WO1992016560A1

WO1992016560A1 - Cns cell differentiation factor, its preparation and use

Info

Publication number: WO1992016560A1
Application number: PCT/GB1992/000500
Authority: WO
Inventors: Mark David Noble; Heidi Hoffmann; Paul Stroobant
Original assignee: Ludwig Institute For Cancer Research
Priority date: 1991-03-22
Filing date: 1992-03-20
Publication date: 1992-10-01
Also published as: IE920868A1; ZA922001B; AU1561992A; PT100274A; GB9106103D0

Abstract

The invention provides a new differentiation-promoting factor of molecular weight 20-25 kilodaltons when isolated by SDS-PAGE. The factor causes O-2A progenitor cells to differentiate expressing GFAP. The new factor is a secreted protein. The invention provides therapeutic formulations comprising the new factor, and the use of the factor and those formulations in treating conditions which involve a factor-sensitive or factor-responsive cell type.

Description

CNS Cell Differentiation Factor, its Preparation and Use

This invention relates to a new polypeptide factor which can promote differentiation in cells of the vertebrate central nervous system (CNS) . The invention is also concerned with a process for the preparation of such factor, and with the use thereof. The factor has been termed "Differin", and will be referred to as such hereinafter.

The glial cells of vertebrates constitute the specialized connective tissue of the central and peripheral nervous systems. In the rat optic nerve, two major classes of glial cells, oligodendrocytes and type-2 astrocytes, structurally and functionally support the axons in the nerve. While oligodendrocytes form an insulating myelin sheath, type-2 astrocytes may enwrap bare axons at the nodal gaps between adjacent myelin sheaths. Both cell types develop from a common oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell. Oligodendrocytes first appear at birth, while the type-2 astrocytes only appear in the second postnatal week. Differentiation of 0-2A progenitor cells into oligodendrocytes appears to be controlled by an internal "clock", whereas differentiation into type-2 astrocytes seems to require the action of at least one exogenous inducing factor (Raff et al. , Science 243 (1989), 1450-1455)- _.

Various factors are known to have an effect on CNS cell division or development, and one of these is known as Ciliary Neutrotrophic Factor (CNTF) . CNTF is a 23 kilodalton protein which has recently been purified, cloned and seguenced from rabbit and rat sciatic nerve (Lin et al., Science 246 (1989), 1023-1025; Stockli et al., Nature 342 (1989), 920-923). It was defined as a neurotrophic factor on the basis of its ability to promote survival of parasympathetic ciliary ganglion neurones (Manthorpe et al., Brain Research 367 (1986), 282-286), and is also capable of modulating various processes of differentiation within the nervous system (Ernsberger et al., Neuron 2 (1989), 1275-1284; Sadaat et al., J.Cell. Biol. 108 (1989), 1807-1816) . Moreover, CNTF is partially responsible for promoting the differentiation of bipotential 0-2A progenitor cells to type-2 astrocytes in vitro (Hughes et al.. Nature 335 (1988), 70-72).

In Trans. Roy. Microscop, Soc. , 1 (1990), 361-364, Entwistle et al discuss techniques for dealing with, inter alia, anchorage-dependent rhabdo yosarcoma derived cells. It was shown that most cells within aggregates of such cells were alive. Analysis of a human rhabdomyosarcoma cell line RD (isolated by McAllister et al., Cancer 24 (1969) , 520-526) has now revealed that it is a source for an astrocyte differentiation-inducing factor. In experiments using immunofluorescence staining techniques, it has now been found that medium conditioned by RD cells induces 0-2A progenitor cells to express Glial Fibrillary Acidic Protein (GFAP) (Bignami et al., (1972), Brain Research 43, 429-425), a characteristic of astrocyte intermediate filaments and, as such, a marker of astrocyte differentiation. Since, as indicated, it is known that cells within aggregates of RD cells will be alive, the active factor within the conditioned medium can clearly be regarded as not derived from cell lysis, but is a secreted substance.

The present invention accordingly relates to an isolated protein factor which is a 20-25 kilodalton molecule having a differentiation-inducing activity similar to CNTF in that it has been found that 20-30% of cells of the 0-2A lineage were rapidly induced to express GFAP. Like CNTF, the new protein promotes the survival of chicken early sympathetic neurones and parasympathetic ciliary ganglion neurones. Like CNTF, these activities distinguish the new protein from any other known neurotrophic factors like Nerve Growth Factor (NGF) , Brain-Derived Growth Factor (BDNF) or, most recently, Neurotrophin 3 (Maisonpierre et al., Science 247 (1990), 1446-1451; Hohn et al. , Nature 344 (1990), 339- 341) .

The present new protein is secreted, since (see above) activity exists predominantly in the culture medium of producing cells, and cannot be accounted for by cell lysis. In contrast, recombinant CNTF expressed in HeLa cells was not released into the culture medium (Stockli et al., supra) , and similar results have been obtained for a CNTF rabbit cDNA clone expressed in COS cells (Lin et al., supra) , consistent with the absence of CNTF of an amino terminal signal sequence typical of those found for secreted proteins.

Thus, the invention provides in one significant aspect a polypeptide factor which has, if obtained from ATCC CCL136 as a source, an observed molecular weight of about 20-25 KD on SDS-polyacrylamide gel electrophoresis in the absence of reducing agent, using the following prestained molecular weight standards:

Lysozyme 14,300

B-lactoglobulin 18,400

Carbonic anhydrase 29,000

Ovalbumin 43,000 Bovine serum albumin 68,000 Phosphorylase B 97,400

Myosin (H-chain) 200,000;

which factor is obtainable, if desired, by secretion from the human rhabdomyosarcoma cell line obtainable from ATCC CCL136, and which factor induces oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells to express GFAP. It will be appreciated that the molecular weight range limits quoted are not exact, but are subject to slight variation depending, inter alia, 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.

An unusual characteristic of the new polypeptide factor is its hydrophobicity. Thus, it is eluted from reversed- phase HPLC by 56-60% acetonitrile.

A process for the preparation of a polypeptide defined in claim 1, comprising preparing serum-free medium conditioned with human rhabdomyosarcoma cells, preparing a concentrate from said conditioned medium, subjecting said concentrate to chromatography comprising reversed-phase HPLC employing an acetonitrile eluant and thereafter to SDS-polyacrylamide gel electrophoresis and collecting that fraction therefrom which has an observed molecular weight of about 20-25 kD if subjected to SDS-polyacrylamide gel electrophoresis in the absence of reducing agent using the following prestained molecular weight standards:

Lysozyme 14,300 B-lactoglobulin 18,400

Carbonic anhydrase 29,000

Ovalbumin 43,000

Bovine serum albumin 68,000

Phosphorylase B 97,400 Myosin (H-chain) 200,000.

Preferably, the reversed-phase HPLC is preceded by hydrophobic interaction chromatography, which hydrophobic interaction chromatography is optionally preceded by ion exchange chromatography. The acetonitrile eluant used in the reversed-phase HPLC is optionally 56-60% acetonitrole. A further aspect of the invention is an assay for differentiation-inducing ability, comprising incubating immortalized 0-2A progenitor cells with the substance under assay and determining GFAP production, if any, in the resulting cells. The determination can take the form of an immunoassay, preferably an enzyme-linked im unoassay, on the resulting cells for GFAP production using a labelled anti-GFAP antibody. In a preferred ELISA version of this assay, the label can be an enzyme capable of reacting with a substrate to generate a fluorescence effect. Rabbit anti-GFAP is commercially available from Dako Ltd. , High Wycombe, England. Preferably, the 0-2A progenitor cells are obtained from a young (the term "young" usually meaning 1 to 7 days old, e.g. 7 days old) rat, and these cells may be immortalized by infection with a retroviral construct encoding (simian virus 40) SV40 large T antigen (Ridley et al EMBO J 7(1988), 1635-1645). A detailed description of performance of an embodiment of the present assay appears in Example 1 hereinafter, from which description usable techniques will be readily apparent to the skilled reader.

The invention also provides a polypeptide factor of observed molecular weight about 20-25 kD on SDS- polyacrylamide gel electrophoresis in the absence of reducing agent, using the following prestained molecular weight standards:

Lysozyme 14,300

B-lactoglobulin 18,400

Carbonic anhydrase 29,000

Ovalbumin 43,000

Bovine serum albumin 68,000

Phosphorylase B 97,400

Myosin (H-chain) 200,000;

said factor obtained by isolation from a secretion product of a cell which produces it, said factor further characterized by inducing oligodendrocyte-type 2 astrocyte progenitor cells to express GFAP.

In other respects, the invention provides pharmaceutical or veterinary formulations comprising a polypeptide 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 factor 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, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.

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, lactide/gl colide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the factor. Other potentially useful parenteral delivery systems for the factor 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 factor can be used as the sole active agent 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 factor 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 factor 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 μg/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 discussed above, the polypeptide factor of the invention stimulates 0-2A progenitor cell differentiation. The present factor, in promoting differentiation, necessarily stops mytosis, i.e. has an anti-cell proliferation effect. It is thus a candidate for achieving antitumour effects, and the invention includes such use.

Furthermore, in stimulating cell differentiation the present factor does give rise to an overall increase in the number of differentiated cells of the relevant type, pushing 0-2A progenitor cells towards differentiation into type-2 astrocytes (cells responsible for maintaining the appropriate homeostatic ionic environment in the region of the Nodes of Ranvier) . Multiple sclerosis, which is characterized by demyelination of nerve fibres of the CNS, is a candidate disease for treatment by administering substances which inhibit binding of the present factor to its recepto (s) ; such inhibition prevents the factor from encouraging 0-2A progenitor differentiation to type-2 astrocytes and away from oligodendrocyte formation, thus giving a tendency to increased myelination.

It is also believed that the factor of the invention, like CNTF, promotes survival of chσlinergic neurones, e.g. ciliary ganglion cells, and promotion of neuronal cell survival is an important aspect of the uses of the present factor. The most important medical condition which involves death of cholinergic neurones is Alzheimer's Disease, for which the present factor thus provides a treatment opportunity.

Another major category of cholinergic neurones which might be treated to prevent death using the present factor are motor neurones, i.e. neurones which are involved in diseases such as amyotropic lateral sclerosis.

Autonomic neuropathies involving ciliary ganglion cells can also be_.treated using the present factor. In general, the present factor can be used to treat any pathophysiological condition of the nervous system in which a factor-sensitive or factor-responsive cell type is involved.

The invention accordingly includes the use of present polypeptide factor in the propylaxis or treatment of any pathophysiological condition of the nervous system in which a factor-sensitive or factor-responsive cell type is involved. Specific embodiments are thus generating an antitumour effect, addressing conditions involving the death of cholinergic neurones e.g. Alzheimer's Disease or conditions involving motor neurone death, or addressing autonomic neuropathies involving ciliary ganglion cells, in addition to the enhancement of myelination (e.g. in the context of multiple sclerosis) as noted above.

The invention also includes the use of the polypeptide factor of the invention in the manufacture of a medicament, e.g. a medicament for use in the treatment of a nervous disease or disorder.

The polypeptide factor of the invention can also be used as an immunogen for making antibodies such as monoclonal antibodies, following standard techniques. These antibodies can, in turn, be used as diagnostic aids or in diagnosis. Thus, conditions perhaps associated with abnormal levels of the factor, eg multiple sclerosis, may be tracked by using such antibodies. In vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods can also be employed in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques employed in the art of, for example, tumour imaging.

Also included in the invention are the use of a factor of the invention in a competitive assay to identify or quantify molecules having receptor binding characteristics corresponding to those of said polypeptide. The polypeptide may be labeled, optionally with a radioisotope.

In another aspect, the invention provides the use of a factor of the invention in an affinity isolation process, optionally affinity chromatography, for the separation of a 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 factor of the present invention. For example, in relation to IL-6 and IFN-gam a the reader is referred to Novick, D. et al., J Chromatogr; 1990 June 27; 510; 331-7, in relation to gonadotropin releasing hormone reference is made to Hazum, E., J Chromatogr; 1990 June 27; 510; 233-8, in relation to G-CSF reference is made to Fukunaga, R., et al., J Biol Che ; 1990 Aug 15; 265(23); 14008-15, in relation to vasoactive intestinal peptide reference is made to Couvineau, A., et al., J Biol Chem; 1990 Aug 5; 265(22); 13386-90, in relation to IL-2 reference is made to Smart, J. E. , et al., J Invest Dermatol; 1990 Jun; 94(6 Suppl) ; 158S-163S, and in relation to human IFN-gamma reference is made to Stefanos, S. et al., J Interferon Res; 1989 Dec; 9(6); 719-30.

It will be appreciated that the invention includes modifications or equivalents of the polypeptide factor of the invention which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially affecting activity are included. By way of illustration, in EP-A-109748 muteins of native proteins are disclosed in which the possibility of unwanted -S-S- bonding is avoided by replacing any cysteine in the native sequence 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 modifications or equivalents of the above-described factor being part of the invention.

in the following Examples, techniques for obtaining the present polypeptide factor are described which give guidance to the skilled reader without limiting the invention. First, however, an embodiment of the new assay of the invention is described in detail.

EXAMPLE 1

To simplify the assay for biological activity during a protein purification process and to make it suitable for handling multiple column fractions, an enzyme-linked immunoassay was developed. The assay system utilizes a cell line, established by taking 0-2A progenitor cells from a 7-day old rat and immortalizing them by transfection with SV40 large T. This cell line (6521 - for the preparation of which see below) can be grown indefinitely in a chemically-defined medium (SATO) in presence of Fibroblast Growth Factor (FGF) and Platelet-Derived Growth Factor (PDGF) . 6521 cells were routinely plated in 96-well plates coated with poly-D-lysine at a density of 2.5 - 10 x 10³ cells in 100 ul culture medium per well. Before the cultures became confluent (on day 3-6) , conditioned medium or assay fractions were added. After a 24 hour incubation time the growth medium was removed, the cells fixed with methanol for 20 minutes at -20°C, washed extensively and labelled with a rabbit anti-GFAP serum. The GFAP- expression was measured reading fluorogenic emission following the reaction of beta-galactosidase coupled to an anti-rabbit antibody with 4-methyl-umbelliferyl-beta-D- galactopyranoside (4MUG) as a substrate with a fluorescence icroplate reader. The cell line is induced by Differin to express GFAP, but not by a factor present in foetal calf serum (FCS) , the inducer that was originally discovered in rat optic nerve (Raff et al., Nature 303 (1983), 390-396). The known factor CNTF, obtained from rat sciatic nerve, is strongly positive in this assay, and gives a half maximum stimulation at 200 pg/ l.

Preparation of Line 6521

0-2A progenitor cells were immortalized by incubating cells with a retroviral vector which causes insertion of SV40 large T antigen gene into the genome of infected cells. This retroviral vector has been widely utilized in the generation of cell lines by many laboratories (e.g. Ridley et al. EMBO J. 7 (1988), 1635-1645; Jat and Sharp J. Virology 59 (1986), 746-750; Fredericksen et al. Neuron 1 (1988), 439-448).

All infections were carried out by the standard techniques described (for example, in Ridley et al.) . 0-2A progenitor cells were grown in a combination of PDGF (lOng/ml) and bFGF (5ng/ml) to enhance initial growth (Bogler et al. PNAS USA 87 (1990) , 6368-6372) . Once cells expressed SV4Q large T antigen, they could be grown in bFGF alone without undergoing terminal differentiation. However, to maximize growth, cells were maintained in chemically defined medium containing PDGF and bFGF.

0-2A progenitors immortalized in this manner were found to retain full capacity to express GFAP in response to treatment with either CNTF or Differin.

The line known as 6521 was serially subcloned by limiting dilution cloning through 3 subclonings.

EXAMPLE 2

A. PRODUCTION OF CONDITIONED MEDIUM CONTAINING DIFFERIN

As the starting material for the purification of Differin, serum-free medium conditioned by the RD cell line from ATCC CCL136 was used. This cell line grows with a doubling time of about 24 hours and can be maintained, even in the absence of serum, at high cell numbers. It is therefore a reproducible source of the differentiation-inducing activity. In general the cells are stored, grown, and handled using standard techniques. However, in order to obtain large amounts of conditioned medium, conditions were established for high yield suspension cultures of RD cell aggregates in 1 litre Techne glass spinning flasks. Aggregate cultures of the anchorage-dependent RD cell line were obtained by first growing the cells in flasks in Dulbecco's Modified Eagle's Medium (DMEM) with sodium pyruvate and 1000 mg/ml glucose, supplemented with 10% FCS. When the cells were confluent they were transferred as single cells following dissociation into siliconized 1 litre Techne culture vessels in the presence of 10% FCS, gassed with 5% C0₂/95% air and stirred at 30 rpm. Under these conditions clusters were formed within a period of 3 days. This technique made it possible to obtain up to 1.5 x 10⁹ eelIs/litre culture medium. The percentage of FCS in the medium was gradually lowered over a period of three weeks until there was no longer any serum present, and then the conditioned medium collected by a weekly exchange of 75% of the culture medium. It has been found that cells could be maintained for more than twelve months in culture while still secreting Differin into their medium.

B. PURIFICATION PROCEDURE

The initial steps are carried out at 2-4°C and all fractions stored at -20°C as soon as possible, although the activity was found to be very stable, as shown by incubating conditioned medium for up to 9 days at room temperature, or up to 6 hours at 60°C with no apparent decrease in activity. Approximately 70% of the activity was destroyed however, when conditioned medium was boiled for 20 minutes. The activity could also be maintained at a wide range of pH's (2-11) in various acids and buffers, including 6M guanidine and 6M urea, as well as organic solvents, such as isopropanol (90%) and acetonitrile (90%) . These properties were used to treat the conditioned medium with 0.2 M formic acid after collecting, to keep the activity soluble before freezing for storage, and to kill any undetected human pathogens originating from the tumour line.

C. CONCENTRATION AND DESALTING OF CONDITIONED MEDIUM

After thawing batches of conditioned medium, any resulting precipitates were removed by centrifugation at 4800 g for 1 hour, or by passing the material through a 0.45 prefilter before concentration. For initial concentration of the conditioned medium a Pellicon tangential-flow filtration system (Millipore) was used with filter units consisting of 10 kD retention membranes. The concentrating process was stopped at an end volume of about 2 litres and used for further concentration with a smaller scale version (Minitan) . The desalting was performed with the same device with a constant volume washing set-up into 50 mM Tris-HCl buffer, pH 7.6 (at 4°C) .

D. ION EXCHANGE CHROMATOGRAPHY

The buffer-exchanged material (500 ml, 38 mg/ml total protein) was then passed through a tangential flow ion exchange cartridge Zetaprep^® 250 (L B or CUNO) at a flow rate of approximately 2 ml/min. In 50 mM Tris-HCl buffer at pH 7.6, 90% of the secreted proteins of the tumour cells bound to a derivatized cellulose containing quaternary amionoethyl (QAE) functional groups but not Differin, leading to an approximately 10-fold purification of Differin. The activity-containing fraction (625 ml, 3.53 mg/ml total protein) could however be bound under the same conditions to a smaller cartridge version (Zetaprep^® 60) with the same solid matrix containing sulphopropyl (SP) functional groups and eluted with approximately 70 ml 0.5 M NaCl in 50 mM Tris-HCl (pH 7.6) to give a further purification of approximately 5 fold.

E. HYDROPHOBIC INTERACTION CHROMATOGRAPHY

The active Zetaprep^® pool (69 ml, 2.43 mg/ml total protein was combined with 48 ml (9 mg/ml protein) of a second active SP-Zetaprep^® pool and a solution of 4 M ammonium sulfate was added to give a final concentration of 0.6 M. Any precipitate was removed by centrifugation at 10,000 g for 10 minutes and contained about 1-3% of the biological activity. The supernatant was applied in multiple loadings (run 1 : 7.5 mg, run 2 : 13 mg, run 3 : 20.5 mg, run 4 : 82 mg, run 5 : 41 mg, and run 6 : 41 mg, protein) to a Phenyl- Superose HRlO/10 column (Pharmacia), equilibrated with 0.6 M ammonium sulfate in 50 mM Tris-HCl buffer, pH 7.6. The flow rate was 1 ml/min. The column was washed each time with a volume of approximately 45 ml of the same buffer or until the absorbance of the effluent at 280 n returned to baseline. Bound proteins were eluted with a 60 ml linear gradient running from 0.6 M to 0 M ammonium sulfate. 1 ml fractions were collected and assayed for biological activity. The activity eluted in one or two broad peaks (dependent on the amount of total protein loaded) ranging from 0.2 M to 0 M ammonium sulfate (run 1 : 15 ml, fraction 92-106, corresponding to min 52-66 of the elution programme, run 2 : 21 ml, fraction 48-68, corresponding to min 48-68, run 3 : 29 ml, fraction 42-70, corresponding to min 42-70, run 4 : 31 ml, fraction 40-70, corresponding to min 40-70, run 5 and 6 : 27 ml, fraction 40-66, corresponding to min 40-66) resulting in a total volume of 150 ml. Washing the column sequentially after each run with 6 M urea resulted in the elution of a further 4% of the activity loaded onto the column together with about a tenth of the A280 absorbing material. F. REVERSED-PHASE HIGH PERFORMANCE CHROMATOGRAPHY

Reversed-phase HPLC was performed using a 7 μm microbore C8 reversed-phase HPLC column (220 x 4.6 mm, Brownlee) with 0.1% aqueous trifluoroacetic acid (TFA) as Solvent A and 90% acetonitrile in A as Solvent B at a flow rate of 1 l/min. The active pool eluted from the Phenyl-Superose HRlO/10 column was processed in three different runs. Each time after the sample was applied to the column this was washed with 20-30 ml 0.1 M ammonium sulfate and then with 40% B until a stable baseline at an absorbance of 214 n was reached (approximately 20 ml) . Proteins were then eluted with a linear gradient from 40-100% B and l ml fractions collected in prelubricated (to avoid adsorption losses) polypropylene tubes (Bioquote Ltd.). About 60% of the biological activity could be recovered and eluted with 56-60% acetonitrile (fraction 31 to 36 of each run) , leading to a several-thousand fold purification in this single step.

G. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS: MOLECULAR WEIGHT OF DIFFERIN

A Differin sample (corresponding to 1000 activity units in the above-described ELISA assay - see Example 1) from the reversed-phase HPLC was dried down in a speed-vac concentrator and dissolved in Laemmli SDS-gel- electrophoresis sample buffer but without reducing agent. Samples were applied to a 12.5% SDS-polyacrylamide resolving slab gel with prestained molecular weight standards on both ends of the slab. Standards used were as noted earlier herein, being prestained protein molecular weight standards, high range, catalogue no. 560-6041 LA, from Gibco BRL, Uxbridge, England. After electrophoresis the sample lane was cut into 2.5 mm strips, which were washed twice with 0.5 ml for 15 minutes with PBS containing 2% BSA and then incubated for another 6-8 hours in 50 μl of the same buffer and bioassayed. Activity was found in a molecular weight range of about 20-25 kD.

Claims

CLAIMS :

1. A polypeptide factor which has, if obtained from ATCC CCL136 as a source, an observed molecular weight of about 20-25 kD on SDS-polyacrylamide gel electrophoresis in the absence of reducing agent, using the following prestained molecular weight standards:

Lysozyme 14,300 B-lactoglobulin 18,400

Carbonic anhydrase 29,000

OvaIbumin 43,000

Bovine serum albumin 68,000

Phosphorylase B 97,400 Myosin (H-chain) 200,000;

which factor is obtainable, if desired, by secretion from the human rhabdomyosarcoma cell line obtainable from ATCC CCL136, and which factor induces oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells to express GFAP.

2. A polypeptide as claimed in claim 1 and which may be eluted from a reversed-phase HPLC by 56-60% acetonitrile.

3. A process for the preparation of a polypeptide defined in claim 1, comprising preparing serum-free medium conditioned with human rhabdomyosarcoma cells, preparing a concentrate from said conditioned medium, subjecting said concentrate to chromatography comprising reversed-phase HPLC employing an acetonitrile eluant and thereafter to SDS-polyacrylamide gel electrophoresis and collecting that fraction therefrom which has an observed molecular weight of about 20-25 kD if subjected to SDS-polyacrylamide gel electrophoresis in the absence of reducing agent using the following prestained molecular weight standards:

Lysozyme 14,300 B-lactoglobulin 18,400

Carbonic anhydrase 29,000

Ovalbumin 43,000

Bovine serum albumin 68,000 Phosphorylase B 97,400

Myosin (H-chain) 200,000

4. A process as claimed in claim 3, wherein the human rhabdomyosarcoma cells are those obtainable from ATCC CCL136.

5. A process as claimed in claim 3 or claim 4, wherein the reversed phase HPLC is preceded by hydrophobic interaction chro otography, which hydrophobic interaction chromotography is optionally preceded by ion exchange chromotography.

6. A process as claimed in any one of claims 3 to 5, wherein the acetonitrile eluant used in the reversed-phase HPLC is 56-60% acetonitrile.

7. A process for the preparation of a polypeptide factor substantially as hereinbefore described in Example 2.

8. An assay for differentiation inducing ability, comprising incubating immortalized 0-2A progenitor cells with the substance under assay and determining GFAP production, if any, in the resulting cells.

9. An assay as claimed in claim 8 wherein said determination is performed by an immunoassay using an anti- GFAP antibody.

10. An assay as claimed in claim 9, wherein the immunoassay is an ELISA relying upon a fluorescence effect to determine GFAP production.

11. An assay as claimed in any one of claims 8 to 10, wherein the immortalized 0-2A progenitor cells have been made by transfection of 0-2A progenitor cells, optionally obtained from a young rat, with SV40 large T antigen- encoding DNA.

12. An assay for 0-2A progenitor cell differentiation- inducing ability substantially as hereinbefore described in Example 1.

13. A pharmaceutical or veterinary formulation comprising a polypeptide as defined in claim 1 or claim 2 formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.

14. A method for the prophylaxis or treatment of pathophysiological condition of the nervous system in which a cell type is involved which is sensitive or responsive to a polypeptide as defined in claim 1 or claim 2 comprising administering an effective amount of said polypeptide.

15. A method as claimed in claim 14, wherein the condition is a tumour, a condition involving death of cholinergic neurones, optionally Alzheimer's Disease, or an autonomic neuropathy involving ciliary ganglion cells.

16. A method for the prophylaxis or treatment of a condition which involves demyelination or inadequate myelination, for example multiple sclerosis, which comprises administering an effective amount of a substance which inhibits the binding of a factor as defined in claim 1 or claim 2 to a receptor therefor.

17. The use of a polypeptide as defined in claim 1 or claim 2 in the manufacture of a medicament.

18. The use of a polypeptide as defined in claim 1 or claim 2 in inducing cell differentiation.

19. The use of a polypeptide as defined in claim 1 or claim 2 as an immunogen in making antibodies, optionally antibodies for use as diagnostic aids or in diagnosis.

20. The use of a polypeptide as defined in claim 1 or claim 2 in a competitive assay to identify or quantify molecules having receptor binding characteristics corresponding to those of said polypeptide.

21. The use of claim 20, wherein said polypeptide is labeled, optionally with a radioisotope.

22. The use of a polypeptide as defined in claim 1 or claim 2 in an affinity isolation process, optionally affinity chromatography, for the separation of a corresponding receptor.