WO1994000140A1 - Glial mitogenic factors, their preparation and use - Google Patents

Glial mitogenic factors, their preparation and use Download PDF

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Publication number
WO1994000140A1
WO1994000140A1 PCT/US1993/006228 US9306228W WO9400140A1 WO 1994000140 A1 WO1994000140 A1 WO 1994000140A1 US 9306228 W US9306228 W US 9306228W WO 9400140 A1 WO9400140 A1 WO 9400140A1
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WIPO (PCT)
Prior art keywords
polypeptide
εaid
seq
amino acid
glial cell
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PCT/US1993/006228
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French (fr)
Inventor
Andrew Goodearl
Paul Stroobant
Luisa Minghetti
Michael Waterfield
Mark Marchioni
Maio Su Chen
Ian Hiles
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Ludwig Institute For Cancer Research
Canbridge Neuroscience
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Application filed by Ludwig Institute For Cancer Research, Canbridge Neuroscience filed Critical Ludwig Institute For Cancer Research
Priority to KR1019940704870A priority Critical patent/KR100307943B1/en
Priority to EP93918139A priority patent/EP0653940B1/en
Priority to JP50265594A priority patent/JP4127567B2/en
Priority to KR1020007004157A priority patent/KR100344006B1/en
Priority to DE69333731T priority patent/DE69333731T2/en
Priority to AT93918139T priority patent/ATE286126T1/en
Priority to AU47694/93A priority patent/AU688270B2/en
Publication of WO1994000140A1 publication Critical patent/WO1994000140A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • C07K14/4705Regulators; Modulating activity stimulating, promoting or activating activity
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/4756Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82Translation products from oncogenes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates to polypeptides found in vertebrate species, which polypeptides are mitogenic growth factors for glial cells, including Schwann cells.
  • the invention is also concerned with processes capable of producing such factors, and the therapeutic application of such factors.
  • the glial cells of vertebrates constitute the specialized connective tissue of the central and peripheral nervous systems.
  • Important glial cells include Schwann cells which provide metabolic support for neurons and which provide myelin sheathing around the axons of certain peripheral neurons, thereby forming individual nerve fibers.
  • Schwann cells support neurons and provide a sheath effect by forming concentric layers of membrane around adjacent neural axons, twisting as they develop around the axons.
  • myelin sheaths are a susceptible element of many nerve fibers, and damage to Schwann cells, or failure in growth and development, can be associated with significant demyelination or nerve degeneration characteristic of a number of peripheral nervous system diseases and disorders.
  • GGF Glial Growth Factor
  • Benveniste et al. (PNAS, 8 (1985) , 3930-3934) describes a T lymphocyte-derived glial growth promoting factor. This factor, under reducing conditions, exhibits a change in apparent molecular weight on SDS gels.
  • Kimura et al. (Nature, 348 (1990) , 257-260) describe a factor they term Schwannoma-derived growth factor (SDGF) which is obtained from a sciatic nerve sheath tumor. The authors state that SDGF does not stimulate the incorporation of tritium-labelled TdR into cultured Schwann cells under conditions where, in contrast, partially purified pituitary fraction containing GGF is active. SDGF has an apparent molecular weight of between 31,000 and 35,000. Davis and Stroobant (J. Cell. Biol., 110 (1990),
  • the pl85 erbB2 protein is a 185 kilodalton membrane spanning protein with tyrosine kinase activity.
  • the protein is encoded by the erbB2 proto-oncogene (Yarden and Ullrich Ann. Rev. Bioche . 5_7: 443 (1988)) .
  • the erbB2 gene also referred to as HER-2 (in human cells) and neu (in rat cells) , is closely related to the receptor for epidermal growth factor (EGF) .
  • EGF epidermal growth factor
  • pl85 erbB2 binding proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins, which are of different lengths and contain some common peptide sequences and some unique peptide sequences. This is supported by the differentially-spliced RNA transcripts recoverable from human breast cancer (MDA-MB-231) (Holmes et al. Science 256: 1205 (1992) ) . Further support derives from the wide size range of proteins which act as (as disclosed herein) ligands for the pl85 e ⁇ B2 receptor (see below) .
  • the invention provides methods for stimulating glial cell (in particular, Schwann cell and glia of the central nervous system) mitogenesis, as well as new proteins exhibiting such glial cell mitogenic activity.
  • glial cell in particular, Schwann cell and glia of the central nervous system
  • DNA encoding these proteins and antibodies which bind these and related proteins are provided.
  • novel proteins of the invention include alternative splicing products of sequences encoding known polypeptides.
  • these known proteins are members of the GGF/pl85 erbB2 family of proteins.
  • the invention provides polypeptides of a specified formula, and DNA sequences encoding those polypeptides.
  • the polypeptides have the formula WYBAZCX wherein WYBAZCX is composed of the amino acid sequences shown in Figure 31 (SEQ ID Nos.
  • W comprises the polypeptide segment F, or is absent; wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segments C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D'
  • the invention includes the DNA sequence comprising coding segments 5 FBA 3' as well as the with corresponding polypeptide segments having the a ino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 139); the DNA sequence comprising the coding segments 5 FBA' 3' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 140) ; the DNA sequence comprising the coding segments 5 FEBA 3 as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos.
  • the invention further includes peptides of the formula FBA, FEBA, FBA' FEBA' and DNA sequences encoding these peptides wherein the polypeptide segments correspond to amino acid sequences shown in Figure 31, SEQ ID Nos. (136, 138 and 139), (136-139) and (136, 138 and 140) and (136-138 and 140) respectively.
  • the polypeptide purified GGF-II polypeptide (SEQ ID No. 167) is also included as a part of the invention.
  • peptides and DNA encoding such peptides which are useful for the treatment of glia and in particular oligodendrocytes, microglia and astrocytes, of the central nervous system and methods for the administration of these peptides.
  • the invention further includes vectors including DNA sequences which encode the amino acid sequences, as defined above. Also included are a host cell containing the isolated DNA encoding the amino acid sequences, as defined above. The invention further includes those compounds which bind the pl85 crbB2 receptor and stimulate glial cell mitogenesis in vivo and/or in vitro .
  • antibodies to the novel peptides described herein are also a part of the invention.
  • antibodies to any of the peptides described herein may be used for the purification of polypeptides described herein.
  • the antibodies to the polypeptides may also be used for the therapeutic inhibitor of glial cell mitogenesis.
  • the invention further provides a method for stimulating glial cell mitogenesis comprising contacting glial cells with a polypeptide defined by the formula
  • WYBAZCX wherein WYBAZCX is composed of the polypeptide segments shown in Figure 31 (SEQ ID Nos. 136-139, 141- 147, 160, 161); wherein W comprises the polypeptide segment F, or is absent wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segment C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D D' D' HL, C/D D' HKL, C/D' D' H, C/
  • the invention also includes a method for the preparation of a glial cell mitogenic factor which consist of culturing modified host cells as defined above under conditions permitting expression of the DNA sequences of the invention.
  • the peptides of the invention can be used to make a pharmaceutical or veterinary formulation for pharmaceutical or veterinary use.
  • the formulation may be used together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
  • a method for stimulating mitogenesis of a glial cell by contacting the glial cell with a polypeptide defined above as a glial cell mitogen in vivo or in vitro is also an aspect of the invention.
  • a method for producing a glial cell mitogenic effect in a vertebrate (preferably a mammal, more preferably a human) by administering an effective amount of a polypeptide as defined is also a component of the invention.
  • Methods for treatment of diseases and disorders using the polypeptides described are also a part of the invention.
  • a method of treatment or prophylaxis for a nervous disease or disorder can be effected with the polypeptides described.
  • a method for the prophylaxis or treatment of a pathophysiological condition of the nervous system in which a cell type is involved which is sensitive or responsive to a polypeptide as defined are a part of the invention.
  • treatment when the condition involves peripheral nerve damage; nerve damage in the central nervous system; neurodegenerative disorders; demyelination in peripheral or central nervous system; or damage or loss of Schwann cells, oligodendrocytes, microglia, or astrocytes.
  • a neuropathy of sensory or motor nerve fibers; or the treatment of a neurodegenerative disorder are included.
  • treatment consists of administering an effective amount of the polypeptide.
  • the invention also includes a method for inducing neural regeneration and/or repair by administering an effective amount of a polypeptide as defined above.
  • a medicament is made by administering the polypeptide with a pharmaceutically effective carrier.
  • the invention includes the use of a polypeptide as defined above in the manufacture of a medicament.
  • the invention further includes the use of a polypeptide as defined above
  • an affinity isolation process optionally affinity chromatography, for the separation of a corresponding receptor.
  • the invention also includes a method for the prophylaxis or treatment of a glial tumor.
  • This method consists of administering an effective amount of a substance which inhibits the binding of a factor as defined by the peptides above.
  • the invention includes a method of stimulating glial cell mitogenic activity by the application to the glial cell of a
  • ARIA -42 kD polypeptide factor
  • the invention further includes a method for the use of the EGFL1, EGFL2, EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, Figure 38 to 43 and SEQ ID Nos. 154 to 159, respectively, for the stimulation of glial cell mitogenesis in vivo and in vitro.
  • GGF-II polypeptide whose sequence is shown in Figure 45 for the stimulation of glial cell mitogenesis.
  • An additional aspect of the invention includes the use of the above-referenced peptides for the purpose of stimulating Schwann cells to produce growth factors which may, in turn, be harvested for scientific or therapeutic use.
  • the peptides described herein may be used to induce central glial proliferation and remyelination for treatment of diseases, e.g., MS, where re-myelination is desired.
  • the novel polypeptides described herein may be used to stimulate the synthesis of acetylcholine receptors.
  • the invention provides new glial growth factors from mammalian sources, including bovine and human, which are distinguished from known factors. These factors are mitogenic for Schwann cells against a background of fetal calf plasma (FCP) .
  • FCP fetal calf plasma
  • the invention also provides processes for the preparation of these factors, and an improved method for defining activity of these and other factors. Therapeutic application of the factors is a further significant aspect of the invention.
  • important aspects of the invention are:
  • a basic polypeptide factor having glial cell mitogenic activity more specifically, Schwann cell mitogenic activity in the presence of fetal calf plasma, a molecular weight of from about 30 kD to about 36 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
  • Schwann cells in the presence of fetal calf plasma, has a molecular weight of from about 55 kD to about 63 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
  • novel peptide sequences set out above derived from the smaller molecular weight polypeptide factor, and from the larger molecular weight polypeptide factor, are also aspects of this invention in their own right. These sequences are useful as probe sources for polypeptide factors of the invention, for investigating, isolating or preparing such factors (or corresponding gene sequences) from a range of different species, or preparing such factors by recombinant technology, and in the generation of corresponding antibodies, by conventional technologies, preferably monoclonal antibodies, which are themselves useful investigative tools and are possible therapeutics.
  • the invention also includes an isolated glial cell mitogenic activity encoding gene sequence, or fragment thereof, obtainable by the methods set out above for the novel peptide sequences of the invention.
  • the invention further embraces a polypeptide factor having glial cell mitogenic activity and including an amino acid sequence encoded by:
  • the invention further includes sequences which have greater than 60%, preferably 80%, sequence identity of homology to the sequences indicated above.
  • DNA probes may be labelled to high specific activity (approximately 10 8 to 10 32 Pdmp/ ⁇ g) by nick-translation or by PCR reactions according to Schowalter and So mer (Anal. Biochem. , 177:90-94, 1989) and purified by desalting on G-150 Sephadex columns.
  • Probes may be denatured (10 minutes in boiling water followed by immersion into ice water) , then added to hybridization solutions of 80% buffer B (2g polyvinylpyrolidine, 2g Ficoll-400, 2g bovine serum albumin, 50ml 1 M Tris HCL (pH 7.5), 58g NaCl, lg sodium pyrophosphate, lOg sodium dodecyl sulfate, 950ml H 2 0) containing 10% dextran sulfate at 10 6 dpm 32 P per ml and incubated overnight (approximately 16 hours) at 60°C.
  • the filters may then be washed at 60°C, first in buffer B for 15 minutes followed by three 20-minute washes in 2X SSC, 0.1% SDS then one for 20 minutes in lx SSC, 0.1% SDS.
  • the invention provides: (a) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, whether in reducing conditions or not, of from about 30kD to about 36kD on SDS-polyacrylamide gel electrophoresis using the following molecular weight standards:
  • Phosphorylase B (rabbit muscle) 97,400; which factor has glial cell mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of said activity after 10 weeks incubation in 0.1% trifluoroacetic acid at 4 ⁇ C; and
  • Phosphorylase B (rabbit muscle) 97,400; which factor the human equivalent of which is encoded by DNA clone GGF2HBS5 described herein and which factor has glial cell mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of the activity after 4 days incubation in 0.1% trifluoroacetic acid at 4'C.
  • GGF-I the lower molecular weight and higher molecular weight factors of this invention are referred to hereafter as "GGF-I" and "GGF-II", respectively.
  • the "GGF2" designation is used for all clones isolated with peptide sequence data derived from GGF-II protein (i.e. , GGF2HBS5, GGF2BPP3) .
  • Another important aspect of the invention is a DNA sequence encoding a polypeptide having glial cell mitogenic activity and comprising:
  • Another aspect of the present invention uses the fact that the Glial Growth Factors and pl85 erbB2 ligand proteins are encoded by the same gene.
  • a variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show pl85 c ⁇ B2 binding and activation.
  • Several of the (GGF-II) gene products have been used to show Schwann cell mitogenic activity.
  • This invention provides a use for all of the known products of the GGF/pl85 erbB2 ligand gene (described in the references listed above) as Schwann cell mitogens.
  • This invention also relates to other, not yet naturally isolated splicing variants of the Glial Growth Factor gene.
  • Figure 30 shows the known patterns of splicing derived from polymerase chain reaction experiments (on reverse transcribed RNA) and analysis of cDNA clones (as presented within) and derived from what has been published as sequences encoding pl85 erbB2 ligands (Peles et al., Cell .69:205 (1992) and Wen et al., Cell j59_:559 (1992)). These patterns, as well as additional ones disclosed herein, represent probable splicing variants which exist.
  • Another aspect of the present invention relates to the nucleotide sequences encoding novel protein factors derived from this gene. The invention also provides processes for the preparation of these factors. Therapeutic application of these new factors is a further aspect of the invention.
  • the amino acid sequence is shown in Figure 32, SEQ ID No. 148, and is encoded by the bovine DNA sequence shown in Figure 32, SEQ ID No. 148.
  • the novel human peptide sequences described above and presented in Figures 31, 32, 33 and 34, SEQ ID Nos. 136-150, respectively, represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAs) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.
  • peptides which bind specifically to the pl85 crbB2 receptor can also be used according to the invention as a glial cell mitogen.
  • a candidate compound can be routinely screened for pl85 erbB2 binding, and, if it binds, can then be screened for glial cell mitogenic activity using the methods described herein.
  • the invention includes any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included.
  • modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included.
  • mutations of native proteins are disclosed in which the possibility of unwanted disulfide bonding is avoided by replacing any cysteine in the native sequence which is not necessary for biological activity with a neutral amino acid.
  • the statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.
  • the new sequences of the invention open up the benefits of recombinant technology.
  • the invention thus also includes the following aspects:
  • DNA constructs comprising DNA sequences as defined above in operable reading frame position within vectors (positioned relative to control sequences so as to permit expression of the sequences) in chosen host cells after transformation thereof by the constructs (preferably the control sequence includes regulatable promoters, e.g. Trp) .
  • control sequence includes regulatable promoters, e.g. Trp
  • host cells modified by incorporating constructs as defined in (a) immediately above so that said DNA sequences may be expressed in said host cells - the choice of host is not critical, and chosen cells may be prokaryotic or eukaryotic and may be genetically modified to incorporate said constructs by methods known in the art; and,
  • the Schwann cell assay used to characterize the present factors employs a background of fetal calf plasma.
  • the assay can be the same as that described by Brockes et al. in Meth. Enz., supra, but with 10% FCP replacing 10% FCS. This difference in assay techniques is significant, since the absence of platelet-derived factors in fetal calf plasma (as opposed to serum) enables a more rigorous definition of activity on Schwann cells by eliminating potentially spurious effects from some other factors.
  • the invention also includes a process for the preparation of a polypeptide as defined above, extracting vertebrate brain material to obtain protein, subjecting the resulting extract to chromatographic purification by hydroxylapatite HPLC and then subjecting these fractions to SDS-polyacrylamide gel electrophoresis.
  • the fraction which has an observed molecular weight of about 30kD to 36 kD and/or the fraction which has an observed molecular weight of about 55kD to 63 kD is collected. In either case, the fraction is subjected to SDS-polyacrylamide gel electrophoresis using the following molecular weight standards:
  • the SDS-polyacrylamide gel is run in non-reducing conditions in reducing conditions or, and in the case of the larger molecular weight fraction the gel is run under non-reducing conditions.
  • the fractions are then tested for activity stimulating the division of rat Schwann cells against a background of fetal calf plasma.
  • the above process starts by isolating a relevant fraction obtained by carboxymethyl cellulose chromatography, e.g. from bovine pituitary material.
  • hydroxylapatite HPLC, cation exchange chromatography, gel filtration, and/or reversed-phase HPLC be employed prior to the SDS-Polyacrylamide gel electrophoresis.
  • activity may be determined using Schwann cell incorporation of radioactive iododeoxyuridine as a measure in an assay generally as described by Brockes in Meth. Enz., supra, but modified by substituting 10% FCP for 10% FCS.
  • assay is an aspect of the invention in its own substance for CNS or PNS cell, e.g. Schwann cell, mitogenic effects.
  • the invention also includes an assay for glial cell mitogenic activity in which a background of fetal calf plasma is employed against which to assess DNA synthesis in glial cells stimulated (if at all) by a substance under assay.
  • Another aspect of the invention is a pharmaceutical or veterinary formulation comprising any factor as defined above formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
  • conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions.
  • formulations of this invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, opthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.
  • parenteral administration for example, intravenous, subcutaneous, intramuscular, intraorbital, opthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.
  • formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA of the instant invention or by the use of surgical implants which release the formulations of the invention.
  • Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes
  • biocompatible, biodegradable lactide polymer or polyoxyethylene-polyoxypropylene copolymers
  • Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
  • the present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.
  • concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
  • the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration.
  • General dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day.
  • the preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of the formulation, and the route of administration.
  • Schwann cells are stimulated to divide in the presence of the factors of the invention.
  • Schwann cells of the peripheral nervous system are involved in supporting neurons and in creating the myelin sheath around individual nerve fibers. This sheath is important for proper conduction of electrical impulses to muscles and from sensory receptors.
  • Neuropathies There are a variety of peripheral neuropathies in which Schwann cells and nerve fibers are damaged, either primarily or secondarily. There are many neuropathies of both sensory and motor fibers (Adams and Victor, Principles of Neurology) . The most important of those neuropathies are probably the neuropathies associated with diabetes, multiple sclerosis, Landry-Guillain-Barr syndrome, neuropathies caused by carcinomas, and neuropathies caused by toxic agents (some of which are used to treat carcinomas) .
  • the invention envisages treatment or prophylaxis of conditions where nervous system damage has been brought about by any basic cause, e.g. infection or injury.
  • any basic cause e.g. infection or injury.
  • glial growth factors can be valuable in the treatment of disorders of the nervous system that have been caused by damage to the peripheral nerves.
  • the regeneration process is led by the growth or the re-establishment of Schwann cells, followed by the advancement of the nerve fibre back to its target. By speeding up the division of Schwann cells one could promote the regenerative process following damage.
  • glial cells there are a variety of tumors of glial cells the most common of which is probably neurofibromatosis, which is a patchy small tumor created by ⁇ overgrowth of glial cells. Also, it has been found _t&at an activity very much like GGF can be found in some Schwann cell tumors, and therefore inhibitors of the action of the present factors on their receptors provides a therapy of a glial tumor, which comprises administering an effective amount of a substance which inhibits the binding of a factor, as defined above, to a receptor.
  • the invention includes the use of present polypeptide factors in the prophylaxis or treatment of any pathophysiological condition of the nervous system in which a factor-sensitive or factor-responsive cell type is involved.
  • the polypeptide factors of the invention can also be used as immunogens for making antibodies, such as monoclonal antibodies, following standard techniques. Such antibodies are included within the present invention. These antibodies can, in turn, be used for therapeutic or diagnostic purposes. Thus, conditions perhaps associated with abnormal levels of the factor may be tracked by using such antibodies.
  • In vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques for the art of tumour imaging may also be employed.
  • the invention also includes the general use of the present factors as glial cell mitogens in vivo or in vitro, and the factors for such use.
  • One specific embodiment is thus a method for producing a glial cell mitogenic effect in a vertebrate by administering an effective amount of a factor of the invention.
  • a preferred embodiment is such a method in the treatment or prophylaxis of a nervous system disease or disorder.
  • a further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a nervous disease or disorder, or for neural regeneration or repair. Also included in the invention are the use of the factors of the invention in competitive assays to identify or quantify molecules having receptor binding characteristics corresponding to those of said polypeptides.
  • the polypeptides may be labelled, optionally with a radioisotope.
  • a competitive assay can identify both antagonists and agonists of the relevant receptor.
  • the invention provides the use of each one of the factors of the invention in an affinity isolation process, optionally affinity chromatography, for the separation of a respective corresponding receptor.
  • affinity isolation process optionally affinity chromatography
  • Such processes for the isolation of receptors corresponding to particular proteins are known in the art, and a number of techniques are available and can be applied to the factors of the present invention.
  • the reader is referred to Novick, D.; et al., J. Chromatogr. (1990) 510: 331-7.
  • gonadotropin releasing hormone reference is made to Hazum, E., J. (1990)
  • FIGS 1 to 8 relate to Example 1, and are briefly described below:
  • Pig. 1 is the profile for product from carboxymethyl cellulose chromatography
  • Pig. 2 is the profile for product from hydroxylapatite HPLC
  • Fig. 3 is the profile for product from Mono S FPLC
  • Pig. 4 is the profile for product from Gel filtration FPLC
  • Figs. 5 and 6 depict the profiles for the two partially purified polypeptide products from reversed-phase HPLC.
  • Figs. 7 and 8 depict dose-response curves for the GGF-I and GGF-II fractions from reversed-phase HPLC using either a fetal calf serum or a fetal calf plasma background;
  • Figs. 9 to 12 depict the peptide sequences derived from GGF-I and GGF-II, SEQ ID Nos. 1-20, 22-29, 32-53 and 169, (see Example 2 hereinafter), Figures 10 and 12 specifically depict novel sequences:
  • Panel A the sequences of GGF-I peptides used to design degenerate oligonucleotide probes and degenerate PCR primers are listed (SEQ ID Nos. 20, 1, 22- 29, and 17). Some of the sequences in Panel A were also used to design synthetic peptides.
  • Panel B is a listing of the sequences of novel peptides that were too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID Nos. 17 and 52) ;
  • Panel A is a listing of the sequences of GGF-II peptides used to design degenerate oligonucleotide probes and degenerate PCR primers (SEQ ID Nos. 45-52) . Some of the sequences in Panel A were used to design synthetic peptides.
  • Panel B is a listing of the novel peptide that was too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID No. 53) ;
  • Figures 13 to 20 relate to Example 3, below and depict the mitogenic activity of factors of the invention
  • Figures 21 to 28 relate to Example 4, below and are briefly described below:
  • Fig. 21 is a listing of the degenerate oligonucleotide probes (SEQ ID Nos. 54-88) designed from the novel peptide sequences in Figure 10, Panel A and Figure 12, Panel A;
  • Pig. 22 depicts a stretch of the putative bovine GGF-II gene sequence from the recombinant bovine genomic phage GGF2BG1, containing the binding site of degenerate oligonucleotide probes 609 and 650 (see Figure 21, SEQ ID NOs. 69 and 72, respectively) .
  • the figure is the coding strand of the DNA sequence and the deduced amino acid sequence in the third reading frame.
  • the sequence of peptide 12 from factor 2 (bold) is part of a 66 amino acid open reading frame (nucleotides 75272) ;
  • Fig. 23 is the degenerate PCR primers (Panel A, SEQ IS Nos. 90-108) and unique PCR primers (Panel B, SEQ ID Nos. 109-119) used in experiments to isolate segments of the bovine GGF-II coding sequences present in RNA from posterior pituitary;
  • Fig. 24 depicts of the nine distinct contiguous bovine GGF-II cDNA structures and sequences that were obtained in PCR amplification experiments using the list of primers in Figure 7, Panels A and B, and RNA from posterior pituitary.
  • the top line of the Figure is a schematic of the coding sequences which contribute to the cDNA structures that were characterized;
  • Pig. 25 is a physical map of bovine recombinant phage of GGF2BG1.
  • the bovine fragment is roughly 20 kb in length and contains two exons (bold) of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, Spel, Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map. Shaded portions correspond to fragments which were subcloned for sequencing;
  • Fig. 26 is.a ;schematic of the structure of three alternative gene products of the putative bovine GGF-II gene.7..Exons are listed A through E in the order of their discovery.
  • the alternative splicing patterns 1, 2 and 3 generate three overlapping deduced protein structures (GGF2BPP1, 2, and 3), which are displayed in the various Figures 28a, b, c (described below) ;
  • Pig. 27 (SEQ ID Nos. 120-132) is a comparison of the GGF-I and GGF-II sequences identified in the deduced protein sequences shown in Figures 28a, 28b and 28c (described below) with the novel peptide sequences listed in Figures 10 and 12. The Figure shows that six of the nine novel GGF-II peptide sequences are accounted for in these deduced protein sequences. Two peptide sequences similar to GGF-I sequences are also found;
  • Fig. 28a is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 1 in Figure 26.
  • This partial cDNA of the putative bovine GGF-II gene encodes a protein of 206 amino acids in length. Peptides in bold were those identified from the lists presented in Figures 10 and 12.
  • Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) ;
  • Pig. 28b (SEQ ID No. 134) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 2 in Figure 26.
  • This partial cDNA of the putative bovine GGF-II gene encodes a protein of 281 amino acids in length. Peptides in bold are those identified from the lists presented in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) ;
  • Fig. 28c (SEQ ID No. 135) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 3 in Figure 26.
  • This partial cDNA of the putative bovine GGF-II gene encodes a protein of 257 amino acids in length. Peptides in bold are those identified from the lists in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) .
  • Fig. 29, which relates to Example 6 hereinafter, is an autoradiogram of a cross hybridization analysis of putative bovine GGF-II gene sequences to a variety of mammalian DNAs on a southern blot.
  • the filter contains lanes of EcoRI-digested DNA (5 ⁇ g per lane) from the species listed in the Figure.
  • the probe detects a single strong band in each DNA sample, including a four kilobase fragment in the bovine DNA as anticipated by the physical map in Figure 25. Bands of relatively minor intensity are observed as well, which could represent related DNA sequences.
  • the strong hybridizing band from each of the other mammalian DNA samples presumably represents the GGF-II homologue of those species.
  • Fig. 30 is a diagram of representative splicing variants.
  • the coding segments are represented by F, E, B, A, G, C, C/D, C/D', D, D', H, K and L.
  • the location of the peptide sequences derived from purified protein are indicated by "o".
  • Fig. 31 (SEQ ID Nos. 136-147, 160, 161) is a listing of the DNA sequences and predicted peptide sequences of the coding segments of GGF.
  • Line 1 is a listing of the predicted amino acid sequences of bovine GGF
  • line 2 is a listing of the nucleotide sequences of bovine GGF
  • line 3 is a listing of the nucleotide sequences of human GGF (heregulin) (nucleotide base matches are indicated with a vertica_ line)
  • line 4 is a listing of the predicted amino acid sequences of human GGF/heregulin where it differs from the predicted bovine sequence.
  • Coding segments E, A' and K represent only the bovine sequences.
  • Coding segment D' represents only the human (heregulin) sequence.
  • Fig. 32 (SEQ ID No. 148) is the predicted GGF2 amino acid sequence and nucleotide sequence of BPP5.
  • the upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence.
  • Pig. 33 (SEQ ID No. 149) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP2.
  • the upper line is the nucleotide sequence and the lower lihe is the predicted amino acid sequence.
  • Fig. 34 (SEQ ID No. 150) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP4.
  • the upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence.
  • Fig. 35 depicts the alignment of two GGF peptide sequences (GGF2bpp4 and GGF2bpp5) with the human EGF (hEGF) . Asterisks indicate positions of conserved cysteines.
  • Fig. 36 depicts the level of GGF activity (Schwann cell mitogenic assay) and tyrosine phosphorylation of a ca. 200kD protein (intensity of a 200 kD band on an autoradiogram of a Western blot developed with an antiphosphotyrosine polyclonal antibody) in response to increasing amounts of GGF.
  • Fig. 37 is a list of splicing variants derived from the sequences shown in Figure 31.
  • Pig. 38 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL1 (SEQ ID No.
  • Fig. 39 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL2 (SEQ ID No.
  • Fig. 40 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL3 (SEQ ID No.
  • Fig. 41 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL4 (SEQ ID No. 157) .
  • Fig. 42 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL5 (SEQ ID No.
  • Fig. 43 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL6 (SEQ ID No.
  • Fig. 44 is a scale coding segment map of the clone.
  • T3 refers to the bacteriophage promoter used to produce mRNA from the clone.
  • R flanking EcoRI restriction enzyme sites.
  • 5' UT refers to the 5' untranslated region.
  • E, B, A, C, C/D', and D refer to the coding segments.
  • O the translation start site.
  • the 5' limit of the region homologous to the bovine E segment (see example 6) and 3' UT refers to the 3' untranslated region.
  • Pig. 45 is the predicted amino acid sequence
  • the bottom (intermittent) sequence represents peptide sequences derived from GGF-II preparations (see Figures 11, 12) .
  • Fig. 46 is a graph depicting the Schwann cell mitogenic activity of recombinant human and bovine glial growth factors.
  • Fig. 47 is a dose-response curve depicting Schwann cell proliferation activity data resulting from administration of different size aliquots of CHO cell conditioned medium.
  • Pig. 48 is a dose-response curve depicting Schwann cell mitogenic activity secreted into the extracellular medium by SF9 insect cells infected with baculovirus containing the GGF2HBS5 cDNA clone.
  • Fig. 49 is a Western blot of recombinant CHO cell conditioned medium using a GGF peptide antibody.
  • Pig. 50 (A) is a graph of Schwann cell proliferation activity of recombinant (COS cell produced) human GGF-II (rhGGF-II) peak eluted from the cation exchange column;
  • (B) is an immunoblot against recombinant GGFII peak using polyclonal antibody made against specific peptide of rhGGFII;
  • FIG. 51 is a graph showing the purification of rhGGF-II (CHO cell produced) on cation exchange column by fraction;
  • (B) is a photograph of a Western blot using fractions as depicted in (A) and a rhGGF-II specific antibody.
  • Pig. 52 is a photograph of a gel depicting tyrosine phosphorylation in Schwann cells treated with recombinant glial growth factors.
  • Fig. 53 is the sequences of GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides (SEQ ID NOS: 170, 171, and 172).
  • Fig. 54 is a map of the CHO cell-expression vector pcDHFRpolyA.
  • the invention pertains to the isolation and purification of novel Glial Growth factors and the cloning of DNA sequences encoding these factors.
  • Other components of the invention are several gene splicing variants which potentially encode a series of glial growth factors, in particular the GGF2HBS5 in particular a variant which encodes the human equivalent of bovine GGF-II. It is evident that the gene encoding GGF's and pl85 erbB2 binding proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins, which are of different lengths and contain some common peptide sequences and some unique peptide sequences.
  • NDF neuro differentiation factor
  • the invention includes any protein which is substantially homologous to the coding segments in Figure 31 (SEQ ID No. ⁇ 136-147, 160, and 161) as well as other naturally occurring GGF polypeptides. Also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid naturally occurring (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference) ; and polypeptides or proteins specifically bound by antisera to GGF polypeptide.
  • the term also includes chimeric polypeptides that include the GGF polypeptides comprising sequences from Figure 31.
  • FGFs fibroblast growth factors
  • Hydroxylapatite HPLC Hydroxylapatite HPLC is not a technique hitherto used in isolating glial growth factors, but proved particularly efficacious in this invention.
  • the material obtained from the above CM-cellulose chromatography was filtered through a 0.22 ⁇ m filter (Nalgene) , loaded at room temperature on to a high performance hydroxylapatite column (50 x 50 mm, Biorad) equipped with a guard column (15 x 25 mm, Biorad) and equilibrated with 10 mM potassium phosphate pH 6.0. Elution at room temperature was carried out at a flow rate of 2 ml.minute "1 using the following programmed linear gradient:
  • Mono S FPLC enabled a more concentrated material to be prepared for subsequent gel filtration.
  • This step commenced the separation of the two factors of the invention prior to final purification, producing enriched fractions.
  • a preparative Supero ⁇ e 12 FPLC column (510 x 20 mm, Pharmacia) was packed according to the manufacturers' instructions.
  • a theoretical plates measurement was made according to the manufacturers' instructions, giving a value of 9,700 theoretical plates.
  • the pool of Mono S eluted material was applied at room temperature in 2.5 Ml aliquots to this column in 50mM sodium phosphate, 0.75 NaCl pH 6.0 (previously passed through a C18 reversed phase column (Sep-pak, Millipore) at a flow rate of 1.0 mL/minute "1 . 1 mL (0.5 minute) fractions were collected from 35 minutes after each sample was applied to the column. Fractions 27 to 41 (GGF-II) and 42 to 57 (GGF-I) inclusive from each run were pooled.
  • the GGF-I and GGF-II pools from the above Superose 12 runs were each divided into three equal aliquots. Each aliquot was loaded on to a C8 reversed-phase column (Aquapore RP-300 7 ⁇ C8 220 x 4.6 mm, Applied Biosystems) protected by a guard cartridge (RP-8, 15 x 3.2 mm, Applied Biosystems) and equilibrated to 40°C at 0.5 mL.minute. Protein was eluted under these conditions using the following programmed linear gradient:
  • GGFII GGFII inclusive from the reversed-phase runs were individually pooled. 7 ⁇ L of the pooled material was boiled in an equal volume of 0.0125 M Tris-Cl, 4% SDS, 20% glycerol, and 10% 3-mercaptoethanol for GGF-I, for 5 minutes and loaded on to an 11% polyacrylamide Laemmli gel with a 4% stacking gel and run at a constant voltage of 50 V for 16 hours. This gel was then fixed and stained using a silver staining kit (Amersham) . Under these conditions, the factors are each seen as a somewhat diffuse band at relative molecular weights 30,000 to 36,000 Daltons (GGF-I) and 55,000 to 63,000 Daltons (GGFII) as defined by molecular weight markers. From the gel staining, it is apparent that there are a small number of other protein species present at equivalent levels to the GGF-I and GGF-II species in the material pooled from the reversed-phase runs.
  • GGF-I Material from the reversed-phase HPLC, in the presence of 0.1% TFA and acetonitrile, was assayed within 12 hours of the completion of the column run and then after 10 weeks incubation at 40°C. Following incubation, the GGF-I had at least 50% of the activity of that material assayed directly off the column.
  • GGF-II Material from the reversed-phase HPLC, in the presence of 0.1% TFA and acetonitrile, and stored at -20°C, was assayed after thawing and then after 4 days incubation at 40°C. Following incubation, the GGF-II had at least 50% of the activity of that material freshly thawed.
  • [ 125 I]IUdR was added for the final 24 hours after the test solution addition.
  • the background (unstimulated) incorporation to each assay was less than 100 cpm, and maximal incorporation was 20 to 200 fold over background depending on Schwann cell batch and passage number.
  • Peptides were obtained by lysyl endopeptida ⁇ e and protease V8 digests, carried out on reduced and carboxymethylated sample ⁇ , with the ly ⁇ yl endopeptida ⁇ e dige ⁇ t of GGF-II carried out on material eluted from the 55-65 RD region of a 11% SDS-PAGE (MW relative to the above-quoted markers) .
  • a total of 21 peptide sequences (see Figure 9, SEQ ID Nos. 1-20, 169) were obtained for GGF-I, of which 12 peptides ( ⁇ ee Figure 10, SEQ ID Nos. 1, 22-29, 17, 19, and 32) are not present in current protein database ⁇ and therefore represent unique sequences.
  • a total of 12 peptide sequences (see Figure 11, SEQ ID Nos. 33-44) were obtained for GGF-II, of which 10 peptides (see Figure 12, SEQ ID No ⁇ .
  • X S S is consistent with the presence of an N linked carbohydrate moiety on an asparagine at the position denoted by X.
  • X represent ⁇ an unknown residue denoting a sequencing cycle where a single position could not be called with certainty either becau ⁇ e there was more than one signal of equal size in the cycle or becau ⁇ e no ⁇ ignal wa ⁇ pre ⁇ ent.
  • a ⁇ asterisk denotes those peptides where the last amino acid called corresponds to the last amino acid present in that peptide.
  • the ⁇ ignal ⁇ trength after the la ⁇ t amino acid called wa ⁇ in ⁇ ufficient to continue ⁇ equence calling to the end of that peptide.
  • the right hand column indicate ⁇ the re ⁇ ult ⁇ of a computer database search using the GCG package FASTA and TFASTA program ⁇ to analyze the NBRF and EMBL sequence databases.
  • the name of a protein in this column denote ⁇ identity of a portion of it ⁇ ⁇ equence with the peptide amino acid sequence called allowing a maximum of two mismatches.
  • a question mark denote ⁇ three mismatches allowed.
  • the abbreviations u ⁇ ed are a ⁇ follows:
  • re ⁇ idual acid wa ⁇ neutralized by filling the wells with 0.1 M borate buffer, pH 9.0, and the cells were washed with phosphate buffered saline (PBS) . Cells were then treated with 50 ⁇ l of blocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goat serum) for 15 min at 37 °C. After aspiration, monoclonal mouse anti-BrdU antibody (Dako Corp., Santa Barbara, CA) (50 ⁇ l/well, 1.4 ⁇ g/ l diluted in blocking buffer) wa ⁇ added and incubated for two hours at 37 °C.
  • PBS phosphate buffered saline
  • Unbound antibodies were removed by three wa ⁇ he ⁇ in PBS containing 0.1% Triton X-100 and peroxidase-conjugated goat ant-mou ⁇ e IgG antibody (Dako Corp., Santa Barbara, CA) (50 ⁇ l/well, 2 ⁇ g/ml diluted in blocking buffer) wa ⁇ added and incubated for one hour at 37°C. After three washe ⁇ in PBS/Triton and a final rin ⁇ e in PBS, well ⁇ received 100 ⁇ l/well of 50 mM pho ⁇ phate/citrate buffer, pH 5.0, containing 0.05% of the ⁇ oluble chromogen o-phenylenediamine (OPD) and 0.02% H 2 0 2 .
  • PPD ⁇ oluble chromogen o-phenylenediamine
  • the reaction wa ⁇ terminated after 5-20 min at room temperature, by pipetting 80 ⁇ l from each well to a clean plate containing 40 ⁇ l/well of 2N sulfuric acid. The absorbance was recorded at 490nm using a plate reader (Dynatech Lab ⁇ ) .
  • the assay plates containing the cell monolayers were wa ⁇ hed twice with PBS and immunocytochemically stained for BrdU-DNA by adding 100 ⁇ l/well of the substrate diaminobenzidine (DAB) and 0.02% H 2 0 2 to generate an insoluble product. After 10-20 min the staining reaction was stopped by washing with water, and BrdU-positive nuclei observed and counted u ⁇ ing an inverted micro ⁇ cope. occasionally, negative nuclei were counterstained with 0.001% Toluidine blue and counted a ⁇ before.
  • DAB diaminobenzidine
  • Cell line ⁇ used for Mitogene ⁇ i ⁇ A ⁇ av ⁇ Swi ⁇ 3T3 Fibroblast ⁇ Cell ⁇ , from Flow Lab ⁇ , were maintained in D' ⁇ M supplemented with 10% FCS, penicillin and streptomycin, at 37 °C in a humidified atmosphere of 10% C0 2 in air. Cells were fed or subcultured every two day ⁇ . For mitogenic assay, cells were plated at a density of 5,000 cells/well in complete medium and incubated for a week until cell ⁇ were confluent and quiescent. The serum containing medium was removed and the cell monolayer washed twice with serum free-medium.
  • BHK (Baby Hamster Kidney) 21 C13 Fibroblast ⁇ Cells from European Collection of Animal Cell Culture ⁇ (ECACC) , were maintained in Gla ⁇ gow Modified Eagle Medium (GMEM) supplemented with 5% tryptose phosphate broth, 5% FCS, penicillin and ⁇ treptomycin, at 37'C in a humidified atmo ⁇ phere of 5% C0 2 in air. Cell ⁇ were fed or subcultured every two to three days. For mitogenic assay, cells were plated at a density of 2,000 cell/well in complete medium for 24 hour ⁇ . The serum containing medium was then removed and after washing with ⁇ erum free medium, replaced with 100 ⁇ l of 0.1% FCS containing GMEM or GMEM alone.
  • GMEM Gla ⁇ gow Modified Eagle Medium
  • GGF ⁇ and FCS or bFGF a ⁇ po ⁇ itive control ⁇ were added, coincident with lO ⁇ M BrdU, and incubated for 48 hour ⁇ . Cell cultures were then proces ⁇ ed a ⁇ de ⁇ cribed for Schwann cell ⁇ .
  • C6 Rat Glioma Cell Line Cell ⁇ , obtained at pa ⁇ age 39, were maintained in DMEM containing 5% FCS, 5% Horse serum (HS) , penicillin and streptomycin, at 37 °C in a humidified atmosphere of 10% C0 2 in air. Cells were fed or subcultured every three days. For mitogenic a ⁇ ay, cell ⁇ were plated at a den ⁇ ity of 2,000 cell ⁇ /well in complete medium and incubated for 24 hour ⁇ . Then medium wa ⁇ replaced with a mixture of 1:1 DMEM and F12 medium containing 0.1% FCS, after wa ⁇ hing in ⁇ erum free medium. Do ⁇ e response ⁇ to GGF ⁇ , FCS and ⁇ FGF were then performed and cell ⁇ were proce ⁇ ed through the ELISA as previously described for the other cell types.
  • HS Horse serum
  • PC12 Rat Adrenal Pheochromocytoma Cell ⁇
  • ECACC ECACC
  • RPMI 1640 RPMI 1640 supplemented with 10% HS, 5% FCS, penicillin and streptomycin, in collagen coated flask ⁇ , at 37°C in a humidified atmosphere of 5% C0 2 in air. Cells were fed every three days by replacing 80% of the medium.
  • mitogenic assay cells were plated at a den ⁇ ity of 3,000 cell ⁇ /well in complete medium, on collagen coated plate ⁇ (50 ⁇ l/well collagen, Vitrogen Collagen Corp., diluted 1 : 50, 30 min at 37°C) and incubated for 24 hour ⁇ .
  • the medium was then placed with fre ⁇ h RPMI either alone or containing 1 mM in ⁇ ulin or 1% FCS. Do ⁇ e re ⁇ pon ⁇ es to FCS/HS (1:2) as positive control and to GGFs were performed as before. After 48 hour ⁇ cell ⁇ were fixed and the ELISA performed a ⁇ previou ⁇ ly de ⁇ cribed.
  • Figure 13 ⁇ how ⁇ the compari ⁇ on of data obtained with the two a ⁇ ay ⁇ , performed in the same cell culture condition ⁇ (5,000 cell ⁇ /well, in 5% FBP/DMEM, incubated in pre ⁇ ence of GGF ⁇ for 48hrs) .
  • the re ⁇ ult ⁇ are comparable, but BrdU incorporation a ⁇ ay appears to be slightly more sensitive, as sugge ⁇ ted by the ⁇ hift of the curve to the left of the graph, i.e. to lower concentration ⁇ of GGFS.
  • the BrdU incorporation a ⁇ ay can therefore provide additional u ⁇ eful information about the biological activity of polypeptide ⁇ on Schwann cell ⁇ when compared to the (125) I-UdR incorporation a ⁇ ay.
  • the data reported in Figure 15 show that GGFs can act on Schwann cells to induce DNA synthe ⁇ i ⁇ , but at lower do ⁇ e ⁇ to increa ⁇ e the number of negative cell ⁇ pre ⁇ ent in the icroculture after 48 hour ⁇ .
  • the total number of cells after 48 hours in presence of higher doses of GGFs is at least doubled, confirming that GGFs induce DNA ⁇ ynthe ⁇ i ⁇ and proliferation in BHK21 C13 cell ⁇ .
  • cells maintained for 48 hours in the presence of 2% FCS ⁇ howed an increa ⁇ e of about ⁇ ix fold (not ⁇ hown) .
  • C6 glioma cells have provided a useful model to ⁇ tudy glial cell propertie ⁇ .
  • the phenotype expressed seems to be dependent on the cell passage, the cell ⁇ more closely resembling an astrocyte phenotype at an early stage, and an oligodendrocyte phenotype at later stage ⁇ (beyond pa ⁇ age 70) .
  • C6 cell ⁇ used in these experiments were from passage 39 to pas ⁇ age 52.
  • C6 cell ⁇ are a highly proliferating population, therefore the experimental condition ⁇ were optimized to have a very low background of BrdU incorporation.
  • the pre ⁇ ence of 0.1% ⁇ erum was neces ⁇ ary to maintain cell viability without ⁇ ignificantly affecting the mitogenic responses, as shown by the do ⁇ e re ⁇ pon ⁇ e to FCS ( Figure 19) .
  • DNA ⁇ equence and polypeptide sequence should be obtainable by this means as with GGF-II, and al ⁇ o DNA con ⁇ tructs and expression vectors incorporating such DNA sequence, host cells genetically altered by incorporating such constructs/vectors, and protein obtainable by cultivating such host cells.
  • the invention envisage ⁇ ⁇ uch ⁇ ubject matter.
  • DNA oligomer probes were de ⁇ igned by backtran ⁇ lating the amino acid ⁇ equence ⁇ (derived from the peptide ⁇ generated from purified GGF protein) into nucleotide ⁇ equence ⁇ . Oligomer ⁇ repre ⁇ ented either the coding strand or the non-coding ⁇ trand of the DNA ⁇ equence.
  • ⁇ erine, arginine or leucine were included in the oligomer de ⁇ ign, then two ⁇ eparate ⁇ ynthe ⁇ e ⁇ were prepared to avoid ambiguitie ⁇ .
  • ⁇ erine was encoded by either TCN or AGY as in 537 and 538 or 609 and 610.
  • DNA oligomer ⁇ were ⁇ ynthe ⁇ ized on a Bio ⁇ earch 8750 4-column DNA synthesizer using ⁇ - cyanoethyl chemi ⁇ try operated at 0.2 micromole scale synthe ⁇ i ⁇ . Oligomer ⁇ were cleaved off the column (500 angstrom CpG resin ⁇ ) and deprotected in concentrated ammonium hydroxide for 6-24 hour ⁇ at 55-60°C.
  • oligomer ⁇ were dried under vacuum (Speedvac) and purified by electrophore ⁇ i ⁇ in gel ⁇ of 15% acrylamide (20 mono : 1 bi ⁇ ) , 50 mM Tri ⁇ -borate-EDTA buffer containing 7M urea. Full length oligomer ⁇ were detected in the gel ⁇ by UV ⁇ hadowing, then the band ⁇ were exci ⁇ ed and DNA oligomer ⁇ eluted into 1.5 l ⁇ H20 for 4-16 hours with shaking. The eluate was dried, redissolved in 0.1 ml H 2 0 and ab ⁇ orbance measurements were taken at 260nm. Concentrations were determined according to the following formula:
  • PCR primers were prepared by essentially the same procedures that were used for probes with the following modifications.
  • Linker ⁇ of thirteen nucleotide ⁇ containing restriction ⁇ ites were included at the 5' ends of the degenerate oligomer ⁇ for u ⁇ e in cloning into vector ⁇ .
  • DNA synthesis was performed at 1 micromole ⁇ cale u ⁇ ing 1,000 ang ⁇ trom CpG re ⁇ ins and inosine wa ⁇ u ⁇ ed at positions where all four nucleotides were incorporated normally into degenerate probe ⁇ .
  • Purifications of PCR primers included an ethanol precipitation following the gel electrophoresis purification.
  • a bovine genomic DNA library wa ⁇ purcha ⁇ ed from Stratagene (Catalogue Number: 945701) .
  • the library contained 2 x 10 6 15-2Okb Sau3Al partial bovine DNA fragment ⁇ cloned into the vector lambda Da ⁇ hll.
  • a bovine total brain CDNA library wa ⁇ purcha ⁇ ed from Clonetech (Catalogue Number: BL 10139) .
  • Complementary DNA libraries were constructed (In Vitrogen; Stratagene) from mRNA prepared from bovine total brain, from bovine pituitary and from bovine po ⁇ terior pituitary. In Vitrogen prepared two cDNA libraries: one library wa ⁇ in the vector lambda glO, the other in vector pcDNAI (a pla ⁇ mid library) .
  • the Stratagene librarie ⁇ were prepared in the vector lambda unizap. Collectively, the cDNA libraries contained 14 million primary recombinant phage.
  • the bovine genomic library was plated on E. coli K12 host strain LE392 on 23 x 23 cm plate ⁇ (Nunc) at 150,000 to 200,000 phage plaques per plate. Each plate represented approximately one bovine genome equivalent. Following an overnight incubation at 37 "c, the plates were chilled and replicate filter ⁇ were prepared according to procedure ⁇ of Maniati ⁇ et al. (2:60-81). Four plaque lift ⁇ were prepared from each plate onto uncharged nylon membrane ⁇ (Pall Biodyne A or MSI Nitropure) .
  • DNA probes were labelled using T4 polynucleotide kinase (New England Biolabs) with gamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to the ⁇ pecification ⁇ of the ⁇ uppliers. Briefly, 50 pmols of degenerate DNA oligomer were incubated in the presence of 600 ⁇ Ci gamma 32 P-ATP and 5 units T4 polynucleotide kinase for 30 minutes at 37 °C.
  • GMC buffer 0.52 M NaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1 M NaCl 10 mg/ml tRNA
  • Wa ⁇ hing wa ⁇ performed in oligowa ⁇ h (160 ml 1 M Na 2 HP0 4 , 200 ml 20% SDS, 8.0 ml 0.5 M EDTA, 100 ml 5M NaCl, 3632 ml H20) .
  • 20 filter ⁇ 400 ⁇ q. centimeter ⁇ each) representing replicate copies of ten bovine genome equivalent ⁇ were incubated in 200 ml hybridization ⁇ olution with 100 pmol ⁇ of degenerate oligonucleotide probe (128-512 fold degenerate) .
  • Hybridization wa ⁇ allowed to occur overnight at 5°C below the minimum melting temperature calculated for the degenerate probe. The calculation of minimum melting temperature assumes 2°C for an AT pair and 4°C for a GC pair.
  • Filters were washed in repeated changes of oligowa ⁇ h at the hybridization temperature ⁇ four to five hour ⁇ and finally, in 3.2M tetramethylammonium chloride, 1% SDS twice for 30 min at a temperature dependent on the DNA probe length.
  • the final wa ⁇ h temperature wa ⁇ 60°C.
  • Filter ⁇ were mounted, then expo ⁇ ed to X-ray film (Kodak XAR5) u ⁇ ing inten ⁇ ifying screens (Dupont Cronex Lightening Plus) .
  • X-ray film Kodak XAR5
  • u ⁇ ing inten ⁇ ifying screens Upont Cronex Lightening Plus
  • filters could be stripped and reprobed. Filters were stripped by incubating through two succe ⁇ ive cycles of fifteen minute ⁇ in a microwave oven at full power in a solution of 1% SDS containing lOmM EDTA pH8. Filters were taken through at least three to four cycles of stripping and reprobing with variou ⁇ probe ⁇ .
  • DNA was first depurinated in the gel by treatment with 0.125 N HC1, denatured in 0.5 N NaOH and transferred in 2Ox SSC (3M ⁇ odium chloride, 0.03 M ⁇ odium citrate) to uncharged nylon membrane ⁇ . Blotting was done for 6 hours up to 24 hours, then the filters were neutralized in 0.5 Tris HC1 pH 7.5, 0.15 M sodium chloride, then rinsed briefly in 50 mM Tris-borate EDTA. For cro ⁇ -linking, the filter ⁇ were wrapped fir ⁇ t in tran ⁇ parent pla ⁇ tic wrap, then the DNA ⁇ ide expo ⁇ ed for five minutes to an ultraviolet light.
  • Hybridization and wa ⁇ hing wa ⁇ performed a ⁇ de ⁇ cribed for library ⁇ creening ( ⁇ ee ⁇ ection 2 of this Example) .
  • For hybridization analy ⁇ i ⁇ to determine whether ⁇ imilar genes exi ⁇ t in other ⁇ pecie ⁇ ⁇ light modification ⁇ were made.
  • the DNA filter wa ⁇ purcha ⁇ ed from Clonetech (Catalogue Number 7753-1) and contain ⁇ 5 microgram ⁇ of EcoRI dige ⁇ ted DNA from variou ⁇ species per lane.
  • the probe was labelled by PCR amplification reaction ⁇ a ⁇ described in section 2 above, and hybridization ⁇ were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 g Ficoll-400, 2 g bovine serum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 g NaCl, 1 g ⁇ odium pyropho ⁇ phate, 10 g sodium dodecyl sulfate, 950ml H 2 0) containing 10% dextran sulfate.
  • the probes were denatured by boiling for ten minutes then rapidly cooling in ice water.
  • the probe was added to the hybridization buffer at 10 6 dpm 32 P per ml and incubated overnight at 60°C.
  • the filters were washed at 60°C first in buffer B followed by 2X SSC, 0.1% SDS then in lx SSC, 0.1% SDS. For high stringency, experiments, final washes were done in 0.1 x SSC, 1% SDS and the temperature raised to 65'C. Southern blot data were u ⁇ ed to prepare a re ⁇ triction map of the genomic clone and to indicate which subfragment ⁇ hybridized to the GGF- probe ⁇ (candidate ⁇ for ⁇ ubcloning) .
  • DNA digest ⁇ (e.g. 5 micrograms) were loaded onto 1% agarose gel ⁇ then appropriate fragment ⁇ exci ⁇ ed from the gel ⁇ following staining.
  • the DNA was purified by adsorption onto glass beads followed by elution using the protocol de ⁇ cribed by the ⁇ upplier (Bio 101) .
  • Recovered DNA fragment ⁇ (100-200 ng) were ligated into linearized depho ⁇ phorylated vectors, e.g. pT3T7 (Ambion) , which is a derivative of pUC18, using T4 ligase (New England Biolabs) .
  • Thi ⁇ vector carrie ⁇ the E.
  • coli ⁇ lactama ⁇ e gene hence, tran ⁇ formant ⁇ can be ⁇ elected on plate ⁇ containing ampicillin.
  • a portion of the ligation reactions was used to transform E. coli K12 XL1 blue competent cell ⁇ (Stratagene Catalogue Number: 200236) and then the tran ⁇ for ant ⁇ were ⁇ elected on LB plates containing 50 microgra s per ml ampicillin.
  • White colonie ⁇ were ⁇ elected and pla ⁇ mid mini preps were prepared for DNA digestion and for DNA sequence analysi ⁇ . Selected clone ⁇ were rete ⁇ ted to determine if their insert DNA hybridized with the GGF probes.
  • Double ⁇ tranded pla ⁇ mid DNA templates were prepared from 5 ml cultures according to ⁇ tandard protocol ⁇ . Sequencing wa ⁇ by the dideoxy chain termination method u ⁇ ing Sequena ⁇ e 2.0 and a dideoxynucleotide ⁇ equencing kit (US Biochemical) according to the manufacturer ⁇ protocol (a modification of Sanger et al. PNAS; USA 7 . 4:5463 (1977)].
  • ⁇ equencing wa ⁇ done in a DNA thermal cycler (Perkin Elmer, model 4800) u ⁇ ing a cycle ⁇ equencing kit (New England Biolabs; Bethesda Re ⁇ earch Laboratorie ⁇ ) and wa ⁇ performed according to manufacturer ⁇ in ⁇ truction ⁇ u ⁇ ing a 5'-end labelled primer.
  • Sequence primers were either those supplied with the sequencing kits or were synthe ⁇ ized according to ⁇ equence determined from the clone ⁇ . Sequencing reactions were loaded on and resolved on 0.4mm thick sequencing gels of 6% polyacrylamide. Gel ⁇ were dried and exposed to X-Ray film.
  • RNA Preparation and PCR Amplification were incorporated when ⁇ tandard ⁇ equencing kits were used and a 32P end labelled primer was used for cycle sequencing reactions. Sequences were read into a DNA sequence editor from the bottom of the gel to the top (5' direction to 3' ) and data were analyzed using program ⁇ ⁇ upplied by Genetic ⁇ Computer Group (GCG, University of Wisconsin) . VII. RNA Preparation and PCR Amplification
  • RNA was prepared from frozen bovine ti ⁇ ue (Pelfreeze) according to the guanidine neutral-C ⁇ Cl procedure (Chirgwin et. al. Biochemi ⁇ try 18:5294(1979).) Polyadenylated RNA wa ⁇ ⁇ elected by oligo-dT cellulo ⁇ e column chromatography (Aviv and Leder PNAS (USA) 69:1408 (1972)).
  • the primer ⁇ either were plu ⁇ ⁇ trand unique ⁇ equence ⁇ a ⁇ used in 3' RACE reactions (Frohman et. al., PNAS (USA) 85:8998 (1988)) or were oligo dT primers with restriction sites attached if the second target site had been added by terminal transferase tailing first strand reaction products with dATP (e.g. 5' race reaction ⁇ , Frohman et. al., ibid).
  • a ⁇ in anchored PCR reaction ⁇ the ⁇ econd ⁇ trand primer ⁇ were degenerate, hence, representing particular peptide sequence ⁇ .
  • the amplification profiles followed the following general scheme: 1) five minute ⁇ soak file at 95°C; 2) thermal cycle file of 1 minute, 95°C; 1 minute ramped down to an annealing temperature of 45°C, 50°C or 55"C; maintain the annealing temperature for one minute; ramp up to 72 °C over one minute; extend at 72 °C for one minute or for one minute plus a 10 second auto extension; 3) extension cycle at 72 °C, five minutes, and; 4) soak file 4°C for infinite time.
  • Thermal cycle files (#2) u ⁇ ually were run for 30 cycles.
  • DNA amplification products could be identified in the blotting experiments and their position ⁇ used as a guide to purification and reamplification.
  • the remaining portions of selected samples were loaded onto preparative gels, then following electrophore ⁇ is four to five slices of 0.5 mm thicknes ⁇ (bracketing the expected position of the ⁇ pecific product) were taken from the gel.
  • the crushed agarose wa ⁇ centrifuged for two minute ⁇ and the aqueou ⁇ pha ⁇ e wa ⁇ transferred to fresh tubes.
  • Reamplification was done on five microliters (roughly 1% of the product) of the eluted material u ⁇ ing the ⁇ ame ⁇ et ⁇ of primer ⁇ and the reaction profile ⁇ as in the original reaction ⁇ .
  • ⁇ ample ⁇ were extracted with chloroform and tran ⁇ ferred to fresh tubes.
  • Concentrated restriction enzyme buffers and enzymes were added to the reactions in order to cleave at the restriction sites present in the linkers.
  • the digested PCR products were purified by gel electrophoresi ⁇ , then subcloned into vector ⁇ as de ⁇ cribed in the ⁇ ubcloning section above. DNA sequencing was done described as above.
  • DNA sequence ⁇ were assembled using a fragment a ⁇ embly program and the amino acid ⁇ equence ⁇ deduced by the GCG programs GelA ⁇ semble, Map and Translate.
  • the deduced protein sequences were used a ⁇ a query sequence to search protein ⁇ equence databases u ⁇ ing WordSearch.
  • Analy ⁇ i ⁇ wa ⁇ done on a VAX Station 3100 work ⁇ tation operating under VMS 5.1.
  • the database search was done on Swis ⁇ Prot release number 21 using GCG Version 7.0.
  • GGF-II 12 SEQ ID No. 44
  • a peptide generated via lysyl endopeptida ⁇ e dige ⁇ tion of a purified GGF-II preparation showed strong amino acid ⁇ equence homology with GGF-I 07 (SEQ ID No. 39) , a tryptic peptide generated from a purified GGF-I preparation.
  • GGF-II 12 wa ⁇ thu ⁇ u ⁇ ed to create ten degenerate oligonucleotide probes (see oligos 609, 610 and 649 to 656 in Figure 21, SEQ ID Nos. 69, 70, 71 and 79, respectively) .
  • Hybridization signal ⁇ were ob ⁇ erved, but, only one clone hybridized to both probe sets.
  • the clone (designated GGF2BG1) wa ⁇ purified.
  • RNA and oligo dT- ⁇ elected (poly A containing) RNA were prepared from bovine total pituitary, anterior pituitary, posterior pituitary, and hypothalamus.
  • U ⁇ ing primer ⁇ from the li ⁇ t ⁇ hown in Figure 23, SEQ ID No ⁇ . 109-119, one- ⁇ ided PCR reactions (RACE) were used to amplify cDNA end ⁇ in both the 3' and 5' direction ⁇ , and anchored PCR reaction ⁇ were performed with degenerate oligonucleotide primer ⁇ repre ⁇ enting additional GGF-II peptide ⁇ .
  • Figure 24 ⁇ ummarize ⁇ the contiguou ⁇ DNA ⁇ tructure ⁇ and ⁇ equence ⁇ obtained in tho ⁇ e experiment ⁇ . From the 3' RACE reaction ⁇ , three alternatively ⁇ pliced cDNA ⁇ equence ⁇ were produced, which have been cloned and ⁇ equenced. A 5' RACE reaction led to the di ⁇ covery of an additional exon containing coding ⁇ equence for at lea ⁇ t 52 amino acid ⁇ . Analy ⁇ is of that deduced amino acid sequence revealed peptide ⁇ GGF-II-6 and a ⁇ equence ⁇ imilar to GGF-I-18 ( ⁇ ee below) .
  • the anchored PCR reaction ⁇ led to the identification of (cDNA) coding ⁇ equence ⁇ of peptide ⁇ GGF-II-1, 2, 3 and 10 contained within an additional cDNA ⁇ egment of 300 bp.
  • the 5' limit of thi ⁇ ⁇ egment i.e., ⁇ egment E, ⁇ ee Fig. 31
  • i ⁇ defined by the oligonucleotide which encodes peptide GGF-II-1 and which was used in the PCR reaction (additional 5' sequence data exists as de ⁇ cribed for the human clone in Example 6) .
  • this clone contain ⁇ nucleotide ⁇ equence ⁇ encoding six out of the existing total of nine novel GGF-II peptide sequence ⁇ .
  • the cloned gene was characterized fir ⁇ t by con ⁇ tructing a phy ⁇ ical map of GGF2BG1 that allowed u ⁇ to po ⁇ ition the coding ⁇ equence ⁇ a ⁇ they were found ( ⁇ ee below, Figure 25) .
  • DNA probes from the coding sequences described above have been used to identify further DNA fragments containing the exon ⁇ on thi ⁇ phage clone and to identify clone ⁇ that overlap in both direction ⁇ .
  • the putative bovine GGF-II gene i ⁇ divided into at least 5 coding segments. Coding segments are defined as discrete lengths of DNA sequence which can be translated into polypeptide sequence ⁇ using the univer ⁇ al genetic code.
  • the coding segments described in Figure 31 and referred to in the present application are: 1) particular exons present within the GGF gene (e.g. coding segment a) , or 2) derived from ⁇ ets of two or more exons that appear in specific ⁇ ub-group ⁇ of RNA ⁇ , where each set can be tran ⁇ lated into the ⁇ pecific polypeptide ⁇ egment ⁇ a ⁇ in the gene product ⁇ ⁇ hown.
  • the polypeptide ⁇ egments referred to in the claims are the translation products of the analogous DNA coding ⁇ egment ⁇ . Only coding ⁇ egment ⁇ A and B have been defined a ⁇ exon ⁇ and ⁇ equenced and mapped thu ⁇ far.
  • GGF2BPP1.CDS GGF2BPP2.CDS
  • GGF2BPP3.CDS GGF2BPP3.CDS
  • Figures 28a SEQ ID No. 133)
  • 28b SEQ ID No. 134
  • 28c SEQ ID No. 135)
  • the deduced amino acid sequence of the three cDNAs i ⁇ al ⁇ o given in Figure ⁇ 28a, (SEQ ID No. 133) , 28b (SEQ ID No. 134) , and 28c SEQ ID No. 135) .
  • the three deduced ⁇ tructure ⁇ encode protein ⁇ of length ⁇ 206, 281 and 257 amino acid ⁇ .
  • the fir ⁇ t 183 re ⁇ idue ⁇ of the deduced protein sequence are identical in all three gene products. At position 184 the clone ⁇ differ significantly.
  • a codon for glycine GGT in GGF2BPP1 also ⁇ erve ⁇ a ⁇ a ⁇ plice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exons C, C/D, C/D' and D or C, C/D and D, respectively, and shown in figure 33, SEQ ID No. 149) .
  • Thi ⁇ repre ⁇ ent ⁇ coding ⁇ egment A' in figure 31 (SEQ ID No. 140) .
  • All three of these molecules contain six of the nine novel GGF-II peptide sequence ⁇ (see Figure 12) and another peptide is highly homologous to GGF-I-18 ( ⁇ ee Figure 27) .
  • Thi ⁇ finding give ⁇ a high probability that thi ⁇ recombinant molecule encode ⁇ at lea ⁇ t a portion of bovine GGF-II.
  • the calculated i ⁇ oelectric point ⁇ for the three peptide ⁇ are con ⁇ istent with the physical propertie ⁇ of GGF-I and II. Since the molecular ⁇ ize of GGF-II i ⁇ roughly 60 kD, the longest of the three cDNAs ⁇ hould encode a protein with nearly one-half of the predicted number of amino acid ⁇ .
  • a probe encompa ⁇ ing the B and A exon ⁇ was labelled via PCR amplification and used to screen a cDNA library made from RNA isolated from bovine posterior pituitary.
  • One clone (GGF2BPP5) showed the pattern indicated in figure 30 and contained an additional DNA coding ⁇ egment (G) between coding ⁇ egment ⁇ A and C.
  • the entire nucleic acid ⁇ equence is shown in figure 32 (SEQ ID No. 148) .
  • the predicted translation product from the longest open reading frame is 241 amino acids.
  • a portion of a second cDNA (GGF2BPP4) was also isolated from the bovine posterior pituitary library using the probe de ⁇ cribed above. This clone showed the pattern indicated in figure 30.
  • BPP4 also displays a novel 3' end with regions H, K and L beyond region C/D.
  • Clone ⁇ detected with the ⁇ e probe ⁇ were further analyzed by hybridization.
  • a probe derived from coding ⁇ egment A (see Figure 21) , which was produced by labeling a polymerase chain reaction (PCR) product from ⁇ egment A, wa ⁇ al ⁇ o u ⁇ ed to screen the primary library.
  • PCR polymerase chain reaction
  • Several clones that hybridized with both A and E derived probes were selected and one particular clone, GGF2HBS5, was selected for further analy ⁇ i ⁇ .
  • Thi ⁇ clone i ⁇ repre ⁇ ented by the pattern of coding segments (EBACC/D'D as shown in Figure 31) .
  • E segment in thi ⁇ clone i ⁇ the human equivalent of the truncated bovine ver ⁇ ion of E ⁇ hown in Figure 37.
  • RNA encoding the GGF2HBS5 protein was produced in an in vitro transcription ⁇ y ⁇ tem driven by the bacteriophage T7 promoter re ⁇ ident in the vector (Blue ⁇ cript SK [Stratagene Inc.] ⁇ ee Figure 44) containing the GGF2HBS5 insert.
  • This RNA wa ⁇ translated in a cell free (rabbit reticulocyte) tran ⁇ lation ⁇ y ⁇ tem and the ⁇ ize of the protein product wa ⁇ 45 Kd. Additionally, the cell-free product has been as ⁇ ayed in a Schwann cell mitogenic a ⁇ ay to confirm biological activity.
  • Schwann cells treated with conditioned medium show both increased proliferation as measured by incorporation of I25 I-Uridine and phosphorylation on tyrosine of a protein in the 185 kilodalton range. Thu ⁇ the size of the product encoded by GGF2HBS5 and the presence of DNA sequences which encode human peptides highly homologous to the bovine peptides shown in Figure 12 confirm that GGF2HBS5 encodes the human equivalent of bovine GGF2. The fact that conditioned media prepared from cells tran ⁇ formed with thi ⁇ clone elicit ⁇ Schwann cell mitogenic activity confirm ⁇ that the GGFIIHBS5 gene produce (unlike the BPP5 gene product) is secreted.
  • GGFIIBPP5 gene product seems to mediate the Schwann cell proliferation respon ⁇ e via a receptor tyrosine kinase such as pi85 erbB2 or a closely related receptor ( ⁇ ee Example 14) .
  • the GGF2HBS5 cDNA clone encoding human GGF2 (as de ⁇ cribed in Example 6 and al ⁇ o referred to herein as HBS5) was cloned into vector pcDL-SR ⁇ 296 (Takebe et al. Mol. Cell. Biol. 8 . :466-472 (1988) and C0S-7 cells were transfected in 100 mm di ⁇ hes by the DEAE-dextran method (Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed. CSH Laboratory NY (1989) . Cell lysates or conditioned media from tran ⁇ iently expressing COS cell ⁇ were harvested at 3 or 4 day ⁇ po ⁇ t-tran ⁇ fection.
  • Conditioned media or cell lysate ⁇ ample ⁇ were te ⁇ ted in the Schwann cell proliferation a ⁇ ay a ⁇ de ⁇ cribed in Example 3.
  • the mitogenic activity data are ⁇ hown in Fig. 46.
  • the cDNA, GGF2HBS5, encoding GGF2 directed the ⁇ ecretion of the protein product to the medium.
  • GGF2HFB1 and GGFBPP5 cDNA' ⁇ failed to direct the ⁇ ecretion of the product to the extracellular medium.
  • GGF activity from the ⁇ e clone ⁇ wa ⁇ detectable only in cell ly ⁇ ate ⁇ (Fig. 46) .
  • GGF2 wa ⁇ al ⁇ o expre ⁇ ed in CHO cell ⁇ The GGF2HBS5 cDNA encoding GGF2 wa ⁇ cloned into the EcoRI ⁇ ite of vector pcdhfrpolyA (Fig. 54) and tran ⁇ fected into the DHFR negative CHO cell line (DG44) by the calcium pho ⁇ phate coprecipitation method (Graham and Van Der Eb,
  • Fig. 49 lane 12
  • Recombinant GGF2 was al ⁇ o expre ⁇ ed in insect cell ⁇ u ⁇ ing Baculoviru ⁇ expre ⁇ ion.
  • Sf9 in ⁇ ect cell ⁇ were infected with baculoviru ⁇ containing the GGF2HBS5 cDNA clone at a multiplicity of 3-5 (10 6 cells/ml) and cultured in Sf900-11 medium (Gibco) .
  • Schwann cell mitogenic activity was secreted into the extracellular medium (Fig. 48) .
  • Different volumes of insect cell conditioned medium were te ⁇ ted in the Schwann cell proliferation a ⁇ say in the absence of forskolin and the data used to produce the dose response curve shown in Fig. 48.
  • Schwann cell mitogenic activity of recombinant human and bovine glial growth factors was determined as follows: Mitogenic response ⁇ of cultured Schwann cell ⁇ were measured in the presence of 5 ⁇ M forskolin using crude recombinant GGF preparations obtained from transient mammalian expression experiments. Incorporation of [ 125 I]-Uridine wa ⁇ determined following an 18-24 hour exposure to materials obtained from tran ⁇ fected or mock transfected COS cells a ⁇ de ⁇ cribed in the Method ⁇ . The mean and standard deviation of four set ⁇ of data are ⁇ hown.
  • the mitogenic re ⁇ pon ⁇ e to partially purified native bovine pituitary GGF (carboxymethyl cellulo ⁇ e fraction; Goodearl et al., ⁇ ubmitted) i ⁇ ⁇ hown (GGF) a ⁇ a ⁇ tandard of one hundred percent activity.
  • cDNA ⁇ (Fig. 53) were cloned into pcDL-SR ⁇ .296 (Takebe et al., Mol. Cell Biol. 8:466-472 (1988))
  • COS-7 cell ⁇ were tran ⁇ fected in 100 mm di ⁇ hes by the DEAE- dextran method (Sambrook et al., In Molecular Cloning. A Laboratory Manual , 2nd . ed .
  • Cell ly ⁇ ate ⁇ or conditioned media were harvested at 3 or 4 days post-transfection.
  • To prepare ly ⁇ ate ⁇ cell monolayers were washed with PBS, ⁇ craped from the di ⁇ hes, and lysed by three freeze/than cycle ⁇ in 150 ⁇ l of 0.25 M Tris-HCl, pH 8. Cell debri ⁇ wa ⁇ pelleted and the ⁇ upernate recovered.
  • Conditioned media samples (7 mis) were collected, then concentrated and buffer exchanged with 10 mM Tri ⁇ , pH 7.4 u ⁇ ing Centriprep-10 and Centricon-10 units as de ⁇ cribed by the manufacturer (A icon, Beverly, MA) .
  • Rat ⁇ ciatic nerve Schwann cell ⁇ were a ⁇ ayed for incorporation of DNA ⁇ ynthe ⁇ i ⁇ precur ⁇ or ⁇ , a ⁇ de ⁇ cribed (Davi ⁇ and Stroobant, J. Cell Biol. 110:1353-1360 (1990); Brocke ⁇ et al., Brain Re ⁇ . 165:105-118 (1979) ) .
  • GGF related sequence ⁇ from human sources can also be easily isolated by using DNA probes derived_ from bovine GGF sequences.
  • DNA probes derived_ from bovine GGF sequences can also be easily isolated by using DNA probes derived_ from bovine GGF sequences.
  • the procedure described by Holmes et al. (Science 256: 1205 (1992)) can be used.
  • a human protein (heregulin ⁇ ) which bind ⁇ to and activate ⁇ the pl85 erbB2 receptor (and i ⁇ related to GGF) , is purified from a tumor cell line and the derived peptide sequence i ⁇ u ⁇ ed to produce oligonucleotide probe ⁇ which we borrowede utilized to clone the cDNA' ⁇ encoding heregulin.
  • Percell Biolytica microcarrier bead ⁇ (Hyclone Lab ⁇ ) .
  • the filtrate was applied to a heparin Sepharose column (Pharmacia) and the protein ⁇ were eluted with ⁇ teps of 0.3, 0.6, and 0.9 M NaCl in phosphate-buffered saline.
  • MCF-7 cells were plated in 24-well Costar plate ⁇ in F12 (50%) Dulbecco's minimum es ⁇ ential medium (50%) containing ⁇ erum (10%) (10 5 cell ⁇ per well) , and allowed to attach for at lea ⁇ t 24 hour ⁇ . Prior to a ⁇ ay, cell ⁇ were tran ⁇ ferred into medium without ⁇ erum for a minimum of 1 hour. Column fraction ⁇ (10 to 100 ⁇ l) were incubated for 30 min. at 37°.
  • Blots were probed with a monoclonal antibody (l:loon dilution) to phosphotyro ⁇ ine (Up ⁇ tate Biotechnology) for a minimum of 1 hour at room temperature. Blots were washed with TBST, probed with an antibody to mouse immunoglobulin G conjugated to alkaline phosphata ⁇ e (Promega) (diluted 1:7500) for a minimum of 30 min. at room temperature. Reactive bands were visualized with 5-bromo-4-chloro-3-indoyl-l-pho ⁇ phate and nitro-blue tetrazolium. Immunoblot ⁇ were ⁇ canned with a Scan Jet Plu ⁇ (Hewlett-Packard) den ⁇ ito eter.
  • Scan Jet Plu ⁇ Hewlett-Packard
  • a peak of activity (at -0.45 M NaCl) was further fractionated on a C4 reversed-pha ⁇ e column (SynChropak RP-4) equilibrated in buffer containing TFA (0.1%) and acetonitrile (15%). Protein ⁇ were eluted from thi ⁇ column with an acetonitrile gradient from 25 to 40% over 60 min. Fraction ⁇ (1 ml) were collected, a ⁇ ayed for activity, and analyzed by SDS-PAGE on tri ⁇ -glycine gel ⁇ (4-20%, Novex) .
  • [A]AEKEKTF[C]VNGGEXFMVKDLXNP (SEQ ID No. 162) .
  • Re ⁇ idues in bracket ⁇ were uncertain and an X repre ⁇ ent ⁇ a cycle in which it wa ⁇ not po ⁇ ible to identify the amino acid.
  • the initial yield was 8.5 pmol and the ⁇ equence did not correspond to any known protein.
  • Residue ⁇ 1, 9, 15, and 22 were later identified in the cDNA sequence as cysteine.
  • Direct ⁇ equencing of the -45-kD band from a gel that had been overloaded and blotted onto a PVDF membrane revealed a low abundance sequence XEXKE[G] [R]GK[G]K[G]KKKEXGXG[K] (SEQ ID No.
  • HRB-01 cDNA wa ⁇ identified by probing a ⁇ econd oligo(dT) -primed ⁇ gtlO library made from MDA-MB-231 cell mRNA with ⁇ equence ⁇ derived from both the 5' and 3' end ⁇ of proHRG- ⁇ .
  • a sequence corresponding to the 5' end of clone 13 as the probe was u ⁇ ed to identify proHRG32 and proHRG/33 in a third oligo(dT) -primed ⁇ gtlO library derived from MDA-MB-231 cell mRNA.
  • Two cDNA clone ⁇ encoding each of the four HRG ⁇ were ⁇ equenced (F. Sanger, S. Milken, A. R. Coul ⁇ on, Proc. Natl. Acad. Sci.U.S.A. .74 . , 5463 1977]).
  • Another cDNA de ⁇ ignated clone 84 ha ⁇ an amino acid ⁇ equence identical to proHRG/32 through amino acid 420.
  • EXAMPLE 9 I ⁇ olation of a Further Splicing Variant
  • the method ⁇ in Example 6 produced four clo ⁇ ely related ⁇ equences (heregulin , ⁇ l , ⁇ 2 , ⁇ 3 ) which arise as a result of splicing variation.
  • Peles et al. Cell 69, 205 (1992)
  • Wen et al. Cell 6£, 559 (1992)
  • the cDNA clone was obtained a ⁇ follow ⁇ (via the purification and ⁇ equencing of a pl85 ert,B2 binding protein from a tran ⁇ formed rat fibroblast cell line) .
  • a pl85 erbB2 binding protein was purified from conditioned medium as follows. Pooled conditioned medium from three harvests of 500 roller bottles (120 liters total) was cleared by filtration through 0.2 ⁇ filter ⁇ and concentrated 31-fold with a Pelicon ultrafiltration ⁇ y ⁇ tem using membranes with a 20kd molecular size cutoff. All the purification steps were performed by using a Pharmacia fast protein liquid chromatography system. The concentrated material was directly loaded on a column of heparin-Sepharose (150 ml, preequilibrated with phosphate-buffered saline (PBS) ) . The column wa ⁇ washed with PBS containing 0.2 M NaCl until no absorbance at 280 nm wavelength could be detected.
  • PBS phosphate-buffered saline
  • the pooled material was loaded on a phenyl-Superose column (HRlO/10, Pharmacia) .
  • the column was developed with a 45 ml gradient of (NH 4 ) 2 S0 4 (from 1.7 M to no salt) in 0.1 M Na 2 P0 4 (pH 7.4) , and 2 ml fractions were collected and a ⁇ ayed (0.002 ml per ⁇ ample) for kina ⁇ e ⁇ timulation (a ⁇ de ⁇ cribed in Example 6) .
  • the major peak of activity wa ⁇ pooled and dialyzed again ⁇ t 50 mM sodium pho ⁇ phate buffer (pH 7.3) .
  • T27.7 The fraction eluted after 27.7 min. (T27.7) contained mixed amino acid ⁇ equence ⁇ and was further rechromatographed after reduction as follows: A 70% aliquot of the peptide fraction was dried in vacuo and recon ⁇ tituted in 100 ⁇ l of 0.2 M ammonium bicarbonate buffer (pH 7.8) . DTT (final concentration 2 mM) wa ⁇ added to the ⁇ olution, which wa ⁇ then incubated at 37 °C for 30 min. The reduced peptide mixture was then separated by reverse-pha ⁇ e HPLC u ⁇ ing a Vydac column (2.1 mm i.d. x 15 cm) .
  • the protein wa ⁇ loaded onto a trifluoroacetic acid-treated glass fiber disc precycled with polybrene and NaCl.
  • the PTH-amino acid analysi ⁇ was performed with a micro liquid chromatography system (Model 120) using dual syringe pump ⁇ and rever ⁇ e-pha ⁇ e (C-18) narrow bore column ⁇ (Applied Bio ⁇ y ⁇ tems, 2.1 mm x 250 mm).
  • the synthetic oligonucleotides were end-labeled with [ ⁇ - 32 P]ATP with T4 polynucleotide kina ⁇ e and used to screen replicate set ⁇ of nitrocellulose filters.
  • the hybridization solution contained 6 x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% ⁇ odium pyropho ⁇ phate, 2 x Denhardt' ⁇ ⁇ olution, 50 ⁇ g/ml ⁇ almon ⁇ perm DNA, and 20% formamide (for probe 1) or no forma ide (for probe 2) .
  • the filter ⁇ were wa ⁇ hed at either 50°C with 0.5 x SSC, 0.2% SDS, 2 mM EDTA (for probe 1) or at 37 °C with 2 x SSC, 0.2% SDS, 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These clone ⁇ were purified by replating and probe hybridization as described above. The cDNA clones were ⁇ equenced u ⁇ ing an Applied Bio ⁇ ystems 373A automated DNA ⁇ equencer and Applied Bio ⁇ y ⁇ tem ⁇ Taq DyeDeoxyTM Terminator cycle ⁇ equencing kit ⁇ following the manufacture' ⁇ in ⁇ truction ⁇ .
  • the ⁇ e variant ⁇ can be obtained by coding segment specific probing of cDNA libraries derived from different ti ⁇ ue ⁇ and by PCR experiment ⁇ u ⁇ ing primer pair ⁇ ⁇ pecific to particular coding ⁇ egment ⁇ .
  • the variant ⁇ can be as ⁇ e bled from specific cDNA clones, PCR product ⁇ or genomic DNA region ⁇ via cutting and ⁇ plicing technique ⁇ known to one ⁇ killed in the art.
  • a rare re ⁇ triction enzyme cutting ⁇ ite in a common coding segment can be u ⁇ ed to connect the FBA amino terminus of GGF2BPP5 to carboxy terminal sequences of GGF2BPP1, GGFBPP2, GGFBPP3, or GGFBPP4. If the presence or the absence of coding ⁇ egment E and/or G provide benefit for contemplated and ⁇ tated u ⁇ e ⁇ , then the ⁇ e coding ⁇ egment ⁇ can be included in expre ⁇ ion con ⁇ truct ⁇ .
  • the ⁇ e variant ⁇ equence ⁇ can be expre ⁇ ed in recombinant ⁇ ystems and the recombinant product ⁇ can be a ⁇ ayed to determine their level of Schwann cell mitogenic activity a ⁇ well as their ability to bind and activate the pl85 erbB2 receptor.
  • EXAMPLE 11 Ide " ication of Functional Elements of GGF e . c od structure ⁇ of the family of GGF ue s in ⁇ , ⁇ e that the longe ⁇ t form ⁇ (as repre ⁇ ented by GGF2BPP4) encode tran ⁇ membrane protein ⁇ where the extracellular part contain ⁇ a domain which re ⁇ e ble ⁇ epidermal growth factor (see Carpenter and Wahl in Peptide Growth Factors and Their Receptors I pp. 69-133, Springer-Verlag, NY 1991) .
  • the positions of the cy ⁇ teine residues in coding segment ⁇ C and C/D or C/D' peptide ⁇ equence are con ⁇ erved with respect to the analogous residue ⁇ in the epidermal growth factor (EGF) peptide sequence (see Figure 35, SEQ ID Nos. 151-153) .
  • Several of the variant forms lack the H, K, and L coding segment ⁇ and thu ⁇ may be expre ⁇ ed a ⁇ ⁇ ecreted, diffu ⁇ ible biologically active protein ⁇ .
  • GGF DNA ⁇ equence ⁇ encoding polypeptide ⁇ which encompass the EGF- like domain (EGFL) can have full biological activity for stimulating glial cell mitogenic activity.
  • Membrane bound version ⁇ of thi ⁇ protein may induce Schwann cell proliferation if expre ⁇ ed on the ⁇ urface of neuron ⁇ during embryogene ⁇ i ⁇ or during nerve regeneration (where the ⁇ urfaces of neuron ⁇ are intimately a ⁇ ociated with the ⁇ urfaces of proliferating Schwann cells) .
  • Secreted (non membrane bound) GGFs may act as classically diffusible factors which can interact with Schwann cell ⁇ at ⁇ ome di ⁇ tance from their point of ⁇ ecretion.
  • Other form ⁇ may be relea ⁇ ed from intracells by sources via tis ⁇ ue injury and cell di ⁇ ruption.
  • GGF2HBS5 An example of a ⁇ ecreted GGF i ⁇ the protein encoded by GGF2HBS5 ( ⁇ ee example 6) ; this i ⁇ the only GGF known which ha ⁇ been found to be directed to the exterior of the cell (example 7) .
  • Secretion i ⁇ probably mediated via an N-terminal hydrophobic ⁇ equence found only in region E, which i ⁇ the N-terminal domain contained within recombinant GGF-II encoded by GGF2HBS5.
  • GGF's appear to be non-secreted (see example 6) . These GGFs may be injury respon ⁇ e form ⁇ which are relea ⁇ ed a ⁇ a con ⁇ equence of ti ⁇ ue damage. Other regions of the predicted protein structure of GGF-II (encoded by GGF2HBS5) and other proteins containing regions B and A exhibit ⁇ imilaritie ⁇ to the human ba ⁇ ement membrane heparan ⁇ ulfate proteoglycan core protein (ref.).
  • the peptide ADSGEY which i ⁇ located next to the ⁇ econd cy ⁇ teine of the C2 immunoglobulin fold in the ⁇ e GGF' ⁇ , occurs in nine of twenty-two C-2 repeats found in that basal lamina protein.
  • This evidence strongly suggest ⁇ that the ⁇ e protein ⁇ may as ⁇ ociate with matrix protein ⁇ ⁇ uch a ⁇ tho ⁇ e a ⁇ ociated with neuron ⁇ and glia, and may ⁇ ugge ⁇ t a method for ⁇ equestration of glial growth factors at target ⁇ ite ⁇ .
  • EXAMPLE 12 Purification of GGF ⁇ from Recombinant Cell ⁇
  • the proteins can be overproduced using cloned DNA.
  • a recombinant E. coli cell containing the sequence ⁇ de ⁇ cribed above can be con ⁇ tructed.
  • Expre ⁇ ion systems such as pNH8a or pHH16a (Stratagene, Inc.) can be used for this purpose by following manufacturers procedures.
  • these sequence ⁇ can be in ⁇ erted in a mammalian expre ⁇ ion vector and an overproducing cell line can be con ⁇ tructed.
  • Tran ⁇ ient expre ⁇ ion can be exa i: d or G418-re ⁇ istant clone ⁇ can be grown in tne pre ⁇ ence of methotrexate to select for cells that amplify the dhfr gene (contained on the pMSXND vector) and, in the process, co-amplify the adjacent GGF protein encoding ⁇ equence.
  • Becau ⁇ e CHO cell ⁇ can be maintained in a totally serum-free, protein-free medium (Hamilton and Ham, In Vitro H, 537-547 (1977)), the desired protein can be purified from the medium.
  • Western analysis u ⁇ ing the anti ⁇ era produced in Example 9 can be u ⁇ ed to detect the pre ⁇ ence of the de ⁇ ired protein in the conditioned medium of the overproducing cell ⁇ .
  • GGF-II purification on cation exchange columns was performed as follows: CHO cell conditioned media expre ⁇ ing rGGFII wa ⁇ loaded on the cation exchange column at 10 ml/min. The column wa ⁇ equilibrated with PBS pH 7.4. The elution wa ⁇ achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50 mM Hepe ⁇ 1M NaCl pH 8.0 re ⁇ pectively. All fraction ⁇ were analyzed u ⁇ ing the Schwann cell proliferation a ⁇ ay (CPM) described herein. The protein concentration (mg/ml) was determined by the Bradford as ⁇ ay u ⁇ ing BSA a ⁇ the ⁇ tandard.
  • recombinant material may be isolated from other variant ⁇ according to Wen et al. (Cell J59 . , 559 (1992)) who expre ⁇ ed the ⁇ plicing variant Neu differentiation factor (NDF) in COS-7 cell ⁇ .
  • cDNA clone ⁇ in ⁇ erted in the pJT-2 eukaryotic pla ⁇ mid vector are under the control of the SV40 early promoter, and are 3'-flanked with the SV40 termination and polyadenylation ⁇ ignal ⁇ .
  • COS-7 cells were transfected with the pJT-2 plasmid DNA by electroporation a ⁇ follows: 6 x 10 6 cell ⁇ (in 0.8 ml of DMEM and 10% FEBS) were tran ⁇ ferred to a 0.4 cm cuvette and mixed with 20 ⁇ g of pla ⁇ mid DNA in 10 ⁇ l of TE ⁇ olution (10 mM Tri ⁇ -HCl (pH 8.0) , 1 mM EDTA) . Electroporation wa ⁇ performed at room temperature at 1600 V and 25 ⁇ F using a Bio-Rad Gene Pulser apparatus with the pul ⁇ e controller unit ⁇ et at 200 ohms.
  • the cell ⁇ were then diluted into 20 ml of DMEM, 10% FBS and tran ⁇ ferred into a T75 fla ⁇ k (Falcon) . After 14 hr. of incubation at 37 °C, the medium wa ⁇ replaced with DMEM, 1% FBS, and the incubation continued for an additional 48 hr.
  • Conditioned medium containing recombinant protein which wa ⁇ harvested from the cells demonstrated biological activity in a cell line expres ⁇ ing the receptor for thi ⁇ protein.
  • This cell line (cultured human breast carcinoma cell line AU 565) was treated with recombinant material.
  • the treated cell ⁇ exhibited a morphology change which i ⁇ characteri ⁇ tic of the activation of the erbB2 receptor.
  • Conditioned medium of this type also can be tested in the Schwann cell proliferation as ⁇ ay.
  • Conditioned media collections were carried u ⁇ ing well-known procedures.
  • the media was concentrated 100-fold in an Amicon ultra-filtration cell (YM5 membrane) (Amicon, Danvers, MA) . Once clarified and concentrated, the media were stored at -20°C while consecutive collections were made during the following days.
  • the concentrated media were dialyzed using Spectra/por® 3 tubing (Spectrum Medical Industrie ⁇ , Lo ⁇ Angeles, CA) against 100 volumes of 0.1 M acetic acid over a two day period at 4°C.
  • the material that precipitated during dialysi ⁇ wa ⁇ removed by centrifugation at 4000 rpm for 30 min. at 4°C; protea ⁇ e inhibitor ⁇ were added.
  • the clarified ⁇ ample wa ⁇ then lyophilized.
  • Lyophilized conditioned medium was dissolved in 1 M acetic acid to a final concentration of about 25 mg/ml total protein. Insoluble material wa ⁇ removed by centrifugation at 10,000 rpm for 15 minutes. The sample was then loaded onto a Sephadex G-100 column (XK 16, Pharmacia, Pi ⁇ cataway, NJ) , was equilibrated and was subjected to elution with 1 M acetic acid at 4°C with an upward flow of 30 ml/hr. 100 ng of protein was proces ⁇ ed from 4 ml of 100-fold concentrated medium. Fraction ⁇ containing 3 ml of eluate were lyophilized and re ⁇ u ⁇ pended in 300 ⁇ l PBS for assay and served as a source for further purification.
  • a second HPLC ⁇ tep involved a ⁇ hallow acetonitrile gradient.
  • the pool of active fraction ⁇ from the previous HPLC ⁇ tep wa ⁇ rechromatographed over the ⁇ ame column.
  • Elution wa ⁇ performed with a 0-18% acetonitrile gradient in 0.05% TFA over a 5 minute period followed by a linear 18-45% acetonitrile gradient in 0.05% TFA over a 30 minute period.
  • the flow rate wa ⁇ 1.0 ml/min. and 1 ml fractions were collected.
  • Human TGF ⁇ -like factor wa ⁇ eluted at a 30-32% acetonitrile concentration a ⁇ a ⁇ ingle peak detectable by RRA.
  • the purified gp30 protein wa ⁇ te ⁇ ted in an a ⁇ ay to determine if it bound to pl85 crbB2 .
  • a competition a ⁇ ay with a monoclonal antibody again ⁇ t pl85 crbB2 .
  • Schwann cell proliferation activity of gp30 can also be demon ⁇ trated by treating Schwann cell culture ⁇ with purified gp30 u ⁇ ing the a ⁇ ay procedure de ⁇ cribed in Example ⁇ 1-3.
  • Pele ⁇ et al. (Cell 69 . , 205 (1992)) have al ⁇ o purified a l85 erbB2 stimulating ligand from rat cells, (NDF, ⁇ ee Example 8 for method) .
  • Holme ⁇ et al. (Science 256. 1205 (1992)) have purified Heregulin ⁇ from human cell ⁇ which bind ⁇ and ⁇ timulate ⁇ l85 erbB2 ( ⁇ ee example 6) .
  • the conditioned medium wa ⁇ cleared by low- ⁇ peed centrifugation and concentrated 100-fold in an Amicon ultrafiltration cell with a YM2 membrane (molecular weight cutoff of 2000) .
  • Biochemical analy ⁇ e ⁇ of the neu ⁇ timulatory activity in conditioned medium indicate that the ligand i ⁇ a 35-kD glycoprotein that it i ⁇ heat ⁇ table but ⁇ ensitive to reduction.
  • the factor is precipitable by either high salt concentrations or acidic alcohol.
  • Partial purification of the molecule by selective precipitation, heparin-agarose chromatography, and gel filtration in dilute acid resulted in an active ligand, which is capable of ⁇ timulating the protooncogenic receptor but i ⁇ ineffective on the oncogenic neu protein, which i ⁇ con ⁇ titutively active.
  • the purified fraction retained the ability to ⁇ timulate al ⁇ o the related receptor for EGF, ⁇ ugge ⁇ ting that the ⁇ e two receptor ⁇ are functionally coupled through a bidirectional mechani ⁇ .
  • the pre ⁇ umed ligand interact ⁇ ⁇ imultaneou ⁇ ly with both receptor ⁇ .
  • the pre ⁇ ented biochemical characteristic of the factor may be u ⁇ ed to enable a completely purified factor with which to explore the ⁇ e pos ⁇ ibilitie ⁇ .
  • ATL-2 cell line i ⁇ an IL-2-independent HTLV-1 (+) T cell line.
  • Mycopla ⁇ m-free ATL-2 cell ⁇ were maintained in RPMI 1640 medium containing 10% FCB a ⁇ the culture medium (10% FCS-RPMI 1640) at 37 °C in a humidified atmosphere with 5% C0 2 .
  • ATL-2 cell ⁇ were wa ⁇ hed twice in 1 x PBS and cultured at 3 x 10 5 ml in ⁇ erum-free RPMI 1640 medium/2 M L-glutamine for seventy-two hours followed by pelleting of the cells.
  • the culture supernatant so produced i ⁇ termed "conditioned medium" (CM.).
  • concentrated CM. containing components greater than 1000 MW were rediluted to original volume with RPMI medium.
  • Gel electrophore ⁇ is u ⁇ ing a polyacrylamide gradient gel (Integrated Separation Sy ⁇ tem ⁇ , Hyde Park, MD or Phoreca ⁇ t Sy ⁇ tem by Amer ⁇ ham, Arlington Height ⁇ , IL) followed by ⁇ ilver ⁇ taining of ⁇ ome of thi ⁇ two column purified material from the one liter preparation revealed at lea ⁇ t four to five band ⁇ of which the lOkD and 20kD band ⁇ were unique to thi ⁇ material. Pa ⁇ ed CM. containing component ⁇ le ⁇ than 1000 NW were used without dilution.
  • Concentrated conditioned medium was filter sterilized with a .45 ⁇ uniflo filter (Schleicher and Schuell, Keene, NH) and then further purified by application to a DEAE-SW anion exchange column (Waters, Inc., Milford, MA) which had been preequilibrated with lOmM Tris-Cl, pH 8.1 Concentrated CM. protein ⁇ repre ⁇ enting one liter of original ATL-2 conditioned medium per HPLC run were ab ⁇ orbed to the column and then eluted with a linear gradient of O M to 40mM NaCl at a flow rate of 4 ml/min.
  • Fraction ⁇ were a ⁇ ayed u ⁇ ing an in vitro immune complex kinase as ⁇ ay with 10% of the appropriate DEAE fraction (1 column purified material) or 1% of the appropriate C18 fractions (two column purified material) .
  • the proteins in the active fractions were concentrated and pooled, concentrated and subjected to C18 (million matrix) reverse phase chromatography (Waters, Inc., Milford, MA) (referred to as the C18+1 step or two column purified material) .
  • Elution wa ⁇ performed under a linear gradient of 2-propanol against 0.1% TFA. All the fractions were dialyzed against RPMI 1640 medium to remove the 2-propanol and as ⁇ ayed u ⁇ ing the jLn vitro immune complex kina ⁇ e a ⁇ ay, described below, and a 1% concentration of the appropriate fraction.
  • a twenty liter preparation employed the same purification strategy.
  • the DEAE active fractions 35-41 were pooled and subjected to cl8 chromatography as discu ⁇ sed above.
  • C18#l fractions 11-13 and 21-24 both had dose-dependent activity.
  • fractions 11-13 and 21-24 had activity.
  • the dose respon ⁇ e of fraction 23 a ⁇ determined by ijn vitro immune complex kinase as ⁇ ay a ⁇ de ⁇ cribed in Example 8 may be obtained upon addition of 0.005% by volume fraction 23 and 0.05% by volume fraction 23. Thi ⁇ repre ⁇ ent ⁇ the greate ⁇ t purity achieved.
  • Molecular weight range ⁇ were determined ba ⁇ ed on gel filtration chromatography and ultrafiltration membrane analysis. Near equal amounts of tyro ⁇ ine kina ⁇ e activity were retained and pa ⁇ ed by a 10,000 molecular weight cut off filter. Almo ⁇ t all activity wa ⁇ pa ⁇ ed by a 30,000 molecular weight cut off filter. Molecular weight range ⁇ for active chromatographic fraction ⁇ were determined by comparing fraction ⁇ containing dose-dependent neu-activating activity to the elution profiles of a ⁇ et of protein molecular weight ⁇ tandard ⁇ (Sigma Chemical Co., St. Loui ⁇ , MO) generated u ⁇ ing the ⁇ ame running condition ⁇ . A low molecular weight region of activity wa ⁇ identified between 7,000 and 14,000 daltons.
  • a second range of activity ranged from about 14,000 to about 24,000 daltons.
  • silver staining of the three-column purified material (cl8#2) was done with a commercially available silver staining kit (BioRad, Rockville Centre, NY) . Fraction 21, 22, 23, and 24 from cl8#2 purification of the twenty liter preparation were run with markers.
  • Fractions 22 and 23 showed the most potent dose re ⁇ pon ⁇ e in the l85 erbB2 (neu) kina ⁇ e a ⁇ ay ( ⁇ ee below) .
  • the fact that ⁇ elected molecular weight fraction ⁇ interact with 185 e ⁇ tB 2 was demon ⁇ trated with an immune complex kina ⁇ e a ⁇ ay.
  • Superdex 75 (fa ⁇ t protein liquid chromatography) .
  • Immune complex assay NDF for ligand binding to pl85 crb B2 This as ⁇ ay reflect ⁇ the difference ⁇ in the autopho ⁇ phorylation activity of immunoprecipitated pl85 driven by pre-incubation of PN-NR6 cell ly ⁇ ate with varying amount ⁇ of ATL-2 conditioned medium (C.H.) or proteinaceous sub ⁇ tance and is referred to hereinafter as neu-activating activity.
  • Cell line ⁇ were all maintained in DMEM medium containing 5% FCS a ⁇ the culture medium (5% FCS-DMEM) at 37°C in a humidified atmo ⁇ phere with 5% C0 2 .
  • Den ⁇ e culture ⁇ of cells in 150 mm dishes were washed twice with cold PBS, ⁇ craped into 10 ml of freeze-thaw buffer (150 mM NaCl, 1 mM MgCl 2 , 20 mM Hepe ⁇ , pH 7.2, 10% Glycerol, 1 mM EDTA, 1% Aprotinin) , and centrifuged (600 x 6, 10 minute ⁇ ) . Cell pellet ⁇ were re ⁇ u ⁇ pended in
  • the cell ly ⁇ ate ⁇ were incubated for fifteen minute ⁇ with 50 ⁇ l of 50% (volume/volume) Protein A-sepharose (Sigma Chemical Co., St. Loui ⁇ , Missouri) , and centrifugated for two minutes to preclear the ly ⁇ ate ⁇ . 50 ⁇ l aliquot ⁇ of precleared cell ly ⁇ ate were incubated on ice for fifteen minute ⁇ with conditioned medium, proteinaceous sub ⁇ tance, or other factor ⁇ a ⁇ ⁇ pecified, in a final volume of 1 ml with ly ⁇ i ⁇ buffer.
  • 50% (volume/volume) Protein A-sepharose Sigma Chemical Co., St. Loui ⁇ , Missouri
  • the sample was then incubated with 5 ⁇ g of 7.16.4 monoclonal antibody, which recognizes the extracellular domain of the pl85neu and pl85c-neu, or other appropriate antibodies, for twenty minutes on ice, followed by a twenty minute incubation with 50 ⁇ l of 50% (vol/vol) protein A-Sepharose with rotation at 4°C Immune complexes were collected by centrifugation, wa ⁇ hed four time ⁇ with 500 ⁇ l of wa ⁇ hing buffer (50 mM Hepe ⁇ , pH 7.5, 0.1%, Brij 35, 150 mM NaCl, 2 mM EDTA, 1% Aprontinin, 30 ⁇ m Na 3 V0 4 ) , then twice with reaction buffer (20 mM Hepes (pH 7.4), 3 mM MnCl 2 and 0.1% Brij 35, 30 ⁇ m Na 3 V0 4 ) .
  • wa ⁇ hing buffer 50 mM Hepe ⁇ , pH 7.5, 0.1%, Brij 35, 150 mM NaCl
  • Pellets were re ⁇ uspended in 50 ⁇ l of reaction buffer and (Gamma- 32 P]-ATP (Amersham, Arlington Height ⁇ , IL) wa ⁇ added giving a final concentration of 0.2 ⁇ .
  • the ⁇ ample ⁇ were incubated at 27°C for twenty minute ⁇ or at 4°C for 25 minute ⁇ with purer ⁇ ample ⁇ .
  • the reaction ⁇ were terminated by addition of 3 x SDS ⁇ ample buffer containing 2 mM ATP and 2 mM EDTA and then incubating them at 100°C for five minutes.
  • the samples were then subjected to SDS-PAGE analysi ⁇ on 10% acrylamide gel ⁇ . Gel ⁇ were ⁇ tained, dried, and expo ⁇ ed to Kodak XAR or XRP film with inten ⁇ ifying screens.
  • ARIA a 42 kD protein which stimulates acetylcholine receptor synthesis, has been isolated in the laboratory of Gerald Fi ⁇ chbach (Fall ⁇ et al., Cell 72:801-815 (1993)) .
  • ARIA induce ⁇ tyro ⁇ ine phosphorylation of a 185 Kda mu ⁇ cle tran ⁇ membrane protein which resembles pl85 erB2 , and stimulate ⁇ acetylcholine receptor ⁇ ynthe ⁇ i ⁇ in cultured embryonic myotube ⁇ .
  • Sequence analy ⁇ is of cDNA clones which encode ARIA shows that ARIA is a member of the GGF/erbB2 ligand group of protein ⁇ , and this i ⁇ potentially u ⁇ eful in the glial cell mitogene ⁇ i ⁇ stimulation and other applications of, e.g., GGF2 de ⁇ cribed herein.
  • Rat Schwann cells following treatment with sufficient levels of Glial Growth Factor to induce proliferation, show stimulation of protein tyro ⁇ ine pho ⁇ phorylation (figure 36) .
  • Varying amounts of partially purified GGF were applied to a primary culture of rat Schwann cell ⁇ according to the procedure outlined in Example 3.
  • Schwann cell ⁇ were grown in DMEM/10% fetal calf ⁇ erum/5 ⁇ M forskolin/0.5 ⁇ g per mL GGF-CM (0.5mL per well) in poly D-lysine coated 24 well plate ⁇ . When confluent, the cell ⁇ were fed with DMEM/10% fetal calf ⁇ erum at 0.5mL per well and left in the incubator overnight to quie ⁇ ce.
  • the cell ⁇ were fed with 0.2mL of DMEM/10% fetal calf ⁇ erum and left in the incubator for 1 hour. Te ⁇ t ⁇ ample ⁇ were then added directly to the medium at different concentrations and for different lengths of time as required.
  • the cell ⁇ were then ly ⁇ ed in boiling ly ⁇ i ⁇ buffer ( ⁇ odium phosphate, 5mM, pH 6.8; SDS, 2%, /3-mercapteothanol, 5%; dithiothreitol, 0.1M; glycerol, 10%; Bromophenol Blue, 0.4%; sodium vanadate, lOmM) , incubated in a boiling water bath for 10 minutes and then either analyzed directly or frozen at -70"C Samples were analyzed by running on 7.5% SDS-PAGE gels and then electroblotting onto nitrocellulo ⁇ e u ⁇ ing ⁇ tandard procedure ⁇ a ⁇ de ⁇ cribed by Towbin et al. (1979) Proc. Natl.
  • the dose re ⁇ pon ⁇ e ⁇ of protein pho ⁇ phorylation and Schwann cell proliferation are very ⁇ imilar (figure 36) .
  • Similar re ⁇ ult ⁇ were obtained when Schwann cell ⁇ were treated with conditioned media prepared from COS cell ⁇ tran ⁇ late ⁇ with the GGF2HBS5 clone.
  • the ⁇ e re ⁇ ult ⁇ correlate well with the expected interaction of the GGF ⁇ with and activation of -l85 erbB2 .
  • Schwann cell proliferation i ⁇ mediated by conditioned medium derived from the human breast cancer cell line MDA-MB-231 was plated in 100 ⁇ l of Dulbecco's Modified Eagle' ⁇ medium ⁇ upplemented with 5% fetal bovine pla ⁇ a per well in a 96 well microtiter plate.
  • 10 ⁇ l of conditioned medium was added to each well of the microtiter plate.
  • the number of Schwann cells per plate was determined using an acid phosphata ⁇ e a ⁇ ay (according to the procedure of Connolly et al. Anal. Biochem. 152 : 136 (1986)) .
  • the plate was incubated at 37 °C for two hours and the reaction was stopped by the addition of 10 ⁇ l of IN NaOH.
  • the optical density of each sample was read in a spectrophotometer at 410 nm.
  • a 38% stimulation of cell number over Schwann cells treated with conditioned medium from a control cell line (HS-294T, a non-producer of erbB-2 ligand) was observed. This result show ⁇ that a protein ⁇ ecreted by the MDA-MB-231 cell line (which secretes a pl85 c, B2 binding activity) stimulate ⁇ Schwann cell proliferation.
  • the protein ⁇ equence predicted from the cDNA sequence of GGF-II candidate clone ⁇ GGF2BPP1,2 and 3 contain ⁇ a number of consensu ⁇ N-glyco ⁇ ylation motif ⁇ .
  • a gap in the GGFII02 peptide ⁇ equence coincide ⁇ with the a ⁇ paragine re ⁇ idue in one of the ⁇ e motif ⁇ , indicating that carbohydrate is probably bound at this site.
  • N-glycosylation of the GGF ⁇ wa ⁇ ⁇ tudied by ob ⁇ erving mobility change ⁇ on SDS-PAGE after incubation with N- glycanase an enzyme that cleaves the covalent linkage ⁇ between carbohydrate and a ⁇ pargine re ⁇ idue ⁇ in proteins.
  • N-Glycanase treatment of GGF-II yielded a major band of MW 40-42 kDa and a minor band at 45-48 kDa.
  • Activity elution experiments under non-reducing conditions ⁇ howed a ⁇ ingle active deglyco ⁇ ylated ⁇ pecies at ca 45-50 kDa.
  • Nucleic acid encoding GGF-II (cDNA, GGF2HBS5) protein (Example 6) in a plasmid pBlue ⁇ cript 5k, under the control of the T7 promoter, wa ⁇ depo ⁇ ited in the American Type Culture Collection, Rockville, Maryland, on September 2, 1992, and given ATCC Acce ⁇ sion No. 75298.
  • Applicant acknowledges its responsibility to replace this plasmid ⁇ hould it become non-viable before the end of the term of a patent i ⁇ ued hereon, and it ⁇ re ⁇ pon ⁇ ibility to notify the ATCC of the i ⁇ uance of ⁇ uch a patent, at which time the depo ⁇ it will be made available to the public. Prior to that time the depo ⁇ it will be made available to the Commi ⁇ ioner of Patent ⁇ under the term ⁇ of 37 CFR ⁇ 1.14 and 35 USC ⁇ 112.

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Abstract

Disclosed is the characterization and purification of DNA encoding a numerous polypeptides useful for the stimulation of glial cell (particularly, Schwann cell) mitogenesis and treatment of glial cell tumors. Also disclosed are DNA sequences encoding novel polypeptides which may have use in stimulating glial cell mitogenesis and treating glial cell tumors. Methods for the synthesis, purification and testing of both known and novel polypeptides for their use as both therapeutic and diagnostic aids in the treatment of diseases involving glial cells are also provided. Methods are also provided for the use of these polypeptides for the preparation of antibody probes useful for both diagnostic and therapeutic use in diseases involving glial cells.

Description

GLIAL MITOGENIC FACTORS. THEIR PREPARATION AND USE
Cross Reference to Related Application This application is a continuation-in-part of Serial No. 07/965,173, filed October 23, 1992, Serial No. 07/940,389, filed September 3, 1992, Serial No. 07/907,138, filed June 30, 1992 and Serial No. 07/863,703, filed April 3, 1992.
Background of the Invention This invention relates to polypeptides found in vertebrate species, which polypeptides are mitogenic growth factors for glial cells, including Schwann cells. The invention is also concerned with processes capable of producing such factors, and the therapeutic application of such factors.
The glial cells of vertebrates constitute the specialized connective tissue of the central and peripheral nervous systems. Important glial cells include Schwann cells which provide metabolic support for neurons and which provide myelin sheathing around the axons of certain peripheral neurons, thereby forming individual nerve fibers. Schwann cells support neurons and provide a sheath effect by forming concentric layers of membrane around adjacent neural axons, twisting as they develop around the axons. These myelin sheaths are a susceptible element of many nerve fibers, and damage to Schwann cells, or failure in growth and development, can be associated with significant demyelination or nerve degeneration characteristic of a number of peripheral nervous system diseases and disorders. In the development of the nervous system, it has become apparent that cells require various factors to regulate their division and growth, and various such factors have been identified in recent years, including some found to have an effect on Schwann cell division or development. Thus, Brockes et al., inter alia, in J. Neuroscience, 4. (1984) 75-83 describe a protein growth factor present in extracts from bovine brain and pituitary tissue, which was named Glial Growth Factor (GGF) . This factor stimulated cultured rat Schwann cells to divide against a background medium containing ten percent fetal calf serum. The factor was also described as having a molecular weight of 31,000 Daltons and as readily di erizing. In Meth. Enz., 147 (1987), 217-225, Brockes describes a Schwann cell-based assay for GGF.
Brockes et al., supra, also describes a method of purification of GGF to apparent homogeneity. In brief, one large-scale purification method described involves extraction of the lyophilized bovine anterior lobes and chromatography of material obtained thereby using NaCl gradient elution from CM cellulose. Gel filtration is then carried out with an Ultrogel column, followed by elution from a phosphocellulose column, and finally, small-scale SDS gel electrophoresiε. Alternatively, the CM-cellulose material was applied directly to a phosphocellulose column, fractions from the column were pooled and purified by preparative native gel electrophoresis, followed by a final SDS gel electrophoresis.
Brockes et al. observe that in previously reported gel filtration experiments (Brockes et al., J. Biol. Chem. 255 (1980) 8374-8377) , the major peak of growth factor activity was observed to migrate with a molecular weight of 56,000 Daltons, whereas in the first of the above-described procedures activity was predominantly observed at molecular weight 31,000. It is reported that the GGF dimer is largely removed as a result of the gradient elution from CM-cellulose in this procedure.
Benveniste et al. (PNAS, 8 (1985) , 3930-3934) describes a T lymphocyte-derived glial growth promoting factor. This factor, under reducing conditions, exhibits a change in apparent molecular weight on SDS gels. Kimura et al. (Nature, 348 (1990) , 257-260) describe a factor they term Schwannoma-derived growth factor (SDGF) which is obtained from a sciatic nerve sheath tumor. The authors state that SDGF does not stimulate the incorporation of tritium-labelled TdR into cultured Schwann cells under conditions where, in contrast, partially purified pituitary fraction containing GGF is active. SDGF has an apparent molecular weight of between 31,000 and 35,000. Davis and Stroobant (J. Cell. Biol., 110 (1990),
1353-1360) describe the screening of a number of candidate mitogens. Rat Schwann cells were used, the chosen candidate substances being examined for their ability to stimulate DNA synthesis in the Schwann cells in the presence of 10% FCS (fetal calf serum) , with and without forskolin. One of the factors tested was GGF-carboxymethyl cellulose fraction (GGF-CM) , which was itogenic in the presence of FCS, with and without forskolin. The work revealed that in the presence of forskolin, inter alia, platelet derived growth factor (PDGF) was a potent itogen for Schwann cells, PDGF having previously been thought to have no effect on Schwann cells.
Holmes et al. Science (1992) 256; 1205 and Wen et al. Cell (1992) 69_ 559 demonstrate that DNA sequences which encode proteins binding to a receptor (pl85ert)B2) are associated with several human tumors.
The pl85erbB2 protein is a 185 kilodalton membrane spanning protein with tyrosine kinase activity. The protein is encoded by the erbB2 proto-oncogene (Yarden and Ullrich Ann. Rev. Bioche . 5_7: 443 (1988)) . The erbB2 gene, also referred to as HER-2 (in human cells) and neu (in rat cells) , is closely related to the receptor for epidermal growth factor (EGF) . Recent evidence indicates that proteins which interact with (and activate the kinase of) pl85erbB2 induce proliferation in the cells bearing pl85erbB2 (Holmes et al. Science 256: 1205 (1992); Dobashi et al. Proc. Natl. Acad. Sci. 88.: 8582 (1991); Lupu et al. Proc. Natl. Acad. Sci. 89.: 2287 (1992)). Furthermore, it is evident that the gene encoding pl85erbB2 binding proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins, which are of different lengths and contain some common peptide sequences and some unique peptide sequences. This is supported by the differentially-spliced RNA transcripts recoverable from human breast cancer (MDA-MB-231) (Holmes et al. Science 256: 1205 (1992) ) . Further support derives from the wide size range of proteins which act as (as disclosed herein) ligands for the pl85eΛB2 receptor (see below) .
Summary of the Invention
In general the invention provides methods for stimulating glial cell (in particular, Schwann cell and glia of the central nervous system) mitogenesis, as well as new proteins exhibiting such glial cell mitogenic activity. In addition, DNA encoding these proteins and antibodies which bind these and related proteins are provided.
The novel proteins of the invention include alternative splicing products of sequences encoding known polypeptides. Generally, these known proteins are members of the GGF/pl85erbB2 family of proteins.
Specifically, the invention provides polypeptides of a specified formula, and DNA sequences encoding those polypeptides. The polypeptides have the formula WYBAZCX wherein WYBAZCX is composed of the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141- 147, 160, 161); wherein W comprises the polypeptide segment F, or is absent; wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segments C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D' HKL; provided that, either a) at least one of F, Y, B, A, Z, C, or X is of bovine origin; or b) Y comprises the polypeptide segment E; or c) X comprises the polypeptide segments C/D HKL, C/D
D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, C/D'H, C/D C/D'H, or C/D C/D' HL.
In addition, the invention includes the DNA sequence comprising coding segments 5FBA3' as well as the with corresponding polypeptide segments having the a ino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 139); the DNA sequence comprising the coding segments 5FBA'3' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 140) ; the DNA sequence comprising the coding segments 5FEBA3 as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139) ; the DNA sequence comprising the coding segments 5FEBA 3' as well as the corresponding polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-138, 140); and the DNA sequence comprising the polypeptide coding segments of the GGF2HBS5 cDNA clone (ATCC Deposit No. 75298, deposited September 2, 1992).
The invention further includes peptides of the formula FBA, FEBA, FBA' FEBA' and DNA sequences encoding these peptides wherein the polypeptide segments correspond to amino acid sequences shown in Figure 31, SEQ ID Nos. (136, 138 and 139), (136-139) and (136, 138 and 140) and (136-138 and 140) respectively. The polypeptide purified GGF-II polypeptide (SEQ ID No. 167) is also included as a part of the invention.
Further included as an aspect of the invention are peptides and DNA encoding such peptides which are useful for the treatment of glia and in particular oligodendrocytes, microglia and astrocytes, of the central nervous system and methods for the administration of these peptides.
The invention further includes vectors including DNA sequences which encode the amino acid sequences, as defined above. Also included are a host cell containing the isolated DNA encoding the amino acid sequences, as defined above. The invention further includes those compounds which bind the pl85crbB2 receptor and stimulate glial cell mitogenesis in vivo and/or in vitro .
Also a part of the invention are antibodies to the novel peptides described herein. In addition, antibodies to any of the peptides described herein may be used for the purification of polypeptides described herein. The antibodies to the polypeptides may also be used for the therapeutic inhibitor of glial cell mitogenesis.
The invention further provides a method for stimulating glial cell mitogenesis comprising contacting glial cells with a polypeptide defined by the formula
WYBAZCX wherein WYBAZCX is composed of the polypeptide segments shown in Figure 31 (SEQ ID Nos. 136-139, 141- 147, 160, 161); wherein W comprises the polypeptide segment F, or is absent wherein Y comprises the polypeptide segment E, or is absent; wherein Z comprises the polypeptide segment G or is absent; and wherein X comprises the polypeptide segment C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D' HKL.
The invention also includes a method for the preparation of a glial cell mitogenic factor which consist of culturing modified host cells as defined above under conditions permitting expression of the DNA sequences of the invention.
The peptides of the invention can be used to make a pharmaceutical or veterinary formulation for pharmaceutical or veterinary use. Optionally, the formulation may be used together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
A method for stimulating mitogenesis of a glial cell by contacting the glial cell with a polypeptide defined above as a glial cell mitogen in vivo or in vitro is also an aspect of the invention. A method for producing a glial cell mitogenic effect in a vertebrate (preferably a mammal, more preferably a human) by administering an effective amount of a polypeptide as defined is also a component of the invention.
Methods for treatment of diseases and disorders using the polypeptides described are also a part of the invention. For instance, a method of treatment or prophylaxis for a nervous disease or disorder can be effected with the polypeptides described. Also included are a method for the prophylaxis or treatment of a pathophysiological condition of the nervous system in which a cell type is involved which is sensitive or responsive to a polypeptide as defined are a part of the invention.
Included in the invention as well, are methods for treatment when the condition involves peripheral nerve damage; nerve damage in the central nervous system; neurodegenerative disorders; demyelination in peripheral or central nervous system; or damage or loss of Schwann cells, oligodendrocytes, microglia, or astrocytes. For example, a neuropathy of sensory or motor nerve fibers; or the treatment of a neurodegenerative disorder are included. In any of these cases, treatment consists of administering an effective amount of the polypeptide.
The invention also includes a method for inducing neural regeneration and/or repair by administering an effective amount of a polypeptide as defined above. Such a medicament is made by administering the polypeptide with a pharmaceutically effective carrier.
The invention includes the use of a polypeptide as defined above in the manufacture of a medicament.
The invention further includes the use of a polypeptide as defined above
-to immunize a mammal for producing antibodies, which can optionally be used for therapeutic or diagnostic purposes
-in a competitive assay to identify or quantify molecules having receptor binding characteristics corresponding to those of the polypeptide; and/or
-for contacting a sample with a polypeptide, as mentioned above, along with a receptor capable of binding specifically to the polypeptide for the purpose of detecting competitive inhibition of binding to the polypeptide.
-in an affinity isolation process, optionally affinity chromatography, for the separation of a corresponding receptor.
The invention also includes a method for the prophylaxis or treatment of a glial tumor. This method consists of administering an effective amount of a substance which inhibits the binding of a factor as defined by the peptides above.
Furthermore, the invention includes a method of stimulating glial cell mitogenic activity by the application to the glial cell of a
-30 kD polypeptide factor isolated from the MDA - MB 231 human breast cell line; or -35 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line to the glial cell or
-75 kD polypeptide factor isolated from the SKBR-3 human breast cell line; or
-44 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line; or
-25kD polypeptide factor isolated from activated mouse peritoneal macrophages; or
-45 kD polypeptide factor isolated from the MDA - MB 231 human breast cell; or
-7 to 14 kD polypeptide factor isolated from the ATL-2 human T-cell line to the glial cell; or
-25 kD polypeptide factor isolated from the bovine kidney cells; or
-42 kD polypeptide factor (ARIA) isolated from brains.
The invention further includes a method for the use of the EGFL1, EGFL2, EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, Figure 38 to 43 and SEQ ID Nos. 154 to 159, respectively, for the stimulation of glial cell mitogenesis in vivo and in vitro.
Also included in the invention is the administration of the GGF-II polypeptide whose sequence is shown in Figure 45 for the stimulation of glial cell mitogenesis.
An additional aspect of the invention includes the use of the above-referenced peptides for the purpose of stimulating Schwann cells to produce growth factors which may, in turn, be harvested for scientific or therapeutic use. Furthermore, the peptides described herein may be used to induce central glial proliferation and remyelination for treatment of diseases, e.g., MS, where re-myelination is desired.
In an additional aspect of the invention, the novel polypeptides described herein may be used to stimulate the synthesis of acetylcholine receptors. As mentioned above, the invention provides new glial growth factors from mammalian sources, including bovine and human, which are distinguished from known factors. These factors are mitogenic for Schwann cells against a background of fetal calf plasma (FCP) . The invention also provides processes for the preparation of these factors, and an improved method for defining activity of these and other factors. Therapeutic application of the factors is a further significant aspect of the invention. Thus, important aspects of the invention are:
(a) a basic polypeptide factor having glial cell mitogenic activity, more specifically, Schwann cell mitogenic activity in the presence of fetal calf plasma, a molecular weight of from about 30 kD to about 36 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
F K G D A H T E A S L A D E Y E Y M X K T E T S S S G L X L K A S L A D E Y E Y M R K
A G Y F A E X A R T T E M A S E Q G A A K E A L A A L K F V L Q A K K
E T Q P D P G Q I L K K V P M V I G A Y T E Y K C L K F K W F K K A T V M E X K F Y V P K L E F L X A K; and
(b) a basic polypeptide factor which stimulates glial cell mitogenesis, particularly the division of
Schwann cells, in the presence of fetal calf plasma, has a molecular weight of from about 55 kD to about 63 kD, and including within its amino acid sequence any one or more of the following peptide sequences:
V H Q V W A A K
Y I F F M E P E A X S S G
L G A W G P P A F P V X Y W F V V I E G K
A S P V S V G S V Q E L Q R
V C L L T V A A L P P T
K V H Q V W A A K
K A S L A D S G E Y M X K
D L L L X V
E G K V H P Q R R G A L D R K
P S C G R L K E D S R Y I F F M E
E L N R K N K P Q N I K I Q K K The novel peptide sequences set out above, derived from the smaller molecular weight polypeptide factor, and from the larger molecular weight polypeptide factor, are also aspects of this invention in their own right. These sequences are useful as probe sources for polypeptide factors of the invention, for investigating, isolating or preparing such factors (or corresponding gene sequences) from a range of different species, or preparing such factors by recombinant technology, and in the generation of corresponding antibodies, by conventional technologies, preferably monoclonal antibodies, which are themselves useful investigative tools and are possible therapeutics. The invention also includes an isolated glial cell mitogenic activity encoding gene sequence, or fragment thereof, obtainable by the methods set out above for the novel peptide sequences of the invention.
The availability of short peptides from the highly purified factors of the invention has enabled additional sequences to be determined (see Examples to follow) .
Thus, the invention further embraces a polypeptide factor having glial cell mitogenic activity and including an amino acid sequence encoded by:
(a) a DNA sequence shown in any one of Figures 28a, 28b or 28c, SEQ ID Nos. 133-135, respectively;
(b) a DNA sequence shown in Figure 22, SEQ ID No. 89; (c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in Figure 28a, SEQ ID No. 133; or
(d) a DNA sequence hybridizable to any one of the DNA sequences according to (a) , (b) or (c) .
The invention further includes sequences which have greater than 60%, preferably 80%, sequence identity of homology to the sequences indicated above.
While the present invention is not limited to a particular set of hybridization conditions, the following protocol gives general guidance which may, if desired, be followed:
DNA probes may be labelled to high specific activity (approximately 108 to 1032Pdmp/μg) by nick-translation or by PCR reactions according to Schowalter and So mer (Anal. Biochem. , 177:90-94, 1989) and purified by desalting on G-150 Sephadex columns. Probes may be denatured (10 minutes in boiling water followed by immersion into ice water) , then added to hybridization solutions of 80% buffer B (2g polyvinylpyrolidine, 2g Ficoll-400, 2g bovine serum albumin, 50ml 1 M Tris HCL (pH 7.5), 58g NaCl, lg sodium pyrophosphate, lOg sodium dodecyl sulfate, 950ml H20) containing 10% dextran sulfate at 106 dpm 32P per ml and incubated overnight (approximately 16 hours) at 60°C. The filters may then be washed at 60°C, first in buffer B for 15 minutes followed by three 20-minute washes in 2X SSC, 0.1% SDS then one for 20 minutes in lx SSC, 0.1% SDS.
In other respects, the invention provides: (a) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, whether in reducing conditions or not, of from about 30kD to about 36kD on SDS-polyacrylamide gel electrophoresis using the following molecular weight standards:
Lysozyme (hen egg white) 14,400
Soybean trypsin inhibitor 21,500 Carbonic anhydrase (bovine) 31,000 Ovalbu in (hen egg white) 45,000
Bovine serum albumin 66,200
Phosphorylase B (rabbit muscle) 97,400; which factor has glial cell mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of said activity after 10 weeks incubation in 0.1% trifluoroacetic acid at 4βC; and
(b) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, under non-reducing conditions, of from about 55 kD to about 63 kD on SDS-polyacryla ide gel electrophoresis using the following molecular weight standards:
Lysozyme (hen egg white) 14,400
Soybean trypsin inhibitor 21,500
Carbonic anhydrase (bovine) 31,000 Ovalbumin (hen egg white) 45,000
Bovine serum albumin 66,200
Phosphorylase B (rabbit muscle) 97,400; which factor the human equivalent of which is encoded by DNA clone GGF2HBS5 described herein and which factor has glial cell mitogenic activity including stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least 50% of the activity after 4 days incubation in 0.1% trifluoroacetic acid at 4'C. For convenience of description only, the lower molecular weight and higher molecular weight factors of this invention are referred to hereafter as "GGF-I" and "GGF-II", respectively. The "GGF2" designation is used for all clones isolated with peptide sequence data derived from GGF-II protein (i.e. , GGF2HBS5, GGF2BPP3) .
It will be appreciated that the molecular weight range limits quoted are not exact, but are subject to slight variations depending upon the source of the particular polypeptide factor. A variation of, say, about 10% would not, for example, be impossible for material from another source.
Another important aspect of the invention is a DNA sequence encoding a polypeptide having glial cell mitogenic activity and comprising:
(a) a DNA sequence shown in any one of Figures 28a, 28b or 28c, SEQ ID Nos. 133-135: (b) a DNA sequence shown in Figure 22, SEQ ID No.
89;
(c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in Figure 28a, SEQ ID No. 133; or
(d) a DNA sequence hybridizable to any one of the DNA sequences according to (a) , (b) or (c) .
Another aspect of the present invention uses the fact that the Glial Growth Factors and pl85erbB2 ligand proteins are encoded by the same gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show pl85cΛB2 binding and activation. Several of the (GGF-II) gene products have been used to show Schwann cell mitogenic activity. This invention provides a use for all of the known products of the GGF/pl85erbB2 ligand gene (described in the references listed above) as Schwann cell mitogens.
This invention also relates to other, not yet naturally isolated splicing variants of the Glial Growth Factor gene. Figure 30, shows the known patterns of splicing derived from polymerase chain reaction experiments (on reverse transcribed RNA) and analysis of cDNA clones (as presented within) and derived from what has been published as sequences encoding pl85erbB2 ligands (Peles et al., Cell .69:205 (1992) and Wen et al., Cell j59_:559 (1992)). These patterns, as well as additional ones disclosed herein, represent probable splicing variants which exist. Thus another aspect of the present invention relates to the nucleotide sequences encoding novel protein factors derived from this gene. The invention also provides processes for the preparation of these factors. Therapeutic application of these new factors is a further aspect of the invention.
Thus other important aspects of the invention are :
(a) A series of human and bovine polypeptide factors having glial cell mitogenic activity including stimulating the division of Schwann cells. These peptide sequences are shown in Figures 31, 32, 33 and 34, SEQ ID Nos. 136-137, respectively.
(b) A series of polypeptide factors having glial cell mitogenic activity including stimulating the division of Schwann cells and purified and characterized according to the procedures outlined by Lupu et al. Science 249: 1552 (1990) ; Lupu et al. Proc. Natl. Acad. Sci USA 139: 2287 (1992); Holmes et al. Science 256: 1205 (1992); Peles et al. 69.: 205 (1992); Yarden and Peles Biochemistry 3J): 3543 (1991) ; Dobashi et al. Proc. Natl. Acad. Sci. B8_: 8582 (1991) ; Davis et al. Biochem. Biophys. Res. Commun. 179: 1536 (1991); Beaumont et al., patent application PCT/US91/03443 (1990) ; Greene et al. patent application PCT/US91/02331 (1990); Usdin and Fischbach, J. Cell. Biol. 103:493-507 (1986) ; Falls et al., Cold Spring Harbor Symp. Quant. Biol. 55: 397-406 (1990) ; Harris et al., Proc. Natl. Acad. Sci. USA £8:7664-7668 (1991) ; and Falls et al., Cell 72:801-815 (1993) . (c) A polypeptide factor (GGFBPP5) having glial cell mitogenic activity including stimulating the division of Schwann cells. The amino acid sequence is shown in Figure 32, SEQ ID No. 148, and is encoded by the bovine DNA sequence shown in Figure 32, SEQ ID No. 148. The novel human peptide sequences described above and presented in Figures 31, 32, 33 and 34, SEQ ID Nos. 136-150, respectively, represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAs) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.
Other compounds in particular, peptides, which bind specifically to the pl85crbB2 receptor can also be used according to the invention as a glial cell mitogen. A candidate compound can be routinely screened for pl85erbB2 binding, and, if it binds, can then be screened for glial cell mitogenic activity using the methods described herein.
The invention includes any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. By way of illustration, in EP-A 109748 mutations of native proteins are disclosed in which the possibility of unwanted disulfide bonding is avoided by replacing any cysteine in the native sequence which is not necessary for biological activity with a neutral amino acid. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.
The new sequences of the invention open up the benefits of recombinant technology. The invention thus also includes the following aspects:
(a) DNA constructs comprising DNA sequences as defined above in operable reading frame position within vectors (positioned relative to control sequences so as to permit expression of the sequences) in chosen host cells after transformation thereof by the constructs (preferably the control sequence includes regulatable promoters, e.g. Trp) . It will be appreciated that the selection of a promoter and regulatory sequences (if any) are matters of choice for those of skill in the art;
(b) host cells modified by incorporating constructs as defined in (a) immediately above so that said DNA sequences may be expressed in said host cells - the choice of host is not critical, and chosen cells may be prokaryotic or eukaryotic and may be genetically modified to incorporate said constructs by methods known in the art; and,
(c) a process for the preparation of factors as defined above comprising cultivating the modified host cells under conditions permitting expression of the DNA sequences. These conditions can be readily determined, for any particular embodiment, by those of skill in the art of recombinant DNA technology. Glial cell mitogens prepared by this means are included in the present invention.
None of the factors described in the art has the combination of characteristics possessed by the present new polypeptide factors.
As indicated, the Schwann cell assay used to characterize the present factors employs a background of fetal calf plasma. In all other respects, the assay can be the same as that described by Brockes et al. in Meth. Enz., supra, but with 10% FCP replacing 10% FCS. This difference in assay techniques is significant, since the absence of platelet-derived factors in fetal calf plasma (as opposed to serum) enables a more rigorous definition of activity on Schwann cells by eliminating potentially spurious effects from some other factors.
The invention also includes a process for the preparation of a polypeptide as defined above, extracting vertebrate brain material to obtain protein, subjecting the resulting extract to chromatographic purification by hydroxylapatite HPLC and then subjecting these fractions to SDS-polyacrylamide gel electrophoresis. The fraction which has an observed molecular weight of about 30kD to 36 kD and/or the fraction which has an observed molecular weight of about 55kD to 63 kD is collected. In either case, the fraction is subjected to SDS-polyacrylamide gel electrophoresis using the following molecular weight standards:
Lysozyme (hen egg white) 14,400
Soybean trypsin inhibitor 21,500
Carbonic anhydrase (bovine) 31,000 Ovalbumin (hen egg white) 45,000
Bovine serum albumin 66,200
Phosphorylase B (rabbit muscle) 97,400 In the case of the smaller molecular weight fraction, the SDS-polyacrylamide gel is run in non-reducing conditions in reducing conditions or, and in the case of the larger molecular weight fraction the gel is run under non-reducing conditions. The fractions are then tested for activity stimulating the division of rat Schwann cells against a background of fetal calf plasma. Preferably, the above process starts by isolating a relevant fraction obtained by carboxymethyl cellulose chromatography, e.g. from bovine pituitary material. It is also preferred that hydroxylapatite HPLC, cation exchange chromatography, gel filtration, and/or reversed-phase HPLC be employed prior to the SDS-Polyacrylamide gel electrophoresis. At each stage in the process, activity may be determined using Schwann cell incorporation of radioactive iododeoxyuridine as a measure in an assay generally as described by Brockes in Meth. Enz., supra, but modified by substituting 10% FCP for 10% FCS. As already noted, such as assay is an aspect of the invention in its own substance for CNS or PNS cell, e.g. Schwann cell, mitogenic effects.
Thus, the invention also includes an assay for glial cell mitogenic activity in which a background of fetal calf plasma is employed against which to assess DNA synthesis in glial cells stimulated (if at all) by a substance under assay.
Another aspect of the invention is a pharmaceutical or veterinary formulation comprising any factor as defined above formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form. In using the factors of the invention, conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions.
Thus, the formulations of this invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, opthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.
The formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA of the instant invention or by the use of surgical implants which release the formulations of the invention.
Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are to be found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
The present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.
The concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
In general terms, the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. General dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of the formulation, and the route of administration.
As indicated above, Schwann cells (the glial cells of the peripheral nervous system) are stimulated to divide in the presence of the factors of the invention. Schwann cells of the peripheral nervous system are involved in supporting neurons and in creating the myelin sheath around individual nerve fibers. This sheath is important for proper conduction of electrical impulses to muscles and from sensory receptors.
There are a variety of peripheral neuropathies in which Schwann cells and nerve fibers are damaged, either primarily or secondarily. There are many neuropathies of both sensory and motor fibers (Adams and Victor, Principles of Neurology) . The most important of those neuropathies are probably the neuropathies associated with diabetes, multiple sclerosis, Landry-Guillain-Barr syndrome, neuropathies caused by carcinomas, and neuropathies caused by toxic agents (some of which are used to treat carcinomas) .
The invention, however, envisages treatment or prophylaxis of conditions where nervous system damage has been brought about by any basic cause, e.g. infection or injury. Thus, in addition to use of the present factors in the treatment of disorders or diseases of the nervous system where demyelination or loss of Schwann cells is present, such glial growth factors can be valuable in the treatment of disorders of the nervous system that have been caused by damage to the peripheral nerves. Following damage to peripheral nerves, the regeneration process is led by the growth or the re-establishment of Schwann cells, followed by the advancement of the nerve fibre back to its target. By speeding up the division of Schwann cells one could promote the regenerative process following damage.
Similar approaches could be used to treat injuries or neurodegenerative disease of the central nervous system (brain and spinal cord) .
Furthermore, there are a variety of tumors of glial cells the most common of which is probably neurofibromatosis, which is a patchy small tumor created by~overgrowth of glial cells. Also, it has been found _t&at an activity very much like GGF can be found in some Schwann cell tumors, and therefore inhibitors of the action of the present factors on their receptors provides a therapy of a glial tumor, which comprises administering an effective amount of a substance which inhibits the binding of a factor, as defined above, to a receptor.
In general, the invention includes the use of present polypeptide factors in the prophylaxis or treatment of any pathophysiological condition of the nervous system in which a factor-sensitive or factor-responsive cell type is involved. The polypeptide factors of the invention can also be used as immunogens for making antibodies, such as monoclonal antibodies, following standard techniques. Such antibodies are included within the present invention. These antibodies can, in turn, be used for therapeutic or diagnostic purposes. Thus, conditions perhaps associated with abnormal levels of the factor may be tracked by using such antibodies. In vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques for the art of tumour imaging may also be employed.
The invention also includes the general use of the present factors as glial cell mitogens in vivo or in vitro, and the factors for such use. One specific embodiment is thus a method for producing a glial cell mitogenic effect in a vertebrate by administering an effective amount of a factor of the invention. A preferred embodiment is such a method in the treatment or prophylaxis of a nervous system disease or disorder.
A further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a nervous disease or disorder, or for neural regeneration or repair. Also included in the invention are the use of the factors of the invention in competitive assays to identify or quantify molecules having receptor binding characteristics corresponding to those of said polypeptides. The polypeptides may be labelled, optionally with a radioisotope. A competitive assay can identify both antagonists and agonists of the relevant receptor.
In another aspect, the invention provides the use of each one of the factors of the invention in an affinity isolation process, optionally affinity chromatography, for the separation of a respective corresponding receptor. Such processes for the isolation of receptors corresponding to particular proteins are known in the art, and a number of techniques are available and can be applied to the factors of the present invention. For example, in relation to IL-6 and IFNγ the reader is referred to Novick, D.; et al., J. Chromatogr. (1990) 510: 331-7. With respect to gonadotropin releasing hormone reference is made to Hazum, E., J. (1990)
Chromatogr. 510:233-8. In relation to G-CSF reference is made to Fukunaga, R. , et al., J. Biol. Chem. ,
265: 13386-90. In relation to IL-2 reference is made to
Smart, J.E., et al., (1990) J. Invest. Dermatol.,
£4.:158S-163S, and in relation to human IFN-gamma reference is made to Stefanos, S, et al. , (1989) J.
Interferon Res., 9_:719-30.
Brief Description of the Drawings The drawings will first be described. Drawings
Figures 1 to 8 relate to Example 1, and are briefly described below:
Pig. 1 is the profile for product from carboxymethyl cellulose chromatography;
Pig. 2 is the profile for product from hydroxylapatite HPLC; Fig. 3 is the profile for product from Mono S FPLC;
Pig. 4 is the profile for product from Gel filtration FPLC;
Figs. 5 and 6 depict the profiles for the two partially purified polypeptide products from reversed-phase HPLC; and
Figs. 7 and 8 depict dose-response curves for the GGF-I and GGF-II fractions from reversed-phase HPLC using either a fetal calf serum or a fetal calf plasma background;
Figs. 9 to 12 depict the peptide sequences derived from GGF-I and GGF-II, SEQ ID Nos. 1-20, 22-29, 32-53 and 169, (see Example 2 hereinafter), Figures 10 and 12 specifically depict novel sequences:
In Fig. 10, Panel A, the sequences of GGF-I peptides used to design degenerate oligonucleotide probes and degenerate PCR primers are listed (SEQ ID Nos. 20, 1, 22- 29, and 17). Some of the sequences in Panel A were also used to design synthetic peptides. Panel B is a listing of the sequences of novel peptides that were too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID Nos. 17 and 52) ;
In Fig. 12, Panel A, is a listing of the sequences of GGF-II peptides used to design degenerate oligonucleotide probes and degenerate PCR primers (SEQ ID Nos. 45-52) . Some of the sequences in Panel A were used to design synthetic peptides. Panel B is a listing of the novel peptide that was too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID No. 53) ;
Figures 13 to 20 relate to Example 3, below and depict the mitogenic activity of factors of the invention;
Figures 21 to 28 (a, b and c) relate to Example 4, below and are briefly described below:
Fig. 21 is a listing of the degenerate oligonucleotide probes (SEQ ID Nos. 54-88) designed from the novel peptide sequences in Figure 10, Panel A and Figure 12, Panel A;
Pig. 22 (SEQ ID No. 89) depicts a stretch of the putative bovine GGF-II gene sequence from the recombinant bovine genomic phage GGF2BG1, containing the binding site of degenerate oligonucleotide probes 609 and 650 (see Figure 21, SEQ ID NOs. 69 and 72, respectively) . The figure is the coding strand of the DNA sequence and the deduced amino acid sequence in the third reading frame. The sequence of peptide 12 from factor 2 (bold) is part of a 66 amino acid open reading frame (nucleotides 75272) ;
Fig. 23 is the degenerate PCR primers (Panel A, SEQ IS Nos. 90-108) and unique PCR primers (Panel B, SEQ ID Nos. 109-119) used in experiments to isolate segments of the bovine GGF-II coding sequences present in RNA from posterior pituitary;
Fig. 24 depicts of the nine distinct contiguous bovine GGF-II cDNA structures and sequences that were obtained in PCR amplification experiments using the list of primers in Figure 7, Panels A and B, and RNA from posterior pituitary. The top line of the Figure is a schematic of the coding sequences which contribute to the cDNA structures that were characterized;
Pig. 25 is a physical map of bovine recombinant phage of GGF2BG1. The bovine fragment is roughly 20 kb in length and contains two exons (bold) of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, Spel, Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map. Shaded portions correspond to fragments which were subcloned for sequencing;
Fig. 26 is.a ;schematic of the structure of three alternative gene products of the putative bovine GGF-II gene.7..Exons are listed A through E in the order of their discovery. The alternative splicing patterns 1, 2 and 3 generate three overlapping deduced protein structures (GGF2BPP1, 2, and 3), which are displayed in the various Figures 28a, b, c (described below) ;
Pig. 27 (SEQ ID Nos. 120-132) is a comparison of the GGF-I and GGF-II sequences identified in the deduced protein sequences shown in Figures 28a, 28b and 28c (described below) with the novel peptide sequences listed in Figures 10 and 12. The Figure shows that six of the nine novel GGF-II peptide sequences are accounted for in these deduced protein sequences. Two peptide sequences similar to GGF-I sequences are also found;
Fig. 28a (SEQ ID No. 133) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 1 in Figure 26. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 206 amino acids in length. Peptides in bold were those identified from the lists presented in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) ; Pig. 28b (SEQ ID No. 134) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 2 in Figure 26. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 281 amino acids in length. Peptides in bold are those identified from the lists presented in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) ;
Fig. 28c (SEQ ID No. 135) is a listing of the coding strand DNA sequence and deduced amino acid sequence of the cDNA obtained from splicing pattern number 3 in Figure 26. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 257 amino acids in length. Peptides in bold are those identified from the lists in Figures 10 and 12. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA) . Fig. 29, which relates to Example 6 hereinafter, is an autoradiogram of a cross hybridization analysis of putative bovine GGF-II gene sequences to a variety of mammalian DNAs on a southern blot. The filter contains lanes of EcoRI-digested DNA (5 μg per lane) from the species listed in the Figure. The probe detects a single strong band in each DNA sample, including a four kilobase fragment in the bovine DNA as anticipated by the physical map in Figure 25. Bands of relatively minor intensity are observed as well, which could represent related DNA sequences. The strong hybridizing band from each of the other mammalian DNA samples presumably represents the GGF-II homologue of those species.
Fig. 30 is a diagram of representative splicing variants. The coding segments are represented by F, E, B, A, G, C, C/D, C/D', D, D', H, K and L. The location of the peptide sequences derived from purified protein are indicated by "o".
Fig. 31 (SEQ ID Nos. 136-147, 160, 161) is a listing of the DNA sequences and predicted peptide sequences of the coding segments of GGF. Line 1 is a listing of the predicted amino acid sequences of bovine GGF, line 2 is a listing of the nucleotide sequences of bovine GGF, line 3 is a listing of the nucleotide sequences of human GGF (heregulin) (nucleotide base matches are indicated with a vertica_ line) and line 4 is a listing of the predicted amino acid sequences of human GGF/heregulin where it differs from the predicted bovine sequence. Coding segments E, A' and K represent only the bovine sequences. Coding segment D' represents only the human (heregulin) sequence.
Fig. 32 (SEQ ID No. 148) is the predicted GGF2 amino acid sequence and nucleotide sequence of BPP5. The upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence.
Pig. 33 (SEQ ID No. 149) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP2. The upper line is the nucleotide sequence and the lower lihe is the predicted amino acid sequence.
Fig. 34 (SEQ ID No. 150) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP4. The upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence.
Fig. 35 (SEQ ID Nos. 151-152) depicts the alignment of two GGF peptide sequences (GGF2bpp4 and GGF2bpp5) with the human EGF (hEGF) . Asterisks indicate positions of conserved cysteines.
Fig. 36 depicts the level of GGF activity (Schwann cell mitogenic assay) and tyrosine phosphorylation of a ca. 200kD protein (intensity of a 200 kD band on an autoradiogram of a Western blot developed with an antiphosphotyrosine polyclonal antibody) in response to increasing amounts of GGF.
Fig. 37 is a list of splicing variants derived from the sequences shown in Figure 31.
Pig. 38 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL1 (SEQ ID No.
154) .
Fig. 39 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL2 (SEQ ID No.
155) .
Fig. 40 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL3 (SEQ ID No.
156) .
Fig. 41 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL4 (SEQ ID No. 157) .
Fig. 42 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL5 (SEQ ID No.
158) .
Fig. 43 is the predicted amino acid sequence, bottom, and nucleic sequence, top, of EGFL6 (SEQ ID No.
159) . Fig. 44 is a scale coding segment map of the clone. T3 refers to the bacteriophage promoter used to produce mRNA from the clone. R = flanking EcoRI restriction enzyme sites. 5' UT refers to the 5' untranslated region. E, B, A, C, C/D', and D refer to the coding segments. O = the translation start site. Λ = the 5' limit of the region homologous to the bovine E segment (see example 6) and 3' UT refers to the 3' untranslated region. Pig. 45 is the predicted amino acid sequence
(middle) and nucleic sequence (top) of GGF2HBS5 (SEQ ID No. 167) . The bottom (intermittent) sequence represents peptide sequences derived from GGF-II preparations (see Figures 11, 12) .
Fig. 46 is a graph depicting the Schwann cell mitogenic activity of recombinant human and bovine glial growth factors.
Fig. 47 is a dose-response curve depicting Schwann cell proliferation activity data resulting from administration of different size aliquots of CHO cell conditioned medium.
Pig. 48 is a dose-response curve depicting Schwann cell mitogenic activity secreted into the extracellular medium by SF9 insect cells infected with baculovirus containing the GGF2HBS5 cDNA clone.
Fig. 49 is a Western blot of recombinant CHO cell conditioned medium using a GGF peptide antibody.
Pig. 50 (A) is a graph of Schwann cell proliferation activity of recombinant (COS cell produced) human GGF-II (rhGGF-II) peak eluted from the cation exchange column;
(B) is an immunoblot against recombinant GGFII peak using polyclonal antibody made against specific peptide of rhGGFII;
Fig. 51 (A) is a graph showing the purification of rhGGF-II (CHO cell produced) on cation exchange column by fraction; (B) is a photograph of a Western blot using fractions as depicted in (A) and a rhGGF-II specific antibody.
Pig. 52 is a photograph of a gel depicting tyrosine phosphorylation in Schwann cells treated with recombinant glial growth factors.
Fig. 53 is the sequences of GGFHBS5, GGFHFB1 and GGFBPP5 polypeptides (SEQ ID NOS: 170, 171, and 172).
Fig. 54 is a map of the CHO cell-expression vector pcDHFRpolyA.
Detailed Description
The invention pertains to the isolation and purification of novel Glial Growth factors and the cloning of DNA sequences encoding these factors. Other components of the invention are several gene splicing variants which potentially encode a series of glial growth factors, in particular the GGF2HBS5 in particular a variant which encodes the human equivalent of bovine GGF-II. It is evident that the gene encoding GGF's and pl85erbB2 binding proteins produces a number of variably-sized, differentially-spliced RNA transcripts that give rise to a series of proteins, which are of different lengths and contain some common peptide sequences and some unique peptide sequences. This is supported by the differentially-spliced sequences which are recoverable from bovine posterior pituitary RNA (as presented herein) , human breast cancer (MDA-MB-231) (Holmes et al. Science 256: 1205 (1992) and chicken brain RNA (Falls et al. Cell 71:1-20 (1993)) . Further support derives from the wide size range of proteins which act as both mitogens for Schwann cells (as disclosed herein) and as ligands for the pl85erbB2 receptor (see below) .
Further evidence to support the fact that the genes encoding GGF and pl85erbB2 are homologous comes from nucleotide sequence comparison. Science, 256 (1992) , 1205-1210) Holmes et aJL. demonstrate the purification of a 45-kilodalton human protein (Heregulin-α) which specifically interacts with the receptor protein pl85erbB2, which is associated with several human malignancies. Several complementary DNA clones encoding Heregulin-α were isolated. Peles et al. (Cell 69.:205 (1992)) and Wen et al (Cell <69_:559 (1992)) describe a complementary DNA isolated from rat cells encoding a protein called "neu differentiation factor" (NDF) . The translation product of the NDF cDNA has pl85ert>B2 binding activity. Usdin and Fischbach, J. Cell. Biol. 101:493-507 (1986); Falls et al., Cold Spring Harbor Symp. Quant. Biol. 55:397-406 (1990); Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991); and Falls et al., Cell 72:801-815 (1993) demonstrate the purification of a 42 Kd glycoprotein which interacts with a receptor protein pl85ert>B2 and several complementary cDNA clones were isolated (Falls et al. Cell 7_2:801-815 (1993). Several other groups have reported the purification of proteins of various molecular weights with pi85erbB2 binding activity. These groups include Lupu et al. (1992) Proc. Natl. Acad. Sci. USA 81:2287; Yarden and Peles (1991) Biochemistry .30.:3543; Lupu et al. (1990) Science 249: 1552) ; Dobashi et al. (1991) Biochem. Biophys. Res. Comm. 179: 1536; and Huang et al. (1992) J. Biol. Chem. 257:11508-11512.
Other Embodiments The invention includes any protein which is substantially homologous to the coding segments in Figure 31 (SEQ ID No.ε 136-147, 160, and 161) as well as other naturally occurring GGF polypeptides. Also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid naturally occurring (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6, hereby incorporated by reference) ; and polypeptides or proteins specifically bound by antisera to GGF polypeptide. The term also includes chimeric polypeptides that include the GGF polypeptides comprising sequences from Figure 31.
The following examples are not intended to limit the invention, but are provided to usefully illustrate the same, and provide specific guidance for effective preparative techniques.
As will be seen from Example 3, below, the present factors exhibit mitogenic activity on a range of cell types. The activity in relation to fibroblasts indicates a wound repair ability, and the invention encompasses this use. The general statements of invention above in relation to formulations and/or medicaments and their manufacture should clearly be construed to include appropriate products and uses. This is clearly a reasonable expectation for the present invention, given reports of similar activities for fibroblast growth factors (FGFs) . Reference can be made, for example, to Sporn et al., "Peptide Growth Factors and their Receptors I", page 396 (Baird and Bohlen) in the section headed "FGFs in Wound Healing and Tissue Repair".
EXAMPLE 1 Purification of GGF-I and GGF-II from bovine Pituitaries I. Preparation of Factor-CM Fraction
4,000 frozen whole bovine pituitaries (c.a. 12 kg) were thawed overnight, washed briefly with water and then homogenized in an equal volume of 0.15 M ammonium sulphate in batches in a Waring Blender. The homogenate was taken to pH 4.5 with 1.0 M HC1 and centrifuged at 4,900g for 80 minutes. Any fatty material in the supernatant was removed by passing it through glass wool. After taking the pH of the supernatant to 6.5 using 1.0 M NaOH, solid ammonium sulphate was added to give a 36% saturated solution. After several hours stirring, the suspension was centrifuged at 4,900 g for 80 minutes and the precipitate discarded. After filtration through glass wool, further solid ammonium sulphate was added to the supernatant to give a 75% saturated solution which was once again centrifuged at 4,900 g for 80 minutes after several hours stirring. The pellet was resuspended in c.a. 2 L of 0.1 M sodium phosphate pH 6.0 and dialyzed against 3 x 40 L of the same buffer. After confirming that the conductivity of the dialysate was below 20.0 μSiemens, it was loaded onto a Bioprocess column (120 x 113 mm, Pharmacia) packed with carboxymethyl cellulose (CM-52, Whatman) at a flow rate of 2 ml min"1. The column was washed with 2 volumes of 0.1 M sodium phosphate pH 6.0, followed by 2 volumes of 50 mM NaCl, and finally 2 volumes of 0.2 M NaCl both in the same buffer. During the final step, 10 mL (5 minute) fractions were collected. Fractions 73 to 118 inclusive were pooled, dialyzed against 10 volumes of 10 mM sodium phosphate pH 6.0 twice and clarified by centrifugation at 100,000 g for 60 minutes.
II. Hydroxylapatite HPLC Hydroxylapatite HPLC is not a technique hitherto used in isolating glial growth factors, but proved particularly efficacious in this invention. The material obtained from the above CM-cellulose chromatography was filtered through a 0.22 μm filter (Nalgene) , loaded at room temperature on to a high performance hydroxylapatite column (50 x 50 mm, Biorad) equipped with a guard column (15 x 25 mm, Biorad) and equilibrated with 10 mM potassium phosphate pH 6.0. Elution at room temperature was carried out at a flow rate of 2 ml.minute"1 using the following programmed linear gradient:
time (min) %B Solvent A: 10 mM potassium phosphate pH 6.0 0.0 0 Solvent B: 1.0 M potassium phosphate pH 6.0 5.0 0 7.0 20 70.0 20 150.0 100 180.0 100 185.0 0
6.0 mL (3 minutes) fractions were collected during the gradient elution. Fractions 39-45 were pooled and dialyzed against 10 volumes of 50 mM sodium phosphate pH 6.0.
III. Mono S FPLC
Mono S FPLC enabled a more concentrated material to be prepared for subsequent gel filtration.
Any particulate material in the pooled material from the hydroxylapatite column was removed by a clarifying spin at 100,000 g for 60 minutes prior to loading on to a preparative HRlO/10 Mono S cation exchange column (100 x 10 mm, Pharmacia) which was then re-equilibrated to 50mM sodium phosphate pH 6.0 at room temperature with a flow rate of 1.0 ml/minute"1. Under these conditions, bound protein was eluted using the following programmed linear gradient:
time (min) %B Solvent A: 50 mM potassium phosphate pH 6.0 0.0 0 Solvent B: 1.2 M sodium chloride, 50 mm
70.0 30 sodium phosphate pH 6.0
240.0 100
250.0 100 260.0 0
1 mL (1 minute) fractions were collected throughout this gradient program. Fractions 99 to 115 inclusive were pooled. IV. Gel Filtration FPLC
This step commenced the separation of the two factors of the invention prior to final purification, producing enriched fractions.
For the purposes of this step, a preparative Superoεe 12 FPLC column (510 x 20 mm, Pharmacia) was packed according to the manufacturers' instructions. In order to standardize this column, a theoretical plates measurement was made according to the manufacturers' instructions, giving a value of 9,700 theoretical plates.
The pool of Mono S eluted material was applied at room temperature in 2.5 Ml aliquots to this column in 50mM sodium phosphate, 0.75 NaCl pH 6.0 (previously passed through a C18 reversed phase column (Sep-pak, Millipore) at a flow rate of 1.0 mL/minute"1. 1 mL (0.5 minute) fractions were collected from 35 minutes after each sample was applied to the column. Fractions 27 to 41 (GGF-II) and 42 to 57 (GGF-I) inclusive from each run were pooled.
V. Reversed-Phase HPLC
The GGF-I and GGF-II pools from the above Superose 12 runs were each divided into three equal aliquots. Each aliquot was loaded on to a C8 reversed-phase column (Aquapore RP-300 7 μ C8 220 x 4.6 mm, Applied Biosystems) protected by a guard cartridge (RP-8, 15 x 3.2 mm, Applied Biosystems) and equilibrated to 40°C at 0.5 mL.minute. Protein was eluted under these conditions using the following programmed linear gradient:
time (min) %B Solvent A: 0.1% trifluoroacetic acid (TFA)
0 Solvent B: 90% acetonitrile, 0.1% TFA
60 66.6
62.0 100
72.0 100
75.0 0 200 μL (0.4 minute) fractions were collected in siliconized tubes (Multilube tubes, Bioquote) from 15.2 minutes after the beginning of the programmed gradient.
VI. SDS-Polvacrylamide Gel Electrophoresis
In this step, protein molecular weight standards, low range, catalogue no. 161-0304, from Bio-Rad Laboratories Limited, Watford, England were employed. The actual proteins used, and their molecular weight standards, have been listed herein previously. Fractions 47 to 53 (GGF-I) and fractions 61 to 67
(GGFII) inclusive from the reversed-phase runs were individually pooled. 7 μL of the pooled material was boiled in an equal volume of 0.0125 M Tris-Cl, 4% SDS, 20% glycerol, and 10% 3-mercaptoethanol for GGF-I, for 5 minutes and loaded on to an 11% polyacrylamide Laemmli gel with a 4% stacking gel and run at a constant voltage of 50 V for 16 hours. This gel was then fixed and stained using a silver staining kit (Amersham) . Under these conditions, the factors are each seen as a somewhat diffuse band at relative molecular weights 30,000 to 36,000 Daltons (GGF-I) and 55,000 to 63,000 Daltons (GGFII) as defined by molecular weight markers. From the gel staining, it is apparent that there are a small number of other protein species present at equivalent levels to the GGF-I and GGF-II species in the material pooled from the reversed-phase runs.
VII. Stability in Trifluoroacetic Acid
Stability data were obtained for the present Factors in the presence of trifluoroacetic acid, as follows:- GGF-I: Material from the reversed-phase HPLC, in the presence of 0.1% TFA and acetonitrile, was assayed within 12 hours of the completion of the column run and then after 10 weeks incubation at 40°C. Following incubation, the GGF-I had at least 50% of the activity of that material assayed directly off the column. GGF-II: Material from the reversed-phase HPLC, in the presence of 0.1% TFA and acetonitrile, and stored at -20°C, was assayed after thawing and then after 4 days incubation at 40°C. Following incubation, the GGF-II had at least 50% of the activity of that material freshly thawed.
It will be appreciated that the trifluoroacetic acid concentration used in the above studies is that most commonly used for reversed-phase chromatography.
VIII. Activity Assay Conditions
Unless otherwise indicated, all operations were conducted at 37"C, and, with reference to Figures 1 to 6, activity at each stage was determined using the Brockes (Meth. Enz., supra) techniques with the following modifications. Thus, in preparing Schwann cells, 5 μM forskolin was added in addition to DMEM (Dulbecco's modified Eagle's medium) , FCS and GGF. Cells used in the assay were fibroblast-free Schwann cells at passage number less than 10, and these cells were removed from flasks with trypsin and plated into flat-bottomed 96-well plates at 3.3 thousand cells per microwell.
[125I]IUdR was added for the final 24 hours after the test solution addition. The background (unstimulated) incorporation to each assay was less than 100 cpm, and maximal incorporation was 20 to 200 fold over background depending on Schwann cell batch and passage number.
In the case of the GGF-I and GGF-II fractions from reversed-phase HPLC as described above, two dose response curves were also produced for each factor, using exactly the above method for one of the curves for each factor, and the above method modified in the assay procedure only by substituting foetal calf plasma for fetal calf serum to obtain the other curve for each factor. The results are in Figures 7 and 8. EXAMPLE 2 Amino acid sequences of purified GGF-1 and GGF-II Amino acid sequence analysis studies were performed using highly purified bovine pituitary GGF-I and GGF-II. The conventional single letter code was used to describe the sequences. Peptides were obtained by lysyl endopeptidaεe and protease V8 digests, carried out on reduced and carboxymethylated sampleε, with the lyεyl endopeptidaεe digeεt of GGF-II carried out on material eluted from the 55-65 RD region of a 11% SDS-PAGE (MW relative to the above-quoted markers) .
A total of 21 peptide sequences (see Figure 9, SEQ ID Nos. 1-20, 169) were obtained for GGF-I, of which 12 peptides (εee Figure 10, SEQ ID Nos. 1, 22-29, 17, 19, and 32) are not present in current protein databaseε and therefore represent unique sequences. A total of 12 peptide sequences (see Figure 11, SEQ ID Nos. 33-44) were obtained for GGF-II, of which 10 peptides (see Figure 12, SEQ ID Noε. 45-53) are not preεent in current protein databaεeε and therefore repreεent unique εequences (an exception is peptide GGF-II 06 which εhowε identical sequences in many proteins which are probably of no significance given the small number of residues) . These novel sequences are extremely likely to correspond to portions of the true amino acid sequences of GGFs I and II.
Particular attention can be drawn to the sequences of GGF-I 07 and GGF-II 12, which are clearly highly related. The similaritieε indicate that the εequences of these peptideε are almost certainly those of the asεigned GGF εpecies, and are most unlikely to be derived from contaminant proteins.
In addition, in peptide GGF-II 02, the sequence X S S is consistent with the presence of an N linked carbohydrate moiety on an asparagine at the position denoted by X. In general, in Figureε 9 and 11, X representε an unknown residue denoting a sequencing cycle where a single position could not be called with certainty either becauεe there was more than one signal of equal size in the cycle or becauεe no εignal waε preεent. Aε asterisk denotes those peptides where the last amino acid called corresponds to the last amino acid present in that peptide. In the remaining peptideε, the εignal εtrength after the laεt amino acid called waε inεufficient to continue εequence calling to the end of that peptide. The right hand column indicateε the reεultε of a computer database search using the GCG package FASTA and TFASTA programε to analyze the NBRF and EMBL sequence databases. The name of a protein in this column denoteε identity of a portion of itε εequence with the peptide amino acid sequence called allowing a maximum of two mismatches. A question mark denoteε three mismatches allowed. The abbreviations uεed are aε follows:
HMG-1 High Mobility Group protein-1 HMG-2 High Mobility Group protein-2
LH-alpha Luteinizing hormone alpha subunit
LH-beta Luteinizing hormone beta εubunit
EXAMPLE 3 Mitogenic Activity of Purified GGF-I and GGF-II
The mitogenic activity of a highly purified εa ple containing both GGFs I and II was studied using a quantitative method, which allowε a εingle microculture to be examined for DNA εyntheεis, cell morphology, cell number and expresεion of cell antigenε. Thiε technique haε been modified from a method previously reported by
Muir et al., Analytical Biochemistry 185, 377-382, 1990. The main modifications are: 1) the uεe of uncoated microtiter plateε, 2) the cell number per well, 3) the uεe of 5% Foetal Bovine Plaεma (FBP) instead of 10% Foetal Calf Serum (FCS) , and 4) the time of incubation in presence of mitogens and bromodeoxyuridine (BrdU) , added simultaneously to the cultures. In addition the cell monolayer waε not waεhed before fixation to avoid loεε of cellε, and the incubation time of monoclonal mouse anti-BrdU antibody and peroxidase conjugated goat anti-mouεe immunoglobulin (IgG) antibody were doubled to increaεe the senεitivity of the assay. The assay, optimized for rat sciatic nerve Schwann cellε, haε alεo been used for εeveral cell lines, after appropriate modifications to the cell culture conditions.
I. Methodε of Mitogeneεis Teεting
On day 1, purified Schwann cellε were plated onto uncoated 96 well plateε in 5% FBP/Dulbecco'ε Modified Eagle Medium (DMEM) (5,000 cells/well) . On day 2, GGFs or other test factorε were added to the cultures, as well as BrdU at a final concentration of 10 μm. After 48 hours (day 4) BrdU incorporation was terminated by aεpirating the medium and cellε were fixed with 200 μl/well of 70% ethanol for 20 min at room temperature. Next, the cellε were waεhed with water and the DNA denatured by incubation with 100 μl 2N HCl for 10 min at 37 °C. Following aεpiration, reεidual acid waε neutralized by filling the wells with 0.1 M borate buffer, pH 9.0, and the cells were washed with phosphate buffered saline (PBS) . Cells were then treated with 50 μl of blocking buffer (PBS containing 0.1% Triton X 100 and 2% normal goat serum) for 15 min at 37 °C. After aspiration, monoclonal mouse anti-BrdU antibody (Dako Corp., Santa Barbara, CA) (50 μl/well, 1.4 μg/ l diluted in blocking buffer) waε added and incubated for two hours at 37 °C. Unbound antibodies were removed by three waεheε in PBS containing 0.1% Triton X-100 and peroxidase-conjugated goat ant-mouεe IgG antibody (Dako Corp., Santa Barbara, CA) (50 μl/well, 2 μg/ml diluted in blocking buffer) waε added and incubated for one hour at 37°C. After three washeε in PBS/Triton and a final rinεe in PBS, wellε received 100 μl/well of 50 mM phoεphate/citrate buffer, pH 5.0, containing 0.05% of the εoluble chromogen o-phenylenediamine (OPD) and 0.02% H202. The reaction waε terminated after 5-20 min at room temperature, by pipetting 80 μl from each well to a clean plate containing 40 μl/well of 2N sulfuric acid. The absorbance was recorded at 490nm using a plate reader (Dynatech Labε) . The assay plates containing the cell monolayers were waεhed twice with PBS and immunocytochemically stained for BrdU-DNA by adding 100 μl/well of the substrate diaminobenzidine (DAB) and 0.02% H202 to generate an insoluble product. After 10-20 min the staining reaction was stopped by washing with water, and BrdU-positive nuclei observed and counted uεing an inverted microεcope. occasionally, negative nuclei were counterstained with 0.001% Toluidine blue and counted aε before.
II. Cell lineε used for Mitogeneεiε Aεεavε Swiεε 3T3 Fibroblastε : Cellε, from Flow Labε, were maintained in D'ΕM supplemented with 10% FCS, penicillin and streptomycin, at 37 °C in a humidified atmosphere of 10% C02 in air. Cells were fed or subcultured every two dayε. For mitogenic assay, cells were plated at a density of 5,000 cells/well in complete medium and incubated for a week until cellε were confluent and quiescent. The serum containing medium was removed and the cell monolayer washed twice with serum free-medium. 100 μl of εerum free medium containing mitogenε and lOμM of BrdU were added to each well and incubated for 48 hourε. Doεe reεponεeε to GGFε and εerum or PDGF (aε a positive control) were performed.
BHK (Baby Hamster Kidney) 21 C13 Fibroblastε : Cells from European Collection of Animal Cell Cultureε (ECACC) , were maintained in Glaεgow Modified Eagle Medium (GMEM) supplemented with 5% tryptose phosphate broth, 5% FCS, penicillin and εtreptomycin, at 37'C in a humidified atmoεphere of 5% C02 in air. Cellε were fed or subcultured every two to three days. For mitogenic assay, cells were plated at a density of 2,000 cell/well in complete medium for 24 hourε. The serum containing medium was then removed and after washing with εerum free medium, replaced with 100 μl of 0.1% FCS containing GMEM or GMEM alone. GGFε and FCS or bFGF aε poεitive controlε were added, coincident with lOμM BrdU, and incubated for 48 hourε. Cell cultures were then procesεed aε deεcribed for Schwann cellε.
C6 Rat Glioma Cell Line : Cellε, obtained at paεεage 39, were maintained in DMEM containing 5% FCS, 5% Horse serum (HS) , penicillin and streptomycin, at 37 °C in a humidified atmosphere of 10% C02 in air. Cells were fed or subcultured every three days. For mitogenic aεεay, cellε were plated at a denεity of 2,000 cellε/well in complete medium and incubated for 24 hourε. Then medium waε replaced with a mixture of 1:1 DMEM and F12 medium containing 0.1% FCS, after waεhing in εerum free medium. Doεe responseε to GGFε, FCS and αFGF were then performed and cellε were proceεεed through the ELISA as previously described for the other cell types.
PC12 (Rat Adrenal Pheochromocytoma Cellε) : Cells from ECACC, were maintained in RPMI 1640 supplemented with 10% HS, 5% FCS, penicillin and streptomycin, in collagen coated flaskε, at 37°C in a humidified atmosphere of 5% C02 in air. Cells were fed every three days by replacing 80% of the medium. For mitogenic assay, cells were plated at a denεity of 3,000 cellε/well in complete medium, on collagen coated plateε (50 μl/well collagen, Vitrogen Collagen Corp., diluted 1 : 50, 30 min at 37°C) and incubated for 24 hourε. The medium was then placed with freεh RPMI either alone or containing 1 mM inεulin or 1% FCS. Doεe reεponεes to FCS/HS (1:2) as positive control and to GGFs were performed as before. After 48 hourε cellε were fixed and the ELISA performed aε previouεly deεcribed.
III. Reεults of Mitogenesis Aεεavε: All the experiments presented in this Example were performed using a highly purified sample from a Sepharose 12 chromatography purification step (see Example 1, section D) containing a mixture of GGF-I and GGF-II (GGFs) .
First, the reεultε obtained with the BrdU incorporation aεεay were compared with the claεεical mitogenic aεεay for Schwann cellε baεed on [125]I-UdR incorporation into DNA of dividing cells, described by J.P.Brockes (Methodε Enzymol. 147:217, 1987).
Figure 13 εhowε the compariεon of data obtained with the two aεεayε, performed in the same cell culture conditionε (5,000 cellε/well, in 5% FBP/DMEM, incubated in preεence of GGFε for 48hrs) . As clearly shown, the reεultε are comparable, but BrdU incorporation aεεay appears to be slightly more sensitive, as suggeεted by the εhift of the curve to the left of the graph, i.e. to lower concentrationε of GGFS.
Aε deεcribed under the εection "Methodε of Mitogeneεiε Teεting", after the immunoreactive BrdU-DNA haε been quantitated by reading the intenεity of the εoluble product of the OPD peroxidaεe reaction, the original aεεay plateε containing cell monolayerε can undergo the εecond reaction reεulting in the insoluble DAB product, which stainε the BrdU poεitive nuclei. The microcultureε can then be examined under an inverted microεcope, and cell morphology and the numberε of BrdU-positive and negative nuclei can be obεerved.
In Figure 14a and Figure 14b the BrdU-DNA immunoreactivity, evaluated by reading abεorbance at 490 nm, iε compared to the number of BrdU-poεitive nuclei and to the percentage of BrdU-positive nuclei on the total number of cells per well, counted in the same cultures. Standard deviations were lesε than 10%. The two evaluation methodε εhow a very good correlation and the diεcrepancy between the valueε at the higheεt doεe of GGFε can be explained by the different extent of DNA εynthesiε in cellε detected aε BrdU-poεitive.
The BrdU incorporation aεεay can therefore provide additional uεeful information about the biological activity of polypeptideε on Schwann cellε when compared to the (125) I-UdR incorporation aεεay. For example, the data reported in Figure 15 show that GGFs can act on Schwann cells to induce DNA syntheεiε, but at lower doεeε to increaεe the number of negative cellε preεent in the icroculture after 48 hourε.
The aεεay haε then been uεed on several cell lines of different origin. In Figure 16 the mitogenic responses of Schwann cells and Swisε 3T3 fibroblaεtε to GGFε are compared; deεpite the weak reεponεe obtained in 3T3 fibroblaεtε, εome clearly BrdU-positive nuclei were detected in theεe cultures. Control cultures were run in parallel in presence of εeveral doεeε of FCS or human recombinant PDGF, εhowing that the cells could respond to appropriate stimuli (not εhown) .
The ability of fibroblaεtε to reεpond to GGFε waε further inveεtigated uεing the BHK 21 C13 cell line. These fibroblastε, derived from kidney, do not exhibit contact inhibition or reach a quieεcent εtate when confluent. Therefore the experimental conditionε were designed to have a very low background proliferation without compromising the cell viability. GGFε have a εignificant mitogenic activity on BHK21 C13 cellε as shown by Figure 17 and Figure 18. Figure 17 showε the Brdu incorporation into DNA by BHK 21 C13 cellε εtimulated by GGFS in the preεence of 0.1% FCS. The good mitogenic reεponεe to FCS indicateε that cell culture conditions were not limiting. In Figure 18 the mitogenic effect of GGFs is expresεed aε the number of BrdU-positive and BrdU-negative cellε and aε the total number of cells counted per well. Data are repreεentative of two experimentε run in duplicates; at least three fields per well were counted. Aε obεerved for Schwann cellε in addition to a proliferative effect at low doεeε, GGFs also increase the numberε of nonreεponding cellε εurviving. The percentage of BrdU poεitive cellε iε proportional to the increaεing amountε of GGFs added to the cultures. The total number of cells after 48 hours in presence of higher doses of GGFs is at least doubled, confirming that GGFs induce DNA εyntheεiε and proliferation in BHK21 C13 cellε. Under the same conditions, cells maintained for 48 hours in the presence of 2% FCS εhowed an increaεe of about εix fold (not εhown) .
C6 glioma cells have provided a useful model to εtudy glial cell propertieε. The phenotype expressed seems to be dependent on the cell passage, the cellε more closely resembling an astrocyte phenotype at an early stage, and an oligodendrocyte phenotype at later stageε (beyond paεεage 70) . C6 cellε used in these experiments were from passage 39 to pasεage 52. C6 cellε are a highly proliferating population, therefore the experimental conditionε were optimized to have a very low background of BrdU incorporation. The preεence of 0.1% εerum was necesεary to maintain cell viability without εignificantly affecting the mitogenic responses, as shown by the doεe reεponεe to FCS (Figure 19) .
In Figure 20 the mitogenic reεponεes to aFGF (acidic Fibroblast growth factor) and GGFε are expreεεed aε the percentages of maximal BrdU incorporation obtained in the presence of FCS (8%) . Values are averages of two experiments, run in duplicates. The effect of GGFs waε comparable to that of a pure preparation of aFGF. aFGF haε been deεcribed aε a εpecific growth factor for C6 cellε (Lim R. et al., Cell Regulation 1:741-746, 1990) and for that reason it was used as a positive control. The direct counting of BrdU poεitive and negative cellε waε not poεsible because of the high cell density in the microcultureε. In contraεt to the cell lineε εo far reported, PC12 cells did not show any evident responεiveneεε to GGFS, when treated under culture conditionε in which PC12 could reεpond to sera (mixture of FCS and HS as uεed routinely for cell maintenance) . Nevertheleεε the number of cellε plated per well εeemε to affect the behavior of PC12 cells, and therefore further experimentε are required.
EXAMPLE 4 Iεolating and Cloning of Nucleotide Sequences encoding proteinε containing GGF-I and GGF-II peptides Iεolation and cloning of the GGF-II nucleotide εequences was performed as outlined herein, using peptide εequence information and library εcreening, and waε performed aε εet out below. It will be appreciated that the peptides of Figureε 4 and 5 can be uεed aε the starting point for isolation and cloning of GGF-I sequences by following the techniques described herein. Indeed, Figure 21, SEQ ID Nos. 54-88) εhowε poεsible degenerate oligonucleotide probeε for this purpose, and Figure 23, SEQ ID Noε. 90-119, liεts posεible PCR primerε. DNA εequence and polypeptide sequence should be obtainable by this means as with GGF-II, and alεo DNA conεtructs and expression vectors incorporating such DNA sequence, host cells genetically altered by incorporating such constructs/vectors, and protein obtainable by cultivating such host cells. The invention envisageε εuch εubject matter.
I. Design and Syntheεis of oligonucleotide Probes and Primers
Degenerate DNA oligomer probes were deεigned by backtranεlating the amino acid εequenceε (derived from the peptideε generated from purified GGF protein) into nucleotide εequenceε. Oligomerε repreεented either the coding strand or the non-coding εtrand of the DNA εequence. When εerine, arginine or leucine were included in the oligomer deεign, then two εeparate εyntheεeε were prepared to avoid ambiguitieε. For example, εerine was encoded by either TCN or AGY as in 537 and 538 or 609 and 610. Similar codon splitting waε done for arginine or leucine (e.g. 544, 545). DNA oligomerε were εyntheεized on a Bioεearch 8750 4-column DNA synthesizer using β- cyanoethyl chemiεtry operated at 0.2 micromole scale syntheεiε. Oligomerε were cleaved off the column (500 angstrom CpG resinε) and deprotected in concentrated ammonium hydroxide for 6-24 hourε at 55-60°C. Deprotected oligomerε were dried under vacuum (Speedvac) and purified by electrophoreεiε in gelε of 15% acrylamide (20 mono : 1 biε) , 50 mM Triε-borate-EDTA buffer containing 7M urea. Full length oligomerε were detected in the gelε by UV εhadowing, then the bandε were exciεed and DNA oligomerε eluted into 1.5 lε H20 for 4-16 hours with shaking. The eluate was dried, redissolved in 0.1 ml H20 and abεorbance measurements were taken at 260nm. Concentrations were determined according to the following formula:
(A 260 x units/ml) (60.6/length = x μM)
All oligomers were adjusted to 50 μM concentration by addition of H20.
Degenerate probes deεigned as above are εhown in Figure 21, SEQ ID Noε. 54-88.
PCR primers were prepared by essentially the same procedures that were used for probes with the following modifications. Linkerε of thirteen nucleotideε containing restriction εites were included at the 5' ends of the degenerate oligomerε for uεe in cloning into vectorε. DNA synthesis was performed at 1 micromole εcale uεing 1,000 angεtrom CpG reεins and inosine waε uεed at positions where all four nucleotides were incorporated normally into degenerate probeε. Purifications of PCR primers included an ethanol precipitation following the gel electrophoresis purification.
II. Library Construction and Screening
A bovine genomic DNA library waε purchaεed from Stratagene (Catalogue Number: 945701) . The library contained 2 x 106 15-2Okb Sau3Al partial bovine DNA fragmentε cloned into the vector lambda Daεhll. A bovine total brain CDNA library waε purchaεed from Clonetech (Catalogue Number: BL 10139) . Complementary DNA libraries were constructed (In Vitrogen; Stratagene) from mRNA prepared from bovine total brain, from bovine pituitary and from bovine poεterior pituitary. In Vitrogen prepared two cDNA libraries: one library waε in the vector lambda glO, the other in vector pcDNAI (a plaεmid library) . The Stratagene librarieε were prepared in the vector lambda unizap. Collectively, the cDNA libraries contained 14 million primary recombinant phage. The bovine genomic library was plated on E. coli K12 host strain LE392 on 23 x 23 cm plateε (Nunc) at 150,000 to 200,000 phage plaques per plate. Each plate represented approximately one bovine genome equivalent. Following an overnight incubation at 37 "c, the plates were chilled and replicate filterε were prepared according to procedureε of Maniatiε et al. (2:60-81). Four plaque liftε were prepared from each plate onto uncharged nylon membraneε (Pall Biodyne A or MSI Nitropure) . The DNA was immobilized onto the membranes by crosε-linking under UV light for 5 minuteε or, by baking at 80°C under vacuum for two hours. DNA probes were labelled using T4 polynucleotide kinase (New England Biolabs) with gamma 32P ATP (New England Nuclear; 6500 Ci/mmol) according to the εpecificationε of the εuppliers. Briefly, 50 pmols of degenerate DNA oligomer were incubated in the presence of 600 μCi gamma 32P-ATP and 5 units T4 polynucleotide kinase for 30 minutes at 37 °C. Reactions were terminated, gel electrophoresis loading buffer was added and then radiolabelled probes were purified by electrophoreεiε. 32P labelled probeε were exciεed from gel εliceε and eluted into water. Alternatively, DNA probeε were labelled via PCR amplification by incorporation of α-32P-dATP or α-32P dCTP according to the protocol of Schowalter and Sommer, Anal. Biochem 177:90-94 (1989). Probeε labelled in PCR reactionε were purified by deεalting on Sephadex G-150 columnε. Prehybridization and hybridization were performed in
GMC buffer (0.52 M NaPi, 7% SDS, 1% BSA, 1.5 mM EDTA, 0.1 M NaCl 10 mg/ml tRNA) . Waεhing waε performed in oligowaεh (160 ml 1 M Na2HP04, 200 ml 20% SDS, 8.0 ml 0.5 M EDTA, 100 ml 5M NaCl, 3632 ml H20) . Typically, 20 filterε (400 εq. centimeterε each) representing replicate copies of ten bovine genome equivalentε were incubated in 200 ml hybridization εolution with 100 pmolε of degenerate oligonucleotide probe (128-512 fold degenerate) . Hybridization waε allowed to occur overnight at 5°C below the minimum melting temperature calculated for the degenerate probe. The calculation of minimum melting temperature assumes 2°C for an AT pair and 4°C for a GC pair.
Filters were washed in repeated changes of oligowaεh at the hybridization temperatureε four to five hourε and finally, in 3.2M tetramethylammonium chloride, 1% SDS twice for 30 min at a temperature dependent on the DNA probe length. For 20merε, the final waεh temperature waε 60°C. Filterε were mounted, then expoεed to X-ray film (Kodak XAR5) uεing intenεifying screens (Dupont Cronex Lightening Plus) . Usually, a three to five day film expoεure at minus 80°C waε εufficient to detect duplicate εignalε in theεe library screens. Following analysis of the reεultε, filters could be stripped and reprobed. Filters were stripped by incubating through two succeεεive cycles of fifteen minuteε in a microwave oven at full power in a solution of 1% SDS containing lOmM EDTA pH8. Filters were taken through at least three to four cycles of stripping and reprobing with variouε probeε.
III. Recombinant Phage Iεolation. Growth and DNA Preparation
Theεe procedureε followed εtandard protocol as described in Recombinant DNA (Maniatis et al 2:60-2:81).
IV. Analvεiε of Iεolated Cloneε Uεing DNA Digeεtion and Southern Blotε Recombinant Phage DNA εampleε (2 microgramε) were digested according to conditions recommended by the restriction endonuclease supplier (New England Biolabs) . Following a four hour incubation at 37 °C, the reactions products were precipitated in the preεence of 0.1M εodium acetate and three volumes of ethanol. Precipitated DNA was collected by centrifugation, rinεed in 75% ethanol and dried. All reεuεpended εampleε were loaded onto agaroεe gels (typically 1% in TAE buffer; 0.04M Tris acetate, 0.002M EDTA) . Gel runs were at 1 volt per centimeter from 4 to 20 hourε. Markers included lambda Hind III DNA fragments and/or 0X174HaeIII DNA fragments (New England Biolabs) . The gels were εtained with 0.5 microgramε/ml of ethidium bromide and photographed. For southern blotting, DNA was first depurinated in the gel by treatment with 0.125 N HC1, denatured in 0.5 N NaOH and transferred in 2Ox SSC (3M εodium chloride, 0.03 M εodium citrate) to uncharged nylon membraneε. Blotting was done for 6 hours up to 24 hours, then the filters were neutralized in 0.5 Tris HC1 pH 7.5, 0.15 M sodium chloride, then rinsed briefly in 50 mM Tris-borate EDTA. For croεε-linking, the filterε were wrapped firεt in tranεparent plaεtic wrap, then the DNA εide expoεed for five minutes to an ultraviolet light. Hybridization and waεhing waε performed aε deεcribed for library εcreening (εee εection 2 of this Example) . For hybridization analyεiε to determine whether εimilar genes exiεt in other εpecieε εlight modificationε were made. The DNA filter waε purchaεed from Clonetech (Catalogue Number 7753-1) and containε 5 microgramε of EcoRI digeεted DNA from variouε species per lane. The probe was labelled by PCR amplification reactionε aε described in section 2 above, and hybridizationε were done in 80% buffer B(2 g polyvinylpyrrolidine, 2 g Ficoll-400, 2 g bovine serum albumin, 50 ml 1M Tris-HCl (pH 7.5) 58 g NaCl, 1 g εodium pyrophoεphate, 10 g sodium dodecyl sulfate, 950ml H20) containing 10% dextran sulfate. The probes were denatured by boiling for ten minutes then rapidly cooling in ice water. The probe was added to the hybridization buffer at 106 dpm 32P per ml and incubated overnight at 60°C. The filters were washed at 60°C first in buffer B followed by 2X SSC, 0.1% SDS then in lx SSC, 0.1% SDS. For high stringency, experiments, final washes were done in 0.1 x SSC, 1% SDS and the temperature raised to 65'C. Southern blot data were uεed to prepare a reεtriction map of the genomic clone and to indicate which subfragmentε hybridized to the GGF- probeε (candidateε for εubcloning) .
V. Subcloning of Segmentε of DNA Homologouε to Hybridization Probeε
DNA digestε (e.g. 5 micrograms) were loaded onto 1% agarose gelε then appropriate fragmentε exciεed from the gelε following staining. The DNA was purified by adsorption onto glass beads followed by elution using the protocol deεcribed by the εupplier (Bio 101) . Recovered DNA fragmentε (100-200 ng) were ligated into linearized dephoεphorylated vectors, e.g. pT3T7 (Ambion) , which is a derivative of pUC18, using T4 ligase (New England Biolabs) . Thiε vector carrieε the E. coli β lactamaεe gene, hence, tranεformantε can be εelected on plateε containing ampicillin. The vector alεo εupplies β- galactoεidaεe complementation to the hoεt cell, therefore non-recombinants (blue) can be detected using isopropylthiogalactoside and Bluogal (Betheεda Research Labs) . A portion of the ligation reactions was used to transform E. coli K12 XL1 blue competent cellε (Stratagene Catalogue Number: 200236) and then the tranεfor antε were εelected on LB plates containing 50 microgra s per ml ampicillin. White colonieε were εelected and plaεmid mini preps were prepared for DNA digestion and for DNA sequence analysiε. Selected cloneε were reteεted to determine if their insert DNA hybridized with the GGF probes.
VI. DNA Seguencing
Double εtranded plaεmid DNA templates were prepared from 5 ml cultures according to εtandard protocolε. Sequencing waε by the dideoxy chain termination method uεing Sequenaεe 2.0 and a dideoxynucleotide εequencing kit (US Biochemical) according to the manufacturerε protocol (a modification of Sanger et al. PNAS; USA 7.4:5463 (1977)]. Alternatively, εequencing waε done in a DNA thermal cycler (Perkin Elmer, model 4800) uεing a cycle εequencing kit (New England Biolabs; Bethesda Reεearch Laboratorieε) and waε performed according to manufacturerε inεtructionε uεing a 5'-end labelled primer. Sequence primers were either those supplied with the sequencing kits or were syntheεized according to εequence determined from the cloneε. Sequencing reactions were loaded on and resolved on 0.4mm thick sequencing gels of 6% polyacrylamide. Gelε were dried and exposed to X-Ray film. Typically, 35S was incorporated when εtandard εequencing kits were used and a 32P end labelled primer was used for cycle sequencing reactions. Sequences were read into a DNA sequence editor from the bottom of the gel to the top (5' direction to 3' ) and data were analyzed using programε εupplied by Geneticε Computer Group (GCG, University of Wisconsin) . VII. RNA Preparation and PCR Amplification
Open reading frames detected in the genomic DNA and which contained sequence encoding GGF peptides were extended via PCR amplification of pituitary RNA. RNA was prepared from frozen bovine tiεεue (Pelfreeze) according to the guanidine neutral-CεCl procedure (Chirgwin et. al. Biochemiεtry 18:5294(1979).) Polyadenylated RNA waε εelected by oligo-dT celluloεe column chromatography (Aviv and Leder PNAS (USA) 69:1408 (1972)). Specific DNA target εequenceε were amplified beginning with either total RNA or polyadenylated RNA εampleε that had been converted to cDNA uεing the Perkin Elmer PCR/RNA Kit Number: N808-0017. Firεt εtrand reverse tranεcription reactions used 1 μg template RNA and either primers of oligo dT with reεtriction enzyme recognition site linkerε attached or εpecific antisenεe primers determined from cloned εequenceε with reεtriction εiteε attached. To produce the εecond εtrand, the primerε either were pluε εtrand unique εequenceε aε used in 3' RACE reactions (Frohman et. al., PNAS (USA) 85:8998 (1988)) or were oligo dT primers with restriction sites attached if the second target site had been added by terminal transferase tailing first strand reaction products with dATP (e.g. 5' race reactionε, Frohman et. al., ibid). Alternatively, aε in anchored PCR reactionε the εecond εtrand primerε were degenerate, hence, representing particular peptide sequenceε.
The amplification profiles followed the following general scheme: 1) five minuteε soak file at 95°C; 2) thermal cycle file of 1 minute, 95°C; 1 minute ramped down to an annealing temperature of 45°C, 50°C or 55"C; maintain the annealing temperature for one minute; ramp up to 72 °C over one minute; extend at 72 °C for one minute or for one minute plus a 10 second auto extension; 3) extension cycle at 72 °C, five minutes, and; 4) soak file 4°C for infinite time. Thermal cycle files (#2) uεually were run for 30 cycles. A sixteen μl sample of each 100 μl amplification reaction waε analyzed by electrophoreεiε in 2% Nuεieve 1% agaroεe gelε run in TAE buffer at 4 volts per centimeter for three hours. The gels were stained, then blotted to uncharged nylon membranes which were probed with labelled DNA probes that were internal to the primers.
Specific setε of DNA amplification products could be identified in the blotting experiments and their positionε used as a guide to purification and reamplification. When appropriate, the remaining portions of selected samples were loaded onto preparative gels, then following electrophoreεis four to five slices of 0.5 mm thicknesε (bracketing the expected position of the εpecific product) were taken from the gel. The agaroεe waε cruεhed, then εoaked in 0.5 ml of electrophoresis buffer from 2-16 hours at 40°C. The crushed agarose waε centrifuged for two minuteε and the aqueouε phaεe waε transferred to fresh tubes.
Reamplification was done on five microliters (roughly 1% of the product) of the eluted material uεing the εame εetε of primerε and the reaction profileε as in the original reactionε. When the reamplification reactionε were completed, εampleε were extracted with chloroform and tranεferred to fresh tubes. Concentrated restriction enzyme buffers and enzymes were added to the reactions in order to cleave at the restriction sites present in the linkers. The digested PCR products were purified by gel electrophoresiε, then subcloned into vectorε as deεcribed in the εubcloning section above. DNA sequencing was done described as above.
VIII. DNA Seguence Analysis
DNA sequenceε were assembled using a fragment aεεembly program and the amino acid εequenceε deduced by the GCG programs GelAεsemble, Map and Translate. The deduced protein sequences were used aε a query sequence to search protein εequence databases uεing WordSearch. Analyεiε waε done on a VAX Station 3100 workεtation operating under VMS 5.1. The database search was done on SwisεProt release number 21 using GCG Version 7.0.
IX. Results of Cloning and Sequencing of genes encoding GGF-I and GGF-II
As indicated above, to identify the DNA sequence encoding bovine GGF-II degenerate oligonucleotide probes were designed from GGF-II peptide εequences. GGF-II 12 (SEQ ID No. 44) , a peptide generated via lysyl endopeptidaεe digeεtion of a purified GGF-II preparation (see Figures 11 and 12) showed strong amino acid εequence homology with GGF-I 07 (SEQ ID No. 39) , a tryptic peptide generated from a purified GGF-I preparation. GGF-II 12 waε thuε uεed to create ten degenerate oligonucleotide probes (see oligos 609, 610 and 649 to 656 in Figure 21, SEQ ID Nos. 69, 70, 71 and 79, respectively) . A duplicate set of filters were probed with two εetε (εet 1=609, 610; set 2=649-5656) of probes encoding two overlapping portions of GGF-II 12. Hybridization signalε were obεerved, but, only one clone hybridized to both probe sets. The clone (designated GGF2BG1) waε purified.
Southern blot analyεis of DNA from the phage clone GGF2BG1 confirmed that both setε of probes hybridized with that bovine DNA sequence, and showed further that both probes reacted with the same set of DNA fragments within the clone. Based on those experiments a 4 kb Eco RI εub-fragment of the original clone waε identified, εubcloned and partially sequenced. Figure 22 showε the nucleotide εequence, SEQ ID No. 89) and the deduced amino acid εequence of the initial DNA εequence readingε that included the hybridization εiteε of probeε 609 and 650, and confirmed that a portion of thiε bovine genomic DNA encoded peptide 12 (KASLADSGEYM) .
Further sequence analyεiε demonεtrated that GGF-II 12 reεided on a 66 amino acid open reading frame (see below) which haε become the starting point for the isolation of overlapping sequences representing a putative bovine GGF-II gene and a cDNA.
Several PCR procedures were used to obtain additional coding sequenceε for the putative bovine GGF-II gene. Total RNA and oligo dT-εelected (poly A containing) RNA εamples were prepared from bovine total pituitary, anterior pituitary, posterior pituitary, and hypothalamus. Uεing primerε from the liεt εhown in Figure 23, SEQ ID Noε. 109-119, one-εided PCR reactions (RACE) were used to amplify cDNA endε in both the 3' and 5' directionε, and anchored PCR reactionε were performed with degenerate oligonucleotide primerε repreεenting additional GGF-II peptideε. Figure 24 εummarizeε the contiguouε DNA εtructureε and εequenceε obtained in thoεe experimentε. From the 3' RACE reactionε, three alternatively εpliced cDNA εequenceε were produced, which have been cloned and εequenced. A 5' RACE reaction led to the diεcovery of an additional exon containing coding εequence for at leaεt 52 amino acidε. Analyεis of that deduced amino acid sequence revealed peptideε GGF-II-6 and a εequence εimilar to GGF-I-18 (εee below) . The anchored PCR reactionε led to the identification of (cDNA) coding εequenceε of peptideε GGF-II-1, 2, 3 and 10 contained within an additional cDNA εegment of 300 bp. The 5' limit of thiε εegment (i.e., εegment E, εee Fig. 31) iε defined by the oligonucleotide which encodes peptide GGF-II-1 and which was used in the PCR reaction (additional 5' sequence data exists as deεcribed for the human clone in Example 6) . Thuε this clone containε nucleotide εequenceε encoding six out of the existing total of nine novel GGF-II peptide sequenceε.
The cloned gene was characterized firεt by conεtructing a phyεical map of GGF2BG1 that allowed uε to poεition the coding εequenceε aε they were found (εee below, Figure 25) . DNA probes from the coding sequences described above have been used to identify further DNA fragments containing the exonε on thiε phage clone and to identify cloneε that overlap in both directionε. The putative bovine GGF-II gene iε divided into at least 5 coding segments. Coding segments are defined as discrete lengths of DNA sequence which can be translated into polypeptide sequenceε using the univerεal genetic code. The coding segments described in Figure 31 and referred to in the present application are: 1) particular exons present within the GGF gene (e.g. coding segment a) , or 2) derived from εets of two or more exons that appear in specific εub-groupε of RNAε, where each set can be tranεlated into the εpecific polypeptide εegmentε aε in the gene productε εhown. The polypeptide εegments referred to in the claims are the translation products of the analogous DNA coding εegmentε. Only coding εegmentε A and B have been defined aε exonε and εequenced and mapped thuε far. The εummary of the contiguouε coding εequenceε identified is shown in Figure 26. The exonε are liεted (alphabetically) in the order of their diεcovery. It is apparent from the intron/exon boundaries that exon B may be included in cDNAs that con., -ct coding εegment E and coding εegment A. That iε, exon B cannot be εpliced out without compromiεing the reading frame. Therefore, we εuggeεt that three alternative splicing patterns can produce putative bovine GGF-II cDNA sequenceε 1, 2 and 3. The coding sequences of these, designated GGF2BPP1.CDS, GGF2BPP2.CDS and GGF2BPP3.CDS, respectively, are given in Figures 28a (SEQ ID No. 133) , 28b (SEQ ID No. 134) , and 28c (SEQ ID No. 135) , respectively. The deduced amino acid sequence of the three cDNAs iε alεo given in Figureε 28a, (SEQ ID No. 133) , 28b (SEQ ID No. 134) , and 28c (SEQ ID No. 135) . The three deduced εtructureε encode proteinε of lengthε 206, 281 and 257 amino acidε. The firεt 183 reεidueε of the deduced protein sequence are identical in all three gene products. At position 184 the cloneε differ significantly. A codon for glycine GGT in GGF2BPP1 also εerveε aε a εplice donor for GGF2BPP2 and GGF2BPP3, which alternatively add on exons C, C/D, C/D' and D or C, C/D and D, respectively, and shown in figure 33, SEQ ID No. 149) . GGFIIBPP1 iε a truncated gene product which is generated by reading paεt the coding εegment A εplice junction into the following intervening εequence (intron) . Thiε repreεentε coding εegment A' in figure 31 (SEQ ID No. 140) . The tranεcript endε adjacent to a canonical AATAAA polyadenylation εequence, and we εuggeεt that thiε truncated gene product repreεentε a bona fide mature tranεcript. The other two longer gene productε εhare the εame 3' untranslated sequence and polyadenylation site.
All three of these molecules contain six of the nine novel GGF-II peptide sequenceε (see Figure 12) and another peptide is highly homologous to GGF-I-18 (εee Figure 27) . Thiε finding giveε a high probability that thiε recombinant molecule encodeε at leaεt a portion of bovine GGF-II. Furthermore, the calculated iεoelectric pointε for the three peptideε are conεistent with the physical propertieε of GGF-I and II. Since the molecular εize of GGF-II iε roughly 60 kD, the longest of the three cDNAs εhould encode a protein with nearly one-half of the predicted number of amino acidε.
A probe encompaεεing the B and A exonε was labelled via PCR amplification and used to screen a cDNA library made from RNA isolated from bovine posterior pituitary. One clone (GGF2BPP5) showed the pattern indicated in figure 30 and contained an additional DNA coding εegment (G) between coding εegmentε A and C. The entire nucleic acid εequence is shown in figure 32 (SEQ ID No. 148) . The predicted translation product from the longest open reading frame is 241 amino acids. A portion of a second cDNA (GGF2BPP4) was also isolated from the bovine posterior pituitary library using the probe deεcribed above. This clone showed the pattern indicated in figure 30. This clone is incomplete at the 5' end, but is a splicing variant in the sense that it lacks coding segments G and D. BPP4 also displays a novel 3' end with regions H, K and L beyond region C/D. The sequence of BPP4 iε shown in figure 34 (SEQ ID No. 150) .
EXAMPLE 5 GGF Sequences in Variouε Species Database searching haε not revealed any meaningful εimilaritieε between any predicted GGF tranεlation productε and known protein sequences. This εuggeεtε that GGF-II iε the firεt member of a new family or εuperfamily of proteins. In high stringency cross hybridization studies (DNA blotting experiments) with other mammalian DNAs we have εhown, clearly, that DNA probeε from this bovine recombinant molecule can readily detect specific sequenceε in a variety of εampleε teεted. A highly homologouε εequence iε alεo detected in human genomic DNA. The autoradiogram iε shown in figure 29. The signalε in the laneε containing rat and human DNA repreεent the rat and human equivalentε of the GGF gene, the εequenceε of εeveral cDNA'ε encoded by thiε gene have been recently reported by Holmeε et al. (Science 256: 1205 (1992)) and Wen et al. (Cell 9: 559 (1992)).
EXAMPLE 6
Isolation of a Human Seguence Encoding Human GGF2
Several human clones containing sequenceε from the bovine GGFII coding εegment E were iεolated by εcreening a human cDNA library prepared from brain εtem (Stratagene catalog #935206) . Thiε εtrategy waε pursued based on the strong link between most of the GGF2 peptideε (unique to
GGF2) and the predicted peptide sequence from clones containing the bovine E segment. Thiε library waε εcreened as described in Example 4 , Section II uεing the oligonucleotide probeε 914-919 listed below. 914TCGGGCTCCATGAAGAAGATGTA 915TCCATGAAGAAGATGTACCTGCT 916ATGTACCTGCTGTCCTCCTTGA 917TTGAAGAAGGACTCGCTGCTCA 918AAAGCCGGGGGCTTGAAGAA 919ATGARGTGTGGGCGGCGAAA
Cloneε detected with theεe probeε were further analyzed by hybridization. A probe derived from coding εegment A (see Figure 21) , which was produced by labeling a polymerase chain reaction (PCR) product from εegment A, waε alεo uεed to screen the primary library. Several clones that hybridized with both A and E derived probes were selected and one particular clone, GGF2HBS5, was selected for further analyεiε. Thiε clone iε repreεented by the pattern of coding segments (EBACC/D'D as shown in Figure 31) . The E segment in thiε clone iε the human equivalent of the truncated bovine verεion of E εhown in Figure 37. GGF2HBS5 iε the moεt likely candidate to encode GGF-II of all the "putative" GGF-II candidates described. The length of coding sequence segment E iε
786 nucleotideε pluε 264 bases of untranslated sequence. The predicted size of the protein encoded by GGF2HBS5 iε approximately 423 amino acidε (approximately 45 kilodaltons, see Figure 45, SEQ ID NO: 167), which is similar to the size of the deglycosylated form of GGF-II (εee Example 16) . Additionally, seven of the GGF-II peptides listed in Figure 27 have equivalent sequenceε which fall within the protein εequence predicted from region E. Peptideε II-6 and 11-12 are exceptionε, which fall in coding εegment B and coding εegment A, reεpectively. RNA encoding the GGF2HBS5 protein was produced in an in vitro transcription εyεtem driven by the bacteriophage T7 promoter reεident in the vector (Blueεcript SK [Stratagene Inc.] εee Figure 44) containing the GGF2HBS5 insert. This RNA waε translated in a cell free (rabbit reticulocyte) tranεlation εyεtem and the εize of the protein product waε 45 Kd. Additionally, the cell-free product has been asεayed in a Schwann cell mitogenic aεεay to confirm biological activity. Schwann cells treated with conditioned medium show both increased proliferation as measured by incorporation of I25I-Uridine and phosphorylation on tyrosine of a protein in the 185 kilodalton range. Thuε the size of the product encoded by GGF2HBS5 and the presence of DNA sequences which encode human peptides highly homologous to the bovine peptides shown in Figure 12 confirm that GGF2HBS5 encodes the human equivalent of bovine GGF2. The fact that conditioned media prepared from cells tranεformed with thiε clone elicitε Schwann cell mitogenic activity confirmε that the GGFIIHBS5 gene produce (unlike the BPP5 gene product) is secreted. Additionally the GGFIIBPP5 gene product seems to mediate the Schwann cell proliferation responεe via a receptor tyrosine kinase such as pi85erbB2 or a closely related receptor (εee Example 14) .
EXAMPLE 7 Expression of Human Recombinant GGF2 in Mammalian and Insect Cells
The GGF2HBS5 cDNA clone encoding human GGF2 (as deεcribed in Example 6 and alεo referred to herein as HBS5) was cloned into vector pcDL-SRα296 (Takebe et al. Mol. Cell. Biol. 8.:466-472 (1988) and C0S-7 cells were transfected in 100 mm diεhes by the DEAE-dextran method (Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed. CSH Laboratory NY (1989) . Cell lysates or conditioned media from tranεiently expressing COS cellε were harvested at 3 or 4 dayε poεt-tranεfection. To prepare lyεates, cell monolayers were waεhed with PBS, εcraped from the diεheε lyεed by three freeze/thaw cycleε in 150 μl of 0.25 M Tris-HCl, pH8. Cell debris waε pelleted and the εupernatant recovered. Conditioned media εamples (7 ml.) were collected, then concentrated and buffer exchanged with 10 mM Tris, pH 7.4 uεing Centiprep-10 and Centricon-10 unitε aε deεcribed by the manufacturer (Amicon, Beverly, MA) . Rat nerve Schwann cells were asεayed for incorporation of DNA εyntheεiε precurεors, as described (see Example 3) . Conditioned media or cell lysate εampleε were teεted in the Schwann cell proliferation aεεay aε deεcribed in Example 3. The mitogenic activity data are εhown in Fig. 46. The cDNA, GGF2HBS5, encoding GGF2 directed the εecretion of the protein product to the medium. A εmall proportion of total activity waε detectable inεide the cellε aε determined by assayε uεing cell lyεateε. GGF2HFB1 and GGFBPP5 cDNA'ε failed to direct the εecretion of the product to the extracellular medium. GGF activity from theεe cloneε waε detectable only in cell lyεateε (Fig. 46) .
Recombinant GGF2 waε alεo expreεεed in CHO cellε. The GGF2HBS5 cDNA encoding GGF2 waε cloned into the EcoRI εite of vector pcdhfrpolyA (Fig. 54) and tranεfected into the DHFR negative CHO cell line (DG44) by the calcium phoεphate coprecipitation method (Graham and Van Der Eb,
Virology 52:456-467 (1973). Cloneε were εelected in nucleotide and nucleoεide free a medium (Gibco) in 96- well plateε. After 3 weekε, conditioned media samples from individual clones were screened for expreεεion of GGF by the Schwann cell proliferation aεεay as described in Example 3. Stable clones which secreted significant levels of GGF activity into the medium were identified. Schwann cell proliferation activity data from different volume aliquots of CHO cell conditioned medium were uεed to produce the dose response curve shown in Fig. 47
(Graham and Van Der Eb, Virology 52:456, 1973) . This material waε analyzed on a Weεtern blot probed with polyclonal antisera raised againεt a GGF2 εpecific peptide. A broad band of approximately 69-90 Kd (the expected εize of GGF2 extracted from pituitary and higher molecular weight glycoforms) is εpecifically labeled
(Fig. 49, lane 12) . Recombinant GGF2 was alεo expreεεed in insect cellε uεing Baculoviruε expreεεion. Sf9 inεect cellε were infected with baculoviruε containing the GGF2HBS5 cDNA clone at a multiplicity of 3-5 (106 cells/ml) and cultured in Sf900-11 medium (Gibco) . Schwann cell mitogenic activity was secreted into the extracellular medium (Fig. 48) . Different volumes of insect cell conditioned medium were teεted in the Schwann cell proliferation aεsay in the absence of forskolin and the data used to produce the dose response curve shown in Fig. 48.
This material was also analyzed on a Weεtern blot (Fig. 47) probed with the GGF II εpecific antibody deεcribed above. A band of 45 Kd, the εize of deglycoεylated GGF-II (εee Example 16) waε seen.
The methods uεed in this example were as follows:
Schwann cell mitogenic activity of recombinant human and bovine glial growth factors was determined as follows: Mitogenic responseε of cultured Schwann cellε were measured in the presence of 5 μM forskolin using crude recombinant GGF preparations obtained from transient mammalian expression experiments. Incorporation of [125I]-Uridine waε determined following an 18-24 hour exposure to materials obtained from tranεfected or mock transfected COS cells aε deεcribed in the Methodε. The mean and standard deviation of four setε of data are εhown. The mitogenic reεponεe to partially purified native bovine pituitary GGF (carboxymethyl celluloεe fraction; Goodearl et al., εubmitted) iε εhown (GGF) aε a εtandard of one hundred percent activity. cDNAε (Fig. 53) were cloned into pcDL-SRα.296 (Takebe et al., Mol. Cell Biol. 8:466-472 (1988)) , and COS-7 cellε were tranεfected in 100 mm diεhes by the DEAE- dextran method (Sambrook et al., In Molecular Cloning. A Laboratory Manual , 2nd . ed . (Cold Spring Harbor Laboratory Preεε, Cold Spring Harbor, NY, 1989)). Cell lyεateε or conditioned media were harvested at 3 or 4 days post-transfection. To prepare lyεateε, cell monolayers were washed with PBS, εcraped from the diεhes, and lysed by three freeze/than cycleε in 150 μl of 0.25 M Tris-HCl, pH 8. Cell debriε waε pelleted and the εupernate recovered. Conditioned media samples (7 mis) were collected, then concentrated and buffer exchanged with 10 mM Triε, pH 7.4 uεing Centriprep-10 and Centricon-10 units as deεcribed by the manufacturer (A icon, Beverly, MA) . Rat εciatic nerve Schwann cellε were aεεayed for incorporation of DNA εyntheεiε precurεorε, aε deεcribed (Daviε and Stroobant, J. Cell Biol. 110:1353-1360 (1990); Brockeε et al., Brain Reε. 165:105-118 (1979) ) .
Western blots of recombinant CHO cell conditioned medium were performed as follows: A recombinant CHO clone waε cultured in 7 ml. of MCDB302 protein-free medium for 3 dayε. 2 ml of conditioned medium waε concentrated, buffered exchanged againεt 10 mM Triε-HCl, pH 7.4 and lyophilized to drynesε. The pellet waε reεuspended in SDS-PAGE sample buffer, εubjected to reducing SDS gel electrophoreεiε and analyzed by Weεtern blotting with a GGF peptide antibody. A CHO control was done by using conditioned medium from untransfected CHO- DG44 host and the CHO HBS5 levels were asεayed using conditioned medium from a recombinant clone.
EXAMPLE 8 Isolation of Other Human Sequences Related to Bovine GGF
The result in Examples 5 and 6 indicate that GGF related sequenceε from human sources can also be easily isolated by using DNA probes derived_ from bovine GGF sequences. Alternatively the procedure described by Holmes et al. (Science 256: 1205 (1992)) can be used. In this example a human protein (heregulin α) , which bindε to and activateε the pl85erbB2 receptor (and iε related to GGF) , is purified from a tumor cell line and the derived peptide sequence iε uεed to produce oligonucleotide probeε which we„e utilized to clone the cDNA'ε encoding heregulin. The biochemical aεεay for pl85eιtB2 receptor activation iε diεtinguiεhed from Schwann cell proliferation. This is a similar approach to that used in examples 1-4 for the cloning of GGF sequences from pituitary cDNAs. The heregulin protein and complementary DNAε were iεolated from tumor cell lineε according to the following procedureε. Heregulin waε purified from medium conditioned by MDA-MB-231 breaεt cancer cellε (ATCC #HTB 26) grown on
Percell Biolytica microcarrier beadε (Hyclone Labε) . The medium (10 literε) waε concentrated ~25-fold by filtration through a membrane (10-kD cutoff) (Millipore) and clarified by centrifugation and filtration through a filter (0.22 μm) . The filtrate was applied to a heparin Sepharose column (Pharmacia) and the proteinε were eluted with εteps of 0.3, 0.6, and 0.9 M NaCl in phosphate-buffered saline. Activity in the various chromatographic fractions was measured by quantifying the increaεe in tyroεine phosphorylation of pl85eΛB2 in MCF-7 breast tumor cells (ATCC # HTB 22) . MCF-7 cells were plated in 24-well Costar plateε in F12 (50%) Dulbecco's minimum esεential medium (50%) containing εerum (10%) (105 cellε per well) , and allowed to attach for at leaεt 24 hourε. Prior to aεεay, cellε were tranεferred into medium without εerum for a minimum of 1 hour. Column fractionε (10 to 100 μl) were incubated for 30 min. at 37°. Supernatantε were then aεpirated and the reaction waε εtopped by the addition of SDS-PAGE εample buffer 100 μl) . Sampleε were heated for 5 min. at 100°C, and portionε (10 to 15 μl) were applied to a triε-glycine gel (4 to 20%) (Novex) . After electrophoreεis, proteins were electroblotted onto a polyvinylidenedifluoride (PVDF) membrane and then blocked with bovine εerum albumin (5%) in triε-buffered εaline containing Tween-20 (0.05%) (TBST) . Blots were probed with a monoclonal antibody (l:loon dilution) to phosphotyroεine (Upεtate Biotechnology) for a minimum of 1 hour at room temperature. Blots were washed with TBST, probed with an antibody to mouse immunoglobulin G conjugated to alkaline phosphataεe (Promega) (diluted 1:7500) for a minimum of 30 min. at room temperature. Reactive bands were visualized with 5-bromo-4-chloro-3-indoyl-l-phoεphate and nitro-blue tetrazolium. Immunoblotε were εcanned with a Scan Jet Pluε (Hewlett-Packard) denεito eter. Signal intenεitieε for unεtimulated MCF-7 cellε were 20 to 30 unitε. Fully stimulated pl85eΛB2 yielded signalε of 180 to 200 unitε. The 0.6 M NaCl pool, which contained most of the activity, was applied to a polyaεpartic acid (PolyLC) column equilibrated in 17 mM εodium phoεphate (pH 6.8) containing ethanol (30%). A linear gradient from 0.3 M to 0.6 M NaCl in the equilibration buffer waε uεed to elute bound proteins. A peak of activity (at -0.45 M NaCl) was further fractionated on a C4 reversed-phaεe column (SynChropak RP-4) equilibrated in buffer containing TFA (0.1%) and acetonitrile (15%). Proteinε were eluted from thiε column with an acetonitrile gradient from 25 to 40% over 60 min. Fractionε (1 ml) were collected, aεεayed for activity, and analyzed by SDS-PAGE on triε-glycine gelε (4-20%, Novex) . HPLC-purified HRG-α waε digested with lysine C in SDS (0.1%), 10 mM dithiothreitol, 0.1 M NH4HC03 (pH 8.0) for 20 hourε at 37 "c and the reεultant fragments were reεolved on a Synchrom C4 column (4000A0, 0.2 by 10 cm) . The column waε equilibrated in 0.1% TFA and eluted with a 1-propanol gradient in 0.1% TFA (W. J. Henzel, J. T. Stultε, C. Hsu, D. W. Aswad, J. Biol. Chem. 264 , 15905 (1989)) . Peakε from the chromatographic run were dried under vacuum and εequenced. One of the peptideε (eluting at -24% 1-propanol) gave the εequence
[A]AEKEKTF[C]VNGGEXFMVKDLXNP (SEQ ID No. 162) . Reεidues in bracketε were uncertain and an X repreεentε a cycle in which it waε not poεεible to identify the amino acid. The initial yield was 8.5 pmol and the εequence did not correspond to any known protein. Residueε 1, 9, 15, and 22 were later identified in the cDNA sequence as cysteine. Direct εequencing of the -45-kD band from a gel that had been overloaded and blotted onto a PVDF membrane revealed a low abundance sequence XEXKE[G] [R]GK[G]K[G]KKKEXGXG[K] (SEQ ID No. 163) with a very low initial yield (0.2 pmol) . This correεponded to amino acid reεidueε 2 to 22 of heregulin-α (Fig. 31) , εuggesting that serine 2 is the NH2-terminus of proHRG-α. Although the NH2 ter inuε waε blocked, it was observed that occasionally a small amount of a normally blocked protein may not be poεt-tranεlationally modified. The NH2 terminal aεεignment waε confirmed by maεε εpectrometry of the protein after digeεtion with cyanogen bromide. The COOH-terminuε of the iεolated protein haε not been definitely identified; however, by mixture εequencing of proteolytic digeεtε, the mature εequence doeε not appear to extend paεt reεidue 241. Abbreviations for amino residueε are: A, Ala; C, Cyε; D, Aεp; E, Glu; F, Phe; G, Gly; H, Hiε; I, lie; K, Lyε; L, Leu; M, Met; N, Aεn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Aε a source of cDNA clones, an oligo(dT) -primed λgtlO (T. V. Huynn, R. A. Young, R. W. Davis, λgtlO and λgtll DNA Cloning Techniques: A Practical Approach, D. Glover, Ed. (IRC Press, Oxford, (1984)) cDNA library was conεtructed (U. Gubler and B. J. Hoffman, Gene 2b., 263 (1983)) with mRNA purified (J. M. Chirwin, A. E. Przbyla, R. J. MacDonald, W. J. Rutter, Biochemiεtry 18., 5294 (1979)) from MDA-MB-231 cellε. The following eightfold degenerate antiεense deoxyoligonucleotide encoding the 13-amino acid sequence AEKEKTFCVNGGE (SEQ ID No. 164) (13) was deεigned on the baεiε of human codon frequency optima (R. Lathe, J. Mol. Biol. 183. 1 (1985)) and chemically εyntheεized:
5'-CTCGCC (G OR T) CC (A OR G) TTCAC (A OR G) CAGAAGGTCTTCTCCTTCTCAGC-3 ' (SEQ ID No. 165) . For the purpoεe of probe deεign a cyεteine waε aεεigned to an unknown reεidue in the amino acid εequence . The probe waε labeled by phoεphorylation and hybridized under low-εtringency conditionε to the cDNA library. The proHRG-α protein waε identified in thiε library. HRB-01 cDNA waε identified by probing a εecond oligo(dT) -primed λgtlO library made from MDA-MB-231 cell mRNA with εequenceε derived from both the 5' and 3' endε of proHRG-α. Clone 13 (Fig. 2A) waε a product of εcreening a primed (5'-CCTCGCTCCTTCTTCTTGCCCTTC-3' primer (SEQ ID No. 166) ; proHRG-α antiεenεe nucleotideε 33 to 56) MDA-MB-231 λgtlO library with 5' HRG-α sequence. A sequence corresponding to the 5' end of clone 13 as the probe was uεed to identify proHRG32 and proHRG/33 in a third oligo(dT) -primed λgtlO library derived from MDA-MB-231 cell mRNA. Two cDNA cloneε encoding each of the four HRGε were εequenced (F. Sanger, S. Milken, A. R. Coulεon, Proc. Natl. Acad. Sci.U.S.A. .74., 5463 1977]). Another cDNA deεignated clone 84 haε an amino acid εequence identical to proHRG/32 through amino acid 420. A εtop codon at poεition 421 iε followed by a different 3'-untranεlated εequence.
EXAMPLE 9 Iεolation of a Further Splicing Variant The methodε in Example 6 produced four cloεely related εequences (heregulin , βl , β2 , β3 ) which arise as a result of splicing variation. Peles et al. (Cell 69, 205 (1992)) , and Wen et al. (Cell 6£, 559 (1992)) have isolated another splicing variant (from rat) using a similar purification and cloning approach to that described in Examples 1-4 and 6 involving a protein which binds to pl85ert>B2. The cDNA clone was obtained aε followε (via the purification and εequencing of a pl85ert,B2 binding protein from a tranεformed rat fibroblast cell line) .
A pl85erbB2 binding protein was purified from conditioned medium as follows. Pooled conditioned medium from three harvests of 500 roller bottles (120 liters total) was cleared by filtration through 0.2 μ filterε and concentrated 31-fold with a Pelicon ultrafiltration εyεtem using membranes with a 20kd molecular size cutoff. All the purification steps were performed by using a Pharmacia fast protein liquid chromatography system. The concentrated material was directly loaded on a column of heparin-Sepharose (150 ml, preequilibrated with phosphate-buffered saline (PBS) ) . The column waε washed with PBS containing 0.2 M NaCl until no absorbance at 280 nm wavelength could be detected. Bound proteins were then eluted with a continuouε gradient (250 ml) of NaCl (from 0.2 M to 1.0 M) , and 5 ml fractions were collected. Samples (0.01 ml of the collected fractions were used for the quantitative aεεay of the kinase stimulatory activity. Active fractions from three column runs (total volume = 360 ml) were pooled, concentrated to 25 ml by using a YM10 ultrafiltration membrane (Amicon, Danvers, MA) , and ammonium sulfate was added to reach a concentration of 1.7 M. After clearance by centrifugation (10,000 x g, 15 min.), the pooled material was loaded on a phenyl-Superose column (HRlO/10, Pharmacia) . The column was developed with a 45 ml gradient of (NH4)2S04 (from 1.7 M to no salt) in 0.1 M Na2P04 (pH 7.4) , and 2 ml fractions were collected and aεεayed (0.002 ml per εample) for kinaεe εtimulation (aε deεcribed in Example 6) . The major peak of activity waε pooled and dialyzed againεt 50 mM sodium phoεphate buffer (pH 7.3) . A Mono-S cation-exchange column (HR5/5, Pharmacia) waε preequilibrated with 50 mM sodium phosphate. After loading the active material (0.884 mg of protein; 35 ml) , the column was washed with the εtarting buffer and then developed at a rate of 1 ml/min. with a gradient of NaCl. The kinase stimulatory activity waε recovered at 0.45-0.55 M salt and was spread over r fractions of 2 ml each. These were pooled and " 3d directly on a Cu+2 chelating columns (1.6 ml, HR2/5 chelating Superoεe, Pharmacia) . Moεt of the proteinε adεorbed to the reεin, but they gradually eluted with a 30 ml linear gradient of ammonium chloride (0-1 M) . The activity eluted in a εingle peak of protein at the range of 0.05 to 0.2 M NH4C1. Samples from various steps of purification were analyzed by gel electrophoresiε followed by εilver εtaining uεing a kit from ICN (Coεta Mesa, CA) , and their protein contents were determined with a Coomaεεie blue dye binding assay using a kit from Bio-Rad (Richmond, CA) .
The p44 protein (10 μg) waε reconεtituted in 200 μl of 0.1 M ammonium bicarbonate buffer (pH 7.8) . Digeεtion waε conducted with L-1-toεyl-amide 2-phenylethyl chloromethyl ketone-treated trypεin (Serva) at 37 °C for 18 hr. at an enzyme-to-εubεtrate ratio of 1:10. The reεulting peptide mixture waε εeparated by reverεe-phaεe HPLC and monitored at 215 nm uεing a Vydac C4 micro column (2.1 mm i.d. x 15 cm, 300 A) and an HP 1090 liquid chromatographic εyεtem equipped with a diode-array detector and a workεtation. The column waε equilibrated with 0.1% trifluoroacetic acid (mobile phase A), and elution waε effected with a linear gradient from 0%-55% mobile phaεe B (90% acetonitrile in 0.1% trifluoroacetic acid) over 70 min. The flow rate waε 0.2 ml/min. and the column temperature waε controlled at 25°C. One-third aliquotε of the peptide peakε collected manually from the HPLC εyεtem were characterized by N-terminal εequence analyεiε by Edman degradation. The fraction eluted after 27.7 min. (T27.7) contained mixed amino acid εequenceε and was further rechromatographed after reduction as follows: A 70% aliquot of the peptide fraction was dried in vacuo and reconεtituted in 100 μl of 0.2 M ammonium bicarbonate buffer (pH 7.8) . DTT (final concentration 2 mM) waε added to the εolution, which waε then incubated at 37 °C for 30 min. The reduced peptide mixture was then separated by reverse-phaεe HPLC uεing a Vydac column (2.1 mm i.d. x 15 cm) . Elution conditionε and flow rat'were identical to thoεe deεcribed above. Amino acid sequence analyεiε of the peptide waε performed with a Model 477 protein sequencer (Applied Biosyεtems, Inc. , Foεter City, CA) equipped with an on-line phenylthiohydantoin (PTH) amino acid analyzer and a Model 900 data analyεiε system (Hunkapiller et al. (1986) In Methods of Protein Microcharacterization. J.E. Shively, ed. (Clifton, New Jersey: Humana Presε p. 223-247) . The protein waε loaded onto a trifluoroacetic acid-treated glass fiber disc precycled with polybrene and NaCl. The PTH-amino acid analysiε was performed with a micro liquid chromatography system (Model 120) using dual syringe pumpε and reverεe-phaεe (C-18) narrow bore columnε (Applied Bioεyεtems, 2.1 mm x 250 mm).
RNA was iεolated from Ratl-EJ cellε by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York (1982) and poly (A) + waε selected using an mRNA Separator kit (Clontech Lab, Inc., Palo Alto, CA) . cDNA was syntheεized with the Superεcript kit (from BRL Life
Technologieε, Inc., Bethesda, MD) . Column-fractionated double-strand cDNA was ligated into an Sail- and Notl-digested pJT-2 plasmid vector, a derivative of the pCD-X vector (Okayama and Berg, Mol. Cell Biol. 3.: 280 (1983)) and transformed into DH10B E. coli cells by electroporation (Dower et al., Nucl. Acidε Reε. 16.: 6127 (1988)) . Approximately 5 x 105 primary tranεformantε were εcreened with two oligonucleotide probeε that were derived from the protein εequenceε of the N-terminuε of NDF (reεidueε 5-24) and the T40.4 tryptic peptide
(reεidueε 7-12) . Their reεpective sequences were as follows (N indicates all 4 nt) :
(1) 5'-ATA GGG AAG GGC GGG GGA AGG GTC NCC CTC NGC A T AGG GCC GGG CTT GCC TCT GGA GCC TCT-3' (2) 5'-TTT ACA CAT ATA TTC NCC-3' C G G C
(1: SEQ ID No. 167; 2: SEQ ID No. 168)
The synthetic oligonucleotides were end-labeled with [γ-32P]ATP with T4 polynucleotide kinaεe and used to screen replicate setε of nitrocellulose filters. The hybridization solution contained 6 x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% εodium pyrophoεphate, 2 x Denhardt'ε εolution, 50 μg/ml εalmon εperm DNA, and 20% formamide (for probe 1) or no forma ide (for probe 2) . The filterε were waεhed at either 50°C with 0.5 x SSC, 0.2% SDS, 2 mM EDTA (for probe 1) or at 37 °C with 2 x SSC, 0.2% SDS, 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These cloneε were purified by replating and probe hybridization as described above. The cDNA clones were εequenced uεing an Applied Bioεystems 373A automated DNA εequencer and Applied Bioεyεtemε Taq DyeDeoxy™ Terminator cycle εequencing kitε following the manufacture'ε inεtructionε. In εome inεtances, sequences were obtained using [35S]dATP (Amersham) and Sequenase™ kits from U.S. Biochemicals following the manufacturer'ε inεtructionε. Both εtrandε of the cDNA clone 44 were εequenced by uεing εynthetic oligonucleotideε as primerε. The εequence of the moεt 5' 350 nt waε determined in εeven independent cDNA clones. The resultant clone demonstrated the pattern shown in figure 30 (NDF) . EXAMPLE 10 Strategies for Detecting Other Posεible Splicing Variants Alignment of the deduced amino acid sequenceε of the cDNA clones and PCR products of the bovine, and the publiεhed human (Fig. 31) and rat εequenceε show a high level of similarity, indicating that these sequences are derived from homologous genes within the three species. The variable number of mesεenger RNA transcripts detectable at the cDNA/PCR product level is probably due to extenεive tissue-specific splicing. The patternε obtained and εhown in Figure 30 suggestε that other εplicing variantε exiεt. A liεt of probable εplicing variantε iε indicated in Figure 37. Many of theεe variantε can be obtained by coding segment specific probing of cDNA libraries derived from different tiεεueε and by PCR experimentε uεing primer pairε εpecific to particular coding εegmentε. Alternatively, the variantε can be asεe bled from specific cDNA clones, PCR productε or genomic DNA regionε via cutting and εplicing techniqueε known to one εkilled in the art. For example, a rare reεtriction enzyme cutting εite in a common coding segment (e.g., A), can be uεed to connect the FBA amino terminus of GGF2BPP5 to carboxy terminal sequences of GGF2BPP1, GGFBPP2, GGFBPP3, or GGFBPP4. If the presence or the absence of coding εegment E and/or G provide benefit for contemplated and εtated uεeε, then theεe coding εegmentε can be included in expreεεion conεtructε. Theεe variant εequenceε can be expreεεed in recombinant εystems and the recombinant productε can be aεεayed to determine their level of Schwann cell mitogenic activity aε well as their ability to bind and activate the pl85erbB2 receptor.
EXAMPLE 11 Ide "ication of Functional Elements of GGF e . c od structureε of the family of GGF ue s inα , ~e that the longeεt formε (as repreεented by GGF2BPP4) encode tranεmembrane proteinε where the extracellular part containε a domain which reεe bleε epidermal growth factor (see Carpenter and Wahl in Peptide Growth Factors and Their Receptors I pp. 69-133, Springer-Verlag, NY 1991) . The positions of the cyεteine residues in coding segmentε C and C/D or C/D' peptide εequence are conεerved with respect to the analogous residueε in the epidermal growth factor (EGF) peptide sequence (see Figure 35, SEQ ID Nos. 151-153) . Thiε εuggeεtε that the extracellular domain functionε aε receptor recognition and biological activation εites. Several of the variant forms lack the H, K, and L coding segmentε and thuε may be expreεεed aε εecreted, diffuεible biologically active proteinε. GGF DNA εequenceε encoding polypeptideε which encompass the EGF- like domain (EGFL) can have full biological activity for stimulating glial cell mitogenic activity.
Membrane bound versionε of thiε protein may induce Schwann cell proliferation if expreεεed on the εurface of neuronε during embryogeneεiε or during nerve regeneration (where the εurfaces of neuronε are intimately aεεociated with the εurfaces of proliferating Schwann cells) . Secreted (non membrane bound) GGFs may act as classically diffusible factors which can interact with Schwann cellε at εome diεtance from their point of εecretion. Other formε may be releaεed from intracells by sources via tisεue injury and cell diεruption. An example of a εecreted GGF iε the protein encoded by GGF2HBS5 (εee example 6) ; this iε the only GGF known which haε been found to be directed to the exterior of the cell (example 7) . Secretion iε probably mediated via an N-terminal hydrophobic εequence found only in region E, which iε the N-terminal domain contained within recombinant GGF-II encoded by GGF2HBS5.
Other GGF's appear to be non-secreted (see example 6) . These GGFs may be injury responεe formε which are releaεed aε a conεequence of tiεεue damage. Other regions of the predicted protein structure of GGF-II (encoded by GGF2HBS5) and other proteins containing regions B and A exhibit εimilaritieε to the human baεement membrane heparan εulfate proteoglycan core protein (ref.). The peptide ADSGEY, which iε located next to the εecond cyεteine of the C2 immunoglobulin fold in theεe GGF'ε, occurs in nine of twenty-two C-2 repeats found in that basal lamina protein. This evidence strongly suggestε that theεe proteinε may asεociate with matrix proteinε εuch aε thoεe aεεociated with neuronε and glia, and may εuggeεt a method for εequestration of glial growth factors at target εiteε.
EXAMPLE 12 Purification of GGFε from Recombinant Cellε In order to obtain full length or portionε of GGFε to assay for biological activity, the proteins can be overproduced using cloned DNA. Several approaches can be used. A recombinant E. coli cell containing the sequenceε deεcribed above can be conεtructed. Expreεεion systems such as pNH8a or pHH16a (Stratagene, Inc.) can be used for this purpose by following manufacturers procedures. Alternatively, these sequenceε can be inεerted in a mammalian expreεεion vector and an overproducing cell line can be conεtructed. Aε an example, for thiε purpoεe DNA encoding a GGF, clone GGF2BPP5 haε been expressed in both COS cells and Chineεe hamεter ovary cells (see Example 7) (J. Biol. Chem. 263 , 3521-3527, (1981)) . This vector containing GGF DNA εequences can be transfected into host cells using eεtabliεhed procedureε.
Tranεient expreεεion can be exa i: d or G418-reεistant cloneε can be grown in tne preεence of methotrexate to select for cells that amplify the dhfr gene (contained on the pMSXND vector) and, in the process, co-amplify the adjacent GGF protein encoding εequence. Becauεe CHO cellε can be maintained in a totally serum-free, protein-free medium (Hamilton and Ham, In Vitro H, 537-547 (1977)), the desired protein can be purified from the medium. Western analysis uεing the antiεera produced in Example 9 can be uεed to detect the preεence of the deεired protein in the conditioned medium of the overproducing cellε.
The deεired protein (rGGF-II) waε purified from the medium conditioned by tranεiently expreεεing coε cellε aε followε. rGGF-II waε harveεted from the conditioned medium and partially purified uεing Cation Exchange
Chromatography (POROS-HS) . The column waε equilibrated with 33.3 mM MES pH 6.0. Conditioned media waε loaded at flow rate of 10 ml/min. The peak containing Schwann cell proliferation activity and immunoreactive (uεing the polyclonal antisera was against a GGFII peptide described above) was eluted with 50 mM Tris, 1M NaCl pH 8.0. (Figure 50A and 50B respectively) . rGGF-II is also expresεed uεing a εtable Chineεe Ovary Hamεter cell line. rGGF-II from the harveεted conditioned media waε partially purified uεing Cation
Exchange Chromatograph (POROS-HS) . The column waε equilibrated with PBS pH 7.4. Conditioned media waε loaded at 10 ml/min. The peak containing the Schwann Cell Proliferative activity and immunoreactivity (uεing GGFII polyclonal antiεera) waε eluted with 50 mM Hepeε, 500 mM NaCl pH 8.0. An additional peak waε observed at 50 mM Hepes, 1M NaCl pH 8.0 with both proliferation aε well aε immunoreactivity (Fig. 51) . rGGF-II can be further purified uεing Hydrophobic Interaction Chromatography aε a high reεolution step;
Cation exchange/Reεerve phaεe Chromatography (if needed aε εecond high reεolution εtep) ; A viral inactivation εtep and a DNA removal εtep such as Anion exchange chromatography.
Detailed description of procedures used are as follows: Schwann Cell Proliferation Activity of the recombinant GGF-II peak eluted from the Cation Exchange column waε determined aε follows: Mitogenic responses of the cultured Schwann cells were meaεured in the preεence of 5 M Forεkolin uεing the peak eluted by 50 mM Triε 1 M NaCl pH 8.0. The peak waε added at 20 1, 10 1 (1:10) 10 1 and (1:100) 10 1. Incorporation of 125I-Uridine waε determined and expressed as (CPM) following an 18-24 hour exposure. An immunoblot using polyclonal antibody raised against a peptide of GGF-II was carried out as follows: 10 μl of different fractionε were ran on 4-12% gradient gelε. The gelε were tranεferred on to Nitrocelluloεe paper, and the nitrocelluloεe blotε were blocked with 5% BSA and probed with GGF-II-εpecific antibody (1:250 dilution). 125I protein A (1:500 dilution, Specific Activity = 9.0/Ci/g) waε used as the secondary antibody. The immunoblots were expoεed to Kodax X-Ray fil ε for 6 hourε. The peak fractionε eluted with 1 M NaCl εhowed a broad im unoreactive band at 65-90 Kd which iε the expected εize range for GGFII and higher molecular weight glycoformε.
GGF-II purification on cation exchange columns was performed as follows: CHO cell conditioned media expreεεing rGGFII waε loaded on the cation exchange column at 10 ml/min. The column waε equilibrated with PBS pH 7.4. The elution waε achieved with 50 mM Hepes 500 mM NaCl pH 8.0 and 50 mM Hepeε 1M NaCl pH 8.0 reεpectively. All fractionε were analyzed uεing the Schwann cell proliferation aεεay (CPM) described herein. The protein concentration (mg/ml) was determined by the Bradford asεay uεing BSA aε the εtandard.
A Weεtern blot uεing 10 μl of each fraction was performed. As indicated in Figure 51A and 5IB, immunoreactivity and the Schwann cell activity co- migrates. The Schwann cell mitogenic aεεay deεcribed herein may be uεed to aεεay the expreεεed product of the full length clone or any biologically active portionε thereof. The full length clone GGF2BPP5 haε been expreεεed tranεiently in COS cells. Intracellular extracts of transfected COS cells show biological activity when assayed in the Schwann cell proliferation assay described in Example 1. In addition, the full length close encoding GGF2HBS5 haε been expreεεed tranεiently in CHO and inεect (Example 7) cells. In thiε caεe both cell extract and conditioned media show biological activity in the Schwann cell proliferation asεay deεcribed in Example 1. Any member of the family of εplicing variant complementary DNA'ε derived from the GGF gene (including the Heregulinε) can be expressed in this manner and assayed in the Schwann cell proliferation assay by one skilled in the art.
Alternatively, recombinant material may be isolated from other variantε according to Wen et al. (Cell J59., 559 (1992)) who expreεεed the εplicing variant Neu differentiation factor (NDF) in COS-7 cellε. cDNA cloneε inεerted in the pJT-2 eukaryotic plaεmid vector are under the control of the SV40 early promoter, and are 3'-flanked with the SV40 termination and polyadenylation εignalε. COS-7 cells were transfected with the pJT-2 plasmid DNA by electroporation aε follows: 6 x 106 cellε (in 0.8 ml of DMEM and 10% FEBS) were tranεferred to a 0.4 cm cuvette and mixed with 20 μg of plaεmid DNA in 10 μl of TE εolution (10 mM Triε-HCl (pH 8.0) , 1 mM EDTA) . Electroporation waε performed at room temperature at 1600 V and 25 μF using a Bio-Rad Gene Pulser apparatus with the pulεe controller unit εet at 200 ohms. The cellε were then diluted into 20 ml of DMEM, 10% FBS and tranεferred into a T75 flaεk (Falcon) . After 14 hr. of incubation at 37 °C, the medium waε replaced with DMEM, 1% FBS, and the incubation continued for an additional 48 hr. Conditioned medium containing recombinant protein which waε harvested from the cells demonstrated biological activity in a cell line expresεing the receptor for thiε protein. This cell line (cultured human breast carcinoma cell line AU 565) was treated with recombinant material. The treated cellε exhibited a morphology change which iε characteriεtic of the activation of the erbB2 receptor. Conditioned medium of this type also can be tested in the Schwann cell proliferation asεay.
EXAMPLE 13
Purification and Aεεay of Other Proteinε which bind p l 8 5 erbB2 Receptθr
I. Purification of gp30 and p70
Lupu et al. (Science 249. 1552 (1990)) and Lippman and Lupu (patent application number PCT/US91/03443 (1990)), hereby incorporated by reference, have purified a protein from conditioned media of a human breaεt cancer cell line MDA-MB-231, aε followε.
Conditioned media collections were carried uεing well-known procedures. The media was concentrated 100-fold in an Amicon ultra-filtration cell (YM5 membrane) (Amicon, Danvers, MA) . Once clarified and concentrated, the media were stored at -20°C while consecutive collections were made during the following days. The concentrated media were dialyzed using Spectra/por® 3 tubing (Spectrum Medical Industrieε, Loε Angeles, CA) against 100 volumes of 0.1 M acetic acid over a two day period at 4°C. The material that precipitated during dialysiε waε removed by centrifugation at 4000 rpm for 30 min. at 4°C; proteaεe inhibitorε were added. The clarified εample waε then lyophilized.
Lyophilized conditioned medium was dissolved in 1 M acetic acid to a final concentration of about 25 mg/ml total protein. Insoluble material waε removed by centrifugation at 10,000 rpm for 15 minutes. The sample was then loaded onto a Sephadex G-100 column (XK 16, Pharmacia, Piεcataway, NJ) , was equilibrated and was subjected to elution with 1 M acetic acid at 4°C with an upward flow of 30 ml/hr. 100 ng of protein was procesεed from 4 ml of 100-fold concentrated medium. Fractionε containing 3 ml of eluate were lyophilized and reεuεpended in 300 μl PBS for assay and served as a source for further purification. Sephadex G-100 purified material was run on reversed-phaεe high preεεure liquid chromatography (HPLC) . The firεt εtep involved a εteep acetonitrile gradient. Steep acetonitrile gradient and all other HPLC εtepε were carried out at room temperature after equilibration of the C3-Reversed phase column with 0.05% TFA (Trifluoroacetic acid) in water (HPLC-grade) . The sampleε were loaded and fractionε were eluted with a linear gradient (0-45% acetonitrile in 0.05% TFA) at a flow rate of 1 ml/min. over a 30 minute period. Abεorbance was monitored at 280 nm. One ml fractions were collected and lyophilized before analyεis for EGF receptor-competing activity.
A second HPLC εtep involved a εhallow acetonitrile gradient. The pool of active fractionε from the previous HPLC εtep waε rechromatographed over the εame column. Elution waε performed with a 0-18% acetonitrile gradient in 0.05% TFA over a 5 minute period followed by a linear 18-45% acetonitrile gradient in 0.05% TFA over a 30 minute period. The flow rate waε 1.0 ml/min. and 1 ml fractions were collected. Human TGFα-like factor waε eluted at a 30-32% acetonitrile concentration aε a εingle peak detectable by RRA.
Lupu et al. (Proc. Natl. Acad. Sci. 89., 2287 (1992)) purified another protein which bindε to the pl85erbB2 receptor. Thiε particular protein, p75, waε purified from conditioned medium used for the growth of SKBr-3 (a human breast cancer cell line) propagated in improved Eagle's medium (IMEM: GIBCO) supplemented with 10% fetal bovine serum (GIBCO) . Protein p75 was purified from concentrated (100X) conditioned medium uεing a pl85eΛB2 affinity column. The 94 Kilodalton extracellular domain of pl85erbB2 (which bindε p75) waε produced via recombinant expreεεion and waε coupled to a polyacrylamide hydrazido-Sepharoεe affinity chromatography matrix. Following coupling the matrix was washed extensively with ice cold 1.0 M HC1 and the beads were activated with 0.5 M NaN02. The temperature was maintained at O'C for 20 minutes and thiε waε followed by filtration and waεhing with ice cold 0.1 M HC1. 500 ml of concentrated conditioned medium waε run through the beadε by gravity. The column waε waεhed and eluted εtepwise with 1.0 M citric acid at pH values from 4.0 to 2.0 (to allow disεociation of the erbB2 and p75) . All fractionε were deεalted on Pharmacia PD10 columnε. Purification yielded a homogeneouε polypeptide of 75kDa at 3.0-3.5 elution pH (confirmed by analyεiε on SDS/PAGE by εilver εtaining) .
II. Binding of gp30 to p!85crbB2
The purified gp30 protein waε teεted in an aεεay to determine if it bound to pl85crbB2. A competition aεεay with a monoclonal antibody againεt pl85crbB2. The gp30 protein diεplaced antibody binding to pl85erbB2 in SK-BR-3 and MDA-MB-453 cellε (human breaεt carcinoma cell lines expreεεing the pl85rbB2 receptor) . Schwann cell proliferation activity of gp30 can also be demonεtrated by treating Schwann cell cultureε with purified gp30 uεing the aεεay procedure deεcribed in Exampleε 1-3.
III. Binding of P75 to pl85erfaB2
To aεεess whether the 75-kDa polypeptide (p75) obtained from SKBr-3 conditioned medium was indeed a ligand for the erbB2 oncoprotein in SKBr-3 cells, a competition asεay as described above for gp30 was used. It waε found that the p75 exhibited binding activity, whereaε material from other chromatography fractions did not show such activity (data not shown) . The flow-through material showed some binding activity. Thiε might be due to the preεence of εhed erbB2 ECD.
IV. Other Pl85erbB2 ligandε
Peleε et al. (Cell 69., 205 (1992)) have alεo purified a l85erbB2 stimulating ligand from rat cells, (NDF, εee Example 8 for method) . Holmeε et al. (Science 256. 1205 (1992)) have purified Heregulin α from human cellε which bindε and εtimulateε l85erbB2 (εee example 6) . Tarakovεky et al. Oncogene 6:218 (1991) have demonεtrated bending of a 25 kD polypeptide isolated from activated macrophages to the Neu receptor, a pl85crbB2 homology, herein incorporated by reference.
VI. NDF Iεolation
Yarden and Peleε (Biochemiεtry 3__, 3543 (1991)) have identified a 35 kilodalton glycoprotein which will εtimulate the l85crt>B2 receptor. The protein waε identified in conditioned medium according to the following procedure. Rat I-EJ cells were grown to confluence in 175-cm2 flaskε (Falcon) . Monolayerε were waεhed with PBS and left in εerum-free medium for 10-16 h. The medium waε diεcarded and replaced by freεh εerum-free medium that waε collected after 3 dayε in culture. The conditioned medium waε cleared by low-εpeed centrifugation and concentrated 100-fold in an Amicon ultrafiltration cell with a YM2 membrane (molecular weight cutoff of 2000) . Biochemical analyεeε of the neu εtimulatory activity in conditioned medium indicate that the ligand iε a 35-kD glycoprotein that it iε heat εtable but εensitive to reduction. The factor is precipitable by either high salt concentrations or acidic alcohol. Partial purification of the molecule by selective precipitation, heparin-agarose chromatography, and gel filtration in dilute acid resulted in an active ligand, which is capable of εtimulating the protooncogenic receptor but iε ineffective on the oncogenic neu protein, which iε conεtitutively active. The purified fraction, however, retained the ability to εtimulate alεo the related receptor for EGF, εuggeεting that theεe two receptorε are functionally coupled through a bidirectional mechaniε . Alternatively, the preεumed ligand interactε εimultaneouεly with both receptorε. The preεented biochemical characteristic of the factor may be uεed to enable a completely purified factor with which to explore theεe posεibilitieε.
In other publicationε, Davis et al. (Biochem. Biophys. Reε. Commun. 179. 1536 (1991) , Proc. Natl. Acad. Sci. .88., 8582 (1991) and Greene et al., PCT patent application PCT/US91/02331 (1990)) deεcribe the purification of a protein from conditioned medium of a human T-cell (ATL-2) cell line. ATL-2 cell line iε an IL-2-independent HTLV-1 (+) T cell line. Mycoplaεm-free ATL-2 cellε were maintained in RPMI 1640 medium containing 10% FCB aε the culture medium (10% FCS-RPMI 1640) at 37 °C in a humidified atmosphere with 5% C02.
For purification of the proteinaceous εubεtance, ATL-2 cellε were waεhed twice in 1 x PBS and cultured at 3 x 105 ml in εerum-free RPMI 1640 medium/2 M L-glutamine for seventy-two hours followed by pelleting of the cells. The culture supernatant so produced iε termed "conditioned medium" (CM.).
CM. waε concentrated 100 fold, from 1 liter to 10 ml, uεing a YM-2 Diaflo membrane (Amicon, Boεton, MA) with a lOOOd cutoff. For uεe in some assays, concentrated CM. containing components greater than 1000 MW were rediluted to original volume with RPMI medium. Gel electrophoreεis uεing a polyacrylamide gradient gel (Integrated Separation Syεtemε, Hyde Park, MD or Phorecaεt Syεtem by Amerεham, Arlington Heightε, IL) followed by εilver εtaining of εome of thiε two column purified material from the one liter preparation revealed at leaεt four to five bandε of which the lOkD and 20kD bandε were unique to thiε material. Paεεed CM. containing componentε leεε than 1000 NW were used without dilution.
Concentrated conditioned medium was filter sterilized with a .45μ uniflo filter (Schleicher and Schuell, Keene, NH) and then further purified by application to a DEAE-SW anion exchange column (Waters, Inc., Milford, MA) which had been preequilibrated with lOmM Tris-Cl, pH 8.1 Concentrated CM. proteinε repreεenting one liter of original ATL-2 conditioned medium per HPLC run were abεorbed to the column and then eluted with a linear gradient of O M to 40mM NaCl at a flow rate of 4 ml/min. Fractionε were aεεayed uεing an in vitro immune complex kinase asεay with 10% of the appropriate DEAE fraction (1 column purified material) or 1% of the appropriate C18 fractions (two column purified material) . The activity which increased the tyrosine kinase activity of pl85c-neu in a doεe-dependent manner uεing the in vitro immune complex kinaεe aεεay waε eluted aε one dominant peak acroεε 4 to 5 fractions (36-40) around 220 to 240 mM of NaCl. After HPLC-DEAE purification, the proteins in the active fractions were concentrated and pooled, concentrated and subjected to C18 (million matrix) reverse phase chromatography (Waters, Inc., Milford, MA) (referred to as the C18+1 step or two column purified material) . Elution waε performed under a linear gradient of 2-propanol against 0.1% TFA. All the fractions were dialyzed against RPMI 1640 medium to remove the 2-propanol and asεayed uεing the jLn vitro immune complex kinaεe aεεay, described below, and a 1% concentration of the appropriate fraction. The activity increasing the tyrosine kinaεe activity of pl85c-neu waε eluted in two peakε. One eluted in fraction 11-13, while a second, slightly lesε active peak of activity eluted in fractionε 20-23. Theεe two peakε correεpond to around 5 to 7% of isopropanol and 11 to 14% isopropanol respectively. C18#l generated fractions 11-13 were used in the characterization studies. Active fractions obtained from the εecond chromatographic εtep were pooled, and deεignated aε the proteinaceouε subεtance sample.
A twenty liter preparation employed the same purification strategy. The DEAE active fractions 35-41 were pooled and subjected to cl8 chromatography as discuεsed above. C18#l fractions 11-13 and 21-24 both had dose-dependent activity. The pool of fractionε 11-13 waε subjected to an additional C18 chromatographic step (referred to as C18#2 or three column purified material) . Again, fractions 11-13 and 21-24 had activity. The dose responεe of fraction 23 aε determined by ijn vitro immune complex kinase asεay aε deεcribed in Example 8 may be obtained upon addition of 0.005% by volume fraction 23 and 0.05% by volume fraction 23. Thiε repreεentε the greateεt purity achieved.
Molecular weight rangeε were determined baεed on gel filtration chromatography and ultrafiltration membrane analysis. Near equal amounts of tyroεine kinaεe activity were retained and paεεed by a 10,000 molecular weight cut off filter. Almoεt all activity waε paεεed by a 30,000 molecular weight cut off filter. Molecular weight rangeε for active chromatographic fractionε were determined by comparing fractionε containing dose-dependent neu-activating activity to the elution profiles of a εet of protein molecular weight εtandardε (Sigma Chemical Co., St. Louiε, MO) generated uεing the εame running conditionε. A low molecular weight region of activity waε identified between 7,000 and 14,000 daltons. A second range of activity ranged from about 14,000 to about 24,000 daltons. After gel electrophoresiε uεing a polyacrylamide gradient gel (Integrated Separation Syεtems, Hyde Park, MD or Phorecase Syεtem by Amerεham, Arlington Heightε, IL) , silver staining of the three-column purified material (cl8#2) was done with a commercially available silver staining kit (BioRad, Rockville Centre, NY) . Fraction 21, 22, 23, and 24 from cl8#2 purification of the twenty liter preparation were run with markers. Fractions 22 and 23 showed the most potent dose reεponεe in the l85erbB2 (neu) kinaεe aεεay (εee below) . The fact that εelected molecular weight fractionε interact with 185 eιtB 2 was demonεtrated with an immune complex kinaεe aεεay.
Huang et al. (1992, J. Biol. Chem. 257:11508-11512), hereby incorporated by reference, have iεolated an additional neu/erb B2 ligand growth factor from bovine kidney. The 25 kD polypeptide factor waε iεolated by a procedure of column fractionation, followed by εequential column chromatography on DEAE/celluloεe (DE52) , Sulfadex (εulfated Sephadex G-50) , heparin-Sepharoεe 4B, and
Superdex 75 (faεt protein liquid chromatography) . The factor, NEL-GF, εtimulateε tyroεine-εpecific autophoεphorylation of the neu/erb B2 gene product.
VII. Immune complex assay NDF for ligand binding to pl85crbB2 : This asεay reflectε the differenceε in the autophoεphorylation activity of immunoprecipitated pl85 driven by pre-incubation of PN-NR6 cell lyεate with varying amountε of ATL-2 conditioned medium (C.H.) or proteinaceous subεtance and is referred to hereinafter as neu-activating activity.
Cell lines used in the immune complex kinaεe aεsay were obtained, prepared and cultured according to the methods discloεed in Kokai et al., Cell 5_5, 287-292 (July 28, 1989) the diεclosures of which are hereby incorporated by reference aε if fully εet forth herein, and U.S. application εerial number 386,820 filed July 27, 1989 in the name of Mark I. Green entitled "Methods of Treating Cancerouε Cellε with Anti-Receptor Antibodieε", the diεcloεureε of which are hereby incorporated by reference aε if fully εet forth herein.
Cell lineε were all maintained in DMEM medium containing 5% FCS aε the culture medium (5% FCS-DMEM) at 37°C in a humidified atmoεphere with 5% C02.
Denεe cultureε of cells in 150 mm dishes were washed twice with cold PBS, εcraped into 10 ml of freeze-thaw buffer (150 mM NaCl, 1 mM MgCl2, 20 mM Hepeε, pH 7.2, 10% Glycerol, 1 mM EDTA, 1% Aprotinin) , and centrifuged (600 x 6, 10 minuteε) . Cell pelletε were reεuεpended in
1 ml Lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 3% Brij 35, 1 mM EDTA, 1.5 mM MgCl2, 1% Aprotinin, 1 mM EGTA,
20 μM Na3V04, 10% Glycerol) and rotated for thirty minuteε at 4°C All chemicalε were from Sigma Chemical Co., St. Louiε, Mo, unleεε otherwise indicated. The insoluble materials were removed by centrifugation at 40,000 x g for thirty minuteε. The clear εupernatant which waε εubεequently uεed iε deεignated aε cell lyεate.
The cell lyεateε were incubated for fifteen minuteε with 50 μl of 50% (volume/volume) Protein A-sepharose (Sigma Chemical Co., St. Louiε, Missouri) , and centrifugated for two minutes to preclear the lyεateε. 50 μl aliquotε of precleared cell lyεate were incubated on ice for fifteen minuteε with conditioned medium, proteinaceous subεtance, or other factorε aε εpecified, in a final volume of 1 ml with lyεiε buffer. The sample was then incubated with 5 μg of 7.16.4 monoclonal antibody, which recognizes the extracellular domain of the pl85neu and pl85c-neu, or other appropriate antibodies, for twenty minutes on ice, followed by a twenty minute incubation with 50 μl of 50% (vol/vol) protein A-Sepharose with rotation at 4°C Immune complexes were collected by centrifugation, waεhed four timeε with 500 μl of waεhing buffer (50 mM Hepeε, pH 7.5, 0.1%, Brij 35, 150 mM NaCl, 2 mM EDTA, 1% Aprontinin, 30 μm Na3V04) , then twice with reaction buffer (20 mM Hepes (pH 7.4), 3 mM MnCl2 and 0.1% Brij 35, 30 μm Na3V04) . Pellets were reεuspended in 50 μl of reaction buffer and (Gamma-32P]-ATP (Amersham, Arlington Heightε, IL) waε added giving a final concentration of 0.2 μ . The εampleε were incubated at 27°C for twenty minuteε or at 4°C for 25 minuteε with purer εampleε. The reactionε were terminated by addition of 3 x SDS εample buffer containing 2 mM ATP and 2 mM EDTA and then incubating them at 100°C for five minutes. The samples were then subjected to SDS-PAGE analysiε on 10% acrylamide gelε. Gelε were εtained, dried, and expoεed to Kodak XAR or XRP film with intenεifying screens.
VIII. Purification of acetylcholine receptor inducing activity (ARIA)
ARIA, a 42 kD protein which stimulates acetylcholine receptor synthesis, has been isolated in the laboratory of Gerald Fiεchbach (Fallε et al., Cell 72:801-815 (1993)) . ARIA induceε tyroεine phosphorylation of a 185 Kda muεcle tranεmembrane protein which resembles pl85erB2, and stimulateε acetylcholine receptor εyntheεiε in cultured embryonic myotubeε. Sequence analyεis of cDNA clones which encode ARIA shows that ARIA is a member of the GGF/erbB2 ligand group of proteinε, and this iε potentially uεeful in the glial cell mitogeneεiε stimulation and other applications of, e.g., GGF2 deεcribed herein.
EXAMPLE 14 Protein tyrosine phosphorylation mediated by GGF in Schwann cells
Rat Schwann cells, following treatment with sufficient levels of Glial Growth Factor to induce proliferation, show stimulation of protein tyroεine phoεphorylation (figure 36) . Varying amounts of partially purified GGF were applied to a primary culture of rat Schwann cellε according to the procedure outlined in Example 3. Schwann cellε were grown in DMEM/10% fetal calf εerum/5 μM forskolin/0.5μg per mL GGF-CM (0.5mL per well) in poly D-lysine coated 24 well plateε. When confluent, the cellε were fed with DMEM/10% fetal calf εerum at 0.5mL per well and left in the incubator overnight to quieεce. The following day, the cellε were fed with 0.2mL of DMEM/10% fetal calf εerum and left in the incubator for 1 hour. Teεt εampleε were then added directly to the medium at different concentrations and for different lengths of time as required. The cellε were then lyεed in boiling lyεiε buffer (εodium phosphate, 5mM, pH 6.8; SDS, 2%, /3-mercapteothanol, 5%; dithiothreitol, 0.1M; glycerol, 10%; Bromophenol Blue, 0.4%; sodium vanadate, lOmM) , incubated in a boiling water bath for 10 minutes and then either analyzed directly or frozen at -70"C Samples were analyzed by running on 7.5% SDS-PAGE gels and then electroblotting onto nitrocelluloεe uεing εtandard procedureε aε deεcribed by Towbin et al. (1979) Proc. Natl. Acad. Sci. USA 76:4350-4354. The blotted nitrocellulose was probed with antiphosphotyrosine antibodieε uεing εtandard methods as described in Kamps and Selton (1988) Oncogene 2:305-315. The probed blots were exposed to autoradiography film overnight and developed using a εtandard laboratory proceεεor.. Denεitometric meaεurementε were carried out uεing an Ultraεcan XL enhanced laεer denεitometer (LKB) . Molecular weight aεεignmentε were made relative to preεtained high molecular weight standards (Sigma) . The dose reεponεeε of protein phoεphorylation and Schwann cell proliferation are very εimilar (figure 36) . The molecular weight of the phoεphorylated band iε very cloεe to the molecular weight of pl85crbB2. Similar reεultε were obtained when Schwann cellε were treated with conditioned media prepared from COS cellε tranεlateε with the GGF2HBS5 clone. Theεe reεultε correlate well with the expected interaction of the GGFε with and activation of -l85erbB2.
This experiment has been repeated with recombinant GGF-II. Conditioned medium derived from a CHO cell line stably transformed with the GGF-II clone (GGF2HBS5) stimulateε protein tyrosine phosphorylation using the aεεay deεcribed above. Mock tranεfected CHO cellε fail to εtimulate this activity (Fig. 52) .
EXAMPLE 15 Asεav for Schwann cell Proliferation by Protein Factor from the MDA-MB-231 cell line.
Schwann cell proliferation iε mediated by conditioned medium derived from the human breast cancer cell line MDA-MB-231. On day 1 of the aεsay, 104 primary rat Schwann cells were plated in 100 μl of Dulbecco's Modified Eagle'ε medium εupplemented with 5% fetal bovine plaε a per well in a 96 well microtiter plate. On day 2 of the aεεay, 10 μl of conditioned medium (from the human breaεt cancer cell line MDA-MB-231, cultured as described in Example 6) was added to each well of the microtiter plate. One day 6, the number of Schwann cells per plate was determined using an acid phosphataεe aεεay (according to the procedure of Connolly et al. Anal. Biochem. 152 : 136 (1986)) . The plate waε waεhed with 100 μl of phoεphate buffered εaline (PBS) and 100 μl of reaction buffer (0.1M εodium acetate, (pH 5.5)) , 0.1% Triton X-100, and 10 mM p-nitrophenyl phoεphate) was added per well. The plate was incubated at 37 °C for two hours and the reaction was stopped by the addition of 10 μl of IN NaOH. The optical density of each sample was read in a spectrophotometer at 410 nm. A 38% stimulation of cell number over Schwann cells treated with conditioned medium from a control cell line (HS-294T, a non-producer of erbB-2 ligand) was observed. This result showε that a protein εecreted by the MDA-MB-231 cell line (which secretes a pl85c, B2 binding activity) stimulateε Schwann cell proliferation.
EXAMPLE 16 N-glycoεylation of GGF
The protein εequence predicted from the cDNA sequence of GGF-II candidate cloneε GGF2BPP1,2 and 3 containε a number of consensuε N-glycoεylation motifε. A gap in the GGFII02 peptide εequence coincideε with the aεparagine reεidue in one of theεe motifε, indicating that carbohydrate is probably bound at this site.
N-glycosylation of the GGFε waε εtudied by obεerving mobility changeε on SDS-PAGE after incubation with N- glycanase, an enzyme that cleaves the covalent linkageε between carbohydrate and aεpargine reεidueε in proteins.
N-Glycanase treatment of GGF-II yielded a major band of MW 40-42 kDa and a minor band at 45-48 kDa. Activity elution experiments under non-reducing conditions εhowed a εingle active deglycoεylated εpecies at ca 45-50 kDa.
Activity elution experiments with GGF-I also demonstrate an increase in electrophoretic mobility when treated with N-Glycanase, giving an active εpecieε of MW 26-28 kDa. Silver εtaining confirmed that there iε a mobility εhift, although no N-deglycoεylated band could be assigned because of background staining in the εample uεed.
Deposit
Nucleic acid encoding GGF-II (cDNA, GGF2HBS5) protein (Example 6) in a plasmid pBlueεcript 5k, under the control of the T7 promoter, waε depoεited in the American Type Culture Collection, Rockville, Maryland, on September 2, 1992, and given ATCC Acceεsion No. 75298. Applicant acknowledges its responsibility to replace this plasmid εhould it become non-viable before the end of the term of a patent iεεued hereon, and itε reεponεibility to notify the ATCC of the iεεuance of εuch a patent, at which time the depoεit will be made available to the public. Prior to that time the depoεit will be made available to the Commiεεioner of Patentε under the termε of 37 CFR §1.14 and 35 USC §112.

Claims

What is claimed is:
1. A DNA sequence encoding a polypeptide of the formula
WYBAZCX wherein WYBAZCX iε co poεed of the polypeptide εegmentε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 147, 160, 161, and 163); wherein W co priεeε polypeptide segment F, or is absent; wherein Y compriseε polypeptide εegment E, or iε abεent; wherein Z comprises polypeptide segment G or is absent; and wherein X compriseε polypeptide εegmentε C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, or C/D' D' HL; provided that, either a) at leaεt one of F, Y, B, A, Z, C, or X iε of bovine origin; or b) Y compriεes polypeptide segment E; or c) X comprises polypeptide segments C/D HKL, C/D D, C/D' HKL, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, C/D'H, C/D C/D'H, or C/D C/D'HL.
2. The DNA sequence of claim 1, wherein X compriεes polypeptide segments C/D HKL having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141-142, 146, 147, 160, 161).
3. The DNA sequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D' H having the amino acid εequenceε εhown in Figure 31 (SEQ ID Nos. 136-139, 141, 143, 146, 160) .
4. The DNA sequence of claim 1, wherein X comprises polypeptide segmentε C/D D having the amino acid εequences shown in Figure 31 (SEQ ID Noε. 136-139, 141, 142, 144, 160).
5. The DNA εequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D' HKL having the amino acid εequences shown in Figure 31 (SEQ ID Nos. 136-139, 141, 143, 146, 147, 160, 161).
6. The DNA sequence of claim 1, wherein X compriseε polypeptide εegmentε C/D C/D' HKL having the amino acid εequenceε shown in Figure 31 (SEQ ID Nos. 136- 139, 141-143, 146, 147, 160, 161).
7. The DNA sequence of claim 1, wherein X compriseε polypeptide εegmentε C/D C/D' H having the amino acid sequences shown in Figure 31 (SEQ ID Noε. 136- 139, 141-143, 146, 160).
8. The DNA εequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D C/D' HL having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136- 139, 141-143, 146, 147, 160) .
9. The DNA εequence of claim 1, wherein X compriεeε polypeptide segments C/D C/D' D having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136- 139, 141-144, 160).
10. The DNA εequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D D'H having the amino acid εequences shown in Figure 31 (SEQ ID Nos. 136-139, 141-142, 145, 146, 160) .
11. The DNA sequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D D'H L having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141-142, 145, 146, 147, 160) .
12. The DNA εequence of claim 1, wherein X compriεeε polypeptide segments C/D D'H K L having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136- 139, 141-142, 145-147, 160, 161).
13. The DNA εequence of claim 1, wherein X compriεeε polypeptide εegments C/D' D' H having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141, 143, 145, 146, 160).
14. The DNA εequence of claim 1, wherein X compriεeε polypeptide εegmentε C/D' D' H K L having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136- 139, 141, 143, 145-147, 160, 161).
15. The DNA εequence of claim 1, wherein X compriεeε polypeptide segments C/D C/D' D' H having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136- 139, 141-143, 145, 146, 160).
16. The DNA sequence of claim 1, wherein X compriseε polypeptide segments C/D C/D' D' H L having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136- 139, 141-143, 145-147, 160) .
17. The DNA εequence of claim 1, wherein X compriεeε polypeptide segments C/D C/D' D' H K L having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141-143, 145-147, 160, 161) .
18. The DNA sequence compriεing coding εegmentε 5FBA3' coding for polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136, 138, 139) .
19. The DNA εequence compriεing coding εegmentε 5FBA'3' coding for polypeptide εegmentε having the amino acid sequences εhown in Figure 31 (SEQ ID Nos. 136, 138, 140) .
20. The DNA sequence comprising coding εegmentε 5FEBA3' coding for polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 163) .
21. The DNA εequence comprising coding segmentε 5FEBA'3' coding for polypeptide segmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-138, 140, 163) .
22. Purified DNA encoding GGF2HBS5.
23. A polypeptide of the formula
WYBAZCX wherein WYBAZCX iε compoεed of the polypeptide segments shown in Figure 31 (SEQ ID Nos. 136-139, 141- 147, 160, 161, 163); wherein W compriseε polypeptide segment F, or is absent; wherein Y comprises polypeptide segment E, or is absent; wherein Z compriεeε polypeptide εegment G or iε abεent; and wherein X compriεes peptide segmentε C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, or C/D' D' HL; provided that, either a) at leaεt one of F, Y, B, A, Z, C, or X iε of bovine origin; or b) Y compriεeε polypeptide εegment E; or c) X compriεeε polypeptide εegmentε C/D HKL, C/D' HKL, C/D D, C/D C/D' HKL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, C/D C/D' D' HKL, C/D'H, C/D C/D'H, or C/D C/D'HL.
24. A polypeptide of claim 23, wherein X compriseε C/D HKL polypeptide segmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 142, 146, 147, 160, 161) .
25. A polypeptide of claim 23, wherein X compriεes C/D D polypeptide εegmentε having the amino acid εequences shown in Figure 31 (SEQ ID Nos. 136-139, 141,
142, 144, 160).
26. A polypeptide of claim 23, wherein X compriεes C/D' H polypeptide segmentε having the amino acid εequences shown in Figure 31 (SEQ ID Nos. 136-139, 141,
143, 146, 160).
27. A polypeptide of claim 23, wherein X compriεeε C/D' HKL polypeptide εegmentε having the amino acid sequences shown in Figure 31 (SEQ ID Noε. 136-139, 141, 143, 146, 147, 160, 161).
28. A polypeptide of claim 23, wherein X compriseε C/D C/D' HKL polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 143, 146, 147, 160, 161) .
29. A polypeptide of claim 23, wherein X compriεeε C/D C/D' H polypeptide εegmentε having the amino acid εequenceε shown in Figure 31 (SEQ ID Nos. 136-139, 141- 143, 146, 160) .
30. A polypeptide of claim 23, wherein X compriseε C/D C/D' HL polypeptide segmentε having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 143,146, 147, 160) .
31. A polypeptide of claim 23, wherein X compriεeε C/D C/D' D, polypeptide εegments having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 144, 160).
32. A polypeptide of claim 23, wherein X compriseε C/D D'H polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141, 142, 145, 146, 160).
33. A polypeptide of claim 23, wherein X compriεeε C/D D'H L polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Nos. 136-139, 141, 142, 145-147, 160).
34. A polypeptide of claim 23, wherein X compriseε C/D D'H K L polypeptide εegmentε having the amino acid sequences shown in Figure 31 (SEQ ID Noε. 136-139, 141,
142, 145-147, 160, 161) .
35. A polypeptide of claim 23, wherein X compriεeε C/D' D' H polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 141,
143, 145, 146, 160) .
36. A polypeptide of claim 23, wherein X compriseε C/D' D' H K L polypeptide εegments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141, 143, 145-147, 160, 161) .
37. A polypeptide of claim 23, wherein X comprises C/D C/D' D' H polypeptide segmentε having the amino acid εequences εhown in Figure 31 (SEQ ID Nos. 136-139, 141- 143, 145, 146, 160) .
38. A polypeptide of claim 23, wherein X compriεeε C/D C/D' D' H L polypeptide segments having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136-139, 141-143, 145-147, 160) .
39. A polypeptide of claim 23, wherein X compriseε C/D C/D' D' H K L polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Nos. 136-139, 141-143, 145-147, 160, 161).
40. A polypeptide comprising FBA polypeptide segmentε having the amino acid sequences shown in Figure 31 (SEQ ID Nos. 136, 138, 139).
41. A polypeptide comprising FEBA polypeptide segmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 163).
42. A polypeptide compriεing FBA' polypeptide segments having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136, 139, 140).
43. A polypeptide compriεing FEBA' polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 140, 163).
44. Purified GGF2HBS5 polypeptide.
45. A method for εtimulating mitogeneεiε of a glial cell, said method comprising contacting εaid glial cell with a polypeptide defined by the formula
WYBAZCX wherein WYBAZCX iε compoεed of the polypeptide εegmentε εhown in Figure 31 (SEQ ID Noε. 136-139, 141- 147, 160, 161, 163) ; wherein W compriεes polypeptide segment F, or is abεent; wherein Y compriεeε polypeptide εegment E, or iε abεent; wherein Z comprises polypeptide segment G or is abεent; and wherein X comprises polypeptide segmentε C/D HKL, C/D H, C/D HL, C/D D, C/D' HL, C/D' HKL, C/D' H, C/D' D, C/D C/D' HKL, C/D C/D' H, C/D C/D' HL, C/D C/D' D, C/D D' H, C/D D' HL, C/D D' HKL, C/D' D' H, C/D' D' HL, C/D' D' HKL, C/D C/D' D' H, C/D C/D' D' HL, or C/D C/D' D' HKL.
46. A method for εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide compriεing FBA polypeptide εegmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136, 138, 139) .
47. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide compriεing FBA' polypeptide segments having the amino acid sequenceε εhown in Figure 31 (SEQ ID Noε. 136, 138, 140) .
48. A method of εtimulating mitogenesis of a glial cell, said method comprising contacting said glial cell with a polypeptide comprising FEBA polypeptide segmentε having the amino acid εequenceε εhown in Figure 31 (SEQ ID Noε. 136-139, 163) .
49. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide compriεing FEBA' polypeptide εegmentε having the amino acid εequences corresponding to polypeptide segmentε shown in Figure 31 (SEQ ID Nos. 136- 138, 140, 163) to glial cells.
50. A method of stimulating mitogenesiε of a glial cell, εaid method compriεing contacting εaid glial cell with GGF2HBS5 polypeptide.
51. A method of εtimulating mitogeneεiε of a glial cell εaid method compriεing contacting εaid glial cell with a compound which specifically binds the pl85eri,B2 receptor of glial cells.
52. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide, compriεing EGFL1, having the amino acid εequence εhown Fig. 38, Seq. ID No. 154.
53. A method of εtimulating mitogeneεiε of a glial cell, εaid method comprising contacting said glial cell with a polypeptide, comprising EGFL2, having the amino acid sequence εhown in Figure 39, Seq. ID No. 155.
54. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide, compriεing EGFL 3, with the amino acid εequence εhown in Fig. 40, Seq. ID No. 156.
55. A method of εtimulating mitogenesis of a glial cell, said method comprising contacting εaid glial cell with a polypeptide, compriεing EGFL4, with the amino acid εequence εhown in Fig. 41, Seq. ID No. 157.
56. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide, comprising EGFL5, with the amino acid sequence εhown in Fig. 42, Seq. ID No. 158, to glial cellε.
57. A method of εtimulating mitogeneεiε of a glial cell, εaid method compriεing contacting εaid glial cell with a polypeptide, comprising EGFL6, with the amino acid sequence shown Fig. 43, Seq. ID No. 159.
58. A method for the prophylaxis or treatment of a pathophyεiological condition of the nervous system in a mammal in which said condition involves a cell type which iε εenεitive or reεponεive to a polypeptide of claim 1, 18, 19, 20, 21 or 22, εaid method compriεing administering to εaid mammal an effective amount of εaid polypeptide.
59. A method as claimed in claim 58, wherein εaid condition involveε peripheral nerve damage.
60. The method aε claimed in claim 58, wherein said condition involves glia of the central nervous εyεtem.
61. A method of εtimulating mitogenic activity in a glial cell, εaid method compriεing applying 35 kD polypeptide factor isolated from the rat I-EJ tranεformed fibroblaεt cell line to said glial cell.
62. A method of stimulating mitogenic activity in a glial cell, said method comprising applying 75 kD polypeptide factor iεolated from the SKBR-3 human breast cell line to said glial cell.
63. A method of stimulating mitogenic activity in a glial cell, said method comprising applying 44 kD polypeptide factor iεolated from the rat I-EJ tranεformed fibroblaεt cell line to εaid glial cell.
64. A method of stimulating mitogenic activity in a glial cell, said method compriεing applying 45 kD polypeptide factor isolated from the MDA - MB 231 human breast cell line to said glial cell.
65. A method of εtimulating mitogenic activity in a glial cell, εaid method compriεing applying 7 to 14 kD polypeptide factor iεolated from the ATL-2 human T-cell line to εaid glial cell.
66. A method of εtimulating mitogenic activity in a glial cell, εaid method compriεing applying 25 kD polypeptide factor iεolated from activated mouse peritoneal macrophages to said glial cell.
67. A method of εtimulating mitogenic activity in a glial cell, εaid method compriεing applying a 25 kD polypeptide factor iεolated from bovine kidney to εaid glial cell.
68. A method of εtimulating mitogenic activity in a glial cell, said method comprising applying ARIA polypeptide to said glial cell.
69. A polypeptide having a glial cell mitogenic activity wherein said polypeptide encoded by the DNA εequence of claim 1, εaid polypeptide obtained by a method compriεing cultivating modified hoεt cells under conditions permitting expreεεion of εaid DNA sequence.
70. A polypeptide haing a glial cell mitogenic activity wherein said polypeptide is encoded by the DNA sequence of claim 18, 19, 20, 21 or 22, εaid polypeptide obtained by a method compriεing cultivating modified hoεt cellε under conditionε permitting expreεεion of εaid DNA εequence.
71. Method for identifying the preεence of a receptor for the polypeptide of claim 23, 40, 41, 42, 43, 44 or 69 in a εample comprising contacting said sample to εaid polypeptide and determining binding therebetween, wherein εaid binding iε indicative of the preεence of said receptor.
72. A method for the prophylaxis or treatment of a glial tumor in a patient, εaid method compriεing adminiεtering to εaid patient an effective amount of a εubεtance which inhibitε the binding of a polypeptide of claim 23, 40 41, 42, 43, 44 or 69 to a receptor therefor.
73. A pharmaceutical or veterinary formulation compriεing a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69 formulated for pharmaceutical or veterinary uεe, respectively, together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
74. A method for stimulating mitogenesiε of a glial cell, εaid method compriεing contacting said glial cell with a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
75. A method for stimulating mitogenesiε of a glial cell in a vertebrate, εaid method compriεing contacting εaid glial cell with an effective amount of a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
76. A method for the prophylaxiε or treatment of pathophyεiological condition of the nervouε εyεtem in a mammal in which εaid condition involveε a cell type which iε senεitive or responsive to a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69, said method compriεing adminiεtering an effective amount of εaid polypeptide.
77. A method for the treatment of a condition which involves peripheral nerve damage in a mammal, said method comprising contacting said peripheral nerves with an effective amount of a polypeptide of claim 23, 40, 41, 42, 43, 44, or 69.
78. A method for the prophylaxiε or treatment of a condition in a mammal in εaid condition involveε demyelination or damage or loεs of Schwann cellε, for example a neuropathy of εenεory or motor nerve fiberε, εaid method compriεing contacting εaid Schwann an effective amount of a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
79. A method for the prophylaxis or treatment of a neurodegenerative disorder in a mammal, said method comprising contacting glial cells in a mammal with an effective amount of a polypeptide of claim 23, 40, 41, 42, 44 or 69.
80. A method for inducing neural regeneration and/or repair in a mammal, said method comprising contacting glial cellε in a mammal with an effective amount of a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
81. A method of inducing fibroblaεt proliferation, said method comprising contacting said fibroblastε with a polypeptide, of claim 23, 40, 41, 42, 43, 44 or 69.
82. A method of wound repair in mammals, εaid method compriεing contacting εaid wound with a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
83. A method of making a medicament compriεing admixing a polypeptide of claim 23, 41, 42, 43, 44 or 69 with a pharmaceutically acceptable carrier.
84. A method for producing an antibody, εaid method compriεing immunizing a mammal with a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
85. A method for detecting a receptor which iε capable of binding to a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69, εaid method compriεing carrying out affinity iεolation on εaid εample uεing a εaid peptide aε the affinity ligand.
86. A method for the prophylaxiε or treatment of a glial tumor in a patient, εaid method compriεing adminiεtering to εaid patient an effective amount of a substance which inhibits the binding of a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69 to a receptor therefor.
87. A method of inveεtigating, iεolating or preparing a glial cell mitogen or gene εequence encoding said glial cell mitogen, said method comprising contacting tisεue preparations or samples with an antibody, said antibody prepared as defined in claim 84.
88. A method for iεolating a nucleic acid εequence coding for a molecule having glial cell mitogenic activity, εaid method compriεing contacting a cell containing sample with a glial cell mitogen specific antibody to determine expresεion of said mitogen in said sample and iεolating εaid nucleic acid sequence from the cells exhibiting said expresεion.
89. The purified GGF2 polypeptide compriεing the amino acid εequence εhown in Fig. 45 (SEQ ID No. 167) .
90. A purified GGF2 DNA encoding the GGF2 polypeptide whoεe εequenceε iε shown in Fig. 45 (SEQ ID No. 167).
91. A method for inducing myelination of a neural cell by a Schwann cell, said method comprising contacting said Schwann cell with a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
92. A method for inducing acetylcholine receptor εyntheεiε in a cell, εaid method compriεing contacting of εaid cell with a polypeptide of claim 23, 40, 41, 42, 43, 44 or 69.
93. An antibody to a polypeptide aε defined in claim 23.
94. An antibody to a polypeptide as defined in claim 40.
95. An antibody to a polypeptide as defined in claim 41.
96. An antibody to a polypeptide as defined in claim 42.
97. An antibody to a polypeptide aε defined in claim 43.
98. An antibody to a polypeptide aε defined in claim 44.
99. An antibody to a polypeptide as defined in claim 69.
100. A method of purifying a protein with glial cell mitogenic activity, said method comprising contacting a cell extract with an antibody of claim 93 ~ 94, 95, 96, 97, 98, or 99.
101. A method for purifying a protein with glial cell mitogenic activity, said method comprising contacting a cell extract with an antibody to a basic polypeptide factor having mitogenic activity, εtimulating the diviεion of Schwann cellε in the preεence of fetal calf plaεma, εaid polypeptide having a molecular weight of from about 30 kD to about 36 kD, said polypeptide including within its amino acid sequence at least one of the following polypeptide sequenceε:
F K G D A H T E
A S L A D E Y E Y M X K
T E T S S S G L X L K
A S L A D E Y E Y M R K
A G Y F A E X A R T T E M A S E Q G A
A K E A L A A L K
F V L Q A K K
E T Q P D P G Q I L K K V P M V I G A Y T
E Y K C L K F K W F K K A T V M
E X K F Y V P
K L E F L X A K
102. A method for purifying a protein with glial cell mitogenic activity, said method compriεing contacting a cell extract with an antibody to a baεic polypeptide factor having mitogenic activity εtimulating the diviεion of Schwann cells in the presence of fetal calf plasma, said polypeptide having a molecular weight of from about 55 kD to about 63 kD, and said polypeptide including within its amino acid sequence at least one of the following peptide sequenceε:
V H Q V W A A K
Y I F F M E P E A X S S G
L G A W G P P A F P V X Y
W F V V I E G K
A S P V S V G S V Q E L V Q R
V C L L T V A A L P P T
K V H Q V W A A K
K A S L A D S G E Y M X K
D L L L X V
E G K V H P Q R R G A L D R K
P S C G R L K E D S R Y I F F M E
E L N R K N K P Q N I K I Q K K
103. A method for purifying a protein with glial cell mitogenic activity, εaid method compriεing contacting a cell extract with an antibody to a polypeptide factor having glial cell mitogenic activity and including an amino acid εequence encoded by:-
(a) a DNA εequence εhown in Figures 28a, 28b, 28c (SEQ ID Nos. 133-135, respectively). (b) a DNA sequence shown in Figure 22 (SEQ ID No. 89);
(c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in Figure 28a.
(d) a DNA sequence which hybridizes to the DNA sequence of (a) , (b) or (c) .
104. A method for purifying a protein with glial cell mitogenic activity, said method comprising contacting a cell extract with an antibody to a basic polypeptide factor having a molecular weight, whether in reducing conditions or not, of from about 30 kD to about 36 kD on SDS-polyacrylamide gel electrophoresiε, said polypeptide factor having mitogenic activity stimulating the division of rat Schwann cells in the presence of fetal calf plasma, and when iεolated uεing reverεed-phaεe HPLC retaining at leaεt 50% of εaid activity after 10 weekε incubation in 0.1% trifluoroacetic acid at 4°C
105. A method for purifying a protein with glial cell mitogenic activity, εaid method compriεing contacting a cell extract with an antibody to baεic polypeptide factor having a molecular weight, under non- reducing conditionε, of from about 55 kD to about 63 kD on SDS-polyacrylamide gel electrophoreεiε, εaid polypeptide factor having mitogenic activity stimulating the division of rat Schwann cellε in the preεence of fetal calf plasma, and when isolated using reversed-phase HPLC retains at least about 50% of εaid activity after 4 dayε incubation in 0.1% trifluoroacetic acid at 4°C
106. A method of treating a mammal εuffering from a diεease of glial cell proliferation, εaid method compriεing adminiεtering to said mammal an antibody of Claim 93, 94, 95, 96, 97, 98, or 99.
107. A method of treating a mammal suffering from a diseaεe of glial cell proliferation, εaid method comprising administering to said mammal an antibody to a basic polypeptide factor having mitogenic activity εtimulating the diviεion of Schwann cellε in the preεence of fetal calf plaεma, εaid polypeptide having a molecular weight of from about 30 kD to about 36 kD, εaid polypeptide including within itε amino acid εequence at leaεt one of the following polypeptide εequenceε:
F K G D A H T E
A S L A D E Y E Y M X K
T E T S S S G L X L K
A S L A D E Y E Y M R K
A G Y F A E X A R
T T E M A S E Q G A
A K E A L A A L K
F V L Q A K K
E T Q P D P G Q I L K K V P M V I G A Y T
E Y K C L K F K W F K K A T V M
E X K F Y V P
K L E F L X A K
108. A method of treating a mammal εuffering from a diεeaεe of glial cell proliferation, εaid method compriεing adminiεtering to εaid mammal an antibody to a baεic polypeptide factor having mitogenic activity εtimulating the diviεion of Schwann cellε in the presence of fetal calf plasma, said polypeptide having a molecular weight of from about 55 kD to about 63 kD, and εaid polypeptide including within itε amino acid εequence at leaεt one of the following peptide εequenceε:
V H Q V W A A K
Y I F F M E P E A X S S G
L G A W G P P A F P V X Y
W F V V I E G K
A S P V S V G S V Q E L V Q R
V C L L T V A A L P P T K V H Q V W A A K
K A S L A D S G E Y M X K
D L L L X V
E G K V H P Q R R G A L D R K
P S C G R L K E D S R Y I F F M E
E L N R K N K P Q N I K I Q K K
109. A method of treating a mammal suffering from a disease of glial cell proliferation, said method comprising administering to εaid mammal an antibody to a polypeptide factor having glial cell mitogenic activity and including an amino acid εequence encoded by:-
(a) a DNA εequence εhown in Figureε 28a, 28b or 28c (SEQ ID Noε. 133-135, reεpectively) .
(b) a DNA sequence shown in Figure 22 (SEQ ID No. 89);
(c) the DNA sequence repreεented by nucleotideε 281-557 of the εequence εhown in Figure 28a.
(d) a DNA sequence which hybridizes to the DNA εequence of (a) , (b) or (c) .
110. A method of treating a mammal εuffering from a diεeaεe of glial cell proliferation, said method comprising administering to εaid mammal an antibody to a baεic polypeptide factor having a molecular weight, whether in reducing conditions or not, of from about 30 kD to about 36 kD on SDS-polyacrylamide gel electrophoreεiε, εaid polypeptide factor having mitogenic activity εtimulating the diviεion of rat Schwann cellε in the preεence of fetal calf plasma, and when iεolated uεing reverεed-phase HPLC retaining at leaεt 50% of εaid activity after 10 weekε incubation in 0.1% trifluoroacetic acid at 4°C
111. A method of treating a mammal εuffering from a diεeaεe of glial cell proliferation, εaid method compriεing adminiεtering to εaid mammal an antibody to basic polypeptide factor having a molecular weight, under non-reducing conditions, of from about 55 kD to about 63 kD on SDS-polyacrylamide gel electrophoresiε, said polypeptide factor having mitogenic activity stimulating the division of rat Schwann cellε in the presence of fetal calf plasma, and when iεolated uεing reverεed-phaεe HPLC retainε at leaεt about 50% of said activity after 4 days incubation in 0.1% trifluoroacetic acid at 4°C
112. A vector comprising a DNA sequence of claim 1, 18, 19, 20, 21 or 22.
113. A polypeptide of claim 23, 40, 41, 42, 43, 44, or 69 for use as a glial cell mitogen.
PCT/US1993/006228 1992-06-30 1993-06-29 Glial mitogenic factors, their preparation and use WO1994000140A1 (en)

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JP50265594A JP4127567B2 (en) 1992-06-30 1993-06-29 Glial mitogenic factor and its preparation and use
KR1020007004157A KR100344006B1 (en) 1992-06-30 1993-06-29 Glial mitogenic factors, and their use
DE69333731T DE69333731T2 (en) 1992-06-30 1993-06-29 GLIALMITOGENIC FACTORS, THEIR MANUFACTURE AND USE
AT93918139T ATE286126T1 (en) 1992-06-30 1993-06-29 GLIALMITOGENIC FACTORS, THEIR PRODUCTION AND USE
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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0656069A1 (en) * 1992-08-10 1995-06-07 Cambridge Neuroscience, Inc. Inhibitors of cell proliferation, their preparation and use
WO1996031599A1 (en) * 1995-04-06 1996-10-10 Amgen Inc. Ndf peptides
WO1997003345A2 (en) * 1995-07-13 1997-01-30 Royal Free Hospital School Of Medicine Detection and quantification of human herpesvirus 7 by enzymic amplification dna virus dna
EP0762885A1 (en) * 1994-05-26 1997-03-19 Ludwig Institute For Cancer Research Glial mitogenic factors, their preparation and use
EP0784488A1 (en) * 1994-11-17 1997-07-23 Cambridge Neuroscience, Inc. Use of neuregulins as modulators of cellular communication
US5667780A (en) * 1994-11-14 1997-09-16 Genentech, Inc. Antibodies to SMDF
EP0820467A1 (en) * 1995-03-27 1998-01-28 Cambridge Neuroscience, Inc. Methods of treating disorders of the eye
US5721139A (en) * 1995-05-10 1998-02-24 Genentech, Inc. Isolating and culturing schwann cells
US5849585A (en) * 1995-05-10 1998-12-15 Genetech, Inc. Isolating and culturing Schwann cells
US5912326A (en) * 1995-09-08 1999-06-15 President And Fellows Of Harvard College Cerebellum-derived growth factors
WO1999031505A1 (en) * 1997-12-15 1999-06-24 Shire Laboratories Inc. Neurotrophic factor assay
WO2000006713A2 (en) * 1998-07-28 2000-02-10 Bayer Corporation Methods for recombinant protein production in mammalian cells comprising co-expression with fetuin
US6033660A (en) * 1995-05-10 2000-03-07 Genentech, Inc. Method of treating a nervous system injury with cultured schwann cells
US6096873A (en) * 1996-07-12 2000-08-01 Genentech, Inc. Gamma-heregulin
US6136558A (en) * 1997-02-10 2000-10-24 Genentech, Inc. Heregulin variants
AU735055B2 (en) * 1996-10-08 2001-06-28 Hartford Hospital Induction of a cellular stress response with heavy metal salts
US6387638B1 (en) 1997-02-10 2002-05-14 Genentech, Inc. Heregulin variants
EP1289553A1 (en) * 2000-05-23 2003-03-12 Cenes Pharmaceuticals, Inc. Nrg-2 nucleic acid molecules, polypeptides, and diagnostic and therapeutic methods
US6696290B2 (en) 1996-07-12 2004-02-24 Genentech, Inc. ErbB2 and ErbB4 Chimeric Heteromultimeric Adhesins
US7153828B2 (en) 1998-02-04 2006-12-26 Genentech, Inc. Use of heregulin as a growth factor
US7226907B1 (en) 1998-12-21 2007-06-05 Zensun (Shanghai) Science & Technology Limited Cardiac muscle function and manipulation
US7662772B2 (en) 1999-04-23 2010-02-16 Acorda Therapeutics, Inc. Methods for treating congestive heart failure
US7795212B2 (en) 2002-05-24 2010-09-14 Zensun (Shanghai) Science & Technology Limited Neuregulin based methods and compositions for treating cardiovascular diseases
US8026213B2 (en) 1993-05-06 2011-09-27 Acorda Therapeutics, Inc. Methods for treating muscle diseases and disorders
US8476405B2 (en) 2005-12-02 2013-07-02 Zensun (Shanghai) Science & Technology Ltd. Neuregulin variants and methods of screening and using thereof
US11179323B2 (en) 2013-05-22 2021-11-23 Zensun (Shanghai) Science & Technology, Co., Ltd. Extended release of neuregulin for treating heart failure
US11253573B2 (en) 2011-10-10 2022-02-22 Zensun (Shanghai) Science & Technology, Co., Ltd. Compositions and methods for treating heart failure
US11638746B2 (en) 2005-12-30 2023-05-02 Zensun (Shanghai) Science & Technology, Co., Ltd. Extended release of neuregulin for improved cardiac function

Families Citing this family (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5530109A (en) * 1991-04-10 1996-06-25 Ludwig Institute For Cancer Research DNA encoding glial mitogenic factors
US20050106666A1 (en) * 1991-04-10 2005-05-19 Cambridge Neuroscience Glial mitogenic factors, their preparation and use
US7319019B1 (en) * 1991-04-10 2008-01-15 Acorda Therapeutics, Inc. Glial mitogenic factors lacking an N-terminal signal sequence
US7037888B1 (en) * 1992-04-03 2006-05-02 Acorda Therapeutics, Inc. Methods for treating muscle diseases and disorders
JP2001519400A (en) * 1997-10-14 2001-10-23 ケンブリッジ ニューロサイエンス インク. Treatment including neuregulin use
US6110718A (en) * 1998-03-20 2000-08-29 Wayne State University Mammalian putative phosphatidylinositol-4-phosphate-5-kinase
US6933122B1 (en) * 1999-05-14 2005-08-23 The Trustees Of Columbia University In The City Of New York A-form of cytoplasmic domain of nARIA (CRD-neuregulin) and uses thereof
EP1252186B1 (en) 2000-02-11 2006-01-11 ProteoSys AG Use of neuregulin-beta as an indicator and/or target
US6933370B2 (en) * 2000-03-27 2005-08-23 Genetics Institute, Llc Methods for purifying highly anionic proteins
EP1417230A2 (en) * 2001-08-06 2004-05-12 ProteoSys AG Neuregulin-beta isoforms associated with neuronal processes
US20090131308A1 (en) * 2003-08-19 2009-05-21 Agos Biotech Ltd. Splice Variants of ErbB Ligands, Compositions and Uses Thereof
TWI276685B (en) * 2003-12-30 2007-03-21 Ind Tech Res Inst Conditional medium for culturing Schwann cells
JP2008511726A (en) * 2004-08-27 2008-04-17 アブゾーバント テクノロジーズ インコーポレイテッド Superabsorbent polymers in agricultural applications.
KR100680479B1 (en) * 2005-04-11 2007-02-08 주식회사 하이닉스반도체 Method for verifying successful programming non-volatile memory device
US9580515B2 (en) 2006-08-21 2017-02-28 Zensun (Shanghai) Science & Technology, Co., Ltd. Neukinase, a downstream protein of neuregulin
US7521535B2 (en) * 2006-09-20 2009-04-21 The Board Of Regents For Oklahoma State University Anti-microbial defensin-related peptides and methods of use
AU2008251822B2 (en) 2007-05-10 2014-02-06 Acorda Therapeutics, Inc. Methods for detecting cardiac damage
US20090156488A1 (en) 2007-09-12 2009-06-18 Zensun (Shanghai) Science & Technology Limited Use of neuregulin for organ preservation
CA2705328C (en) * 2007-11-16 2018-01-02 Proteosys Ag Active soluble post-translationally modified neuregulin isoforms
EP2262527B1 (en) * 2008-02-29 2016-08-03 Acorda Therapeutics, Inc. Compositions for achieving desired glial growth factor 2 plasma levels
US9348975B2 (en) * 2008-05-02 2016-05-24 Baxter International Inc. Optimizing therapy outcomes for peritoneal dialysis
US8882700B2 (en) 2008-05-02 2014-11-11 Baxter International Inc. Smart patient transfer set for peritoneal dialysis
US8057679B2 (en) 2008-07-09 2011-11-15 Baxter International Inc. Dialysis system having trending and alert generation
AU2009292216B2 (en) 2008-07-17 2015-03-05 Acorda Therapeutics, Inc. Therapeutic dosing of a neuregulin or a subsequence thereof for treatment or prophylaxis of heart failure
ES2827923T3 (en) 2008-08-15 2021-05-25 Acorda Therapeutics Inc Compositions and treatment methods during non-acute periods after CNS neurological injury
WO2010060266A1 (en) * 2008-11-28 2010-06-03 Zensun (Shanghai) Science & Technology Limited Neuregulin and cardiac stem cells
EP2370458B1 (en) 2008-11-28 2014-10-15 Zensun (Shanghai) Science and Technology Limited Neuregulin peptides and their use
EP2391404B1 (en) * 2009-01-30 2013-01-16 Baxter International Inc. Transfer sets for therapy optimization
US8282829B2 (en) 2009-05-20 2012-10-09 Baxter International Inc. System and method for automated data collection of twenty-four hour ultrafiltration and other patient parameters using wired or wireless technology
EP2440236B1 (en) 2009-06-09 2016-05-04 Zensun (Shanghai) Science and Technology Limited Neuregulin based methods for treating heart failure
ES2748886T3 (en) 2009-06-09 2020-03-18 Zensun Shanghai Science & Tech Co Ltd Neuregulin-based methods for the treatment of heart failure
JP6096262B2 (en) 2009-08-25 2017-03-15 ゼンサン (シャンハイ) サイエンス アンド テクノロジー,シーオー.,エルティーディー. Treatment of heart failure based on neuregulin
MX2012004386A (en) * 2009-10-14 2012-05-23 Acorda Therapeutics Inc Use of a neuregulin to treat peripheral nerve injury.
US9020827B2 (en) 2009-10-16 2015-04-28 Baxter International Inc. Peritoneal dialysis optimized using a patient hand-held scanning device
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WO2014138502A1 (en) 2013-03-06 2014-09-12 Acorda Therapeutics, Inc. Therapeutic dosing of a neuregulin or a fragment thereof for treatment or prophylaxis of heart failure
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5019559A (en) * 1986-11-14 1991-05-28 President And Fellows Of Harvard College Wound healing using PDGF and IGF-II
US5057494A (en) * 1988-08-03 1991-10-15 Ethicon, Inc. Method for preventing tissue damage after an ischemic episode
US5202428A (en) * 1990-06-20 1993-04-13 The Salk Institute For Biological Studies DNA encoding neurotropic growth factor
US5214034A (en) * 1986-09-26 1993-05-25 Mitsui Toatsu Chemicals, Incorporated Catechol derivatives, and preventive and remedial preparations for regressive disorders in the central nervous system containing the same
US5235043A (en) * 1990-04-06 1993-08-10 Synergen, Inc. Production of biologically active, recombinant members of the ngf/bdnf family of neurotrophic proteins

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4935341A (en) * 1986-06-04 1990-06-19 Whitehead Institute For Biomedical Research Detection of point mutations in neu genes
US4968603A (en) * 1986-12-31 1990-11-06 The Regents Of The University Of California Determination of status in neoplastic disease
WO1989006692A1 (en) * 1988-01-12 1989-07-27 Genentech, Inc. Method of treating tumor cells by inhibiting growth factor receptor function
DE69031120T2 (en) * 1989-05-19 1998-01-15 Genentech, Inc., South San Francisco, Calif. HER2 EXTRACELLULAR DOMAIN
ATE153858T1 (en) * 1990-04-06 1997-06-15 Univ Pennsylvania LIGAND FOR THE NEW GENE PRODUCT
EP0597845A1 (en) * 1990-05-25 1994-05-25 Georgetown University GROWTH FACTOR WHICH INHIBITS THE GROWTH OF CELLS OVEREXPRESSING THE HUMAN ONCOGENE erbB-2
CA2100549A1 (en) * 1991-01-14 1992-07-15 Marc E. Lippman Ligand growth factors that bind to the erbb-2 receptor protein and induce cellular responses
GB9107566D0 (en) * 1991-04-10 1991-05-29 Ludwig Inst Cancer Res Glial mitogenic factors,their preparation and use
US5530109A (en) * 1991-04-10 1996-06-25 Ludwig Institute For Cancer Research DNA encoding glial mitogenic factors
IL101943A0 (en) * 1991-05-24 1992-12-30 Genentech Inc Structure,production and use of heregulin
US5367060A (en) * 1991-05-24 1994-11-22 Genentech, Inc. Structure, production and use of heregulin
US5237056A (en) * 1991-05-29 1993-08-17 President And Fellows Of Harvard College DNA encoding a protein which copurifies with acetylcholine receptor inducing activity and uses therefor
AU676476B2 (en) * 1992-04-29 1997-03-13 Georgetown University Binding peptides which interact with ligand growth factors of the epidermal growth factor receptor and erbB-2-receptor
KR950701379A (en) * 1992-04-29 1995-03-23 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 RECOMBINANT STIMULATING FACTOR OF THE neu RECEPTOR

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5214034A (en) * 1986-09-26 1993-05-25 Mitsui Toatsu Chemicals, Incorporated Catechol derivatives, and preventive and remedial preparations for regressive disorders in the central nervous system containing the same
US5019559A (en) * 1986-11-14 1991-05-28 President And Fellows Of Harvard College Wound healing using PDGF and IGF-II
US5057494A (en) * 1988-08-03 1991-10-15 Ethicon, Inc. Method for preventing tissue damage after an ischemic episode
US5235043A (en) * 1990-04-06 1993-08-10 Synergen, Inc. Production of biologically active, recombinant members of the ngf/bdnf family of neurotrophic proteins
US5202428A (en) * 1990-06-20 1993-04-13 The Salk Institute For Biological Studies DNA encoding neurotropic growth factor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Cell, Vol. 45, No. 2, issued 25 April 1986, BROCKES et al., "Glial Growth Factor and Nerve-Dependent Proliferation in the Regeneration Blastema of Urodele Amphibians", pages 301-306, see entire document. *

Cited By (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6087323A (en) * 1992-04-03 2000-07-11 Cambridge Neuroscience, Inc. Use of neuregulins as modulators of cellular communication
US5876973A (en) * 1992-08-10 1999-03-02 Cambridge Neuroscience, Inc. Method for making antibodies which specifically bind to glial growth factors
EP0656069A1 (en) * 1992-08-10 1995-06-07 Cambridge Neuroscience, Inc. Inhibitors of cell proliferation, their preparation and use
EP0656069A4 (en) * 1992-08-10 1996-07-10 Cambridge Neuroscience Inc Inhibitors of cell proliferation, their preparation and use.
US8026213B2 (en) 1993-05-06 2011-09-27 Acorda Therapeutics, Inc. Methods for treating muscle diseases and disorders
EP0762885A1 (en) * 1994-05-26 1997-03-19 Ludwig Institute For Cancer Research Glial mitogenic factors, their preparation and use
EP0762885A4 (en) * 1994-05-26 1999-04-28 Ludwig Inst Cancer Res Glial mitogenic factors, their preparation and use
US5667780A (en) * 1994-11-14 1997-09-16 Genentech, Inc. Antibodies to SMDF
US5763213A (en) * 1994-11-14 1998-06-09 Genentech, Inc. Sensory and motor neuron derived factor (SMDF)
US5770567A (en) * 1994-11-14 1998-06-23 Genentech, Inc. Sensory and motor neuron derived factor (SMDF)
US5756456A (en) * 1994-11-14 1998-05-26 Genentech, Inc. Methods involving sensory and motor neuron derived factor (SMDF)
EP0784488A4 (en) * 1994-11-17 1998-12-16 Cambridge Neuroscience Inc Use of neuregulins as modulators of cellular communication
EP0784488A1 (en) * 1994-11-17 1997-07-23 Cambridge Neuroscience, Inc. Use of neuregulins as modulators of cellular communication
EP0820467A4 (en) * 1995-03-27 2001-12-05 Cambridge Neuroscience Inc Methods of treating disorders of the eye
EP0820467A1 (en) * 1995-03-27 1998-01-28 Cambridge Neuroscience, Inc. Methods of treating disorders of the eye
AU713384B2 (en) * 1995-03-27 1999-12-02 Cenes Pharmaceuticals, Inc. Methods of treating disorders of the eye
US6750196B1 (en) 1995-03-27 2004-06-15 Acorda Therapeutics Methods of treating disorders of the eye
US5849705A (en) * 1995-04-06 1998-12-15 Amgen Inc. Method for the treatment of Schwann cells in vivo
WO1996031599A1 (en) * 1995-04-06 1996-10-10 Amgen Inc. Ndf peptides
US5670342A (en) * 1995-04-06 1997-09-23 Amgen Inc. NDF peptides
US5686415A (en) * 1995-04-06 1997-11-11 Amgen Inc. Method for the treatment of colon epithelial cells in vivo
US5929032A (en) * 1995-04-06 1999-07-27 Amgen Inc. Method for treating Schwann and colon cells in vitro
US5721139A (en) * 1995-05-10 1998-02-24 Genentech, Inc. Isolating and culturing schwann cells
US5849585A (en) * 1995-05-10 1998-12-15 Genetech, Inc. Isolating and culturing Schwann cells
US6033660A (en) * 1995-05-10 2000-03-07 Genentech, Inc. Method of treating a nervous system injury with cultured schwann cells
WO1997003345A3 (en) * 1995-07-13 1997-03-13 Royal Free Hosp School Med Detection and quantification of human herpesvirus 7 by enzymic amplification dna virus dna
US6331417B1 (en) 1995-07-13 2001-12-18 Royal Free Hospital School Of Medicine Detection and quantification of human herpes virus 7 by enzymic amplification
WO1997003345A2 (en) * 1995-07-13 1997-01-30 Royal Free Hospital School Of Medicine Detection and quantification of human herpesvirus 7 by enzymic amplification dna virus dna
US5912326A (en) * 1995-09-08 1999-06-15 President And Fellows Of Harvard College Cerebellum-derived growth factors
US7659368B2 (en) 1996-07-12 2010-02-09 Genentech, Inc. ErbB2 and ErbB3 chimeric heteromultimer adhesins
US8226935B2 (en) 1996-07-12 2012-07-24 Genentech, Inc. ErbB2 and ErbB3 chimeric heteromultimer receptors
US6096873A (en) * 1996-07-12 2000-08-01 Genentech, Inc. Gamma-heregulin
US7585673B2 (en) 1996-07-12 2009-09-08 Genentech, Inc. γ-heregulin
US6916624B2 (en) 1996-07-12 2005-07-12 Genentech, Inc. Antibodies that bind gamma-heregulin
US6696290B2 (en) 1996-07-12 2004-02-24 Genentech, Inc. ErbB2 and ErbB4 Chimeric Heteromultimeric Adhesins
AU735055B2 (en) * 1996-10-08 2001-06-28 Hartford Hospital Induction of a cellular stress response with heavy metal salts
US6387638B1 (en) 1997-02-10 2002-05-14 Genentech, Inc. Heregulin variants
US7563583B2 (en) 1997-02-10 2009-07-21 Genentech, Inc. Heregulin variants
US6136558A (en) * 1997-02-10 2000-10-24 Genentech, Inc. Heregulin variants
US7063961B2 (en) 1997-02-10 2006-06-20 Genentech, Inc. Heregulin variants
WO1999031505A1 (en) * 1997-12-15 1999-06-24 Shire Laboratories Inc. Neurotrophic factor assay
US7153828B2 (en) 1998-02-04 2006-12-26 Genentech, Inc. Use of heregulin as a growth factor
WO2000006713A2 (en) * 1998-07-28 2000-02-10 Bayer Corporation Methods for recombinant protein production in mammalian cells comprising co-expression with fetuin
WO2000006713A3 (en) * 1998-07-28 2001-08-09 Bayer Ag Methods for recombinant protein production in mammalian cells comprising co-expression with fetuin
US6051401A (en) * 1998-07-28 2000-04-18 Bayer Corporation Methods and constructs for protein expression
US7964555B2 (en) 1998-12-21 2011-06-21 Zensun (Shanghai) Sci & Tech Co., Ltd. Cardiac muscle function and manipulation
US7612164B2 (en) 1998-12-21 2009-11-03 Zensun (Shanghai) Science and Technologies Ltd. Cardiac muscle function and manipulation
US7226907B1 (en) 1998-12-21 2007-06-05 Zensun (Shanghai) Science & Technology Limited Cardiac muscle function and manipulation
US8394761B2 (en) 1999-04-23 2013-03-12 Beth Israel Deaconess Medical Center Methods for treating congestive heart failure
US8076283B2 (en) 1999-04-23 2011-12-13 Acorda Therapeutics, Inc. Methods for treating congestive heart failure
US7662772B2 (en) 1999-04-23 2010-02-16 Acorda Therapeutics, Inc. Methods for treating congestive heart failure
US10232016B2 (en) 1999-04-23 2019-03-19 Acorda Therapeutics, Inc. Methods for treating congestive heart failure
US10065999B2 (en) 2000-05-23 2018-09-04 Acorda Therapeutics, Inc. Methods for protecting dopaminergic neurons from stress and promoting proliferation and differentiation of oligodendrocyte progenitors by NRG-2
EP2357004A1 (en) * 2000-05-23 2011-08-17 CeNeS Pharmaceuticals, Inc. NRG-2 nucleic acid molecules, polypeptides and diagnostic and therapeutical methods
EP1289553A1 (en) * 2000-05-23 2003-03-12 Cenes Pharmaceuticals, Inc. Nrg-2 nucleic acid molecules, polypeptides, and diagnostic and therapeutic methods
US8114838B2 (en) 2000-05-23 2012-02-14 Acorda Therapeutics, Inc. Methods for protecting dopaminergic neurons from stress and promoting proliferation and differentiation of oligodendrocyte progenitors by NRG-2
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US10717775B2 (en) 2000-05-23 2020-07-21 Acorda Therapeutics, Inc. Methods for cardiomyocyte survival, proliferation or differentiation by NRG-2
US8785387B2 (en) 2002-05-24 2014-07-22 Zensun (Shanghai) Science & Technology Limited Neuregulin based methods and compositions for treating cardiovascular disease
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