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WO1998048011A1 - Novel chimeric molecules - Google Patents

Novel chimeric molecules Download PDF

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Publication number
WO1998048011A1
WO1998048011A1 PCT/AU1998/000282 AU9800282W WO9848011A1 WO 1998048011 A1 WO1998048011 A1 WO 1998048011A1 AU 9800282 W AU9800282 W AU 9800282W WO 9848011 A1 WO9848011 A1 WO 9848011A1
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Prior art keywords
polypeptide
chain
domain
derivative
haemopoietin
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PCT/AU1998/000282
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French (fr)
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WO1998048011A9 (en
Inventor
Meredith Jane Layton
Catherine Mary Owczarek
Nicos Antony Nicola
Donald Metcalf
Yu Zhang
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The Walter And Eliza Hall Institute Of Medical Research
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Priority to AU70141/98A priority Critical patent/AU7014198A/en
Publication of WO1998048011A1 publication Critical patent/WO1998048011A1/en
Publication of WO1998048011A9 publication Critical patent/WO1998048011A9/en

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to chimeric molecules and more particularly to interspecies cytokine receptor ⁇ -chain chimeras. Even more particularly, the present invention provides interspecies leukaemia inhibitory factor receptor ⁇ -chain chimeras.
  • the chimeric molecules of the present invention are useful inter alia as antagonists of human cytokine activities in vivo.
  • LIF is a glycoprotein initially identified, purified and genetic sequences encoding same cloned based on its ability to induce differentiation in the mouse leukaemic cell line Ml (reviewed by Metcalf, 1991). Subsequently, LEF has been shown to have a wide variety of actions in many different cell types and tissues including adipocytes, osteoblasts, megakaryocytes, hepatocytes, neurons, embryonal stem cells and primordial germ cells (reviewed by Hilton, 1992). A critical role of LEF in the implantation process has been implicated, since mice in which the LEF gene has been ablated are essentially normal but are unable to implant their otherwise viable blastocysts (Stewart et al., 1992).
  • LEF exerts its biological actions through high affinity receptors which are expressed on the surface of LIF-responsive cells.
  • the high affinity LIF receptor (“LIFR") complex is composed of two components: the LIFR ⁇ -chain, which binds LIF with low affinity, and gpl30 which does not itself bind LIF but is essential for high affinity complex formation and signal transduction (Gearing et al, 1992).
  • the LIFR ⁇ -chain and gpl30 are components of other receptor systems including those of oncostatin-M (Gearing and Bruce, 1992; Gearing et al, 1992), ciliary neurotrophic factor [CNTF] (Ip et al, 1992) and the cytokine cardiotrophin-1 [CT-1] (Pennica et ⁇ /., 1995a; Pennica et ⁇ /., 1995b).
  • interleukin-6 [EL- 6] (Hibi et al, 1990) and interleukin-11 [IL-11] Fourcin et al, 1994; Hilton et al, 1994) receptors also have gpl30 as part of their high affinity receptors and this use of common receptor components may provide a basis for the overlapping biological activities and functional redundancy of these cytokines.
  • Targeted disruption of the LIF receptor ⁇ -chain results in mutant animals with neuronal, musculo-skeletal, placental and metabolic defects (Ware et al, 1995). Mice carrying the LIFR nullizygous mutation died shortly after birth indicating that the LEFR ⁇ -chain is necessary for normal development and survival.
  • the LIFR ⁇ -chain is a member of the haemopoietin family of receptors (Bazan, 1990). In contrast to the majority of the members of this family, the LIFR ⁇ -chain contains in its extracellular domain two copies of the haemopoietin domain, which are separated by an immunoglobulin-like domain (Cosman, 1993; Gearing et al, 1991).
  • the L FR ⁇ -chain Similar to the G-CSF receptor (Fukunaga et al, 1990a; Fukunaga et al, 1990b) and gp 130 (Hibi et al, 1990), the L FR ⁇ -chain also contains three fibronectin type in (FNIII) repeats that are located C-terminal to the membrane proximal haemopoietin domain. Mutagenesis studies of the G-CSFR (Fukunaga et al, 1991) and gpl30 (Horsten et al, 1995) have indicated that the FNHI repeats are not essential for ligand binding.
  • FNIII fibronectin type in
  • mLIF murine LIF
  • hLIF human LIF
  • mLIFR high and low affinity mouse LEFRs
  • mLIFR mouse LEFRs
  • hLIF also binds to both a naturally occurring soluble form of the mLIFR ⁇ -chain and mLIF-binding protein ["mLBP”] (Layton et al, 1992).
  • hLIF binds to mLIFR ⁇ -chain with a much higher affinity (K d ⁇ 10-20 nM) than it does to the isologous hLIFR ⁇ -chain or than mLIF binding to the mLIFR ⁇ -chain.
  • Cross- competition studies using the mLIFR ⁇ -chain reveal that the competition curves are dependent on which LEF is used as the radioactive tracer and this behaviour is interpreted as an interference by each type of LEF in the binding of the other.
  • LEF binding protein occurs at high levels (2 ⁇ g/ml) in normal mouse serum and is dramatically elevated in pregnancy (Layton et al, 1992; Tomida et al, 1993).
  • the very high binding affinity of this receptor for hLEF makes it a potent biological inhibitor of hLIF (Layton et al, 1994a), and suggests that it could be useful in clinical situations such as for treating inflammatory diseases where LIF levels may be expected to be elevated.
  • LIF leukaemia inhibitory factor
  • MH human LIF receptor hybrids
  • Sequence identity numbers for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
  • One aspect of the present invention provides a polypeptide or a derivative or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine binding properties.
  • polypeptide or derivative or chemical equivalent thereof comprising first and second covalently linked portions wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof such that said polypeptide has LEF binding properties.
  • Yet another aspect of the present invention provides a polypeptide or a derivative or a chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a LIFR ⁇ -chain haemopoietin domain and said other portion comprises at least two LIFR ⁇ -chain Ig-like domains such that said polypeptide has LIF binding properties.
  • Still another aspect of the present invention contemplates a polypeptide or derivative or chemical equivalent thereof having the structure:
  • X, and X 3 are located distally and proximally, respectively, to the transmembrane domain of the LEFR ⁇ -chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
  • X 2 is an Ig-like domain or a functional derivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
  • Still yet another aspect of the present invention provides a polypeptide or derivative or chemical equivalent thereof having the structure: - ⁇ .
  • X, and X 3 may be the same or different and each is a LIFR ⁇ -chain haemopoietin domain; X 2 is a LIFR ⁇ -chain Ig-like domain; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
  • Another aspect of the present invention provides a chimera comprising a LIFR ⁇ -chain haemopoietin domain or a functional derivative thereof and a LIFR ⁇ -chain Ig-like domain or a functional derivative thereof wherein binding of LEF to the chimera gives rise to a two- contact state and a single kinetic dissociation rate according to the Scatchard transformation of LEF binding to its receptor at equilibrium:
  • B is the specifically bound LIF concentration
  • F is the free LEF concentration
  • R ⁇ is the total concentration of LEF receptors
  • K is the equilibrium affinity constant for the first contact site of LIF with its receptor and K-, is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LEF.
  • nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide comprising first and second portions wherein one portion comprises a haemopoietin domain or a functional derivative then of and said other portion comprises an Ig-like domain or a functional derivative thereof wherein said polypeptide exhibits cytokine binding properties.
  • Figure 1 is a schematic representation of a proposed model of the LIFR ⁇ -chain interacting with both hLIF and mLIF. Both mLIF and hLEF first contact site A on the LIFR ⁇ -chain, which is mainly dependent on the association rate (k onI ) and the dissociation rate (k om ). For hLIF binding the primary to mLIFR ⁇ , interaction leads to full receptor isomerisation which results in further interactions on site B on the receptor. The isomerisation process is determined by the isomerisation constant (1/K C ).
  • Figure 2 is a representation of amino acid sequence specifications for recombinant LIF Receptors.
  • Amino acid sequences are numbered according to the LEFR sequence described by (Gearing et al, 1991).
  • the letters M and H denote that amino acids are derived from mLIFR sequences or hLEFR sequences respectively. Because gaps were introduced in the mLEFR amino acid to maximise the alignment, the numbers refer to the specific hLEFR or mLIFR amino acid sequence.
  • the amino acid sequences of the recombinant receptors are shown as beginning at residues 52 (hLIFR) or 50 (mLIFR) the N-termini were modified as described in the Examples.
  • Figure 3 is a representation of analyses of mouse-human hybrid LIF receptors expressed in Pichia pastoris.
  • A Photographic representation of Western blotting of Recombinant receptors from P. pastoris. Culture supematants were separated by 0.1% w/v SDS-10% w/v PAGE under reducing conditions, transferred to PVDF membranes and hybridised to 12CA5 antibody as described in the Examples.
  • B Photographic representation of chemical cross- linking of Recombinant receptors from P. Pastoris. Culture supernatant were cross-linked to 125 H ⁇ LIF in the absence or presence of excess unlabelled hLIF as described in the Examples.
  • C Graphical representation showing a gel filtration profile of recombinant LIF receptors from P.
  • Figure 4 is a graphical representation of a Scatchard analyses of 125 D ⁇ LEF binding to chimeric LIF receptor variants. Saturation binding was performed by incubating aliquots of P. pastoris culture supernatant containing recombinant LEF receptors with increasing concentrations of 125 IhLIF. Specific binding assays and Scatchard transformations were performed as described in the Examples. These Scatchard binding data are representative for several independently performed experiments and the resulting Y ⁇ values are shown in Table ⁇ .
  • the receptor variants are as follows: (A) mLIFR; (B) MH1LIFR; (C) MH2LIFR; (D) MH3LIFR; (E) MH4LEFR; (F) MH5LIFR; (G) MH6LIFR; (H) MH7LIFR; (I) MH8LIFR, and (J) hLIFR.
  • Figure 5 is a graphical representation of kinetic dissociation of 1 5 IhLIF from chimeric LIFRs. Each chimeric LIFR (0.01-0.02 nM) was incubated at room temperature for 3-4 hours with 125 IhLIF and kinetic dissociation assays performed as described in the Examples. The plot of the natural log of the ratio of the amount of 125 IhLIF remaining bound after a given time (SB,) to the amount bound initially (SB 0 ) versus time is shown. Estimates of the kinetic rate constant governing dissociation (k d ) of ligand and receptor were made using the curve-fitting program KINETIC and shown in Table II.
  • the receptor variants are as follows: (A) mLIFR; (B) MH1LIFR; (C) MH2LIFR; (D) MH3LIFR; (E) MH4LIFR; (F) MH5LIFR; (G) MH6LIFR; (H) MH7LIFR; (I) MH8LIFR, and (J) hLIFR.
  • Figure 6 is a graphical representation of displacement curves for unlabelled mLfF ( ⁇ ) and hLIF (O) competing for binding with 125 IhLIF to the mLIFR, hLIFR and hybrid LIF receptors.
  • the receptor variants and concentrations are as follows: (A) mLEFR (0.067 nM); (B) MH1LIFR (0.033nM); (C) MH2LIFR (0.142 nM); (D) MH3LIFR (0.014 nM); (E) MH4LIFR (0.027 nM); (F) MH5LEFR (0.033 nM); (G) MH6LIFR (0.039 nM); (H) MH7LIFR (0.014 nM); (i) MH8LIFR (0.059 nM), and (J) hLIFR (0.033 nM).
  • Figure 7 is a photographic representation of the effect of chimeric LIFRs on hLIF-induced STAT-3 tyrosine phosphorylation. Ml cells were incubated at 37°C for 5 min in the presence of either 1 ng of hLIF, or 1 ng of hLIF together with 11 ng of chimeric LIFR, or 11 ng of chimeric LEFR alone and analysed by immunoprecipitation and Western blotting as described in the Examples.
  • the present invention is predicated in part on the exploitation of the structural homology of mouse and human LEF receptors and their differing binding characteristics for mouse and human LEF to define the structural elements involved in LIF binding.
  • one aspect of the present invention provides a polypeptide or a derivative or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an immunoglobulin (Ig) -like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine binding properties.
  • the first portion comprises at least two haemopoietin domains.
  • the present invention is hereafter described in relation to the first and second portions being covalently linked together by a peptide bond.
  • the first and second portions may be linked by ionic bonds, hydrogen bonds, ie. electrostatic interaction, molecular bridging, molecular association or other interactive bonding mechanisms including other covalent bonding systems such as disulphide bridges.
  • Reference to a first and second portion is not intended to exclude third or subsequent portions which are encompassed by the present invention.
  • the polypeptide is a chimera encoded by single nucleotide sequence. A "chimera" has a similar meaning herein to a "fusion" molecule.
  • the cytokine is LIF although the present invention extends to functional derivatives, homologues or analogs of LIF as well as other cytokines.
  • cytokines contemplated by the present invention include, but are not limited to, interleukins, colony stimulating factors.
  • the present invention is hereinafter described in relation to chimeras involving LIFR or molecules having LIF binding properties. This is done, however, with the understanding that the present invention extends to other cytokine receptors or molecules having other cytokine binding properties.
  • another aspect of the present invention is directed to a polypeptide or derivative or chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof such that said chimera has LIF binding properties.
  • the haemopoietin domain comprises a LEFR ⁇ -chain haemopoietin domain and the Ig-like domain comprises a LEFR ⁇ -chain Ig-like domain.
  • the first portion comprises at least two haemopoietin domains.
  • polypeptide or a derivative or a chemical equivalent thereof comprising first and second covalently linked portions wherein one portion comprises a LEFR ⁇ -chain haemopoietin domain and said other portion comprises at least two LIFR ⁇ -chain Ig-like domain such that said polypeptide has LEF binding properties.
  • one of said portions or a functional derivative or chemical equivalent thereof is from one source and said other portion or functional derivative or chemical equivalent thereof is from another source.
  • sources include, but are not limited to, different species or allelic variants within a single species.
  • one of said portions is from a murine LEFR (mLIFR) ⁇ -chain and said other portion is from a human LEFR (hLIFR) ⁇ -chain.
  • mLIFR murine LEFR
  • hLIFR human LEFR
  • Such a heterologous molecule is useful for example, for humanising a mLEFR ⁇ -chain.
  • the LEFR ⁇ -chain Ig-like domain is from mLIFR ⁇ -chain or hLIFR ⁇ -chain and the LIFR ⁇ -chain haemopoietin domain is from mLIFR ⁇ -chain or hLIFR ⁇ -chain.
  • said polypeptide or derivative or chemical equivalent thereof comprises at least three portions, wherein two portions comprise haemopoietin domains and one portion comprises an Ig-like domain.
  • the polypeptide or derivative or chemical equivalent thereof comprises a LIFR ⁇ - chain Ig-like domain flanked by at least two LIFR ⁇ -chain haemopoietin domains.
  • the binding of LIF to the chimera is thought to lead to ligand-dependent receptor isomerisation.
  • the predominant involvement of the Ig-like domain is in determining ligand binding specificity and conferring high affinity LEF binding.
  • the chimera is selected from the listing consisting of MH1LIFR, MH2LEFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as defined in Figure 2.
  • the chimera is MH3LEFR ( Figure 2) and comprises at the membrane-distal position, an hLIFR ⁇ -chain haemopoietin domain, an mLIFR ⁇ -chain Ig domain and at the membrane-proximal position an mLIFR ⁇ -chain haemopoietin domain.
  • the chimera is MH4LEFR ( Figure 2) and comprises at the membrane-distal position an mLEFR ⁇ -chain haemopoietin domain, mLIFR ⁇ -chain Ig domain and at the membrane-proximal position an hLIFR ⁇ -chain haemopoietin domain.
  • the chimera is MH5LIFR ( Figure 2) and comprises at the membrane-distal position an hLIFR ⁇ -chain haemopoietin domain, an mLEFR ⁇ -chain Ig domain and at the membrane-proximal position an hLIFR ⁇ -chain haemopoietin domain.
  • Chimeras MH3LIFR, MH4LIFR and MH5LDFR all contain an intact Ig-like domain from mouse LEF receptor chain and high affinity 125 IhLIF binding ⁇ K d ⁇ 11-60 pM) similar to that seen for hLEF binding to the mLIFR (Fig. 4, Table II). This indicates that the immunoglobulin-like domain from the mouse LEF receptor has the most important influence in conferring the high affinity binding of hLIF.
  • polypeptide or derivative or chemical equivalent thereof having the structure:
  • X [ and X 3 are located distally and proximally, respectively, to the transmembrane domain of the LEFR ⁇ -chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
  • X 2 is an Ig-like domain or a functional derivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
  • the present invention provides a polypeptide or derivative thereof having the structure: wherein:
  • X, and X 3 may be the same or different and each is a LIFR ⁇ -chain haemopoietin domain;
  • X 2 is a LIFR ⁇ -chain Ig-like domain; and wherein the polypeptide or derivative thereof is capable of binding, interacting, influencing or otherwise associating with LEF.
  • X, and X 3 are derived from mLIFR ⁇ -chain or hLIFR ⁇ -chain.
  • X 2 is derived from mLIFR ⁇ -chain or hLIFR ⁇ -chain or is either composed of hLEFR ⁇ -chain amino acid residues at the N-terminal region, to approximately half way down the Ig-like domain, and mLEFR ⁇ -chain amino acid residues at the C-terminal region of the Ig-like domain or is composed in the converse.
  • both haemopoietin domains or their functional derivative are of murine or human origin or one each from a human or murine source domain or functional derivative thereof and are capable of binding, interacting, influencing or otherwise associating with LIF.
  • the chimeras, or functional derivatives thereof selected from MH1LIFR, MH2LIFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as set forth in Figure 2.
  • Still more preferred are the chimeras, or functional derivatives thereof, MH3LEFR, MH4LIFR and MH5LEFR as set forth in Figure 2.
  • the present invention further contemplates a range of derivatives of the polypeptides of the present invention.
  • Derivatives include fragments, parts, portions, mutants, homologues and analogs of the chimera and corresponding genetic sequence.
  • Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the chimera or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding the chimeras.
  • "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences.
  • Analogues of said polypeptides include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ⁇
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminol ixanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and or D- isomers of amino acids.
  • the use of unnatural amino acids provides a means of stabilising the polypeptide structure especially when the polypeptide is used in vitro or for diagnostic purposes.
  • a list of unnatural amino acid, contemplated herein is shown in Table 1.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D- ⁇ -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D- ⁇ -methylglutamine Dmgln N-(2- aminoethyl)glycine Naeg
  • peptides can be conformationally constrained by, for 5 example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C a and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates chemical analogues of the polypeptides of the present invention capable of acting as antagonists or agonists of said polypeptides or which can act as functional analogues of said polypeptides.
  • Chemical analogues may not necessarily be derived from said polypeptides but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain
  • Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.
  • mLIF binds to the mLEFR ⁇ -chain with low affinity but does not detectably interact with the hLIFR ⁇ -chain (Layton et al, 1994b; Owczarek et al, 30 1993).
  • Human LIF binds to the mLIFR ⁇ -chain and does so with a much higher affinity than mLIF.
  • the higher affinity binding of hLEF to the mLEFR ⁇ -chain is found to be due almost exclusively to a slower kinetic dissociation rate compared to mLIF.
  • the binding affinity for hLEF is most strongly dependent on the presence of an intact mLEFR Ig- like domain irrespective of the species origin of the haemopoietin domain(s).
  • the species origin of the membrane proximal haemopoietin domain is more important than the distal haemopoietin domain in determining hLEF binding affinity.
  • the dissociation kinetics are predominantly a single class with slow dissociation rate (off-rate).
  • the dissociation kinetics are biphasic with variable ratio of fast-off and slow-off components depending on the affinity.
  • B is the specifically bound LIF concentration
  • F is the free LIF concentration
  • R ⁇ is the total concentration of LEF receptors
  • K is the equilibrium affinity constant for the first contact site of LIF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF.
  • said chimera exhibits an apparent equilibrium dissociation constant for binding to hLEF of about 300 pM. More preferably, said chimera exhibits an affinity binding to hLEF of about 150 pM and even more preferably, about 10 pM affinity binding to hLEF.
  • said chimera exhibits a bi-phasic dissociation rate for hLIF with one phase being of about k 0j fQ.16 min "1 and the second phase of about k ojg ⁇ 0.002 min "1 . More preferably said chimera exhibits a bi-phasic dissociation rate for hLIF of about k OJ[ -0.07 min "1 and a second dissociation phase of about fc o ⁇ 0.001 min "1 and even more preferably a single slow dissociation rate for hLIF of about k oj f-0.00l min "1 .
  • a chimera comprising a LEFR ⁇ -chain haemopoietin domain or a functional derivative thereof and a LEFR ⁇ -chain Ig-like domain or a functional derivative thereof wherein binding of LIF to the chimera gives rise to a two-contact state and a single kinetic dissociation rate according to the Scatchard transformation of LEF binding to its receptor at equilibrium:
  • a further aspect of the present invention contemplates the use of chimeras as therapeutic agents in relation to human disease conditions.
  • the LEF binding properties of the chimeras of the present invention are particularly useful, but in no way limited to, use as a biological inhibitor of LEF.
  • LEF is bound by the chimera and thereby blocked from binding to any other unoccupied LEFR.
  • blocking of hLEF induced STAT-3 tyrosine phosphorylation in Ml cells is measured.
  • the differentiation of Ml cells is dependent upon the binding of LEF to the Ml cell surface LEFR.
  • STAT-3 activation is a critical step in gpl30-mediated terminal differentiation of Ml cells.
  • Tyrosine phosphorylation of STAT-3 is increased by hLIF stimulation of Ml cells within five minutes.
  • STAT-3 tyrosine phosphorylation is almost completely blocked by pre-incubation of hLIF with chimeric molecule MH3LIFR.
  • the chimeric LIFR could therefore be useful as a therapeutic agent in clinical situations such an inflammatory diseases where LIF levels are expected to be elevated.
  • a polypeptide, derivative or chemical equivalent thereof, comprising, but not limited to, X,X 2 X 3 , as defined above, is designed and constructed such that it binds, interacts or otherwise associates with LIF activity.
  • binding, interaction or association of said polypeptide with LIF results in inhibition of LEF activity.
  • a mLIFR ⁇ -chain or derivative or chemical equivalent thereof comprising said X,X 2 X 3 is "humanised” by the substitution of sufficient of the mLIFR ⁇ -chain Ig-like domain (or part thereof) or haemopoietin domains with hLEFR ⁇ - chain Ig-like domains (or part thereof) or haemopoietin domains, respectively, to result in a chimeric LEFR ⁇ -chain exhibiting a high affinity for hLIF binding.
  • Such humanised mLIFR could act as a specific and potent antagonist of hLEF.
  • a "sufficient" substitution is the minimum required to result in said "humanised" chimera exhibiting at least 10-100 pM hLIF binding affinity.
  • the present invention contemplates said chimeras or derivatives or chemical equivalents thereof and one or more pharmaceutically acceptable carriers and/or diluents.
  • the polypeptides of the present invention may be produced by recombinant DNA means or by chemical synthetic processes. With respect to the former this aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin domain or functional derivative thereof and an Ig-like domain or functional derivative thereof.
  • the nucleic acid molecule comprises a sequence of nucleotides which encode or are complementary to nucleotide sequences which encode the polypeptides of the present invention.
  • the nucleic acid molecule of the present invention encodes said polypeptides, said nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule comprising a sequence of nucleotides substantially encoding said polypeptides
  • nucleic acid molecule comprising a sequence of nucleotides having at least about
  • nucleic acid molecule capable of hybridising under low stringency conditions at 42°C to the nucleotide sequence encoding said polypeptides.
  • the nucleotide molecule is preferably derivable from the human genome but genomes and nucleotide sequences from non-human animals are also encompassed by the present invention.
  • Non-human animals contemplated by the present invention include livestock animals (e.g. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals (e.g. dogs, cats), birds (e.g. chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals (e.g. foxes, kangaroos, dingoes).
  • livestock animals e.g. sheep, cows, pigs, goats, horses, donkeys
  • laboratory test animals e.g. mice, rats, guinea pigs, hamsters, rabbits
  • domestic companion animals e.g. dogs
  • Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • 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 (eg. E. coli) or a eukaryotic cell (eg. an animal or mammalian cell).
  • the nucleic acid molecules encodes a fusion molecule comprising a haemopoietin domain or functional derivative thereof or an Ig-like domain or functional derivative thereof. Expression of the nucleic acid molecule of the present invention leads to synthesis of a fusion molecule.
  • polypeptides and nucleic acid molecules of the present invention are preferably in isolated form, having undergone at least one purification step from their original source.
  • the present invention further contemplates use of the polypeptides herein described in the manufacture of a medicament for the treatment of a condition requiring the antagonsim of LIF.
  • a cDNA encoding a soluble mouse LIFR ⁇ -chain was modified to encode an Xhol site and an in-frame 12CA5 epitope (YPYDVPDYA) [SEQ. ID NO: 1] (Wilson et al, 1984).
  • the 3' end of the mLIFR cDNA was modified to encode an Xbal site, and a stop codon was introduced after amino acid residue 531 in te amino acid sequence described in (Gearing et al, 1991).
  • a cDNA encoding the hLIFR ⁇ -chain (Owczarek et al, 1993) wv also altered at its 5' end to encode an Xhol site and an in-frame 12CA5 epitope.
  • the 3' end was also modified to encode an Xbal site, and a stop codon was introduced after position 536 in the amino acid sequence described by (Gearing et al, 1991).
  • the sequence at the N- terminus of the recombinant MLEFR was GVQ YPYDVPDYA [SEQ. ED NO: 2], and trie sequence at the N. terminus of the recombinant hLIFR was GAPYPYDVPDYA [SEQ. ID NO: 3].
  • the recombinant LIFRs therefore lacked the cytoplasmic domain, transmembrane domain and all three FNHI-like domains.
  • the resulting cDNAs were subsequently ligated into the Pichia pastoris expression vector pPIC9, that was digested with Xhol and AvrEl, as Xhol-Xbal fragments.
  • Mutagenesis of the LIFR cDNAs and construction of hybrid mouse- human LIFRs was carried out using a PCR-based technique, splicing by overlap extension (Ho et al, 1989), and Pfu polymerase (Strategene).
  • All cDNAs were expressed as soluble secreted proteins in the methylotrophic yeast Pichia pastoris.
  • This expression system uses the promoter from the methanol-induced alcohol oxidase gene, AOXI. Stably expressing clones are selected using the HIS4 gene as a selectable marker.
  • the recombinant plasmids were digested with either Bgl I or Sail and integrated into host cells by tri. isforming his4 (GS115) P. pastoris sphaeroplasts as described (Cregg et al, 1985). Digestion of a plasmid with Bglll disrupts the AOXI gene and results in a strain that is phenotypically His + Mut s (Methanol utilisation sensitive).
  • plasmids MH1LIFR, MH3LEFR, MH5LIFR and MH7LIFR contained Bglll sites, they were digested with Sail prior to transformation into P. pastoris sphaeroplasts. The resulting strains were His + Mut + . His + transformants were patched first onto a nitrocellulose filter overlayed onto an agar plate (MM) containing 0.5% (v/v) methanol, 1.34% (w/v) Yeast Nitrogen Base (YNB) and 4xl0 "5 % (w/v) biotin, and then onto another agar plate (MD) containing 1% (w/v) dextrose instead of methanol as the carbon source.
  • MM agar plate
  • MD agar plate
  • Clones identified in this way were grown in a shaking incubator at 30°C to an OD ⁇ of 2-6 in 10 ml of medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, lOOmM potassium phosphate (pH 6), 1.34% (w/v) YNB, 4xl0 "5 % (w/v) biotin, and 1% (v/v) glycerol. After 5 -fold concentration by centrifugation the cultures were resuspended in medium that contained 0.5% (v/v) methanol instead of glycerol to induce the cells to express the heterologous protein.
  • Proteins separated by SDS-PAGE were electrophoretically transferred onto pre- wetted 5 polyvinylidene diflouride (PVDF-Plus, Micron Separations Inc.) membrane using a transfer buffer containing 20mM Tris-HCI, 150 mM glycine pH 8.2, and 20% (v/v) methanol in a Mini-Protean II system. Blots were blocked in 1% BSA (w/v) in PBS containing 0.1% (v/v) Tween-20, followed by incubation with mouse 12CA5 antibody and then horseradish peroxidase-conjugated rabbit-anti-mouse antibody (DAKO, Denmark). The receptor 0 proteins were visualised using an ECL substrate kit (Amersham) followed by autoradiography.
  • P. pastoris expression supernatant was concentrated t- to 50- fold using a Centricon-50 microconcentrator (Amicon). Aliquots (200-500 ⁇ l) of each sample were injected onto a Superose-12 10/30 (Pharmacia) column equilibriated in PBS containing 0.02% (v/v) Tween- 20, 0.02% (w/v) sodium azide and 5% (v/v) glycerol. Elution was carried out isoctratically using the same buffer and monitored by absorbance at 280 nm. The 0.5-ml fractions were collected at a flow rate of 0.5 ml per min. An aliquot of each fraction was tested for 125 IhLIF binding as previously described.
  • Each chimeric LIF receptor sample (0.25-0.5 nM) was mixed with approximately 1.6 nM 125 IhLIF (200,000 cpm) in 20 ⁇ l of PBS containing 0.02% (v/v) Tween-20 and 0.02% (w/v) sodium azide, in the presence or absence of 100 ng of unlabelled hLIF, and the binding reaction was performed for 90 min at room temperature.
  • Ml cells (10 7 per sample) were stimulated for 5 min at 37°C with either 1 ng of hLIF, 1 ng of hLIF together with 11 ng of each chimeric LEFR, or 11 ng of each chimeric LIFR alone and then lysed in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2 MM EDTA, 1% (v/v) Triton X-100, ImM Na 3 VO 4 and proteinase inhibitors.
  • the immune complexes were washed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (w/v) NP-40, ImM Na 3 VO 4 and proteinase inhibitors, eluted from the sepharose beads by boiling in SDS sample buffer under reducing conditions for 5 min before being subjected to 4-20% (w/v) polyacrylamide SDS-PAGE and then transferred to a pre- wetted polyvinylidene diflouride membranae (PVDF-Plus, Micron Separations Inc.).
  • PVDF-Plus polyvinylidene diflouride membranae
  • the membranae was incubated with an anti-STAT-3 polyclonal antibody (K-15, Santa Cruz Biotechnology), followed by incubation with a goat anti-rabbit immunoglobulin polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark).
  • an anti-STAT-3 polyclonal antibody K-15, Santa Cruz Biotechnology
  • DAKO horseradish peroxidase
  • the phosphorylated STAT-3 protein was visualised by autoradiography using an ECL system (Amersham). Quantitation of STAT-3 phosphorylation levels was performed by densitometric analysis of the band intensities using Imagequant version 3.0 software.
  • Mouse LIFR and human LIFR were initially expressed as soluble proteins that were truncated 13 amino acid residues after the predicted membrane proximal haemopoietin domain. These receptors therefore did not contain the cytoplasmic domain, the transmembrane domain and all three fibronectin type Ell repeats that are present in native cellular LEF receptors.
  • the recombinant proteins were modified at their N-termini to encode a 12CA5 epitope tag (Wilson et al, 1984) in order to monitor their expression, and contained the yeast ⁇ -factor signal peptide to enable the proteins to be secreted into the culture medium after transformation into yeast.
  • the molecular weight of these recombinant receptors is predicted to be approximately 65 kDa.
  • Fig. 3B Chemical cross-linking (Fig. 3B) of the soluble receptor variants with 125 IhLIF demonstrated that only the species with molecular weights higher than 70 kDa could specifically interact with 125 IhLEF. Furthermore, the position of the 125 IhLEF binding peak at 70-10 kDa (Fig. 3C) by size-exclusion chromatography of soluble receptor samples indicated that the hybrid LIFRs have the apparent molecular weight of 70-100 kDa and exist as monomers. The expression levels of the different receptors were variable, ranging from 10 ⁇ g to 1 mg of receptor protein per litre of expression medium as determined by Scatchard analysis.
  • Hybrid LEFRs MH4 and MH5 were found to be difficult to detect by Western blot analysis which may be due to either extremely low expression levels, or cleavage of the 12CA5 epitope tag during protein production. However, the behaviour of these two hybrid receptors was similar to that of the other recombinant receptors with respect to both chemical cross- linking with 125 D ⁇ LIF and size-exclusion chromatography.
  • the mouse LIFR ⁇ -chain binds hLIF with high affinity whereas the human LEFR ⁇ -chain binds hLIF with low affinity (Layton et al, 1994a).
  • the hybrid LIF receptors were characterised by performing binding assays and subsequent Scatchard analyses to determine their affinities of interaction with 125 IhLEF> As shown in Fig. 4 and Table II, the recombinant' mouse and human LIFRs had K d values of 10-46 pM and 0.3-0.9 nM respectively, which were similar to those observed for the naturally-occurring soluble mouse LIF receptor and a soluble form of human receptor ⁇ -chain expressed in COS cell-conditioned medium (Layton et al. 1994a), respectively.
  • Hybrids MH3LIFR, MH4LEFR and MH5LIFR all contain an intact Ig-like domain from mouse LEF receptor but have either one haemopoietin domain (MH3 and MH4) or two haemopoietin domains (MH5) from the human LEF receptor.
  • all of these three hybrids exhibited high affinity 125 IhLEF binding (K d ⁇ 11-60 pM) similar to that seen for hLEF binding to the mLEFR (Fig. 4, Table II). This strongly suggested that the immunoglobulin- like domain from the mouse LIF receptor has the most important influence in conferring the high affinity binding of hLEF.
  • hybrid MH1LIFR the N-terminal region, to approximately halfway down the Ig-like domain, was composed of hLIFR residues and the C-terminal half was composed of mLEFR residues while hybrid MH2LEFR was the converse.
  • riant hybrid LIF receptors were tested for binding of 125 IhLIF by Scatchard analysis both had intermediate affinities (K d ⁇ 190-400 pM and 150-440 pM respectively) (Fig. 4, Table II).
  • the relative contributions of the membrane-distal and membrane-proximal haemopoietin domains from the mLIFR to 125 IhLIF binding were investigated next.
  • Hybrid MH6LIFR was composed almost entirely of mLEFR residues except that the Ig-like domain was derived from the hLEFR and it bound 125 IhLIF with intermediate affinity ⁇ K d ⁇ 260 pM).
  • MH7LEFR in which only the membrane-proximal haemopoietin domain was composed of mLIFR residues, also bound 125 IhLIF with intermediate affinity (K d ⁇ 300 pM) (Fig. 4, Table II). This result indicated that of the two mLEFR haemopoietin domains the major contribution to high affinity 125 IhLEF binding was from the membrane-proximal haemopoietin domain.
  • MH8LIFR which contained only the membrane-distal haemopoietin domain derived from mLIFR residues, had an almost identical binding affinity for 125 IhLIF to the hLEFR (K d ⁇ 2 nM), indicating that the mouse LIFR membrane-distal haemopoietin domain is not involved in high affinity I25 IhLIF binding (Fig. 4, Table II).
  • the difference in hLEF-binding affinities of chimeric LIFRs was further explored by performing kinetic dissociation experiments (Fig. 5).
  • the LIF receptor variants which had high affinity binding for hLEF based on Scatchard analysis, including mLIFR, MH3LIFR, MH4LIFR and MH5LEFR, showed single slow dissociation rates (K off ⁇ 0.16-0.2 min "1 ) and the other slow (K off ⁇ 0.001-0.002 min "1 ).
  • hybrid receptors which contained either an intact mLIFR Ig-like domain (hybrids MH3LIFR, MH4LIFR and MH5LIFR) or part of an mLIFR Ig-like domain (hybrids MH1LIFR and MH2LIFR) (Fig. 6).
  • the ID 50 values for either hLEF or mLEF competing with 125 IhLEF binding to these hybrid receptors were essentially the same.
  • 125 ImLEF was able to detectably bind to MH3LIFR, MH4LEFR and MH5LH-R but only at 10- to 50-fold higher receptor concentrations compared to those used for 125 IhLEF binding (data not shown).
  • mLIF was unable to compete with 125 IhLIF even at high ligand concentrations (100 ⁇ g/ml).
  • the JD 50 values for hLIF competing with 125 IhLIF bound to these receptors were 2- to 10-fold higher compared to that obtained with the mLIFR. This is essentially consistent with the K j values obtained from the Scatchard analysis (Table II).
  • a short term assay was employed which involved stimulation of STAT-3 tyrosine phosphorylation by hLEF in Ml cells.
  • STAT-3 activation is a critical step in gpl SO- mediated terminal differentiation of Ml cells (Minami et al, 1996) and, as shown in Fig. 7, tyrosine phosphorylation of STAT-3 was dramatically increased by hLIF stimulation of Ml cells within 5 minutes. This STAT-3 phosphorylation was almost completely blocked by preincubation of hLEF with recombinant mouse LEFR and hybrid MH3LIFR (Fig. 7).
  • hybrids MH4LEFR, H5LIFR and MH6LIFR also showed a moderately inhibitory effect (65%) on hLEF-induced STAT-3 phosphorylation although it was not as significant as that seen for mLEFR and MH3 LIFR.
  • STAT-3 phosphorylation in Ml cells was not affected by addition of chimeric LEFRs alone (Fig. 7).
  • Owczarek CM., Layton, M.J., Metcalf, D., Lock, P., Willson, T.A., Gough, N.M. and
  • Wilson I.A., Niman, H.L., Houghten, R.A., Cherenson, A.R., Connolly, M.L. and -Lerner,
  • ATTORNEY/AGENT INFORMATION (A) NAME: HUGHES, DR E JOHN L (C) REFERENCE/DOCKET NUMBER: EJH/AF

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Abstract

The present invention relates generally to chimeric molecules and more particularly to interspecies cytokine receptor α-chain chimeras. Even more particularly, the present invention provides interspecies leukaemia inhibitory factor (LIF) receptor α-chain chimeras. The chimeric molecules of the present invention are useful inter alia as antagonists of human cytokine activities in vivo. In one embodiment, the present invention provides a polypeptide or a derivative or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an immunoglobulin (Ig)-like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine, such as LIF binding properties.

Description

NOVEL CHIMERIC MOLECULES
FIELD OF THE INVENTION
The present invention relates generally to chimeric molecules and more particularly to interspecies cytokine receptor α-chain chimeras. Even more particularly, the present invention provides interspecies leukaemia inhibitory factor receptor α-chain chimeras. The chimeric molecules of the present invention are useful inter alia as antagonists of human cytokine activities in vivo.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
LIF is a glycoprotein initially identified, purified and genetic sequences encoding same cloned based on its ability to induce differentiation in the mouse leukaemic cell line Ml (reviewed by Metcalf, 1991). Subsequently, LEF has been shown to have a wide variety of actions in many different cell types and tissues including adipocytes, osteoblasts, megakaryocytes, hepatocytes, neurons, embryonal stem cells and primordial germ cells (reviewed by Hilton, 1992). A critical role of LEF in the implantation process has been implicated, since mice in which the LEF gene has been ablated are essentially normal but are unable to implant their otherwise viable blastocysts (Stewart et al., 1992).
LEF exerts its biological actions through high affinity receptors which are expressed on the surface of LIF-responsive cells. The high affinity LIF receptor ("LIFR") complex is composed of two components: the LIFR α-chain, which binds LIF with low affinity, and gpl30 which does not itself bind LIF but is essential for high affinity complex formation and signal transduction (Gearing et al, 1992). The LIFR α-chain and gpl30 are components of other receptor systems including those of oncostatin-M (Gearing and Bruce, 1992; Gearing et al, 1992), ciliary neurotrophic factor [CNTF] (Ip et al, 1992) and the cytokine cardiotrophin-1 [CT-1] (Pennica et α/., 1995a; Pennica et α/., 1995b). The interleukin-6 [EL- 6] (Hibi et al, 1990) and interleukin-11 [IL-11] Fourcin et al, 1994; Hilton et al, 1994) receptors also have gpl30 as part of their high affinity receptors and this use of common receptor components may provide a basis for the overlapping biological activities and functional redundancy of these cytokines. Targeted disruption of the LIF receptor α-chain results in mutant animals with neuronal, musculo-skeletal, placental and metabolic defects (Ware et al, 1995). Mice carrying the LIFR nullizygous mutation died shortly after birth indicating that the LEFR α-chain is necessary for normal development and survival.
Human and mouse low affinity LEF receptors have been biochemically characterised and cDNA clones which encode these receptors have been described (Gearing et al, 1991; Tomida et al, 1994). The LIFR α-chain is a member of the haemopoietin family of receptors (Bazan, 1990). In contrast to the majority of the members of this family, the LIFR α-chain contains in its extracellular domain two copies of the haemopoietin domain, which are separated by an immunoglobulin-like domain (Cosman, 1993; Gearing et al, 1991). Similar to the G-CSF receptor (Fukunaga et al, 1990a; Fukunaga et al, 1990b) and gp 130 (Hibi et al, 1990), the L FR α-chain also contains three fibronectin type in (FNIII) repeats that are located C-terminal to the membrane proximal haemopoietin domain. Mutagenesis studies of the G-CSFR (Fukunaga et al, 1991) and gpl30 (Horsten et al, 1995) have indicated that the FNHI repeats are not essential for ligand binding.
Whilst murine LIF ("mLIF") is unable to bind to the human LIF ("hLIF") receptor (hLIFR), hLIF is able to bind to both high and low affinity mouse LEFRs ("mLIFR"), and is fully biologically active on mouse cells. Interestingly, hLIF also binds to both a naturally occurring soluble form of the mLIFR α-chain and mLIF-binding protein ["mLBP"] (Layton et al, 1992).
This unusual cross-species reactivity has been exploited to map the binding epitope on hLIF that is responsible, on the one hand, for binding to the hLIFR α-chain and on the other for binding with high-affinity to the mLIFR α-chain (Layton et al, 1994b; Owczarek et al, 1993). The interaction of LIF with its receptor α-chain is complex. The primary binding affinities of both mouse and human LIF for their respective α-chains are relatively low (Kd~l-2 nM) but, whereas mLIF binds to its receptor α-chain with apparent single site kinetics, hLIF binds to its receptor α-chain with biphasic kinetics. This led the subject inventors speculating that hLIF binding to the α-chain is associated with a receptor isomerisation process, the two forms of which, correspond to fast or slow kinetic dissociation rates (Layton et al, 1994a).
Surprisingly, hLIF binds to mLIFR α-chain with a much higher affinity (Kd ~ 10-20 nM) than it does to the isologous hLIFR α-chain or than mLIF binding to the mLIFR α-chain. Cross- competition studies using the mLIFR α-chain reveal that the competition curves are dependent on which LEF is used as the radioactive tracer and this behaviour is interpreted as an interference by each type of LEF in the binding of the other.
A LEF binding protein (mLBP) occurs at high levels (2μg/ml) in normal mouse serum and is dramatically elevated in pregnancy (Layton et al, 1992; Tomida et al, 1993). The very high binding affinity of this receptor for hLEF makes it a potent biological inhibitor of hLIF (Layton et al, 1994a), and suggests that it could be useful in clinical situations such as for treating inflammatory diseases where LIF levels may be expected to be elevated.
Understanding the way in which LIF interacts with receptors is required for the rational design of antagonists to LEF action. In work leading up to the present invention, the inventors exploited the structural homology of mouse and human LIF receptors and their differing binding characteristics for mouse and human LIF to generate inter-species receptor chimeras for the purpose of defining the structural elements involved in LIF binding. The present invention provides an approach for the generation of LEF receptor α-chains that retain their high affinity binding for human LIF. SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Certain aspects of the present invention relate to hybrids between leukaemia inhibitory factor (LIF) receptor α chain of human (h) and murine (m) origin. Specific murine and human LIF receptor hybrids (MH) referred to in the specification are defined in Figure 2.
Sequence identity numbers (SEQ ED Nos) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
One aspect of the present invention provides a polypeptide or a derivative or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine binding properties.
Another aspect of the present invention is directed to a polypeptide or derivative or chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof such that said polypeptide has LEF binding properties.
Yet another aspect of the present invention provides a polypeptide or a derivative or a chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a LIFR α-chain haemopoietin domain and said other portion comprises at least two LIFR α-chain Ig-like domains such that said polypeptide has LIF binding properties. Still another aspect of the present invention contemplates a polypeptide or derivative or chemical equivalent thereof having the structure:
-Λ. i - -2--r 3 wherein:
X, and X3 are located distally and proximally, respectively, to the transmembrane domain of the LEFR α-chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
X2 is an Ig-like domain or a functional derivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
Still yet another aspect of the present invention provides a polypeptide or derivative or chemical equivalent thereof having the structure: -Λ.|- -.2-- 3 wherein:
X, and X3 may be the same or different and each is a LIFR α-chain haemopoietin domain; X2 is a LIFR α-chain Ig-like domain; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
Another aspect of the present invention provides a chimera comprising a LIFR α-chain haemopoietin domain or a functional derivative thereof and a LIFR α-chain Ig-like domain or a functional derivative thereof wherein binding of LEF to the chimera gives rise to a two- contact state and a single kinetic dissociation rate according to the Scatchard transformation of LEF binding to its receptor at equilibrium:
^=(K,+KcKy)(Rτ-B) r
where B is the specifically bound LIF concentration, F is the free LEF concentration, Rτ is the total concentration of LEF receptors, K, is the equilibrium affinity constant for the first contact site of LIF with its receptor and K-, is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LEF.
Yet another aspect of the present invention relates to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide comprising first and second portions wherein one portion comprises a haemopoietin domain or a functional derivative then of and said other portion comprises an Ig-like domain or a functional derivative thereof wherein said polypeptide exhibits cytokine binding properties.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic representation of a proposed model of the LIFR α-chain interacting with both hLIF and mLIF. Both mLIF and hLEF first contact site A on the LIFR α-chain, which is mainly dependent on the association rate (konI) and the dissociation rate (kom). For hLIF binding the primary to mLIFRα, interaction leads to full receptor isomerisation which results in further interactions on site B on the receptor. The isomerisation process is determined by the isomerisation constant (1/KC).
Figure 2 is a representation of amino acid sequence specifications for recombinant LIF Receptors. Amino acid sequences are numbered according to the LEFR sequence described by (Gearing et al, 1991). The letters M and H denote that amino acids are derived from mLIFR sequences or hLEFR sequences respectively. Because gaps were introduced in the mLEFR amino acid to maximise the alignment, the numbers refer to the specific hLEFR or mLIFR amino acid sequence. Although the amino acid sequences of the recombinant receptors are shown as beginning at residues 52 (hLIFR) or 50 (mLIFR) the N-termini were modified as described in the Examples.
Figure 3 is a representation of analyses of mouse-human hybrid LIF receptors expressed in Pichia pastoris. (A) Photographic representation of Western blotting of Recombinant receptors from P. pastoris. Culture supematants were separated by 0.1% w/v SDS-10% w/v PAGE under reducing conditions, transferred to PVDF membranes and hybridised to 12CA5 antibody as described in the Examples. (B) Photographic representation of chemical cross- linking of Recombinant receptors from P. Pastoris. Culture supernatant were cross-linked to 125HιLIF in the absence or presence of excess unlabelled hLIF as described in the Examples. (C) Graphical representation showing a gel filtration profile of recombinant LIF receptors from P. Pastoris culture supernatant: Absorbance (solid lines) and specific 125IhLIF binding activity (shaded area) of fractions based on a concanavalin A-Sepharose binding assay are shown. The receptor variants are as follows: (A) mLIFR; (B) MH1LIFR; (C) MH2LIFR; (D) MH3LIFR; (E) MH4LEFR; (F) MH5LIFR; (G) MH6LEFR; (H) MH7LIFR; (I) MH8LIFR, and (J) hLIFR.
Figure 4 is a graphical representation of a Scatchard analyses of 125DιLEF binding to chimeric LIF receptor variants. Saturation binding was performed by incubating aliquots of P. pastoris culture supernatant containing recombinant LEF receptors with increasing concentrations of 125IhLIF. Specific binding assays and Scatchard transformations were performed as described in the Examples. These Scatchard binding data are representative for several independently performed experiments and the resulting Y^ values are shown in Table π. The receptor variants are as follows: (A) mLIFR; (B) MH1LIFR; (C) MH2LIFR; (D) MH3LIFR; (E) MH4LEFR; (F) MH5LIFR; (G) MH6LIFR; (H) MH7LIFR; (I) MH8LIFR, and (J) hLIFR.
Figure 5 is a graphical representation of kinetic dissociation of 1 5IhLIF from chimeric LIFRs. Each chimeric LIFR (0.01-0.02 nM) was incubated at room temperature for 3-4 hours with 125IhLIF and kinetic dissociation assays performed as described in the Examples. The plot of the natural log of the ratio of the amount of 125IhLIF remaining bound after a given time (SB,) to the amount bound initially (SB0) versus time is shown. Estimates of the kinetic rate constant governing dissociation (kd) of ligand and receptor were made using the curve-fitting program KINETIC and shown in Table II. The receptor variants are as follows: (A) mLIFR; (B) MH1LIFR; (C) MH2LIFR; (D) MH3LIFR; (E) MH4LIFR; (F) MH5LIFR; (G) MH6LIFR; (H) MH7LIFR; (I) MH8LIFR, and (J) hLIFR. Figure 6 is a graphical representation of displacement curves for unlabelled mLfF (♦) and hLIF (O) competing for binding with 125IhLIF to the mLIFR, hLIFR and hybrid LIF receptors.
Competitive inhibition assays were carried out by incubating aliquots of P. pastoris culture supernatant containing recombinant LEF receptors with a constant concentration of 125-thLIF (105 cpm) and increasing concentrations of either unlabelled hLEF or mLIF. The ID50 values are shown in Table II. The receptor variants and concentrations are as follows: (A) mLEFR (0.067 nM); (B) MH1LIFR (0.033nM); (C) MH2LIFR (0.142 nM); (D) MH3LIFR (0.014 nM); (E) MH4LIFR (0.027 nM); (F) MH5LEFR (0.033 nM); (G) MH6LIFR (0.039 nM); (H) MH7LIFR (0.014 nM); (i) MH8LIFR (0.059 nM), and (J) hLIFR (0.033 nM).
Figure 7 is a photographic representation of the effect of chimeric LIFRs on hLIF-induced STAT-3 tyrosine phosphorylation. Ml cells were incubated at 37°C for 5 min in the presence of either 1 ng of hLIF, or 1 ng of hLIF together with 11 ng of chimeric LIFR, or 11 ng of chimeric LEFR alone and analysed by immunoprecipitation and Western blotting as described in the Examples.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the exploitation of the structural homology of mouse and human LEF receptors and their differing binding characteristics for mouse and human LEF to define the structural elements involved in LIF binding.
Accordingly, one aspect of the present invention provides a polypeptide or a derivative or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an immunoglobulin (Ig) -like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine binding properties.
Preferably, the first portion comprises at least two haemopoietin domains. The present invention is hereafter described in relation to the first and second portions being covalently linked together by a peptide bond. However, the first and second portions may be linked by ionic bonds, hydrogen bonds, ie. electrostatic interaction, molecular bridging, molecular association or other interactive bonding mechanisms including other covalent bonding systems such as disulphide bridges. Reference to a first and second portion is not intended to exclude third or subsequent portions which are encompassed by the present invention. Preferably, the polypeptide is a chimera encoded by single nucleotide sequence. A "chimera" has a similar meaning herein to a "fusion" molecule.
Preferably, the cytokine is LIF although the present invention extends to functional derivatives, homologues or analogs of LIF as well as other cytokines. Examples of other cytokines contemplated by the present invention include, but are not limited to, interleukins, colony stimulating factors. The present invention is hereinafter described in relation to chimeras involving LIFR or molecules having LIF binding properties. This is done, however, with the understanding that the present invention extends to other cytokine receptors or molecules having other cytokine binding properties.
Accordingly, another aspect of the present invention is directed to a polypeptide or derivative or chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof such that said chimera has LIF binding properties.
In a preferred embodiment, the haemopoietin domain comprises a LEFR α-chain haemopoietin domain and the Ig-like domain comprises a LEFR α-chain Ig-like domain. Preferably, the first portion comprises at least two haemopoietin domains.
In a preferred aspect of the present invention, there is provided a polypeptide or a derivative or a chemical equivalent thereof, said polypeptide comprising first and second covalently linked portions wherein one portion comprises a LEFR α-chain haemopoietin domain and said other portion comprises at least two LIFR α-chain Ig-like domain such that said polypeptide has LEF binding properties.
In another aspect of the present invention, one of said portions or a functional derivative or chemical equivalent thereof is from one source and said other portion or functional derivative or chemical equivalent thereof is from another source. Examples of different sources include, but are not limited to, different species or allelic variants within a single species.
In a preferred embodiment one of said portions is from a murine LEFR (mLIFR) α-chain and said other portion is from a human LEFR (hLIFR) α-chain. Such a heterologous molecule is useful for example, for humanising a mLEFR α-chain.
In a most preferred embodiment the LEFR α-chain Ig-like domain is from mLIFR α-chain or hLIFR α-chain and the LIFR α-chain haemopoietin domain is from mLIFR α-chain or hLIFR α-chain.
In a related aspect of the present invention, to ensure binding of LEF to the polypeptide or derivative thereof said polypeptide or derivative or chemical equivalent thereof comprises at least three portions, wherein two portions comprise haemopoietin domains and one portion comprises an Ig-like domain.
Preferably, the polypeptide or derivative or chemical equivalent thereof comprises a LIFR α- chain Ig-like domain flanked by at least two LIFR α-chain haemopoietin domains. Although not intending to limit the present invention to only one theory or mode of action, the binding of LIF to the chimera is thought to lead to ligand-dependent receptor isomerisation. The predominant involvement of the Ig-like domain is in determining ligand binding specificity and conferring high affinity LEF binding.
In one embodiment, the chimera is selected from the listing consisting of MH1LIFR, MH2LEFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as defined in Figure 2. In a particularly preferred embodiment, the chimera is MH3LEFR (Figure 2) and comprises at the membrane-distal position, an hLIFR α-chain haemopoietin domain, an mLIFR α-chain Ig domain and at the membrane-proximal position an mLIFR α-chain haemopoietin domain.
In another preferred embodiment, the chimera is MH4LEFR (Figure 2) and comprises at the membrane-distal position an mLEFR α-chain haemopoietin domain, mLIFR α-chain Ig domain and at the membrane-proximal position an hLIFR α-chain haemopoietin domain.
In yet another most preferred embodiment, the chimera is MH5LIFR (Figure 2) and comprises at the membrane-distal position an hLIFR α-chain haemopoietin domain, an mLEFR α-chain Ig domain and at the membrane-proximal position an hLIFR α-chain haemopoietin domain.
Chimeras MH3LIFR, MH4LIFR and MH5LDFR all contain an intact Ig-like domain from mouse LEF receptor chain and high affinity 125IhLIF binding {Kd ~ 11-60 pM) similar to that seen for hLEF binding to the mLIFR (Fig. 4, Table II). This indicates that the immunoglobulin-like domain from the mouse LEF receptor has the most important influence in conferring the high affinity binding of hLIF.
Another aspect of the present invention contemplates a polypeptide or derivative or chemical equivalent thereof having the structure:
-- .jΛ2-Λ.3 wherein:
X[ and X3 are located distally and proximally, respectively, to the transmembrane domain of the LEFR α-chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
X2 is an Ig-like domain or a functional derivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF. Preferably, the present invention provides a polypeptide or derivative thereof having the structure:
Figure imgf000014_0001
wherein:
X, and X3 may be the same or different and each is a LIFR α-chain haemopoietin domain;
X2 is a LIFR α-chain Ig-like domain; and wherein the polypeptide or derivative thereof is capable of binding, interacting, influencing or otherwise associating with LEF.
Preferably, X, and X3 are derived from mLIFR α-chain or hLIFR α-chain.
Preferably, X2 is derived from mLIFR α-chain or hLIFR α-chain or is either composed of hLEFR α-chain amino acid residues at the N-terminal region, to approximately half way down the Ig-like domain, and mLEFR α-chain amino acid residues at the C-terminal region of the Ig-like domain or is composed in the converse.
In the preferred chimeras both haemopoietin domains or their functional derivative are of murine or human origin or one each from a human or murine source domain or functional derivative thereof and are capable of binding, interacting, influencing or otherwise associating with LIF. Even more preferred are the chimeras, or functional derivatives thereof, selected from MH1LIFR, MH2LIFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as set forth in Figure 2. Still more preferred are the chimeras, or functional derivatives thereof, MH3LEFR, MH4LIFR and MH5LEFR as set forth in Figure 2.
The present invention further contemplates a range of derivatives of the polypeptides of the present invention. Derivatives include fragments, parts, portions, mutants, homologues and analogs of the chimera and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the chimera or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding the chimeras. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences.
Analogues of said polypeptides include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH^ amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBHφ
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminol ixanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and or D- isomers of amino acids. The use of unnatural amino acids provides a means of stabilising the polypeptide structure especially when the polypeptide is used in vitro or for diagnostic purposes. A list of unnatural amino acid, contemplated herein is shown in Table 1.
TABLE 1
Non-conventional Code Non-conventional Code amino acid amino acid
α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaUne Nmnva
D-glutamic acid Dglu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-or ithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl-γ -aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa
D- α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen
D- α-methylasparagine Dmasn α-methyl- α-napthylalanine Manap
D-α-methylaspartate Dmasp α-methylpenicillamine Mpen
D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2- aminoethyl)glycine Naeg
D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-α-methylisoleucine Dmile N-amino- α-methylbutyrate Nmaabu
D-α-methylleucine Dmleu α-napthylalanine Anap
D-α-memyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D- α-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D- α-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-α-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3 ,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg D-N-methylgluta ate Dnmglu N-( 1 -hydroxyethy l)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-α-methylalanine Mala
L-α-methylarginine Marg L-α-methylasparagine Masn
L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug
L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu
L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe
L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-α-methylleucine Mleu L-α-methyllysine Mlys
L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn
L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr
L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine l-carboxy-l-(2,2-diphenyl- Nmbc 5 ethylamino)cyclopropane
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- 0 bifunctional crosslinkers such as the bifunctional iniido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for 5 example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
0 The present invention further contemplates chemical analogues of the polypeptides of the present invention capable of acting as antagonists or agonists of said polypeptides or which can act as functional analogues of said polypeptides. Chemical analogues may not necessarily be derived from said polypeptides but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain
25 physiochemical properties of said polypeptides. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.
It has previously been reported that mLIF binds to the mLEFR α-chain with low affinity but does not detectably interact with the hLIFR α-chain (Layton et al, 1994b; Owczarek et al, 30 1993). Human LIF binds to the mLIFR α-chain and does so with a much higher affinity than mLIF. The higher affinity binding of hLEF to the mLEFR α-chain is found to be due almost exclusively to a slower kinetic dissociation rate compared to mLIF. In fact, the binding affinity for hLEF is most strongly dependent on the presence of an intact mLEFR Ig- like domain irrespective of the species origin of the haemopoietin domain(s).
However, in the context of an hLIFR Ig-like domain, the species origin of the membrane proximal haemopoietin domain is more important than the distal haemopoietin domain in determining hLEF binding affinity. In all cases where high-affinity binding of hLEF is observed the dissociation kinetics are predominantly a single class with slow dissociation rate (off-rate). In cases where intermediate- or low-affinity binding of hLEF is observed the dissociation kinetics are biphasic with variable ratio of fast-off and slow-off components depending on the affinity.
Although not intending to limit the present invention to any one theory or mode of action, the binding of LIF to the chimeric molecule leads to ligand dependent receptor isomerisation. A model of the LEFR in which there are two potential ligand contact sites on the receptor requires ligand dependent receptor isomerisation to occur for both contacts to be made.
For hLIF binding to the hLIFR, isomerisation is inefficient, resulting in two kinetically distinguishable bound states of the receptor (1 contact or 2 contact) but for hLEF binding to the mLEFR, isomerisation to the two contact state is nearly complete giving rise to high- affinity and a single kinetic dissociation rate. Analysis of this model for the binding of LEF to its receptor at equilibrium gives for Scatchard transformation (Boynaens and Dumont, 1980):
B
=(K. +K K.)(RT-B)
where B is the specifically bound LIF concentration, F is the free LIF concentration, Rτ is the total concentration of LEF receptors, K, is the equilibrium affinity constant for the first contact site of LIF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF.
The form of this equation shows that, regardless of the value of K-,, Scatchard plots of LEF equilibrium binding data will all be apparent one site linear curves (see Fig. 3). However, the slopes of such curves will not be true affinity constants but the combined constants K.+K..K,.
In a particularly preferred embodiment said chimera exhibits an apparent equilibrium dissociation constant for binding to hLEF of about 300 pM. More preferably, said chimera exhibits an affinity binding to hLEF of about 150 pM and even more preferably, about 10 pM affinity binding to hLEF.
In a most preferred embodiment, said chimera exhibits a bi-phasic dissociation rate for hLIF with one phase being of about k0jfQ.16 min"1 and the second phase of about kojg~0.002 min"1. More preferably said chimera exhibits a bi-phasic dissociation rate for hLIF of about kOJ[ -0.07 min"1 and a second dissociation phase of about fco ~0.001 min"1 and even more preferably a single slow dissociation rate for hLIF of about kojf-0.00l min"1.
According to another aspect of the present invention there is provided a chimera comprising a LEFR α-chain haemopoietin domain or a functional derivative thereof and a LEFR α-chain Ig-like domain or a functional derivative thereof wherein binding of LIF to the chimera gives rise to a two-contact state and a single kinetic dissociation rate according to the Scatchard transformation of LEF binding to its receptor at equilibrium:
Figure imgf000022_0001
where B is the specifically bound LIF concentration, F is the free LEF concentration, Rτ is the total concentration of LIF receptors, K, is the equilibrium affinity constant for the first contact site of LEF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF. Chimeras MH3LEFR, MH4LIFR and MH5LIFR all contain an intact Ig-like domain from mouse LIF receptor and high affinity 125IhLIF binding (Kd ~ 11-60 pM) similar to that seen for hLIF binding to the mLIFR (Fig. 4, Table I).
A further aspect of the present invention contemplates the use of chimeras as therapeutic agents in relation to human disease conditions. For example, the LEF binding properties of the chimeras of the present invention are particularly useful, but in no way limited to, use as a biological inhibitor of LEF. LEF is bound by the chimera and thereby blocked from binding to any other unoccupied LEFR. To investigate the potential of chimeric LEFRs functioning as antagonists of LEF biological activity, blocking of hLEF induced STAT-3 tyrosine phosphorylation in Ml cells is measured. The differentiation of Ml cells is dependent upon the binding of LEF to the Ml cell surface LEFR. STAT-3 activation is a critical step in gpl30-mediated terminal differentiation of Ml cells. Tyrosine phosphorylation of STAT-3 is increased by hLIF stimulation of Ml cells within five minutes. However, STAT-3 tyrosine phosphorylation is almost completely blocked by pre-incubation of hLIF with chimeric molecule MH3LIFR. The chimeric LIFR could therefore be useful as a therapeutic agent in clinical situations such an inflammatory diseases where LIF levels are expected to be elevated.
In a preferred embodiment a polypeptide, derivative or chemical equivalent thereof, comprising, but not limited to, X,X2X3, as defined above, is designed and constructed such that it binds, interacts or otherwise associates with LIF activity. In a particularly preferred embodiment, binding, interaction or association of said polypeptide with LIF results in inhibition of LEF activity.
In a most preferred embodiment, a mLIFR α-chain or derivative or chemical equivalent thereof comprising said X,X2X3 is "humanised" by the substitution of sufficient of the mLIFR α-chain Ig-like domain (or part thereof) or haemopoietin domains with hLEFR α- chain Ig-like domains (or part thereof) or haemopoietin domains, respectively, to result in a chimeric LEFR α-chain exhibiting a high affinity for hLIF binding. Such humanised mLIFR could act as a specific and potent antagonist of hLEF. A "sufficient" substitution is the minimum required to result in said "humanised" chimera exhibiting at least 10-100 pM hLIF binding affinity.
In a preferred embodiment it may be necessary to stabilise said chimera such that degredation does not occur.
Accordingly, the present invention contemplates said chimeras or derivatives or chemical equivalents thereof and one or more pharmaceutically acceptable carriers and/or diluents.
The polypeptides of the present invention may be produced by recombinant DNA means or by chemical synthetic processes. With respect to the former this aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding a haemopoietin domain or functional derivative thereof and an Ig-like domain or functional derivative thereof. The nucleic acid molecule comprises a sequence of nucleotides which encode or are complementary to nucleotide sequences which encode the polypeptides of the present invention. Preferably, the nucleic acid molecule of the present invention encodes said polypeptides, said nucleic acid molecule selected from the list consisting of:
(i) a nucleic acid molecule comprising a sequence of nucleotides substantially encoding said polypeptides;
(ii) a nucleic acid molecule comprising a sequence of nucleotides having at least about
70% similarity to the nucleotide sequence encoding said polypeptides; (iii) a nucleic acid molecule capable of hybridising under low stringency conditions at 42°C to the nucleotide sequence encoding said polypeptides.
The nucleotide molecule is preferably derivable from the human genome but genomes and nucleotide sequences from non-human animals are also encompassed by the present invention. Non-human animals contemplated by the present invention include livestock animals (e.g. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals (e.g. dogs, cats), birds (e.g. chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals (e.g. foxes, kangaroos, dingoes).
Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.
The genetic sequences may be cDNA or mRNA and may be single or double stranded, linear or covalently closed, circular molecules. Conveniently, the genetic molecules are part of an expression vector capable of expression in a prokaryotic cell (eg. E. coli) or a eukaryotic cell (eg. an animal or mammalian cell).
Generally, the nucleic acid molecules encodes a fusion molecule comprising a haemopoietin domain or functional derivative thereof or an Ig-like domain or functional derivative thereof. Expression of the nucleic acid molecule of the present invention leads to synthesis of a fusion molecule.
The polypeptides and nucleic acid molecules of the present invention are preferably in isolated form, having undergone at least one purification step from their original source.
The present invention further contemplates use of the polypeptides herein described in the manufacture of a medicament for the treatment of a condition requiring the antagonsim of LIF.
The present invention is further described by the following non-limiting Examples. EXAMPLE 1 CONSTRUCTION OF SOLUBLE LIFR AND HYBRID MHLIFR cDNAs
The 5 ' end of a cDNA encoding a soluble mouse LIFR α-chain was modified to encode an Xhol site and an in-frame 12CA5 epitope (YPYDVPDYA) [SEQ. ID NO: 1] (Wilson et al, 1984). The 3' end of the mLIFR cDNA was modified to encode an Xbal site, and a stop codon was introduced after amino acid residue 531 in te amino acid sequence described in (Gearing et al, 1991). A cDNA encoding the hLIFR α-chain (Owczarek et al, 1993) wv also altered at its 5' end to encode an Xhol site and an in-frame 12CA5 epitope. The 3' end was also modified to encode an Xbal site, and a stop codon was introduced after position 536 in the amino acid sequence described by (Gearing et al, 1991). The sequence at the N- terminus of the recombinant MLEFR was GVQ YPYDVPDYA [SEQ. ED NO: 2], and trie sequence at the N. terminus of the recombinant hLIFR was GAPYPYDVPDYA [SEQ. ID NO: 3]. The recombinant LIFRs therefore lacked the cytoplasmic domain, transmembrane domain and all three FNHI-like domains. The resulting cDNAs were subsequently ligated into the Pichia pastoris expression vector pPIC9, that was digested with Xhol and AvrEl, as Xhol-Xbal fragments. Mutagenesis of the LIFR cDNAs and construction of hybrid mouse- human LIFRs was carried out using a PCR-based technique, splicing by overlap extension (Ho et al, 1989), and Pfu polymerase (Strategene).
The nucleotide sequences of the resulting constructs were confirmed by dideoxy sequencing (S anger et al, 1977) using either a PRISM Ready Reaction DeyDeoxy Terminator Cycle sequencing kit on an Applied Biosystems 373 DNA sequencer or a T7 -based Pharmacia Dideoxy sequencing kit. EXAMPLE 2 EXPRESSION OF SOLUBLE LIFRs IN PICHIA PASTORIS.
All cDNAs were expressed as soluble secreted proteins in the methylotrophic yeast Pichia pastoris. This expression system uses the promoter from the methanol-induced alcohol oxidase gene, AOXI. Stably expressing clones are selected using the HIS4 gene as a selectable marker. The recombinant plasmids were digested with either Bgl I or Sail and integrated into host cells by tri. isforming his4 (GS115) P. pastoris sphaeroplasts as described (Cregg et al, 1985). Digestion of a plasmid with Bglll disrupts the AOXI gene and results in a strain that is phenotypically His+Muts (Methanol utilisation sensitive). Because plasmids MH1LIFR, MH3LEFR, MH5LIFR and MH7LIFR contained Bglll sites, they were digested with Sail prior to transformation into P. pastoris sphaeroplasts. The resulting strains were His+Mut+. His+ transformants were patched first onto a nitrocellulose filter overlayed onto an agar plate (MM) containing 0.5% (v/v) methanol, 1.34% (w/v) Yeast Nitrogen Base (YNB) and 4xl0"5% (w/v) biotin, and then onto another agar plate (MD) containing 1% (w/v) dextrose instead of methanol as the carbon source. The lates were incubated at 30°C. After 48 hours the clones on the MD agar plate were placed at 4°C. The nitrocellulose filters containing the His+ transformants were then lifted off the MM plates and incubated in 10% (w/v) skim milk powder in PBS. Antibodies Colonies that expressed recombinant LIF receptors were then detected using a 12CA5 antibody. Clones identified in this way were grown in a shaking incubator at 30°C to an OD^ of 2-6 in 10 ml of medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, lOOmM potassium phosphate (pH 6), 1.34% (w/v) YNB, 4xl0"5% (w/v) biotin, and 1% (v/v) glycerol. After 5 -fold concentration by centrifugation the cultures were resuspended in medium that contained 0.5% (v/v) methanol instead of glycerol to induce the cells to express the heterologous protein. Expression of the recombinant receptors was analysed by SDS- PAGE of the culture supernatant, followed by both Western blotting and detection with 12CA5 antibody, and binding assays to 125IhLIF. EXAMPLE 3 WESTERN-BLOTTING
Proteins separated by SDS-PAGE were electrophoretically transferred onto pre- wetted 5 polyvinylidene diflouride (PVDF-Plus, Micron Separations Inc.) membrane using a transfer buffer containing 20mM Tris-HCI, 150 mM glycine pH 8.2, and 20% (v/v) methanol in a Mini-Protean II system. Blots were blocked in 1% BSA (w/v) in PBS containing 0.1% (v/v) Tween-20, followed by incubation with mouse 12CA5 antibody and then horseradish peroxidase-conjugated rabbit-anti-mouse antibody (DAKO, Denmark). The receptor 0 proteins were visualised using an ECL substrate kit (Amersham) followed by autoradiography.
EXAMPLE 4 RADIOIODINATION OF LIGANDS 5
Recombinant mLIF or hLIF produced in E. coli was purified and iodinated as previously described (Hilton et al, 1988).
EXAMPLE 5 0 BINDING OF LIF TO RECOMBINANT SOLUBLE RECEPTORS
Equilibrium binding experiments for soluble receptors were performed using concanavalin A-Sepharose beads to precipitate the soluble receptor complexes. Non-specific binding, and separation of bound and free labelled LEF were determined as previously described (Layton 25 et al, 1994a). Scatchard analyses of saturation binding isotherms were performed using the curve-fitting program LIGAND (McPherson, 1985; Munson and Rodbard, 1980).
Experiments to determine the kinetic dissociation rate (k0^) were carried out by pre- incubating soluble receptors, immobilised on concanavalin A-Sepharose beads, with 30 125LhLEF at a final concentration of 105 cpm/60 μl in the presence and absence of 8 μg/ml unlabelled hLIF. When the specific interaction had reached an equilibrium, the precipitated receptor complexes were collected by rapid centrifugation (3 sec). Dissociation of the 125IhLEF was initiated by immediately resuspending the receptor complexes in the same volume of ice-cold KHF (KDS-RPMI medium plus 10% (v/v) FSC) containing 20 μg/ml labelled hLEF. At various times thereafter, 60 μl aliquots of suspension were removed, and bound and free 125IhLEF were separated and counted as previously described (Layton et al, 1994a).
EXAMPLE 6 SIZE-EXCLUSION CHROMATOGRAPHY
P. pastoris expression supernatant was concentrated t- to 50- fold using a Centricon-50 microconcentrator (Amicon). Aliquots (200-500 μl) of each sample were injected onto a Superose-12 10/30 (Pharmacia) column equilibriated in PBS containing 0.02% (v/v) Tween- 20, 0.02% (w/v) sodium azide and 5% (v/v) glycerol. Elution was carried out isoctratically using the same buffer and monitored by absorbance at 280 nm. The 0.5-ml fractions were collected at a flow rate of 0.5 ml per min. An aliquot of each fraction was tested for 125IhLIF binding as previously described.
EXAMPLE 7 CHEMICAL CROSS-LINKING
Each chimeric LIF receptor sample (0.25-0.5 nM) was mixed with approximately 1.6 nM 125IhLIF (200,000 cpm) in 20 μl of PBS containing 0.02% (v/v) Tween-20 and 0.02% (w/v) sodium azide, in the presence or absence of 100 ng of unlabelled hLIF, and the binding reaction was performed for 90 min at room temperature. After incubation, 10 μl of 7.5 mM Bis-(sulfosuccinimidyl)-suberate (BS3) (Pierce), which was dissolved in PBS containing 0.02% (v/v) Tween-20, was added as a chemical cross-linker, and the mixture was incubated for 30 minutes on ice. The reaction was terminated by the addition of SDS sample buffer. The cross-linked proteins were analysed by 10% (w/v) poly acly amide gel electrophoresis (PAGE) in the presence of 0.1% (v/v) SDS under non-reducing conditions, followed by autoradiography. EXAMPLE 8 TYROSINE PHOSPHORYLATION ANALYSIS OF STAT-3
Ml cells (107 per sample) were stimulated for 5 min at 37°C with either 1 ng of hLIF, 1 ng of hLIF together with 11 ng of each chimeric LEFR, or 11 ng of each chimeric LIFR alone and then lysed in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2 MM EDTA, 1% (v/v) Triton X-100, ImM Na3 VO4 and proteinase inhibitors. After pelleting insoluble material and protein standardisation, the supernatant was incubated with protein A- sepharose beads (Pharmacia Biotech.) for 1 hour, then immunoprecipitated overnight at 4°C in the presence of 4G10 anti-phosphotyrosine mAb (Upstate Biotechnology Inc.) and protein A-Sepharose beads. The immune complexes were washed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (w/v) NP-40, ImM Na3 VO4 and proteinase inhibitors, eluted from the sepharose beads by boiling in SDS sample buffer under reducing conditions for 5 min before being subjected to 4-20% (w/v) polyacrylamide SDS-PAGE and then transferred to a pre- wetted polyvinylidene diflouride membranae (PVDF-Plus, Micron Separations Inc.). After blocking, the membranae was incubated with an anti-STAT-3 polyclonal antibody (K-15, Santa Cruz Biotechnology), followed by incubation with a goat anti-rabbit immunoglobulin polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark).
The phosphorylated STAT-3 protein was visualised by autoradiography using an ECL system (Amersham). Quantitation of STAT-3 phosphorylation levels was performed by densitometric analysis of the band intensities using Imagequant version 3.0 software.
EXAMPLE 9
CONSTRUCTION AND EXPRESSION OF RECOMBINANT mLIFR, hLIFR AND HYBRID MHLIFRs
Mouse LIFR and human LIFR were initially expressed as soluble proteins that were truncated 13 amino acid residues after the predicted membrane proximal haemopoietin domain. These receptors therefore did not contain the cytoplasmic domain, the transmembrane domain and all three fibronectin type Ell repeats that are present in native cellular LEF receptors. The recombinant proteins were modified at their N-termini to encode a 12CA5 epitope tag (Wilson et al, 1984) in order to monitor their expression, and contained the yeast α-factor signal peptide to enable the proteins to be secreted into the culture medium after transformation into yeast. The molecular weight of these recombinant receptors is predicted to be approximately 65 kDa. Scatchard analysis of 125ImLIF to the recombinant mLEFR α-chain showed a single class of mLEF binding site (Kd ~ 6-7 nM) which is essentially the same as that for mLBP (Kd ~ 1-4 nM) and the low affinity mLIFR formed by detergent solubilisation of mLIFRs present on liver membranes or activated macrophages (Hilton and Nicola, 1992). The binding of 125IhLIF to the recombinant soluble mLEFR α-chain displayed a Kd value of 0.3-0.93 nM which again was in the normal range for 125IhLIF binding (Table I). These results indicated that the entire ligand-binding domain of both the human and mouse LIFR α-chains is included within the membrane-distal and membrane-proximal haemopoietin domains plus the intervening Ig-like domain.
As shown in Fig. 3A several receptor protein bands could be detected in the expression medium of the majority of the transformant clones. In addition to differentially glycosylated forms of receptor, the extra bands with molecular weights below 60KDa were present early in the time course of expression and also in the cell lysate (data not shown), suggesting that they may either represent prematurely terminated translation products or, more likely, be proteolytically degraded products.
Chemical cross-linking (Fig. 3B) of the soluble receptor variants with 125IhLIF demonstrated that only the species with molecular weights higher than 70 kDa could specifically interact with 125IhLEF. Furthermore, the position of the 125IhLEF binding peak at 70-10 kDa (Fig. 3C) by size-exclusion chromatography of soluble receptor samples indicated that the hybrid LIFRs have the apparent molecular weight of 70-100 kDa and exist as monomers. The expression levels of the different receptors were variable, ranging from 10 μg to 1 mg of receptor protein per litre of expression medium as determined by Scatchard analysis. Hybrid LEFRs MH4 and MH5 were found to be difficult to detect by Western blot analysis which may be due to either extremely low expression levels, or cleavage of the 12CA5 epitope tag during protein production. However, the behaviour of these two hybrid receptors was similar to that of the other recombinant receptors with respect to both chemical cross- linking with 125DιLIF and size-exclusion chromatography.
EXAMPLE 10
THE MOUSE LIFR IMMUNOGLOBULIN-LIKE DOMAIN DETERMINES HIGH AFFINITY hLIF BINDING TO HYBRID LIFE RECEPTORS
The mouse LIFR α-chain binds hLIF with high affinity whereas the human LEFR α-chain binds hLIF with low affinity (Layton et al, 1994a). The hybrid LIF receptors were characterised by performing binding assays and subsequent Scatchard analyses to determine their affinities of interaction with 125IhLEF> As shown in Fig. 4 and Table II, the recombinant' mouse and human LIFRs had Kd values of 10-46 pM and 0.3-0.9 nM respectively, which were similar to those observed for the naturally-occurring soluble mouse LIF receptor and a soluble form of human receptor α-chain expressed in COS cell-conditioned medium (Layton et al. 1994a), respectively.
Hybrids MH3LIFR, MH4LEFR and MH5LIFR all contain an intact Ig-like domain from mouse LEF receptor but have either one haemopoietin domain (MH3 and MH4) or two haemopoietin domains (MH5) from the human LEF receptor. Suφrisingly, all of these three hybrids exhibited high affinity 125IhLEF binding (Kd ~ 11-60 pM) similar to that seen for hLEF binding to the mLEFR (Fig. 4, Table II). This strongly suggested that the immunoglobulin- like domain from the mouse LIF receptor has the most important influence in conferring the high affinity binding of hLEF.
In hybrid MH1LIFR the N-terminal region, to approximately halfway down the Ig-like domain, was composed of hLIFR residues and the C-terminal half was composed of mLEFR residues while hybrid MH2LEFR was the converse. When these recomb; riant hybrid LIF receptors were tested for binding of 125IhLIF by Scatchard analysis both had intermediate affinities (Kd ~ 190-400 pM and 150-440 pM respectively) (Fig. 4, Table II). The relative contributions of the membrane-distal and membrane-proximal haemopoietin domains from the mLIFR to 125IhLIF binding were investigated next. Hybrid MH6LIFR was composed almost entirely of mLEFR residues except that the Ig-like domain was derived from the hLEFR and it bound 125IhLIF with intermediate affinity {Kd ~ 260 pM). MH7LEFR, in which only the membrane-proximal haemopoietin domain was composed of mLIFR residues, also bound 125IhLIF with intermediate affinity (Kd ~ 300 pM) (Fig. 4, Table II). This result indicated that of the two mLEFR haemopoietin domains the major contribution to high affinity 125IhLEF binding was from the membrane-proximal haemopoietin domain. MH8LIFR, which contained only the membrane-distal haemopoietin domain derived from mLIFR residues, had an almost identical binding affinity for 125IhLIF to the hLEFR (Kd ~ 2 nM), indicating that the mouse LIFR membrane-distal haemopoietin domain is not involved in high affinity I25IhLIF binding (Fig. 4, Table II).
Interestingly, when either mLEFR haemopoietin domain was present in conjunction with the mouse LIFR Ig-like domain, as in hybrids MH3LEFR and MH4LIFR, there was no increase in binding affinity for 125HιLIF when compared to hybrid MH5LIFR, which had only the mouse LEFR Ig-like domain, further suggesting that the Ig-like domain from the mouse LIFR plays the dominant role in determining the high affinity binding for hLEFR (Fig. 4, Table II). The presence of the membrane-distal haemopoietin domain from mouse LEFR had no effect in increasing the affinity of 125IhLEF binding, as indicated by the similar kd values of hybrids MH6LEFR and MH7LIFR, and hybrid MH8LIFR and the hLIFR (Fig. 4, Table II).
The difference in hLEF-binding affinities of chimeric LIFRs was further explored by performing kinetic dissociation experiments (Fig. 5). The LIF receptor variants, which had high affinity binding for hLEF based on Scatchard analysis, including mLIFR, MH3LIFR, MH4LIFR and MH5LEFR, showed single slow dissociation rates (Koff ~ 0.16-0.2 min"1) and the other slow (Koff ~ 0.001-0.002 min"1). In the receptor variants (MH1LEFR, MH2LIFR, MH6LEFR and MH7LIFR) which had intermediate hLIF-binding affinity, curvilinear kinetic dissociation curves were observed, which comprised a slow dissociation rate {Koff ~ 0.0003- 0.001 min"1) and a fast dissociation rate {Koff ~ 0.02-0.11 min 1) (Fig. 5, Table II). EXAMPLE 11 BINDING OF mLIF TO MOUSE-HUMAN HYBRID LIF RECEPTORS
The binding of mLIF and hLIF to each of the hybrid receptors was also evaluated by performing competitive inhibition assays. When 125IhLIF was used as a tracer, mLEF was able to compete with 125IhLIF for binding only on hybrid receptors which contained either an intact mLIFR Ig-like domain (hybrids MH3LIFR, MH4LIFR and MH5LIFR) or part of an mLIFR Ig-like domain (hybrids MH1LIFR and MH2LIFR) (Fig. 6). The ID50 values for either hLEF or mLEF competing with 125IhLEF binding to these hybrid receptors were essentially the same. However, the effective concentration of mLIF required to displace 125IhLEF bound to these receptors was 2000- to 3000- fold higher than that of hLEF (Fig. 6, Table H). 125ImLEF was able to detectably bind to MH3LIFR, MH4LEFR and MH5LH-R but only at 10- to 50-fold higher receptor concentrations compared to those used for 125IhLEF binding (data not shown).
In those receptors which did not contain the mLEFR Ig-like domain, including hLIFR and hybrid receptors MH6LIFR, MH7LIFR and MH8LIFR, mLIF was unable to compete with 125IhLIF even at high ligand concentrations (100 μg/ml). The JD50 values for hLIF competing with 125IhLIF bound to these receptors were 2- to 10-fold higher compared to that obtained with the mLIFR. This is essentially consistent with the Kj values obtained from the Scatchard analysis (Table II). These data indicate that the mouse LIFR Ig-like domain was primarily responsible for the species-specific interaction of mLIF with the mLIFR.
EXAMPLE 12
BLOCKING OF hLIF-INDUCED STAT-3 PHOSPHORYLATION
A short term assay was employed which involved stimulation of STAT-3 tyrosine phosphorylation by hLEF in Ml cells. STAT-3 activation is a critical step in gpl SO- mediated terminal differentiation of Ml cells (Minami et al, 1996) and, as shown in Fig. 7, tyrosine phosphorylation of STAT-3 was dramatically increased by hLIF stimulation of Ml cells within 5 minutes. This STAT-3 phosphorylation was almost completely blocked by preincubation of hLEF with recombinant mouse LEFR and hybrid MH3LIFR (Fig. 7). In the same experiment hybrids MH4LEFR, H5LIFR and MH6LIFR also showed a moderately inhibitory effect (65%) on hLEF-induced STAT-3 phosphorylation although it was not as significant as that seen for mLEFR and MH3 LIFR. The same applied to hybrids MHl LIFR, MH2LEFR and MH7LEFR, but to a lesser extent. Little or no inhibition of hLEF-stimulated STAT-3 phosphorylation was observed for both MH8LIFR and hLIFR, which could be correlated with their low binding affinity for hLEF. STAT-3 phosphorylation in Ml cells was not affected by addition of chimeric LEFRs alone (Fig. 7).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 5
(2) INFORMATION FOR SEQ ID NO : 2 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Gly Val Gin Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 5 10
( 2 ) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
Iii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Gly Ala Pro Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 5 10

Claims

CLAIMS:
1. A polypeptide or a derivatives or chemical equivalent thereof comprising first and second portions linked, bound or otherwise associated together wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an immunoglobulin (Ig)-like domain or a functional derivative thereof whereas said polypeptide exhibits cytokine binding properties.
2. A polypeptide or derivative or chemical equivalent thereof according to claim 1 wherein the first portion comprises at least two haemopoietin domains or functional derivatives thereof.
3. A polypeptide or derivative or chemical equivalent thereof according to claim 1 or 2 wherein the first and second portions are linked by covalent bonds, ionic bonds, hydrogen bonds, molecular bridging, molecular association or disulphide bridges.
4. A polypeptide or derivative or chemical equivalent thereof according to claim 3 wherein the first and second portions are covalently linked together by a peptide bond.
5. A polypeptide or derivative or chemical equivalent thereof according to claim 1 wherein said polypeptide exhibits leukemia inhibitoiy factor (LEF) binding properties.
6. A polypeptide or derivative or chemical equivalent thereof according to claim 5 wherein the haemopoietin domain comprises a LEF receptor (LEFR) ╬▒-chain haemopoietin domain or a functional derivative thereof and the Ig-like domain comprises a LIFR ╬▒-chain Ig-like domain.
7. A polypeptide or derivative or chemical equivalent thereof according to claim 6 wherein the first and second portions are derived from LIFR molecules from different species or from different allelic variants within a single species.
8. A polypeptide or derivative or chemical equivalent thereof according to claim 7 wherein one of said first and second portions is derived from a murine LIFR (mLIFR) ╬▒ chain and the other of said first and second portions is from a human LEFR (hLIFR) ╬▒-chain.
9. A polypeptide or derivative or chemical equivalent thereof according to claim 8 wherein the LIFR ╬▒-chain haemopoietin domain is from mLIFR ╬▒-chain or hLIF ╬▒-chain and the LIFR ╬▒-chain Ig-like domain is from mLEFR ╬▒-chain or hLIFR ╬▒-chain.
10. A polypeptide or derivative or chemical equivalent thereof according to claim 8 or 9 wherein said polypeptide comprises at least three portions wherein two portions comprise haemopoietin domains and one portion comprises an Ig-like domain.
11. A polypeptide or derivative or chemical equivalent thereof wherein said polypeptide comprises an LIFR ╬▒-chain Ig-like domain flanked by at least two LEFR ╬▒-chain haemopoietin domains.
12. A polypeptide or derivative or chemical equivalent thereof according to claim 11 wherein said polypeptide is MH2LEFR as defined in Figure 2.
13. A polypeptide or derivative or chemical equivalent thereof according to claim 11 wherein said polypeptide is MH4LIFR as defined in Figure 2.
14. A polypeptide or derivative or chemical equivalent thereof according to claim 11 wherein said polypeptide is MH5LEFR as defined in Figure 2.
15. A polypeptide or derivative or chemical equivalent thereof having the structure:
wherein
X, and X3 are located distally and proximally, respectively, to the transmembrane domain of LIFR ╬▒-chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
X2 is an Ig-like domain or a functional ╬▒erivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LEF.
16. A polypeptide or derivative or chemical equivalent thereof according to claim 15 wherein X, and X3 may be the : ime or different and each is a LEFR ╬▒-chain haemopoietin domain; and X2 is a LIFR ╬▒-chain Ig-like domain.
17. A polypeptide or derivative or chemical equivalent thereof according to claim 16 wherein Xt and X3 are derived from mLIF ╬▒-chain and/or hLIF ╬▒-chain.
18. A polypeptide or derivative or chemical equivalent thereof selected from the list consisting of MH1LIFR, MH2LIFR, MH3LIFR, MH4LIFR, MH5LEFR, MH6LIFR, MH7LEFR and MH8LEFR as defined in Figure 2.
19. A polypeptide or derivative or chemical equivalent thereof comprising a LEFR ╬▒- chain haemopoietin domain or a functional derivative thereof and a LEFR ╬▒-chain Ig-like domain or a functional derivative thereof wherein binding of LEF to the polypeptide gives rise to a two-contact state and a single kinetic dissociation rate according to Scatchard transformation of LIF binding to its receptor at equilibrium:
B
■■(K] +K KΛ(RT-B)
where B is the specifically bound LIF concentration, F is the free LIF concentration, Rτ is the total concentration of LEF receptors, Kj is the equilibrium affinity content for the first contact site of LIF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF.
20. A polypeptide or derivative or chemical equivalent thereof according to claim 19 wherein the polypeptide is selected from MH3LIFR, MH4LIFR and MH5LIFR as defined in Figure 2.
21. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide comprising first and second portions wherein one portion comprises a haemopoietin domain or a functional derivative thereof and said other portion comprises an Ig-like domain or a functional derivative thereof wherein said polypeptide exhibits cytokine binding properties.
22. A nucleic acid molecule according to claim 21 wherein the first portion comprises at least two haemopoietin domains or functional derivatives thereof.
23. A nucleic acid molecule according to claim 22 wherein said polypeptide exhibits LIF-binding properties.
24. A nucleic acid molecule according to claim 23 wherein the haemopoietin domain comprises a LIF receptor (LIFR) ╬▒-chain haemopoietin domain or a functional derivative thereof and the Ig-like domain comprises a LIFR ╬▒-chain Ig-like domain.
25. A nucleic acid molecule according to claim 24 wherein the first and second portions are derived from LEFR molecules from different species or from different allelic variants within a single species.
26. A nucleic acid molecule according to claim 25 wherein one of said first and second portions is derived from a murine LEFR (mLIFR) ╬▒-chain and the other of said first and second portions is from a human LEFR (hLIFR) ╬▒-chain.
27. A nucleic acid molecule according to claim 26 wherein the LEFR ╬▒-chain haemopoietin domain is from MLIFR ╬▒-chain or hLEF ╬▒-chain and the LIFR ╬▒-chain Ig-like domain is from mLIFR ╬▒-chain or hLIFR ╬▒-chain.
28. A nucleic acid molecule according to claim 26 and 27 wherein said polypeptide comprises at least three portions wherein two portions comprise haemopoietin domains and one portion comprises an Ig-like domain.
29. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide wherein said polypeptide comprises a LEFR ╬▒-chain Ig-like domain flanked by at least two LEFR ╬▒-chain haemopoietin domains.
30. A nucleic acid molecule according to claim 29 wherein said polypeptide is MH3 LIFR defined in Figure 2.
31. A nucleic acid molecule according to claim 29 wherein said polypeptide is MH4 LEFR defined in Figure 2.
32. A nucleic acid molecule according to claim 29 wherein said polypeptide is MH5 LEFR defined in Figure 2.
33. A nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a polypeptide having the structure:
Figure imgf000045_0001
wherein
X, and X3 are located distally and proximally, respectively, to the transmembrane domain of
LIFR ╬▒-chain and may be the same or different and each is a haemopoietin domain or a functional derivative thereof;
X2 is an Ig-like domain or a functional derivative thereof; and wherein the polypeptide or derivative or chemical equivalent thereof is capable of binding, interacting, influencing or otherwise associating with LIF.
34. A nucleic acid molecule according to claim 33 wherein X, and X3 may be the same or different and each is a LIFR ╬▒-chain haemopoietin domain; and X2 is a LIFR ╬▒-chain Ig- like domain.
35. A nucleic acid molecule according to claim 34 wherein X, and X3 are derived from mLEFR ╬▒-chain and/or hLIF ╬▒-chain.
36. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide selected from the list consisting of MHl LEFR, MH2LIFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as defined in Figure 2.
37. A nucleic acid molecule according to claim 36 encoding MH3LIFR as defined in Figure 2.
38. A nucleic acid molecule according to claim 36 encoding MH4LEFR as defined in Figure 2.
39. A nucleic acid molecule according to claim 36 encoding MH5LIFR as defined in Figure 2.
40. A nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide comprising a LIFR ╬▒-chain haemopoietin domain or a functional derivative thereof and a LIFR ╬▒-chain Ig-like domain or a functional derivative thereof wherein binding of LEF to the polypeptide gives rise to a two-contact state and a single kinetic dissociation rate according to Scatchard transformation of LEF binding to its receptor at equilibrium:
B
XKχ +KcKχ)(Rτ-B)
where B is the specifically bound LIF concentration, F is the free LIF concentration, Rτ is the total concentration of LEF receptors, K, is the equilibrium affinity content for the first contact site of LIF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF with its receptor and K,. is the equilibrium isomerisation constant for receptor isomerisation to form the second contact with LIF.
41. A nucleic acid molecule according to claim 37 wherein the polypeptide is selected from MH3LEFR, MH4LIFR and MH5LIFR as defined in Figure 2.
42. Use of a polypeptide or derivative or chemical equivalent thereof according to any one of claims 1 to 20 in the manufacture of a medicament for the treatment of a condition requiring antagonism of LIF.
43. A composition comprising a polypeptide or derivative or chemical equivalent thereof according to any one of claims 1 to 20 and one or more pharmaceutically acceptable carriers and/or diluents.
PCT/AU1998/000282 1997-04-21 1998-04-21 Novel chimeric molecules WO1998048011A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3823983A4 (en) * 2018-07-20 2022-05-18 The Board of Trustees of the Leland Stanford Junior University Soluble polypeptides and methods of using same for inhibiting leukemia inhibitory factor activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HILTON DOUGLAS J., "LIF: Lots of Interesting Functions", TRENDS IN BIOCHEMICAL SCIENCES, Vol. 17(2), February 1992, pp. 72-76. *
NICOLA NICOA A. (Edit), "Guidebook to Cytokines and Their Receptors", OXFORD UNIVERSITY PRESS, 1994, pp. 130-135. *
OWCZAREK CATHARINE et al., "The Unusual Species Cross-Reactivity of the Leukemia Inhibitory Factor Receptor-alpha-Chain is Determined Primarily by the Immunoglobulin-Like Domain", THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 272, No. 38, 19 September 1997, pp. 23976-23985. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3823983A4 (en) * 2018-07-20 2022-05-18 The Board of Trustees of the Leland Stanford Junior University Soluble polypeptides and methods of using same for inhibiting leukemia inhibitory factor activity
US12049490B2 (en) 2018-07-20 2024-07-30 The Board Of Trustees Of The Leland Stanford Junior University Soluble leukemia inhibitory factor receptor polypeptides and methods of using same for inhibiting leukemia inhibitory factor activity

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