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  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
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  • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
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  • C12N15/09—Recombinant DNA-technology
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  • C07K2319/00—Fusion polypeptide

Definitions

• This invention relates to Leukaemia Inhibitory Factor, to its production and uses.
• LIF Leukaemia Inhibitory Factor
• LIF is a normal embryonic regulatory factor.
• DIA differentiation inhibiting activity
• ES cells can be cultured and maintained in vitro without loss pluripotentiality. Even after many generations in vitro, these cells can be reintroduced into the mouse blastyocyst where they may contribute differentiated progeny to all tissues including the germ line (Bradley et al 1984).
• EP-A-285 448 discloses the complete amino acid sequence of a form of murine LIF and also discloses a sequence, incomplete at the N-terminus, of the human form of this factor.
• the murine LIF disclosed by EP-A-285 448 has the N-terminal sequence NH 2 -met-lys-val-leu-ala-ala-gly (KVL-LIF) .
• Stahl et al J.Biol Che . , 2 ⁇ 5(15),8833 (May 1990) disclose the genomic sequence of human LIF.
• RCR-LIF is therefore a localised, as opposed to diffusible, determinant of differentiation.
• RCR-LIF is thus distinguished from KVL-LIF by the following criteria: l. LIF activity is physically confined to the extracellular matrix (ECM) and is thus only acts upon cells which are in direct physical proximity to ECM containing RCR-LIF. 2. ECM associated RCR-LIF source has superior stability to KVL-LIF.
• RCR-LIF activity is confined to cells which are capable of physically associating with the RCR-LIF associated ECM preparation and therefore its range of biological activities in vivo and jLn vitro can be targeted more specifically than KVL-LIF.
• RCR-LIF can be presented in a more concentrated form (as it is not freely diffusible) with improved biological efficacy compared to KVL-LIF derived from non-ECM associated sources.
• RCR-LIF as defined by these parameters has the following properties:
• neurotransmitters for example, choline acetyltransferase and acetylcholine esterase, in neuronal preparations.
• the novel N-terminal sequence of the LIF of the present invention provides a signal sequence for directing and anchoring molecules such as proteins or peptides to a mammalian ECM. Therefore, the tetrapeptide sequence NH2- met-arg-cys-arg may be attached by chemical or recombinant means to proteins or peptides to cause the molecules to become attached to or associated with the ECM. C-terminal derivatives of this tetrapeptide sequence corresponding to further LIF sequence, eg. NH2-met-arg-cys-arg-ile-val, may also be used. It is to be understood that changes in the tetrapeptide sequence NH2 ⁇ Met-Arg-Cys-Arg which do not substantially alter the ability of this sequence to direct association with the ECM are within the scope of the invention.
• LIF of the present invention can be used for the propagation of embryonic stem (ES) cells and for the maintenance of ES cell pluripotentiality (as defined by the ability to form functional gametes in chimeras) .
• ES embryonic stem
• LIF of the invention and N-terminal fragments thereof, and polyclonal or monoclonal antibodies or fragments thereof against LIF and its N-terminal fragments have potential utility for the following applications:
• the present invention thus provides a novel LIF which is capable of associating with a mammalian extracellular matrix.
• this novel LIF is characterised by an N-terminal sequence NH2- met-arg-cys-arg.
• the invention also provides novel forms of LIF containing sequences which are substantially identical to the sequence met-arg-cys-arg which are capable of causing LIF to be associated with the ECM.
• homologues of this LIF sequence in which 2 of the first 5 amino acid residues from the N-terminus are substituted are within the scope of the invention.
• a further embodiment of the invention provides a DNA coding for any of these novel forms of LIF, N-terminal signal sequences, or recombinant proteins or peptides described above.
• the DNA may be used to produce a labelled probe by conventional means using radioactive or non- radiaoactive labels, or the DNA may be cloned into a vector.
• a further embodiment of the invention provides vectors for the replication and expression of the said DNA.
• the vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said DNA and optionally a regulator of the promoter.
• the vector may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
• the vector may be used in vitro, for example for the production of RNA corresponding to the DNA, or used to transfect or transform a host cell.
• a further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of the said LIF DNA.
• the cells will be chosen to be compatible with the vector and may for example be bacterial, yeast or mammalian.
• the invention also provides N-terminally directed monoclonal or polyclonal antibodies, or fragments thereof, to the novel proteins or peptides, or N-terminal fragments thereof, of the invention.
• the invention also provides a process for the production of monoclonal or polyclonal antibodies to the novel proteins or peptides of the invention.
• Monoclonal antibodies may be prepared by conventional hybridoma technology using the novel proteins or peptides, or fragments thereof, as an immunogen.
• Polyclonal antibodies may also be prepared by conventional means which comprise innoculating a host animal, for example a rat or a rabbit, with a protein or peptide of the invention and recovering immune serum.
• the present invention also provides pharmaceutical compositions containing the LIF proteins or peptides of the invention, or fragments thereof, and antibodies or fragments thereof to the proteins, peptides and fragments thereof for the treatment and/or regulation of conditions, including proliferative diseases, associated with abnormal (eg at an unusually high or low level) and/or aberrant (eg due to a mutation in the gene sequence) expression of LIF in a mammal, including man, or for the treatment of conditions or diseases of a mammal, including man which benefit from an alteration in naturally occuring levels of LIF.
• proliferative diseases associated with abnormal (eg at an unusually high or low level) and/or aberrant (eg due to a mutation in the gene sequence) expression of LIF in a mammal, including man, or for the treatment of conditions or diseases of a mammal, including man which benefit from an alteration in naturally occuring levels of LIF.
• the invention also provides the above protein, peptides and antibodies and fragments thereof, and DNA coding for the said proteins and peptides (or fragments thereof) for the therapy or diagnosis of conditions, including proliferative diseases, associated with abnormal and/or aberrant expression of LIF in a mammal, including man.
• the invention provides peptides of the sequence NH2-met-arg-cys-arg, C-terminal derivatives thereof, eg NH 2 -met-arg-cys-arg-ile-val, and substantially identical sequences as defined above for use as N-terminal signal sequences for naturally occurring molecules lacking such a sequence or any form of recombinant molecules. Such sequences will cause such molecules, eg proteins or peptides, to become associated with the ECM.
• the present invention thus further provides recombinant protein or peptide which comprises, at its N- terminus, the sequence:
• the N terminal sequence comprises: NH2 ⁇ U-arg-X-arg-ile-val-pro-; or NH 2 -U-arg-X-arg-ile-val-pro-leu-leu- where U and X are as defined above, and substantially identical variants thereof. It will be understood that substantially identical sequences are those which do not substantially alter the ability of the N-terminal sequence to direct the protein or peptide to which the sequence is attacted to the ECM.
• the protein or peptide to which the N-terminal sequences of the invention may be attached may, for example, be a hormone, growth factor or cytokine. Attachment of sequences according to the invention to such proteins and peptides will cause the resulting recombinant proteins and peptides to become attached to the ECM. This will enable the action of these proteins and peptides to be localized. It will be appreciated by those of skill in the art that, for example, a recombinant hormone which comprises an N-terminal sequence according to the invention together with all, or an active fragment of, the hormone, will still be considered a hormone for the purposes of the invention. Similarly, other naturally occuring proteins or peptides modified according to the invention will be considered equivalent to the native protein or peptide if their function is substantially unaltered.
• modified proteins or peptides may be used, where appropriate, in the treatment or therapy of the human or animal body.
• the invention further provides recombinant DNA encoding a protein or peptide according to the invention.
• the invention also provides a recombinant replicable expression vector comprising such DNA, a host cell transformed or transfected with such a vector, and a polyclonal or monoclonal antibody directed against the N- terminal of a protein or peptide according to the invention.
• a recombinant replicable expression vector comprising such DNA, a host cell transformed or transfected with such a vector, and a polyclonal or monoclonal antibody directed against the N- terminal of a protein or peptide according to the invention.
• the present invention also provides a peptide of the formula: NH 2 -U-arg-X-arg-ile-val-pro-leu-leu- where U is the residue of a peptide of 20 or fewer amino acids or a covalent bond and X is any amino acid, and substantially identical variants thereof.
• the invention provides a method of causing a protein or peptide to become attached to the ECM of a mammalian cell comprising contacting the ECM with a protein or peptide having an N-terminus of the sequence: NH 2 -U-arg-X-arg-; or NH2-U-arg-X-arg-ile-val-pro-leu-leu- where U is the residue of a peptide of 20 or fewer amino acids or a covalent bond and X is any amino acid, and substantially identical variants thereof.
• the variants of all the sequences mentioned above will retain the motif n -arg-X-arg", where X is as defined above.
• ECM-associated LIF from a murine source.
• ECM-associated LIF from other sources e.g. human or porcine are within the scope of the present invention and could be isolated in an analogous manner.
• EXAMPLE 1 MATERIALS & METHODS CELL CULTURE AND BIOLOGICAL ASSAYS Cell culture was performed in a humidified 7.5% C0 2 atmosphere in DMEM:Ham's F12 (50:50) containing antibiotics, 10 ⁇ M 2- mercaptoethanol and 10% (v/v) foetal bovine serum (selected batches) .
• DIA/LIF Diffusible DIA/LIF was assayed by plating ES cells at an initial density of IO 4 eelIs/ml/16mm well and culturing for 4 days in the presence of experimental samples. The samples were then fixed and stained with Leishman's and inhibition of morphological differentiation was assessed by microscopic inspection (Smith and Hooper, 1987) .
• Feeder layers were prepared by treatment of confluent cultures with mitomycin C (lO ⁇ g/ml) for 2-3 hours, followed by trypsinisation and replating (Martin and Evans, 1975) .
• the collagenous membrane inserts (Transwell ⁇ M , Costar) were transparent with a pore size of 0.4 ⁇ m.
• Cell-free ECM preparations were obtained by hypotonic lysis with 0.02M NH 4 OH (Gospodarowicz, 1984) or by detachment of cells with 0.02% EDTA in PBS (Rheinwald and Green, 1975) .
• a cDNA encompassing the entire murine LIF coding region was obtained by the poly erase chain reaction (PCR) on first strand cDNA generated from Ehrlich ascites RNA from Ehrlich ascites cells. Oligo dT was used to prime reverse transcription of 2.5 ⁇ g of poly A + RNA (cDNA Synthesis Kit, Amersham). After phenol/ chloroform extraction, RNA was hydrolysed by incubation at 4 ⁇ C overnight in 0.25M NaOH, the solution was neutralised by the addition of HC1, and the cDNA was purified by Sephadex G-50 chromatography and ethanol precipitation.
• PCR poly erase chain reaction
• 20ng of the cDNA was used as a template for PCR amplification using the mLIF specific primers LIFI 5 ' ...AAGAATTCCATAATGAAGGTCTTGG...3 ' and LIF2 5• ...AAGAATTCAGTCCATGGTACATTCAAGA...3 ' .
• These primers which both contain EcoRI restriction sites, hybridise to the mLIF cDNA at residues 11 - 28 and 657 - 636 respectively.
• PCR conditions were as recommended by Perkin Elmer-Cetus. Amplification proceeded through a cycle of denaturation for 2 minutes at 94° C, annealing for 2 minutes at 60° C and polymerisation for 3 minutes at 72° C.
• Amplified DNA was digested with EcoRI, purified from agarose gels using Geneclean (BIO 101, San Diego, CA) and cloned into the EcoRI restriction site of pBluescript II KS+ (pDRl) and the expression vector pXMT2 (pDRlO) .
• a probe specific for the 5' end of the mouse LIF open reading frame was constructed by digestion of pDRl with Smal and religation to give pDR2 which lacks mLIF sequences downstream of the mLIF Smal restriction site.
• a cDNA encoding the matrix-associated form of DIA/LIF was constructed in the expression vector pXMT2.
• DNA derived by RACE PCR cloning was removed from pDRlOl by Xhol/Smal digestion and cloned into Xhol/Smal cut pDRlO to produce the plasmid pDRll.
• RNA ANALYSIS High specific activity riboprobes were synthesised by in vitro transcription of murine LIF cDNA fragments cloned into pBluescript II KS+ (Stratagene) . Antisense probes were generated by transcription with T7 polymerase (New England Biolabs) after linearisation of the template by digestion with Hindlll (pDRl, pDR2) or Xhol (pDRlOO, pDRlOl) .
• a riboprobe specific for the 3' end of the mLIF open reading frame was produced by transcription of pDRl with T7 RNA polymerase after linearisation of the plasmid by digestion with Ddel which cuts at nucleotide 353 in the mLIF cDNA.
• the reaction conditions were as described by Krieg and Melton (1988) except that 6.25 ⁇ l of ⁇ - 32 P-UTP (800 Ci/mmol, 40mCi/ml, Amersham) was used in a 15 ⁇ l reaction volume.
• Ribonuclease protection assays were carried out essentially as described by Krieg and Melton (1988) . 15 ⁇ g of cytoplasmic RNA (Edwards et al, 1985) was hybridised with 6 x IO 4 cpm probe (specific activity 3 x 10 s cpm/ ⁇ g) in the absence of added tRNA. Hybridisations were carried out at 45°C for 16-20 hours. RNA hybrids were digested with 40 ⁇ g/ml RNase A (Boehringer) and 2 ⁇ g/ l RNase TI (BRL) at 4°C for 30 minutes. Protected fragments were analysed on a 5% polyacrylamide/ 8M urea sequencing gel which was dried before autoradiography.
• PCR was carried out using a mouse LIF specific oligonucleotide 5' ...TTCTGGTCCCGGGTGATATTGGTCA...3' (mLIF residues 389 - 365) and an anchor oliognucleotide 5 ' ...CCATGGCCTCGAGGGCCCCCCCCCCCC...3 ' .
• the anchor oligonucleotide includes the restriction sites Apal, Ncol, Xhol and Sfil while the mLIF oliogonucleotide includes the unique Smal restriction site at nucleotide 379 in the mLIF open reading frame.
• PCR was carried out in a final volume of 50 ⁇ l containing 7 ⁇ l of the cDNA template and 25 pmol of each oligonucleotide. Reaction conditions were as recommended by Perkin Elmer-Cetus except that a final concentration of 3mM MgCl2 was used. DNA was denatured prior to the addition of Taq polymerase (Perkin Elmer- Cetus) by heating the reaction mixture to 94°C for 5 minutes. Each PCR cycle consisted of denaturation for 2 minutes at 94°C, annealing for 2 minutes at 55°C and elongation for 3 minutes at 72°C.
• Taq polymerase Perkin Elmer- Cetus
• Products of this reaction were analysed by Southern blot hybridisation using probes derived from pDRl, pDR2 or the LIFI oligonucleotide. After the final elongation (30 minutes at 72°C) samples were ethanol precipitated, digested with Smal and Xhol, and analysed by agarose gel electrophoresis. DNA was purified from agarose gels using Geneclean and cloned into Xhol/Smal digested pBluescript II KS+ (Stratagene) . Suitable recombinant plasmids were purified by the alkaline lysis method. The clone corresponding to the diffusible form of DIA/LIF was designated pDRlOO and that corresponding to the matrix-associated form of DIA/LIF was designated pDRlOl.
• the maintainance of ES cells in an undifferentiated state can be achieved by growing the cells in medium conditioned by preincubation with BRL cells. This results from secretion of soluble DIA/LIF protein into the medium by BRL cells.
• medium conditioned by different feeder cell lines may be unable to prevent or be relatively inefficient at preventing ES cell differentiation. This suggests that at least part of the DIA/LIF activity associated with feeder cells is not diffusible but is localised to the cell in some way. This could involve attachment to either the feeder cell membrane or extracellular matrix.
• 10T1/2 cells were removed from the culture by treatment with either ammonium hydroxide, which causes osmotic lysis, or EDTA, which results in the detachment of intact cells from the extracellular matrix. Since cells grow above the secreted extracellular matrix in vitro. the effect of these treatments was to yield a substrate composed almost exclusively of extracellular matrix. ES cells seeded onto this substrate were maintained in an undifferentiated state ( Figure 1, iv, v) as judged by their overt morphology or the presence of the SSEA-1 antigen.
• the probe used for Northern blots and the initial ribonuclease protections was derived from a cDNA fragment that contained the entire murine LIF (mLIF) open reading frame as defined by Gearing et al. (1987) .
• This cDNA was obtained by PCR on RNA from Ehrlich ascites cells which had previously been shown to secrete soluble DIA/LIF.
• To test for biological activity the mLIF fragment was cloned into the unique EcoRI restriction site of pXMT2, an expression vector which contains the SV40 origin of replication.
• RNA from Ehrlich ascites cells was also screened for DIA/LIF expression by ribonuclease protection.
• Two transcripts were detected by this technique ( Figure 3A) .
• the similar sizes of the two protected bands argues that they are identical throughout the majority of the mLIF coding region since the ribonuclease protection technique identifies differences between probe and substrate by cleavage at the region of dissimilarity.
• riboprobes specific for either end of the open reading frame were produced. Of these, only the 5* probe protected two different transcripts (Figure 3B) .
• the identity of the two transcripts was established by analysis of DIA/LIF transcription in two embryonic fibroblast feeder cell lines, 10T1/2 and STO, as well as in EA and PYS-2 (a parietal yolk sac cell line) ( Figure 4A) .
• the upper transcript, D corresponds to the secreted and diffusible form of the DIA/LIF protein since it shares complete ho ology with the riboprobe throughout the LIF coding region. This identification is consistent with the presence of significant levels of this transcript in RNA from Ehrlich ascites and STO cells which secrete DIA/LIF activity but not in 10T1/2 cells which do not secrete DIA/LIF activity.
• the identity of the lower transcript, M could be deduced from a comparison of the ribonuclease protection pattern and known biological properties of the cell lines ( Figure 4B) .
• the appearance of this transcript in both STO and 10T1/2 cells suggests that it encodes the matrix-associated form of the DIA/LIF protein which allows these cell lines to act as ES cell feeders. This correlation is most apparent in I0T1/2 cells where only the lower transcript is present at significant levels and where only the matrix-associated foi of the protein can be detected.
• oligonucleotide complementary to residues 500 - 484 of the mLIF cDNA was used to prime cDNA synthesis from Ehrlich ascites total RNA.
• the cDNA was tailed with dGTP residues at the 3' end using terminal deoxynucleotide transferase and the resultant single stranded DNA was used as a template for PCR using an oligonucleotide complementary to residues 389-365 of the mLIF sequence and an anchor oligonucleotide,
• the internal oligonucleotide was chosen both to increase the specificity of the PCR reaction and because it incorporated the unique Smal restriction site of the mLIF open reading frame. This was used later in reconstruction of the complete open reading frame.
• THE PCR did not yield the expected two bands but rather a smear of material between 400 and 600 nucleotides which was homologous to mLIF as ascertained by Southern blot.
• the products of an entire PCR reaction were digested with Xhol and Smal and separated on a 2% agarose gel.
• Zones of DNA corresponding to mLIF sequences were excised from the gel and cloned into Xhol/Smal cut pBluescript II KS+S. Recombinant colonies were screened for the size of the inserted fragment and the largest inserts among two distinct size classes were subcloned into M13 and sequenced. The sequences of these two clones upstream of residue 37 are shown in Figure 5 with the deduced amino acid sequences of the respective proteins.
• the larger cDNA (pDRlOO) encodes the diffusible form of the DIA/LIF protein.
• the sequence shown in Figure 5 is identical to that of Gearing et al (1988) but extends 122 nucleotides upstream of the ATG initiation codon.
• the smaller clone (pDRlOl) diverges from the known mLIF sequence at precisely the point deduced to be the start of the second exon and is therefore a good candidate to encode the matrix-associated form of the DIA/LIF protein.
• the amino acid sequence of the protein encoded by this cDNA differs from that of the diffusible from of DIA/LIF in that the first part of the leader sequence.
• MKVLAAG is replaced with the sequence MRCR.
• the identity of the ATG initiation codon is confirmed by the presence of an in-frame termination codon, UAG, 30 nucleotides upstream of the ATG.
• the matrix-associated protein retains the signal sequence of the diffusible mLIF protein between amino acid 8 and the cleavage site after residue 23.
• This leader sequence contains the hydrophobic core sequence that is required for translocation of the LIF proteins across the cell membrane.
• the sequence of the pDRlOl cDNA extends 31 nucleotides upstream of the ATG. This may represent the 5• end of the RNA encoding the matrix-associated DIA/LIF protein.
• riboprobes derived from pDRlOO and pDRlOl were used to protect cytoplasmic RNA from Ehrlich ascites cells.
• Antisense riboprobes extending from the 5' ends of the pDRlOO and pDRlOl cDNAs to the Smal restriction site within the mLIF open reading frame were produced by m vitro transcription of these clones.
• the protection patterns obtained with these riboprobes were compared with the known pattern generated by the 5' riboprobe derived from pDR2.

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