WO1994013800A2 - Mk protein preparation and use in cell culture - Google Patents

Abstract

The invention provides a method of growing neurectodermal cells which comprises culturing said cells in vitro in the presence of MK protein. A suitable form of MK protein is the protein of Seq.ID No. 1. Cells grown in the presence of MK protein, or the protein itself, may be used to treat conditions in which damage to nerve cells has occurred.

Description

MK PROTEIN PREPARATION AND USE IN CELL CULTURE

The present invention relates to a pluripotential embryonic stem cell-derived neuroregulatory factor, the MK protein, and more particularly to its use in the regulation of a variety of cell types.

MK protein was first described as a retinoic acid-induced protein in HM-1 embryonal carcinoma (EC) cells (Kadomatsu et al (1988) Biochem. Biophys. Res. Commun. 151, 1312- 1318). The amino acid sequence of human MK is shown in Seq. ID No. 1. The murine homologue of this protein has about 85% homology at the amino acid level and is shown as Seq. ID No. 2. The function of MK was unknown although in situ hybridisation studies showed that the MK gene is transcribed in a number of embryonic cell types including the egg cylinder, nervous system, lung and kidney ([Kadomatsu et al (1990) J. Cell. Biol. 110, 607-616).

It has also been found that MK has about a 65% homology at the amino acid level to the 18 kD protein isolated by heparin affinity chromatography from adult brain as neurite outgrowth- promoting factor for foetal rat CNS neurons. The protein has been termed B-GAM (Rauvala et al (1989)EMBO J. 8, 2933-2941), pleiotropin (Li et al (1990) Science 250, 1690-1694) or heparin-binding neurotrophic factor (Bohlen et al (1990) Growth Factors 41, 97-107).

It has now been found that MK protein has heparin binding activity and is a mitogen for neurectodermal precursor cell types. Precursor cell types include peripheral neurons and glia cells. We have also found that MK activity is potentiated by heparin.

Thus, the present invention provides a method for growing neurectodermal cells in vitro which comprises culturing the neurectodermal cells in the presence of MK protein. Usually, the concentration of MK factor effective to achieve growth and/or differentiation of the cells will be from 50pM to lOnM, preferably from 500pM to 5nM. Optionally and desirably, an effective amount of heparin to potentiate the action of MK protein will be used in combination with the MK protein. The amount of heparin which will be effective may be determined by experiment although typically from 1 to 20 μg per ml may be used. The MK protein may be the protein described by Kadomatsu (ibid) or mammalian homologues thereof, eg. the murine homologue. Allelic variations of the protein may be used, as well as synthetic variants which substantially retain MK activity. Such variants may be those which are about at least 75%, eg. 80%, 85%, 90% or even 95% homologous (where homology is measured by amino acid identity) at the amino acid level. Fragments of MK or variants thereof may also be used provided that such fragments retain the mitogenic activity, and preferably also the heparin binding activity of MK. Reference to MK protein herein will be understood to encompass such variants and fragments thereof.

The MK may be prepared by culture of cells which naturally produce MK, eg. EC cells, or produced by synthetic or recombinant means. In a further aspect of the invention, there is provided a method for producing MK protein which comprises expressing a gene encoding MK protein in a host cell, collecting the proteins expressed by the host cell and purifying MK protein by a method which comprises contacting the unpurified MK protein with heparin fixed to a solid support under conditions in which MK protein will bind heparin, washing the sample to remove material not bound to heparin, and eluting said protein from the heparin support.

Conveniently, this process may be carried out by heparin affinity chromatography. Chromatography columns comprising heparin fixed to a solid phase are commercially available (see Examples, part A(4) below). To purify MK factor on a heparin affinity column a sample containing the factor is applied to the column under conditions which allow binding of the MK factor to heparin. The column is washed to remove material not bound to the heparin, and the MK factor is then eluted using a high salt buffer. A suitable salt buffer is sodium chloride at a concentration of about 1.5 to 2.0 M.

The gene encoding MK protein is desirably carried by a vector adapted to be compatible with the host cell for replication of the vector and for expression of the MK gene. The cells may be stably transformed or transfected with a construct designed to transiently express the MK protein.

In a further aspect of the invention, the invention provides a host cell stably transformed with an expression vector compatible with the host cell, said expression vector adapted to express MK protein. The host cell may be prokaryotic or eukaryotic. Suitable eukaryotic cells include yeast, insect and mammalian cells.

There are a number of diseases associated with neurodegenerative disorders, for example multiple sclerosis, Parkinson's Disease and Alzheimer's disease, and other medical conditions associated with damage to nerve cells, for example spinal injuries which result in paralysis of part of all of the body. Nerve cells may also be damaged through mechanical injuries.

In the treatment of such diseases or injuries one method of therapy proposed is the regeneration of neuronal cells to combat the degeneration of such cells associated with the disease or the treatment of neuronal cells in damaged tissues. Such treatment may involve either the implantation of cells into the affected area of the body (ie. where cell degeneration is occurring) or the provision of a factor to such an area. If cells are implanted, the implant may be allogenic, autogenic or homotypic. An allogenic implant comprises cells or tissue which have a different genetic constitution than the host tissue, this is because they are obtained from a subject other than the recipient (eg. a foetus). An autogenic implant comprises cells or tissue originating within the body of the recipient (with the same genetic constitution). Finally, a homotypic implant comprises cells of the same genetic constitution and the same type and function.

The results in the examples which follow shows MK protein to have both mitogenic and neurotrophic effects on a variety of neuronal cell types in vitro. Thus, in a further aspect, the invention provides a method of treating diseases of the human or animal body which involve neurodegeneration or other damage to nerve tissue which method comprises treating neurectodermal cells or their immature precursors in vitro with an effective amount of MK protein to provide for the growth and/or differentiation of such cells, and implanting such treated cells into the human or animal subject.

The introduction of transformed or modified mammalian cells into a human patient has been proposed as a method of gene therapy to combat a number of conditions, eg tumours (see for example Culver et al, (1992) Science 256; 1550-1552 or Schinstine et al, 1991 (see refs below)). Furthermore, the introduction of fetal cells into human brains has been attempted as a means of combatting degenerative diseases (for a review see "Rescuing Minds from Disease and Decay", New Scientist, 14 November 1992, pages 2-8). Such techniques may be followed or modified by routine experimentation to determine the optimum methods of treatment for patients in which it is desired to transplant neurectodermal cells for the treatment of disease. Typically, an effective amount of cells will be in the range of from 10⁶ to 10⁸ cells per dose. The cells will normally be introduced into the patient by injection although any other suitable route may be used.

The invention also provides cells grown in vitro in the presence of MK protein for use in a method of treatment or therapy of the human or animal body. Such treatment includes the treatment of the conditions mentioned above.

In a further aspect of the invention, neuronal cells within the human or animal body may be treated directly by the introduction of MK protejn into the human or animal body at a location where the MK protein can act upon the cells, in order to induce neuronal growth. Accordingly, the present invention provides MK protein for use in a method of treatment or therapy of the human or animal body. The present invention also provides pharmaceutical compositions comprising MK protein together with a pharmaceutically acceptable carrier or diluent. The invention further provides such compositions for use in a method of treatment or therapy of the human or animal body.

The invention further comprises a method of treatment of the human or animal body which comprises administration to a subject in need of treatment an effective amount of MK protein or a pharmaceutical composition comprising MK protein. The effective amount of protein and the route of administration will ultimately be at the discretion of the physician, taking account of the state of the patient and the nature of the disease being treated. However, an effective dose will typically be in the range of from 1 to 100 μg of protein per kg of body weight of the patient, for example about lOμg/kg. The protein may be administered by any convenient route, for example parenterally by injection or orally. If by injection, it may be into the blood or directly at the site of the body where the action of the MK protein is required, eg in the brain, spinal column or site of tissue damage.

It is also envisaged that the MK protein may be introduced into the human or animal body by introducing cells into the region of the body to be treated a cell or cells adapted to express MK protein. Such cells can be prepared by transformation of cells which do not normally express MK protein, or cells which do not express the protein in significant amounts. Alternatively, methods of gene therapy have been proposed in the art which comprise direct infection of target cells (in this case such cells include neural cells) with a vector such as a retroviral vector in order that such target cells produce a particular factor. Thus, the present invention further provides a method of treatment of the human or animal body which comprises gene therapy with an vector adapted to express MK protein in target cells. Such vectors include retroviral vectors. The treatment may be performed in accordance with the discussion above in connection with the transplant of cells grown in vitro for reimplantation into a patient. Similar doses of cells will be required, and the cells may also be administered by injection or other suitable route. Methods of in situ retroviral-mediated gene transfer are disclosed by Ram et al, (1993) Cancer Research 53; 83-88.

The invention also provides a vector adapted to express MK protein in target cells within the human or animal body, and such a vector for use in a method of treatment of the human or animal body.

Description of the drawings.

Fig. 1. Northern blot analysis of MK expression in murine EC and ES cells. Total cytoplasmic RNA was probed with ³²P-labelled MK cDNA (MK) and the blot reprobed with a murine glyceraldehyde phosphate dehydrogenase cDNA (mGAP) as a loading control. Samples are; P19= P19 EC cell RNA, P19 D+RA= Differentiated P19 EC cells derived by RA treatment. E14= E14 ES cells, E14 D+RA =E14 ES cells differentiated by exposure to RA, E14 D-RA = E14 ES cells differentiated by withdrawal of Leukaemia inhibitory factor, P19 -RA = P19 EC cells propagated in retinoid depleted FCS and 10Tl/2= 10T1/2 fibroblast RNA.

Fig. 2. SDS-PAGE of HPLC purified rMK. S50 and S100 = 50 ng and 100 ng of protein standards. M= 50 ng of rMK. The apparent molecular mass of protein standards is indicated in kilodaltons. Fig. 3. Induction of DNA synthesis measured by pH]thymidine incorporation in 10T1/2 fibroblasts by recombinant FGF-4 (gift of Dr D Rogers, Genetics Institute) and rMK.

Fig. 4. Induction of DNA synthesis measured by pH]thymidine incorporation in 1009 EC cells treated with RA for 4 days by rMK.

Fig. 5. Effects of rMK and substrate preparations on chick E12 sympathetic neuron survival in vitro. Neurons were plated into tissue-culture dishes coated wityh poly-L-lysine and laminin. Cell numbers were determined after 3 hours, 24 hours and 48 hours culture. Additives are;

Con= control COS-cell-conditioned media; PL= none; MK[sol] = rMK added to the liquid phase of the tissue culture media; MKfinsol] = rMK was incubated on prepared substrates prior to addition of cells. Heparin + MKfinsol] = substrata were incubated with heparin (10 μg/ml) followed by rMK at the deisgnated concentrations followed by addition of the test cells; NGF = Nerve growth factor added at 50 ng/ml to the liquid phase of the culture.

Fig. 6. Morphological appearance of E12 sympathetic neurons exposed to MK and experimental substratum preparations after 48 hours culture. Neurons were plated into tissue culture dishes treated with poly-L-lysine and laminin. (A) Matrix preparation from 10T1/2 cells expressing MK from transfected plasmid

(B) 1 ng/ml rMK coated on substratum preparations in the presence of heparin.

(C) 10 ng/ml rMK coated on substratum preparations in the presence of heparin.

(D) 10 ng/ml rMK added to the liquid phase of the culture medium. (E) 50 ng/ml NGF. (F) Control COS-cell-conditioned medium.

Fig. 7. E12 sympathetic neuron survival on ECM derived from MK-transfected 10T1/2 clones. 10³ E12 neurons were plated onto ECM preparations from 10T1/2 cell clones which expressed MK from a transfected plasmid (clones 1, 6, 10 and 11) or from a clone recovered in the same experiment in which the MK expression plasmid had not integrated (clone 15). C, non-transfected 10T1/2 cells. Cell numbers were determined after 3 hours and 24 hours.

The following examples illustrate the invention. Part A - Material and methods

1. Cell culture and reagents

Cell culture medium comprised DME.F12 (50:50 vol:vol; Gibco) supplemented with foetal calf serum (10% by volume, selected batches). All cells were cultured in a humidified incubator at 37 °C in an atmosphere of 5% CO₂ in air. ES cell culture medium was additionally supplemented (unless otherwise indicated) with 10 ng/ml recombinant leukemia inhibitory factor (Smith et al., 1988) and 10^ M betamercaptoethanol (Sigma, tissue culture grade). In some experiments the FCS used for cell culture was charcoal stripped to remove endogenous RA by the method described by VanderBurg et al., (1988).

Swiss 3T3 (Flow laboratories) and 10T1/2 fibroblasts were propagated as mass cultures according to the '3T3' regime described by Todaro and Green (1963). Embryonal carcinoma cells (P19 and 1009) and embryonic stem cells (E14TG2a) were maintained as described (Rathjen et al., 1990).

P19 EC cell differentiation was induced by exposure to RA as described by Rudnicki and McBurney (1987). E14TG2a ES cell differentiation was achieved either by exposure to 10"⁷M RA (all trans: Sigma, added from a 10^"2 M stock dissolved in tissue culture trade DMSO) or by culture of cells at low density in the absence of leukemia inhibitory factor (Smith et al., 1988). 1009 EC cell differentiation was achieved by plating 1009 cells at 10" cells/ well into 5 mm 6-well cluster dishes in 4 ml of medium (DME;F12 10%FCS). RA was added from a 10^"2 M stock (dissolved in DMSO) after 24 hours to a final concentration of 5xl0"⁷ M.

2. Cell multiplication

Induction of DNA synthesis in experimental cell populations was determined by [³H]thymidine incorporation as described by Heath (1987). 3. Stable cell lines

6x10° 10T1/2 fibroblasts were co-transfected with pXMT2-MK by the calcium phosphate method of Chen and Okyama (1987) using 10 μg of pXMT2-MK and 10 μg of PGK-Neo/3. 5 The day after transfection the cells were plated into 10x10 cm. tissue culture dishes and cultured in the presence of 300 μg/ml G418 (Sigma) for 14 days. A random sample of G418 resistant colonies were picked under a dissecting microscope and expanded in mass culture for further analysis. A matrix preparation was obtained from selected clones by release of cells with EDTA as described by Rathjen et al., (1990). 10

4. Purification of rMK

(i) Conditioned media

15 rMK was purified from media conditioned by COS cells transfected with the MK expression plasmid pXMT2MK. 5xl0⁷ COS cells were transfected with 50 μg of the PXMT2MK by electroporation (Biorad gene pulser, 330 V, 500 μF) and plated into 175 cm² tissue-culture flasks in 75 ml of culture medium. The following day the medium was changed to 100 ml DME:F12 supplemented with 10 μg/ml transferrin (Sigma) and 10 μg/ml Heparin (BDH).

20 The cells were cultured for a further 48 hours and the cell culture medium collected and passed through a 0.22 μm filter prior to processing for purification.

COS-cell conditioned medium (200 ml) was pumped onto a 1 ml Hitrap heparin affinity column (Pharmacia) at a flow rate of 200 μl/minute at 4°C. The column was washed with 25 10 ml of 50 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and the rMK eluted by washing the column with 5 ml of phosphate buffer (pH 7.4) containing 2 M NaCl. The 5 ml heparin affinity eluate was then desalted (pharmacia PD-10 desalting column) and used for biological assays.

30 Final purification was obtained by reverse phase chromatography on a Brownlee rp-300 microbore column. The heparin affinity eluate was loaded directly onto the column at a flow rate of 1 ml/minute and then washed with solvent A (0.1 % triflouracetic acid in water) for 5 minutes at a flow rate of 1 ml/minute. The flow rate was adjusted to 0J ml. minute over 1 minute and protein eluted by a gradient (0-60% at 1 %/minute) of solvent B (0.1 % triflouracetic acid in acetonitrile (Far UV grade BDH). rMK purified by reverse phase electrophoresis was analysed by SDS-PAGE (Phastgel system pharmacia 20% linear gels). An initial standard sample of rMK purified by rpHPLC was subjected to amino acid analysis for protein quantification. In subsequent experiments rMK was quantified by comparison of peak height (280 nM) with data obtained from the standard sample. The yield of rMK produced by these methods varied from 18 μg to 23 μg (3 experiments from 200 mis of initial conditioned media) and agreed well with the protein concentration of the heparin affinity eluate determined by dye binding assay (Biorad, bovine serum albumin standard). Recoveries were not determined.

(ii) Tissue culture substrata and media

Tissue culture substrata were coated where indicated with a solution containing 0.1 mg/ml poly-L-lysine in distilled water for 5 minutes. Substrata were rinsed three times in water and allowed to dry. In some experiments the substrata were further coated with either EHS laminin or fibronectin (Sigma 10 μg/ml in PBS, 2 hours at room temperature) and rinsed with DMEM immediately before use, or heparin (10 μg/ml (BDH) in PBS, 2 hours at room temperature), or a mixture of both. In some experiments, MK was coated by incubation for 2 hours, at room temperature, over prepared substrata at designated concentrations to a maximum of 100 ng/ml. Growth factors were added to the culture medium at the nominated concentrations.

(iii) Assay for neurotrophic activity

The sympathetic ganglia from embryonic day-12 (E12) chicks were dissected out and dissociated into single cells using standard techniques ( Wakade et al., 1982). Briefly, the ganglia were trypsinised for 30 minutes in Ca²⁺ and Mg²⁺ -free phosphate-buffered saline, triturated, and the resultant single-cell suspension pre-plated over tissue culture plastic in DMEM: 10% FCS for a period of 45 minutes to enrich for neurons. They were plated at a concentration of 2.0X10³ cells per 16 mm well of a 24-well cluster under the appropriate experimental conditions. After incubation at 37°C for 2 days in the presence or absence of MK, the resulting cell survival, and the proportion of neuronal cell bodies with neurites longer than 2 cell diameters were monitored under phase contrast microscopy.

5. Isolation of RNA

Cell pellets (10⁷-10⁸ cells) were washed in phosphate-buffered saline (PBS) and lysed in 5 ml of 30 mM Tris pH 7.5, 150 mM NaCl, 15mM MgCl₂, 0.4% NP40. Nuclei were removed by centrifugation for 15 minutes at 3000g. An equal volume of TUNES (10 mM Tris, pH 7.5, 7 M urea, 0.35 M NaCl, 1 mM EDTA, 2% SDS) was added to the supernatant. The cytoplasmic RNA was extracted twice with methanol/chloroform and once with chloroform before ethanol precipitation and recovery by centrifugation. The pellet was washed with 70% ethanol before being dissolved in DEPC-treated water.

6. 1st strand DNA synthesis

2.5 μg of cytoplasmic RNA in 40 μl DEPC-treated water was heated at 65°C for 3 minutes, quenched on ice and added to 5 μl 10X RTC buffer (BRL), 2.5 μl 10 mM dNTP mix, 1 μl (40 units) of RNasin (Promega), 0.5 μl (0.5μg) oligo dT 12-18 (Pharmacia) and 1 μl (200 units) cloned MuLV-1 reverse transcriptase (BRL). The reaction was heated at 37°C for one hour and terminated by heating at 95 °C for 5 minutes.

7. Oligonucleotides

Oligonucleotides were synthesised on an applied biosystems 380A DNA synthesiser. 5'MK had the sequence 5'-GCAATTCATGAGCACCGAGGTTCTT-3'. The ATG underlined represents the initiation codon of murine MK (Matsubara et al., 1990, Tomomura et al 1990b). The preceding nucleotides constitute an EcoRI site used to facilitate cloning of the amplified material.3 'MK had the sequence 5'-AAGTCGACGGCCTCCTGACTTAGTCCTT- 3'. The first 8 residues constitute a Sail site used to facilitate cloning the amplified material. The rest of the oligonucleotide represents residues 415-434 of murine MK (Kadomatsu et al 1988). 8. Polvmerase chain reaction and cloning

1 μl of cDNA was amplified in a 50 μl reaction volume containing 50 mM KC1, 10 mM Tris- HC1 pH 8.3, 1.5 mM MgCl₂, 0.01 % (w/v) gelatin, 200 μM each dNTP, 0.5 μM 5'MK and 3'MK oligonucleotide primers, 2 units Taq polymerase (Cetus). PCR was performed according to the following protocol: an initial denaturation for 5minutes at 95 °C was followed by 30 cycles of 1.5 mins denaturation, annealing at 64°C for 1.5 minutes and extension for 1.5 minutes at 72 °C. Following amplification the reaction was increased to 100 ul by addition of reaction buffer to which was added 5 μl 10% SDS and 1 μl of proteinase K (1 mg/ml). The reaction was heated at 55 °C for 30minutes before extraction with phenol/chloroform and ethanol precipitation. The amplified material was digested with EcoRI and Sail and subjected to electrophoresis in a 1.5% agarose gel. For cloning, the amplified band was excised from the gel and purified by means of a gene clean kit. The purified insert was cloned into EcoRI/Sall cut pBS KS+ (Stratagene).

MK was cloned into the expression plasmid pXMT2 in both orientations. For the sense orientation the pBSMK plasmid was cut with Sail, the ends were blunted by Klenow fill in and the insert released by digestion with Pstl. The pXMT2 vector was prepared by cutting with EcoRI followed by blunting with Klenow and digestion with Pstl. For the antisense orientation the insert was released from pBS/MK as an EcoRI/Sall fragment and cloned into EcoRI/XhoI cut pXMT2.

9. Northern blotting

RNA was analysed by electrophoresis in a formaldehyde/Mops-buffered agarose gel. Typically 15 μg of cytoplasmic RNA was run on a 1.25% agarose gel containing 2.22% formaldehyde. RNA samples were denatured by heating at 60°C for 10 minutes in loading buffer containing 50% formamide, 6.66% formaldehyde, 10% glycerol, 0.025% bromophenol blue and 10 μg/ml ethidium bromide. Samples were transferred to HYBOND N +(Amersham) by capillary blotting for 5 hours in the presence of 40 mM NaOH. Probes for hybridisation were prepared by separating cloned inserts from plasmid vectors by agarose gel electrophoresis followed by purification using a Gene Clean Kit. DNA was labelled with ³²P by random priming (Boehr iger). Prehybridisation was for 1 hour at 64°C in 0.5 M sodium phόsphate (pH 7.2), 7% SDS, 1 mM EDTA. Radiolabelled probe (10⁸-10⁹ cts/minutes per μg) was added to the prehybridisation solution and hybridisation was allowed to proceed for 18 hours at 65 °C. Filters were washed to a final stringency of 0.2xSSC/0J % SDS at 64°C and exposed to X-ray film at -70°C with intensifying screens.

Part B - Results

1. Expression of MK transcripts in ES and EC cells

The presence of MK and B-GAM transcripts in ES cells was examined by polymerase chain reaction (PCR) amplification of cDNA derived from E14TG2a ES cell polyA-f mRNA using primers corresponding to the nucleotide sequence of the predicted amino terminus and carboxy terminus of both proteins. A strong MK amplification product was obtained after 30 cycles of amplification. Significant amplification of an B-GAM product was also observed although at lower levels. The PCR-amplified cDNAs were cloned into plasmid vectors for further analysis of MK gene expression.

Northern blot analysis of total RNA from P19EC or E14TG2a ES cells with the MK cDNA revealed a strong signal of apparent size 900 bp (Fig. 1) which was in accord with the previous reported size of the MK transcript (Tomomura et al., 1990b). No detectable signal was observed upon northern hybridisation of the same RNAs with the cloned B-GAM cDNA. It thus appears that MK (but not B-GAM) is an abundant transcript in both EC and ES cells. These results were unexpected since MK transcripts were reported to be present at very low levels in HM-1 EC cells (Kadomatsu et al., 1988, Huang et al., 1990) and increased significantly upon exposure of the cells to retinoic acid. Northern hybridisation of P19 and E14TG2a cells differentiated by exposure to RA revealed that MK mRNA expression was not significantly altered by either exposure to 10^"7 M retinoic acid or differentiation (by withdrawal of LIF) of ES cells in the absence of retinoic acid. One possible explanation for these results is that MK expression is induced in these cells by trace levels of retinoids present in FCS. However, the expression of MK mRNA in P19 cells grown in the presence of FCS depleted of retinoids by charcoal stripping was equivalent to that observed in untreated FCS (Fig. 1). The results show that MK is expressed in both undifferentiated stem cells and their differentiated derivatives and that its expression, in these cell types, is not detectably affected by exposure to RA. Twelve full-length MK cDNAs were also isolated by screening approximately 100,000 clones of a P19 EC cell cDNA library with the PCR-derived MK cDNA. This finding is in accord with the results of the northern hybridisation analysis and indicates that MK is a relatively abundant transcript in EC and ES cells.

2. Biological actions of MK protein

The biological function(s) of MK has not previously been clearly defined. Muramatsu and his colleagues have shown that MK protein derived from expression in eukaryotic cells has neurite-promoting actions on PC12 cells (Muramatsu and Muramatsu, 1991) although this activity was only observed at relatively high protein concentrations (100 ng/ml) where the possibility of contamination by other bioactive factors cannot be firmly excluded. It has recently been shown that bacterially expressed MK has neurite outgrowth promoting effects on spinal cord neurons but again the biological actions were manifest at high concentrations in which cross-reactivity with receptor systems for other neurotrophic factors cannot be firmly excluded.

3. Production of recombinant MK protein

The MK cDNA described above was cloned into the eukaryotic expression vector PXMT2 which was then transfected into COS cells. Serum-free conditioned media was collected from the COS cells and the MK protein purified by heparin affinity chromatography. Characterisation of heparin affinity-purified rMK by reverse phase HPLC (rpHPLC) yielded a major biologically active protein product of approximate relative molecular mass lό.SOOxlO³ (Fig. 2). The identity of this species was confirmed by immunoblotting using an antibody directed against MK expressed in bacteria and amino acid analysis.

A second protein species detected by SDS PAGE of rpHPLC fractionated material had an apparent Mr of 65x10^s and was devoid of both biological activity and anti-MK immunoreactivity. This species was also detected in conditioned media derived from COS cells transfected with the PXMT2 expression plasmid cloned in the antisense orientation. In addition, no biological activity could be detected in high salt eluates of heparin affinity chromatography of media conditioned by cells expressing the antisense MK expression construct.

The use of rpHPLC as a purification step resulted in a substantial loss of MK bioactivity resulting, at least in part, from exposure to acid solvents. Similar findings have been reported for HBNF (Bohlen et al., 1990). It was also noted in the course of these experiments that biological activity present in either COS-cell-conditioned media or heparin affinity-purified samples was very unstable upon storage between -20° and +4°C. In order to circumvent this problem, MK preparations for biological assays described below were used within 5 days of purification and not subjected to the rpHPLC purification step (which was thereafter employed for quantitation of rMK protein concentration present in the heparin affinity eluates).

4. Mitogenic actions on fibroblast cell lines

Since FGF-4 does not account for the totality of heparin-binding mitogens present in EC cell conditioned medium (Heath et al., 1989), the ability of rMK protein to induce DNA synthesis in quiescent 10T1/2 and Swiss 3T3 fibroblast cells was examined. Unlike rFGF-4, MK was completely inactive as a mitogen for Swiss 3T3 cells. This observation demonstrates that COS cell-derived rMK is not detectably contaminated with FGF-like biological activities (such as bFGF) which might have been expected to co-purify with MK. rMK was, however, able to induce DNA synthesis in quiescent 10T1/2 cells (Fig 3). The maximal induction of DNA synthesis observed was, however, approximately one fifth of that observed with rFGF-4. Half-maximal induction of DNA synthesis was manifest at approximately 1 nM rMK. This shows that rMK is a significant but weak (in comparison to FGF-4) growth factor for 10T1/2 cells.

5. rMK is a mitogen for EC cell-derived neurectodermal cells

In view of the reported actions of MK on PC12 cells and the effects of the related factor B- GAM on CNS neurons, the action of rMK in two neuronal cell systems was examined. 1009 EC cells differentiate into neurons with cholinergic properties (as well as an additional non- neuronal cell type) upon exposure to retinoic acid (Pfeiffer et al., 1981). RA-treated 1009 cells therefore represent a population of neuronal cells which, in view of their EC derivation, resemble the neurectodermal cell types of the early embryo.

rMK is a significant growth factor for neurectodermal cells derived by RA treatment of 1009 EC cells (Fig. 4) for 4 days. The induction of DNA synthesis observed was comparable to that found for rFGF-4, with a half maximal response at approximately 1 nM. No mitogenic effect of rMK was observed on undifferentiated 1009 EC cells.

RA-treated 1009 cells progressively form extensive networks of non-dividing neurons after culture for longer periods of time (7-14 days). No significant effect of exogenous rMK on DNA synthesis, cell numbers or neuron morphology could be observed after culture for 10 days in the presence of RA. This finding would suggest that the mitogenic effects of rMK on these cells are restricted to a cell type formed during the early phases of RA-induced differentiation, which gives rise to more mature cell types that are not overtly responsive to exogenous rMK.

6. rMK is a neurotrophic factor for sympathetic neurons

Since rMK was found to have significant biological actions on EC cell-derived neuronal cell types in vitro we examined the action of rMK on a number of other neuronal cell types from different sources. A striking finding from these investigations was that MK was able to enhance the survival of E12 chick sympathetic neurons in vitro, as well as promoting neurite outgrowth. The potency of rMK in this assay was, however, significantly affected by both the nature of the tissue culture substratum employed and the mechanisms of rMK presentation to responding cells. Only minor effects of rMK on neuron survival and neurite outgrowth were observed when neurons were plated on poly-lysine/laminin and the rMK was added to the liquid phase of the cell cultures (Figs. 5, 6D). However, marked increases in response were observed when the tissue culture plates were first coated with heparin and the rMK presented to cells by prebinding it to the tissue culmre plate (Figs. 5, 6B, C). The effect of heparin was minimal at low (1 ng/ml) concentrations of rMK but became significant at rMK concentrations of lOng/ml (O.όnM) and above. Under these conditions the response to rMK approached that obtained with nerve growth factor (NGF) added to cells in the soluble phase (Figs. 5, 6E). These findings indicate that rMK can be a potent neurotrophic factor for sympathetic neurons if presented to cells in the insoluble phase in the presence of heparin.

These effects of cell substratum on MK function were further examined by testing the biological properties of a 'natural' substratum preparation which might be expected to more closely resemble the physiological presentation of MK in vivo. 10T1/2 fibroblasts were co- transfected with the MK expression plasmid pXMT2MK and a PGK-Neo drug resistance plasmid. Stable cell lines were isolated by selection in G418 and a number of G418 resistant clones isolated by colony selection and expanded for analysis of expression of MK transcripts by PCR. No significant effects of MK expression on either cell morphology or growth rates were observed in these cell lines, in keeping with its weak effect on 10T1/2 cell proliferation when added exogenously. Four 10T1/2 clones which were found to express MK by PCR, a control clone (in which MK expression could not be detected by PCR) and non transfected 10T1/2 cells were grown to confluence in vitro and a 'conditioned matrix' preparation obtained by removal of the cells from the tissue-culmre dish by treatment with EDTA (Rathjen et al., 1990). Replating E12 sympathetic neurons onto the conditioned matrix produced a significant effect on both 24 hour neuronal survival and neurite outgrowth when sympathetic neurons were plated onto matrix derived from clones expressing MK (Fig 6a, Fig 7), but not in the non-expressing control clone or non-transfected 10T1/2 cells (Fig 7). Some interclonal variation was observed in the ability to support neuronal survival which may reflect differing levels of MK expression between clones. These findings indicate that MK can be physically associated with matrix components in a biologically active form when expressed continuously in stably transformed fibroblast cell lines.

Part C - Discussion

1. Expression of MK

The present invention demonstrates that MK is a major gene product of EC and ES cells and their immediate differentiated derivatives. This finding contrasts with the original results of Muramatsu and colleagues (1988) who isolated MK from HM-1 EC cells by differential screening for genes induced by retinoic acid. The present data indicate that MK mRNA levels are not appreciably changed either by the presence or absence of retinoic acid. The basis of this difference is not completely clear; it seems that the effects of RA on MK gene expression in HM-1 cells are only transient. In particular, Huang et al (1990) have shown that MK induction after RA treatment peaks after 48 hours of exposure and then subsides to non- induced levels after 96 hours, which are the equivalent of the time points examined here. The distinction between the two sets of data may therefore reflect the relatively low level of MK expression in HM-1 cells compared to EC or pluripotent ES cells. The fact that MK transcripts can be detected in embryonic ectoderm by in situ hybridisation techniques suggests that MK is normally expressed by pluripotential stem cells in vivo. The persistence of MK expression in differentiated cells also accords with the finding that MK is not confined to any single germ layer during early post-implantation development.

The physiological significance of the transient burst of MK expression in HM-1 cells in response to retinoic acid is not wholly clear. Since nuclear run-on experiments (Huang et al. , 1990) indicate that MK gene transcription is only modestly induced by RA treatment of HM-1 cells (in contrast to steady state mRNA levels) it may be concluded that the MK mRNA is for some reason especially unstable in HM-1, cells and the increase in steady state MK mRNA levels has little general physiological significance. It is unlikely that MK protein acts as a mediator of some of the biological effects of RA (Urios et al., 1991) since the present data clearly show that it can be expressed in ES and EC cells in the absence of exogenous RA.

2. Biological function of rMK

The results reported above show that rMK produced by expression in COS cells and purified by heparin affinity chromatography has biological effects on three distinct cell types.

Firstly, rMK is a significant but extremely weak mitogen for 10T1/2 fibroblasts, while at the same time being inactive towards 3T3 cells. Furthermore, stable 10T1/2 cell lines were isolated that express MK but did not exhibit any overt evidence of morpholological or proliferative changes associated with cell transformation. This contrasts with the effects of FGF-4 which is both a potent mitogen for these cells and powerful transforming gene when expressed under equivalent conditions. Therefore, it appears that the effects of MK on fibroblast cell proliferation are influenced by the exact identity of the target cell line although the molecular basis of these differences is currently obscure. This may explain why some controversy exists regarding the mitogenic actions of rB-GAM/HBNF/PTN on fibroblast cell lines (Li et al. , 1990, Bohlen et al., 1990); the activity observed may entirely depend on the fibroblast cell line employed. These findings also indicate that, at least in the expression system employed here, MK is not a transforming gene for mouse fibroblasts. This does not of course exclude the possibility that MK expression may be associated with oncogenic transformation of other cell types. Taken together, these observations show that the biological properties of MK are different from that of the heparin-binding growth factor activity isolated from P19 EC cells (Heath et al., 1989) which was very active in fibroblast mitogen assays. It is possible that the conditions of isolation of rMK expressed by COS cells results in a significant loss of activity, although this would seem unlikely in view of the potent effects of this factor in other systems and the absence of transforming activity in vitro. The most likely explanation for this discrepancy is that EC cells express an additional heparin-binding mitogen which is not a member of either the FGF or MK/B-GAM family of growth factors. It is intriguing in this respect that the mitogenic activity of B-GAM preparations derived from adult rat brain have been ascribed to a structurally distinct contaminating mitogenic factor designated pl7.

The major biological effects of MK reported here involve actions on cells of direct or indirect neurectodermal origins. Thus, rMK proves to be a potent mitogen for neurectodermal cells derived from 1009 EC cells by prior treatment with RA for 4 days. This effect is transient, since no mitogenic or morphological effects could be observed at later stages when the cultures had formed extensive networks of non-dividing neurons. It is, however, possible that rMK has more subtle effects on this population in, for example, controlling neurotransmitter expression by differentiated 1009-derived neurons. In light of the embryonic origins of 1009 cells and the expression of MK in both the primitive ectoderm and neurectoderm of the normal embryo (Kadomatsu et al., 1990), this finding indicates that MK is involved in the control of neurectodermal proliferation in the early development of the in vivo nervous system, and may function as a transforming gene in neuroectodermal tumours and cell lines.

The effects of rMK on neuronal cell behaviour are, however, clearly cell-type dependant, since the factor has potent neurotrophic and neurite outgrowth promoting effects on sympathetic neurons from later developmental stages. Together these findings indicate that MK is multifunctional regulator of neuronal cell function with distinct actions on different cell types within the developing nervous system.

3. MK and the extracellular matrix

Previous studies of the biological actions of both MK and B-GAM/HBNF/PTN have been difficult to interpret due to the low specific activity of the protein jn vivo. It appears that, at least in the case of MK, this is due to at least two distinct phenomena. Firstly the biological activity of the rMK protein produced in COS cells diminishes upon storage. The biochemical basis of this instability is at present unknown but may be due to the removal of co-factors required for protein stability during the purification procedure. In this respect the availability of stable cell lines expressing biologically active MK protein continuously may be a useful tool in further in vitro studies of MK function, as well as in defining the biological actions of MK in the adult nervous system by jn vivo grafting techniques (Schinstine et al., 1991).

Further determinants of MK bioactivity are the identity of the cellular substratum and the means by which rMK is presented to responding cells in vitro. This is most clearly demonstrated by the neurotrophic and neurite outgrowth promoting effects of rMK on sympathetic neurons. In this case, the effects of rMK were potentiated both by the addition of heparin to the tissue culmre substratum, and by allowing rMK to associate with the tissue- culture dish prior to plating the cells, rather than adding the soluble protein directly to the culmre media. It is probable that these two effects are related since a 'conditioned matrix' tissue culmre substratum preparation derived from 10T1/2 cells expressing MK from a transfected expression plasmid also exhibited potent neurotrophic and outgrowth promoting actions. Since rMK exhibits a high affinity for immobilised heparin, it seems highly likely that MK protein requires to be physically associated with heparin in the extracelluar matrix in order to exhibit maximal biological activity. This phenomenon is reminiscent of at least some members of the FGF family of heparin-binding growth factors in which, in at least one case, expression of heparin containing glycosoaminoglycans has been found to be required for FGF function in vitro (Ornitz et al 1992). The requirement for a physical association between MK and heparin (presumably in the form of HSPGs) for maximal activity could reflect either a stabilisation of the three dimensional structure of MK in a form available for receptor interaction, or a direct participation of HSPGs in the process of MK/receptor interaction.

Nevertheless, freshly prepared rMK presented to cells by substrate attachment in the presence of heparin is a very potent bioregulatory polypeptide with half maximal effects on sympathetic neuron survival and outgrowth at between 10-50 pM. This is consistent with action via association with a high affinity, signal-transducing receptor. It follows that these requirements for MK biological activity must be taken into account in attempting to characterise the identity of putative MK signal transducing receptor(s). The studies reported here also identify candidate target cell types that respond to physiologically plausible concentrations of MK in which such receptors may be characterised.

References

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Li, Y. S., Milner, P. G., Chauhan, A., Watson, M., Hoffman, R., Kodner, C. M., Milbrandt, J., and Duel, T (1990). Cloning and expression of a developmentally regulated protein that induces mitogenic and neurite outgrowth activity. Science 250, 1690-1694.

Matsubara S, Tomomura M, Kadomatsu K, Muramatsu T. (1990). Structure of a retinoic acid-responsive gene, MK, which is transiently activated during the differentiation of embryonal carcinoma cells and the mid-gestation period of mouse embryogenesis. J.Biol.Chem. 265, 9441-9443.

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Tsutsui-J, Uehara-K, Kadomatsu-K, Matsubara-S, Muramatsu-T. (1991) A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse. Biochem-Biophys-Res-Commun 176:792-7

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(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Cancer Research Campaign Technology Limited

(B) STREET: 6-10 Cambridge Terrace, Regent's Park

(C) CITY: London

(D) STATE: London

(E) COUNTRY: GB

(F) POSTAL CODE (ZIP): NW1 4JL

(A) NAME: Heath, John Kaye

(B) STREET: Dept. Biochemistry, South Parks Road

(C) CITY: Oxford

(D) STATE: Oxfordshire

(E) COUNTRY: GB

(F) POSTAL CODE (ZIP): OX1 3QU

(ii) TITLE OF INVENTION: Growth Factor (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.2 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 143 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Gi n His Arg Gly Phe Leu Leu Leu Thr Leu Leu Al a Leu Leu Al a 1 5 10 15 Leu Thr Ser Ala Val Ala Lys Lys Lys Asp Lys Val Lys Lys Gly Gly 20 25 30

Pro Gly Ser Glu Cys Ala Glu Trp Ala Trp Gly Pro Cys Thr Pro Ser 35 40 45

Ser Lys Asp Cys Gly Val Gly Phe Arg Glu Gly Thr Cys Gly Ala Gin 50 55 60

Thr Gin Arg He Arg Cys Arg Val Pro Cys Asn Trp Lys Lys Glu Phe 65 70 75 80

Gly Ala Asp Cys Lys Tyr Lys Phe Glu Asn Trp Gly Ala Cys Asp Gly 85 90 95

Gly Thr Gly Thr Lys Val Arg Gin Gly Thr Leu Lys Lys Ala Arg Tyr 100 105 110

Asn Ala Gin Cys Gin Glu Thr He Arg Val Thr Lys Pro Cys Thr Pro 115 120 125

Lys Thr Lys Ala Lys Ala Lys Ala Lys Lys Gly Lys Gly Lys Asp 130 135 140

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 140 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gi n His Arg Gly Phe Phe Leu Leu Ala Leu Leu Al a Leu Leu Val 1 5 10 15

Val Thr Ser Al a Val Ala Lys Lys Lys Glu Lys Val Lys Lys Gly Ser 20 25 30

Gl u Cys Ser Glu Trp Thr Trp Gly Pro Cys Thr Pro Ser Ser Lys Asp 35 40 45

Cys Gly Met Gly Phe Arg Glu Gly Thr Cys Gly Al a Gin Thr Gin Arg 50 55 60

Val His Cys Lys Val Pro Cys Asn Trp Lys Lys Glu Phe Gly Al a Asp 65 70 75 80

Cys Lys Tyr Lys Phe Gl u Ser Trp Gly Al a Cys Asp Gly Ser Thr Gly 85 90 95 Thr Lys Ala Arg Gin Gly Thr Leu Lys Lys Ala Arg Tyr Asn Ala Gin 100 105 110

Cys Gin Glu Thr He Arg Val Thr Lys Pro Cys Thr Ser Lys Thr Lys 115 120 125

Ser Lys Thr Lys Ala Lys Lys Gly Lys Gly Lys Asp 130 135 140

Claims

1. A method of growing neurectodermal cells which comprises culturing said cells in vitro in the presence of MK protein.

2. A method according to claim 1 wherein the neurectodermal cells are peripheral neurons or glia cells.

3. A method according to claim 1 or 2 wherein the MK protein is selected from:

(a) the protein of Seq. ID NoJ;

(b) a mammalian homologue thereof; and

(c) an allelic variant of (a) or (b).

4. A method according to any one of claims 1 to 3 wherein the concentration of MK protein is from 50pM to lOnM.

5. A method according to any one of claims 1 to 4 wherein heparin is added to the culmre.

6. A method of recovering MK protein from a sample containing said protein which comprises contacting said sample with heparin fixed to a solid support under conditions in which MK protein will bind heparin, washing the sample to remove material not bound to heparin, and eluting said protein.

7. A method according to claim 6 wherein the eluting buffer is sodium chloride.

8. A recombinant expression vector comprising a DNA sequence encoding MK protein.

9. A vector according to claim 8 wherein the protein is:

(a) the protein of Seq. ID NoJ;

(b) a mammalian homologue thereof; and

(c) an allelic variant of (a) or (b).

10. A host cell transformed with a vector according to claim 8 or 9.

11. A cell according to claim 10 which is a human cell.

12. A cell according to claim 10 or 11 for use in a method of treatment of the human or animal body.

13. A neurectodermal cell which has been cultured in vitro in the presence of MK protein for use in a method of treatment of the human or animal body.

14. A pharmaceutical composition comprising MK protein together with a pharmaceutically acceptable carrier or diluent.

15. MK protein or a composition according to claim 14 for use in a method of treatment of the human or animal body.

16. The use of MK protein for the manufacmre of a medicament for the treatment of neurodegenerative disorders or damaged nerve tissue.

17. A method of treatment of neurodegenerative disorders or damaged nerve tissue which comprises administering to an individual in need of treatment an effective amount of a medicament selected from:

(a) a cell according to claim 10 or 11;

(b) a neurectodermal cell which has been cultured in vitro in the presence of MK protein; and

(c) MK protein.

18. A method according to claim 16 wherein the medicament is a neurectodermal cell which has been cultured in vitro in the presence of MK protein, wherein said cell is autogenic to said patient.