Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5415—Leukaemia inhibitory factor [LIF]

Definitions

• the present invention relates generally to molecules carrying one or more bindin determin.ants for the ⁇ -chain of human Leukaemia Inhibitory Factor binding recepto and to genetic sequences encoding same.
• LIF leukaemia inhibitory facto
• LIF is a glycoprotein that was originaUy purified and cloned on the basis of its abilit to induce terminal macrophage differentiation of the Ml myeloid leukaemic cell lin (Hilton et al 1988a). It has since been shown to have a variety of activities on wide range of cell types including megakaryocytes, osteoblasts, hepatocytes adipocytes, neurons, embryonal stem cells and primordial germ cells (Metcaff, 1991)
• LIF transduces its biological signal via a multi-subunit membrane bound receptor.
• the receptor for LIF is a member of the hemopoietin or cytokin family of receptors, which generally have roles in cell growth and differentiatibn These receptors are characterised by their extracellular domain, which contains a least one copy of an approximately 200 amino acid hemopoietin domain (Cosman 1990).
• a number of prim.ai'y amino acid sequence motifs distinguish this domain including pairs of disulfide-bonded -cysteine residues .and the Trp-Ser-X-Trp-Ser moti (where X is any amino acid).
• the over.all second ⁇ and tertiary fold of th hemopoietin domain is predicted to be similar in each member of the recepto f-amily (Bazan, 1990a).
• the LLF recepto consists of two known subunits, both of which are members of the hemopoieti receptor family.
• the LLF receptor ⁇ -chain binds LIF with low-affinity and contain two hemopoietin domains (Gearing et al, 1991).
• the ⁇ -chain of the LLF receptor ha been identified as gpl30 (Gearing et al, 1992) which is also a component of th interleukin (IL)-6, IL-11, oncostatin M (OSM) and ciliary neurotrophic facto receptor (C ⁇ TF) complexes.
• IL interleukin
• OSM oncostatin M
• C ⁇ TF ciliary neurotrophic facto receptor
• the ligands for this family of receptors are unrelated at the primary amino aci sequence level, but secondary and tertiary structural predictions indicate that all known ligands for hemopoietin receptors have a similar overaU fold, suggestin evolution from a common ancestor (Bazan, 1990b).
• Members of this family o ligands form an anti-parallel, four ⁇ -helical bundle (the helices are designated A, B, C and D ordered from the N-terminus), that is characterised by one short and cwo long connecting loops (labelled by the helices they join).
• G-CSF granulocyte colony- stimulating factor
• GM- CSF granulocyte-macrophage CSF
• GH growth hormone
• IL-2 Brainndhuber et al 1987
• IL-4 Powers et al, 1992
• IL-5 Milburn et al, 1993
• the three-dimensional structure of LIF is predicted to be most similar to the structures of OSM, C ⁇ TF, IL-6 and G-CSF, amongst others.
• hLIF murine LIF
• hLIF-R human LLF receptor
• mLIF-R high- and low-affinity mouse LLF receptors
• mLIF-R high- and low-affinity mouse LLF receptors
• hLIF binds to both the naturally occurring soluble form of the mLIF-R ⁇ -chain, mLIF-binding protein (mLBP) (Layton et al, 1992), and the high-affinity mLIF-R on PC.13 cells with a higher affinity than mLIF, due to markedly different dissociation kinetics.
• mLBP mLIF-binding protein
• hLIF human LIF
• Th present invention provides, for the first time, a rational approach to the generatio of a new range of therapeutics based on LIF by providing non-naturaUy occurrin molecules capable of binding to hLIF receptor.
• Such therapeutics may be bot proteinaceous and non-proteinaceous molecules.
• one aspect of the present invention contemplates a non-naturall occurring molecule comprising a tertiary structure which presents a functiona binding face for the ⁇ -chain of hLIF binding receptor.
• This aspect of the present invention is predicated in part on the identification of th amino acid residues on hLLF which constitute the binding face for the hLIF recepto and in particular the ⁇ -chain of the hLIF receptor. Since a major contribution to th binding face is the three dimensional arrangement of chemically interactive group on the side chains of the amino acid residues, a preferred aspect of the presen invention relates to the non-naturally occurring molecule wherein the functiona binding face comprises the chemically interactive groups of the amino acid residue which constitute the binding determinant on hLIF for the ⁇ -chain of hLLF bindin receptor.
• the most preferred chemically interactive groups including groups selecte from at least one of a methyl group, a hydroxyl group, an imino-nitrogen group an a positively charged nitrogen group or chemical equivalents homologues or analogue thereof. These chemical groups are in spatial arrangement such that they presen a binding face for the ⁇ -chain of the LIF binding receptor.
• the chemical groups are presented on the amino acid residue themselves, or their chemical homologues, analogues or equivalents, which amin acid residues are selected from Gin 21 to Lys 160 of hLIF.
• the amino acid residue are more particularly selected from at least one of each of the following regions o hLIF:
• amino acids are Ser 107, His 112, Ser 113, Nal 155 and Ly 158 of hLIF or chemical homologues, .analogues or equivalents thereof.
• the critical aspect of these amino acids is th chemically interactive group(s) on the side chains of each residue which, in define spatial arrangement, constitute the binding face of hLLF for the ⁇ -chain of the hLL binding receptor. It is clear, therefore, that the binding face does not need' t comprise amino acid residues but the chemical interactive groups thereon.
• the non-naturally occurring molecule may be any convenient carrier as describe below including a solid support or matrix, or a non-proteinaceous molecule, or protein, polypeptide or peptide.
• the non-naturall occurring molecule in the form of a carrier is a mammalian cytokine such as but no limited to mLIF carrying the hLIF receptor-binding face.
• the non-naturally occurring molecule may not necessarily carry the exact hLI receptor-binding face of hLIF but will contain an amount of the hLLF receptor binding face of hLIF to enable the non-naturally occurring molecule to exhibit a least about 35% and more preferably at least about 50% hLLF-like activity a described below.
• a non-naturall occurring chemical entity such as a proteinaceous or non-proteinaceous molecul and/or a solid or non-immobilised support, said chemical entity comprising a tertiar structure which presents one or more chemically interactive groups selected from a least one of each of a methyl group, a hydroxyl group, an imino-nitrogen group an a positively charged nitrogen group as a functional binding face for the ⁇ -chain o hLIF binding receptor.
• the non-naturally occurrin chemical entity exhibits at least about 50% hLIF-like activity determined -a described below.
• the chemica ⁇ y interactive groups are presented on amino acid residue selected from one or more of Ser, His, Nal and Lys or their chemical equivalents homologues or analogues. More preferably, the chemica ⁇ y interactive group correspond to those on Ser 107, His 112, Ser 113, Nal 155 and Lys 158 of hLIF o chemical homologues, analogues or equivalents thereof.
• Another .aspect of the present invention is directed to a molecule which is non naturally occurring and which comprises a carrier portion and an active portio wherein said active portion comprises amino acid residues or chemical equivalent thereof which constitute a binding determinant for the ⁇ -chain of the hLIF bindin receptor.
• molecule is used in its broadest sense to refer to .any chemical entity o compound, .or a protein, polypeptide, or peptide or a non-peptide analogue an which is capable of carrying a sufficient number of amino acid residues or thei chemical equivalents to constitute an effective binding determinant for the ⁇ -chai of the hLIF binding receptor.
• a "sufficient number” of amino acid residues is th minimum number required to exhibit at least 35% hLIF-like activity in terms of th ability to compete for 125 I-hLIF binding to murine LIF-binding protein (mLBP) whic is determined as described below.
• a "chemical equivalent” includes active an interactive groups present on a particular amino acid residue and, hence, extends t non-peptide or non-amino acid mimics of the amino acid residues involved in th binding face.
• the "molecule” is considered a "carrier” of the amino acid residues or their analogue which constitute all or a functional part of a binding determinant for the ⁇ -chain o the hLIF binding receptor.
• the selection of an appropriate carrier molecule i determined by its tertiary structure.
• the amino acid residues or their analogues ar required to form a receptor binding face in the tertiary structure of the carrie molecule.
• the present invention provides, therefore, a carrier molecule having tertiary structure such that a sufficient number of amino acid residues or thei analogues including chemical equivalents, constituting a binding determinant for th ⁇ -ch n of the hLIF binding receptor, when introduced into s.aid carrier molecule t form a receptor-binding face capable of at least 35% hLLF-like activity.
• the carrier molecule is a protein, polypeptide or peptide or a molecul substantially consisting of amino acid residues linked via peptide bonds.
• Th molecules may also be engineered to contain non-naturally occurring amino aci residues such as some D-amino acids, non-naturally occurring substituted amino acid or chemical equivalents of amino acids which direct or otherwise influence th terti.ary structure of the molecule.
• a carrier molecule such as a protein polypeptide or protein may be engineered to contain an amino acid, chemica derivative thereof or other chemical entity e.xhibiting conf ormational restraints on th overall molecule or a part thereof. Molecules exhibiting pre-determined tertiar conformations are particularly useful in their selection as carrier molecules i accordance with the present invention.
• the carrier molecule is a mammalian cytokine such as but not limited to a non-human LIF, a colony stimulating factor (CSF) (e.g. G-CSF, GM-CSF, growth hormone), an interleukin (e.g. IL-2, IL-4, IL-6), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) or other factor (e.g. a haemopoieti growth factor, interferon).
• CSF colony stimulating factor
• IL-2, IL-4, IL-6 interleukin
• OSM oncostatin M
• CNTF ciliary neurotrophic factor
• Preferred non-human LIF molecules are those fro livestock animals (e.g. from sheep, cattle, donkeys, horses, deer, goats and pigs) laboratory test animals (e.g.
• mice mice, rats and guinea pigs), companion animals (e.g. dogs and cats) or captive wild animals (e.g. kangaroos, foxes, dingos, wild boar an emus).
• the preferred non-human LIF is murine LIF.
• the preferred cytokine is G- CSF.
• the present invention extends t a carrier molecule carrying amino acid residues or their chemical equivalents o derivatives or amino acid equivalents, homologues or analogues of the .amino acid in hLIF which constitute the binding determinant for the ⁇ -chain of the hLLF bindin receptor, wherein the number of amino acid residues and their proximity ar sufficient for the molecule to exhibit at least about 35% hLIF-like activity and mor preferably at least about 50% hLIF-like activity.
• the .amino acid residues constituting the binding determinant are selected from Gi 48 to Lys 160 of hLIF using the numbering system depicted in Figure 4.
• at least one amino acid residue is selected from each of the following regions o hLIF: (i) a region between the A and B helices; (ii) a region between the B and C helices; (iii) the C helix; and (iv) a region between the C and D helices.
• the amino acid residues are selected from one or more of a Ser, His, Nal and/or Lys or chemical equivalents thereof including chemically interactive groups thereon.
• the actual binding determin.ant on LLF comprises Ser 107, His 112, Ser 113, Nal 155 and Lys 158 which, in a most preferred embodiment, forms the same arrangement or a functional equivalent arranged in the non-naturally occurring molecule.
• These amino acids are arranged in the carrier molecule such that at least two and more preferably at least three and even more preferably at least four residues are non-contiguous relative to each other. Reference is made herein to the subject molecule having at least about 35% hLLF like activity.
• the molecule has at least about 50% hLLF-like activit and even more preferably at least about 70% hLIF-like activity.
• hLLF-like activit is defined in terms of the ability for a molecule to compete for 125 I-hLIF binding t mLBP based on the equation:
• ⁇ is the dose of unlabelled mLIF
• X jj is the dose of unlabelled hLLF
• p i the dose of the molecule (e.g. protein, polypeptide or peptide) required to give 50% inhibition of 125 I-hLIF binding to mLBP.
• a preferred embodiment of the present invention contemplates a hybrid molecule comprising:
• ⁇ is a dose of unlabelled mLIF
• j is a dose of unlabelled hLLF
• x is a dose of hybrid molecule required to give 50% inhibition of 125 I-hLLF binding to murine LIF binding protein (mLBP).
• the molecule is a hybrid between a carrier molecule in the form of a polypeptide and the amino acid residues forming the binding determinant for the ⁇ -chain of the hLIF-binding receptor.
• the polypeptide is a mammalian cytokine as hereinbefore described.
• the amino acid residues are selected from hLIF as hereinbefore described. This definition is also applicable when the carrier molecule anu/or the binding interface are non-proteinaceous in a form such as a chemical compound capable of mimicking a polypeptide or peptide.
• This embodiment of the present invention is conveniently elucidated by the following diagram which shows the competitive inhibition of mLIF, hLLF .and a hybrid LLF peptide or polypeptide with iodinated hLIF for the mLIF receptor:
• a “peptide” is taken herein to be a molecule comprising ten or less amino acid residues.
• a "polypeptide” is a molecule comprising eleven or more amino acid residues and includes a protein.
• the peptides and polypeptides are preferably recombinant or synthetic molecules but extend to fragments or parts of naturally occurring LIF molecules and to hybrid molecules comprising regions from two or more LIF molecules from different species.
• the peptides and polypeptides are "isolated” meaning that they have undergone at least one step toward biological purity.
• a peptide or polypeptide is deemed herein to be “isolated” or “biologically pure” where a preparation comprises at least 35%, preferably at least 45%, more preferably at least 55-60%, still more preferably at least 65-75% and even more preferably at least 80-90% of the peptide or polypeptide relative to other components in the preparation as determined by weight, LIF receptor-binding activity, amino acid content or other convenient means.
• the present invention is particularly exemplified using a soluble murine LIF-binding protein (mLBP) (Layton et al, 1992) or a recombinant human LLF receptor which are convenient since human LIF readily binds or otherwise associates with these molecules. It is not intended, nowever, for the present invention to be limited solely to the murine receptor or to the elucidation of the binding determinant solely on the human LIF molecule.
• mLBP soluble murine LIF-binding protein
• the molecules of the present invention are useful inter alia in rendering a LIF molecule from one species more like a LIF molecule from .another species.
• the hybrid LEF molecule is also useful in designing drug .analogues, agonists and antagonists.
• the hybrid LLF molecule is between non-human LIF and human LLF and most preferably between murine LIF and human LIF.
• the hybrid LIF molecule is s d to be a "humanised" form of non-human LIF and, in such a case, the humanised non- human LIF molecule may be more efficacious in humans and/or may provoke no or a reduced immune response compared to the non-human LLF molecule per se.
• the present invention is particularly useful in designing .analogues, antagonists and agonists based on the chemically interactive groups which constitute the binding face on hLIF for. hLIF receptor. This permits a rational design of such molecules which can be readily assayed in vitro and in vivo.
• the present invention extends to aU such analogues, antagonists and/or agonists capable of binding or otherwise interacting with the hLIF receptor binding face on a non-naturally occurring molecule, chemical entity or hybrid molecule as hereinbefore described.
• Another embodiment of the present invention is directed to genetic sequences comprising a sequence of nucleotides which encode or are complementary to nucleotide sequences which encode the peptides and polypeptides encompassed by the present invention.
• the genetic sequences may be cDNA or mRNA .and may be single or double stranded, linear or covalently closed, circular molecules.
• the genetic molecules are part of an expression vector capable of expression in a prokaryotic cell (e.g. E. colt) or a eukaryotic cell (e.g. an animal or ma.mmali.an cell).
• Tixe present invention further contemplates a method for mapping LIF receptor binding activity associated with a mammalian LLF to one or more amino aci residues or to a sequence of amino acid residues on said LLF, said metho comprising substituting for regions on said mammalian LLF, structurally simila regions from a LIF from a different species of mammal wherein both LIF molecule before substitution differ in respect of the activity to be mapped, assaying th substituted LIF molecules so obtained and identifying regions on one or both of sai LIF molecules responsible for LIF receptor-binding activity.
• the substitutions are based on secondary structural predictions (se gener.ally Bazan, 1991) such that the hybrids so constructed differ in the activity't be mapped.
• the substitution of regions are at the DNA level whic are then expressed to form recombinant hybrid LIF molecules.
• This aspect of the present invention is exemplified .and described hereinafter b determining the LIF receptor-binding determinant on human LLF using a series o mouse-human LIF hybrid molecules some of which retain hLIF-like activity a previoxisly described.
• the present invention extends to the use of LI molecules from other mammalian species.
• the preferred hybrids comprise a murine LIF carrier molecule and a sufficient o effective number of amino acid residues from hLIF to constitute a receptor bindin face for the ⁇ -chain of hLIF-binding receptor.
• Even more preferred amino aci residues or peptides are those which carry the hLIF binding determinant in MH5, MH6, MH16, MH17, MH18, MH31, MH19, MH20, MH21, MH22, MH23, MH24, MH25, MH29, MH30, MH32, MH33, MH34, MH35, MH36, MH37, MH38, MH39, MH40 and MH41.
• Still more preferred amino acids or peptides are those in MH33, MH35, MH36, MH37, MH38, MH40 and MH41.
• the most preferred amino acids or peptides are those in MH33, i.e. Ser 107, His 112, Ser 113, Nal 155 and Lys 158.
• the present invention is further described by the following non-limiting Figures an Examples.
• Figure 1 is a summary of data from biological assays and competitive binding assay of mLIF, hLIF and chimeric LLF proteins.
• Mouse LLF sequence is represented b blue boxes and hLIF sequence is represented by yellow boxes.
• the hybrid protein are numbered from 1 to 41 and are prefixed by "MH" to denote that they contai both mLIF and hLIF amino acid sequence.
• the relative positions of the predicte ⁇ -helices and connecting loops in the hybrid proteins can be determined by visua alignment of the schematic representations with the diagr.am immediately below
• the percentage human score of mLIF, hLIF and hybrid proteins is calculated fro the ID ⁇ Q for inhibition of [ 125 I]hLIF binding to mLIF-binding protein. Most assay were performed two or more times with essentially identical results. N.D., no determined.
• the exact amino acid construction of each hybrid according to SEQ I NO. 2 is indicated in Table 1.
• Figure 2 (A) Competitive inhibition of [ 125 I]mLIF binding to mLBP (soluble mLLF- ⁇ -chain) by mLIF, hLIF and a selection of m-hLIF hybrids, (•) mLIF; (o) hLIF ( A) MH1; ( ⁇ ) MH2; (B) MH3; (D) MH4; ( ⁇ ) MH5; (0) MH6; ( T) MH14. Result for all competition assays were expressed as the number of counts bound to th receptor at a particular concentration of unlabelled competitor (B) divided by th number of counts bound to the receptor when no unlabelled competitor was presen (B 0 ).
• (B) Competitive inhibition of [ 125 I]hLIF binding to mLBP by mLLF, hLIF an a selection of m-hLIF hybrids.
• (C Competitive inhibition of [ 125 I]hLIF biding to mLBP by mLIF, hLIF .and a selection of m-HLEF hybrids, ( ⁇ ) mLIF; (o) hLLF; (B) MH3; ( G) MH4; ( ⁇ ) MH5; ( 0) MH6; ( ⁇ ) MH14.
• Figure 3 (A) Competitive inhibition of [ 125 I]hLLF binding to COS cells transfected with the hLIF-R ⁇ -chain by mLLF, hLLF .and a selection of m-hLIF hybrids, (•) mLLF; (o) hLEF; ( ⁇ ) MHl; ( ⁇ ) MH2; (D) MH4; ( ⁇ ) MH5; (0) MH6; (D) MH14.
• FIG. 4 Comparison of mouse, human and porcine LIF and G-CSF amino acid sequences. Amino acid residues that are identical between mLIF .and hLLF are indicated by asterisks and predicted ⁇ -helices A, B, C and D in hLIF are marked within boxes. Numbering of the amino acid residues of each protein is indicated as starting at the first serine of the mature protein. The first glycine is -1 and is derived by thrombin cleavage of GST-LIF fusion. The serine + 1 is equiv.alent to serine +24 of the immature native protein.
• the figure also shows the alignment of LIF proteins to hG-CSF and the secondary structure assignments for G-CSF. (H, ⁇ -helrx; G, 3 10 -helix turn; T, turn; S, bend).
• Figure 5 is a graphical representation showing:
• FIG. 6 is a pictorial representation showing a ribbon diagram of the model of hLLF with the receptor binding site amino acid residues shown in CPK form.
• Hybrid cDNAs in which portions of mouse LLF were replaced with homologous regions of human LIF were either synthesized from discrete restriction fragments of the two LIF cDNA molecules, or they were generated by the "splicing by overlap extension" method (Ho et al, 1989) which is a PCR-based technique using Pfu polymerase (Stratagene).
• the cDNA encoding porcine LIF was constructed from a genomic porcine LIF clone (Willson et al 1992) in which the second intron was removed using the above PCR-based technique.
• the mutated cDNAs were subcloned into the E. coli expression vector pGEX-2T. All constructs were sequenced in both directions using T7 DNA polymerase (Pharmacia) and a Promega dideojcy sequencing kit.
• Plasmid pmLIF was cut with the restriction enzymes Smal and Hindlll to produce fragments of approjcimately 257 bp and 5241 bp. Plasmid phLIF was also cut with the restriction enzymes Smal and Hindlll to produce fragments of approximately 258 bp and approximately 247 bp. The approximately 293 bp Smal-Hindlll mLLF fragment was ligated with the approximately 247 bp Smal-Hindlll hLLF fragment to give construct MH1.
• a 4.1 kb cDNA encoding the human LIF receptor was isolated from a foetal liver cDNA library (Clonetech) by plaque hybridisation with a 32 P-radiolabelled DNA fragment corresponding to amino acids 13-177.
• the nucleotide sequence corresponding to the coding region of this cDNA was determined by the dideoxynucleotide chain termination method (Sanger et al, 1977) using synthetic oligonucleotide primers. With the exception of a T to C nucleotide substitution at the third position of codon 555, which does not alter the predicted amino acid sequence, the sequence of this cDNA was otherwise identical to that previously reported (Gearing et al, 1991).
• the LIF-R cDNA was subcloned into the m.ammalian expression vector pCDM8 (Seed, 1987) and designated pCDM8/16C.
• the pl.asmid pCDM8/16CP encoding a truncated soluble form of the hLLF-R that includes both haemopoietin domains and two of the three fibronectin repeat structures, was constructed from pCDM8/16C by deletion of a Pstl fragment that codes for 97 membrane proximal residues, the transmembrane and cytoplasmic domains.
• the C-terminus of the soluble LIF-R encoded by pCDM8/16CP includes 10 residues encoded by vector sequences.
• Transient expression of the hLIF-R in COS cells was achieved by electroporation with plasmid DNA. Briefly, 2 x 10 7 cells were harvested in 0.8 ml of phosphate- buffered saline (PBS), mixed with 20 ⁇ g of pCDM8/16C DNA at 4 °C and subjected to electroporation at 300 N and 500 ⁇ FD. Viable cells were harvested by centrifugation through a cushion of FCS and incubated in 50 ml of medium at 37 °C in an atmosphere of 10% v/v C0 2 .
• PBS phosphate- buffered saline
• the functional activity of the m-hLLF hybrids was assayed by their ability to induce differentiation in murine Ml leukaemic colonies. Ml differentiation assays were performed as described (Metcalf et al, 1988). The specific activity of each hybrid was expressed as the units of Ml differentiation-inducing activity per milligr.am of protein.
• each LLF preparation was determined by titration in cultures of Ml cells. Aliquots of normal mouse serum (containing approximately 5 ⁇ g/ml mLBP) were then added in serial 2-fold dilutions to cultures of Ml cells that also contained a just maximal concentration (200 U) Of mLIF, hLLF or m-hLLF hybrid. Assays including mLLF and hLLF gave identical results when either crude mouse serum or a highly purified preparation of mLBP was used. Hybrids were assessed for mLIF or hLLF character from the dilution of serum required to block 50% of their Ml cell differentiation-inducing activity.
• Normal mouse serum was vised as a source of mLBP.
• 50 ⁇ l aliquots of unlabelled LIF or a mouse-human LLF hybrid were added to 96-well filtration assay plates containing a 0.65 micron Durapore membrane (Millipore, MA, USA) with 20 ⁇ l aliquots of a 1/10 to 1/20 dilution of normal mouse serum, 10 ⁇ l radiolabelled ligand and 25 ⁇ l Concanavalin-A Seph-arose 4B (ConA-Sepharose, Pharmacia, Uppsala) (diluted 1:4 in 100 mM sodium acetate pH6.0 containing 1 mM MnCl 2 , 1 mM MgCl 2 and 1 mM CaCl 2 ; this and all subsequent buffers contained 0.02% w/v sodium azide and 0.02% v/v Tween 20) and incubated at room temperature overnight with agitation.
• a cDNA encoding the hLIF-R was isolated, subcloned and expressed in COS cells as described in the above examples.
• LLF molecule is an anti-parallel, four ⁇ -helical bundle, a topology common to a number of growth factors and cytokines (Diedrichs et al, 1991; Parry et al, 1991; de Nos et al, 1992).
• the model of Bazan (1991) was used to divide the LIF amino acid sequence into a series of modules of predicted ⁇ -helices and connecting loops.
• a series of plasmids was then designed to encode mouse-human chimeric LLF (m-hLLF) molecules (Figure 1) in which regions of hLIF sequence were incorporated into a mLLF molecular framework.
• the protein concentration of each purified sample was determined by amino acid analysis, and the amount of biologically active protein in each sample was estimated by its ability to induce differentiation in mouse Ml myeloid leukaemic colonies. .An internal standard of mLIF (10 4 U/ml) was used to normalise .all Ml cell bioassays (50 U/ml of LIF is defined as the concentration of LIF required for half ma ⁇ dm ⁇ stimulation).
• the first feature of hLIF that distinguishes it from mLLF i.e. its ability to bind to the hLLF-R ⁇ -chain
• the second feature of hLIF that distinguishes it from mLIF was evaluated by the ability of each hybrid to compete with [ 125 I]hLIF for binding to COS cells expressing the hLIF-R ⁇ -chain (COS hLIF-R cells).
• the second feature of hLIF that distinguishes it from mLIF i.e. its ability to bind to the mLIF-R with a higher affinity than mLIF, was evaluated by the ability of each hybrid to compete with [ 125 I]hLIF for binding to mLBP and by its sensitivity to biological inhibition by mLBP.
• the 1000- to 5000- fold difference in the ability of mLIF and hLLF to compete with [ 125 I]hLIF for binding to mLBP provided a large window in which to measure the degree of hLIF-like specific binding of each chimeric protein.
• the doses of hLIF, mLIF and m-hLLF hybrid required to inhibit 50% of [ 125 I]hLIF binding to mLBP (ID 50 ) were measured, and hLLF and mLLF were defined as having 100% and 0% hLIF-like binding activity, respectively.
• .Assays could then be norm ised for inter-assay variations by using a logarithmic scale to convert the ID for hLIF and mLLF to a score of 100% and 0%, respectively, then converting th ED JO for each hybrid to a percentage score between these two extremes.
• Hybrids MH3, MH4, MH5 and MH6 were constructed to resolve which structural modules were involved in the receptor binding site.
• the hybrid had no hLIF-like binding to mLBP.
• the hybrid displayed 26 ⁇ 1% hLIF-like activity in its ability to compete for [ 125 I]hLIF binding to mLBP ( Figure 2C).
• Hybrids MH7, MH8 and MH9 were constructed in order to test whether the D-helix co-operated with either the C-helix or the C-D loop to enhance hLLF-like binding specificity.
• Hybrid MH9 which contained hLIF residues in the C-helix and the D-helix, behaved like hybrid MH4, in which only the C-helix comprised hLIF sequence.
• FIG. 3A A typical example of a competitive binding assay between the chimeras and [ 125 I]hLIF on COS hLIF R cells is shown in Figure 3A.
• the hierarchy was the same in each assay ( Figure 1) indicating that the two features of hLIF that distinguish it from mLIF map to the same regions on the hLLF molecule.
• Hybrid MH10 which comprised an MH8 framework with a residue in the D-helix, K168, swapped to its mLIF equivalent (T168), showed that changing the least conserved residue on the external face of the D-helix did not affect hLIF-like binding.
• Hybrid MH14 which was based on hybrid MH5 but with additional substitutions Q112H and V113S, showed 71 ⁇ 2% hLIF-like binding to mLBP, indicating that these two residues define the contribution of the C-helix to hLIF-specific binding ( Figure 2C).
• Hybrid MH13 which was identical to hybrid MH14 except for an additional T168K mutation, also had 73 ⁇ 3% hLLF-specific binding to mLBP, confirming that the mutated residues in the C-helix were sufficient to restore MH6-like mLBP binding.
• These chimeras were also tested for their ability to compete with [ 125 I]hLIF binding to both mLBP and COS hLDF-R cells and gave qualitatively the same results in both assays (Figure 1). This strategy has, therefore, identified two residues within the C-helix, H112, and S113, as critical for hLLF-like binding to both mLBP and the hLLF receptor.
• a 1:4 to 1:8 dilution of normal mouse serum (equivalent to - 0.5-1 ⁇ g/ml mLBP) is required to block 50% of the Ml differentiation-inducing activity of up to 200 U/ml mLIF, whereas, due to the higher affinity of hLIF for mLBP, a 1:8192 dilution of serum ( - 0.6 ng/ml mLBP) is sufficient to block 50% of the Ml activity of up to 200 U/ml hLIF.
• This ability of mLBP to inhibit the differentiation-inducing activity of LIF in a species-specific manner was utilized as a second assay that distinguished hLIF from mLIF.
• Hybrids were also assessed for hLIF-like activity in this assay according to the dose of serum required to inhibit 50% of their Ml cell differentiation-inducing activity when they were present in the culture dish at a just maximal stimulatory concentration ( - 200 U/ml).
• An example of a typical serum dilution Ml assay is shown in Figure 5B. All data from this assay were consistent with the data obtained from the competitive binding assays with mLBP and [ 125 I]hLLF ( Figure 3B), thus eliminating the possibility that the higher affinity of hLIF for mLBP was an artefact of the binding assays themselves or of the use of iodinated hLIF.
• Hybrids in which the C-D loop had been subdivided were constructed.
• Hybrids MH16 and MH17 are based on a MH4 framework, with MH16 having the first half of the C-D loop (residues 131-153) substituted for hLIF residues .and MH17 having the second h-alf of the C-D loop (residues 154-160) swapped (see Table 1).
• the strategy for mutagenesis was to divide the LIF molecule into predicted secondary structural units of ⁇ -helix and connecting loop, test the contribution of each structural unit to hLIF-specific binding, then to ascertain the individual amino acids in that region that were responsible for that contribution.
• Residues in the C-D loop were initially investigated.
• the assays used were found to be best able to discriminate between hybrids in the middle of their range, so hybrid MH6 which had 78 ⁇ 2 % hLIF-like activity, was chosen as a basis for hybrids MH18-25 and MH31.
• .Amino acid residues in the C-D loop that were different between mLIF and hLLF were individually swapped from the hLLF residue to the equivalent mLIF residue, and the resulting chimeras tested for a decrease in hLLF-like activity relative to MH6.
• Hybrids MH24 .and MH25 showed a significant decrease in their % human score and their affinity for the hLIF-R ⁇ -chain relative to hLIF, and thus defined residues Val 155 and Lys 158 as being the two residues in the C-D loop that were responsible for its contribution to the overall hLIF-like activity.
• hybrid MH33 was constructed in order to test that the residues identified in the C-helix, the C-D loop and the B-C loop could be substituted onto a mLLF framework, and reconstitute hLIF-like activity.
• Hybrid MH33 was a mouse framework molecule that contained the human LIF residues His 112 and Ser 113 from the C-helix, Val 155 and Lys 158 from the C-D loop, and had 72 ⁇ 3 % hLIF- like activity (Figure 5B) and had only an 8-fold lower affinity for the hLIF-R ⁇ -chain compared to hLIF ( Figure 5C), indicating that only 5 residues out of 180 conferred approximately three-quarters of the binding energy of hLLF.
• the helices of the 4 ⁇ -helical bundle were designated A, B, C and D (ordered from the N-terminus) .and the loops were labelled according to the helices they join.
• the helical segments in G-CSF were treated as structurally conserved regions and their co-ordinates copied to the LIF model.
• the region Cys 131- Ser 135 was deemed not to be part of the C-helix so as not to constrain the disulfide bridges.
• mLIF, hLIF and all m-hLIF chimeras had an approximately equd ability to compete with [ 125 I]mLIF binding to the soluble mLIF-R ⁇ -chain, and had nearly equal biological activities in a mouse cell bioassay they must contain a common binding site for the ⁇ -ch n of the mLIF-R. This binding site on the lig-and (site a) presumably comprises conserved amino acid residues in the mLIF and hLIF proteins and so is invisible in our assay system.
• site b is comprised of a small number of residues in the predicted C-D loop, C-helix and B-C loop of hLLF and is proposed to mediate the exclusive properties of hLLF. These residues form a cluster on one face of the predicted three-dimensional structure of hLLF ( Figure 6).
• the primary interaction site on the LIF ligand for its isologous receptor ⁇ -chain is not the same in the human and mouse systems. It is proposed that hLLF but not mLLF is able to recognise a site which is conserved in both the hLLF and mLLF receptor ⁇ -chains (site B), while the primary binding site for mLIF on the mLLF-R ⁇ -chain (site A), is not present on the hLIF-R ⁇ -chain.
• MOLECULE TYPE protein - murine LIF; the mature protein begins at Ser 1
• MOLECULE TYPE protein - human LIF; the mature protein begins at Ser 1
• MOLECULE TYPE protein - porcine LIF; the mature protein begins at Ser 1

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