WO1998048011A9 - Nouvelles molecules chimeres - Google Patents

Nouvelles molecules chimeres

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Publication number
WO1998048011A9
WO1998048011A9 PCT/AU1998/000282 AU9800282W WO9848011A9 WO 1998048011 A9 WO1998048011 A9 WO 1998048011A9 AU 9800282 W AU9800282 W AU 9800282W WO 9848011 A9 WO9848011 A9 WO 9848011A9
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Prior art keywords
polypeptide
chain
domain
lifr
derivative
Prior art date
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PCT/AU1998/000282
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English (en)
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WO1998048011A1 (fr
Inventor
Meredith Jane Layton
Catherine Mary Owczarek
Nicos Antony Nicola
Donald Metcalf
Yu Zhang
Original Assignee
Inst Medical W & E Hall
Meredith Jane Layton
Catherine Mary Owczarek
Nicos Antony Nicola
Donald Metcalf
Yu Zhang
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Application filed by Inst Medical W & E Hall, Meredith Jane Layton, Catherine Mary Owczarek, Nicos Antony Nicola, Donald Metcalf, Yu Zhang filed Critical Inst Medical W & E Hall
Priority to AU70141/98A priority Critical patent/AU7014198A/en
Publication of WO1998048011A1 publication Critical patent/WO1998048011A1/fr
Publication of WO1998048011A9 publication Critical patent/WO1998048011A9/fr

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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, LIF 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 LIF in the implantation process has been implicated, since mice in which the LIF gene has been ablated are essentially normal but are unable to implant their otherwise viable blastocysts (Stewart et al, 1992).
  • LIF exerts its biological actions through high affinity receptors which are expressed on the surface of LLF-responsive cells.
  • the high affinity LIF receptor (“LIFR") complex is composed of two components: the LIFR ⁇ -chain, which binds LLF 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 gp 130 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 [IL- 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 LIFR ⁇ -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 LIFR ⁇ -chain Similar to the G-CSF receptor (Fukunaga et al, 1990a; Fukunaga et al, 1990b) and gp 130 (Hibi et al, 1990), the LIFR ⁇ -chain also contains three fibronectin type DI (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 FNIH repeats are not essential for ligand binding.
  • FNIII fibronectin type DI
  • mLIF murine LIF
  • hLIF human LIF
  • mLBFR high and low affinity mouse LIFRs
  • 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 rnL ⁇ F binding to the mLIFR ⁇ -chain.
  • Cross- competition studies using the mLIFR ⁇ -chain reveal that the competition curves are dependent on which LIF is used as the radioactive tracer and this behaviour is interpreted as an interference by each type of LIF in the binding of the other.
  • LIF 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 ⁇ /., 1993).
  • the very high binding affinity of this receptor for hLIF 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 LIF 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 LIFR ⁇ -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 10 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: 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 20 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 25 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 LIF binding to its receptor at equilibrium:
  • B is the specifically bound LIF concentration
  • F is the free LIF concentration
  • R ⁇ is the total concentration of LIF 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.
  • 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 hLIF first contact site A on the LIFR ⁇ -chain, which is mainly dependent on the association rate (k onI ) and the dissociation rate (k finger m ). For hLIF binding die primary to rnLIFR ⁇ , 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 LIFR sequence described by (Gearing et al, 1991).
  • the letters M and H denote that amino acids are derived from mLIFR sequences or hLIFR sequences respectively. Because gaps were introduced in the mLIFR amino acid to maximise the alignment, the numbers refer to the specific hLIFR 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 crosslinking of Recombinant receptors from P. Pastoris. Culture supernatant were cross-linked to 125 IhLIF 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 H ⁇ LIF binding to chimeric LIF receptor variants. Saturation binding was performed by incubating aliquots of P. pastoris culture supernatant containing recombinant LIF receptors with increasing concentrations of 125 D ⁇ LIF. 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 Kj values are shown in Table ⁇ .
  • 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 5 is a graphical representation of kinetic dissociation of 125 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 t ) 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) MH2LEFR; (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 mLIF ( ⁇ ) 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) mLIFR (0.067 nM); (B) MH1LIFR (0.033nM); (C) MH2LIFR (0.142 nM); (D) MH3LIFR (0.014 nM); (E) MH4LIFR (0.027 nM); (F) MH5LIFR (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 hLEF-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 LIFR 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 LIF receptors and their differing binding characteristics for mouse and human LIF 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 LIFR ⁇ -chain haemopoietin domain and the Ig-like domain comprises a LIFR ⁇ -chain Ig-like domain.
  • the first portion comprises at least two haemopoietin domains.
  • a polypeptide or a derivative or a chemical equivalent thereof 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 domain such that said polypeptide has LIF 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 LIFR (mLIFR) ⁇ -chain and said other portion is from a human LIFR (hLIFR) ⁇ -chain.
  • mLIFR murine LIFR
  • hLIFR human LIFR
  • Such a heterologous molecule is useful for example, for humanising a mLIFR ⁇ -chain.
  • the LIFR ⁇ -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 LIF binding.
  • the chimera is selected from the listing consisting of MH1LIFR, MH2LIFR, MH3LIFR, MH4LIFR, MH5LIFR, MH6LIFR, MH7LIFR and MH8LIFR as defined in Figure 2.
  • the chimera is MH3LIFR ( 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 MH4LIFR ( Figure 2) and comprises at the membrane-distal position an mLIFR ⁇ -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 mLIFR ⁇ -chain Ig domain and at the membrane-proximal position an hLIFR ⁇ -chain haemopoietin domain.
  • Chimeras MH3LIFR, MH4LIFR and MH5LIFR all contain an intact Ig-like domain from mouse LIF receptor chain and high affinity 125 H ⁇ LIF binding (K d ⁇ 11-60 pM) similar to that seen for hLIF binding to the mLIFR (Fig. 4, Table II). This indicates that the immunoglobulin-like domain from the mouse LIF receptor has the most important influence in conferring the high affinity binding of hLIF.
  • polypeptide or derivative or chemical equivalent thereof having the structure: wherein:
  • X, and X 3 are located distally and proximally, respectively, to the transmembrane domain of the LIFR ⁇ -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:
  • 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 LIF.
  • 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 hLIFR ⁇ -chain amino acid residues at the N-terminal region, to approximately half way down the Ig-like domain, and mLIFR ⁇ -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, MH3LIFR, MH4LIFR and MH5LIFR 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);
  • 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 carboxy methylation 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-amino! ixanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, 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 15 example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ 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 mLIFR ⁇ -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 mLIFR ⁇ -chain is found to be due almost exclusively to a slower kinetic dissociation rate compared to mLIF.
  • the binding affinity for hLIF is most strongly dependent on the presence of an intact mLIFR 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 hLIF 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 LIF receptors
  • K is the equilibrium affinity constant for the first contact site of LIF with its receptor
  • 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 hLIF of about 300 pM. More preferably, said chimera exhibits an affinity binding to hLIF of about 150 pM and even more preferably, about 10 pM affinity binding to hLIF.
  • said chimera exhibits a bi-phasic dissociation rate for hLIF with one phase being of about k oj ⁇ 0.16 min "1 and the second phase of about £ ⁇ 0.002 min "1 . More preferably said chimera exhibits a bi-phasic dissociation rate for hLIF of about k o]S ⁇ 0.Ql min "1 and a second dissociation phase of about k oj f-0.00l 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 LIFR ⁇ -chain haemopoietin domain or a functional derivative thereof and a LIFR ⁇ -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 LIF 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 LIF binding properties of the chimeras of the present invention are particularly useful, but in no way limited to, use as a biological inhibitor of LIF.
  • LIF is bound by the chimera and thereby blocked from binding to any other unoccupied LIFR.
  • blocking of hLIF induced STAT-3 tyrosine phosphorylation in Ml cells is measured.
  • the differentiation of Ml cells is dependent upon the binding of LIF to the Ml cell surface LIFR.
  • 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 LEF activity.
  • binding, interaction or association of said polypeptide with LIF results in inhibition of LIF activity.
  • a mLIFR ⁇ -chain or derivative or chemical equivalent thereof comprising said XjX 2 X 3 is "humanised” by the substitution of sufficient of the mLIFR ⁇ -chain Ig-like domain (or part thereof) or haemopoietin domains with hLIFR ⁇ - chain Ig-like domains (or part thereof) or haemopoietin domains, respectively, to result in a chimeric LIFR ⁇ -chain exhibiting a high affinity for hLIF binding.
  • Such humanised mLIFR could act as a specific and potent antagonist of hLIF.
  • 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 IM to at least about 2M salt for hybridisation, and at least about IM 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. col ⁇ ) 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) w. 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 MLIFR was GVQ YPYDVPDYA [SEQ. ID NO: 2]
  • 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 FNHJ-like domains.
  • the resulting cDNAs were subsequently ligated into the Pichia pastoris expression vector pPIC9, that was digested with Xhol and Avrll, 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 Bglll or Sail and integrated into host cells by ti . 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, MH3LIFR, 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 LIFR, 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 supematant 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 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 pre-wetted 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 HI repeats that are present in native cellular LIF 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 IhLIF. Furthermore, the position of the 125 IhLIF 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 LIFRs 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 crosslinking with l25 IhLIF and size-exclusion chromatography.
  • the mouse LIFR ⁇ -chain binds hLIF with high affinity whereas the human LIFR ⁇ -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 IhLIF> 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, MH4LIFR and MH5LIFR all contain an intact Ig-like domain from mouse LIF receptor but have either one haemopoietin domain (MH3 and MH4) or two haemopoietin domains (MH5) from the human LIF receptor.
  • all of these three hybrids exhibited high affinity 125 IhLIF binding (K d ⁇ 11-60 pM) similar to that seen for hLIF binding to the mLIFR (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 hLIF.
  • 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 mLIFR residues while hybrid MH2LIFR was the converse.
  • these recomb nant 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 mLIFR residues except that the Ig-like domain was derived from the hLIFR and it bound 125 IhLIF with intermediate affinity (K d ⁇ 260 pM).
  • MH7LIFR 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 mLIFR haemopoietin domains the major contribution to high affinity 125 IhLIF 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 hLIFR (K d ⁇ 2 nM), indicating that the mouse LIFR membrane-distal haemopoietin domain is not involved in high affinity 125 IhLIF binding (Fig. 4, Table II).
  • the difference in hLIF-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 hLIF based on Scatchard analysis, including mLIFR, MH3LIFR, MH4LIFR and MH5LIFR, 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, MH4LEFR and MH5LIFR) or part of an mLIFR Ig-like domain (hybrids MH1LIFR and MH2LEFR) (Fig. 6).
  • the ID 50 values for either hLIF or mLIF competing with 125 IhLIF binding to these hybrid receptors were essentially the same.
  • 125 ImLIF was able to detectably bind to MH3LIFR, MH4LEFR and MH5L1 R but only at 10- to 50-fold higher receptor concentrations compared to those used for 125 IhLIF binding (data not shown).
  • mLIF was unable to compete with 125 IhLIF even at high ligand concentrations (100 ⁇ g/ml).
  • the ID 50 values for hLIF competing with 125 IhLBF 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). These data indicate that the mouse LIFR Ig-like domain was primarily responsible for the species-specific interaction of mLIF with the mLIFR.
  • a short term assay was employed which involved stimulation of STAT-3 tyrosine phosphorylation by hLIF in Ml cells.
  • STAT-3 activation is a critical step in gpl30- 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 hLIF with recombinant mouse LIFR and hybrid MH3LIFR (Fig. 7).
  • hybrids MH4LIFR, MH5LIFR 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 mLIFR and MH3 LIFR.
  • STAT-3 phosphorylation in Ml cells was not affected by addition of chimeric LIFRs alone (Fig. 7).
  • Owczarek CM., Layton, M.J., Metcalf, D., Lock, P., Willson, T.A., Gough, N.M. and
  • ATTORNEY/AGENT INFORMATION (A) NAME: HUGHES, DR E JOHN L (C) REFERENCE/DOCKET NUMBER: EJH/AF

Abstract

La présente invention concerne généralement des molécules chimères, et plus particulièrement des chimères interspécifiques de la chaîne α des récepteurs de cytokine. Plus particulièrement, la présente invention traite des chimères interspécifiques de la chaîne α des récepteurs du facteur inhibiteur des leucémies. Les molécules chimères selon la présente invention peuvent servir, entre autres choses, d'antagonistes des activités de la cytokine humaine in vivo. Dans un mode de réalisation, la présente invention concerne un polypeptide ou un dérivé ou un produit chimique équivalent comprenant des première et deuxième parties reliées, liées ou associées dans lesquelles une partie comprend un domaine d'hématopoïétine ou un dérivé fonctionnel, et l'autre partie comprend un domaine de type immunoglobuline (Ig) ou un dérivé fonctionnel de ce dernier, tandis que ce polypeptide présente de la cytokine, comme des propriétés de liaison au facteur inhibiteur des leucémies.
PCT/AU1998/000282 1997-04-21 1998-04-21 Nouvelles molecules chimeres WO1998048011A1 (fr)

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