AU609128B2 - Leukaemia-inhibitory factor - Google Patents

Description

a L AU-Al-15907/ 88 Pur WORLD Ir INTERNATIONAL APPLICATION PUBLISHE] (51) International Patent Classification 4: (11) International Publication Number: WO 88/ 07548 C07K 13/00, 15/12, 15/14 C12N 1/18, 15/00, C12P 19/34 Al C 12P 21/00, 21/02, C07H 21/04 (Cl2P 19:34, C12R 1:91) (43) International '4b.,ication Date-, 6 October 1988 (06.10.88) (C12P 21/02, C12R 1:865) (21) International Application Number: PCT/AU88/00093 8 West End Road, Warrandyte, VIC 3113 KING, Julie, Ann [AU/AU]; 121 Springvale Road, Nunawading, VIC (22) International Filing Date: 31 March 1988 (31.03.88) 3131 METCALF, Donald [AU/AU]; 268 Union Road, Balwyn, VIC 3103 NICE, Edouard, Collins [GB/AU]; (31) Priority Application Numbers: P1 1209 9 Odessa Street, St. Kilda, VIC 3182 NICOLA, Nicos, PI 3317 Anthony [AU/AU]; 59 Queen Street, Regent, VIC 3073 PI14903 SIMPSON, Richard, John [AU/AU]; 42 Stanley PI 6005 Street, Richmond, VIC 3121 WILLSON, Tracy, Ann [AU/AU]; 26 Fortuna Avenue, North Balwyn, VIC 3104 (32) Priority Dates: 2 April 1987 (02.04.87) (AU).

24 July 1987 (24.07.87) October 1987 (15.10.87) (74) Agents: SLATTERY, John, Michael et al.; Davies Collison, 21 December 1987 (21.12.87) 1 Little Collins Street, Melbourne, VIC 3000 (AU).

(33) Priority Country: AU (81) Designated States: AU, BR, DK, FI, HU, JP, KR, NO, US.

(71) Applicant (for all designated States except US): AMRAD COR- Published PORATION LIMITED [AU/AU]; Unit 6, 663 Victoria With international search report.

Street, Abbotsford, VIC 3067 Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amend- (72) Inventors; and ments.

Inventors/Applicants (for US only) GEARING, David, Paul .P [GB/AU]; 134 Miller Street, North Fitzroy, VIC 3068 D. E~ 1D C 1988 GOUGH, Nicholas, Martin [GB/AU]; 20 Rangeview Grove, North Balwyn, VIC 3104 HILTON, Douglas, James AUSTRALIAN

[AU/AU];

2 NOV 1988 PATENT OFFICE (54) Title: LEUKAEMIA-INHIBITORY FACTOR W-omj (57) Abstract HlsTrpLysHisGlyAlalySerProLeuProlleThrProVatAsnAI 17 CACTOGAAACACOOOOCAOOOi~AOCCCTCTTCCCATCACCCCTOiTAAATOiC So A leukaemia-inhibitory factor (LIF) aThr CysA taIt eA rgH IsPro CysH 1sG IyAsnLeuMet AsnG In I IeL 3t4 is disclosed, together with a method of CACCTOTOCCATACO3CCACCCATGCCACOOCAACCTCATOAACCAOATCA 100 preparation of LIF in essentially pure form. Nucleotide and amino acid, se- ysAsfOlfLeuAbOalfLeuAsnySerAaAsnAloLeuPhelleSer quences are disclosed, together with rec- AOAATCAACTOOCACAOiCTCAATGOCAO COCCAATOCTCTCTTCATTTCC 150 ombiant NA oleclesand ostcell Ty rThrA IaGInG lyG IuPr oPheProAsnAsnVal 01uLysLeuCy 67 for production of polypeptides having LIF TATTACACAOCTCAAGOAGAGCCGTTTCC CAACAACOTGGAAAAOCTATO 200 activity. sA1 aProAsnMe tThr AspPheProSerPheHi sG IyAsnG IyThroluWt 84 TGCGCCTAACATOACAOACTTCCCATCTTTCCATOCCAGOOACAGAGA 250 YsThrLysLeuVa(ltuLeuTyrAr MetVaLAlaT rLeuSerAlaSer 100 This document contains the AOACCAAGTTGOTG-7:TGTATCG ATGGTCGCATZ CCTOAGCGCCTCC 300 amcendmenis made under ecio 4 ad s orec or LeuThrAsnl~eThrArgAsp~irlLysvaL-euAsnProThrAt'ValSe 117 ,Secion49 ad i corectforCTOACCAATATCACCCGGOACCAOAAGGTCCTGAACCCCACTGCCOTGAG 350 rLeu~jlnVatLysLeuAsnAlarhrlI~pa~tr~oye~u 3 CCTCCAGGTCAAOCTCMATOCTACTATAOACOTCATGAGGGC CCTCCTCA 400 erAsnVatLeuCysArgLeuCysAsnLysTyVrArgvaityHisValAsp 150 GCAATGTGCTTTGCCOTCTOTOiCAACAAGTACCOTTOOCCOOA 450 Val ProProValProAspHisSerAspLysoi[UAlaPheGnArgLysLy 167 COTGCCACCTGTCCCC OACCACTCTGACAAAGAAGCCTTCCAAMOOAAAAA 500 sLeu~lyCysGLnLeuLeu~lylhrTyrLysaln 178 OTTGOOOTTOCCAOCTTCTOOOOGACATACAAOCMOG 535 WO 88/07548 PCT/AU88/00093 1 LEUKAEMIA INHIBITORY FACTOR FIELD OF THE INVENTION This invention relates to Leukaemia Inhibitory Factor (LIF), to the production of LIF in substantially pure form, and to the cloning and expression of recombinant LIF.

BACKGROUND OF THE INVENTION Mature blood cells are produced by the clonal proliferation and concomitant differentiation of immature precursor cells (Metcalf,D. (1984) The Hemopoietic Colony Stimulating Factors, Elsevier, Amsterdam.). For normal haemopoietic cells, the two processes of proliferation and differentiation are tightly coupled so that the short-lived mature cells are continually replenished. At 15 least four biochemically distinct growth factors, the colony-stimulating factors (CSFs), have been shown in vitro to stimulate the proliferation and differentiation of precursor cells of granulocytes and macrophages: G-CSF, M-CSF, GM-CSF and Multi-CSF (IL-3) (see Metcalf, D.

2 i i:- WO 88107548 PCT/AU88/00093 2

F

(1987), Proc.R.Soc.B, 230, 389-423, for review).

In contrast to normal cells, leukaemic myeloid cells are characterized by an uncoupling of proliferation and differentiation so that immature progenitor cells accumulate, retain their proliferative capacity and fail to differentiate. There has been considerable controversy concerning the nature of biological factors that are able to induce the differentiation of myeloid leukaemic cells in vitro and whether certain factors are capable of inducing differentiation in the absence of proliferation.

Indeed, it has been proposed that the events of proliferation and differentiation are necessarily mediated by different factors (Sachs, L. (1982), J.Cell.Physiol.Suppl., 1, 151-164). On the one hand, two groups have described and purified an activity (MGI-2 or D-factor) capable of inducing the differentiation of the murine myeloid leukaemic cell line Ml, that does not stimulate the proliferation of normal progenitor'cells (Lipton, J.H. and Sachs, L. (1981), Biochem.Biophys. Acta.

673, 552-569; Tomida, Yamamoto-Yamaguchi, and Hozumi, M. (1984) J.Biol.Chem. 259, 10978-10982). On the other hand, the present inventors have previously shown that one of the CSFs, G-CSF, is a strong differentiation-inducing stimulus for the murine myeloid leukaemic cell line, WEHI-3B D as well as being a proliferative and differentiative stimulus for normal cells (Nicola,N.A., Metcalf, Matsumoto, M. and Johnson, G.R. (1983), J.Biol.Chem. 258, 9017-9023).

Moreover, Tomida, et.al. (Tomida, Yamamoto-Yamaguchi, Hozumi, Okabe, T. and Takaka, F. (1986), FEBS Lett., 207, 271-275) have shown that recombinant G-CSF is also able to induce the macrophage differentiation of Ml cells. The relationship between these factors has been a matter of debate. The situation was further complicated S WO 88/07548 PCT/AU88/00093 3 by the finding that tumour necrosis factor a(TNFa) is capable of stimulating the differentiation of the human myeloblastic cell line ML-1 (Takeda,K., Iwamoto, S., Sugimoto, Takuma,T., Kawatani,N., Noda,M., Masaki,A., Morise,H., Arimura,H. and Konno,K. (1986) Nature 323, 338-340).

In an attempt to resolve the discrepancies between the data of these groups, the present inventors have biochemically fractionated the medium conditioned by Krebs II ascites tumour cells used in a number of previous studies and shown it to contain not only authentic G-CSF and GM-CSF, active on normal progenitor cells as well as WEHI-3B D cells, but also two biochemically distinct, but functionally similar, factors capable of inducing the differentiation of M1 cells. These latter factors have been termed leukaemia inhibitory factor (LIF)-A and LIF-B because of their ability to suppress the proliferation of M1 leukaemic cells in vitro and it has been shown that they do not induce the differentiation of WEHI-3B D cells and do not stimulate the proliferation of normal granulocyte/macrophage progenitor cells.

SUMMARY OF THE INVENTION LIF, in accordance with the present invention, is defined as a molecule that has the following two properties: it has the ability to suppress the proliferation of myeloid leukaemia cells such as Ml cells, with associated differentiation of the leukaemic cells; and it will compete with a molecule having the defined V sequence of murine LIF or human LIF hereein for binding to specific cellular receptors on Ml cells or murine or human macrophages. LIF, has the potential for use as a therapeutic non-proliferative agent for suppressing some forms of myeloid leukaemia as well as a reagent for modifying macrophage function and other responses to 1 -4-

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The term "polypeptide having LIF activity" as used herein denotes a polypeptide or glycopeptide having one or more of the properties of LIF as defined above and more specifically which is bound by cellular receptors which bind LIF and which binding is competitively inhibited by native murine or human LIF, including but not restricted to polypeptides having an amino acid sequence which is fully or partially homologous with the amino acid sequence of either murine or human LIF as disclosed herein. This term also includes polypeptides which are fully or partially homologous with a portion only of the amino acid sequence of murine or human LIF provided that the polypeptide has the properties of LIF as defined above. This term further includes polypeptides or glycopolypeptides produced by expression in a host cell which are inactive when expressed but which are processed by the host cell to yield active molecules, as well as polypeptides or glycopolypeptides which are inactive when expressed but which are selectively cleavable in vitro or in vivo to yield active molecules.

Murine LIF is a molecule having the following biological properties: induction of macrophage differentiation in cells of the murine myeloid leukaemic cell line Ml with loss of proliferative capacity and death of the clonogenic leukaemic cells, an action potentiated by G-CSF; selective binding to high affinity receptors on Ml cells and on normal murine monocytes and macrophages from the peritoneal cavity, spleen and bone marrow, with the number of receptors increasing with macrophage maturation or functional activation, but not to granulocytic, erythroid or lymphoid cell from these tissues; specific binding of 125 I-LIF to high affinity

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I I i I a j S WO 88/07548 PCT/AU88/00093 receptors is not competed for by G-CSF, GM-CSF, Multi-CSF (interleukin-3), M-CSF, interleukins 1,2, 4 or 6, endotoxin or a variety of other growth factors, but is competed for by unlabeled

LIF.

elevation by bacterial endotoxin in the serum of normal or athymic mice, but not in endotoxin-resistant C3H/HeJ mice; production by various tissues including lung, salivary gland, peritoneal cells and bone shaft; reduction of the survival time in vitro of normal granulocyte-macrophage progenitor cells when grown in the absence of CSF;; an inability to suppress proliferation or induce differentiation of WEHI-3B D murine myeloid leukaemic cells, murine myeloid cells transformed to leukaemogenicity by infection with retroviruses expressing GM-CSF or Multi-CSF, the murine leukaemic cell lines WEHI265 and WR19; no ability to stimulate the proliferation of normal progenitor cells of the granulocyte, macrophage, eosinophil, megakaryocyte, erythroid and mast cell lineages, and an inability to suppress the clonal proliferation or alter the quantitative responsiveness to stimulation by CSFs in vitro of progenitors of normal granulocytes, macrophages, megakaryocytes, eosinophils or natural cytotoxic cells or the proliferation of cells of the continuous cell lines 32CD1.13 and FDCP-1; an inability to compete with the iodinated derivatives of granulocyte colony-stimulating factor (G-CSF) for binding to specific cellular receptors despite the ability of G-CSF to induce I 'r WO 88/07548 PCT/AU88/00093

I

differentiation in Ml and WEHI-3B D murine myeloid leukaemic cells and to potentiate the action of LIF on Ml cells; an inability of the action of LIF to be inhibited by antisera specifically raised against tumour necrosis factor (TNF); transcripts for murine LIF are present constitutively in the cytoplasm of LB3 and E9.D4 T cells, are not induced by the lectin concanavalin A in these cells, and are present in a number of other cell types; Murine LIF has also been determined to have the following properties: (1) (m) (n) (o)

(P)

a single subunit glycoprotein with molecular weight of 58,000± 5,000 as determined by electrophoresis in 8-25% gradient polyacrylamide gels containing sodium dodecyl sulphate with or without reducing agent (2-mercaptoethanol or dithiothreitol), and a molecular weight of 23,000 t 5,000 after treatment with the endoglycosidase, N-glycanase; an isoelectric point between 8.6 and 9.3; the primary amino acid sequence detailed in Figures 10, 11 and 15 herein; is encoded by the nucleotide sequence detailed in Figures 10, 11 and 15 herein; a specific activity of 1-2 x 108 units/mg (where 50 units are defined as the amount of LIF which in one millilitre induces a 50% reduction in the clone formation by murine M1 myeloid leukaemic cells); is encoded by a unique gene on murine chromosome 11 (as determined by analysis of a panel of mouse/Chinese hamster ovary somatic cell hybrids) (q) WO 88/07548: PCT/AU88/00093 bounded by restriction endonuclease cleavage sites as illustrated in Figure 19 herein; is homologous to a human gene sequence bounded in chromosomal DNA by restriction endonuclease cleavage sites as illustrated in Figure 27 herein.

Human LIF is a molecule having the following biological properties of the native and/or yeast derived recombinant product: induction of macrophage differentiation in cells of the murine myeloid leukaemia cell line Ml; with loss of proliferative capacity and death of the clonogenic leukaemia cells; no ability to stimulate the proliferation of normal human progenitor cells of the granulocyte, macrophage, eosinophil and erythroid lineages; an ability in combination with G-CSF to partially suppress the proliferation of cells of the human leukaemic cell line U937 and in combination with GM-CSF the proliferation of the human leukaemic cell lines U937 and binds specifically to murine LIF receptors on Ml cells and murine macrophages and competes completely for the binding of murine 125

I-LIF

to such cells.

binds to specific cellular receptors on the human hepatoma cell line Hep-2G.

Human LIF has also been determined to have the following properties: is identical to murine LIF at approximately of amino acid residues in the mature protein; the primary amino acid sequence detailed in Figures 25, 26 and 29 herein; is encoded by a unique gene bounded in chromosomal DNA by restriction endonuclease k B i WO 88/07548 PCT/AU88/00093 8 I cleavage sites as illustrated in Figure 27 herein; has as a portion of the sequence of its gene, the nucleotide sequence detailed in Figure 25 herein.

In a first aspect of the present invention, there is provided leukaemia-inhibitory factor (LIF), in essentially pure form. This invention also provides methods for the production of essentially pure LIF, particularly for the production of essentially pure murine LIF by purification from Krebs II ascites tumour cells, and of essentially pure human LIF by purification from the human bladder carcinoma cell line 5637 (ATCC No. HTB 9).

Essentially pure LIF may also be produced by host cells, such as yeast cells, mammalian cells and E.coli, containing recombinant DNA molecules coding for the amino acid sequence of LIF, or part thereof The references herein to "in essentially pure form" denote a form of polypeptide or glycopolypeptide in which at least 90% of the polypeptide or glycopolypeptide appear as a single band when electrophoresed on an appropriate sodium dodecyl sulphate polyacrylamide gel, said band being coincident with LIF activity.

In another aspect there is provided a method for isolation of recombinant DNA clones containing nucleotide sequences encoding the LIF protein, either completely or in part. The present invention also relates to a nucleotide sequence which has the capacity to encode the unique sequence of amino acids determined to be characteristic of LIF and to furthermore allow the complete amino acid sequence of LIF to be inferred.

0 In yet another aspect, this invention extends to recombinant DNA molecules containing the aforementioned nucleotide sequences encoding LIF (or substantially similar analogues thereof), either completely or in part, in a form in which said nucleotide sequences are able to 5 ic: 4 WO 88/07548 PCT/AU88/00093 9 direct the synthesis and production of LIF, either completely or in part. This aspect of the invention also extends to cloning vectors (such as plasmids) and host cells having such recombinant DNA molecules inserted therein. Furthermore, the invention also extends to synthetic LIF, either complete or in part, or substantially similar analogues thereof, produced by expression of such recombinant DNA molecules or by peptide synthesis.

DETAILED DESCRIPTION OF THE INVENTION In the present investigation of LIF, in particular with a view to providing alternative sources of this glycoprotein of both murine and human origin to enable use in clinical and other applications, murine LIF has been purified from Krebs II ascites tumour cells by an efficient purification procedure and has been subjected to partial amino acid sequence analysis as a first step towards chemical or biosynthetic production of this factor.

125 LIF radioactively derivatized with 125 has been used in a receptor competition assay to identify cells and tissues of murine and human origin that synthesize and produce LIF. As a result of this survey, a recombinant DNA library of DNA copies of mRNA from one of 'these sources has been screened and murine LIF-encoding clones identified by hybridization with radioactivelylabelled oligonucleotides encoding portions of the previously determined murine LIF amino acid sequence, and the aforementioned murine LIF cDNA clones subjected to nucleotide sequence analysis. Moreover, the cloned murine LIF encoding sequence has been used as a hybridization probe to identify a human gene encoding a homologue of murine LIF and hybridization conditions established under which said cross-species hybridization can be effectively performed. As a result, recombinant DNA clones containing k WO 8 /7 4 C i P...8 WO 88/07548 PCr/AU88/00093 DNA sequences encoding the human homologue of murine LIF have been identified.

As a result of this construction of LIF- encoding sequences, recombinant DNA molecules have been constructed that use cloned murine or human LIF- encoding DNA sequences to direct the synthesis of clonally pure LIF, either completely or in part, for example in cultured mammalian cells, in yeast or in bacteria. This invention thus extends to substantially pure LIF produced in this manner.

In one particular embodiment, this invention relates to the expression of the cloned human and murine LIF genes in yeast cells, in fibroblasts and in E.coli, and to the modification of the cloned gene so that it may be so expressed, as well as to the establishment of the biological and biochemical properties of recombinant human and murine LIF.

In this embodiment also, the present invention relates to recombinant DNA molecules containing nucleotide sequences encoding human or murine LIF (or substantially similar analogues thereof), either completely or in part, in a form in which said nucleotide sequences are able to direct the synthesis and production of human or murine LIF in yeast cells, fibroblasts or E.coli, either completely or in part. This invention also provides cloning vectors (such as plasmids) and host cells having such recombinant DNA molecules inserted therein. Furthermore, the invention extends to synthetic human or murine LIF, either complete or in part, or substantially similar homologues thereof, produced by expression of such recombinant DNA molecules in yeast cells. In one embodiment of the present work relating to LIF, the human and murine LIF gene sequences have been modified and installed in a yeast expression vector, YEpsecl. Yeast cells have been i WO 88/07548 PCT /AU88/00093 transformed with the resulting recombinants and the medium conditioned by yeast cells so transformed has been shown to contain a factor with biological properties analogous to those of native human and murine LIF.

In view of the potential of LIF for use in the treatment of patients with some forms of myeloid leukaemia and patients with certain infections, the present invention also extends to pharmaceutical compositions comprising LIF, particularly human LIF, either completely or in part, produced for example using cloned LIF-encoding DNA sequences or by chemical synthesis, and to pharmaceutical compositions of analogues of LIF, for example produced by chemical synthesis or derived by mutagenesis of aforesaid cloned LIF-encoding DNA sequences. The pharmaceutical compositions may also contain at least one other biological regulator of blood cells, such as G-CSF or GM-CSF. Furthermore, the invention also extends to diagnostic reagents for use in detecting genetic rearrangements, alterations or lesions associated with the human LIF gene in diseases of blood cell formation, including leukaemia and congenital diseases associated with susceptibility to infection.

EXAMPLE 1 The accompanying Figures 1 to 5 relate to various steps of the purification method described below, showing where LIF is pooled in each fractionation step and showing evidence of its purity. In the Figures: Figure i: relates to Step 2 of the purification of LIF on DEAE-Sepharose CL-6B. The bar indicates fractions that are pooled for Step 3.

Figure 2: relates to Step 3 of the purification of LIF on Lentil lectin Sepharose 4B. The bar indicates fractions that are pooled for Step 4.

r I /3 "I WO 88/07548 PCr/AU88/O093 12 Figure 3: relates to Step 4 of the purification of LIF on CM-Sepharose CL-6B. The bar indicates fractions that are pooled for Step Figure 4: relates to Step 5 of the purification of LIF on a fatty acid analysis HPLC column. Fractions from 39.6 to 41.3% acetonitrile are pooled.

Figure 5: shows the molecular weight and purity of purified LIF. The upper panel shows the migration of protein standards (93,000; 67,000; 43,000; 30,000; 20,000 and 14,000 molecular weight) and purified LIF (two preparations) on 8-25% polyacrylamide SDS gel. The protein and biological activity are both associated with a protein of molecular weight 58,000.

Step 1 Krebs II tumour cells (1 x 66) are injected intraperitoneally into C57B16/6fj/ WEHI mice and after 7 days the mice are sacrificed and the peritoneal fluid removed. The cells are centrifuged, resuspended at 5 x 6cells/ml in Dulbecco's modified Eagle's medium containing E.coli lipopolysaccharide (200ng/ml) and incubated for 24 hrs at 37°C in a humidified incubator containing 10% CO 2 in air. The cells are again centrifuged, the conditioned medium removed, made up to 0.02% sodium azide and stored at 4°C. Thirty-six litres of conditioned medium are concentrated to 100ml using a HIP10-8 hollow fibre cartridge in an Amicon DC2A concentrator, exchanged into 10mM Tris-HCl buffer containing ImM phenylmethyl sulphonyl fluoride (PMSF), sodium azide w/v) and Tween 20 (0.02%v/v) and re-concentrated to 20ml over a YM-10 membrane in an Amicon stirred cell.

Step 2. The concentrate is applied to a column (2.5 x of DEAE-Sepharose CL-6B (Pharmacia) equilibrated in i ii b, i i -r 1 1 1 1 WO 88/07548 PCT/AU88/00093 13 the same buffer and eluted with 200ml of equilibration buffer followed by a 400ml linear gradient to 0.3M NaC1 in the same buffer. The flow rate is 25ml/hr and 5.8ml fractions are collected and assayed. (See Fig.l) Step 3. The fractions of protein which failed to bind to the gel during the pre-gradient step and which contained activity on murine M1 cells are pooled, concentrated over a YM-10 membrane, exchanged into 100mM sodium acetate buffer pH6.0 containing ImM MgC 2 ImM PMSF, 0.2% sodium azide and 0.02% Tween 20 and applied to a column (1 x 4cm) of Lentil lectin-sepharose 4B (Pharmacia) equilibrated in the same buffer. The column is eluted with 25ml of equilibration buffer and then a 100ml linear gradient to 0.5 M d-methyl-D-mannopyranoside in the same buffer at a flow rate of 10ml/hr. Fractions of 3ml are collected and assayed. (See Fig.2) Step 4. The fractions of proteins which bind to the gel and are eluted by the sugar and have activity on Ml cells are pooled, concentrated and exchanged into 100mM sodium acetate buffer pH5.0- containing ImM PMSF, 0.02% sodium azide and 0.02% Tween 20 and applied to a column (1.0 x of CM-Sepharose CL-6B (Pharmacia) equilibrated in S the same buffer. The column is eluted with equilibration buffer and then a 100ml linear gradient to NaC1 in the same buffer and then by 25ml of 1.OM NaCl adjusted to pHlO with NaOH. The flow rate is 2.0 ml/hr and 3.0ml fractions are collected. (see Fig.3) t Step 5. The fractions of proteins which bind to the gel and are eluted by the NaCl gradient and have activity on Ml cells are pooled, concentrated to 2ml over a membrane and exchanged into 20mM ammonium acetate buffer WO 88/07548 PCT/AU88/00093 14 containing 0.02% Tween 20 and then filtered through a 0.45p Millipore filter. The pool is loaded into the injector of a high performance liquid chromotography system (Beckman) equipped with a Waters fatty acid analysis column and dual pumps with gradient programmer.

The column is equilibrated in water containing 0.1% (v/v) trifluoroacetic acid (TFA), the sample is injected, and the column is then eluted with a 5min linear gradient to 34.8% acetonitrile in water and 0.1% TFA and then eluted with a 50min linear gradient to 49.8% (v/v) acetonitrile in water and 0.1% TFA. The flow rate is 1 ml/min and Iml fractions are collected into polypropylene tubes containing 50pl of 100mM ammonium bicarbonate and 0.4% Tween 20. (see Fig.4) The fractions with activity on Ml cells from step consisted of two overlapping ultraviolent- absorbing peaks. Each peak was associated with activity on Ml cells and each peak corresponded to a single protein band of Mr 58,000 on sodium dodecyl sulphate polyacrylamide gels using silver staining. In each case, the protein band of Mr 58,000 contained the biological activity on Ml cells as assessed by cutting a parallel track on the gel (also loaded with LIF) into 1mm strips, extracting the protein from the gel strips and assaying the extracted protein for its ability to suppress proliferation and induce differentiation in Ml cells in semi-solid agar cultures.

(see EXAMPLE 2 The following Example sets out the steps used in obtaining the N-terminal amino acid sequence and amino acid sequences of various peptides generated by proteolytic cleavage with trypsin or Staphylococcal V8 protease.

S WO 88/07548 PCT/AU88/00093 LIF purified by the procedure of Example 1 was applied to a microbore (2mm) column packed with Brownlee RP300 beads (C8) and eluted on a HPLC system with a linear gradient from 0-60% acetonitrile in 0.1% trifluoroacetic acid. The protein eluted as a single peak in a volumn of 100pl. This was applied to a glass fibre disc and allowed to dry and then placed in the sequencing chamber of an Applied Biosystem (Model 470A) gas phase protein sequencer. The protein was sequenced by Edman chemistry and the phenylthiohydantoin derivatives of the individual amino acids identified by HPLC on an Applied Biosystems 120A PTH analyser.

A separate aliquot of LIF (20pg) also purified by the aforementioned procedure, was diluted to 2ml in 6m guanidine hydrochloride, 0.2M Tris-HCL buffer pH8.5, 2mM dithiothreitol and incubated at 37 0 C for 2 hrs. Then a molar excess of iodoacetic acid was added and the solution incubated at 37 0 C for a further 15min. The solution was applied to the same microbore HPLC column and eluted as described above. The reduced and carboxymethylated derivative of LIF (RCM-LIF) eluted 3min later from the column than the untreated LIF and RCM-LIF (200pl) was diluted to lml with 0.1 M Tris-HCL buffer containing 0.02% Tween 20, ImM CaCl 2 and 2pg of TPCK-treated trypsin. This incubation was allowed to proceed for 18hr at 37 0 C and the mixture was again applied to the microbore column and eluted in the same way as described above. Individual peptides appeared as UVabsorbing peaks, and were collected individually. Some of these peptides were sequenced directly as described above while others were repurified on the same column but using an 0-60% acetonitrile gradient in 0.9% NaC1 pH6.0 before being sequenced.

A second aliquot of RCM-LIF (200pl=10pg) WO 88/07548 PCT/AU88/0009 16 was diluted to imi with 0.05M sodium phosphate buffer, pH 7.8 containing 0.002M EDTA and 0.02% Tween 20, and was digested with 2pq' of Staphylococcus aureus V8 protease for 18 hours at 37 0 C. The reaction mixture was applied to the same microbore column and eluted as described above.

The 26 amino acids defining LIF from the amino terminal in sequence were found to be: N-Pro-Leu-Pro-I le-Thr-Pro-Val-X-Ala-Thr-X-Al a-I le- Arg-His-Pro-Cys-His-Gly-Asn-Leu-Met-Asn-i'2n-Ile-Lys The amino acid sequences of several tryptic peptides were found to be: 1. His-Pro-Cys--His-Gly-Asn-Leu--Met-Asn-Gln-Ile-Lys 2. Met-Val-'Ala-Tyr 3. Gly-Leu-Leu-Ser-Asn-Val-Leu-Cys 4. Leu-Gly-Cys-Gln-Leu-Leu-Gly-Thr-Tyr-Lys Val-Leu-Asn-Pro-Thr-Ala-Val-Ser-Leu-Gln-Val-Lys 6. Asn-Gln-Leu-Ala-Gln-Leu-X-Gly-Ser-Ala--Asn-Ala-Leu- Phe-Leu-Val--Glu-Leu-Tyr 7. Leu-Val--Ile-Ser--Tyr 8. Val-Gly-Hi s-Val-Asp-Val-Pro-Va 1-Pro-Asp--His-Ser- Asp-Lys--Glu-Ala-Phe-Gln.

9. Leu-X-Ala-Thr-Ile--Asp-Val-Met-Arg.

Gln-Val-Ile-Ser-Val-Val-X-Gln-Ala-Phe.

The amino acid sequence of a peptide generated by digestion of LIF with the V8 protease was: Al a-Phe-G ln-Arg-Lys-Lys-Leu-Gly-Cys -G ln-Leu-Leu-G ly-Thr-Tyr- Lys-Gln-Val-Ile-Ser-Val--Val-Val-Gln-Ala-Phe.

EXAMPLE 3 The following Example sets out the steps used to obtain radioactively-derivatized LIF and to identify sources of LIF by a receptor competition assay.

The accompanying Figure 6 relates to Example 3: Binding of radioiodinated-pure LIF (approximately 2 x WO 88/07548 PCT/AU88/00093 17 105 cpm/ng) to Ml myeloid leukaemia cells at 0°C. Ml cells (5 x 106) were incubated with 125 I-LIF (4 x 105 cpm) with or without competing factors for 2 hours after which bound radioactivity was separated from unbound radioactivity by centrifugation through foetal calf serum. A. Titration of unlabelled pure LIF and pure Multi-CSF, GM-CSF, G-CSF or M-CSF as competing factors at the indicated dilutions (10pl of each dilution added to a final volume of The highest concentrations of each were 10 units/ml; 4 4 4 x 10 units/ml; 3 x 104 units/ml; 5 x 10 units/ml; 4 x 104 units/ml; respectively. B.

Ability of pure LIF or lipopolysaccharide (LPS) or concentrated (4-8-fold) media conditioned by various cells or serum from endotoxin-injected mice to compete for the 125 binding of I-LIF to Ml cells as above.

LIF, purified from Krebs II ascites cell conditioned medium as described in Example 1, was 125 radioactively labelled with I on tyrosine residues as described previously (Nicola, N.A. and Metcalf, D., 125 J.Cell.Physiol. 128:180-188, 1986) producing T-LIF with a specific radioactivity of approximately 2 x 105 cpm/ng. 125 I-LIF bound specifically to Ml myeloid leukaemic cells as well as murine adult bone marrow, spleen, thymus and peritoneal exudate cells. Cell 125 autoradiographic analysis indicated that I-LIF bound specifically only to macrophages and their precursor cells, with receptor numbers increasing with cell maturation, and not detectably to any other haemopoietic cell type. This suggests that LIF might have clinically useful applications in modulating macrophage function in a variety of disease states. The specific binding of 125 I-LIF to Ml myeloid leukaemic cells or to peritoneal macrophage populations was inhibited by unlabelled LIF, L i WO 88/07548 PCT/AU88/0009,3 18 g but not by a range of other haemopoietic growth factors, including G-CSF, GM-CSF, M-CSF (CSF-1), Multi-CSF (interleukin interleukins 1,2,4,6, endotoxin or other growth factors (see for example Figure The ability of cell conditioned media or cell extracts to inhibit the 125 binding of 125I-LIF to such cell populations (radioreceptor assay for LIF) (Figure 6B) thus provides a specific assay for the presence of LIF production or storage by various cells and tissues. For example, medium conditioned by lectin-activated LB3 T cells strongly 125 inhibited specific binding of 25 -LIF to Ml cells thus indicating that LB3 cells produce LIF.

EXAMPLE 4 The following Example sets out the steps used to obtain a cloned complementary DNA copy of the mRNA encoding murine LIF.

The accompanying Figures 7 to 10 relate to various steps of the method described below. In the Figures: Figure 7 relates to Step 1 of the cloning of LIF cDNA clones: the accumulation of mRNAs for haemopoietic growth regulators in the cytoplasm of LB3 cells following stimulation with the lectin concanavalin A. Cytoplasmic polyadenylated RNA (5pg) from WEHI-3B D- cells (track T cell clone LB3 cells cultured at 106 cells/ml with concanavalin A for 5 hours (track and unstimulated LB3 cells (track 3) was electrophoresed on formaldehyde agarose gels, transferred to nitrocellulose 332 and hybridized with a P-GM-CSF probe (panel a) or a 3 2 P-Multi-CSF probe (panel b) (for details see Kelso, Metcalf, D. and Gough,N.M. J.Immunol. 136:1718-1725, 1986).

Figure 8 relates to Step 3 of the cloning of LIF cDNA 1 1 W II 1 I 1 S WO 88/07548 PCT/AU88/00093 19 clones: Synthetic oligonucleotide used for identifying LIF clones. A portion of the amino acid sequence of murine LIF near the N-terminus (residues 15-26, see Example 2) is shown on the top line, the possible combinations of mRNA sequences that could encode this peptide in the middle, and the oligonucleotide probe complementary to this mRNA sequence, below. Note that for all amino acid residues except Cys 17 and His 18, the oligonucleotide is complementary only to the codons most S0 frequently used in mammalian coding regions (Grantham et.al., Nucleic Acids Res. 9:43-77, 1981). For Cys 17, .the sequence is complementary to both codons. For His 18, the oligonucleotide is complementary to the less frequent CAU codon. It was considered unlikely that the CAC codon would be used as it would generate, in conjunction with the neighbouring Gly codon, the infrequent CpG dinucleotide (Swartz et.al, J. Biol. Chem. 238:1961-1967, 1962) Fiqure 9 relates to Step 4 of the cloning of LIF cDNA clones; the identification of LIF cDNA clones by hybridization with the oligonucleotide illustrated in Figure 8.

Figure 10 relates to Step 5 of the cloning of LIF cDNA clones: nucleotide sequence of cDNA clone pLIF7.2b and LIF amino acid sequence. The sequence of the mRNA-synonymous strand of the cDNA is listed 5' to 3' with the predicted amino acid sequence of LIF given above; numbers at the end of lines indicate the position of the final residue (amino acid or nucleotide) on that line.

The amino acid sequence is numbered consecutively from the first residue encoded in this clone. Regions of amino acid sequence determined by analysis of the protein are overlined; there are no discordancies between the two.

Pro 9 was the N-terminal residue determined by direct WO 88/07548 PCT/AU88/00093 amino acid sequence analysis (Example Seven potential N-linked glycosylation sites (Neuberger, A. et a- in Glvcoproteins, Gottschalk, A, ed., Elsevier, Amsterdam, pp 450-490, 1972) are indicated with asterisks and four potential O-linked glycosylation sites (Takahashi, N. et al, Proc. Natl. Acad. Sci. USA. 81: 2021-2025, 1984) with hatches.

Step 1: Preparation of RNA containing the LIF mRNA.

Cells of the cloned murine T cell line LB3 (Kelso, A. and Metcalf, D. Exptl.Hematol. 13:7-15, 1985) were stimulated with the lectin concanavalin A for 6 hours to enhance the accumulation in the cytoplasm of mRNAs encoding various factors which regulate the growth and differentiation of haemopoietic cells. (Kelso, A., Matcalf, D. and Gough, N.M. J.Immunol. 136:1718-1725, 1986). (For example, Figure 7 shows the increased production of mRNAs encoding the haemopoietic growth factors GM-CSF and Multi-CSF in cells so stimulated).

Cytoplasmic RNA was prepared from 5 x 108 concanavalin A-stimulated LB3 cells using a technique described previously (Gough, N.M. J.Mol.Biol. 165:683-699, 1983).

Polyadenylated mRNA molecules were partitioned from ribosomal RNA by two cycles of chromatography on oligo-dT cellulose using standard procedures (Maniatis, T. et.al., Molecular Cloning, Cold Spring Harbor, New York, 1982).

It was found subsequently by Northern blot analysis that unlike the mRNAs for GM-CSF and Multi-CSF, the LIF mRNA is present constitutively in LB3 cells and its abundance is not enhanced by concanavalin A stimulation (Gearing D.P. et al. EMBO J. 6: 3995-4002, 1987).

Step 2: Synthesis and cloning of a library of LB3 cDNA WO 88/07548 PCT/AU88/00093 21 molecules.

Double-stranded DNA copies of the LB3 mRNA prepared as described above were synthesized by standard procedures (Maniatis, T. et.al., Molecular Cloning, Cold Spring Harbor, New York, 1982 and Gough, N.M. et.al.

Nature 309:763-767, 1984). Briefly, 10pg of cytoplasmic polyadenylated LB3 mRNA were used as a template for synthesis of single-stranded complementary DNA (cDNA) in a reaction catalyzed by avian myeloblastosis virus reverse transcriptase and primed with oligo-dT. After completion of this reaction, the mRNA was degraded by incubation at 0 C for 1 hour in 0.3 M NaOH, ImM EDTA. After neutralisation of the base and recovery of the cDNA by ethanol precipitation, the single-stranded cDNA was converted to duplex form in a reaction catalyzed by the Klenow fragment of E.coli DNA polymerase I. The "hairpin loop" structure of the cDNA was then cleaved by treatment with the single-strand specific nuclease S1 and then tails of deoxycytidine residues (approximately 20-30 residues long) were appended to each end of the double-stranded cDNA using the enzyme terminal deoxynucleotidyltransferase as described (Michelson, A.M, and Orkin, S. J.Biol.Chem.

257:14773-14782, 1982). The dC-tailed cDNA was fractionated by electrophoresis on a 1.5% agarose gel and molecules greater than 500bp in length recovered and annealed to a plasmid DNA molecule (pJL3, Gough, N.M.

et.al., EMBO J. 4:645:653, 1984) that had been cleaved with the restriction endonuclease Sac 1 and to which tails of deoxyguanosine residues had been appended (Michelson, A.M. and Orkin, J.Biol.Chem. 257:14773-14782, 1982).

The tailed cDNA and plasmid molecules were annealed as described (Gough, N.M. et.al., Biochemistry 19:2702-2710, 1980) and E.coli MC1061 (Casadaban, M. and Cohen, S., J.Mol.Biol. 138:179-207, 1980) transformed with the WO 88/07548 pCT/AU88/000 93 C-terminal residues missing from this construct might WO 88/07548 PCT/AU88/00093 22 annealed cDNA/plasmid mixture and approximately 50,000 independently transformed bacterial colonies selected by growth on agar plates containing 10pg/ml ampicillin.

The transformed bacterial colonies were removed from the S agar plates by washing with liquid growth medium containing 50pg/ml ampicillin and stored as independent pools in 10% glycerol at Step 3: Design of oligonucleotide probes.

A sequence of 26 amino acids at the amino terminus of LIF and residues from 11 different peptides derived by digestion of LIF with trypsin and V8 protease have been determined (see Example These amino acid sequences provided the basis for the design of oligonucleotides complementary to certain defined regions of the mRNA encoding LIF for use as hybridization probes to identify cDNA clones corresponding to the LIF mRNA.

Each naturally occurring amino acid is encoded in its corresponding mRNA by a specific combination of 3 ribonucleoside triphosphates (a codon) (eg Watson, J.

Molecular Biology of the Gene, 3xd ed., Benjamin/Cummings, Menlo Park, Calif., 1976). Certain amino acids are specified by only one codon, whereas others are specified by as many as 6 different codons (eg Watson, J. Molecular bioloqy of the Gene, supra). Since therefore a large number of different combinations of nucleotide sequences could in fact encode any particular amino acid sequence, a large number of different degenerate oligonucleotides would need to be constructed in order to cater for every 3 possible sequence potentially encoding that peptide.

There are however, certain technical constraints in the use of highly degenerate oligonucleotides as hybridization probes. 'However, since for a given amino acid not all codons are employed with equivalent frequency (Grantham, WO 88/07548 PCT/AU88/00093 23 R. et.al. Nucleic Acids Res. 9:43-73, 1981) and since in the mmmmalian genome the CpG dinucleotide is underrepresented, occurring at only 20-25% of the frequency expected from base composition (Swartz, M.N.

et.al., J.Biol.Chem. 238:1961-1967, 1962), it is often possible to predict the likely nucleotide sequence encoding a given peptide and thus reduce the complexity of a given oligonucleotide probe. Given the foregoing considerations, a number of oligonucleotides corresponding to different LIF peptides were designed and synthesized by standard procedures.

Step 4: Screening of an LB3 cDNA library for LIF-encoding clones.

For screening colonies of bacteria by hybridization with oligonucleotide probes, 10,000-15,000 bacterial colonies from each pool of the aforementioned LB3 cDNA library were grown on agar plates (containing ampicilin), transferred to nitrocellulose filter disks and plasmid DNA amplified by incubation of the filters on agar plates containing 200pg/ml chloramphenicol (Hanahan, D. and Meselson, M. Gene 10:63-67, 1980). After regrowth of colonies on the original plate, a second nitrocellulose filter was prepared as above. The master plate was regrown a second time and then stored at 4 0 C. Plasmid DNA was released from the bacterial colonies and fixed to the nitrocellulose filters as previously described (Maniatis, T. et.al. Molecular Cloning, Cold Spring Harbor, New York, 1982). Before hybridization, filters were incubated for several hours at 37°C in 0.9M NaCl, 0.09 M sodium citrate, 0.2% Ficoll., polyvinyl-pyrollidone, 0.2% bovine serum albumin, 50pg/ml heat denatured salmon sperm DNA, E.coli tRNA, 0.1M ATP and 2mM sodium 1 1 1

'-I

WO 88/07548 PCT/AU88/00093 24 pyrophosphate. Hybridization was performed in the same solution, containing in addition 0.1% NP40, at 37 0 C for 18 hours. 500ng of the synthetic oligonucleotide shown in Figure 8, was radioactively labelled in a reaction catalyzed by polynucleotide kinase and containing 32 500 pCi of [y- 32 p]ATP (specific activity 2,000-3,000 Ci/mmol). Unincorporated [y- 32 PATP was then separated from the radioactively labelled oligonucleotide by ion exchange chromatography on a NACS-PREPAC column (Bethesda Research Laboratories) according to the manufacturer's instructions. The radioactively labelled oligonucleotide was included in the hybridization reaction at a concentration of approx.20ng/ml. After hybridization, filters were washed extensively in 0.9M NaC1, 0.09M sodium citrate, 0.1% sodium dodecyl sulphate at various temperatures (as described below) and after each wash, autoradiographed.

The rationale behind performing successive rounds of washing was that at lower temperatures, all clones having even a small degree of homology with the oligonucleotide (as little as approximately nucleotides) would be revealed as autoradiographic spots appearing on duplicate filters. As the temperature is increased, the oligonucleotide probe would be melted from clones with the lowest level of homology and the corresponding autoradiographic signals would thus be lost. Clones with the highest degree of homology (and thus clones which represent the best candidates for containing LIF cDNA sequences) will retain hybridization at the highest temperature. By this strategy it is thus possible to focus directly on the strongest candidate clones. Figure 9 shows the performance of a set of clones on a duplicate pair of filters containing 15,n00 LB3 cDNA clones as the washing temperature was raised from 46°C to U WO 88/07548 PCT/AU88/00093 66 0 C. Several clones retained hybridization to the oligonucleotide only at lower temperatures whereas several retained hybridization even at 66°C (for example, clones 1 and The latter clones were thus selected for further analysis and the corcesponding bacterial colonies removed from the master plates, purified and plasmid DNA prepared from each bacterial clone by standard procedures (Maniatis, T. et.al. Molecular Cloning, Cold Spring Harbor, 1982). Preliminary structural analysis of these clones (by assessing the size of the inserted cDNA, by mapping the locations of cleavage sites for various restriction endonucleases and by testing for hybridization with various oligonucleotides corresponding to different LIF peptides) indicated that each of these clones were in fact identical; that is, they represented different isolates of the same original cloning event. Thus further detailed analysis was performed on only one clone, pLIF7.2b.

Step 5: Determination of the nucleotide sequence of pLIF7.2b.

Nucleotide sequence analysis of the cDNA portion of clone pLIF7.2b was performed by the dideoxy chain termination method (Sanger, F. et.al., Proc.Natl.Acad.Sci.

USA 74:5463-5467, 1977), using alkaline or heat denatured double-stranded plasmid DNA as template, a variety of oligonucleotides complementary both to the regions of the vector (pJL3) flanking the cDNA and to sequences within the cDNA insert as primers, and using both the Klenow fragment of E.coli DNA polymerase I and the avian myeloblastosis virus reverse transcriptase as polymerases in the sequencing reactions. The sequence so determined for the cDNA portion of clone pLIF7.2b is shown in Figure Such analysis confirmed that this clone does indeed 1 1 1 I SWO 88/07548 PCT/AU88/00093 26 contain a DNA copy of a mRNA with the capacity to encode the LIF molecule, since within the only translational reading frame that spans the entire cDNA uninterrupted by stop codons, all of the amino acid sequences previously determined for various peptides of the LIF molecule may be found.. This clone does not however contain a complete copy of the LIF coding region since it does not extend atb the 5' end through a region encoding a presumed hydrophobic leader sequence initiated by a methionine codom and it does not include at its 3' end an in-frame translational stop codon. It does however extend at the 5' end past the start of the region encoding the mature protein, determined by comparison with the previously determined amino-terminal amino acid sequence (residue Pro 9 in Figure EXAMPLE The following sets out the steps used to construct a full length copy of the murine LIF coding region, to install this coding region in a yeast expression vector and to produce murine LIF.

The accompanying Figures relate to various steps of the method described below. In the Figures: Figure-11: relates to Step 1 of the expression of murine LF im yeast cells: C-terminal amino acid sequence of LIF-.. The amino acid sequence at the C-terminus of the p.IIE7I..2b reading frame is shown above the sequence of a Staphylococcal V8 protease and a tryptic peptide derived from LIF purified from Krebs ascites tumour cells. Note that these latter peptides extend the pLIF7.2b LIF sequence by 9 amino acids and terminate at the same residue. The nucleotide and amino acid sequence at. the corresponding region of clone pLIFNK1 is shown at the bottom line, confirming the C-terminus assigned by direct WO 88/07548 PCT/AU88/00093 27 amino acid sequencing. The numbering system is that of Figure Figure 12: relates to step 2 of the expression of murine LIF in yeast cells: reconstruction of the pLIF7.2b cDNA for insertion into the yeast expression vector, YEpsecl. Partial nucleotide sequence of the pLIF7.2b cDNA and its encoded amino acid sequence are shown on the top line. Numbering is according to Figures 10 and 11. The second and third lines show the sequence of pLIFmutl and pLIFmut2 respectively. Asterisks indicate stop codons. Restriction sites (Bam HI, Hind III and Eco Rl) used for cloning into YEpsecl (Baldari. C. et al, EMBO J. 6: 229-234, 1987) and pGEX-2T (Smith, D.B.

and Johnson, Gene, in press, 1988) are indicated.

The bottom line shows partial sequence of the K.lactis killer toxin signal sequence in YEpsecl. The sequence Gly-Ser encoded at the Bam HI restriction site is efficiently recognized by signal peptidase (Baldari, C. et al, EMBO J. 6: 229-234, 1987).

Figure 13: relates to step 4 of the expression of murine LIF in yeast cells: the biological activity of yeast-derived recombinant LIF in cultures of Ml leukaemic cells. Titration in Ml cultures of yeast-derived recombinant LIF versus purified native LIF-A showing similar concentration dependent induction of differentiation in Ml colonies (panel A) and suppression of colony formation (panel Each point represents the mean value from duplicate cultures.

FiQure 14: relates to step 5 of the expression of murine LIF in yeast cells: the ability of different dilutions of authentic native murine LIF-A o conditioned medium from yeast cells containing the LIFmutl/YEpsecl recombinant induced with galactose and conditioned medium from the same yeast cells, but not induced with 1 1 1 j -L,1 wo 88/07548 PCT/AU88/00093 28 galactose to compete for the binding of native 125 1 I-LIF-A to cellular receptors on murine peritoneal cells.

SStep 1: Determination of the amino acid sequence at the C-terminus of murine LIF.

The cDNA clone pLIF7.2b of Example 4 contains an incomplete copy of the murine LIF mRNA. At the end, the cDNA encodes 8 residues N-terminal to the first residue determined by N-terminal amino acid sequence analysis (Pro 9 in Figure 10). At the 3' end however, pLIF7.2b is incomplete as it does not contain an in-frame translational stop codon. Inspection of the amino acid sequence of two LIF peptides determined by direct amino acid sequencing (Example 2) suggested that pLIF7.2b was lacking only 27 nucleotides of coding region at the 3' end. As illustrated in Figure 11, a V8-derived peptide started at residue Ala 162 of the cDNA sequence and extended the amino acid sequence deduced from the cDNA clone by 9 residues. A tryptic peptide contained within the V8 peptide terminated at the same residue, suggesting that Phe 187 is the C-terminal residue of the protein. In order to confirm this conclusion, a LIF cDNA clone overlapping with pLIF7.2b and extending 3'-ward was isolated and subjected to nucleotide sequence analysis.

In order to construct and screen an appropriate cDNA library from which to isolate such a clone, a series of different mRNA samples were screened by Northern blot hybridization to identify the RNA samples with the highest concentration of LIF mRNA molecules. Cytoplasmic polyadenylated RNA, prepared essentially as described previously (Gough, N.M.

J.Mol.Biol. 165: 683-699, 1983), was fractionated on i l WO88/07548 PCT/AU88/00093 29 1% agarose gels containing 20mM morpholinopropane sulfonic acid (MOPS), 5mM sodium acetate, ImM EDTA (pH plus 6% v/v formaldehyde, filters containing RNA were soaked in 2 x SSC, containing 0.2% Ficoll, 0.2% polyvinyl- pyrollidone, 0.2% bovine serum albumin, 2mM sodium pyrophosphate, ImM ATP, 50pg/ml denatured salmon sperm DNA and 50pg/ml E.coli tRNA, at 67 0 C for several hours. Hybridization was in the same buffer plus 0.1% SDS at 670C. The probe used to detect LIF transcripts consisted of an approx.750 bp Eco RI Hind III fragment containing the cDNA insert of pLIF7.2b subcloned in pSP65. This fragment contains not only the cDNA sequence but also G-C tails and approx.150 bp of pJL3 vector sequence. Riboprobes of approx.2 x 109 cpm/pg were derived by transcription of this SP6 subclone using reagents supplied by BRESA (Adelaide). The probe was included in hybridization at approx.2 x cpm/ml. Filters were washed extensively in 2 x SSC, 2mM EDTA, 0.1% SDS at 67 0 C and finally in 0.2 x SSC at 67°C prior to autoradiography.

Such analysis revealed that LIF transcripts were present at low levels in a wide variety of haemopoietic cell lines and that there was considerable variation in the level of LIF mRNA in different batches of Krebs RNA. Two batches of Krebs ascites tumour cell RNA were selected for synthesis of two cDNA libraries.

cDNA libraries were constructed using the reagents supplied by Amersham (Product numbers RPN.1256 and RPN.1257) and using the manufacturer's instructions; the cloning vector was XGT10. Approximately 4 x 105 recombinant clones were obtained and screened by hybridization with an oligonucleotide corresponding to a sequence of 36 residues at the 3' end of the pLIF7.2b sequence: nucleotides 500-535 (inclusive) in Figure y| i i p WO 88/07548 PCT/AU88/0009,3 Phage plaques representing the Krebs cDNA library were grown at a density of approx.50,000 plaques per 10 cm petri dish, transferred in duplicate to nitrocellulose and treated using standard techniques (Maniatis, T. et al, Molecular Cloning, Cold Spring Harbor, 1982). Prior to hybridization, filters were incubated for several hours at 37 0 C in 6 x SSC (SSC 0.15M NaCI, 0.015M sodium citrate), 0.2% Ficoll, 0.2% polyvinyl-pyrollidone, 0.2% bovine serum albumin, 2mM sodium pyrophosphate, 1mM ATP, 50 mg/ml denatured salmon sperm DNA and 50 p/ml E.coli tRNA. Hybridization was in the same solution containing 0.1% NP40, at 37 0 C for 16-18 hours. The aforementioned oligonucleotide probe, 32 radioactively labelled using P] ATP and polynucleotide kinase to a specific activity of 9 cpm/pg and separated from unincorporated label by ion exchange chromatography on a NACS column (Bethesda Research Laboratories), was included in the hybridization at a concentration of approx.20 ng/ml.

After hybridization, filters were extensively washed in 6 x SSC, 0.1% sodium dodecyl sulphate at 60°C. One plaque representing clone XLIFNK1, positive on duplicate filters, was picked and rescreened at lower density, as before.

The cDNA insert of approx.950 bp in XLIFNKI, which hybridized with the aforementioned oligonucleotide, was subcloned into a plasmid vector (pEMBL8+, Dente, L.

et al, NuclAcids Res. 11: 1645-1655, 1983) to generate clone pLIFNKl. Nucleotide sequence analysis of the cDNA insert in pLIFNK1 was performed by the dideoxy chain termination method (Sanger, F. et al, Proc.Natl.Acad.Sci.

USA 74: 5463-5467, 1977)-using alkaline denatured double-stranded plasmid DNA as template (Chen, E.Y. and Seeburg, DNA 4: 165-170, 1985), a variety of 1 i SWO 88/07548 PCT/AU88/00093 31 oligonucleotides complementary both .to regions of the vector flanking the cDNA and to sequences within the cDNA insert as primers, and using both the Klenow fragment of E.coli DNA polymerase and AMV reverse transcriptase as polymerases in the sequencing reactions. The nucleotide sequence of a portion of the cDNA insert in pLIFNK1 is shown in Figure 11. The amino acid sequence specified by pLIFNK1 is identical at the C-terminus with the 9 amino acids predicted by direct amino acid sequencing to constitute the C-terminus of LIF, and confirms that Phe 187 is the C-terminal residue of LIF, since in the pLIFNK1 cDNA sequence the codon for this residue is immediately followed by an in-frame translational stop codon.

Step 2: Construction of a LIF codon region in a yeast expression vector.

Initial production of the protein encoded by the LIF cDNA clones was achieved in a eukaryotic system (yeast), so that the expressed product would be glycosylated, secreted and correctly folded. The expression vector used, YEpsecl (Baldari, C. et al, EMBO J. t: 229-234, 1987), provides an N-terminal leader sequence derived from the killer toxin gene of Kluyveromyces lactis, shown previously to direct the efficient secretion of interleukin 1 (Baldari, C. et al, EMBO J. 6: 229-234, 1987), transcribed from a galactose-inducible hybrid GAL-CYC promoter.

In order to express the protein encoded by pLIF7.2b in this vector, it was necessary to modify the cDNA in several ways (see Figure 12). At the 5' end it was necessary not only to remove the few nucleotides specifying the partial mammalian leader sequence, but also to include an appropriate restriction |1 nodrt xrs tepoenecddb WO 88/07548 PCT/AU88/00093 32 endonuclease cleavage site (Bam HI) to allow insertion in-frame with the K.lactis leader and retain an appropriate signal peptidase cleavage site (Gly-Ser). At the 3' end, two versions of the coding region for LIF were constructed. One version (LIFmutl) was constructed so that a stop codon immediately followed the last codon of pLIF7.2b (Gln 178). The other version (LIFmut2) was constructed to append a sequence encoding the 9 amino acid residues known to be missing 1:01 from pLIF7.2b (see above), followed by an in-frame translational stop codon. An appropriate restriction endonuclease cleavage site (Hind III) completed both constructs. All of these modifications were achieved by oligonucleotide- mediated mutagenesis: The approx.750 bp Eco RI Hind III fragment carrying the 535 bp cDNA insert of pLIF7.2b, bounded by G-C tails and a portion of the pJL3 vector was subcloned into plasmid pEMBL8+ (Dente, L. et al, Nucl.Acids Res. 11: 1645-1655, 1983) and single-stranded DNA was prepared as described (Cesarini, G. and Murray, J.A.H. in Setlow, J.K. and Hollaender, A.

(eds), Genetic Engineering: Principles and Methods.

Plenum Press, New York, Vol. 8, 1987 (in press)). In vitro mutagenesis was performed as described (Nisbet, I.T. and Beilharz, M.W. Gene Anal.Techn. 2: 23-29, 1985), using oligonucleotides of 35, 51 and 61 bases respectively, to modify the 5' end and the 3' end of the cDNA as outline above. Bam HI Hind III fragments containing the modified LIF cDNA sequences were ligated into plasmid YEpsecl and the nucleotide sequence of S the inserts in the resulting recombinants was determined.

Step 3: Introduction of the YEpsecl/LIF recombinants into yeast cells.

WO 88/07548 PCT/AU88/00093 r 33 S.cerevisiae strain GY41 (leu2 ura3 adel his4 met2 trp5 gall cir+; x 4003-5b from Yeast Genetic Stock Centre, Berkeley) was transformed by the polyethylene glycol method (Klebe, R.J. et al, Gene 25: 333-341, 1983). Transformants were selected and maintained on synthetic minimal medium carbon source, 0.67% yeast nitrogen base (Difco) supplemented with 50pg/ml of the required amino acids) under uracil deprivation.

Expression of insert sequences in plasmid YEpsecl was achieved by growing transformants in either non-selective complete medium yeast extract, 2% peptone) or in synthetic minimal medium, each containing 2% galactose.

Step 4: Determination of the biological properties of yeast-derived LIF.

Assays for differentiation-inducing activity and leukaemia-suppressive activity of yeast conditioned medium were performed in 1 ml cultures containing 300 Ml cells (provided by Dr. M. Hozumi, Saitama Cancer Research Centre, Japan) in Dulbecco's Modified Eagle's Medium with a final concentration of 20% foetal calf serum and 0.3% agar. Material to be assayed was added in serially diluted 0.1 ml volumes to the culture dish prior to the addition of the cell suspension in agar medium.

Cultures were incubated for 7 days in a fully humidified atmosphere at 10% CO 2 Cultures were scored using a dissection microscope at x35 magnification, scoring as differentiated any colonies with a corona of 3 dispersed cells or composed wholly of dispersed cells.

Morphological examination of colonies was performed by fixing the entire culture with 1 ml 2.5% glutaraldehyde then staining the dried cultures on microscope slides using acetylcholinesterase/Luxol Fast 3 5 1 v

I

WO 88/07..48 T/ ,A U 9-8 WO 88/07548 PCAU88/0093 34 Blue/Haematoxylin.

Assays for colony stimulating activity were performed using 75,000 C57BL bone marrow cells as described previously (Metcalf, D. The Hemopoietic Colony Stimulating Factors Elsevier, Amsterdam, 1984). Assays for differentiation inducing activity on WEHI-3B D cells were performed as described previously (Nicola, N. et al, J.Biol.Chem. 258: 9017-9023, 1983).

Medium from cultures of yeast containing the full-length coding region (LIFmut2), but not from cultures of non-transformed yeast, yeast containing the vector YEpsecl alone, or yeast containing the incomplete coding region (LIFmutl), was able to induce typical macrophage differentiation in cultures of M1 colonies (Figure 13A). As with purified native LIF, with increasing concentrations the yeast-derived material also progressively reduced the number and size of Ml colonies developing (Figure 13B). Comparison with purified native LIF indicated that the yeast conditioned medium contained up to 16,000 Units/ml (approximately 130ng/ml) of LIF. Yeast-derived LIF, like purified native LIF-A, failed to stimulate colony formation by normal granulocyte-macrophage progenitor cells or to induce differentiation in, or suppress proliferation, of WYHI-3B D+ leukaemic colonies.

Size fractionation on a Sephacryl S-200 column, indicated that yeast-derived LIF co-eluted with proteins of apparent molecular weight 67,000 to 30 150,000 Daltons, compared with 58,000 Daltons for LIF 3O derived from Krebs cells.

The absence of LIF activity in medium conditioned by yeast containing an incomplete coding region (LIFmutl) suggests that the nine hydrophobic *1

P

I

:i ii i i i i Jtr

I

i WO 88/07548 PCT/AU88/00093 C-terminal residues missing from this construct might be required for LIF function. Whilst these residues might interact with the receptor, they may also constitute part of a hydrophobic core within the protein, and thus their absence may prevent proper folding. Alternatively they might in some way be required for efficient secretion of LIF.

Step 5: Receptor binding specificity of yeast- derived murine LIF and purified native LIF-A from Krebs II ascites cell conditioned medium.

Purified murine LIF-A (Example 1) was iodinated as disclosed previously (Example Peritoneal cells were harvested by lavage from mice in which a high level of macrophages had been induced in the peritoneal cavity with thioglycollate. These cells were washed and resuspended at 2.5 x 106/50pl in Hepes-buffered RPMI medium containing 10% foetal calf serum. Cells in aliquots were incubated with 200,000 cpm of 125

I-LIF-A

(10pl, in the same medium) and 10pl of control medium or serial two-fold dilutions of unlabelled pure murine LIF-A or yeast-derived murine LIF. At appropriate concentrations, supernatant from galactose-induced yeast containing the LIFmut2 construct (Figure 12) competed for binding of 125I-LIF-A to its receptors on peritoneal cells while uninduced yeast supernatant did not (Figure 14). The degree of competition was equal to that of authentic native LIF-A, indicating that the yeast-derived LIF-A contained all the information required for binding to LIF-A cellular receptors.

EXAMPLE 6.

The'following example sets out Lhe steps used to The-olloingexamle ets ut he sepsusedto L 11 1 I% h L_ t WO 88/07548 PCT/AU88/00093 36 express recombinant murine LIF in mammalian cells.

The accompanying Figure 15 relates to step 1 of the method described below: The nucleotide sequence of the cDNA portion of clone pLIFNK3. The nucleotide sequence of the mRNA-synonymous strand is presented in the to 3' orientation with the inferred amino acid sequence of LIF given above. The N-terminal amino acid residue previously determined is indicated as The Eco R1 site at the 5' end of the cDNA and the Xba I site spanning the stop codon (used in inserting the LIF coding region into pMP-Zen in step'2) are indicated.

Step 1: Determination of the amino acid sequence of the N-terminal leader sequence of murine LIF.

The cDNA clones pLIF7.2b and pLIFNK1 (see above) contain incomplete copies of the LIF mRNA. Although they contain, together, the complete coding region for the mature portion of the LIF protein, they do not contain the complete hydrophobic leader sequence, required for secretion of LIF from mammalian cells.

In order to isolate a cDNA clone containing the region encoding the hydrophobic leader, a further cDNA clones constructed as above (Example using the same batch of Krebs mRNA as a template, were screened (as above) using as probes two oligonucleotides corresponding to a sequence of 35 residues at the 5' end and 36 residues at the 3' end of the pLIF7.2b sequence (nucleotides 67-102 and 500-535, inclusive, in Figure Two plaques representing clones XLIFNK2 and XLIFNK3, positive on duplicate filters were picked and rescreened at lower density, as previously. Since XLIFNK3 was shown to hybridize with each of the two oligonucleotides used, it was selected for further analysis.

i- 1 1 j WO 88/07548 PCT/AU88/00093 37 The cDNA insert of approx.1400bp in XLIFNK3 was subcloned into the plasmid vector pEMBL 8 (Dente, L., et.al., Nucleic Acids Res. 11:1645-1655, 1983) to generate clone pLIFNK3. Nucleotide sequence analysis of the cDNA insert of pLIFNK3 was performed as for pLIF7.2b and pLIFNK1 (above).

The nucleotide sequence of the cDNA insert in pLIFNK3 is shown in Figure 15, and indicates that pLIFNK3 contains a complete LIF coding region: there is an initiation codon (AUG) at position 23-25 in the pLIFNK3 sequence, preceding a sequence encoding a typical hydrophobic leader sequence of 24 amino acid residues.

The coding region extends to the same translational stop codon as defined previously by pLIFNK1 (above).

Step 2: Introduction of a LIF coding region into a mammalian expression vector.

The mammalian expression vector chosen initially was the retroviral expression vector pMP-Zen. The vector is derived from the Moloney murine leukaemia virus-based vector pZIPNeo SV(X) (Cepko, C.L. et.al., Cell 37:1053-1062, 1984) by deletion of the neo gene together with nearby SV40 and plasmid sequences, leaving an Xho I expression site. The 3' region of the vector is also modified to incorporate the enhancer from the long terminal repeat (LTR) of the myeloproliferative sarcoma virus (MPSV) (Bowtel, D.D.L. et.al., Mol.Biol.Med.

4:229-250, 1987). This vector was chosen, firstly since the LIF/PMP-Zen recombinant may be packaged into helper-free infectious retroviral particles by passage through #2 cells (Mann, et.al. Cell, 33:153-159, 1983). Such viral particles may then be used to efficiently introduce (by infection) the LIF/pMP-Zen recombinant into a wide variety of murine cell types *I 3 5 11 1 1 1 l l 1 i 1 I. 1 WO 88/07548 PCT/AU88/00093 38 (Mann, R. et.al. Cell 33:153-159, 1983).. Secondly, this particular vector, which employs the MPSV LTRs for expression of the foreign coding region, has been shown to direct the efficient expression of certain other haemopoietic growth and differentiation factors including

GM-CSF.

The segment of pLIFNK3 chosen for insertion into pMP-Zen extended from position 1 (an Eco R1 site) to position 630 (an Xba 1 site spanning the stop codon) see Figure 15. This segment was chosen since it contains little more than the coding region of pre-LIF and excludes all of the 3' untranslated region, which may contain sequences conferring mRNA instability Shaw, G. and Kamen, R. Cell 46:659-667, 1986; V&rma, I.M. and Sassone-Corsi, Cell 51 513-514, 1987 In order to insert this segment into pMP-Zen, it was first inserted between the co R1 and Xba I sites of pIC20H (Marsh, J.L.

et.al. Gen_ 32: 481-485, 1984) by standard techniques (Maniatis et.al., 1982, supra). The cDNA insert was then recovered from the polylinker of the pIC20H plasmid by Sal I plus Xho I digestion, thus generating a LIF cDNA fragment with cohesive ends appropriate for inserting into the Xho I cloning site of pMP-Zen. The insertion of the LIF cDNA fragment into pMP-Zen was achieved by standard techniques (Maniatis et.al., 1982, supra).

Step 3: Introduction of the pMP-Zen/LIF recombinant into murine fibroblasts.

pMP-Zen/LIF DNA was introduced into y2 fibroblasts by electroporation (Potter et.al., PNAS 81:7161-7165, 1984). 30pg pMP-Zen/LIF DNA plus 3pg pSV2Neo DNA (Southern, P.J. and Berg, P. J.Mol.App.Genet.

1:327-341, 1982) were mixed with 1 x 106 y2 fibroblasts in iml of DME/10% FCS (Dulbeccos modified Eagles medium containing 10% foetal calf serum) and 3L1 WO 88/07548 PCT/AU88/00093 39 subjected to a pulse of 500v at a capacitance of (using a BioRad Gene-Pulser model No. 1652078).

Transfectants were initially selected on the basis of resistance to the antibiotic G418 conferred by the pSV2Neo DNA. G418-resistant y2 cells were selected in 400g/ml G418 by standard procedures (Mann, R. et al, Cell 33: 153-159, 1983). Of 19 G418-resistant clones examined, 2 also contained the pMP-Zen/LIF construct as assessed by LIF activity detectable in y2 conditioned media.

Step 4: Determination of the biological properties'of 2-derived LIF.

Assays for differentiation-inducing activity and leukaemia _-"pressive activity of conditioned medium from pMP-Zen/LIF ,Ontaining i2 cells were performed as described previously. Medium from cultures of 106 '2 cells in 3ml DME/10% FCS were assayed for differentiation-inducing activity. The results for two positive cultures are presented in the following table: 1 11; TI 3 WO 88ffl7548 PCT/AU88/00093 Ml Differentiation-inducing Activity (Units/10 6 cells/ml C.M.) Clone No.

2-control not detectable y2-lla >16,000 %2-lld >16,000 Thus, significant levels of biologically active, recombinant murine LIF can be produced in this expression system.

Step 5: Transmission of pMP-Zen/LIF retrovirus from r2 cells to haemopoietic cells.

Infectious pMP-Zen/LIF retrovirus could be transferred from the parent %p2 cell lines to haemopoietic cells of the line FDC-P1 (Dexter, T.M.

et.al., J.Exp.Med. 152:1036-1047, 1980) by cocultivation.

106 %2 cells were mixed with 106 FDC-P1 cells in DME/10% FCS containing the optimal concentration of WEHI-3BD- conditioned medium for growth of the FDC-PI cells. Following 2 days incubation, the non-adherant FDC-PI cells were removed, washed free from all adherant T2 cells and grown for 16 hours in 3ml of the same medium. Conditioned medium was harvested and assayed for differentiation-inducing and leukaemia suppressive activity as described previously. The results are shown in the following table: FD Culture derived from Clone No.

Ml Differentiation-Inducing Activity (units/10 6 FDC-P1 cells) '2-control not detectable '2-11a approx. 3,000 '2-lld approx. 1,500 Thus the y2 clones, '2-11a and '2-11d, are capable of transmitting biologically active pMP-Zen/LIF j WO 88/07548 PCT/AU88/00093 41 retrovirus to haemopoietic cells. These clones should therefore be applicable to the infection of normal murine haemopoietic progenitor cells which can be used to reconstitute the haemopoietic system of irradiated mice in order to study the effects of high level expression of native LIF on normal murine haemopoiesis, in a manner analogous to that used for Zen/GM-CSF viral infection.

EXAMPLE 7.

The following sets out the steps used to express recombinant murine LIF in E.coli cells.

The accompanying Figure 16 relates to step 1 of the method described below: The nucleotide sequence at the glutathione S-transferase/thrombin cleavage site/LIF junction in plasmid pGEX-2T/LIF, and the sequence at the junction of the encoded fusion protein. The C-terminus of the glutathione S-transferase, the thrombin cleavage site and the N-terminus of the murine LIF portions of the tripartite fusion protein, along with the nucleotide sequence encoding this amino acid sequence, are shown.

The expected site of cleavage by thrombin is indicated by an arrow.

Step 1: Introduction of a LIF coding region into an E.coli expression vector.

The expression vector used for expressing LIF in E,coli was pGEX-2T (Smith, D.B. and Johnson, Gene (in press), 1988), which directs the synthesis of foreign polypeptides as fusions with the C-terminus of Sj26, a 26kD glutathione S-transferase 2.5.1.18) encoded by the parasitic helminth Schistosoma iaponicum. In the majority of cases, fusion proteins have been shown to be soluble in aqueous solutions and can be purified from crude bacterial, lysates under non-denaturing conditions by ~rurs.

I

8-

C-'

1: WO 88/07548 PCT/AU88/00093 42 affinity chromatography on immobilized glutathione. The particular vector pGEX-2T has been engineered so that the glutathione S-transferase carrier can be cleaved from the fusion protein by digestion with the site-specific protease thrombin.

The complete coding region of murine LIF, derived from plasmid pLIFmut2 (see Example 5 and Figure 12 above) was introduced as a Bam HI EcoRI fragment into the multiple cloning site of pGEX-2T (Smith, D.B. and Johnson, supra), thus positioning the LIF coding region 3' of, and in the same translational reading frame as, that of the glutathione S-transferase and the thrombin cleavage site. Thus the LIF protein would be located C-terminal to these elements in a tripartite glutathione S-transferase/ thrombin cleavage site/LIF fusion protein (see Figure 16). Note that the position of the thrombin cleavage site is such that two amino acid residues (Gly-Ser) will be appended to the N-terminus of the LIF protein after thrombin cleavage. The construction of the aforementioned plasmid, pGEX-2T/LIF, was achieved by standard techniques, and the plasmid was introduced into E.coli NM522 (Gough, J.A. and Murray, J.Mol.Biol. 166: 1-19, 1983) by standard techniques.

Step 2: Expression and purification of glutathione S-transferase/LIF fusion protein.

In order to induce the expression of the glutathione S-transferase/LIF fusion protein, cultures of E.coli NM522 cells containing pGEX-2T/LIF were 30 grown to logarithmic phase in Luria broth, and isopropyl P-D-thiogalactopyranoside added to a concentration of 0.lmM. After a further 4 hours growth, during which time the glutathione S-transferase/LIF gene is expressed, the cells were harvested by centrifugation and resuspended in WO; 88/07548 PCAU8/00093 43 iml mouse tonicity phosphate-buffered saline (MTPBS: 150mM NaCI, 16mM Na 2

HPO

4 4mM NaH 2

PO

4 pH7.3).

Cells were lysed on ice by mild sonication and after adding Triton X-100 to cell debris was removed by centrifugation (10,000g, 5 min, 4 0 The clarified supernatant was mixed at room temperature in a polypropylene tube on a rotating platform with 200pl glutathione-agarose beads (sulphur linkage, Sigma).

(Before use, beads were pre-swollen in MTPBS, washed twice in the same buffer and stored in MTPBS at 4 0 C as a solution After absorption (5 min.), beads were collected by centrifugation (500g, 1 min) and washed three times in MTPBS/Triton X-100. The glutathione S-transferase/LIF fusion protein was then eluted by competition with free glutathione: beads were incubated with 100pl 50mM Tris. HC1, 5mM reduced glutathione (pH for 2 min at room temperature and the beads removed by centrifugation. The elution step was performed twice, and the two 100pl aliquots of eluate pooled.

100pl of the glutathione S-transferase/LIF fusion protein was treated with thrombin as described (Smith, D.B. and Johnson, supra).

pl aliquots of the uncleaved and thrombin-cleaved glutathione S-transferase LIF fusion proteins were electrophoresed on a Pharmacia Phast Gel (8-25% polyacrylamide gradient gel). After staining with Coomassie Blue, a single major protein species of relative molecular weight -46 kDa was revealed in the uncleaved preparation, and in the thrombin-cleaved preparation two major bands of -26 kDa and -20 kDa (corresponding to the expected sizes of the fusion protein (46 kDa), the glutathione S-transferase (26 kDa) and the LIF proteins kDa) respectively). By estimating the mass of the Coomassie-stained bands on this gel, the yield of Si! WO 88/07548 PCT/AU88/00093 44 i glutathione S-transferase/LIF protein from the original E,coli culture was estimated to be Assays for differentiation-inducing activity and leukaemia-suppressive activity of both the glutathione S-transferase/LIF fusion protein and the thrombin cleaved LIF preparation, were performed using murine Ml cells (as described previously). Both preparations of LIF were determined to be biologically active, with specific activities in excess of 7x10 6 units/mg.

EXAMPLE 8 The following sets out the steps used to determine whether the murine genome contains any other genes closely related to the gene encoding the sequence present in clone pLIF7.2b (Example 4) and the derivation of a map of the location of restriction endonuclease cleavage sites around the LIF gene.

The accompanying Figures relate to various steps of the method described below: Figure 17: relates to step 1: hybridization of mouse DNA with a pLIF7.2b-derived probe under high and low stringency conditions. BALB/c liver DNA digested with the indicated restriction enzymes was probed for LIF gene sequences as described in Example 8, step 1. In the left hand panel, hybridization was at 65°C in 2 x SSC, and final washing at 65 0 C in 0.2 x SSC. In the right hand panel, hybridization and washing were both in 6 x SSC at In the left hand panel, linear pLIF7.2b plasmid DNA (5.6 kb) was included at an amount equivalent to and 1 copies per haploid mouse genome (400 pg and 40 pg respectively) assuming a molecular weight for the haploid mouse genome of 3 x 10 bp (Laird, C.D. Chromosoma, 32: 378-406, 1971). The sizes of the hybridizing genomic fragments are indicated.

WO 88/07548 PCT/AU88/00093 Fiqure 18: relates to step 2: Hybr.idization of mouse DNA cleaved with various restriction endonucleases, singly and in pair-wise combinations, with a mouse LIF probe. Digested DNAs were electrophoresed on 0.8% agarose gels and hybridized with the pLIF7.2b cDNA fragment as described in Example 8, step 1, under high stringency conditions.

Figure 19: relates to step 2: Restriction endonuclease cleavage map of the murine LIF gene. The region of chromosomal DNA containing sequences corresponding to the cDNA clone pLIF7.2b is indicated by a box. The restriction sites given above (Eco R5) and below (Xba I, Bam HI, Pst I and Stu I) the central line have not been oriented with respect to the central line. The relative location of sites within the complex below the line is as indicated.

Figure 20: relates to step 3: chromosomal assignment of the murine LIF gene. In lane 1 BALB/c mouse embryo DNA was electrophoresed, in lane 2, DNA from Chinese hamster ovary cells and in lanes 3-8 DNA from the hybrid clones I-18A HAT, I-18A-2a- 8AG; EBS 58; EBS 11; EBS 4; and I-13A-la--8AG (Cory, S. et al, EMBO J. 2: 213-216, 1983). The murine chromosome content of these hybrids is given in Cory, S. et al, (EMBO J. 2: 213-216, 1983).

All DNAs were digested with Bam HI. The position of the 3 kbp Bam HI fragment bearing the murine LIF gene is indicated.

Step 1: Determination of the number of LIF-related genes in the murine genome.

In view of the data (Example 1) that medium conditioned by Krebs II cells contains two biochemically separable but functionally similar factors capable of inducing the differentiation of Ml cells (LIF-A and ii 3

J

WO 88/07548 PCT/AU88/00093 46 LIF-B) we wished to determine how many genes there are in the murine genome related to the species we have purified and cloned. Southern blots of murine genomic DNA digested with various restriction endonucleases were therefore hybridized with a probe derived from pLIF7.2b under conditions of both high and low stringency.

pg aliquots of high molecular weight genomic DNA from BALB/c mouse livers were digested to completion with various restriction endonucleases, fractionated by electrophoresis in 0.8% agarose gels and transferred to nitrocellulose. Prior to hybridization, filters were incubated for several hours at 65 0 C in either 6 x SSC (low stringency) or 2 x SSC (high stringency) (SSC 0.15M NaCI, 0.015M sodium citrate), 0.2% Ficoll, 0.2% polyvinyl-pyrollidone, 0.2% bovine serum albumin, 2mM sodium pyrophosphate, ImM ATP, 50 pg/ml denatured salmon sperm DNA and 50 pg/ml E.coli tRNA. Hybridization was in the same solution containing 0.1% SDS, at 65 0 C for 16-18 hours. Filters were then washed in either 6 x SSC, 0.1% SDS at 65 0 C (low stringency) or in 2 x SSC, 0.1% SDS at 65°C, followed by 0.2 x SSC, 65°C (high stringency) as detailed in the legend to Figure 17. The hybridization probe used was as previously disclosed the approx.750 bp Eco RI Hind III fragment spanning the cDNA insert of pLIF7.2b radiolabelled to a specific activity of approx.4 x 108 cpm/pg by nick-translation and included in the hybridization at a concentration of approx.2 x 107 cpm/ml.

In mouse liver DNA (Figure 17) as well as in DNA from Krebs II cells, LB3 T cells and WEHI263 monocytic cells (not shown) the LIF probe detected unique Eco RI, Bam HI, and Hind III fragments of approximately 11, 3 and 13 kbp respectively. The same pattern of hybridization i> ilI WO 88/07548 PCT/AU88/00093 47 was evident at both high (0.2 x SSC, ,65 0 C) and low stringency (6 x SSC, 65°C) (Figure 17); under low stringency hybridization and washing conditions no additional bands were evident. Similarly, unique hybridizing fragments were also detected in Pst I, Stu I, Sac I, Eco R5 and Bgl II-digested genomic DNA (Figure 17). Moreover, in the experiment illustrated in Figure 16, pLIF7.2b plasmid DNA was included at a concentration equivalent to 10 and 1 copies per haploid mouse genome (tracks 1 and the hybridization intensity of the genomic LIF sequences was not significantly different from the intensity of the unique gene standard (track 2).

Taken together, the foregoing data indicate that the LIF gene is unique in the murine genome, with no close relatives. Thus the two species of LIF are not likely to be products of different genes, but rather are more likely to represent post- transcriptional or posttranslational variants of the same gene product.

Step 2: Derivation of a restriction endonuclease cleavage map of the murine LIF gene.

In order to determine the disposition of restriction endonuclease cleavage sites in and around the murine LIF gene, and thus provide a molecular fingerprint of this gene, DNA from mouse livers or from Krebs ascites tumour cells was digested with various restriction endonucleases and subjected to Southern blot analysis as described in Step 1 (under high stringency hybridization and washing conditions). Examples of such experiments are S shown in Figure 18. Analysis of the size of the digestion products enables one to generate a map of the location of each cleavage site around the LIF gene (Figure 19).

Step 3: Chromosome assignment of the murine LIF gene.

p.

r i I I i i WO 88/07548 PCT/AU88/00093 48 To determine the chromosome on which the mouse LIF gene is located, DNA was examined from six mouse-Chinese hamster ovary somatic cell hybrid cell lines which retain various mouse chromosomes (Cory, S.

et al, EMBO J. 2: 213-216, 1983; Francke, U. et al, Cvtoaenet.Cell.Genet. 12: 57-84, 1977). Southern blot analysis performed as described in Step 1 (using high stringency hybridization and washing conditions) indicated that the 3 kbp Bam HI fragment containing the murine LIF gene was absent from all of the hybrids (Figure 20). Since chromosome 11 is the only mouse chromosome not retained in any of these lines (Cory, S.

et al, EMBO J. 2: 213-216, 1983), a characteristic of mouse-Chinese hamster hybrids (Francke, U. et al, Cytogenet.Cell.Genet. 19: 57- 84, 1977), it is likely that the LIF gene is on this chromosome. On the same basis, the murine GM-CSF gene (Barlow, D.P. et al, EMBO J. 6: 617-623, 1987) and Multi-CSF gene (Ihle, J.N. and Kozak, National Cancer Institute, Frederick Cancer Research Facility, Annual Report, 1984) have also been assigned to chromosome 11, an assignment confirmed by genetic linkage studies (Barlow, D.P. et al, EMBO J. 6: 617-623, 1987).

EXAMPLE 9.

The following example sets out the steps used to establish conditions under which the cloned murine LIF cDNA can be used to identify by hybridization a human gene or mRNA or recombinant DNA clones containing human LIF-encoding sequences.

The accompanying Figure (Figure 21) relates to Step 2 of the method described below: hybridization of 32 P- labelled fragment of cDNA from clone pLIF7.2b under various conditions to genomic DNA of both mouse i r 31: r:I, i r t i i i WO 88/07548 PCT/AU88/00093 49 and human origin. In each case, track 1 contains murine (LB3) DNA and track 2 and 3 contain human DNA (from the cell lines Raji or Ramos, respectively). The hybridization and washing conditions applied to each filter are described in Step 2. The approximately 10 kbp Eco RI fragment containing the murine LIF gene, and the approximately 9 kbp Eco RI fragment containing the human homologue, are arrowed.

Molecular weight standards are given at the left. Two different autoradiographic exposures (16 hours and 62 hours) are shown.

Step 1: Preparation and Radioactive Labelling of a Fragment of cDNA from pLIF7.2b pLIF7.2b plasmid DNA was amplified by growth in E.coli MC1061 (Casadaban, M. and Cohen, S., J.Mol.Biol. 138:179-207, 1980), extracted from the E.coli cells by standard procedures (Maniatis, J. et al, Molecular Cloning, Cold Spring Harbor, New York, 1982) and purified by CsCl-gradient centrifugations.

pLIF7.2b plasmid DNA so purified was cleaved with the restriction endonucleases Eco RI and Hind III to release a cDNA-containing fragment of -770 bp from the vector (pJL3), which was resolved from vector sequences by electrophoresis through a 1.5% agarose gel.

200 ng of pLIF7.2 cDNA fragment thus purified was radioactively'labelled to a specific activity of approximately 3x10 8 cpm/pg by nick-translation (Rigby, Dieckmann, Rhodes, C. and Berg, P., J.Mol.Biol. 113:237-251, 1977) in a reaction containing 100pCi 32]-dATP. The radioactively labelled cDNA fragment was purified from unincorporated label-by precipitation in the presence of 1M sodium perchlorate and 33% isopropanol.

i I WO 88/07548 PCT/AU88/00093 Step 2: Hybridization of Southern Blots of Mouse and Human Genomic DNA with a pLIF7.2b cDNA Probe High molecular weight genomic DNA (15pg) from the murine T cell line LB3 (lane 1) and from two human B cell lines (Raji and Ramos; lanes 2 and 3) cleaved with the restriction endonuclease Eco RI was electrophoresed through a 0.8% agarose gel and transferred to nitrocellulose using standard techniques (Southern, E.

J.Mol.Biol. 98:503-517, 1975). Five identical Southern blots were prepared containing each of these three DNAs. Before hybridization, filters were incubated for several hours at 55*C in either 0.9M NaC1, 0.09M sodium citrate, 0.2% Ficoll, 0.2% polyvinyl-pyrollidone, 0.2% bovine serum albumin, 50pg/ml heat denatured salmon sperm DNA, 50 pg/ml E.coli tRNA, 0.1mM ATP and 2mM sodium pyrophosphate (filters a, b, c, d) or in 0.3M NaC1, 0.03M sodium citrate, with the same additional components (filter Hybridization with 32 the P-labelled pLIF7.2b cDNA prepared in Step 1 was performed in the same solutions as the prehybridization, containing in addition 0.1% sodium dodecyl sulphate, at 55 0 C (filters a, b, or (filters d, e) for 16 hours. The 32P-labelled cDNA was denatured by boiling prior to hybridization and was 7 included in the hybridization reaction at ~10 cpm/ml.

After hybridization the filters were washed in either 0.9M NaCI, 0.09M sodium citrate, 0.1% sodium dodecyl sulphate at 55 0 C (filter a) 60°C (filter b) or 0 C (filters c, d) or in 0.3M NaCI, 0.03M sodium S citrate, 0.1% sodium dodecyl sulphate at 65°C (filter After exhaustive washing, filters were autoradiographed at -70°C using Kodak XAR-5 film and two intensifying screens. Figure 21 shows the results of such WO 88/07548 PCT/AU88/00093 51 an experiment, in which two of the hybr.idization/washing regimes described (filters d and e) allowed both the murine LIF gene (present on an approximately 10 kbp Eco RI fragment) and the human LIF gene (present on an approximately 9 kbp Eco RI fragment) to be detected above residual background non-specific hybridization.

All other conditions tested gave rise to an unacceptably high level of background hybridization (filters, a, b, c).

EXAMPLE The following example sets out the steps used to obtain a cloned human gene homologous to murine LIF.

The accompanying Figures relate to various steps of the method described below. In the Figures: Figure 22:-relates to step 1 of the cloning of the human LIF gene: Detection of the LIF gene by Southern blot hybridization using a mouse LIF cDNA probe. Genomic DNA from the human cell line RAMOS was digested with the indicated restriction endonucleases and hybridized under the conditions described in Example 19, step 1, with a mouse cDNA fragment derived from pLIF7.2b.

Figure 23: relates to step 1 of the cloning of the human LIF gene: detection of the LIF gene by Southern blot hybridization using a mouse LIF cDNA probe under a variety of hybridization conditions. Genomic DNA from the human cell line RAMOS or mouse liver DNA digested with the restriction endonuclease Bam HI were hybridized with a murine LIF cDNA probe (pLIF7.2b) under a variety of S conditions of hybridization and washing. The temperatures and concentrations of SSC used for hybridization are indicated on the top line and the conditions of washing on the second line (see Example 10, step 1).

Figure 24: relates to step 2 of the cloning of the human 41 0

S.

0 o ff *00 .00.

S@ S

S

S

0@*S

OS

S.

S~

WO 88/07548 PCT/AU88/00093 52 LIF gene: restriction endonuclease cleavage of three candidate LIF gene clones. 1 pg of DNA from X phage of the clones XHGLIF1, XHGLIF2 and XHGLIF3 was digested with Sal I, Bam HI or Pst I, electrophoresed on an 0.8% agarose gel (left hand panel), transferred to nitrocellulose and hybridized (as disclosed previously) with pLIF7.2 cDNA. The approximate sizes of the hybridizing fragments are indicated.

Figure 25: relates to step 3 of the cloning of the human LIF gene: The nucleotide sequence of the mRNA- synonymous strand of a 1.3kbp segment of )HGLIF1 spanning the human LIF gene is presented in a 5' to 3' orientation. The corresponding nucleotide sequence of the murine LIF mRNA derived from cDNA clones pLIF7.2b, pLIFNK1 and pLIFNK3 15 is aligned beneath the human gene and given in lower case letters. Identities between the mouse and human sequences are indicated with asterisks. The presumed N-terminal residue of mature human LIF, by analogy with mouse LIF, is designated as +1.

Figure 26: relates to step 3 of the cloning of the human LIF gene: amino acid sequence of human LIF and comparison with mouse LIF. The amino acid sequence of mature murine LIF as determined by direct amino acid sequencing, and analysis of the cDNA clones pLIF7.2b, pLIFNK1 and 2 pLIFNK3 is listed on the top line, with the-corresponding sequence of human LIF as deduced from the sequence of XHGLIF1, listed below. Identities are indicated with dashes; differences are indicated by designation of the amino acid.

Figure 27: relates to step 4 of the cloning of the human LIF gene: restriction endonuclease cleavage map of the human LIF gene. The exons of the human gene homologous with pLIF7.2b (Figure 25) are indicated as boxes. The direction of transcription of the gene is indicated by the arrow below the line.

oV WO 88/07548 PCT/AU88/00093 53 Step 1: Detection of the human LIF gene with a mouse probe.

A method has been previously disclosed for using a radioactively labelled fragment of the mouse LIF cDNA clone pLIF7.2b as a hybridization probe to detect the human LIF gene. Figure 22 demonstrates that this method allows the human LIF gene to be detected in human genomic DNA digested with a variety of restriction endonucleases. Analyses such as this, and other gels not shown, revealed the sizes of DNA fragments generated by a variety of restriction endonucleases on which the human LIF gene is located. Such data is of importance in subsequent steps of this example, not only to establish conditions under which the mouse probe can be used as a hybridization probe to detect the human, but also to provide diagnostic restriction mapping data to aid in identifying human genomic LIF clones.

A high degree of homology between the mouse and human LIF sequences was apparent when mouse and human genceic DNA digested with Bam HI was hybridized with a mouse LIF cDNA probe under a variety of conditions of hybridization and washing (Figure 23). As the stringency of hybridization and washing was raised, so the background smear was reduced, revealing a unique fragment of -3 kbp hybridizing with the mouse probe. Significantly, the human gene retained substantial hybridization even at in 0.2 x SSC.

Step 2: Screening of a human genomic library and isolation of a clone containing the LIF gene.

A library of human genomic DNA, partially digested with Sau 3A and ligated into the lambda phage cloning vector EMBL3A, was screened for LIF gene- WO: 88/07548 PCT/AU88/00093 54 containing clones by hybridization with the mouse cDNA as a probe. The fragment of LIF cDNA was radioactively labelled and the conditions of hybridization were as disclosed previously (Example Briefly, phage plaques representing the genomic library wer grown at a density of -50,000 plaques per 10 cm petri dish and transferred to nitrocellulose as described (Maniatis, T.

et al, Molecular Cloning, Cold Spring Harbor, 1982).

Prior to hybridization, filters were incubated for several hours at 65 0 C in 6 x SSC (SSC 0.15M NaC1, 0.015M sodium citrate), 0.2% Ficoll, 0.2% polyvinyl-pyrollidone, 0.2% bovine serum albumin, 2mM sodium pyrophosphate, ImM ATP, 50 pg/ml denatured salmon sperm DNA and 50 pg/ml E.coli tRNA.

Hybridization as in the same solution containing 0.1% SDS, at 65 0 C for 16-18 hours. The LIF cDNA fragment, radioactively labelled by nick-translation using S32P] dATP to a specific activity of -2 x cpm/pg was included in the hybridization at a 6 concentration of -2 x 10 cpm/ml. After hybridization, filters were extensively washed in 6 x SSC, 0.1% sodium dodecyl sulphate at 65 0 C and then autoradiographed. Plaques positive on duplicate filters were picked and rescreened at lower density, as before.

Three clones were thus identified and purified: XHGLIFl, 2 and 3. In order to determine the relationship between these clones and to determine which, if any, contain the human LIF gene, DNA was prepared from each clone and digested with these restriction endonucleases: Sal I which liberates the entire segment of cloned genomic DNA and Bam HI and Pst I which cleave in and around the LIF gene to generate characteristic fragments of ~3 kbp and 1.8 kbp and 0.6 kbp respectively (as determined above). After digestion of WO 88/07548 PCT/AU88/00093 the recombinant phage DNAs and resolution by electrophoresis on agarose gels (Figure 24, left hand panel), the DNA was transferred to nitrocellulose and hybridized with the mouse LIF cDNA probe (under the conditions outlined above) to reveal the fragments containing the LIF gene (Figure 24, right hand panel). This analysis revealed that all three clones appeared to be identical; all contain an -9 kbp segment of genomic DNA containing a region homologous with the mouse LIF probe; the region of homology with the mouse cDNA is present on a 3 kbp Bam HI fragment and on 1.8 and 0.6 kbp Pst I fragments, characteristics of the human LIF gene (see above). Thus it was concluded that all three clones contain a segment of chromosomal DNA encompassing the human LIF gene.

Step 3: Determination of the nucleotide sequence of the human LIF gene.

The -3 kbp Bam HI fragment of XHGLIF1 shown above to hybridize with the mouse LIF cDNA probe was recloned into the plasmid vector pEMBL8 giving rise to clone pHGLIFBaml, and subjected to nucleotide sequence analysis. Nucleotide sequencing was performed by the dideoxy chain termination method (Sanger, F et al, Pro.Natl.Acad.Sci.USA. 74: 5463-5467, 1977) using alkaline denatured double-stranded plasmid DNA as template, a variety of oligonucleotides complementary to sequences within the gene as primers, and using both the Klenow fragment of E.coli DNA polymerase I and the avian myeloblastosis virus reverse transcriptase as polymerases in the sequencing reactions.

The entire nucleotide sequence of the Bam HI fragment spanning the LIF gene (2840bp) was determined, of k! WO 88/07548 PCT/AU88/00093 56 which 1297bp is shown in Figure 25. Alignment of this sequence with the murine LIF mRNA sequence revealed that the sequences encoding the mature human LIF protein are present on two exons separated by an intron of 693bp (Figure 25). For the region encoding the mature protein there is a high degree of homology between the two species at both the nucleotide and the amino acid sequence level.

Within exon 1 there is 88% nucleic acid sequence homology (114/129 residues compared) and 91% amino acid sequence homology (39/43) for the region encoding the mature protein (position 58-186). Exon 2 is somewhat less homologous, 77% at the nucleotide level (318/411) and 74% at the amino acid level (101/136) within the coding region. Considering the mature protein as a whole, mouse and human LIF sequences determined here are identical at 140 of 179 positions with no insertions or deletions (Figure 26). Moreover, many of the differences are highly conservative substitutions (Lys:Arg, Glu:Asp and Leu:Val:Ala).

5' of the codon for the N-terminal proline residue of mature LIF, (position 58 in Figure 25) the human gene is homologous with the murine LIF mRNA sequence through a region encoding most of the hydrophobic leader.

However, the entire leader is not encoded on this exon, 2 since the mRNA and gene sequences diverge at a typical RNA splice site (TCCCCAG) (Mount, Nucleic Acids Res.

10:459-472, 1982). The exon specifying the untranslated region and the first residues of the leader is not present within 1097 bp 5' of this splice site. In the mouse LIF gene, the exon specifying the first 6 amino acid residues of the hydrophobic leader is located 5' of the analogous splice site.

Step 4: Derivation of a restriction endonuclease cleavage WO 88/07548 PCT/AU88/00093 57 map of the human LIF gene.

In order to determine the disposition of the restriction endonuclease cleavage sites in and around the human LIF gene, and thus provide a molecular fingerprint of this gene, human genomic DNA (from the RAMOS cell line) was digested with various restriction endonucleases singly and in pair-wise combinations and subject to Southern blot analysis as described in Example 8, except that the probe used was the 3 kbp Bam HI fragment derived from pHGLIFBaml described in Step 3 above and radiolabelled by nick-translation. Analysis of the data so-derived (not shown), as well as that shown in Figure 22 and derived from analysis of XHGLIF1 and pHGLIFBaml, gave rise to the restriction endonuclease cleavage map shown in Figure 27.

EXAMPLE 11 The following example details modifications made to the cloned human LIF gene in order to allow expression in yeast cells and determination of the biological and biochemical properties of the recombinant human LIF so derived.

In the Figures, Figure 28: relates to step 1: oligonucleotides used to modify the human LIF gene for incorporation into YEpsecl.

Oligonucleotide corresponds to the 5' end of the coding region (residues 31 to 69 in Figure Oligonucleotide corresponds to the middle of the coding region (residues 163 to 186 and 880 to 903 in Figure 25). The portion of this oligonucleotide complementary to exon 1 is underlined with dashes, and that complementary to exon 2 with dots. Oligonucleotide corresponds to the 3' end of the coding region (from S position 1279 in Figure 25). Oligonucleotides and (b) I l i 1 3

I

WO 88/07548 PCT/AU88/00093 58 introduce the indicated restriction endonuclease cleavage sites.

Figure 29: relates to step 1: nucleotide sequence of, and amino acid sequence encoded by, the synthetic human LIF cDNA derived by mutagent sis of the cloned human LIF gene. The Bam HI and Hind II cleavage sites introduced by oligonucleotides and (Figure 28) are indicated.

The presumed N-terminal amino acid of mature LIF (by analogy with the mouse) is designated as +1.

Figure 30: relates to step 3: Induction of differentiation in colonies of Ml leukaemic cells by dilutions of purified native murine LIF and conditioned medium from yeast cells containing the YEpsecl/HLIF recombinant induced with galactose Medium from uninduced yeast cultures containing the YEpsecl/HLIF recombinant (o o) was inactive. Mean data from replicate 7 day cultures is presented.

Figure 31: relates to step 4: competition of 125 yeast-derived HLIF with native murine 125I-LIF for binding to specific receptors on murine M1 cells.

Dilutions of authentic native murine LIF-A (Example 1) (o recombinant murine LIF and conditioned medium from yeast cells containing the YEpsecl/HLIF construct uninduced U_ and induced with galactose were tested for ability to compete for the binding of native 125 I-LIF-A to cellular receptors on murine M1 cells at 37°C, as disclosed previously.

Step 1: Modification of the human LIF gene for expression in yeast.

The isolation of a recombinant DNA clone containing the human LIF gene and the nucleotide sequence of the aforementioned gene has been previously disclosed WO 88/07548 PCT/AU88/00093 59 (Example 10). The nucleotide sequence of 1297bp of DNA spanning the 2 exons encoding the mature human LIF protein is shown in Figure Murine recombinant LIF has previously been produced in yeast cells using the yeast expression vector, YEpsecl (Example This vector provides an N-terminal leader sequence derived from the killer toxin gene of Kluyveromyces lactis, transcribed from a galactose-inducible hybrid GAL-CYC promoter.

In order to express the protein encoded by the human LIF gene in this vector it was necessary to modify the gene in several ways. At the 5' end of the region encoding the mature protein a cleavage site for the restriction endonuclease Bam HI was introduced to allow insertion in frame with the K.lactis leader and retain an appropriate signal peptidase cleavage site (Gly-Ser).

The same modification as previously applied to the mouse cDNA (pLIF7.2b) was made here. In the middle, the 693 bp intervening sequence was removed, fusing the two exons in the same translational reading frame. At the 3' end, a second translational stop codon was introduced immediately 3' of the natural stop codon, followed by a Hind III site for insertion into YEpsecl. All of the modifications were achieved by oligonucleotide-mediated mutagenesis: the ~3kbp Bam HI fragment spanning the LIF gene was subcloned into the plasmid pEMBL8 (Dente, L. et al, Nucleic Acids Res. 11:1645-1655, 1983) and single-stranded DNA prepared by Fl superinfection. In vitro mutagenesis was performed as described (Nisbet, I.T. and Beilharz, M.W. Gene Anal. Techn. 2:23 29, 1985), using oligonucleotides of 39, 48 and 39 bases respectively to modify the 5' end, the middle and 3' end of the gene as outlined above (see Figure 28). The nucleotide sequence of the modified human LIF coding region is given in Figure 29.

4 t WO 88/07548 PCT/AU88/00093 Step 2: Introduction of the YEpsecl/HLIF recombinant into yeast cells.

S.cerevisiae strain GY1 (leu2 ura3 ade2 trpl ci.r+; from G. Cesareni, EMBL Heidelberg) was transformed by the polyethylene glycol method (Klebe, R.J. et al, Gene 333-341, 1983). Transformants were selected and maintained on synthetic minimal medium carbon source, 0.67% yeast nitrogen base (Difco) supplemented with of the required amino acids) under uracil deprivation. Recombinant HLIF was produced by either of two methods. Either Ura transformants were grown to: stationary phase in non-selective medium containing 2% galactose, and the medium assayed for LIF activity or Ura+ transformants were grown to stationary phase in selective minimal medium containing 2% glucose. Cells were then washed and resuspended in the same volume of selective minimal medium containing 2% ethanol and grown for 8 hours to overcome glucose repression. Transcription of the HLIF insert was then induced by diluting the cells (1:10) into synthetic minimal medium containing 2% galactose. Aliquots of culture supernatant were removed at vazious times after induction, filtered through Millipore filters (0.2im) and assayed for LIF activity directly.

Step 3: Determination of the biological properties of yeast-derived HLIF.

In view of the high degree of sequence similarity between mouse and human LIF, the activity of yeast derived human LIF on murine M1 cells was assessed. Assays for differentiation-inducing activity and leukaemiasuppressive activity of yeast conditioned medium were performed in 1 ml cultures containing 300 murine Ml cells v I 1 WO 88/07548 PCT/AU88/00093 61 (provided by Dr M. Hozumi, Saitama Cancer Research Centre, Japan) in Dulbecco's Modified Eagle's medium with a final concentration of 20% foetal calf serum and 0.3% agar.

Material to be assayed was added in serially diluted 0.1 ml volumes to the culture dish prior to the addition of the cell suspension in agar medium. Cultures were incubated for 7 days in a fully humidified atmosphere of

CO

2 in air. Cultures were scored using a dissection microscope at x35 magnification, scoring as differentiated any colonies with a corona of dispersed cells or composed wholly of dispersed cells. Morphological examination of colonies was performed by fixing the entire culture with 1 ml 2.5% glutaraldehyde then staining the dried cultures on microscope slides using acetylcholinesterase/Luxol Fast Blue/Haematoxylin.

Medium from galactose-induced cultures of yeast containing the human coding region in YEpsecl, but not from uninduced cultures of the same yeast cells, from cultures of non-transformed yeast, or yeast containing the vector YEpsecl alone, was able to induce typical macrophage differentiation in cultures of M1 colonies (Figure 30). As with murine LIF, with increasing concentrations the yeast-derived human material also progressively reduced the number and size of M1 colonies developing. Comparison with purified native murine LIF indicated that the yeast conditioned medium contained up to 50,000 Units/ml of human LIF.

Step 4: Receptor binding specificity of yeast-derived human LIF.

Purified murine LIF-A (Example 1) was iodinated as disclosed previously. Ml cells were washed and resuspended at 2.5 x 10 6 /50pl in Hepes-buffered RPMI medium containing 10% foetal calf serum. Cells in 50 pl WO 88/07548 PCT/AU88/00093 62 aliquots were incubated with 200,000 cpm of 125

I-LIF-A

in the same medium) and 10pl of control medium or serial two-fold dilutions of unlabelled pure murine LIF-A or conditioned medium from galactose-induced or uninduced cultures of yeast transformants containing the YEpsecl/HLIF construct.

Medium conditioned by galactose-induced yeast cells containing the Y T psecl/HLIF recombinant was able to 12.5 compete for the binding of native murine 1I-LIF-A to specific cellular receptors on murine Ml cells to the same extent as native and recombinant murine LIF-A, at 37 0

C.

(Figure 31) and at 0°C (not shown). Medium from uninduced yeast cells and from yeast cells containing the vector YEpsecl alone did not. Thus there appears to be a strong conservation of the receptor binding domain in murine and human LIFs, compatible with the high degree of primary amino acid sequence similarity.

Step 5: Purification, sequencing and iodination of yeastderived human LIF.

Human LIF in medium conditioned by galactoseinduced yeast cells containing the YEpsec 1/HLIF recombinant was purified using steps 2 and 4 of Example 1, except that the LIF activity binding to the DEAE-Sepharose CL-6B column and eluted with the salt gradient was pooled in step 2. Purified yeast-derived human LIF was radioiodinated by incubating lpg of human LIF in of 0.2M sodium phosphate buffer, pH 7.2, with ImCi of Na 125I (2.7pl) and 5pl of 0.2mM IC1 in 2M NaC1 for 125 125 3 sec. I-LIF was separated from unincorporated 125I 3O by passage of the reaction mixture through a column of Sephadex G-25M (Pharmacia) equilibrated in phosphate buffered (20mM, pH 7.4) saline (0.15M) containing 0.02% Tween 20 and 0.02% sodium azide. Human 125I-LIF WO 88/07548 PCT/AU88/00093 63 electrophoresed on a 8-25% gradient sodium dodecyl sulphate polyacrylamide gel as a single broad band with 125 apparent molecular weight of 170,000. Human I-LIF bound specifically to murine M1 cells and bone marrow cells confirming the ability of human LIF to bind to murine LIF receptors. Purified yeast-derived human LIF (approx. 10pg) was subjected to amino-terminal amino acid sequencing as described in Example 2 and gave a single sequence of: Ile-Thr-Pro-Val-X-Ala This is identical to the predicted amino acid sequence of human LIF (Figure 26) except that the sequence begins at the fourth amino acid compared to the start of the murine sequence (see Example 2) which is: Pro-Leu-Pro-Ile-Thr-Pro-Val-Asn-Ala This indicates that the purified yeast-derived human LIF is missing the corresponding first three amino acids of the mouse sequence but is still biologically active and still able to bind to the murine LIF receptor. The first 2 three amino acids thus appear to be dispensable for the biological activity of LIF.

EXAMPLE 12 The following example sets out the steps used to identify and partially purify a putative native human LIF molecule.

The accompanying Figures relate to various steps of the method described below. In the Figures: Fiqure 32: relates to step 2 of the identification of a 3 putative human LIF: the ability of different dilutions of medium conditioned by the human bladder carcinoma cell line 5637 (ATCC No. HTB9) 0 crude or DEAE non-binding fraction or of native murine LIF-A o 125 to compete for the binding of murine I-LIF-A to -r it -i i

I

WO 88/07548 PCT/AU88/00093 64 cellular receptors on murine peritoneal cells. The inability of human G-CSF U to compete for binding is also shown (undiluted Figure 33: relates to step 3 of the identification of a putative human LIF: the fractionation of medium conditioned by the human bladder carcinoma cell line 5637 on a column of DEAE- Sepharose CL-6B,.eluted exactly as for the previously described fractionation of murine LIF-A (Example and the ability of individual fractions from this fractionation to induce the formation of differentiated colonies of murine M1 cells. Panel A shows the salt gradient, Panel B shows the fractionation of 5637 conditioned medium; Panel C shows the fractionation of Krebs II cell conditioned medium for comparison.

Figure 34: relates to step 3 of the identification of a putative human LIF: the fractionation of medium conditioned by the human bladder carcinoma cell line 5637 on a column of lentil-lectin Sepharose 4B eluted as previously described for the fractionation of murine LIF-A (Example 1) and the ability of individual fractions from this fractionation to induce the formation of differentiated colonies of murine Ml cells. Panel A shows the gradient of a-methyl-D-mannopyrannoside, Panel 2 B shows the fractionation of 5637 conditioned medium and Panel C shows the fractionation of Krebs II conditioned medium for comparison.

Step 1: Medium conditioned by several human cell lines 3 were assayed for their capacity to induce the differentiation and inhibit the proliferation of M1 murine myeloid leukaemic cells in semi-solid agar cultures (as described in Example 5, Step 4 of the present application). Several cell lines produced such an i i- WO 88/07548 PCT/AU88/00093 activity, of which the bladder carcinoma cell line 5637 produced the highest levels. However, the 5637 cell line has been shown previously to produce human G-CSF (Nicola, N.A. et al, Nature 314: 625-628, 1985; Welte, K. et al, Proc.Natl.Acad.Sci. USA 82: 1526-1530, 1985) which is also active in inducing differentiation of Ml cells (Tomida, M. et al, FEBS Lett. 207: 271-275, 1986; Neckers, L.M. and Pluznik, D.H. Exp.Hematol. 700-703, 1987). To confirm that 5637 cells produced authentic human LIF (in addition to G-CSF), 5637-conditioned medium was further subjected to Steps 2 and 3 below.

Step 2: Medium conditioned by 5637 cells was concentrated 20- fold and tested for its ability to 125 compete for the binding of native murine I-LIF-A to specific cellular receptors on murine peritoneal cells.

This competition binding assay was as disclosed in Step 5 of Example 5 in the present application. 5637 2 cell-conditioned medium contained activity capable of 125 competing for the binding of murine I-LIF-A to cellular receptors and this activity was concentrated in the DEAE non-binding fraction of 5637 CM (LIF-A) (Figure 32). Since human and murine G-CSF do not compete, even 125 at very high concentrations, for 125I-LIF-A binding sites, this establishes the presence in 5637-conditioned medium of a homologous human LIF activity capable of recognizing specifically the murine LIF receptor. This indicates strong conservation of the murine and human LIFs in their receptor binding do.main, compatible with the high degree of primary amino acid sequence homology.

Step 3: Medium conditioned by human bladder carcinoma 5637 cells (two litres in 10% v/v foetal calf serum) was i _L -i WO 88/07548 PCT/AU88/0009,3 66 concentrated to 40 ml and chromatographed sequentially on DEAE-Sepharose CL-6B and lentil-lectin-Sepharose 4B as described previously for murine LIF from Krebs II ascites cell-conditioned medium. Ml d-fferentiationinducing activity from 5637 cells (putative human LIF) chromatographed in a very similar fashion to murine LIF on DEAE-Sepharose,' with some activity not binding to the column (LIF-A), while the remainder bound and was eluted during the salt gradient (LIF-B) (Figure 33). Similarly, the putative human LIF behaved like murine LIF on lentil-lectin-Sepharose chromatography with a proportion of activity binding to the column indicating the presence of mannose-containing carbohydrates on the glycoprotein (Figure 34). Thus by its cross-reactivity in inducing murine Ml cell differentiation, its ability to recognize specifically the murine LIF cellular receptor, and its biochemical fractionation characteristics, the human activity in 5637 cell conditioned media meets the criteria of the native human analogue of murine LIF.

Native human LIF from 5637 cell conditioned medium was purified by steps 2 and 4 of Example 1, pooling the non-binding LIF activity from step 2 and the binding LIF activity from step 4. This pooled LIF activity was fractionated by reverse-phase HPLC as for step 5 of Example 1 except that a Brownlee RP300 C8 column was used twice, first using a gradient from 0-60% CH 3 CN in 0.1% TFA and then using a gradient from 45-55% CH3CN in 0.1% TFA. In the second gradient human LIF eluted at CH3CN and when electrophoresed on 8-25% gradient sodium dodecyl sulphate polyacrylamide gel- showed a major silver staining band of apparent molecular weight 73,000. Native purified human LIF was radioiodinated as described in step of Example 10 and bound specifically to murine Ml cells and mouse bone marrow cells as described for yeast-derived human 1 2 5 I-LIF (step 5 of Example 11).

i, ~L i i

Claims (28)

1. A composition comprising leukemia-inhibitory factor (LIF) in essentially pure form.

2. The composition of claim 1 wherein the LIF is murine LIF.

3. The composition of claim 1 wherein the LIF is human LIF.

4. A method of purifying glycosylated or non-glycosylated LIF, which comprises chromatographing LIF in crude form on an anion exchange column, and eluting LIF by an increasing salt gradient, chromatographing the product of step on a lectin affinity column, said lectin having an affinity for mannose, and eluting LIF with a mannose derivative, chromatographing the product of step on a cation exchange column and eluting LIF with an increasing salt gradient, (d) chromatographing the product of step under HPLC conditions on a reverse phase column and eluting LIF with an increasing gradient of acetonitrile. The method of claim 4, wherein the LIF in crude form comprises a Krebs II ascited tumour cell conditioned medium, and murine LIF is purified from said medium.

6. The method of claim 4, wherein the LIF in crude form comprises human bladder carcinoma cell line 5637, ATCC Number HTB9, conditioned medium, and human LIF is purified from said medium. -68-

7. A recombinant DNA molecule comprising a nucleotide sequence which codes on expression in a suitable host cell for a polypeptide having LIF activity as hereinbefore defined.

8. A recombinant DNA molecule comprising a nucleotide sequence which codes for a polypeptide having the amino acid sequence selected from the group consisting of those set forth in Figures 15, 26 and 29.

9. The molecule of claim 7, wherein said nucleotide sequence hybridizes to a nucleic acid probe corresponding to a subsequence of a LIF encoding insert selected from o the group consisting of the LIF encoding inserts of clones pLIF7.2b, pLIFNK2, pLIFNK3, and pHGLIFBaml as S..hereinbefore defined.

10. The molecule of claims 7, 8 or 9 wherein the nucleotide sequence codes on expression in a eukaryotic host for a polypeptide which comprises a mature polypeptide which has at least 50% homology with the amino acid sequence selected from the group consisting of those set forth in Figures 15, 26 and 29 and which has LIF activity as hereinbefore defined. S11. The molecule of claim 10, wherein the mature polypeptide is fully homologous with the amino acid sequence set fotth in Figure 29.

12. The molecule of claim 7, wherein said nucleotide codes on expression for a polypeptide which is bound by cellular receptors which bind LIF, said binding being competitively inhibited by native murine or human LIF.

13. The molecule of claim 7, further comprises an origin of replication, whereby said molecule is rendered suitable for use as a cloning vector. RAZ -69-

14. The molecule of claim 13, further comprising a promoter sequence operably linked to said nucleotide sequence whereby said molecule is rendered suitable for use of an expression vector. A host cell transformed with a recombinant DNA molecule according to claim 14, wherein said cell expression a polypeptide having LIF actually under the control of said promoter.

16. A method of producing a polypeptide having LIF activity as hereinbefore defined which comprises: (a) providing host cells according to claim 15, (b) cultivating said cells under conditions conducive to expression of a polypeptide having LIF activity; and (c) recovering said polypeptide.

17. The method of claim 16, wherein the polypeptide comprises a mature polypeptide which has at least homology with the amino acid sequence selected from a group consisting of those set forth in Figures 15, 26 and

29. 18. The method of claim 16, wherein the mature polypeptide is fully homologous with amino acid sequence set forth in Figure 29. S 19. The method of claim 16, wherein the polypeptide is glycosylated by the host cell. The method of claim 16, wherein the host cell is a yeast cell. 0rv~12 'AT LL I: WO 88/07548 PCT/AU88/000f(3 21. The method of claim 16, wherein the promoter is a galactose inducible hybrid GAL-CYC promoter. 22. The method of claim 16, wherein said molecule further comprises a signal sequence operably linked to said nucleotide sequence, said signal sequence coding an expression for a leader sequence directing the secretion of the polypeptide. 23. The method of claim 22, wherein the signal sequence is derived from the signal sequence of the killer toxin gene of Kluvveromyces lactis. 24. The method of claim 20, in which the host cell is of the species Saccharomvces cerevisiae. The method of claim 16, wherei:n the host cell is a mammalian cell. 26. The method of claim 25, wherein the host cell is transformed with a retroviral expression vector. 27. The method of claim 25, wherein the vector is derived from the Moloney murine leukemia virus. *0 0* 9 0@r 0 0 00.. 0000 0 00 0 00 28. The method of claim 27, comprises the LTR enhancer of virus. 29. The method of claim 28, the 3' untranslated region of The method of claim 25, hemopoietic cells. wherein the vector further myeloproliferative sarcoma wherein said vector lacks native LIF mRNA. wherein the host cells are 1~ )0093 I ii II t IWO 88/07548 PCT/AU88/00093 /1/35 m I I -71-

31. The method of claim 16, wherein the host cells are E.coli cells.

32. The method of claim 31, wherein the vector is one which directs the expression of a glutathione S-transferase/LIF fusion protein.

33. The method of claim 32, wherein the vector is one which directs the expression of a glutathione S-transferase/thrombin cleavage site/LIF fusion protein. 34 A polypeptide having LIF activity, prepared by the method of claim 16.

35. A polypeptide having LIF activity as hereinbefore defined, encoded by a recombinant DNA molecule according to claim 7 which is not identical in amino acid sequence to any naturally occurring form of LIF.

36. The polypeptide of claim 35, which has an amino acid sequence comprising amino acid residues 179-187 of natural :murine LIF.

37. The polypeptide of claim 36, which has an N-terminal e* which lacks at least the first three amino acid residues of natural murine LIF.

38. A method of hindering the proliferation of myeloid leukaemia cells which comprise administering a pharmaceutically effective dose of polypeptide having LIF activity as hereinbefore defined in a pharmaceutically acceptable form.

39. The method of claim 38, which further comprises administering a polypeptide having G-CSF or GM-CSF activity. -72- A pharmaceutical composition comprising a polypeptide having LIF activity as hereinbefore defined in association with one or more pharmaceutically acceptable carriers and/or diluents.

41. The composition of claim 40, wherein said polypeptide comprises a mature polypeptide which has at least 50% homology with the amino acid sequence selected from the group consisting of those set forth in Figures 26 and 29.

42. The composition of claim 41, wherein the mature peptide is fully homologous with the amino acid sequence set forth in Figure 29. 4

43. The composition of claim 40, further comprising at least one other biological regulator of blood cells.

44. The composition of claim 43, wherein said other regulator is a polypeptide having G-CSF or GM-CSF activity. A diagnostic reagent or probe comprising at least one nucleic acid sequence corresponding to a sequence or *0 subsequence of an LIF encoding insert selected from the group consisting of the LIF-encoding inserts of clones pLIF7,2b, pLIFNKl, pLIFNK3, and pHGLIFBaml, or to a 'sequence or subsequence of the corresponding mRNAs. 44 DATED this 24th day of December, 1990 AMRAD CORPORATION LIMITED By Its Patent Attorneys DAVIES COLLISON t V7\ 1z L 'y r WO 88/07548 WO 8807548P CT/AUt88/00093 I//iS -4- a) 0 0- 0- 0-4 0 3 0 2 0.1 0 120 10.0 0 100 0 100 0 S120 100 0 40 60 80 100 Fraction number FIG.!/. SUBSTITUITE RHF=FT, 120 140 WO 88/07548 PCT/A U88/00093 I WO 88/07548 PCr/AU88/00093 255S wc-- EO (I) 0 0-- C: a)i C- 0-a o 6 0-4 0-2 0 0-3 0-4 0-3 0-2 0.1 0 100 2 0 6 0 Fraction number F1IG.2. SUBSTITUTE SHEET NVO 88/07548 WO 8807548PCT/AU88/00093 335 10.0 0 100 0 40 Fr~action number F G.J SUBSTITUTE SHEET ~~1 WO 88/07548 P CT/A U88/00093 43J5 C 2 0- 100 0 E C C co 0002 Li, ED 0,002 E 0 £lt 010 Z 005 0 0 34,8 360 372 3B84 396 408 42-0 43-2 444 45-6 478 49-0 Acetonitrite FIG .4. SUBSTITUTE SHEET Differentiated colonies Molecular weight (x10- 3 r-1 4r- ON CC) C) L C C OC)CCDCD C) W C I CD -1 C7% m-- CDCD CD C)1 00 00 WO 88/07548 WO 8807548PCT/AU88/00093 63IJ5 20,000- Multi CSF GM-CSF ~G-CSF M- CSF E 16,000- -~12,000- 0 m0 Li-

8000- -n 1- 0 0 512 1 28 3'2 8 Reciprocni dilution 20,00 E 16,00 12,0( 0 800 -Tj Ll 0- S o cline B CM LIF End Iotoxi rum SCM E9-D4 LPS C M lOpg/ml) n L ce[: EL-4 CM CM Comoetitclr FIG. 6. SUBSTITUTE SHEE WO 88/07548 WO 88/0758P CT/ A 88/00093 735J a. GM-{SF 1 2 3 b. Mult-CSF 1 2 3 28S- 23S- 18S- 16S- .4. q 'S FIG. 7 Hi sProC ysHi sG IyAsnLeuMetAsnG In I teLys CACC C CNU 5CC ACGGNAACUUAAUGAACCAAAUCAAA Ui UU UG 2' TGGGGACEGT4 CCATGATCTj3CAT p7GGAT7GUCAT- E L IF LIF mRNA L IF 0 L kjlC I FIG. 8.I SUB3STITUTE SI-4iF=-- T- WO 88/07548 W O 8807548PCT/AU88/00093 81 4 -J -n 4 G6 7.. 0~ '0 S-U P ST'-'TU-*T-E HEET SHEET WVO 88/07548 PCT/ AU88f00093 9/35 Hi sTrpLysHisG I yA IaG I ySer Pro LeuProl It elh rProVQi[AsnA 1 17 CACTGGAAACACOGGGCAGGGAGCCCTCTTCCCATCACCCCTGTAAATGC 5 0 lT h r Cy sA[ l I Ie A rgH is Pr o C ysH isGLI yAs nL e uM etfA s nGtIn IIeL 34 C A C CT GTG CC ATA C GCC A CC CA T GCC AC G GC AACC T CAT GAA C CAGAT CA 100 ys As n G (nL e u A Ia.GInLe u A sn GIy S e -A IaA snAI aL e uP heI Ile SeFr AGAATCAA[TGGCACAGCTCAATGGCAGCGCCAATGCTCTCTTCATTTCC 150 TATTACACAGCTCAAGGAGAGCCGTTTIC CCAA[AAC GTGGAAAAGCTATG .200 s A [caP r oA snM et Th rAs pP h eP ro SeFrP h eHi s G I y A snG t yTh r Gt uL 84 TGCGCCTAACATGACAGACTTCCCATCTTT CCATGGCAACGGGACAGAGA 250 y slTh rLy sL e uVl I[u Le uT y rA r gM e fV at AIciTy rLe u Se rA( a Se r 100 A GA C CAAGT TG GT G GAG CT GT ATC G GAT G GT C GCATA C CT GAGC G CC T CC 300 L euT hr As n II eT h rA r gA s pIn L y sV t L euA snP r oT h r A[V aIS e 117 CTGACCAATATCACCCGGGACCAGAAGGTCCTGAACCCCACIGCCGTGAG 350 rLeuGtnVaLLysLeuAsnAiQThrleAspVcilMetArgGtv/LeuLeuS 134 CCTCCAGGTCAAGCTCAATGCTACTATAGACGTICATGAGGGGCCTCCTCA 400 e rAsnVQL[ LeuC ysArgLeuC ysAs nLysTyrA rgV~alGlI yH sVQI Asp 150 GCAATG-TGCTTTGCC GTCTGTGCAACAAGTACCGTGTGGGCCACGTGGAT 450 VQ! PFQPr oVoI PFoAs pH sSerAspLysG I uA iPheG InArg LysLy 167 GTGCCACCTGTCCCC GACCACT CTOACAAAGAAGCCTT1CCAAAGGMAAAA sLeuG IyCy sG n LeuLe uL'yThrTy L YSG~n 17E GTTGGGTTiGCCAGCTTCr'TGGG3GACATACAAGCAAG. 5 3 FIG. /0 SUBSTITUTE SHEET I ,I A 4 -07WJ o WO 88/07548 P CT/AUL88/00093 162 A F Q P K K L G CO0 L L G T Y K, A F a K K L GC Q L L 6 T Y K A F 0 RK K LGC QOL L G TY K 613CCTT C CAAAGGAAAAA6 TTGGOTTG [GAG C TTC TGGGACATA CAAG pLIF7. 2b cDNA '0 I S V V V a A F V8 peptide aV I S V V X 0 A F 'C V I S V V V Q A F CAAGTCATAAGTGTGGTGGTCIAG GCCTTCTAG Tryptic peptide pLIFNKI cDNA FIG.!! SUBSTJITUTE SHEET WO 88/07548 WO 88/0758P CT/A U88/00093 /135 pLIF7. 2b p LI Fmut 1 pLlFmuf 2 178 t yAaG(ySerPFoLeu LysG[ n 5 GGGGAGGGA3CQCTT- -AACCAAGi G[I ySerProLeu---------L ysG~n*, GGATC IZ[CTCTT---------AAGCAAT G [y Se rProLeu- LsGt nVi7 GGATCCCCTCTT---------AAGCAAC't YEpsec leaider GI nG I yThrArgGl. ySer- CAAGGTACCCLGGGGATCC Barn Hi IAATAGATATCAAGCTT al I eSerVal Vatl Val 61 nAt a Phe IT 6ATC AGC 6TGGTGGTGC AGGCC TTC TAATAG6ATATC AAG CTTAGCT[GAATTC Hi nd 3 EFco RI SUBSTITUTE SHEET 74O 88/07548 C/ 8/009 PCT/AU88/00093 /123J5 (A S100. 0 ci cJ Li L- ai n~ Dilution of LIF preparation assayed f--IG. IJ -r 6000. 40001 2000il 02 J- Reciorc.--. Ulturl FIG./14. 1UBSTITUTEs -Mona WO 88/07548 WO 88/07548PCT/A U88/00093 U188/ 00093 IWO 88/07 /3/35 Ec oR 1Me fLysVQa[LeuAtaA I cG Iy It eVa [P GAATTCCGGAGTC CAG CC CAT AATGAAGGTC TTGGECCGCAGGGATTGiTGC 1 FoL e uLeu LeuL eu V atLeu H i s I pLys H isG iyA [ta ySer Pr o~e u CC T TAC TG CTGCT GG TTC TG C AC TGGAAAC AC GGGGC AGG GAGCQ-C TCT T Pro I IeT h r Pr oVa.i [AsnA[ ciTh r CysA KI llIIeAroH is ProCyv sH isGit CC CATCAC CCCTGTAAATGC CACCTGTGCCAT ACG CACCCATbGCCACGG yAsn LeuMe tAsnG [i IeL ysAsnG InLeu A[ cG IfLeuAsflG IySe rA CAACCT7CATGAACCAG-ATCAAGAATCAAICTG(CA ACTCATGC/GC C CAAT GC TC T C TTCAT TT CC TAT TA CAC AG C -1CAAG GAG AGC C GTTTCC C A s nA snV a [G [HuL y s L eu C ys A[cP r oQAs n Me t Th rA s p Phe P r o Se r P h A AC AAC G TGGAAAA GC TATG TGC GC C TAA CAT GA CAG AC T TC C C ATC TTT1 e H1 s G IyA s nG 1y Th rG [u L ysT h r Ly s L e u Vo(I[u L euT yr A rg Me t V C CAT GGC AACG GGACAGAGAAGAC CAAGIGGTCGAGCTGTATCGGATGG al[Al ciyrLeuSerAlSerLeulhrAsnl7 teThrArgAsDG~nLysVot TCGCATACCTGAGCGCCTCCCTGACCAATA-tC-ACCC_.rGGGACCAGAAGGTC LeuAsnProThrA~ciVa1SerLeuGHVoLysLeuAsflAtQThrI~eAs C TG AAC CC CACTG CC GTGAGCC TC CAGGTCAAGCTC AATGCTACTATAGA p Va I.Me tAr gGtyLe uLeuSer Asn Vat LeuCy sAF gLeuCyAsrsn'L I CGTCATGAGGGGCCTCC TCAGCAATGT-C-TT7GCCGT"ICTGTGCAACAAG1 y r Ar oVa IG1,y Hi sVcI AsoDV ca1 Pr o Pr o'iatP ro As DH i sSe 'As cLS ACCGTGTGGCCACG GGAGTCAZCCw-A.G/CA- 100 150 200 G IuA [taP GAAG CC I sGl nVal GCAAGTC GT AC C A GCTCAAP CTGGCAC ATGATGC GCCCAG( TGTCTC/ TCAGCC( GCAGAC CATC TG( ATGT GA( TTTT AT' GTACCC' GAGTTC, C AG C TC AACTCG SCIG C C 250 300 350 4.00 450 500 F1 G./IS A. SUBSTITUTE SHEET 8/00093 WO 88/07548 PCTIAL88/00093 xv 0 27/35 WO 88/07548 \VO 8807548PCT/ A U88/00093 GHuAtaPheGtnAr-LysLysLeuGtyCysG~nLeULeuGly-ihrTyFLy GAAG CCTTCC A AAGG AAA AAG-iTTGGGTTGC CA3-C T TCT- GGGACATAC AA 6 C sG InVci [I [eSe r VQIVot V IGtnAtQcPh e~ GCAAGTCATAAGTGTG3TGGTCCAGGIZCTT CTAGAGAGGAGGTC TTGAAT C xbci I GTACCATGGACTGAGGGACCT CAGGAGCAGGATCCGGAGGTGGGGUAG6GG 7 00C GCTCAAAATGTG'CTGGGGTTTGGGACATTGTTAAATGCAAAACGGGGCTG 750 C T GOCAG ACC C CAGGATTCC AGGTAC T C CT 1G C ACTC T 0G3G CT GGGC C ATGATGGAATC TGGC AAAG6TTGAAACTTC CATAGGCAGAGCTICTATACA G C CC A GCAC CAG C TAG AAAT G CAATG AG G GT G-1Ti CGTC -1GAGA GATTTC 900 TGTC TC ACTCAC TCACTCACT CACTCTCACT CACTC ACT CACTIC ACTCAC 950 T CAG C CC CTTGC TT G CT GOGT TAG AACAAGC T GCCA CAAG TTG T CTA CA 1000 GCAGACAGCAAAGGGCTGGGAAGTGTCCTAGACCCCTACA6AGTCACCAT 1050 C AT CTG GTC CT T TOCTGT CT CT C AGAGAA AC TTTG GA AGG CT TG G TTGGG 1100 ATGTI3AGAGAGCTAAGGGGACTGGGATCCAGAAGGAATCCTTTTATTTTA 1150 TTTTATTTTATTTTATTTTATTTTATTTTATAA6TTTTGTGGGTGGAAGG 1200 G TA C CC TGGG GTGA A TGAT GGAAT1GTG0T CTT C-1C 77G AGTTG GATGAGA 12 GAGTTCAGGCTTAGAGAC-TGTCAGAT-GGAAGAGTCTAZCTCALCAGTGTT- 1300 C AGCT CC CAC AGAAG CACAG CGG CCAG CT T CCAGTTGiTC AAAG CCT -AC G 1 AACTCGGTITAGCTTCTATGCAGTTCCCCCCACAGCCiGGC GG3TTGGGG 14+00C TCTGCCAGCTGGACCTAGAGGTGA6GGTGTGTOGAATT i 13 FIG. /58. SUBSTITUTE SHEET WO 88/ 07548 PCT/A1J88/00093 G ttlione S tra nsfe rase Tnrornbin M-LIF k----FF0o Lys Ser Asp Leu Vol Pro Arg Gty Ser Pro Pro TIe-----C LA AAA TCG GAT CTG GTT LWO LGT GGA TLC CLT LCT- CCL Bam HI 5'-LT5LALTG6ALLTTAGGGGLGGGATLLLLLLTLLLLATC--3 Barn HI c5'GCrAATG ILLCTCTITA T-lTCTTATTALALAGLCCAGGGGA--UAGCGTT>3 5 CAGG CCTTCTAGTAAGATA TCAAGCTTTGTGCTGTGAAC -31 H in c 3 SUBSTITUTE SHEET SUBSTITUTE SHEET WO 88/07548 PCT-/AU88/00093 0 0 E -LUJ M LULJ 13 11 9*' 3- 6 k O 2 xSSC 0 C 6xSSC 6 5 C FIG. /7 SUBSTITUTE SHEET SUBSTITUTE SHEET WO 88107548 PCT/AU88/00093 /735s rfl Lfl c~ I I I S RI Bom H3 R I+ BoQrn RI+H3 Born +H3 Bgl 2 Pst Sadc Stu 0 0 BT 2 RI Born Pst Stu Psf+H3 Pst+Born Stu+H3 Sfu +Bor BgI. 2+H3 BgI 2+Pl Bgl 2+Boim FIG. /8. suBS~r]TUTE SHEE I NVO 88/07548 WO 88/07548PCT/AIJ88/00093 /83J5 -BI.2 Eco~- -Hind 3 -Sdc X ha Stu Ps- Pst -Bg[2 SGCI EcoP- -Hind3 -EcoP3 FIG. /9. SUBSTITUTE SHEET WO 88/07548PCT/AU~88f 00093 NVO 88/07548 1/9/35 -Y 3kb- 4w FIG. SUBSTITUTE SHEET W O 88/07548 P T A U8 0 9 PCT/AU88/00093 2035s al 1 23 b 12 3 Kbp 23- 9.4- 4.3- 23 c 12 3 d 2 123 123 It FI G.2!, SUBSTITUTE S~-IEET "1 WO 88/07548 P CT/A L88f 00093 Lfl E cc Er acx LJDL 23- 9.4- 4.3- 2-3- 6xSSC F1IG.?22. SUBSTITUTE SHEET WO 88/07548 PCT/AU88/ 00093 223J5 Bam-digested DNA 55/6 65/6 65/2 65/2 65/6 65/6 65/2 65/0-2 M H M H M H M H Washing: 3kb- FI G. 23 SBSTITUTE SHET SUBSTITUTE SHEET WO 88/07548 PCT/AU88/000 9 3 C u rm"ll" LI) mo ag 0-6 FIG. 24. SUBSTITUTE SHEET WO 88/07548 PCT/AU88/00093 Gl yVatVoI. Pro LeuLeu L2U---Voit H TCCCCAGGAGTTGTGCCCCTGCTGTTG---GTT 1 m ggglttgtgCCCtt~CltQCtgctggtt rgHisPro~ysHisAsnAsnLeurletAsnGW U H GCCACCCATGTCACAACAACCTCATGAACCAGA' M gccccatgccQccggcQocc tccitgCicicgQoc H CTG6GATA[TGACAGGAGATGGCAGGGAGGGGGI H ATGGGGAGA6G6CTTGATTAAACCACiZCCCAGA H AACTGAGTGCAAAGGTGGiGGACCTGGCALTTCT' H ATGCIGTTG AGAGGCAGTGGGCTUGTGGGTGCT,1 H TAGGGCTAGACACCI3AGTTTTCCCTTCTGTCCG H GCAAGGGCACTCACATTACAATTAGTTTTGGCTV H CG[AAGAG[TTGCtZCAAAGGGiTTGGCGGCA6GGG PFQPhePF oAsnAsnLeuAspLysLeuCysGly, H CCGTTCCCCAACAACCTGGACAAGCTATGTGGC! M cccoltttcccQacQ~cg tggcioo.agctcitgtgcg yrArocieVQI[Va[TyFLeu61yThrSerL2UGI H ACCATAGTCGTGTACCTTGGCACCTCCDITGG; M QltCggQ 9ltCgcalCCtgg CCtCCC tgQ' Q~hFA QaASp t eLeuArg6 ty LeuLeuSe rAs H CA[CG'ZCGACATCLCTG CGAGGC C TLC TTAGELAA' V -rC t n7 go ,c aTrggg gc c tcc cogcac SerG AsDVo P heG nLys Ly s LysLeu Ti TGGGTAAGGA-TTCTC[AGAAGAA GAAGCTG SUBSTITUTE SHEETj WO 88/07548 PCT/AUi88/00093 2535 +1 LeuH i sTrpLysH i sG iy At. cGt ,SeFProLeu~ro; CTGC ACTGG AAACATG GGGC G-GGAGCCC C CIC CCC, c tgc cctgoQQac cggggc agggagc cc t ct cc c ~eArgSeFGtInLeuA ciG InLeuAsnG ty SrA tAk, TCAGGAJC CAACTGG CACAGCT CA ATGGC AGT GCC A! tcI~Q~cQioctggcQccigctcQiotggccigcgccQ CTTGTAAATATC ATTAGGGGC TG TCCT GAT CTGGG-1 CTC C TC CACTTCCTG CCC AAG CTTCC CCAGCGGAAGI TATCTT1GTGATTGTCC TGCTi'G'IC GGAGCGAGGGAT1 GGCATGGAGGGGCGT1CCCGGAACAT TGTGAGTGCAG, CT TAGGGT GGTGATG AT GAT GAT GATGATA ATGATG ,CTCATGAC AATTC CAGATGCTTACAGGGCAAGGAGT CTGGGACACTGACCCCTGACTC CCACGTCACCTCCC' ProAsnVatThrAspPleFroPro~neHlsAtQiAsn, CCCAACGTGACGGACTTCCCGCC CTTICCACGCCAAC cctQQcatgacQg~cft CCCGc- tt,-'CcctQgCCE1C, l(yAsnlleThrArgAspG6WLyslleLeuAsnProS' CCAMC AT C AC CCGCCA CC AGAACATCC TCAAC CCCA' c -c CC c ccCcggg CCcc ccc rgcc tg c ccc CC nVcotLeuCysArgLeuCYsSer!-ysTYFrHsVciLC! CCTECTCGTfCCCCCTGTGCAGC AACTACCACrGTCGGC rg C TQCCrg 7 lgtgcC Cra: C Cg trg ggg, GCC7GTC AACT CC -1CC CGG IAT ACACU7 I F1IG. K SUBSTITUTE SHEET C--11 2 HR9ciro-.O- EUitu1ic- FIG. /4. U 8/003WO 88/07548 PCT/AU88/00093 WO 8~ 261JS J eTh rPr oV'aAsnA aTh rC y sA Ia IeA ATCACC CTGTCAACG CCACC TGTGCCATA[ 97 QCtc Qc cc ftcictuCC cCC to gc CQao snAlcaLeuPhelteLeuTyr ATGCCCTCTTTATTCTCTATGTIAAGTTACIZC 197 afgc tctc ttCclttCcct TGAGGGGACCTTTTGGGGCTGGAAGGAGAGA 297 CTTCCCCAGGGTGCCCAGTTAGCAAGGGGAG 397 GGAGGGGAAATG6GCGTGAGGCACC-AGGGAG, 497 IGGATGGAAGTACTTGTG6TGTGGTGCIZCCAGC 597 ACTGCGTGCATGGCTCAGTCTTTIGATCTTTA 697 797 TyrThrA~ciG I.nGtyGt u TTCTGCCCCTCAGTACACAGCCCAGGGGGAG 897 tQC aCQgC tc Qclggcigag iGyThrGl uLysAtQcLysLeuVal (GCtuLeuT GGACGcAGAAGGCAAGCTGGTGGAGCTG, 99? gggQCicgagQicgQCCQagttggtgqgcgtg t 'erALQLeuSeFLeuHI sSerLysLeuAsnAl GTGCC[TLAGCCTC[ACAGCAAGCTCAACGC 1097 ctoCcgtg QQCC tcccigTcclgc tcaitqg yHisVolAsDVo[-IhrTyrG [yProAspThr CCAT GTGGACGT AC TACGGC C CTGACAC 119? CC -CC Og-CY9TgCCQCrtCCcIccQaccr AMcVclLeuAkaG~nAlPheTE I-CL AGCCTAG C AGGAEG 129- FIG2SC. SU13STITUTE SHf-ET WO 88/07548 WO 8807548PCT/AU188/00093 27/Js M H :LeuMetAsnGtnI e AF9SerGtnLeui M S -Se H Ie Leu Tyr TyrThrA161nGly Glu H Pro AsnVat ThrAspP heProPro Phel H: 6[uLeuTyrArgleVo.[VciTyrLeu, M Vo.1 Thr---Vo1 H: LysI1eLeuAsnPro SerAto.LeuSer M H: LeuArgGlyLeuLeuSerAsnVolLeu M ProProVQI.------H's1 H: Vci(AspVo.[ThrTyrG~yProAspThr H: GlyCyslnLeuLeulyLysTyrLys F1IG. 264A SUBSTITUTE SH"EET WO 88/07548 SUBSTITUTE SHEETI P CT/AU188/00093 Thr C IQ lI le Arg His Pro CysH is AsnAsn 1A [QG [nLeu Asn G1 y SerAlcaAsnAl ci LeuPhe Vi. G A t a ProPhePro AsnAslLeu AspLys LeC CysGty H is A to.AsnG lyThr 6 tu LysAliQ LysLeu Vat. Ser Ala-- -ThF Gly ThrSer LeuG y Asn1I eTh rArg AspGIln I I Ie -Val. jLeu H is S erLys Leu Asn AhQThF AItAspII[ e C ys Arg Leu Cys Ser Lys Tyr H is Val Gly H is I--Asp G u SeF (tyLys Asp Val PheG tnLys Lys Lys Leu Voat Ser VQI[Val----- 6 Il n1[e I[e At (a Val[ LeuAli~o Gi[n A[ a Phe FIG. 2 6B. SUBSTITUJTE* HNET SUBSTITUTE SHEET WVO 88/07548 PCT/AU88/00093 2955S kbpl EcoRS Hin Bg c13 cu E ci CORE cu c$ Stul C ind3 StuI _0 4- (A (ii F1G27 SUBSTITUTE SHEET WO 88/07548 WO 8807548PCT/AU88/00093 po/Js +1 GtIySerProLeuPro I IeThrPr OVG[ As n' G AT CC CC CCTCCCC AT[ I ,CC CCTGTCAAC Bum HI AsnAsnLeuMe tAsnG I nil.eAF gSerGt n AACAACCT CATGAACCAGATCAGGAGCCAA LeuPhelteLeuTyrTyFTlrAtQaGlnGly: CT CT ITATTC TC TATTAC ACAGCC IIAGGGG CysG IyProAs nVcitThrAspPheProProI TGTGGCCCCAACGTGAZGGACTTCCCGCCCI LeuVo.IG uLeuTyFArgI IeVo.IVQ I Tyri CTG 5TGGAGCTGTA CC GCATAGTCGTGTACO AspGt nLy slteLeu/'-snProSerAtaLeu GACCAGAAGAT CCTCAACC CCAGTGCCCTC AsolI eLeuArgGlyLeuLeuSerAsnVci( GACATCC TGCGAGGCCTCCTTAGCAACGTG G yHi SV i(AspVQI ThFTyr G IyProAsp GGCCATGTGGACGTGACCTAC GGCCCTGAC L ysLeuG IyC ysG In L euLeuG IyLysTyr ASAGCTGGGCTGTCAAC TCCTGGGGAAGTAT P h e TTCTAGTAAGATATCAAGCTT- H i n d 7 FG. 29A. SUSSTITUTFM -ac-r SUBS-rITUTE SHEET WO 88107548 PCT/AU88/00093 A[ aThrCysA clQTIeArgHis Pro CysH is GCCAL'CTGTGCCATA[CACCTCAL LeuA IcG I nLeu As nGI ySeFA IaA s A I a 'C TGGCACAGCTrC AAT GGCAGT6CCAATGC C3 tuPro PheP roAsnAsn L euAsp.LyvsLe u GIAGCC6TTCCCCAACAACCT TGGACAAGCTpAl Ph eHi s A toAs nG I yThrG Iu Ly sAIa Ly s TTCC ACGCCAA CGGCACGGAGAAGGCCAAG lLe uG IyTh rS erLe u GIyAsn I IeTh r Ar SerLe uH i sSerLysLeuAsnA IcThi-A I AGCCT CCACAGCAAGZTCAAZG CCACCGiCC L eu C y sAr gL e uC y sS e rLy sTy F H s Va I CTI3TGCCGC C TTGCAGCAAGTACCACGVG Thr Ser G IyLy sAspVci IPh eG n LysLys ACCTCG OGTAAGGATGTCTTCCAGAAGAAG L ysG In IIe I IAlc aI LeuAl aG InAlc AAGCAGATCATCGCC~iTGTTGGCCCAGGCC 120 180 240 300 360 420 480 540 561 FG 293. SUBSTITUTE SHEET FIG./9 SUBSTITUTE SHEET WO 88/07548 PCT/A L88/00093 3235s 10.0 4 0k 0' /0 ?n I r '0 I! C' 256 16 4 1 Dilution added FIG.3JO. 600 400 oc '65,536, 4096 256 16 ReciorocaI dilution /C/G J/. SUBSTITUTE SHEET NV 88/07548 PCT/AU88/000 9 3 3555s T I_ U T (I 000-~ L- -j Ll C Co cui U- 4000 20001 C OD 4096 1024 256 64 16 4 Reciprocal Dilution F1/G.3J2. G018STITU TE SHEET SUBSTITUTE S- PCT/AU88/0 0 09 a WVO 88/ 07548 ,343J5 A 0 4- 0 3a 0 0 40 6080 100 Fraction Numb2F FIG.33.- SU13ST-ITUTE SHEET NVO 88/07548 \VO 88/7548 CT/A U88/00093 A 0. 1001 Fraction Numbp.r FIG.3J4. SUBSTITUTE SHEET up' INTERNATIONAL SEARCH REPORT international Apolicairon No PCT/AU 88/00093 1. CLASSIFICATION OF SUBJECT MAT-TER several ci ssic.1igon- symbois apply. Indicate ali) According to International Patent Casiicaton (1PC) or to both Natinial Ciassficailon and PC 4 CO7K 1 3 /0O,A 5/12,1/ C2- 1/~1/Oc2 19/34,21/00,21/02, Int. Ci. C07H 21/041/(C12P 19/31f,C12R 1:91) (C12P 21/02, C12R 1:865) 11. FIELDS SEARCHED Minimum Documentation Searched Ctassiicalion System Clasalficalion Symbols WPI APIL (LEUKEMIA or LEUKAEMi A-or NLfUPLA 11LC and (INHIBIT or INHIBITS or INHIBITING or INHIBITORY) and (FACTORS or PROTEIN or NBRF Database PLPP IE Documentaton Searched other than Minimum Documentaion to the Extent thast such Documents are Included In the Fields Searched AU: IPO C07G 7/00, C07K 15/12,14, C12N 15/00, C07H 21/04 Ill. DOCUMENTS CONSIDERED TO BE RELEVANTO category Citation of Document, wvilh ardic3tion. where appropriate, of the relevant passages 12 Relevant to Claim No. 13 P AU,A, 66756/86 (HOECHST AKTIENGESELLSCHAFT) (1-45) June 1987 (25.06.87) P Patents Abstracts of Japan, 0-482, page 147, (1-45) JP,A, 62-223126 (SA NKYC Co. LTD.) 1 October 1987 (01.10.87) Special categories of cited documents: 0 later document published ar the International iling date document deaining the general state of the art which 16 not or priority date and not In conflict with the application but iicited to understand the principle or theory underlying the considered to be of parlicuiar relevance Invention E erlier document but published on or sher the International X douetfprtutrelvn;th clidivnin fIling data cannot be considered novel or cannot be considered to document which may throw doubts on priority claimis) or involve an Inventive stop ,,hich isocited to estabisah the pubication date of another document of paricular relevance:' the claimed Invention citation or other special reason las sp~cried) cannot be considered to Involve an Inventive step when the 0" document referring to an oral disclosure, uees, exhibition or document is combined with one or more other such docu- other meant mei j, such combination being obvious to a person siled document published prior to the International riling date but In a art. later than the priority date climed document member of the same patent family IV. CERTIFICATION Dal* of the Actual Compltion of the International Search Date of Meiling of this International Search Report 12 July .1988 (12.07.88) (10-69' F /a /9?E international Searching Authority Signature o 1 .A .iiofized OMcer Australian Patent Office R. EVANS Form PCItSAltO (second sheet) (January li SUB3STITUTE -SHEET I 1 1 A1MNE 1 THE =ERNATIONAL SEARCH REPORT ON INERNATIONAL APPLICATION NO. PCT/AU 88/00093 This Annex lists the known publication level patent family members relating to the patent docume~nts cited in the above-mnentioned international search repo~rt, The Auzrralian Patent Office is in no way liable for 'these particulars which are mrerely given for the purpose of information. Patent Docume~nt Cited in Search Patent Family Mmbers Report AU 66756/86 DE 3545568 EP 228018 Fl 865186 PT 83972 ZA 8609557 END OF ANNEX I 23/kk/48/1