.alternative embodiments the tag itself may be an ant1body,for example, the_monoclona1 antibody RP3—l7.

A ‘ It is desirable that the tag not interfere with the biological activity of CNTF and that the methods of detection of the tag would not substantially interfere with the binding of CNTF to its receptor. _ 44 _ The tag may be attached to CNTF using any method known in the art.’ Preferably, the tag is covalently linked to CNTF but in some cases it may be desirable that the tag be attached by noncovalent forces (for example, if the tag comprises an immunoglobulin molecule).

The tag may be of any molecular size suitable for preserving its-detector function without substantially altering the biological activity of the attached CNTF. If the tag is to provide an antigenic determinant, it may be desirable that it comprise at least about 5-15 amino acids.

For purposes of illustration, . .cfiTF may be tagged using a "patch" polymerase chain reaction in which recombinant neurotrophic factor (CNTF) is engineered to carry at its C-terminal end ten amino acids corresponding to a known antigenic determinant.

F0: example, and not by way of limitation, this antigenic determinant may correspond to a defined epitope of the human C-myc proto-oncogene protein.

For example, the "patch" PCR method may be used to attach the ten amino acid myc tag as follows (the present invention provides for any amino acid tag attached by analogous methods). A 5’ PCR primer corresponding to an CNTF sequence upstream of a unique restriction enzyme cleavage site in a bacterial expression construct may be utilized in PCR reaction with a "patch"‘ primer comprising nucleic acid sequence corresponding to 3' fterminal CNTF sequence and nucleic acid sequence encoding the peptide tag, using CDNA from CNTF—responsive cells as template.

The PCR reaction should also comprise a 3’ primer corresponding to the patch primer sequence and including nucleic acid sequence which incorporates_unique restriction endonuclease cleavage sites. Preferably. the _ 45 _ ' and 3’ primers may be used in excess of patch primerk such that PCR amplification between 5’ and patch primers may cease after a few PCR cycles whereas amplification between the 5' and 3' primers may initiate and continue to produce a high yield of full length CNTF/tag sequence. The "patch" technique oyercomes the need for long primers whose-. synthesis may be difficult and time consuming. The amplified CNTF/tag product may be gel purified, digested with restriction enzymes which cleave at the sites engineered into thefitermini of the product, and then subcloned into the corresponding restriction sites of an expression vector. For example, to produce CNTF-myc tag, the following primers may be used: 5’ primer = 5’ GAC TCG AGT CGA CAT CGG AGG CTG ATG GGA TGCC 3’ (SEQ ID N013)? patch primer = 3’ CTA AAG ACT CCT CCT AGA CAT CGC CGG CGT ATCG 5' (SEQ ID NO:4); primers may be used in a ratio of 100 ng 5' primer/100 ng 3’ primer/lng patch primer; for details see Section 6, igfra. The expression of CNTF/tag may be carried out as described for the expression of recombinant CNTF in W0 él/Q4315.

Alternatively, the tag may comprise; an immunoglobulin molecule, or a portion thereof, e.g. an Fc, F(ab)2, or F(ab)’ fragment of an antibody The tag should bind to CNTF, and may be a] polyclonal or monoclonal antibody. molecule.

,. Tagged CNTF maY be incubated with cells under conditions which would promote the binding or attachment of CNTF to said cells. In most cases, this may be achieved under standard culture For example, cells may be incubated for about 30 minutes in If the tag is an antibody conditions. the presence of tagged CNTF. _ 45 _ molecule, it may be preferable to allow CNTF to bind to cells first and subsequently wash cells to remove unbound ligand and then add anti—CNTF antibody tag. i Tagged CNTF on the surface of CNTF-responsive cells, hereafter called target cells, may be detected by rosetting assays in which indicator cells that are capable of binding to the tag are incubated with cells bearing CNTF/tag such that they adhere to CNTFfitag on the target cells and the bound indicator cells form rosette~like clusters around CNTF-tag bearing cells. These rosettes may be visualized by standard microscopic techniques on plated cells, or, 'alternatively, may allow separation of rosetted and non- rosetted cells by density centrifugation.

Target cells, such as neuronal cells, may be harvested and plated at a concentration of about 200 cells/well in a multiple well (e.g. 60 well) culture plate in medium such as RPM1 1640 with 10% fetal bovine serum and 2 mM glutamine. Plated cells may be incubated for about 16 to 24 hours at 37°C in a humidified 5% CO2 atmosphere incubator to allow cells to attach. Next, excess cell culture media may be removed and the cells may be incubated for about.3O minutes at room temperature with tagged CNTF. The cells may then be washed several times with PBS (with calcium and magnesium) supplemented with 1% bovine serum albumin (BSA) to remove unbound ligand and then incubated for about 30 minutes at room temperature with about 10 pg/ml of antibody which Vrecognizes the tag molecule. Cells may then be washed several times with PBS to remove unbound antibody. Then, the target cells (bearing CNTF/tag bound to anti-tag antibody} may be incubated at room temperature for 1 hour with about a 0.2% (v/v) suspension of rosetting indicator cells which bind to the anti~tag antibody (such as indicator cells bearing rabbit-anti-mouse immunoglobulin). — 47 — In alternative embodiments of the invention, tagged CNTF on the surface of target cells may be detected using immunofluorescent techniques in which a molecule which reacts with the tag, preferably an antibody, directly or indirectly produces fluorescent light. The fluorescence may either be observed under a microscope or used to segregate CNTF/tag-bearing cells by fluorescence activated cell sorting techniques.

. For example, target cells may be triturated and resuspended in assay buffer containing CNTF/tag (in excess concentration) and sodium azide (0.05%) for about 30 minutes at 4'C. Cells may then be washed three times in assay buffer by centrifugation at 800 rpm for 5 minutes. Cells may then be incubated with anti-tag antibody at a concentration of - about loipg/ml for about 30 minutes at 4°C, washed as above, and then incubated for about 30 minutes at 4'C with biotinylated anti-immunoglobulin and streptavidin—Texas Red conjugate. The cells may then be washed, resuspended in mounting solution, coverslipped, and then examined by fluorescent microscopy. _ 43 _ Methods also exist for detecting other forms of tags, such as chromogenic tags, catalytic tags, etc. The detection methods for any particular tag will depend on the conditions necessary for producing a signal from the tag, but should be readily discernible by one skilled in the artr Yet another variety of probe which may be used is a monoclonal anti—CNTFR antibody.

According to the invention, CNTFR protein may be used as an immunogen to generate anti-CNTFR antibodies.’ By providing for the production of relatively abundant amounts of CNTFR protein using recombinant techniques for protein synthesis (based upon the CNTFR nucleic acid sequences of the invention), the problem of limited quantities of CNTFR has been obviated.

To further improve the likelihood of producing an anti-CNTFR immune response, the amino acid sequence of CNTFR may be analyzed in order to identify portions of the molecule which may be associated with increased immunogenicity. For example, the amino acid sequence may be subjected to computer analysis to identify surface epitopes which present computer-generated plots of hydrophilicity, surface probability, flexibility, antigenic index, amphiphilic helix, amphiphilic sheet, and secondary structure of CNTFR. Alternatively, the deduced amino acid sequences of CNTFR from different species could be compared, and relatively non-homologous regions identified: , these non—homo1ogous regions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward CNTFR, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein _ 49 _ The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e;g;, Teng et al., 1983, Proc. Natl. Acad. Sci.

.U.S.A. 8027308-7312: Kozbor et al., 1983, Immunology Today 4:72-79: Olsson et al., 1982, Meth. Enzymol. 92:3—16).

Chimeric antibody molecules may be prepared containing a mouse antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 8l:685l, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art mav_be used for the production of antibodies of the invention. iFor the production of antibody, various host animals can be immunized by injection with CNTFR protein of the invention ' including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund’s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as _ lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette—Guerin) and, Corynebacterium parvum.

A molecular clone of an antibody to a CNTFR epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular '—5o— Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) may be used to construct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.

Antibody molecules may be purified by known techniques, e. ., immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof, etci The present invention provides for antibody molecules as well-as fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be .generated by known techniques. For example, such fragments include but are not limited to: the F(ab’)2 can be produced by pepsin digestion of the antibody fragment which molecule; the Fab’ fragments which can be generated by reducing the disulfide bridges of the F(ab’) fragment, and the Fab fragments which can be generated by ireating the antibody molecule with papain and a reducing agent.

[The abovementioned probes may be used experimentally to identify cells or tissues which hitherto had not been shown to express CNTFR. Furthermore, these methods may be used to identify the expression of CNTFR by aberrant tissues, such as malignancies. In additional embodiments, these methods may.be used diagnostically to compare the expression of CNTFR in cells, fluids, or tissue from a patient suffering from a disorder with comparable cells, fluid, or tissue from a healthy person. Fluid is construed to refer to any body fluid, but particularly blood or cerebrospinal fluid. ~A"difference in the levels of l expression of CNTFR in the patient compared to a healthy person may indicate that the patient’s disorder may be primarily or secondarily related to CNTF metabolism. An increase in levels of CNTFR, for example, could either indicate that the patient’s disorder is associated with an _ 51 _ ; increased sensitivity to normal levels of CNTF or, alternatively, may suggest that the patient’s CNTF levels are low such that the number of receptors is increased by way of compensation. These etiologies may be distinguished from one another by administering CNTF to the patient. If his condition worsens, he may suffer from CNTF hypersensitivity: if it improves, he may be suffering from a CNTF deficiency. CNTF or CNTF antagonist-based therapeutic regimens may be chosen accordingly.

Differences in expression can be detected at the protein and/or RNA level; i.e. by measuring amounts of CNTFR protein or CNTFR RNA in a patient relative to those amounts ‘in healthy persons.

The abovementioned probes may also be used to select CNTF—responsive cells for use in assay systems, as described above or according to standard methods of cell selection or cell sorting. .6.4. THERAPEUTIC APPLICATIONS The present invention also provides CNTFR protein of the invention for use in methods of treatment of the human or animal body by therapy, particularly for the treatment of neurological disorders.

Therapeutic methods comprising administering.

CNTFR. CNTFR aqonisfsz and anti-CNTFR antibodies may all be uéed in this way according to the present invention. .

The present invention also provides for pharmaceutical compositions comprising CNTFR protein, of the invention in a suitable pharmacoloqic carrier.

The CNTFR Pr°tei". of the invention 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, ,systemicallyL -52.. intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or by local injection or surgical imp1ant_ Sustained release formulations are also provided for.

As our understanding of neurodegenerative disease/neurotrauma becomes clearer, it may become apparent that it would be beneficial to decrease the trophic effect of endogenous CNTF. Therefore, in areas_of nervous system trauma, it may be desirable to provide aspa CNTF antagonist cell-free CNTFR which may I compete with endogenous cellular receptor for CNTF binding.

Under such circumstances; it may be desirable to provide CNTFR locally at the injury site rather than Use of a CNTFR providing implant may be desirable.

Alternatively, certain conditions may benefit from an increase in CNTF responsiveness. It may therefore be beneficial to increase the number or binding affinity of CNTFRS in patients suffering from such conditions. This could be achieved through gene therapy. Selective expression of recombinant CNTFR in appropriate cells could be achieved using CNTFR genes controlled by tissue specific or inducible promoters or by producing localized infection with replication defective viruses carrying a recombinant CNTFR gene. sensitivity to CNTF include particularly but are not Conditions which may benefit from increased limited to motorneuron disorders including amyotrophic lateral sclerosis, Werdnig—Hoffmann disease, chronic proximal spinal muscular atrophy, and post-polio syndrome.

Such treatment may also be used for treatment of neurological disorders associated with diabetes, Parkinson's disease, Alzheimer's disease, and Huntington's chorea. ~ 53 — expressed. . .

Such disorders include but are not limited to those in which atrophic or dystrophic change of -muscle is the fundamental pathological finding. For example, such muscle atrophy can result from denervation (loss of Contact by the muscle with its nerve) due to nerve trauma; degenerative, metabolic, or inflammatory (e.g., Guillian-Barre syndrome) peripheral neuropathy; or damage to nerves caused by environmental toxins or drugs. In another embodiment, the muscle atrophy results from denervation due to a motor neuronopathy. Such motor 4 neuronopathies include, but are not limited to: adult motor neuron disease, including Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease): infantile and juvenile spinal muscular atrophies, and autoimmune motor neuronopathy with multifocal conduction block. In another embodiment, the muscle atrophy results from Chronic disuse.

Such disuse atrophy may stem from conditions including, but not limited to: paralysis due to stroke, spinal cord injury, brain trauma or other Central Nervous System injury; skeletal immobilization due to trauma (such as fracture, sprain or dislocation) or prolonged bed rest- 1" yet another embodiment, the muscle atrophy results fr0m -54 _ metabolic stress or nutritional insufficiency, including, but not limited to, the cachexia of cancer and other chronic illnesses, fasting or rhabdomyolysis, endocrine disorders such as, but not limited to, disorders of the thyroid gland and diabetes. The muscle atrophy can also be due to a muscular dystrophy syndrome, including but not limited to the Duchenne, Becker, myotonic, Fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal, scapulohumeral, limb girdle, and congenital types, and the dystrophy known as Hereditary Distal Myopathy. In a further embodiment, the muscle atrophy is due to a congenital myopathy, including, but not limited to Benign Congenital Hypotonia, Central Core disease, Nemaline Myopathy, and Myotubular (centronuclear) myopathy. In addition, CNTFR-encoding nucleic acids may be of use in the treatment of acquired (toxic or inflammatory) myopathies.

Myopathies which occur as a consequence of an inflammatory disease of muscle, include, but are not limited to polymyositis and dermatomyositis. Toxic myopathies may be due to agents including, but not limited to amiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol, hydroxychloroquine,.organophosphates, perihexiline, and vincristine. : In a further embodiment, the invention provides for the treatment of patients that suffer from an excess hypersensitivity to CNTF, excess CNTF,etc. anti—sense RNA or antijsense 01igodeoxyribonucleotides corresponding to the CNTFR gene coding region. These decrease expression of CNTFR. _ 55 _ . EXAMPLE: EXPRESSION CLONING OF THE CILIARY NEUROTROPHIC FACTOR RECEPTOR .1. MATERIALS AND METHODS CONSTRUCTION OF A CNTF-RECEPTOR EXPRESSION LIBRARY SH-SY5Y cells (originally obtained from Dr. June .1.1.

Biedler) were used as a source of mRNA for construction of a CDNA librarv using the pCMX expression vector (described in. i .1.2. "PANNING" METHOD The "panning" method developed by Seed and Aruffo -12 were coated with anti-myc mouse monoclonal antibody, ‘diluted to 10 micrograms per ml in 50 mM Tris HCl pH 9.5.‘ ml of antibody was used to coat each 6 cm dish or 10 ml was used per 10 cm dish: plates were exposed to antibody for about 1.5 hrs, then antibody was removed to the next dish, allowed to stand for 1.5 hrs, and then removed again to a third dish. Plates were washed three times with 0- I5 _ 55 _ M Nacl (a wash bottle is convenient for this), and incubated with 3 ml 1 mg/ml BSA in PBS overnight. In particular "panning" was performed as follows: cells were cultured in 100 mm dishes. Medium was aspirated from each dish, and 2 ml PBS/0.5 mH EDTA/0.02% azide was added and the mixture was incubated at 37' for 30 min to detach cells from the dish. The cells were triturated vigorously with a short pasteur pipet, collected from each dish in a I centrifuge tube, and spun 4 min at a setting of 2.5 (200 r g). Cells were resuspended in 0.5-1.0 ml PBS/EDTA/azide/5%, FBS and incubated with CNTF/myc for 30 min on ice. An equal volume of PBS/EDTA/azide was added, layered carefully on 3 ml PBS/EDTA/azide/2% Ficoll, spun 4 min at a setting of 2.5, and the supernatant was aspirated in one smooth movement. The cells were then incubated with 9E10 antibody for 30 minutes on ice, and the spin through PBS/EDTA/azide/2% Ficoll was repeated. The cells were taken up in 0.5 ml PBS/EDTA/azide and aliquots were added to anti—myc mouse monoclonal antibody—coated dishes containing 3 ml PBS/EDTA/azide/5% FBS. Cells were added from at most two 60 mm dishes to one 60 mm antibody coated plate, and allowed to sit at room temperature 1—3 hours.

Excess cells not adhering to dish were removed by gentle washing with PBS/5% serum or with medium (2 or 3 washes of ml were usually sufficient). .1.3. IDENTIFICATION OF CLONES CONTAINING THE CILIARY NEUROTROPHIC FACTOR RECEPTOR GENE Plasmid DNA from the expression library was transfected into COSM5 cells (approximately 250~500 ng per 100 mm dish: 2 dishes were transfected), using DEAE/chloroquine according to standard procedures. Two days after transfection, cells were detached from their — 57 — dishes and subjected to the Aruffo/Seed panning procedure modified as described supra. .1.4. DIRECT l25I-hCNTF BINDING ASSAY COS cells were transfected with plasmid DNA from the ‘library, the enriched library, or individual clones. After hours, the media was removed and replaced with 0.25 ml of binding buffer (RPMl 1640 with 10% FBS and 0.1% NaN3) I—hCNTF alone or with unlabelled hCNTF. 125 containing I—hCNTF were for 60 minutes at room 125I_ Incubations with temperature. After incubations were complete, the hCNTF solution was removed and the cells were washed three _ 53 _ times with 1.0 ml of binding buffer and then lysed with 0.25 ml of 0.1 N NaOH. x 75 mm polystyrene tube and placed in a gamma counter.

This lysate was transferred to a 12 Non—specific binding was determined by the addition of at least 100 fold excess unlabelled hCNTF. After the last wash the plates were autoradiographed. .1.5. FLUORESENCE ACTIVATED CELL-SORTING ANALYSIS Transfected 008 cells were incubated sequentially with CNTF/myc, 9El0 antibody, and FITC-labelled goat anti—mouse antibody. subjected to FACS analysis.

Then they were detached from dishes and The results of transfections ‘with a negative and positive plasmid are depicted in Figure D: COS cells transfected with a CNTF-receptor expressing plasmid contain a large subpopulation displaying greatly increased fluorescence by this assay. .1.6. IODINATION or hCNTF pg hCNTF (560 pg/ml in 10 mM NaPO4pH7.4) was iodinated with 1 mCi 125 INa using lactoperoxidase 6 ng/pl (Sigma) for 15 minutes at 20°C. After 15 minutes the reaction was 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% NaN3. Aliquots were removed for determination of TCA precipitatable counts.

The remainder was loaded onto a BioRad PD 4 10 biogel column equilibrated with 0.05 M NaP04, 0.1 M NaCl, 0.5 mg/ml protamine sulfate and 1 mg/ml BSA. Fractions were A Collected and TCA precipitatable counts determined. .1.7. SEQUENCING OF CNTFR Sequencing was performed using a kit (U.S.

Biochemical) for dideoxy double stranded DNA using Sequenase', according to the manufacturer's instructions. - 59 — .1.8. INDIRECT 1251 GOAT ANTI—MOUSE ANTIBODY BINDING ASSAY COS cells were transfected with plasmid DNA from the library, the enriched library, or individual clones. After 48 hours, cells were incubated sequentially for 30 minutes on ice with PBS (with Ca, Mg)/5% FBS containing: ) 1 pg/ml CNTF-myc ) 10 pg/ml 9El07 ) 1251 goat anti-mouse antibody (GaM) (0.5-1 pci/m1)} Cells were washed 3 x 5 minutes in PBS/5% FBS after each step. After the last wash, the plates were .autoradiographed.

For the individual clones, a quantitative estimate of total radioactivity bound was made with a hand—held gamma counter. .2. RESULTS AND DISCUSSION 6.2.1. RESTRICTION ANALYSIS On restriction analysis, the 14 positive clones fell into four classes: a) I2=I7 (zkb) b) Il=I5=I6 (2kb) C) I4=I8=I9=I1l=Il4=I15 (4kb) d) IlO=I12=I13 (1.6kb) (I3 was negative)) Members of each class produced an identical pattern of bands on digestion with the enzyme ggtl. Further I restriction analysis revealed that the four classes of clones overlapped, and preliminary sequence data confirmed that they shared overlapping sequences at their 5’ ends.

Curiously, class (b) proved to have its insert in the wrong orientation in the vector with respect to the eukaryotic promoter element. As can be seen from Table 1, these clones were low expressors relative to the other clones. _ 50 _ Transcription in these clones may arise from a weak Cryptic promoter in the region downstream of the vector’s polylinker. .2.2. IN VITRO TRANSCRIPTION AND TRANSLATION To characterize the proteins coded for by the four classes of clones, they were all transcribed from the T7 promoter in the 5’ region of the vector polylinker. After -in vitro translation, the products were electrophoresed on a polyacrylamide gel. Class (a) produced no protein, since it is in the wrong orientation with respect to the T7 promoter. The other three classes all produced proteins of -identical sizes (approximately 42 kd), verifying that they encoded the same protein.

TABLE I.

Quantitation of I-GaM binding in CNTFR clones.

Clone CPM bound I1 2000 I2 8500 I3 600 I4 9000 I5 2000 I6 1600 I7 ‘ 6000 I8 ' 7500 I9 7000 I10 4500 Ill 7000 I12 5000' I13 8000 I14 10000 I15 8000 Negative Control 500 Background 2 I5 _ 51 _ .2.3. BINDING ANALYSIS WITH CNTF The results of the indirect CNTF—myc binding assay goat anti-mouse using 9E1O anti—myc antibody and antibody are shown in Figure 1B and 1C as well as in Table I. In Figure 1B, the plate on the left results from itransfection of the unenriched library, while the plate on the right is from transfection of the enriched library plasmid DNA rescued after one round of panning (using approximately the same amount of DNA as for the unenriched library). Note the large number of dark spots seen only in the plate on the right, each representing a single COS cell expressing CNTF~myc binding site detected by radioautography.

For the individual positive clones discussed in Section 6.2.1, a quantitative estimate of total radioactivity was made with a hand-held gamma counter. The results of this assay for the individual clones I1-I15 are shown in Table I and demonstrate that 14 out of 15 clones express CNTF binding sites, as determined by indirect antibody binding assay. In addition, fragments of the plates from some of the individual clones were autoradiographed, as shown in Figure 1C.

A second demonstration of indirect binding utilized CNTF-myc followed by 9E1O antibody, FITC—label1ed goat anti-mouse antibody, and FACS analysis, as shown in Figure 1D. COS cells transfected with positive clones demonstrated a l00—fo1d increase in expression of CNTFR as compared with cells transfected with negative clones.

The indirect binding data obtained using CNTF—myc was verified using direct IZSI-CNTF binding, as shown in Table II. The receptor expressed on transfected COS cells specifically binds to iodinated CNTF as well as to the CNTF-myc ligand, as did the SH-SY5Y cells from which the CNTFR was cloned. Each transfected COS cell expresses about 30-fold more receptor per cell than SH—SY5Y cells. - 52 _ TABLE II.

Binding Analysis With Iodinated CNTF COS I2 SH—SY5Y Egpc. Specific Specific I—CNTF cpm bound cpm/ce1l* cpm bound cpm/cell 2.16 nM ' 1412 2.17 x 10"‘? 1284 4.28 x 1o"3' *COS cells were assayed 48 hours after transfection by DEAE Dextran in which typically only 20-40% of the cells are transfected. Assuming 20% COS cells are transfected, the specific cpm bound indicate that each transfected COS cell expresses about 30-fold more receptors per cell than SH- SYSY cells. .2.4. SEQUENCE OF CNTFR AND HOMOLOGY TO OTHER GROWTH FACTOR RECEPTORS The CNTFR contains motifs which are shared with a variety of other receptors. The extracellular portion of the CNTFR contains both an "immunoglobulin" domain at its N—terminus; as well as-a "cytokine receptor"-domain which is separated from the "immunoglobulin" domain by a short hinge region. Although many receptors have homology to either the "immunoglobulin" or "cytokine receptor" domains (Figure 3), only one receptor — the IL-6 receptor - shares the same particular arrangement of these domains with the _ 53 _ ‘shares important features with the IL-6 receptor that are not found in other known receptors, thus defining a new family of receptors. The similarities between IL-6R and CNTFR suggest that CNTFR is likely to utilize the same signal transducer as the IL-6 receptor, or a related molecule. Finally, the identification of CNTFR-related receptors should aid in the identification of novel ligands that would bind to these receptors.

. EXAMPLE: TISSUE LOCALIZATION OF MESSAGE FOR CNTFR .1. MATERIALS AND METHODS 7.1.1. CNTFR PROBE PREPARATION Molecular cloning of the coding region for hCNTFR into the pCMX expression vector is described in U. S. patent application entitled "Mammalian Expression Vector" filed concurrently herewith, and the resulting expression vector is depicted in Figure 6. A PCR probe extending from base 889 to base 1230 of the CNTFR sequences was synthesized and used as a probe for Northern analysis. -64.. .1.2. RNA PREPARATION AND NORTHERN BLOTS Selected tissues were dissected from Sprague—Dawley rats and immediately frozen in liquid nitrogen. RNAS were isolated by homogenization of tissues in 3 M Licl, 6 M urea, as described in Bothwell et al. 1990 (Methods of Cloning and Analysis of Eukaryotic Genes, Boston, Ms, Jones and Bartlett). RNAs (10 mg) were fractionated by electrophoresis through quadruplicate 1% agarose— formaldehyde gels (Bothwell et al., 1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett) followed by capillary transfer to nylon membranes (MagnaGraph, Micron Separations Inc.) with 10xSSC ‘(pH 7). RNAS were UV-cross-linked to the membranes by exposure to ultraviolet light (stratalinker, Stratagen, Inc.) and hybridized at 68°C with radiolabeled probes in the presence of 0.5 M NaPO4 (pH 7), 1% bovine serum albumin (fraction V, Sigma, Inc.) 7% SDS, 1 mM EDTA (Mahmoudi et al., 1989, Biotechniques 1:331-333), 100 pg/ml sonicated, denatured salmon sperm DNA. Filters were washed at 68°C with 3xSSC, 0.1% SDS and subjected to autoradiography for 1 day to 2 weeks with one or two intensifying screens (Cronex, DuPont) and X~ray film (SAR-5, Kodak) at 70°C.

Ethidium bromide staining of the gels demonstrated that equivalent levels of total RNA were being assayed for the different samples (as in Maisonpierre et al., 1990, Science -1451. .2. RESULTS As shown in Figure 5, CNTFR mRNA was detectable in tissues of the central nervous system at low levels in sciatic nerve and adrenals, and in muscle. This would indicate that CNTF possesses not only neurotrophic activity, but myotrophic activity as well, and may explain the involvement of both the central nervous system and muscle in certain disorders, such as Duchennes muscular ~ dystrophy and congenital myotonic dystrophy, in which patients may suffer from mental retardation. Expression of CNTFR in muscle suggests CNTF may have a role in muscle physiology. may have important action in muscle such as functioning as Thus, in addition to action on neurons, CNTF a myotrophic agent, or otherwise effect muscle development and/or differentiation.

. EXADPLE: EVIDENCE THAT THE CNTF RECEPTOR IS LINKED TO THE CELL SURFACE VIA A GLYCOSYL— PHOSPHATIDYLINOSITOL (GPI) LINKAGE .1. MATERIALS AND METHODS SH-SY5Y cells were cultured in a 24-well plate (Falcon) in RPMI supplemented with 10% inactivated fetal bovine serum. For experiments in which phospholipase (and control) treatments were done prior to CNTF—binding, the media was aspirated, cells were rinsed twice in PBS(+Ca/Mg), and then incubated with PBS(+Ca/Mg) supplemented with or without phosphatidylinositol—specific phospholipase (PI—PLC) at final concentration of 500 mU/ml (purchased from Boehringer Mannheim, catalogue # 1143-069) for 45 minutes at 37°C. with binding buffer (PBS(+Ca/Mg) and 5% fetal bovine serum) and then incubated with 250 microliters binding buffer Cells were then washed three times containing iodinated CNTF (approximately 100 picomolar) with or without a thousand~fo1d excess of unlabelled CNTF for 30 minutes at room temperature. For experiments in which the iodinated CNTF was bound prior to PI—PLC treatment, cells were first incubated in binding buffer "containing iodinated CNTF with or without excess unlabelled CNTF at 37'C for 45 minutes. Cells were then washed two times with PBS(+Ca/Mg) and then incubated for 45 minutes with PBS(+Ca/Mg) supplemented with or without PI—PLC (final concentration 500 mU/ml). Cells were then rinsed three times with binding buffer. In all cases cells were _ 55 _ solubilized prior to counting in 0.1N NaOH, and then counted. .2. RESULTS AND DISCUSSION The sequence of the CNTF receptor revealed that the encoded protein ended within a hydrophobic region that followed the extra—cytoplasmic domains, without any apparent stop transfer sequence or intra-cytoplasmic domain. This structure seemed reminiscent of the C- :terminals found on membrane proteins which lack 'transmembrane domains and are attached to the cell surface via GPI-linkages (Ferguson and Williams, 1988). Thus, .experiments were performed to test whether the CNTF receptor was linked to the cell surface via a GPI-linkage.

As shown in Table III, treatment of SH—SY5Y cells with PI~ PLC completely eliminated the ability of SH—SY5Y cells to subsequently bind CNTF, consistent with the notion that the CNTF receptor is linked to the cell surface via a GP1- linkage. However, CNTF already bound to SH—SY5Y cells cannot be released by PI-PLC treatment (Table III).

Interestingly, a soluble form of the IL-6 receptor can tightly associate with a second membrane protein (GP130) required for IL-6 signal transduction. Thus, prevention of CNTF receptor release by prior binding to CNTF may be due to an association between the CNTF, its receptor, and its signal transducer (GP130 or a GP130 analog).

Alternatively, CNTF-binding may alter the structure of the CNTF receptor, making it less susceptible to PI-PLC v(several GPI-linked proteins have PI-PLC resistant forms).

The finding that the CNTF receptor is attached to the cell surface via a GPI—1inkage has important ramifications.

It represents the first known growth factor receptor to be linked to the membrane in this fashion, raising the possibility that additional receptors may be GPI-linked.

Because several proteins have both GPI-linked forms as well _ 57 _. as forms that contain conventional transmembrane domains, our findings raise the possibility that the CNTF receptor has an alternative C—terminus that could encode a transmembrane domain, and similarly that the IL-6 receptor has a GPI—linked mm. The GPI-linked forms of growth factor receptors may be able to utilize novel mechanisms of receptor regulation and release. For example, down- A regulation of surface receptors could rapidly occur by releasing the GPIjlinked receptors by activating extra- cytoplasmic phospholipase activities. These released receptors might also act on other cells, either alone or when bound to CNTF in much the same way that soluble IL—6 receptor has been shown to bind IL-6 and activate cells expressing GP130.

The possibility that release of CNTF receptors using PI-PLC could block CNTF action may have important implications. It could be used to verify that observed effects of CNTF are due to the cloned CNTF receptor.

Therapeutically, PI-PLC could be used to release CNTF receptors and possibly block CNTF action in cases where CNTF activity is thought to be detrimental.

If the CNTF—blockable PI—PLC release of the CNTF receptor is due to the formation of a tertiary complex between the CNTF, its receptor, and the potentia1.signal transducing protein, then this feature of the receptor could be used to define and molecularly clone the transducing molecule. _68_ TABLE III Analysis Of PI—PLC Treatment On CNTF Binding To SH-SYSY Cells CPM Bound Cold Excess‘ No Cold Excess Pre~Treat with PI—PLC No PI-PLC 1440 370 With PI-PLC 420 310 .Bind CNTF Before PI-PLC No PI-PLC 1250 310 With PI-PLC 1060 300 . THE EFFECTS OF CNTF ON DENERVATED RAT SKELETAL MUSCLE IN VIVO The goal of the experiments described herein was to examine the effects of purified recombinant CNTF on denervated skeletal muscle in yiyg and to determine whether CNTF could prevent some of the phenotypic changes associated with denervation atrophy such as muscle weight and myofibrillar protein loss. We found that the CNTF receptor is expressed in skeletal muscle on both myotubes and myoblasts, and that CNTF prevents the loss of both muscle weight and myofihril protein content associated with , denervation atrophy. .1. THE CNTF RECEPTOR IS EXPRESSED IN SKELETAL MUSCLE ON BOTH MYOTUBES AND MYOBLASTS Northern blot analysis was performed on RNA samples derived from a variety of rat tissues in order to identify the primary cellular targets of CNTF as shown in Figure 5. _ 59 _ A probe derived from the human CNTF receptor coding region identified a 2 kb transcript whose expression was generally restricted to the central nervous system, except for surprisingly high levels found in skeletal muscle and low levels in adrenal gland and sciatic nerve.

A more detailed analysis of the CNTF receptor expression specifically in skeletal muscle was carried out as described sgpra by Northern blotting using mRNA prepared from specific rat muscle types, purified human muscle myotubes, and several skeletal muscle cell lines of both mouse and rat origin (Figure 7). Using a human probe for the CNTF receptor, two mRNA species (2.0 and 1.7 kb) were detected in several muscle RNA samples. Figure 7 demonstrates that the CNTF receptor is expressed in both myotube and myoblast muscle cell lines of either mouse (lanes 1 and 2) or rat (lanes 3 and 4) origin, as well as in both red slow-twitch soleus muscle and white fast-twitch extensor digitorum longus (EDL) muscle of the rat (lanes 5 It appeared that the level of CNTF receptor mRNA was increased in both soleus (lane 12) and and 6, respectively).

EDL muscle (lane 14) that were first denervated for 72 hours relative to their sham-operated contralateral controls (lanes 11 and 13 respectively). Interestingly, the highest level of expression was observed in RNA samples from myotubes derived from human fetal skeletal muscle.

These myotubes were cultured and then purified away from fibroblasts and other non—muscle cells by fluorescence- activated cell sorting prior to RNA isolation (lane 8). We noted that two distinct CNTF receptor mRNA species were identified on the muscle cell Northern blot and that the 1.7 kb CNTF receptor message was preferentially expressed in the myoblast cell line C2C12 mb (lane 1) and may represent an alternatively spliced form of the receptor. _containing 1 mg/ml of BSA (PBS/BSA). _ 70 - .2. CNTF PREVENTS THE 1058 OF BOTH MUSCLE WEIGHT AND MYOFIBRILLAR PROTEIN CONTENT ASSOCIATED WITH DENERVATION ATROPHY .2.1. DENERVATION SURGERY The various animal groups used for these studies are described in Table IV, infra. Generally, three animals comprised a single group. For all experimental groups, an initial incision of approximately 20 cm was made through the skin of the right hindlimb at midthigh level.

Following this surgical procedure, the 20 cm cut was also performed on the left hindlimb at midthigh in order to carry out the sham—operation.

In animal groups 2-6, the soleus muscle of the right hindlimb was denervated by surgically removing a 2~5 mm segment of the right sciatic nerve at midthigh level to leave a distal nerve stump of 32 to 35 mm (labeled as A in Figure 8). The left soleus muscle served as the control in that a sham—operation was performed on this muscle by gently pulling on the sciatic nerve 32 to 35 mm from its point of innervation of the soleus muscle. All surgeries were carried out while the animals were under light chloro-pentobarbitol anesthesia (0.3 g/kg). Animal group 1 (controls) did not receive any denervation and were not injected. All animals weighed between 100 and 150 grams. .2.2. TREATMENTS Animals in groups 1 and 2 were not treated. Animals in group 3 were injected daily for a total of 4 days intramuscular (IM) with phosphate-buffered saline (PBS) Multiple injections were made into the muscles of the midthigh on both sides of the animals. Animals in group 4 were injected daily for 4 days IM with recombinant rat CNTF (1 mg/kg) containing 1 mg/ml of BSA (CNTF/BSA). Multiple injections were made on both sides of the animal as described above. Animals in _ 71 _ group S were also injected daily with rCNTF/BSA but subcutaneously (SC) rather than IM. Animals in group 6 were injected daily (SC) with PBS/BSA.

TABLE IV Surgical Denervation Group # Animals Protocol Time Treatment 1 3 »None 96 hours None 2 R-DenyL-Sham 96 hours None 3 3 R—Den/L-Sham 96 hours PBS/BSA (1 mg/ml) h 3 R-Den/L-Sham 96 hours CNTF/BSA (1 mg/kg) (IM) 3 R-Den/L-Sham 96 hours CNTF/BSA (1 mg/kg) (SC) 6 3 R-Den/L—Sham 96 hours PBS/BSA (SC) -Den=right hindlimb denervated; L-Shamuleft hindlimb sham-operated .2.3. MUSCLE WEIGHT AND PROTEIN ANALYSIS hours after the denervation surgery was performed, the animals were sacrificed by decapitation, and the soleus muscles were carefully excised from tendon to tendon. The soleus muscles were placed on a weigh boat on ice, tendons were removed with a scalpel, and the muscles were then weighed immediately so as to prevent any drying. To prepare myofibrillar protein homogenates, the excised soleus muscles were pooled, minced while on ice in a cold room, and then homogenized in PBS containing 0.32 M sucrose and 3 mm MgCl2 (2.5% w/V). The homogenate was centrifuged ' at approximately 800 x g and the supernatants were assayed for total myofibril protein per muscle by using the Bio—Rad Dye Binding procedure according to the manufacturers recommendations.

Figure 9 demonstrates that denervated soleus muscle decreased significantly (p<0.0l) in wet weight _ 72 _ approximately 25% at 96 hours. Daily injection of PBS/BSA had no effect on this denervation-dependent muscle weight loss. However, denervated soleus muscles from rats injected daily with CNTF (1 mg/kg)/BSA weighed approximately 5% less than their contralateral sham- operated controls. The wet weights of the CNTF treated denervated and sham—operated soleus muscles were not significantly different from unoperated controls. CNTF, when injected SC daily for 4 days (group 5), also appeared to significantly prevent the denervation-induced loss of myofibrillar protein, and the loss of protein paralleled the decrease in muscle wet weight (Table V). ~ 73 — TABLE V Effect Of CNTF On Denervated Soleus Muscle Protein Content Total Hyofibril Protein (mg per Muscle Sample Muscle) % of Sham Group 1 — No denervation 7.5 Group 2 — den. - no injection 5.8 80 — sham — no injection 7.2 Group 3 - dem+PBS (IM) 5.2 75 - sham+PBS(IM) 6.9 Group 4 - den+CNTF (IN) 6.5 83 - sham+CNTF (IM) 7.8 Group 5 - den+CNTF (SC) 6 6.6 93 - sham+CNTF (SC) 7.1 Group 6 - den+PBS (SC) 5.3 67 — sham+PBS (SC) 7.9 (IM) = intramuscular injection; (SC) = subcutaneous injection; data presented represent the total protein content of 3 pooled soleus muscles when injected daily IN, a less pronounced effect of CNTF on total myofibril protein was observed.

We found that the CNTF receptor is expressed in skeletal muscle on both myotubes and myoblasts, and that CNTF prevents the loss of both muscle weight and myofibril protein content associated with denervation atrophy.

. DEPOSIT OF MICROORGANISM The following deposit has_been made on March 26, 1991 with The Agricultural Research Culture Collection (NRRL), 1815 North -University Street, Peoria, Illinois, 61604: E. ggli carrying plasmid pCMX-hCNTFR (I2), an expression plasmid comprising hCNTFR encoding sequences, assigned accession number NRRL B~18789.

Claims (1)

1. An isolated nucleic acid molecule selected from: (a) a nucleic acid molecule having the sequence of