EP0563304A1 - Facteur de differenciation cholinergique neuronal - Google Patents

Facteur de differenciation cholinergique neuronal

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Publication number
EP0563304A1
EP0563304A1 EP92903756A EP92903756A EP0563304A1 EP 0563304 A1 EP0563304 A1 EP 0563304A1 EP 92903756 A EP92903756 A EP 92903756A EP 92903756 A EP92903756 A EP 92903756A EP 0563304 A1 EP0563304 A1 EP 0563304A1
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European Patent Office
Prior art keywords
protein
neurons
expression
vitro
ability
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EP92903756A
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German (de)
English (en)
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EP0563304A4 (en
Inventor
Mahendra S. Rao
Story C. Landis
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Case Western Reserve University
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Case Western Reserve University
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Publication of EP0563304A1 publication Critical patent/EP0563304A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators

Definitions

  • the present invention relates to a target- derived neuronal cholinergic differentiation factor (NCDF) , and the therapeutic and diagnostic uses thereof.
  • NCDF target- derived neuronal cholinergic differentiation factor
  • the invention provides NCDF, and derivatives, analogs, and fragments thereof, pharmaceutical j O compositions of the foregoing, as well as anti-NCDF antibodies.
  • VIP vasoactive intestinal peptide
  • CGRP peptide immunoreactivity
  • NPY neuropeptide Y
  • the superior cervical ganglion which contains noradrenergic sympathetic neurons
  • the neurons innervate the 5 glands, reduce their expression of catecholamine histofluorescence and NPY, and develop immunoreactivity for choline acetyltransferase and VIP (Stevens and Landis, 1990, Dev. Biol. 137, 109-124).
  • cross-innervation experiments provide direct evidence 0 for a target role.
  • footpad skin is transplanted in place of hairy skin in the thoracic region of early postnatal rats, the transplant is innervated by sympathetic neurons whose normal targets are piloerectors and blood vessels.
  • the sympathetic fibers * - > that innervate hairy skin are noradrenergic and do not normally contain choline acetyltransferase activity, acetylcholinesterase staining, or VIP immunoreactivity (Schotzinger and Landis, 1990, Cell Tissue Res. 260, 575-587) .
  • the fibers show a marked reduction in catecholamine fluorescence and express properties characteristic of the innervation of their novel target: they exhibit choline acetyltransferase activity, acetylcholinesterase
  • the cholinergic differentiation factor (CDF) purified 5 from heart cell conditioned medium (Patterson and Chun, 1977, Dev. Biol. 56, 263-280; Fukada, 1985, Proc. Natl. Acad. Sci. USA 82, 8795-8799) has been shown to be identical to leukemia inhibitory factor (LIF) (Yamamori et al., 1989, Science 246, 1412-1416).
  • LIF leukemia inhibitory factor
  • Ciliary 10 neurotrophic factor (CNTF) , originally identified as a survival factor for ciliary neurons (Adler et al., 1979, Science 204, 1434-1436; Barbin et al., 1984, J. Neurochem. 43, 1468-1478; Manthorpe et al., 1986, Brain Res. 367, 282-286), and recently cloned (Lin et al., 151989, Science 246, 1023-1025; Stockli et al., 1989, Nature 342, 920-923), induces cholinergic and reduces catecholaminergic function in cultured sympathetic neurons (Saadat et al., 1989, J. Cell Biol. 108, 1807- 1816) .
  • CNTF Ciliary 10 neurotrophic factor
  • a membrane-associated neurotransmitter- stimulating factor (MANS) has been solubiiized and 0 partially purified from rat spinal cord.
  • the latter activity is associated with a 29 kd protein (Wong and Kessler, 1987, Proc. Natl. Acad. Sci. USA 84, 8726- 8729; Adler et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1080-1083) . It is as yet unclear, however, whether 5 the cholinergic-inducing ability of these factors represents their primary, or even a relevant, function in normal development. Several of these factors have been shown in cell culture systems to have additional functions.
  • CDF/LIF inhibits proliferation 5 and induces macrophage differentiation in the Ml myeloid cell line (Hilton et al., 1988, Anal. Biochem. 173, 359-367) and maintains the developmental potential of embyronic stem cells (Smith et al., 1988, Nature 336, 688-690; Williams et al., 1988, Nature 336, 684- 0687) ; CNTF has trophic activity for ciliary neurons (Barbin et al., 1984, J. Neurochem. 43, 1468-1478; Manthorpe et al., 1986, Brain Res. 367, 282-286) and induces astrocytic properties in 0-2A progenitor cells (Hughes et al., 1988, Nature 335, 50-73).
  • the present invention is directed to a target-derived neuronal cholinergic differentiation factor (NCDF) , and the therapeutic and diagnostic uses thereof.
  • NCDF target-derived neuronal cholinergic differentiation factor
  • the NCDF of the invention is a protein present in extracts of mammalian sweat glands, which exhibits heat and trypsin lability, lack of substantial binding to a heparin-agarose affinity column, an isoelectric point (pi) in the range of approximately 4.8 to 5.2, a non-membrane cellular localization, and an approximate molecular weight in the range of 16 to 32 kilodaltons.
  • NCDF protein its derivatives, analogs, and fragments are able to reduce the expression of tyrosine hydroxylase and of total catecholamines, and increase the expression of choline acetyltransferase and vasoactive intestinal peptide (VIP) , by sympathetic neurons in cell culture (in vitro) .
  • VIP vasoactive intestinal peptide
  • NCDF protein its derivatives, analogs, and fragments, ' can be used to induce cholinergic activity in neurons.
  • Such proteins, derivatives, analogs and fragments can be administered therapeutically to patients with nervous system damage or diseases where it is desirable to support survival and/or cholinergic differentiation of a number of neuronal types.
  • FIG. 1 Soluble protein extracted from sweat glands, hairy skin, parotid gland, liver, or sciatic nerve of adult rats was added to cultures of dissociated sympathetic neurons. Seven days after the addition of extracts, neurons were homogenized and aliquots were assayed for levels of choline acetyltransferase (ChAT) activity by the method of Fonnum (1969, Bi ⁇ chem. J. 115, 465-472). Samples were run in triplicate. In (a) , 250 ⁇ g of protein extracted from the indicated tissues was added.
  • ChAT choline acetyltransferase
  • the data are expressed as the fold induction of ChAT activity compared with that present in control cultures grown without added extract, in (b) 250 ⁇ g of protein extracted from sciatic nerve or sweat gland was added. The data are expressed as the fold induction of specific activity per mg of protein added.
  • Figure 2. (a) Increasing concentrations of sweat gland extracts cause increased induction of choline acetyltransferase activity. Increasing concentrations of soluble protein extracted from sweat glands of adult rats were added to sympathetic neuron cultures. Seven days after the addition of extract, neurons were homogenized and aliquots were assayed for choline acetyltransferase activity by the method of Fonnum (1969, Bioche . J., 115, 465-472). Samples were run in triplicate. Data are expressed as pmol of activity per min per well ⁇ SD.
  • Sweat gland extracts reduce tyrosine hydroxylase.
  • Sympathetic neurons were grown in medium without sweat gland extract (a) or with 100 ⁇ g/ml sweat gland extract (b) .
  • Samples were pooled from several wells, homogenized in sample buffer, electrophoresed, and blotted onto nitrocellulose. The 5 blots were probed with a monoclonal antibody to tyrosine hydroxylase (inset) .
  • the laser densitometer scan (absorbance of 600 nm) of the staining intensity of the bands from control and treated cultures is shown. 10 Figure 4.
  • Sweat gland extracts modulate the expression of VIP.
  • Serial dilutions of the soluble protein extracted from adult rat sweat glands were added to sympathetic neuron cultures. Cultures were
  • Sweat gland extracts reduce the levels of NPY and elevate the levels of VIP.
  • Sweat gland extracts (100 ⁇ g/ml) were added to sympathetic neuron 5 cultures. Cultures were harvested on the eighth day after the addition of extract. Sister wells were assayed for VIP or for NPY by radioimmunoassay. All samples were run in triplicate. Data are expressed as pg of VIP or NPY per well ⁇ SD. 0 Figure 5. Appearance of cholinergic differentiation activity in sweat gland extracts. Sweat gland extracts were prepared from animals at the indicated ages. Approximately equal protein concentrations (100 ⁇ g/ml) were added to sympathetic £ -* neuron cultures.
  • Sweat gland extracts were incubated with protein A-Sepharose (A) , affinity- purified antibodies to the N-terminal sequence of CDF (B) , or affinity-purified antibodies preincubated with the peptide antigen (C) . After immunoprecipitation,
  • FIG. 7. CNTF is not detectable in sweat gland extracts.
  • 10 ng of recombinant CNTF 5 was blotted onto nitrocellulose.
  • 60 ⁇ g of soluble protein (DEAE fractions) from sciatic nerve extract (lane 1) , from hairy skin extract of adult rat (lane 2) or from sweat gland extract of adult (lane 3) or 21 day (lane 4) animals (panels b and c) 10 were blotted onto nitrocellulose.
  • the blots were probed with a polyclonal antiserum raised against recombinant rat CNTF, while in panel c the blot was probed with antibody preincubated with 10 ⁇ m recombinant CNTF.
  • Panel a documents that the 15 antiserum recognizes CNTF (arrowhead) .
  • the antiserum recognizes a 24 kilodalton (kd) band present in sciatic nerve extracts (lane 1 b,c) , but no specific bands were evident in hairy skin extracts (lane 2) or in sweat gland extracts from 21 day (lane 203) or adult (lane 4) animals.
  • Arrowheads in b and c indicate 92, 30 and 22.5 kd standards.
  • CNTF message is not detectable in sweat gland extracts. 30 ⁇ g of total RNA from adult sciatic nerve " (a) , sweat gland (b) , liver (c) and optic 5 nerve (d) was electrophoresed and transferred onto nylon membrane. The membrane was then probed with an oligonucleotide probe to rat CNTF. Arrow shows a positive 1.3 kb band in lane a containing sciatic nerve RNA and a fainter band in the same position in optic 0 nerve (d) . No specific signal is detected in lanes b and c containing sweat gland and liver RNA, respectively.
  • FIG. 9 In Situ Hybridization. Sections of sciatic nerve were probed with an oligonucleotide 5 probe to rat CNTF. Panel a shows specific hybridization to Schwann cells in sciatic nerve sections (with an antisense probe) . Panel b shows the same tissue section stained with ethidium bromide. No binding is seen with the sense (control) probe in Panel c, which shows a random distribution of grains. Panel d shows the same tissue section stained with ethidium bromide.
  • FIG. 10 In situ Hybridization. Sections of sweat gland were probed with an oligonucleotide probe to rat CNTF, as used in Fig. 9. No specific binding is seen in sections of sweat glands (Panel a) . No binding is seen with the sense (control) probe (Panel c) . Panels b and d represent ethidium bromide stained sections. Figure 11. Anion exchange chromatography.
  • the sweat gland extract supernatant was applied to a DEAE ion exchange column, and assayed for choline acetyltransferase (ChAT) inducing activity in sympathetic neurons. Closed squares: ChAT induction. Closed diamonds: NaCl gradient.
  • FIG. 13 (a) SDS gel fractions betwen 22- 26 kd and 26-32 kd were eluted and added to cultures of dissociated sympathetic neurons. Seven days after the addition of extracts, neurons were homogenized and aliquots were assayed for levels of choline acetyltransferase (ChAT) activity by the method of Fonnum. Samples were run in triplicate. The data are expressed as the fold induction of ChAT activity compared to that present in control cultures grown without added extract.
  • ChAT choline acetyltransferase
  • Lane a shows the 22-26 kd (lower arrow) fraction and lane b the 26-32 (upper arrow) kd fraction.
  • the present invention is directed to a 10 target-derived neuronal cholinergic differentiation factor (NCDF) , and the therapeutic and diagnostic uses thereof.
  • NCDF target-derived neuronal cholinergic differentiation factor
  • the invention provides NCDF, and derivatives, analogs, and fragments thereof, pharmaceutical compositions containing the foregoing, as well as anti- 15 NCDF antibodies.
  • the NCDF of the invention is a protein present in extracts of mammalian sweat glands, which exhibits heat and trypsin lability, lack of substantial binding to a heparin-agarose affinity column, an 0 isoelectric point (pi) in the range of approximately 4.8 to 5.2, a non-membrane cellular localization, and an approximate molecular weight in the range of 16 to 32 kilodaltons.
  • the NCDF protein, its derivatives, analogs, and fragments are able to reduce the 5 expression of tyrosine hydroxylase and of total catecholamines, and increase the expression of choline acetyltransferase and vasoactive intestinal peptide (VIP) , by sympathetic neurons in cell culture (in vitro) .
  • the NCDF protein, its derivatives, analogs, and fragments can be used to induce cholinergic activity in neurons.
  • Such proteins, derivatives, analogs and fragments can be administered therapeutically to patients with nervous system damage *** * or diseases where it is desirable to support survival and/or cholinergic differentiation of a number of neuronal types.
  • the NCDF protein is that found in extracts of human sweat glands. In another embodiment, the NCDF protein is that found in extracts of sweat glands from rats.
  • the NCDF protein, its derivatives, analogs, and fragments may induce the expression of additional peptides such as enkephalin, somatostatin, and substance P.
  • NCDF proteins isolated from ovine, bovine, feline, avian, equine, or canine, as well as primate sources and any other species in which NCDF activity exists.
  • the invention also provides for NCDF proteins, fragments and derivatives thereof or their functional equivalents.
  • the invention also provides fragments or derivatives of NCDF proteins which comprise antigenic determinant(s) or which are functionally active.
  • functionally active shall mean having positive activity in assays for known NCDF function, e.g. the ability to increase the expression ' of choline acetyltransferase by sympathetic neurons in vitro.
  • NCDF derivatives, analogs, or fragments of the invention include, but are not limited to, those containing all or part of the primary amino acid sequence contained in the full-length NCDF protein as purified from sweat gland extracts, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • 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.
  • 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.
  • NCDF proteins, fragments, analogs or derivatives thereof which are modified, e.g. f by proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, acetylation, formylation, oxidation, reduction, etc.
  • NCDF may be purified from any available source of mammalian sweat glands using techniques known in the art. Such techniques include but are not limited to chro atography (e.g.. ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any 5 other standard technique for the purification of proteins.
  • NCDF may be isolated from sweat gland extracts according to the following method.
  • Sweat gland extracts may be prepared according to the method set forth in Section 6.3.3. After homogenization and centrifugation as set forth therein, the supernatant may be collected and applied to an anion exchange column (e.g. DEAE, Whatman DE52 5 cellulose equilibrated in phosphate buffer) , and collected therefrom by methods known in the art.
  • Purified extract may then be subjected to sucrose gradient centrifugation by known methods, with the appropriate fraction concentrated by ultra filtration.
  • the purified NCDF may then be subjected to analytic or preparative polyacrylamide gel electrophoresis. If desired, following elution from a polyacrylamide gel, NCDF may be further purified and freed from certain buffer components by use of a HPLC reverse phase 5 column.
  • purified NCDF may be analyzed using a slab SDS- polyacrylamide gel.
  • Purified NCDF or molecular weight standards may be electrophoresed and the gel cut out 0 and processed as follows: the polypeptides may be visualized without fixation by precipitating the protein-associated SDS during an incubation of the gel in 0.25 M KC1 and recording the positions of the standards and NCDF bands. Lanes may then be fixed and ⁇ stained with Coomassie blue. Other lanes may then be cut into slices, and eluted, e.g. by electroelution or by incubation with Triton X-100, and then the eluates may be assayed for NCDF activity.
  • NCDF activity may be evaluated using any NCDF-sensitive In vivo or in vitro systems.
  • assays including including but not limited to those described in Section 6.3., infra, may be used, e.g., those assaying the ability to increase the expression of choline acetyltransferase or increase the expression of vasoactive intestinal peptide, or reduce the expression of tyrosine hydroxylase, or reduce the expression of total catecholamines, by sympathetic neurons in cell culture.
  • NCDF activity may be measured by quantitating 24-hour survival of embryonic (E8) chick ciliary ganglion (CG) neurons in monolayer cultures.
  • E8 chick embryos E8 chick embryos
  • ciliary ganglia may be collected from E8 chick embryos, dissociated (yielding approximately 20,000 cells per ganglion) and then diluted in HEBM medium contain —iVng 20 percent horse serum as described in Varon et ai. (1979, Brain Res. 173, 29-45). About fifty microliters of cell suspension containing 1,000 neurons (2,000 cells) may then be seeded into icrotitre dishes and then putative NCDF activity may be added.
  • Culture plates may then be maintained at 37°C in 5% C0 2 for 24 hours, after which the cultures may be fixed by the addition of 200 ⁇ l 2 per cent glutaraldehyde in HEBM medium, and the number of surviving neurons may be determined visually by direct count under phase contrast microscopy.
  • NCDF protein may be sequenced directly or initially cleaved by any protease or other compound known in the art, including, but not limited to, Staphylococcus aureus V8, trypsin, and cyanogen bromide.
  • Peptides may be sequenced by automated Edman degradation on a gas phase microsequencer according to the method of Hewick et al. (1981, J. Biol. Chem. 256, 7990-7997) and Hunkapiller et al. (1983, Methods Enzymol. 91, 227-236) .
  • Detection of phenylthiohydantoin amino acids may then be performed according to Lottspeich (1985, Chromatography 326, 321- 327) . Overlapping fragments of amino acid sequence may be determined and used to deduce longer stretches of contiguous sequence.
  • NCDF protein may be used as im unogen to generate anti-NCDF antibodies.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of NCDF.
  • various host animals can be immunized by injection with NCDF protein, " or a fragment or derivative thereof, including but not limited to rabbits, mice, rats, etc.
  • 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
  • the amino acid sequence of NCDF may be analyzed in order to identify portions of the molecule 5 which may be associated with increased immunogenicity.
  • the amino acid sequence may be subjected to computer analysis to identify surface epitopes, according to the method of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828).
  • Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828).
  • the monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric hu an- 25 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. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; Olsson et al., 301982, 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. 81, 6851,
  • a molecular clone of an antibody to a NCDF epitope can be prepared by known techniques. Recombinant DNA methodology (see e.g., Maniatis et al., 1982, Molecular 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.g.. im unoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography) , or a combination thereof, etc.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the 2 Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • NCDF RNA cleavage protein
  • peptides peptides, and derivatives, and anti-NCDF antibodies
  • NCDF may be utilized.in diagnostic and therapeutic applications.
  • NCDF protein peptide fragments, or analogs or derivatives produced therefrom, as well as antibodies directed against NCDF protein, peptides, or derivatives, may be utilized to diagnose diseases and disorders of the nervous system which may be associated with alterations in the pattern of NCDF expression.
  • Assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in NCDF expression, including, in particular, conditions resulting in damage and degeneration of neurons which may respond to NCDF, such as parasympathetic neurons, cholinergic neurons, spinal cord neurons, neuroblastoma cells and cells of the adrenal medulla.
  • diseases and conditions may include but are not limited to trauma, infarction, infection, degenerative nerve disease, malignancy, or post-operative changes including but not limited to Alzheimer's Disease, Parkinson's Disease, Huntington's Chorea, and amyotrophic lateral sclerosis.
  • antibodies directed toward NCDF protein, peptide fragments, analogs or derivatives can be used to diagnose diseases and disorders of the nervous system, including, in particular, those neuronal populations and clinical disorders and diseases listed supra.
  • Antibodies directed toward NCDF proteins of the invention can be used, for example, in in situ hybridization techniques using tissue samples obtained from a patient in need of such evaluation.
  • the antibodies of the invention can be used in ELISA procedures to detect and/or measure amounts of NCDF present in tissue or fluid samples; similarly, the antibodies of the invention can be used in Western blot analysis to detect and/or measure NCDF present in tissue or fluid samples.
  • the immunoassays which can be used to detect or measure NCDF protein, its analogs, derivatives or fragments, include but are not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay) , "sandwich” immunoassays, pre ⁇ ipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and immunoelectrophoresis assays, to name but a few.
  • NCDF protein, peptide fragments or derivatives can be used to diagnose diseases and disorders of the nervous system.
  • labeled NCDF protein or peptide fragments can be used to identify tissues or cells which express the NCDF receptor in order to identify aberrancies of NCDF receptor expression and consequently, potential abnormalities in the tissue or cellular response to NCDF.
  • NCDF protein, peptide fragments, analogs or derivatives produced therefrom, as well as to antibodies directed against NCDF protein, peptides, analogs or derivatives may be utilized to treat diseases and disorders of the nervous system which may be associated with alterations in the pattern of NCDF expression or which may benefit from exposure to NCDF or anti-NCDF antibodies.
  • NCDF, and its derivatives, fragments, and analogs can be used to support the survival and cholinergic differentiation of a number of neuronal types, including spinal motor neurons, parasympathetic neurons of the ciliary ganglion, etc.
  • NCDF products of the present invention may have utility in supporting jj ⁇ vivo the survival and differentiation of certain cell populations, including but not limited to, spinal motor neurons, parasympathetic neurons (including ciliary ganglion neurons which innervate the iris, heart, gastrointestinal tract and other visceral structures) .
  • a pharmaceutical preparation containing NCDF or its active derivatives, fragments or analogs can be administered to patients in whom the central nervous system is damaged.
  • a pharmaceutical preparation containing NCDF or its active derivatives, fragments, or analogs, alone or in combination with another neurotrophic factor e.g.
  • CNTF neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • diseases might include, but are not limited to: chronic anhidrosis and hyperhidrosis, cardiac arhythmias, chronic constipation, neurogenic bladder dysfunction and ejaculatory disturbances.
  • NCDF protein, peptide fragments or derivatives can be administered to patients in whom the nervous system has been damaged by trauma, surgery, ischemia, infection (e.g. polio or- A.I.D.S.) , metabolic disease, nutritional deficiency, malignancy, or toxic agents.
  • the invention in particular can be used to treat conditions in which damage has occurred to neurons, by administering effective therapeutic amounts of NCDF protein or peptide fragments or derivatives or analogs.
  • NCDF can be administered to spinal cord neurons which have been damaged, for example, by trauma, infarction, infection, degenerative disease or surgical lesion.
  • NCDF-related peptides or NCDF protein may be administered by adsorption onto a membrane, e.g. a silastic membrane, that could be implanted in the proximity of the damaged nerve.
  • a membrane e.g. a silastic membrane
  • the present invention can also be used for example in hastening the recovery of patients suffering from diabetic neuropathies, e.g. mononeuropathy multiplex or impotence.
  • NCDF protein or peptide fragments or derivatives derived therefrom can be used to treat congenital conditions or neurodegenerative disorders, including, but not limited to, Alzheimer's disease, ageing, peripheral neuropathies, Parkinson's disease, Huntington's c orea and diseases and disorders of motorneurons; in particular, the invention can be used to treat congenital or neurodegenerative disorders associated with cholinergic neuron dysfunction.
  • NCDF protein or peptide fragments or derivatives derived therefrom
  • NCDF * may also be useful in the treatment of a variety of dementias as well as congenital learning disorders.
  • NCDF protein, fragments or derivatives can be used in conjunction with other cytokines to achieve a desired neurotrophic effect.
  • NCDF can be used together with NGF to achieve a stimulatory effect on growth and survival of neurons. It is envisioned that NCDF may function synergistically with other CNS- derived peptide factors yet to be fully characterized, in the growth, development, and survival of a wide array of neuronal subpopulations in the central and peripheral nervous system.
  • antibodies directed toward NCDF protein, or peptide fragments or derivatives thereof can be administered to patients suffering from a variety of neurologic disorders and diseases and who are in need of such treatment.
  • patients who suffer from excessive production of NCDF may be in need of such treatment.
  • Anti-NCDF antibodies can be used in prevention of ' aberrant regeneration of sensory neurons (e.g. post-operatively) , or in the treatment of chronic pain syndromes.
  • compositions of the invention which may comprise all or portions of the NCDF protein, peptide fragments or analogs or derivatives produced therefrom, or antibodies (or antibody fragments) directed toward NCDF protein, peptide fragments, or derivatives, or a combination of NCDF and a second agent (such as NGF) may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • NCDF protein, peptide fragment, derivative, or antibody which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the dose- response curve first in vitro, e.g. in the NCDF bioassay systems described supra. and then in useful animal model systems prior to testing in humans.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal.
  • compositions of the invention may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the invention also provides for pharmaceutical compositions comprising NCDF proteins, peptide fragments, analogs, or derivatives administered via liposomes, microparticles, or microcapsules.
  • compositions comprising NCDF proteins, peptide fragments, analogs, or derivatives administered via liposomes, microparticles, or microcapsules.
  • it may be useful to use such compositions to achieve sustained release of NCDF and NCDF-related products.
  • the sympathetic innervation of rat sweat glands undergoes a target-induced switch from a noradrenergic to a cholinergic and peptidergic phenotype during development.
  • Treatment of cultured sympathetic neurons with sweat gland extracts mimics many of the charges seen in vivo. Extracts induce choline acetyltransferase activity and vasoactive intestinal peptide expression in the neurons in a dose- dependent fashion while reducing catecholaminergic properties and neuropeptide Y.
  • the cholinergic differentiation activity appears in developing glands of postnatal day 5 rats and is maintained in adult glands. It is a heat-labile, trypsin-sensitive, acidic protein that does not bind to heparin-agarose.
  • sweat glands contain a soluble factor(s) with the appropriate spectrum of activities: it reduces the expression of catecholamines and tyrosine hydroxylase and induces the expression of choline acetyltransferase and VIP. This activity is present when the phenotype of the sweat gland innervation is changing.
  • Our 5 initial characterization of the sweat gland-derived choline acetyltransferase-inducing activity permits a comparison with the cholinergic factors previously identified in cell culture.
  • peripheral nerve plexus constitutes only a small proportion of the gland tissue.
  • Sympathetic neurons were cultured in L15-C0 2 either lacking serum or containing 5% rat serum with 300 ⁇ g/ml sweat gland extract. Cells were harvested 7 days after the addition of extracts and aliquots were tested for choline acetyltransferase activity by the method of Fonnum (1969, Biochem. J. 115, 465-472). Samples were run in triplicate. Data are expressed as pmol of choline acetyltransferase per min per well ⁇ SEM.
  • Sweat gland extracts were tested for their ability to support the survival of cultured sympathetic neurons.
  • Table II shows that neurons plated in medium lacking nerve growth factor (NGF) but containing sweat gland extracts at a dose of 1 mg/ml did not survive more than 3 days in culture. Furthermore, there was no significant difference in neuron number in cultures grown with or without sweat gland extract even at extract doses as high as 1 mg/ml in the presence of 50 ng/ml of NGF. Since the levels of choline acetyltransferase activity and acetylcholine synthesis are initially very low in dissociated sympathetic neuron cultures: (Johnson et al., 1976, Nature 262, 308-310; Johnson, 1980, J. Cell Biol.
  • Sympathetic neurons were cultured in 56 well plates in L15-C0 2 with NGF (50 ng/ml) for 2 days. On the second day, the culture medium was replaced with medium containing no NGF (-NGF) , no NGF but with 1 mg/ml sweat gland extract
  • sweat gland extracts contained a factor(s) that played a role in altering neurotransmitter phenotype, one would predict that it would decrease the expression of noradrenergic properties in cultured sympathetic neurons.
  • SDS-PAGE sodium dodecyl sulfate- polyacryla ide gel electrophoresis
  • the catecholamine content was determined in cultures of sympathetic neurons grown with and without sweat gland extract. The total catecholamine content of wells incubated with • sweat gland extracts was reduced compared with that of control cultures (Table III) .
  • Sympathetic neurons were grown with sweat gland extracts (100 ⁇ g/ml, 250 ⁇ g/ml and 1 mg/ml) . Seven days after the addition of extracts, the cultures were harvested and assayed for catecholamine content by high-performance liquid chromatography. Samples were run in triplicate. Data are expressed as mean pmol of catecholamines per dish + SEM. The figures in brackets are the mean fold choline acetyltransferse induction assayed in sister wells by the method of Fonnum (1969, Biochem. J. , 115, 465-472).
  • Sweat gland extracts significantly increased VIP (Figure 4a) ; a dose of 100 ⁇ g/ml causes an induction of 80 pg per well of VIP, a more than 4- fold increase over the levels present in control cultures. The induction of VIP expression increased with increasing concentrations of sweat gland extracts ( Figure 4a) . -,.r
  • extracts were prepared from footpads of animals ranging in age from 2 to 21 days and were assayed for their ability to induce choline acetyltransferase
  • the activity was heat and trypsin labile and retained by a Centricon filter with a 10 kd cutoff, indicating that the activity is a protein.
  • the activity was only partially retained by a Centricon filter with a 30 kd cutoff, suggesting that a low molecular mass protein is responsible for the induction of choline acetyltransferase.
  • the cholinergic-inducing activity was relatively stable; little activity was lost with storage at -20°C and on repeated freeze-thawing.
  • the sweat gland cholinergic factor does not appear to be a heparin binding protein like the 50 kd soluble cholinergic factor from brain (Kessler et al., 1986, Proc. Natl. Acad. Sci. USA 83, 3528-3532). Almost all choline acetyltransferase-inducing activity and 35% of the protein were recovered in the 0.25 M eluate from a DEAE column, indicating that the differentiation activity is an acidic protein(s) and that this can be used as an initial purification step.
  • the 0.25 DEAE eluate not only induced choline acetyltransferase activity, but also increased levels of VIP and reduced levels of tyrosine hydroxylase (data not shown).
  • the several effects of the sweat gland extract on neurotransmitter properties of cultured sympathetic neurons are not readily separated.
  • CDF/LIF One candidate for the cholinergic-inducing activity in sweat gland extracts is CDF/LIF, since it has many of the same effects on the neurotransmitter properties of cultured sympathetic neurons.
  • Antisera generated against a synthetic peptide whose sequence corresponds to the N-terminal peptide sequence of CDF/LIF can immunoprecipitate the cholinergic-inducing activity from a partially purified DEAE fraction from heart cell conditioned medium (Yamamori et al., 1989, Science 246, 1412-1416; Rao et al., 1990, Dev. Biol. 139, 65-74).
  • NCDF IS DISTINCT FROM CNTF
  • ciliary neurotrophic factor CNTF
  • equal amounts of cholinergic inducing activity from sciatic nerve extracts and sweat gland extracts were loaded on an SDS-PAGE gel, electrophoresed and probed with a polyclonal antiserum generated against recombinant rat CNTF (a kind gift of Dr. Mark Furth, Regeneron Pharmaceuticals) .
  • the antiserum recognized recombinant CNTF (Fig. 7a) and a 24 kd band present in the sciatic nerve extracts (Fig. 7b) .
  • RNA from the adult sweat gland and probed the Northern blots for message with a probe against rat CNTF (Fig. 8).
  • the same probe was also used to probe sections of sciatic nerve and sweat gland by in situ hybridization, as a more sensitive assay of the cells 0 type that may be making CNTF/CNTF-like molecule.
  • the 0.25 M DEAE eluate containeci the ChAT and VIP inducing, and tyrosine hydroxylase reducing, 0 activity. Almost all activity and 35% of the protein was recovered in the 0.25 M eluate of a DEAE column, suggesting that this can be used as an initial purification step (Fig. 11) . 5 6.1.11. ISOELECTRIC FOCUSING
  • the DEAE eluate was chromatographed on a MONO P column and 0.5 ml fractions were collected and assayed for cholinergic activity.
  • Fig. 12 shows that the ChAT activity was eluted at between pH 4.8 to 5.2 with a peak of activity at pH 5.0, indicating that the pi of the active protein was in this range. This is similar to the value reported for CNTF purified from sciatic nerve extracts and differs from LIF which is a strongly basic protein.
  • Extracts of sweat glands but not of liver, hairy skin, or parotid gland increase levels of choline acetyltransferase activity and of VIP-like immunoreactivity in a dose-dependent fashion.
  • levels of choline acetyltransferase activity increase in the cultured neurons, there is a concomitant 10 decrease in the catecholamine content and tyrosine hydroxylase.
  • extracts of soluble protein from sweat glands cause many of the changes in cultured sympathetic neurons that characterize the developing
  • Extracts from P5 animals increase choline acetyltransferase activity, and when extracts of glands from animals between P9 and P21 are tested, they alter all three transmitter properties examined: they increase choline aceyltransferase and VIP-like immunoreactivity and reduce tyrosine hydroxylase. 0 Furthermore, since elevated levels of choline acetyltransferase activity are detectable after 2 days of treatment in culture, the extract is able to induce changes with a time course consistent with in vivo studies.
  • Sweat glands of adult as well as developing animals contain NCDF activity.
  • the concentration of cholinergic-inducing activity present in sweat gland extracts is greater than that in spinal cord extracts (Wong and Kessler, 1987, Proc. Natl. Acad. Sci. USA 84, 8726-8729; Adler et al. 1989, Proc. Natl. Acad. Sci. USA 86, 1059-1083) and at least as high as that in sciatic nerve extracts (Sendtner et al. , 1989, Soc. Neurosci. Abs. 15, 710; Rao et al., 1990, Dev. Biol. 139, 65-74).
  • Extracts prepared from animals of different ages influence the several properties assayed, and more importantly, the several effects were not resolved into distinct activities in the preliminary characterization that we have performed. Thus, a single molecule seems likely; however, the possibility that several factors are involved cannot be formally excluded.
  • CDF/LIF Two soluble factors, CDF/LIF and CNTF, are similar in their effects on sympathetic neurons; they increase choline acetyltransferase and VIP expression and reduce tyrosine hydroxylase and catecholamine content (Fukada,
  • CDF/LIF was an attractive candidate; it has a consensus signal sequence, it is glycosylated, and it is secreted by heart cells (Patterson and Chun, 1977, Dev. Biol 56, 263-280; Yamamori et al., 1989, Science
  • CDF/LIF does not bind to a
  • CDF/LIF affinity-purified antibodies raised against the N-terminal region of CDF/LIF can immunoprecipitate the cholinergic-inducing activity from the DEAE or Sephadex fractions of heart cell conditioned medium (Yamamori et al., 1989, Science 246, 1412-1416; Rao et al., 1990, Dev. Biol. 139, 65-74), but these antibodies do not immunoprecipitate the cholinergic-inducing activity from sweat gland extracts. Thus, it is unlikely that the major cholinergic factor in the extract is identical to CDF/LIF. CNTF was another likely candidate.
  • CNTF appears to be a cytosolic protein (Stockli et al., 1989, Nature 342, 920-923; Lin et al., 1989, Science 246, 1023-1025); while a candidate sweat gland differentiation molecule is likely to be secreted to exert an effect on the innervation, suggest that CNTF is an unlikely candidate for the sweat gland-derived cholinergic factor.
  • a cholinergic sympathetic target tissue contains cholinergic differentiating activity that mimics in culture the effects of the target on sympathetic neurons in vivo.
  • our preliminary purification and analysis suggest that the cholinergic-inducing activity present in the sweat gland extracts is not identical to either CDF/LIF or CNTF and that it is a novel factor.
  • the cholinergic-inducing activity present in sweat gland extracts represents an excellent candidate for mediating the target-induced phenotypic changes in the cholinergic sympathetic neurons that innervate sweat glands.
  • the neurons 0 were grown in Leibovitz's L15-C0 2 medium with NGF (100 ng/ml) , 100 U of penicillin, 100 ⁇ g of streptomycin, 10 ⁇ M cytosine arabinoside, and 5% rat serum, and the medium changed every third or fourth day.
  • NGF 100 ng/ml
  • penicillin 100 ⁇ g of streptomycin
  • 10 ⁇ M cytosine arabinoside 10 ⁇ M cytosine arabinoside
  • 5% rat serum 5% rat serum
  • cells were grown without rat serum in L15- 5 C0 2 supplemented with transferrin, selenium, bovine serum albumin, insulin and fatty acids.
  • the tissue extracts were diluted in growth medium, sterilized by passage through a 0.2 ⁇ m filter and added to the neurons from the third day of culture 0 on. Neurons were harvested for assay between days 9 and 14 of culture.
  • TISSUE EXTRACTS To prepare sweat gland extracts, footpads 5 were extracted from rats of various postnatal ages and weighed. Tissue from 20 animals was generally processed at one time. The weight of footpads from 20 rats varied from 0.5 to 5 grams, depending upon the age of the animal. The tissue was homogenized for 5 sec in 0 ⁇ o vol of 10 mM phosphate butter (pH 7.0) with a
  • ASSAYS 10 The induction of cholinergic function was determined by assaying choline acetyltransferase activity in homogenates essentially according to the method of Fonnum (1969, Biochem. J. 115, 465-472). To increase the sensitivity of the assay, an incubation 15 period of 1 hr was used. All activity was inhibitable by 500 ⁇ M napthylvinyl pyridine, a specific inhibitor of choline acetyltransferase activity. Protein concentration was assayed by the method of Lowry using bovine serum albumin as a standard.
  • Catecholamine content was assayed by high performance liquid chromatography (Rittenhouse et al., 1988, Neurosci. 25, 207-215) on a 5 ⁇ m pore reverse- phase C-18 column (Altex Ultrasphere-IP; Beckman, Berkeley, CA) Using a colorometric detector (5100A,
  • the amount of tyrosine hydroxylase present in the cultured neurons was determined by semiquantitative
  • the blots were then sequentially incubated with a biotinylated secondary antibody and avidin conjugated to alkaline phosphatase.
  • the reaction product was developed with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate in 10 mM bicarbonate butter (pH 9.5). After optimal color development, the reaction was stopped by rinsing in distilled water. The blots were allowed to dry, and the color intensity was read on a scanning laser densitometer (Shimadzu) . Comparisons were made between samples run in parallel lanes and treated identically. Neuropeptide levels were determined by radioimmunoassay.
  • a ersham and peptide content was determined by the delayed tracer method. Since the antibody shows only
  • tissue extract sufficient for a cell culture assay were added to buffer (PBS [pH 7.3] with 2% bovine serum albumin, 0.2% Triton X-100, and 0.02% PEG 6000) .
  • buffer PBS [pH 7.3] with 2% bovine serum albumin, 0.2% Triton X-100, and 0.02% PEG 6000
  • Affinity-purified antibodies raised*against a synthetic peptide corresponding to the N-terminal region of CDF (Rao et al., 1990, Dev. Biol. 139, 65-74) were added to each vial to a final concentration of 10 ⁇ M. After an overnight incubation, the antigen- antibody complex was adsorbed to 10 ⁇ l of protein A- Sepharose for an additional 2 hr at room temperature.
  • the bound complexes were separated by centrifugation, and the supernatant was diluted into L15-C0 2 medium and used for cell culture assays. Two controls were performed to ensure that the loss of activity consequent to absorption was due to a specific effect of the antibody. Aliquots of extract were incubated without the antibody and treated as described above, and other aliquots were treated with antibody that had been previously absorbed with 50 ⁇ M synthetic peptide originally used as antigen.
  • CDF/LIF (a kind gift of Dr. Yamamori, California Institute of Technology) was iodinated with Bolton Hunter reagent as described previously (Rao et al., 1990, Dev. Biol. 139, 65-74). About 20,000 cpm were added to buffer or an equal volume of the DEAE fraction of the sweat gland extract and immunoprecipitated with the N-terminal antibody as described above. The counts that were immunoprecipitated were extracted and analyzed by SDS- PAGE.
  • the blots were then sequentially incubated with a biotinylated secondary antibody for an hour and then with avidin-conjugated alkaline phosphatase for 30 min.
  • the bound enzyme was detected with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3 indoyl phosphate in 10 mM bicarbonate buffer, pH 9.5. After optimal color development, the reaction was 5 stopped by rinsing in distilled water.
  • RNA was prepared from liver, sweat gland and sciatic nerve 10 using the single step guanidinium-isothio ⁇ yanate method (Chomczynski and Sacchi, 1987, Analytical Biochem. 162, 156-159) . 30 ⁇ g of total RNA was loaded per lane and transferred to a Genescreen nylon membrane. Blots were probed with a 45 base pair oligonucleotide probe 15 (region 99-144) against rat CNTF radiolabelled with 32 P. Blots were sequentially washed and then examined by autoradiography.

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Abstract

La présente invention se rapporte à un facteur de différenciation cholinergique neuronal (FDCN) dérivé d'une cible, ainsi qu'à ses utilisations thérapeutiques et diagnostiques. L'invention se rapporte au FDCN, à ses dérivés, analogues et fragments, à des compositions pharmaceutiques contenant ceux-ci ainsi qu'à des anticorps agissant contre le FDCN. Le FDCN de l'invention est une protéine présente dans des extraits de glandes sudoripares de mammifères. et qui fait preuve de thermolabilité et de labilité à la trypsine ainsi que d'une absence de liaison sensible à une colonne d'affinité héparine-agarose, et présente un point isoélectrique (pI) compris entre 4,8 et 5,2 environ, une localisation cellulaire non membraneuse et un poids moléculaire compris entre 16 et 32 kilodaltons. La protéine de FDCN, ainsi que ses dérivés, analogues et fragments sont susceptibles de réduire l'expression de tyrosine hydroxylase et de catécholamines entières et d'augmenter l'expression de choline acétyltransférase et du peptide intestinal vasoactif (PIV) par l'intermédiaire des neurones sympathiques dans une culture cellulaire. La protéine de FDCN et ses dérivés, analogues et fragments peuvent être utilisés pour induire une activité cholinergique dans des neurones. De telles protéines, dérivés, analogues et fragments peuvent être administrés à des fins thérapeutiques à des patients souffrant de lésions ou de troubles du système nerveux où il est souhaitable de maintenir la survie et/ou la différenciation cholinergique d'un certain nombre de types neuronaux.
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US7258983B2 (en) 1994-04-25 2007-08-21 Genentech, Inc. Cardiotrophin-1 compositions and methods for the treatment of tumor
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