WO2005030803A1 - Molecules therapeutiques - Google Patents

Molecules therapeutiques Download PDF

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WO2005030803A1
WO2005030803A1 PCT/AU2004/001336 AU2004001336W WO2005030803A1 WO 2005030803 A1 WO2005030803 A1 WO 2005030803A1 AU 2004001336 W AU2004001336 W AU 2004001336W WO 2005030803 A1 WO2005030803 A1 WO 2005030803A1
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Prior art keywords
lif
mutations
class
molecule
lifr
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PCT/AU2004/001336
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English (en)
Inventor
Manuel Baca
Walter Douglas Fairlie
Alessandro Dimitri Uboldi
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The Walter And Eliza Hall Institute Of Medical Research
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Priority claimed from AU2003905300A external-priority patent/AU2003905300A0/en
Application filed by The Walter And Eliza Hall Institute Of Medical Research filed Critical The Walter And Eliza Hall Institute Of Medical Research
Publication of WO2005030803A1 publication Critical patent/WO2005030803A1/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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5415—Leukaemia inhibitory factor [LIF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to Leukemia Inhibitory Factor (LIF) variants. More particularly, the present invention provides cytokine antagonists and agonists. Even more particularly, the present invention provides agonists and antagonists of cytokines which act via the Leukemia Inhibitory Factor (LIF) receptor -chain (LIFR) and gpl30 signalling system.
  • LIF Leukemia Inhibitory Factor
  • the agonists and antagonists are based on modified forms of LIF.
  • the agonists and antagonists of the present invention are also useful as targets for developing or screening for chemical mimetics and in particular small chemical mimetics of the modified LIF molecules.
  • the agonists and antagonists of the present invention are useful agents in the treatment and/or prophylaxis of cytokine-mediated conditions including inflammation, cancer growth, fertility and pregnancy, or for the culturing of embryonic stem cells.
  • the present invention further contemplates a method for modulating cytokine- mediated physiological or disease conditions such as inflammation, cancer, fertility or pregnancy as well as compositions comprising the antagonists.
  • Leukemia Inhibitory Factor is a member of the family of cytokines that includes interleukin (IL-)-6, IL-11, ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), oncostatin M (OSM) and cardiotrophin-like cyto ine/cytokine-like factor- 1 (CLC/CLF), (Bravo et al, EMBO J: 19, 2399-2411, 2000).
  • IL- interleukin
  • CNTF ciliary neurotrophic factor
  • CT-1 cardiotrophin-1
  • OSM oncostatin M
  • CLC/CLF cardiotrophin-like cyto ine/cytokine-like factor- 1
  • LIF displays a wide variety of biological actions on numerous cell types and tissues including neurons, embryonic stem cells, hepatocytes, megakaryocytes, adipocytes and osteoblasts, (Hilton, Trends Biochem. Sci.: 17, 72-76, 1992). All are four ⁇ -helical bundle type cytokines that exert their biological effects following the formation of high affinity receptor complexes involving at least one gpl30 molecule.
  • LIF LIF receptor ⁇ - chain
  • nM low affinity
  • LIF-LIFR LIF receptor ⁇ - chain
  • gpl30 gpl30 signalling complex
  • a phenylalanine and a lysine residue at the N-terminal end of the D-helix were found to be critical for LIFR binding, whilst a number of residues on both the A- and C-helices were important for interaction with gpl30.
  • LIF LIF has been described by Robinson et al, Cell. 77(7):1101- 16, 1994. These receptor binding sites are referred to as Site III and Site II, respectively, following the convention established for IL-6 (Simpson et al, Protein Sci.: 6, 929-955, 1997).
  • LIF LIF has a critical role in embryo implantation that cannot be compensated for by the other family members. It follows, therefore, that antagonists of LIF may be able to act as novel contraceptives. Indeed, in several studies in which anti-LIF antibodies have been injected into both mice and monkeys, a decrease in the pregnancy rate of both animals were observed (Yue et al, Biol. Reprod. : 63, 508-512, 2000, Cai et al, Cytokine: 12, 1676-1682, 2000).
  • LIF belongs to a family of cytokines which act via LIFR and gpl30, these modulators, will also act as modulators for a range of cytokines including CNTF, CT-1, OSM and CLC/CLF.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • a s immary of the sequence identifiers is provided in Table 1.
  • a sequence listing is provided after the claims.
  • XnY amino acid residue X at position n has been substituted by residue Y.
  • LIF Leukemia Inhibitory Factor
  • LIFR LIF receptor ⁇ -chain
  • gpl30 co-receptor used by all members of the IL-6 family of cytokines.
  • substitutions are Q29A, G124R and N128A.
  • the two mutations incorporated into the LIF molecule are either Q29A and G124R or N128A and G124R.
  • the present invention provides modulators of cytokine function, and in particular, agonists and antagonists of cytokines which act via the LIFR/gpl30 signalling pathway.
  • the present invention provides an agonist of a cytokine which induces signalling via LIFR and gpl30, wherein the agonist comprises a LIF molecule comprising mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR, or a homolog or chemical analog of said LIF molecule.
  • the present invention provides an antagonist of a cytokine which induces signalling via LIFR and gpl30, said antagonist comprising a LIF molecule comprising two classes of mutations (class 1 and class 2), or a homolog or chemical analog of said LIF molecule.
  • Class 1 mutations correspond to those which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR.
  • Class 2 mutations result in disruption of binding to gpl30. While modified LIF molecules only containing class 2 mutations are also antagonists of signalling via LIFR and gpl30, combining class 1 together with Class 2 mutations leads to a significant enhancement of antagonistic activity.
  • one aspect of the present invention provides an isolated modulator of signalling via Leukemia Inhibitory Factor (LIF) Receptor (LIFR) and gpl30 wherein said modulator comprises a variant LIF molecule having either agonist or antagonist activity to said signalling wherein: (i) the agonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more Class 1 mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR; (ii) the antagonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more mutations from Class 1 which results in high affinity binding of LIF to LIFR and one or more mutations, from Class 2 which disrupt binding of LIF to gpl30.
  • LIF Leukemia Inhibitory Factor
  • LIFR Leukemia Inhibitory Factor
  • the LIF molecule is preferably of human origin although non-human primate, livestock animal, laboratory test animal and companion animal LIF are also encompassed by the present invention, as are LIF molecules containing mixed sequence derived from more than one species.
  • the Class 1 mutations in LIF resulting in increased, enhanced or otherwise high affinity binding of LIF to LIFR are located at the C-terminal end of helix B, N-terminal end of helix D, or within the A-B and C-D connecting loops proximal to two residues, Phel56 and Lysl59 of human LIF or their non-human equivalents.
  • the Class 2 mutations in LIF resulting in disrupted binding of LIF to gpl30 are located on the A- and C-helices of human LIF or their non-human equivalents.
  • LIF amino acid residues in the vicinity of Phel56 and Lysl59 of human LIF can make contact with LIFR, and that the affinity of this interaction is increased significantly by mutations in this region.
  • Preferred class 1 affinity enhancing substitutions include P53G, N54L, N54M, N54F, N54W, N55L, N55M, N55F, N55W, D57G, D57P, K102W, I103E, I103D, T150W, K153E, D154Y, D154F, D154W, V155T, V155Y, Q157S, K160M, K170V.
  • class 2 mutations which disrupted gpl30 binding together with class 1 mutations
  • highly potent antagonists of LIF bioactivity were generated.
  • Preferred class 2 mutations include Q29A, G124R and N128A. The most active of these antagonists were able to inhibit LIF-induced proliferation of cells at concentrations that were similar to that of the wild type LIF used for stimulation. This represents an increase in potency in the order of 100-2000 fold compared to antagonists LIF molecules containing only Class 2 mutations.
  • Reference to disrupted binding of LIF to gpl30 includes reference to mutations in LIF resulting in abrogated gpl30-mediated signalling.
  • the present invention is further directed to a nucleic acid molecule comprising a nucleotide sequence encoding a sequence of amino acids which define a modulator of signalling via LIFR and gpl30 wherein said modulator comprises a variant LIF molecule having either agonist or antagonist activity to said signalling wherein: (i) the agonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more Class 1 mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR; (ii) the antagonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more mutations from Class 1 which results in high affinity binding of LIF to LIFR and one or more mutations, from Class 2 which disrupt binding of LIF to gpl30.
  • the present invention provides immunointeractive molecules such as but not limited to antibodies and other immunoglobulins including fragments, derivatives, antigen binding portions, recombinant forms, chimeric forms as well as deimmunized including humanized forms thereof directed to the subject modulators.
  • immunointeractive molecules such as but not limited to antibodies and other immunoglobulins including fragments, derivatives, antigen binding portions, recombinant forms, chimeric forms as well as deimmunized including humanized forms thereof directed to the subject modulators.
  • LIF antagonists generated in accordance with the present invention are also likely to be antagonists of signalling by these other cytokines. These antagonists will, therefore, be useful in defining the in vivo function of cytokines which signal through the LIFR, or for treating diseases which result from the biological activity of LIF, CT-1, CNTF, OSM and CLC/CLF.
  • the present invention contemplates a method for modulating cytokine function including cytokine-mediated physiological conditions or disease states, said method comprising administration to a subject of an effective amount of a LIF agonist or antagonist or its homolog or chemical derivative wherein the LIF agonists or antagonists comprises one or both of two classes of mutations, the first class resulting in enhanced LIFR binding affinity, while the 2nd class of mutations disrupts binding of LIF to gpl30.
  • the present invention provides a method for ameliorating the symptoms associated with cytokine dysfunction in a subject said method comprising administering to said subject a modulator of cytokine signalling via LIFR and gpl30 wherein said modulator comprises a modified LIF molecule having either agonist or antagonist activity to said cytokine wherein: (i) the agonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more Class 1 mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR; (ii) the antagonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more mutations from Class 1 which results in high affinity binding of LIF to LIFR and one or more mutations, from Class 2 which disrupt binding of LIF to gpl30; said modulator being administered for a time or under conditions sufficient to ameliorate the symptoms.
  • Conditions contemplated include inflammation, cancer treatment, fertility and pregnancy.
  • the agonist LIF variants may also be useful in the culturing of embryonic stem cells.
  • the antagonist LIF variants are proposed to be useful as contraceptive agents in human and non-human animals. Abbreviations used in the present specification are listed in Table 1.
  • Figure 1 is a diagrammatic representation showing design of mutant LIF libraries.
  • Figure 2 is a graphical representation showing binding analysis of soluble LIF mutants by BIAcore.
  • LIF mutants or wild type proteins were immobilized onto biosensor chips at a density of 1000 + 200 RU.
  • Sensorgrams for each LIF protein are shown following injection of 50 nM recombinant human LIFR.
  • A) Association and initial dissociation data are shown for MH35-BCD (1); MH35-BD (2); MH35-B (3); MH35-D (4); human LIF (5); MH35 (6); murine LIF (7).
  • Figure 3 is a graphical representation showing biological activity of wild type or mutant LIF proteins in the Ba/F3-LIFR/gpl30 assay.
  • Cells were stimulated with the various proteins, and proliferation after 48 h incubation was measured colorimetrically by MTT assay (Mosmann, J. Immunol. Methods: 65, 55-63, 1983). Values represent the mean of triplicate samples. Closed circles, human LIF; open circles, murine LIF; squares, MH35; triangles Q25A/N28A/Q32A MH35; diamonds Q29A/G124R MH35.
  • Figure 4 is a graphical representation showing inhibition of Ba/F3-LIFR/gpl30 cell proliferation by LIF mutants.
  • LIF mutants were titrated in the presence of a constant amount of wild type LIF, and proliferation after 48 h incubation was measured colorimetrically by MTT assay (Mossman, Supra. 1983). Values represent the mean of triplicate samples.
  • Figure 5 is a graphical representation showing inhibition of Ml cell differentiation by LIF mutants.
  • LIF mutants were titrated in the presence of a constant amount of murine LIF (8 pM). Results are expressed as the percentage of Ml colonies, grown in semi-solid agar, that showed a differentiated (disperse) phenotype.
  • the present invention provides modified LIF molecules or homologs or chemical analogs thereof, which act as agonists or antagonists of LIFR/gpl30-mediated signalling.
  • Cytokines which mediate signalling via these receptors include LIF, OSM, CNTF, CT-1 and CLC/CLF.
  • the modified LIF molecules generally comprise two classes of mutations (class 1 and class 2). Class 1 mutations result in increased, enhanced or otherwise high affinity binding of LIF to LIFR. Modified LIF molecules containing these mutations act as agonists of LIFR/gpl30-mediated signalling. Class 2 mutations result in disruption of binding to gpl30. Modified LIF molecules containing class 2 mutations act as antagonists of LIFR/gpl30-mediated signalling. Modified LIF molecules containing both class 1 and class 2 mutations exhibit enhanced potency as antagonists of LIFR/gpl30-mediated signalling relative to modified LIF molecules that only contain class 2 mutations.
  • each mutation class may have a single mutation or multiple mutations such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 mutations in each class, or combinations thereof in both classes.
  • the mutations at the amino acid level may be a substitution, addition and/or a deletion of one or more amino acid residues. Preferably, however, the mutation is a substitution.
  • the present invention further extends to genetic molecules encoding the modified LIF molecules. Such genetic molecules may also be used in therapy (as discussed further below) or may be used to generate commercial quantities of the modified LIF molecules.
  • modified LIF molecule includes homologs thereof and chemical equivalents or analogs thereof.
  • modified LIF molecule acts as an agonist or antagonist of LIFR/gpl30-mediated signalling
  • natural product or chemical library screening or combinatorial chemical approaches may be implemented to find a chemical molecule which mimics at least functionally the agonistic or antagonistic properties of the modified LIF molecule.
  • the modified LIF molecule or its homolog or chemical analog may also be referred to herein as an agonist, antagonist or superantagonist and this extends to antagonizing any cytokine which mediates signalling -via LIFR/gpl30.
  • Other terms used to describe the modified LIF or its homolog or chemical analog include agent, compound and active ingredient.
  • modulator is used to encompass an agonist and an antagonist.
  • Human LIF is particularly preferred although the present invention extends to non-human primate LIF, livestock animal LIF (eg. from sheep, pig, horse, goat, camel or cow), laboratory test animal LIF (eg. from mouse, rat, guinea pig, hamster or rabbit) or companion animal LIF (eg. from dog or cat).
  • Mature LIF protein sequences are highly conserved across species. Examination of mutations G124, Q29 and N128 in the amino acid sequences from a number of species revealed that Q29 is conserved in all species except porcine, G124 is conserved across all species examined and N128 is conserved in all species except rat (Nicola, N. A. (1994) Guidebook to Cytokines and Their Receptors, Oxford Univ.
  • the mature forms of proteins from all species are the same length (180 amino acids) and as such, any numbering scheme used herein in reference to the human sequence, applies to each of the other sequence from different species.
  • the length of the precursor form of LIF does vary in some species due to a single amino acid deletion in some of the signal peptide regions.
  • the preferred class 1 mutations in LLF resulting in high affinity binding to LIFR are in one or more of three regions. These regions include amino acid residues 53-58, 102-103 and 150-160 of human LIF or their equivalents in non-human LIF molecules.
  • Reference to "high affinity binding” means an affinity which is at least about 100 times higher affinity than wild type LIF including at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 16O0, 1700, 1800, 1900 or 2000 times higher binding affinity.
  • one aspect of the present invention provides an isolated modulator of cytokine signalling via LIFR and gp 130 wherein said modulator comprises a modified LIF molecule having either agonist or antagonist activity to said cytokine wherein: (i) the agonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more Class 1 mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR; (ii) the antagonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more mutations from Class 1 which results in high affinity binding of LIF to LIFR and one or more mutations, from Class 2 which disrupt binding of LIF to gpl30.
  • binding affinity-enhancing substitutions defined as class 1 mutations, include P53G, N54L, N54M, N54F, N54W, N55L, N55M, N55F, N55W, D57G, D57P, K102W, I103E, I103D, T150W, K153E, D154Y, D154F, D154W, V155T, V155Y, Q157S, K160M, K170V in the human LIF protein sequence, or equivalent residues in non-human LIF molecules.
  • Class 2 mutations include mutations on the A- and C-helices of human LIF or their equivalent in non-human LIF molecules and preferably at positions Q29, G124 and N128, wherein the amino acids are substituted by any of the other amino acids.
  • Particularly preferred class 2 mutations which abrogate gpl30 binding include Q29A, G124R and N128A.
  • the mutations are Q29A and G124R or N128A and G124R in the human LIF protein sequence, or their equivalents in other LIF molecules.
  • the present invention further provides an antagonist of a cytokine which induces signalling via LIFR and/or gpl30, said antagonist comprising a LIF molecule comprising a first set of mutations selected from a substitution, addition or deletion in an amino acid sequence in any of the regions including amino acid residues 53-58, 102-103 and 150-160 of human LIF or their equivalent in non-human LIF molecules and a second set of mutations in the A- and C-helices of human LIF or equivalent in non-human LIF molecules which abrogates interaction with gpl30 or a homolog or chemical analog or equivalent of said LIF molecule.
  • prefened mutations affecting binding to gpl30 result in abrogated signalling through LIFR/gpl30.
  • Particularly preferred mutations include Q29A together with G124R, or N128A together with G124R.
  • the present invention also contemplates a modified LIF molecule comprising one or more amino acid substitutions selected from P53G, N54L, N54M, N54F, N54W, N55L, N55M, N55F, N55W, D57G, D57P, K102W, I103E, I103D, T150W, K153E, D154Y, D154F, D154W, V155T, V155Y, Q157S, K160M and K170V, together with a second set of amino acid substitutions selected from G124R together with Q29A and/or N128A, or a homolog or chemical analog or equivalent of said modified LIF molecule.
  • the term "antagonist” includes the modified LIF molecule as well as its homologs or chemical equivalent or analogs.
  • the present invention further contemplates nucleic acid molecules including genetic constructs comprising same which encode the subject modulators.
  • another aspect of the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence encoding a sequence of amino acids which define a modulator of signalling via LIFR and gpl30 wherein said modulator comprises a variant LIF molecule having either agonist or antagonist activity to said signalling wherein: (i) the agonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more Class 1 mutations which result in increased, enhanced or otherwise high affinity binding of LIF to LIFR; (ii) the antagonist LIF variant comprises a LIF molecule or a homolog or chemical analog or functional equivalent thereof containing one or more mutations from Class 1 which results in high affinity binding of LIF to LIFR and one or more mutations, from Class 2 which disrupt binding of LIF to gpl30.
  • the nucleic acid molecule of the present invention may be used to generate recombinant forms of the modulators and/or may be used in gene therapy or in a range of diagnostic situations
  • the present invention further contemplates prokaryotic or eukaryotic cells comprising the subject nucleic acid molecules or genetic constructs comprising same.
  • eukaryotic cells include human and mouse cell lines.
  • prokaryotic cells include E. coli.
  • the present invention provides immunointeractive molecules such as but not limited to antibodies and other immunoglobulins including fragments, derivatives, antigen binding portions, recombinant forms, chimeric forms as well as deimmunized including humanized forms thereof directed to the subject modulators.
  • immunointeractive molecules such as but not limited to antibodies and other immunoglobulins including fragments, derivatives, antigen binding portions, recombinant forms, chimeric forms as well as deimmunized including humanized forms thereof directed to the subject modulators.
  • the present invention provides antibodies that bind, interact or otherwise associate with the modified LIF molecules of the present invention.
  • the antibodies maybe monoclonal or polyclonal antibodies, although, monoclonal antibodies are preferred. Generally, the antibodies are in isolated, homogenous or fully or partially purified form.
  • the antibodies are humanized or chimeric or are human antibodies suitable for administration to humans.
  • humanized antibodies prepared, for example, from murine monoclonal antibodies, and human monoclonal antibodies which may be prepared, for example, using transgenic mice as described below, or by phage display.
  • a "humanized” antibody includes a deimmunized antibody.
  • antibodies are raised against a human modified LIF molecules or immunogenic parts thereof.
  • the present invention provides antibodies that are human or humanized monoclonal antibodies that bind to the modified LIF molecules of the present invention.
  • Reference to an "antibody” or “antibodies” includes reference to all the various forms of antibodies, including but not limited to: full antibodies (e.g. having an intact Fc region), including, for example, monoclonal antibodies; antigen-binding antibody fragments , including, for example, Fv, Fab, Fab' and F(ab') 2 fragments; humanized antibodies; human antibodies (e.g., produced in transgenic animals or through phage display); and immunoglobulin-derived polypeptides produced through genetic engineering techniques.
  • the terms "antibody” or “antibodies” and as used herein encompasses both full antibodies and antigen-binding fragments thereof.
  • an antibody of the present invention binds substantially only to its target antigen with no appreciable binding to unrelated proteins.
  • an antibody will be designed or selected to bind to two or more related proteins.
  • a related protein includes different splice variants or fragments of the same protein or homologous proteins from different species. Such antibodies are still considered to have specificity for those proteins and are encompassed by the present invention.
  • the term "substantially” means in this context that there is no detectable binding to a non-target antigen above basal, i.e. nonspecific, levels.
  • the antibodies of the present invention may be prepared by well known procedures. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).
  • One method for producing an antibody of the present invention comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a modified LIF molecule or immunogenic parts thereof whereby antibodies directed against the modified LIF molecule or immunogenic parts are generated in said animal.
  • a non-human animal such as a mouse or a transgenic mouse
  • a modified LIF molecule or immunogenic parts thereof whereby antibodies directed against the modified LIF molecule or immunogenic parts are generated in said animal.
  • Various means of increasing the antigenicity of a particular immunogen such as administering adjuvants or conjugated antigens, comprising the antigen against which an antibody response is desired and another component, are well known to those in the art and may be utilized.
  • Immunizations typically involve an initial immunization followed by a series of booster immunizations. Animals may be bled and the serum assayed for antibody titer. Animals may be boosted until the titer plateaus. Conjugates may be made in.
  • Both polyclonal and monoclonal antibodies can be produced by this method.
  • the methods for obtaining both types of antibodies are well known in the art.
  • Polyclonal antibodies are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of a modified LIF molecule, or immunogenic parts thereof, collecting serum from the animal and isolating modified LIF molecule specific antibodies by any of the known immunoadsorbent techniques.
  • a itibodies produced by this technique are generally less favoured, because of the potential for heterogeneity of the product.
  • monoclonal antibodies are particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • Monoclonal antibodies may be produced by conventional procedures.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature 256:495 (1975), or may be made by recombinant DlSfA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using for example, the techniques described in Clackson et al, Nature 352:624- 628, 1991 and Marks et al., J. Mol. Biol. 222:581-597, 1991.
  • the present invention contemplates a method for producing a hybridoma cell line which comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a modified LIF molecule or immunogenic parts thereof; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line to generate hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a modified LIF molecule.
  • hybridoma cell lines and the anti-modified LIF molecule monoclonal antibodies produced by them are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell lines are purified by conventional techniques.
  • Hybridomas or the monoclonal antibodies produced by them may be screened further to identify monoclonal antibodies with particularly desirable properties.
  • the modified LIF molecule or immunogenic part thereof that may be used to immunize animals in the initial stages of the production of the antibodies of the present invention may be from any mammalian source.
  • Antigen-binding fragments of antibodies of the present invention may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments, including single chain Fv fragments (termed sFv or scFv). Antibody fragments and derivatives produced by genetic engineering techniques, such as disulphide stabilized Fv fragments (dsFv), single chain variable region domain (Abs) molecules, minibodies and diabodies are also contemplated for use in accordance with the present invention.
  • dsFv disulphide stabilized Fv fragments
  • Abs single chain variable region domain
  • Such fragments and derivatives of monoclonal antibodies directed against modified LIF molecules may be prepared and screened for desired properties, by known techniques, including the assays described herein. Certain of the techniques involve isolating DNA encoding a polypeptide chain (or a portion thereof) of a mAb of interest, and manipulating the DNA through recombinant DNA technology. The DNA may be fused to another DNA of interest, or altered (e.g. by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues, for example.
  • DNA encoding antibody polypeptides may be isolated from B-cells of mice that have been immunized with modified LIF molecules.
  • the DNA may be isolated using conventional procedures.
  • Phage display is another example of a known technique whereby derivatives of antibodies may be prepared.
  • polypeptides that are components of an antibody of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides are allowed to assemble to form antibody molecules.
  • Single chain antibodies may be formed by linking heavy and light chain variable region (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain.
  • Fv region heavy and light chain variable region
  • amino acid bridge short peptide linker
  • VL and VH The resulting antibody fragments can form dimers or trimers, depending on the length of a flexible linker between the two variable domains.
  • Techniques developed for the production of single chain antibodies include those described in U.S. Patent No. 4,946,778; Bird (Science 242: 423,
  • Single chain antibodies derived from antibodies provided herein are encompassed by the present invention.
  • the present invention provides antibody fragments or chimeric, recombinant or synthetic forms of the antibodies of the present invention that bind to modified LIF molecules.
  • IgGl or IgG4 monoclonal antibodies may be derived from an IgM monoclonal antibody, for example, and vice versa.
  • Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody.
  • Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g. DNA encoding the constant region of an antibody of the desired isotype.
  • the monoclonal production process described above may be used in animals, for example mice, to produce monoclonal antibodies.
  • Conventional antibodies derived from such animals, for example murine antibodies are known to be generally unsuitable for administration to humans as they may cause an immune response. Therefore, such antibodies may need to be modified in order to provide antibodies suitable for administration to humans.
  • Processes for preparing chimeric and/or humanized antibodies are well known in the art and are described in further detail below.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which the variable domain of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a non-human species (e.g., murine), while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from humans, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl Acad. Sci. USA 81:6851-6855, 1984).
  • a non-human species e.g., murine
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from the non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which the complementarity determining regions (CDRs) of the recipient are replaced by the corresponding CDRs from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired properties, for example specificity, and affinity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired properties, for example specificity, and affinity.
  • framework region residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework region residues are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • CDRs complementarity determining regions
  • transgenic mice into which genetic material encoding one or more human immunoglobulin chains has been introduced may be used to produce the antibodies of the present invention.
  • Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
  • Phage display techniques for generating human antibodies are well known to those skilled in the art, and include the methods used by companies such as Cambridge Antibody Technology and MorphoSys and which are described in International Patent Publication Nos. WO 92/01047, WO 92/20791, WO 93/06213 and WO 93/11236.
  • Antibodies of the present invention may be employed in vitro or in vivo to inhibit a biological activity that results from modified LIF molecule.
  • assays either in vitro or in vivo to detect the presence of modified LIF molecules and immunoaffmity chromatography to purify modified LIF molecules.
  • the antagonist LIF molecules of the present invention are useful in treating cytokine- mediated physiological conditions or disease conditions including, without being limited to, inflammation, cancer, fertility and pregnancy. With respect to the latter, this includes embryo implantation, maintenance, development, conception and spermatogenesis.
  • the antagonists therefore, are useful as contraceptive agents for human and non-human use.
  • the agonist LIF molecules of the present invention are also be useful in treating cytokine- mediated physiological conditions or disease conditions that result from a lack of LIF or from decreased signaling by LIF through the LIFR.
  • the agonist LIF molecules of the present invention are also useful in enhancing physiological conditions arising from LIF signaling.
  • the agonist LIF molecules of the present invention are useful in enhancing embryo implantation and embryo maintenance.
  • the agonist LIF moleculess of the present invention are useful in the culture of embryonic stem cells.
  • the present invention contemplates a method for ameliorating symptoms associated with cytokine-mediated physiological conditions and/or diseases in a subject, said method comprising administering to said subject an effective amount of an antagonist of said cytokine wherein said antagonist consists of a modified LIF molecule comprising one class of mutations which results in enhanced LIFR binding affinity, together with a second class of mutations which disrupts binding of LIF to gpl30.
  • the present invention is directed to a method for ameliorating symptoms associated with cytokine-mediated physiological conditions and/or diseases in a subject said method comprising administering to said subject an effective amount of an antagonist of a cytokine which induces signalling via LIFR and/or gpl30, said antagonist comprising a LIF molecule comprising a first set of mutations selected from a substitution, addition or deletion in an amino acid sequence in any of the regions including amino acid residues 53-58, 102-103 and 150-160 of human LIF or their equivalent in non-human LIF molecules and a second set of mutations in the A- and C-helices of human LIF or equivalent in non-human LIF molecules which abrogates interaction with gpl30 or a homolog or chemical analog or equivalent of said LIF molecule.
  • preferred mutations affecting gpl30 binding include G124R together with Q29A and/or N128A.
  • a most preferred embodiment of the present invention contemplates a method for the treatment or prophylaxis of a cytokine-mediated condition in a subject, said method comprising administering to said subject an effective amount of an antagonist of said cytokine said antagonist comprising a modified LIF molecule comprising one or more amino acid substitutions selected from P53G, N54L, N54M, N54F, N54W, N55L, N55M, N55F, N55W, D57G, D57P, K102W, I103E, I103D, T150W, K153E, D154Y, D154F, D154W, N155T, V155Y, Q157S, K160M and K170V, together with a second set of amino acid substitutions selected from G124R together with Q29A and/or ⁇ 128A, or a homolog or chemical analog or equivalent of said modified LIF molecule.
  • the present invention extends, therefore, to composition, and more particularly pharmaceutical formulations for use in prophylaxis and treatment of subjects.
  • subject refers to an animal, preferably a mammal and more preferably human who can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of animal that could benefit from the present invention. A subject regardless of whether a human or non-human animal may be referred to as an individual, subject, animal, host or recipient.
  • the compounds and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry.
  • an "animal” includes an avian species such as a poultry bird, an aviary bird or game bird. A poultry bird such as a duck is a preferred example of an avian species.
  • an antagonist includes a single antagonist, as well as two or more antagonists
  • an active agent includes a single active agent, as well as two or more active agents
  • agonist used interchangeably herein to refer to an agonist or antagonist of cytokine signalling via LIFR/gpl30 which induces a desired pharmacological and/or physiological effect such as but not limited to controlling inflammation, reducing cancer growth and inducing contraception.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • agonist When the terms "agonist”, “antagonist”, “compound”, “active agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • compound is not to be construed as a chemical compound only but extends to RNA and DNA encoding a modified LIF molecule.
  • Reference to a "modified LIF molecule” includes for example, molecules with a polysaccharide or lipopolysaccharide or polyethylene glycol component.
  • an effective amount and “therapeutically effective amount” of an agonist or antagonist as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological effect such as inhibiting inflammation, reducing the growth or spreading of cancer cells or controlling fertility or pregnancy.
  • Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
  • the present invention extends to a method of treatment or prophylaxis.
  • pharmaceutically acceptable carrier excipient or diluent
  • a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.
  • treating and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms of diseases or disorders or physiological conditions elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms of disease and/or their underlying cause and improvement or remediation of conditions associated with cytokine activity.
  • Treating" a patient may involve prevention of the disorder or disease condition or physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting a disease or disorder or, in the case of contraception, preventing conception or spermatogenesis or implantation.
  • another aspect of the present invention is directed towards therapeutic or prophylactic composition comprising an agonist or antagonist of LIFR/gpl30-mediated signalling wherein said agonist or antagonist is a modified LIF molecule as herein described or a homolog or chemical analog or equivalent thereof.
  • the agonists or antagonists of the present invention can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698.
  • the agonists or antagonists of the present invention may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands or specific nucleic acid molecules. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.
  • a target cell e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.
  • the vector could also be targeted to the target cells.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 731 A and International Patent Publication No. WO 90/07936.
  • modified means an entity which is different from the parent entity but which has a similar biological effect or effect substantially similar to the biological effect of the parent entity.
  • the biological effect of the modified form may be substantially the same or better than that of the parent.
  • a modified LIF molecule may have one or more amino acid substitutions, amino acid modifications, amino acid deletions and/or amino acid additions.
  • an amino acid substitution may be conservative or non-conservative as is well understood in the art.
  • variants of the modified LIF molecule may include molecules having modified amino acid side chains or chemical analogs of amino acid residues.
  • the present invention further includes genetic manipulations to introduce genetic material encoding a cytokine antagonist to a cell. This is generally part of a gene therapy approach. In such a situation, the genetic material will be expressed by the cell from the extrachromosomal location.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation calcium phosphate co-precipitation and viral transduction are known in the art.
  • Gene transfer systems known in the art may be useful in the practice of genetic manipulation. These include viral and non-viral transfer methods.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors including papovaviruses (e.g. SV40, Madzak et al, J. Gen. Virol. 73: 1533-1536, 1992) adenovirus (Berkner, Curr. Top. Microbiol Immunol 158: 39-66, 1992; Berkner et al BioTechniques 6; 616-629, 1988; Gorziglia and Kapikian, J. Virol 66: 4407-4412, 1992: Quantin et al, Proc. Natl Acad. Sci.
  • herpesviruses including HSV and EBV (Margolskee, Curr. Top., Microbiol. Immunol. 158: 67-95, 1992; Johnson et al, J. Virol. 66: 2952-2965, 1992; Fink et al, Hum. Gene Ther. 3: 11-19, 1992; Breakefield and Geller, Mol. Neurobiol 1: 339-371, 1987; Freese et al, Biochem. Pharmacol 40: 2189-2199, 1990; Fink et al, Ann. Rev. Neurosci.
  • Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to particular cells.
  • the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
  • plasmid DNA of any size is combined with a polyly sine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is non-specific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration.
  • the vector also contains a promoter functional in eukaryotic cells.
  • the cloned genetic material is under control of this promoter. Suitable eukaryotic promoters include those described above.
  • the expression vector may also include sequences, such as selectable markers and other sequences described herein.
  • the agonists or antagonists, compounds, agents, medicaments, nucleic acid molecules and other target antagonists or agonists of the present invention can be formulated in pharmaceutical compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • compositions may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. topical, intravenous, oral, intrathecal, epineural or parenteral.
  • compositions comprising a carrier and an agonist or antagonist, or a DNA encoding an agonist or antagonist, according to the invention may be accomplished through the use of a needleless injector.
  • Needleless injectors and their use are well known in the art. For example, Bellhouse et al. in U.S. Pat. Nos. 6,053,889, 6,013,050, 6,010,478, 6,004,286 and 5,899,880 disclose novel needleless injectors.
  • the present invention relates to antibodies and other immunointeractive molecules directed to the cytokine agonists or antagonists. Such antibodies are useful for diagnostic purposes or to inhibit activity of the LIF agonist or antagonist such as when the agonist or antagonist is used locally.
  • Concentrated supernatant containing the recombinant LIFR was loaded onto a lentil-lectin sepharose 4B column (Amersham Biosciences, Uppsala, Sweden), and eluted with 0.3M Methyl- ⁇ -D- mannopyranoside.
  • the eluate was then loaded onto M2 resin (Sigma-Aldrich, Sydney, Australia) and eluted using 100 ⁇ g/ml FLAG peptide in 100 mM NaCl, 20 mM Tris-Cl, pH 7.5, 0.02% w/v sodium azide.
  • the M2 eluate was finally chromatographed on a Superdex 200 size exclusion column in the same buffer.
  • LIF cDNA was subcloned into an in-house phage display vector. Libraries were constructed essentially as described by Sidhu et al, Methods Enzymol : 328, 333-363, 2000.
  • codons for each residue to be randomized were mutated to TGA stop codons by site-directed mutagenesis using the method of Kunkel et al, Methods Enzymol: 204, 125-139, 1991. Randomization of each of these residues was then performed using the same method, but with degenerate oligonucleotides in which an NNS codon was incorporated at each randomized position. Following transformation of electrocompetent SS320 E.
  • Each library was subjected to four rounds of panning against recombinant human LIFR.
  • Five wells of a 96-well microtitre plate were coated with LIFR at 2.5 ⁇ g/mL (rounds 1 and 2) or 1 ⁇ g/mL (rounds 3 and 4) in phosphate-buffered saline (PBS) overnight at 4°C.
  • PBS phosphate-buffered saline
  • the initial phage stocks, or phage from the previous round of panning were diluted 10-fold with PBS containing 0.1% v/v Tween-20 and either 1% w/v bovine serum albumin (rounds 1 and 3) or 0.5% w/v casein (rounds 2 and 4).
  • Diluted phage stock 100 ⁇ L; ⁇ 10 phage was added to each well and incubated for 1.5 h at room temperature.
  • eluted phage were serially diluted and used to infect XLl-Blue cells for 1 h prior to plating onto agar plates containing 100 ⁇ g/mL ampicillin.
  • 1 ⁇ L of culture supernatant from individual colonies grown in the presence of helper phage was used in a PCR to amplify a fragment encompassing the encoded LIF sequence.
  • This PCR product was then treated with ExoSAPit, (USB Corp., Cleveland, OH) to degrade unconsumed deoxynucleotides and primers, and directly sequenced.
  • EXAMPLE 4 Construction of LIF mutants and expression of recombinant proteins
  • the cDNA's for either human or murine LIF, or the human/murine chimera MH35 were subcloned into a modified pGEX-2T vector (Amersham Biosciences, Uppsala, Sweden) in which an fl origin of replication had been inserted to enable single-stranded DNA preparation for mutagenesis. All mutant forms were made using the method of Kunkel et al, Supra, 1991. Recombinant proteins were then expressed from the same vector as glutathione S-transferase (GST) fusion proteins in BL-21 DE3 pLys E.
  • GST glutathione S-transferase
  • Mutants of LIF were prepared according to the method of Kunkel et al, Supra, 1991 in the phg3_l phagemid vector to enable monovalent display on phage as fusions to gene IIIp. These constructs were used to transform XL-1 Blue E. coli and phage were propagated from individual colonies following incubation in the presence M13KO7 helper phage.
  • Phage displaying the various mutant proteins were isolated from culture supernatants by PEG NaCl precipitation as previously described (Sidhu et al, Methods Enzymol. : 328, 333-
  • Ba/F3 an IL-3-dependent cell line, is derived from murine pro B lymphocytes and does not normally express LIFR or gpl30 (Gearing et al, Proc. Natl Acad. Scie. USA: 91, 1119- 1123, 1994).
  • Ml differentiation assays were performed as previously described (Metcalf et al, Leukemia: 2, 216-221, 1988). Cells were stimulated with a fixed, submaximal concentration of recombinant murine LIF (8 pM) in the presence of increasing concentrations of the various antagonists.
  • each library contained between 10 8 and 10 9 transformants, with mutation rates of 40-80%). Whilst this is below the level required for representation of all possible protein sequences within a library containing six randomized positions (2.5 x 10 9 transformants required for a complete library at the 90% confidence level (Sidhu et al, Supra, 2003)), it was considered to provide a high enough level of diversity.
  • a new library was prepared with reduced diversity by limiting the randomized segment to four residues located near the top of the AB loop (amino acids 49-52). Following four rounds of panning with this library, 20/24 clones analyzed contained the wild type sequence. Only three clones contained a sequence which differed from wild type, and this variation only occuned at residue 50. Hence, it is proposed that the sequence of LIF is intolerant to substitution between amino acids 48-53.
  • non-wild type residues were strongly selected at residues 102 and 103, with retention of the wild type sequence at the remaining positions. Tryptophan was found in 7/13 clones in place of the wild type lysine at residue 102, while 11/13 clones contained an acidic residue (predominantly glutamic acid) in place of isoleucine at 103. The wild type sequence was completely retained for residues 104-106, and in 7/13 clones for residue 107.
  • Library E (residues 155-160, N-terminal end of D-helix) included residues 156 and 159 that had previously been shown to be critical for LIFR binding (Hudson et al, Supra, 1996). The wild type sequence was retained at these two positions in all of the clones analyzed. Most clones had a serine at residue 157 in place of glutamine, and methionine at 160 in place of the native lysine. Interestingly, all clones contained a tyrosine at residue 155. This residue was also randomized in library D, where the prefened residue was threonine, although some clones (4/17) from library D did contain a tyrosine.
  • a pentamutant of LIF (P53G/N54L/N55L/L56Q/D57G), designed from the consensus sequence of library B selectants, was used as a template for combining additional mutations derived from the consensus sequences of libraries C (K102W/I103E), D (T150W/G152Q/K153E/D154Y/N155T) or E (V155Y/Q157S/K158Q/K160M). Binding affinities were again measured by competition ELISA, where each of the LIF mutants was expressed monovalently on Ml 3 phage. However, no change in binding affinity was observed for any of these combination mutants (B+C, B+D, B+E, B+C+D, B+C+E) relative to the affinity of the parent library B pentamutant.
  • MH35 is 85% identical to that of human LIF and its three dimensional structure is quite similar to that of the human protein (Robinson et al, Supra 2001; Hinds et al, J. Biol.
  • MH35-BD consensus mutations from libraries B and D
  • MH35-BD the combination of consensus mutations from libraries B and D
  • MH35-BD gave a 1300-fold improvement in binding affinity over wild type MH35, as opposed to improvements of 175- and 34-fold for MH35- B and MH35-D.
  • the lowest IC 50 value obtained was on the order of ⁇ 50 pM. Again, this probably represented the lower limit of IC 50 that could be determined in this assay and may explain why the MH35- BCD mutant had a similar IC 50 to MH35-BD (70 pM and 54 pM, respectively).
  • the MH35 mutants were also tested in a competition phage ELISA to determine their affinities for binding murine LIFR (Table 4).
  • Murine LIFR binds to wild type MH35 and human LIF with unusually high affinity that is significantly higher than for human LIFR (Layton et al, Supra, 1994).
  • each of the affinity matured MH35 mutants MH35-B, -C and -D and -BD exhibited higher affinity binding.
  • the apparent improvements in affinity appeared to be modest, perhaps due to the limitation of the phage ELISA assay to measure IC 50 values below -50 pM. Consequently, the relative enhancements in murine LIFR binding affinities may have been greater than that suggested by these data.
  • Each MH35-based protein was expressed and purified, and its human LIFR binding affinity determined by SPR analysis on a BIAcore instrument.
  • the benefit of BIAcore analysis over ELISA-based assays is that real-time kinetic analysis can be performed, and the equilibrium binding constant (KD) can be calculated from this data.
  • Each LIF mutant was immobilized onto a biosensor chip at similar levels (1000 RU +/- 20%), and LIFR binding analyzed by injection of soluble receptor over the chip surfaces ( Figure 2). For reference, a wild type human LIF was included in this analysis. Due to the very slow off- rates exhibited by the highest affinity mutants, long dissociation times of up to 10 h were used.
  • association/dissociation rate constants and K D determinations for binding of the various proteins to human LIFR are summarized in Table 5.
  • All of the proteins analyzed had very similar association rates with less than 3-fold difference between the slowest (MH35) and fastest (MH35-D) k on values.
  • very large differences were observed in the dissociation rates of the proteins, as is obvious from visual inspection of the sensorgrams, particularly those generated over 10 h ( Figure 2B).
  • the values obtained for k 0ff extend over five orders of magnitude between the fastest (MH35) and the slowest (MH35-BCD) rates.
  • the large differences in binding affinity between wild type MH35 and the affinity matured mutants arise almost entirely from reductions in the rate of dissociation of the LIFR complexes.
  • the K D values obtained for binding of LIFR to MH35, human LIF, MH35-B and MH35-D were all within 4-fold of the IC 50 values obtained for the same proteins in the competition phage ELISA.
  • the ability of the BIAcore analysis to determine K D values down to the very low picomolar range resulted in greater differences between the assays for measured affinities of the combination mutants.
  • the most potent of these, MH35-BCD had a calculated K D that was 150,000-fold lower than that of wild type MH35. Whilst this difference may be correct, some caution needs to be taken with regards to the accuracy of calculating such extremely slow dissociation rates.
  • the present invention aims to provide superantagonists of both human and murine LIF signalling by combining mutations which enhance LIFR binding affinity with those that disrupt binding to the gpl30 co-receptor. Because of the difficulties in recombinantly producing the human LIF mutants, the MH35 backbone was again used for conversion of the high affinity mutants into superantagonists. The ability to make milligram quantities of the MH35-based proteins was particularly advantageous, as a long term aim of this project is to examine the effect of such antagonists in vivo which would likely require relatively large amounts of recombinant protein. Furthermore, because of the similar affinities of MH35 and human LIF for the murine LIF receptor (Layton et al, Supra, 1994), it was not considered disadvantageous to use the MH35 backbone for production of an antagonist for future testing in mice.
  • the Q29A/G124R double mutant was found to be completely inactive as an agonist (up to 20 nM), importantly, both of human or murine LIFR/gpl30 transfected Ba/F3 cells (Figure 3).
  • a similar result was obtained for the G124R/N128A double mutant. It was also shown that these two double mutants of MH35 acted as antagonists of LIF-induced Ba/F3 cell proliferation, and that this response was LIF-specific, as neither mutant had any effect on the ability of IL-3 to induce proliferation of the same cell lines.
  • Q29A/G124R MH35 was found to bind human and murine LIFR with a similar affinity to wild type MH35. The Q29A/G124R double mutation was therefore used to convert the affinity matured MH35 mutants into antagonists.
  • the Q29A/G124R double mutation was introduced into several of the affinity matured MH35 variants and these proteins were recombinantly prepared.
  • These proteins were tested for their ability to inhibit LIF -induced proliferation of Ba/F3 cells expressing human LIFR/gpl30 ( Figure 4A).
  • these mutants of LIF exhibited dose dependent inhibition of proliferation, moreover, differences in the potency of inhibition were well correlated with their anticipated affinities for binding human LIFR. Affinity maturation had a dramatic effect on the ability of these mutants of LIF to act as antagonists.
  • the parent MH35 molecule (Q29A/G124R MH35) had weak activity, showing partial inhibition of LIF-induced proliferation only at the highest concentration tested (2500 nM).
  • the Q29A/G124R-containing forms of the highest affinity MH35 mutants (MH35-BD, MH35- BCD) were potent antagonists of signalling.
  • the Q29A/G124R-containing mutants of MH35 were also potent antagonists of LIF- induced proliferation of Ba/F3 cells expressing murine LIFR/gpl30 (Fig. 4B).
  • MH35 LIF has ⁇ 50-fold higher affinity for binding murine LIFR compared to murine LIF, thus, wild type MH35 represents an affinity matured LIF variant with respect to murine LIFR binding.
  • Q29A/G124R MH35 was considerably more potent as an antagonist of signalling than Q29A/G124R murine LIF. Further improvements in the superantagonist activity of Q29A/G124R MH35 were achieved by incorporating the affinity maturation mutations identified from the phage-display screen.
  • Table 3 Binding affinities of phage-selected clones.
  • Kinetic constants were determined by injection of soluble human LIFR over biosensor chips onto which the various LIF mutant were immobilized.
  • the association (k on ) and dissociation (k 0 f ) rate constants were calculated using the BIAEvealuation software (Biacore).
  • the equilibrium binding constant (K D ) was calculated from the ratio of rate constants (k 0 ff/k 0 n).

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Abstract

La présente invention porte, de manière générale, sur des variants du facteur inhibiteur de la leucémie (FIL) et, en particulier, sur des antagonistes et des agonistes des cytokines, et plus précisément, sur des agonistes et des antagonistes des cytokines qui agissent via la chaîne α du récepteur du facteur inhibiteur de la leucémie) et le système de signalisation de gp130. Les agonistes et les antagonistes sont basés sur des formes modifiées du facteur inhibiteur de la leucémie. Les agonistes et les antagonistes de cette invention sont également utiles comme cibles dans le développement ou le criblage de substances mimétiques chimiques et notamment de petites substances mimétiques chimiques des molécules modifiées du facteur inhibiteur de la leucémie. Les agonistes et les antagonistes de l'invention sont utiles comme agents de traitement et/ou prophylaxie d'états induits par des cytokines tels que les inflammations, la prolifération cancéreuse, la fécondité et la grossesse ou pour la culture de cellules souches embryonnaires. L'invention porte également sur un procédé de modulation d'états physiologiques ou pathologiques induits par des cytokines tels que ceux précités, ainsi que sur des compositions comprenant les antagonistes.
PCT/AU2004/001336 2003-09-29 2004-09-29 Molecules therapeutiques WO2005030803A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010115868A2 (fr) 2009-04-03 2010-10-14 Fundacio Privada Institucio Catalana De Recerca I Estudis Avancats (Icrea) Agents thérapeutiques pour le traitement de maladies associées à une prolifération cellulaire indésirable
WO2010099979A3 (fr) * 2009-03-06 2010-11-18 Charité - Universitätsmedizin Berlin Gliedkörperschaft Der Freien Universität Berlin Und Der Humboldt-Universität Berlin Utilisations et procédés d'identification d'un composé cytoprotecteur comprenant gp130 ou lifrα
EP3067422A3 (fr) * 2015-03-13 2016-11-30 Sabanci Üniversitesi Inhibiteurs ct-1
EP3173483A1 (fr) 2015-11-27 2017-05-31 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Agents pour le traitement de maladies associées à une prolifération cellulaire indésirable
US10968273B2 (en) 2010-04-05 2021-04-06 Fundacio Privada Institut D'investigacio Oncologica Vall D'hebron (Vhio) Antibody recognizing human leukemia inhibitory factor (LIF) and use of anti-LIF antibodies in the treatment of diseases associated with unwanted cell proliferation

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TIMMERMANN A. ET AL.: "A functional role of the membrane-proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor", EUROPEAN JOURNAL OF BIOCHEMISTRY, FEBS, vol. 269, no. 11, 2002, pages 2716 - 2726 *
VERNALLIS A.B. ET AL.: "An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 43, 1997, pages 26947 - 26952 *
ZHANG Y. ET AL.: "The box-1 region of the leukemia inhibitory factor receptor alpha-chain cytoplasmic domain is sufficient for hemopoietic cell proliferation and differentiation", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 51, 1998, pages 34370 - 34383 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099979A3 (fr) * 2009-03-06 2010-11-18 Charité - Universitätsmedizin Berlin Gliedkörperschaft Der Freien Universität Berlin Und Der Humboldt-Universität Berlin Utilisations et procédés d'identification d'un composé cytoprotecteur comprenant gp130 ou lifrα
CN107964043A (zh) * 2009-04-03 2018-04-27 加泰罗尼亚研究和深造学院 用于治疗与有害细胞增殖相关的疾病的治疗剂
WO2010115868A3 (fr) * 2009-04-03 2011-02-03 Fundacio Privada Institucio Catalana De Recerca I Estudis Avancats (Icrea) Agents thérapeutiques pour le traitement de maladies associées à une prolifération cellulaire indésirable
ES2363358A1 (es) * 2009-04-03 2011-08-01 Fundacio Institut De Recerca Lhospital Universitari Vall D'hebron Agentes terapéuticos para el tratamiento de enfermedades asociadas con una proliferación celular indeseable.
CN102574918A (zh) * 2009-04-03 2012-07-11 加泰罗尼亚研究和深造学院 用于治疗与有害细胞增殖相关的疾病的治疗剂
EP3045474A3 (fr) * 2009-04-03 2016-09-28 Fundació Privada Institució Catalana de Recerca I Estudis Avançats Agents thérapeutiques pour le traitement de maladies associées à une prolifération cellulaire indésirable
WO2010115868A2 (fr) 2009-04-03 2010-10-14 Fundacio Privada Institucio Catalana De Recerca I Estudis Avancats (Icrea) Agents thérapeutiques pour le traitement de maladies associées à une prolifération cellulaire indésirable
US10100112B2 (en) 2009-04-03 2018-10-16 Fundacio Privada Institutcio Catalana de Recerca I Estudis Avancats (ICREA) Therapeutic agents for the treatment of diseases associated with undesired cell proliferation
KR20200078668A (ko) * 2009-04-03 2020-07-01 인스티튜시오 카탈라나 드 르세르카 아이 에스투디스 아반카츠 바람직하지 않은 세포 증식과 관련된 질환의 치료를 위한 치료제
KR102373754B1 (ko) * 2009-04-03 2022-03-15 인스티튜시오 카탈라나 드 르세르카 아이 에스투디스 아반카츠 바람직하지 않은 세포 증식과 관련된 질환의 치료를 위한 치료제
US10968273B2 (en) 2010-04-05 2021-04-06 Fundacio Privada Institut D'investigacio Oncologica Vall D'hebron (Vhio) Antibody recognizing human leukemia inhibitory factor (LIF) and use of anti-LIF antibodies in the treatment of diseases associated with unwanted cell proliferation
EP3067422A3 (fr) * 2015-03-13 2016-11-30 Sabanci Üniversitesi Inhibiteurs ct-1
EP3173483A1 (fr) 2015-11-27 2017-05-31 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Agents pour le traitement de maladies associées à une prolifération cellulaire indésirable
WO2017089614A1 (fr) 2015-11-27 2017-06-01 Fundació Privada Institut D'investigació Oncològica De Vall Hebron Agents pour le traitement de maladies associées à une prolifération cellulaire non souhaitée

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