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WO1996001319A1 - Variantes du facteur inhibiteur de la leucemie - Google Patents

Variantes du facteur inhibiteur de la leucemie Download PDF

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Publication number
WO1996001319A1
WO1996001319A1 PCT/GB1995/001528 GB9501528W WO9601319A1 WO 1996001319 A1 WO1996001319 A1 WO 1996001319A1 GB 9501528 W GB9501528 W GB 9501528W WO 9601319 A1 WO9601319 A1 WO 9601319A1
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lif
protein
fragment
human
seq
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PCT/GB1995/001528
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English (en)
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Laura Mary Grey
David Staunton
Keith Ronald Hudson
John Kaye Heath
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Cancer Research Campaign Technology Limited
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Priority to EP95923449A priority Critical patent/EP0769055A1/fr
Priority to AU28007/95A priority patent/AU2800795A/en
Publication of WO1996001319A1 publication Critical patent/WO1996001319A1/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
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • 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]
    • 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 to artificial variants of Leukemia Inhibitory Factor (LIF) with agonist or antagonist activity, and hybrid proteins incorporating domains from LIF
  • LIF Leukemia Inhibitory Factor
  • Leukaemia inhibitory factor is a secreted polyfunctional cytokine which elicits a diversity of biological effects on many cell types. LIF expression has been detected in a variety of cell lines and primary tissues, including primordial germ cells, neurons, embryonic stem cells, adipocytes, hepatocytes and osteoblasts (reviewed Metcalf, 1992 (Growth Factors 7, 169-173); Heath, 1992 (Nature 359, 17)), and stimulated T lymphocytes and monocytes, brain glial cells, liver fibroblasts, bone marrow stromal cells, thymic epithelial cells and uterine endometrial gland cells just prior to blastocyst implantation.
  • LIF Leukaemia inhibitory factor
  • Non-functional mutants have also revealed an essential role for LIF in the process of embryonic implantation in the mouse (Stewart et al., 1992, (Nature 359, 76-79); Escary et al., 1993 (Nature 363, 361-364)).
  • LIF can inhibit proliferation and induce macrophage differentiation in the murine leukemic myeloid cell line M1. It can synergize with IL-3 to stimulate the production of primitive hematopoietic progenitor and megakaryocyte colonies in vitro and increase the number of megakaryocytes and platelets in vivo. LIF can enhance the synthesis of acute phase proteins in liver hepatocytes stimulate the proliferation of myoblasts in culture and inhibit the lipoprotein iipase that mediates the transport to, and accumulation in, adipocytes.
  • LIF is a cholinergic neuronal differentiation factor that up-regulates neuropeptides and acetylcholine synthesis in sympathetic neurons in vitro and in vivo.
  • LIF is a neurotrophic, as well as a survival, factor in sensory neuron development in vitro.
  • LIF is a paracrine or autocrine regulator that acts directly on osteoblasts, and indirectly on osteoclasts to inhibit bone resorption and regulate bone formation. LIF inhibits the differentiation and maintains the pluripotent developmental potential of embryonic stem cells.
  • LIF "knockout" mice females have a specific defect in uterine function that prevents implantation of the blastocyst. Overexpression of LIF in mouse embryos, however, will result in inhibition of differentiation of embryonic ectodermal cells.
  • Both human and mouse LIF cDNAs encode mature LIF polypeptides of 180 amino acid residues with a predicted molecular mass of approximately 20kDa.
  • the mature form of the LIF protein is present as a monomer in solution.
  • LIF is subjected to extensive post-translational glycosylation.
  • Native human and mouse LIF are highly glycosylated (Moreau et al., 1988, (Nature 336, 690-692); Smith et al., 1988 (Nature 336, 688-690)), single chain molecules varying in molecular masses from approximately 38-67 kDa.
  • Both human and murine LIFs have multiple N- and O-linked glycosylation sites and six conserved cysteine residues that are involved in three intramolecular disulfide bridges.
  • the non-glycosylated, E.coli-expressed, recombinant human LIF is indistinguishable from native LIF in its biological activities in vitro.
  • Human and murine mature LIF exhibit a 78% sequence identity at the amino acid level, whereas human LIF is equally active on both human and mouse cells, murine LIF is approximately 1000 times less active on human cells.
  • LIF is a member of a family of ligands, which are characterized by a four a-helix bundle topology.
  • the prototype member of this family is growth hormone (GH) and subsequent structural studies have confirmed that several other proposed members of this set of molecules conform to the predicted four helix structure (reviewed by Sprang and Bazan, 1993, Curr. Op. Struct. Biol. 3, 815-827).
  • GH growth hormone
  • LIF-R LIF receptor subunit
  • LIF-R Gearing et al., 1991, EMBO J. 10, 2839-2848
  • LIF-R a specific LIF receptor subunit
  • LIF-R also contains a cytoplasmic motif which has been found to be common to other signal transducing elements (Taga and Kishimoto, 1992 FASEB J.
  • ciliary neurotrophic factor CNTF: Stockli et al., 1989 Nature 342, 920-923
  • IL-11 interleukin 11
  • IL-11 Paul et al., 1990 Proc. Natl. Acad. Sci. USA 87, 7512-7516; Kawashima et al., 1991 FEBS Lett. 283, 199-202).
  • Interleukin 6 is a multifunctional protein produced by lymphoid and non-lymphoid cells as well as by normal and transformed cells.
  • OSM is a pleiotropic cytokine that can affect the growth and differentiation of a variety of normal and tumor cells.
  • IL-11 is a growth factor with multiple effects on both hematopoietic and nonhematopoietic cell populations.
  • OSM Besides its growth inhibitor activities on the human A375 melanoma and other solid tumor cells, OSM also has grown stimulatory activities on normal fibroblasts, AIDS-Kaposi's sarcoma derived cells, and a human erythroleukemia cell line, TF-1.
  • Other OSM-mediated activities include: the induction of differentiation of the M1 murine myeloid leukemia cell line; the inhibition of differentiation of murine embryonic stem (ES) cells; the stimulation of plasminogen activator activity in cultured bovine aortic endothelial cells as well as in human synovial fibroblasts; the stimulation of acute phase protein production in primary hepatocytes; and LDL uptake and up-regulation of cell surface LDL receptors in HepG2 cells.
  • ES murine embryonic stem
  • OSM is a heat and acid stable, single chain glycoprotein.
  • cDNA derived from U937 lymphoma cells predicts a 252 amino acid (aa) residue peptide containing a 25 amino acid hydrophobic N-terminal signal sequence peptide. Cleavage of the signal sequence results in a 227 amino acid pro-cytokine that undergoes C-terminal processing to form a mature peptide 195 to 196 amino acid in length. Two potential N-glycosylation sites and five cysteine residues are present in the mature protein.
  • IL-6 and CNTF show a more complex involvement of gp130 in ligand mediated signalling.
  • IL-6 interacts with the IL-6 receptor subunit (IL6-R) which is frequently found expressed in a soluble form (reviewed by Kishimoto et al., 1992, Science 258, 593-597; Taga and Kishimoto, 1992, ibid).
  • IL-6/IL6-R complex mediates cellular signalling by formation of gp 130 homodimers (Murakami et al., 1993, Science 260, 1808-1810).
  • the effects of IL-6 on different cells are numerous and varied. The effect on B cells is to stimulate differentiation and antibody secretion.
  • IL-6 also affects T cells, acting as a co-stimulant with sub- optimal concentrations of PHA or Con A to stimulate IL-2 production and IL-2 receptor expression.
  • IL-6 exhibits growth factor activity for mature thymic or peripheral T cells and reportedly enhances the differentiation of cytotoxic T cells in the presence of IL-2 or IFN- ⁇ .
  • the human IL-6 cDNA sequence predicts a precursor protein of 212 amino acids with two potential N-glycosylation sites.
  • the hydrophobic N-terminal 28 amino acid signal peptide is cleaved to produce a mature protein of 184 amino acids with four cysteine residues and a predicted molecular weight of 21 kDA.
  • IL-11 is a pleiotropic cytokine that was originally detected in medium conditioned by an IL-1 ⁇ - stimulated primate bone marrow stromal cell line, PU-34, by its ability to stimulate the proliferation of the IL-6-dependent murine plasmacytoma cell line T1165.85.2.1 in the presence of excess neutralizing anti-IL-6 antibodies. Similar to IL-1, IL-6, G-CSF, and c-kit ligand, IL-11 has blast cell growth factor activity and can synergize with IL-3 and IL-4 to shorten the G o period of early hematopoietic progenitors. Although IL-11 alone will not support the growth of megakaryocyte colonies, it will synergize with IL-3 to increase the number, size and average ploidy value of megakaryocyte colonies formed from bone marrow cells.
  • the human IL-11 cDNA encodes a 199 amino acid precursor polypeptide with a 21 amino acid residue hydrophobic signal peptide that is processed proteolytically to generate the 178 amino acid residue mature form of IL-11.
  • CNTF acts by heterodimerisation of gp130 with LIF-R which is arbitrated by a complex of CNTF and a specific CNTF receptor subunit (CNTF-R: Davis et al., 1991, Science 253, 59-63; Ip et al., 1992, Cell 69, 1121-1132; Davis et al., 1993, Science 260, 1805-1808) which can also exist in a soluble form.
  • a non-signalling, specificity-determining receptor subunit interacts with two transmembrane transducing components which include at least one molecule of gp130.
  • LIF is most similar in sequence to the "LIF-R binding" cytokines OSM and CNTF (Bazan, 1991, Neuron 7, 197- 208), in particular human LIF exhibits about 20% amino acid identity with OSM (Rose and Bruce, 1991, Proc. Natl. Acad. Sci. USA 88, 8641-8645; Bruce et al., 1992, Prog. Growth Factor Res. 4, 157-170).
  • Cardiotrophin (CT) is a cytokine which also binds through the gp130 receptor system.
  • CT induces a hypertrophic (swelling) response in heart cells (Pennica et al, Proc. Natl. Acad. Sci. USA., 1995, 92, 1142-6).
  • ligand-mediated homodimerisation of gp130, or heterodimerisation of gp130 with another transmembrane receptor subunit is essential for signal transduction in this group of ligands (reviewed by Stahl and Yancopoulos, 1993, Cell 74, 587-590).
  • the 'gp130-dependent' set of cytokines should exhibit two functionally distinct sites of interaction; one which is involved in interaction with the ligand specific receptor subunit, and the second which interacts with the common gp130 transducer.
  • the participation of a third specificity conferring receptor subunit in signalling, via the formation of a trimolecular receptor complex, may involve a third site on the ligand.
  • murine LIF, human LIF and OSM exhibit closely related three dimensional structures. It has been established, however, that murine and human LIF exhibit distinct differences in biological specificity. Whilst human LIF is able to bind to murine LIF-R and exert a biological effect on murine cells (Smith et al., 1988, ibid; Moreau et al., 1988, ibid) murine LIF exhibits relatively low affinity for human LIF-R (Owczarek et al., 1993, ibid) and, as described below, exhibits significantly reduced potency in bioassays dependant upon human LIF-R and human gp130.
  • OSM has been shown to bind LIF-R and gp130 albeit with lower affinity than LIF itself (Gearing and Bruce, 1992, New Biol. 4, 61-65; Baumann et al 1993, Biol. Chem. 268, 8414-8417).
  • the 3-dimensional structure of LIF has been established for the first time. This has enabled us to identify those regions of LIF which are crucial to the interaction between LIF and its receptors, i.e. gp130 and LIF-R, and to examine the regions of LIF which may account for the differences between LIF and related molecules mentioned above.
  • Figure 1 A Representation of the three dimensional structure of human LIF determined by crystallisation studies. The positions of residues of particular interest, described in the specification, are indicated.
  • B Representation of LIF from opposite orientation to (A).
  • Figure 2 Sequence alignments for human and murine LIF (SEQ ID Nos. 1 & 2 respectively), human OSM (SEQ ID No. 3), human CNTF (SEQ ID No. 4) and human cardiotrophin (hCT, SEQ ID No. 5) based on structural considerations. conserveed residues are highlighted in dark shading and conservative substitutions are shown in light shading. Large open boxes represent the positions of the helices in murine LIF, and the residue numbers refer to the murine LIF sequence. The graph below the alignment indicates the solvent accessibility of each murine LIF residue in gradations of 20, 40, 60, 80 ⁇ 2
  • Figure 3 A) Composition of human/mouse LIF chimeras. For amino acid numbering refer to figure 2.
  • Figure 4 Antagonism of hLIF by a site 2 variant of hLIF.
  • Figure 5 Antagonism of OSM by a site 2 variant of hLIF
  • Site 3 is from residues 150 to 160
  • site 2 comprises residues 25-38 and 120-128
  • site 1 comprises residues 161 to 180.
  • the numbering herein refers to that contained in the human sequence set out above. It has now been found that sites 1 and 3 are responsible for the interaction of LIF to LIF-R, whereas site 2 is responsible for the interaction of LIF to g ⁇ 130. This has enabled the provision of variants of LIF which have either a higher or lower affinity for the components of the LIF receptor than natural LIF. Such variants can be used as LIF agonists or antagonists depending upon their affinity.
  • sites 1 and 3 provide agonists of LIF which enhanced binding to the receptor.
  • site 2 provide LIF antagonists.
  • the present invention thus provides a protein which comprises the sequence of human Leukemia Inhibitory Factor (hLIF (SEQ ID No. 1)) which has been altered by substitution at, insertion into, or deletion within a portion of said sequence such that the affinity for the LIF receptor is lowered compared to LIF, or a fragment of said protein which contains said alteration.
  • hLIF human Leukemia Inhibitory Factor
  • the protein or fragment thereof has a binding affinity for gp130 which is substantially the same as hLIF.
  • Such proteins desirably contain alterations from the hLIF sequence at or within one or more of the residues of site 1 from amino acids 161 to 180 and/or of the residues of site 3 from 150 to 160.
  • the affinity for such proteins ("site 1 or 3 mutants") for LIF is lowered at least about 2, preferably 10 fold compared to LIF.
  • such proteins thereof will have an EC50 protein / EC50 hLIF binding ratio (as measured in the examples) of at least 40, and preferably greater than 100.
  • Such proteins thereof will also preferably have a EC50 protein / EC50 gp130 binding ratio of between about from 0.01 to 10, e.g. from 0.1 to 5.0 or from 0.5 to 5.0.
  • the invention further provides a protein which comprises the sequence of human Leukemia Inhibitory Factor (hLIF (SEQ ID No. 1)) which has been altered by substitution at, insertion into, or deletion within a portion of said sequence such that the affinity for the gp 130 receptor is lowered compared to LIF, or a fragment of said protein which contains said alteration.
  • hLIF human Leukemia Inhibitory Factor
  • a protein or fragment thereof has a binding affinity for LIF receptor which is substantially the same as hLIF.
  • Such proteins desirably contain alterations from the hLIF sequence at or within one or more of the residues of site 2 from amino acids 25 to 38 and/or from 120 to 128.
  • the affinity for such proteins ("site 2 mutants") for gp130 is lowered at least about 2, preferably 10 fold compared to LIF.
  • such proteins thereof will have an EC50 protein / EC50 gp130 binding ratio (as measured in the examples) of at least 10, and preferably greater than 10, e.g greater than 100.
  • Such proteins thereof will also preferably have a EC50 protein / EC50 hLIF binding ratio of between about from 0.01 to 10, e.g. from 0.1 to 5.0 or from 0.5 to 5.0.
  • Preferred fragments of proteins of the invention will have the ability to bind to the LIF receptor or to gp130 in competition with LIF, or related proteins including murine LIF, oncostatin, ciliary neurotrophic factor or cardiotrophin. This can be measured by assaying the ability of LIF to stimulate the growth of cells in culture in the presence and absence of such fragments.
  • fragments of LIF or its variants will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50, 60 or 100 amino acids in length.
  • the fragments may be made by synthetic methods such as synthesis on a solid phase or by expression of a recombinant DNA in an expression vector in a host cell, wherein the vector comprises DNA encoding the peptide operably linked to a promoter compatible with the host cell.
  • the vector may also contain transcription termination signals.
  • Proteins of the invention may also be made by recombinant DNA technology, as described below.
  • the present findings have enabled us to provide novel hybrid proteins which will require specific combinations of receptor molecules at the cell surface in order to initiate signalling via those receptors. This can provide novel proteins which can be targeted to a specific subset of cell types, in particular those which express a LIF receptor and a second receptor, for example and interleukin receptor.
  • Mammalian homologues or fragments thereof of LIF which have the alterations mentioned above in the corresponding residues of their sequences may also be used in the present invention.
  • Such homologues can be obtained by routine cloning procedures, e.g. by using the hLIF cDNA sequence as a probe to obtain another mammalian LIF from a cDNA library made from cells of the mammal which express LIF.
  • the human and mammalian LIF proteins may be altered using standard techniques of genetic engineering known per se (e.g. see Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989) and which are further illustrated in the examples below.
  • the invention further provides a recombinant protein or fragment thereof which comprises a site (as defined above) from LIF or a variant thereof.
  • the recombinant protein may comprise a protein or fragment thereof of the invention which further comprises, at the N- or C- terminus, all or part of the sequence of a cytokine including a cytokine selected from the group consisting of murine LIF (SEQ ID No. 2), Oncostatin (SEQ ID No. 3), ciliary neurotrophic factor (SEQ ID No. 4) and cardiotrophin (SEQ ID No. 5).
  • recombinant proteins are based upon site 1 or 3 mutants of LIF it is desirable that their affinity for LIF is lowered at least about 2, preferably 10 fold compared to LIF.
  • such proteins thereof will have an EC50 protein / EC50 hLIF binding ratio (as measured m the examples) of at least 40, and preferably greater than 100.
  • Such proteins thereof will also preferably have a EC50 protein / EC50 gp130 binding ratio of between about from 0.01 to 10, e.g. from 0.1 to 5.0 or from 0.5 to 5.0.
  • recombinant proteins based upon site 2 mutants preferably have an affinity for gp130 which is lowered at least about 2, preferably 10 fold compared to LIF.
  • such proteins thereof will have an EC50 protein / EC50 gp130 binding ratio (as measured in the examples) of at least 10, and preferably greater than 10, e.g greater than 100.
  • Such proteins thereof will also preferably have a EC50 protein / EC50 hLIF binding ratio of between about from 0.01 to 10, e.g. from 0.1 to 5.0 or from 0.5 to 5.0.
  • Such recombinant proteins based on LIF proteins of the invention may have the general structure:
  • X 1 is an N-terminal sequence of amino acids or hydrogen
  • A is amino acids 25-38 of LIF or a variant thereof
  • X 2 is a C-terminal sequence or a carboxy group; provided that the protein is not naturally occurring mammalian LIF.
  • X 1 is preferably the sequence of human or murine LIF from the N-terminal to the amino acid 24.
  • X 2 may comprise the sequence of human or murine LIF, or a chimera thereof, from amino acid 39 to the C-terminal of the human or murine LIF, or chimera thereof.
  • a recombinant protein is of the formula:
  • B is amino acids 120-128 of LIF or variant of this region thereof and Z is a sequence of amino acids linking A and B.
  • Z, X 1 and X 2 are preferably derived from the same protein, preferably another cytokine (e.g. OSM, IL-6, IL-11 or CNTF or cardiotrophin (CT)) which also interacts with gp130.
  • another cytokine e.g. OSM, IL-6, IL-11 or CNTF or cardiotrophin (CT)
  • CT cardiotrophin
  • the regions A and B are grafted into the other cytokine in place of the regions of that cytokine which correspond to site 2 of LIF when that cytokine is aligned with LIF.
  • OSM or CNTF this may be done by reference to Figure 2 of the examples.
  • residues 3-16 and/or 96-104 of mature human CNTF may be replaced by the residues 25-38 and/or 120-128 or variants thereof respectively of LIF.
  • residues 12-24 and/or 112-120 of mature human OSM may be replaced.
  • residues 29-42 and/or 127-135 of SEQ ID No. 5 of human cardiotrophin may be replaced.
  • the site 1 and/or site 3 regions of LIF or variants thereof may be grafted into the corresponding regions of other cytokines (including those mentioned above). This will provide further recombinant proteins which interact with the LIF receptor but will also require another receptor at the cell surface in order to mediate their effect. This enables the hybrid molecule to be targeted to a specific subset of cells which express a particular combination of receptors.
  • S1 is site 1 or a variant thereof
  • S3 is site 3 or a variant thereof.
  • residues 125-135 and/or 136-155 of mature human CNTF may be replaced by the residues 150-160 and/or 161-180 or variants thereof respectively of LIF.
  • residues 150-160 and/or 161-180 of mature human OSM (179-189 and/or 190-209 of SEQ ID No. 3) may be replaced.
  • the other cytokine is preferably IL-6, IL-11 OSM, CNTF or CT.
  • the cytokine may also be LIF of a different species, e.g. murine.
  • the fusion proteins may be obtained by expression of recombinant DNA. cDNA sequences of the cytokines are available and can be manipulated to be spliced in-frame to the relevant portion of LIF or a variant thereof using techniques known per se in the art.
  • PCR primers directed to the regions X 1 , SI, Z, S3 and X 2 may be made and these regions amplified separately.
  • the primers can be designed to overlap or to contain restriction sites which may be used to splice the fragments together. In this sense, the process is analogous to preparing DNA encoding recombinant antibodies in which murine CDRs are spliced into a human framework region.
  • Other variant and hybrid proteins according to the invention may be made in this manner. Reference may be made to Sambrook et al (Molecular Cloning: A Laboratory Manual, 1989) for details of such techniques.
  • LIF amino acid substitutions
  • deletions or insertions or 1, 2, 3, 4, 5 or more amino acids are also possible.
  • the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, or 10 substitutions.
  • site 3 T150, S151, K153, F156, K158 and K159.
  • Preferred substitutions are in particular, in site 2, changes which result in an opposite charge where the residue being substituted has a charge.
  • site 2 changes include D120 to K or R.
  • site 1 or 3 changes include K170 to R170; V175 and/or V177 to G, A, L or I; T150 to S; S151 to T; K153 to R; F156 to Y; and K158 to R.
  • any of the residues of sites 1, 2 or 3 may be changed to A.
  • the present invention also provides a nucleic acid, e.g. a DNA, encoding a protein or fragment thereof of the invention, and a vector comprising such nucleic acid.
  • the vector may be an expression vector, wherein said nucleic acid is operably linked to a promoter compatible with a host cell.
  • the invention thus also provides a host cell which contains an expression vector of the invention.
  • the host cell may be bacterial (e.g. E.coli), insect, yeast or mammalian (e.g. hamster or human).
  • Host cells of the invention may be used in a method of making a protein or fragment thereof of the invention which comprises culturing the host cell under conditions in which said protein or fragment thereof is expressed, and recovering the protein or fragment thereof in substantially isolated form.
  • the protein or fragment thereof may be expressed as a fusion protein.
  • the invention further provides pharmaceutical formulations.
  • Such formulations comprise a protein or fragment thereof of the invention together with a pharmaceutically acceptable carrier or diluent.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral or parenteral (e.g. intramuscular or intravenous) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs.
  • Proteins of the invention may be used to target specific groups of cells in the body, in order to either stimulate or inhibit growth.
  • Particular conditions which may be treated include neuronal disorders, including degenerative diseases of the nervous system such as Parkinson's Disease, conditions which require nerve regeneration following trauma, disorders of the blood system including leukemias, bone osteoporosis and weight loss.
  • the proteins may also be useful in assisting embryonic implantation in IVF procedures.
  • Treatment of a patient with a protein or fragment thereof of the invention will comprise administering to a patient in need of treatment an effective amount of the protein or fragment thereof (or composition containing the protein).
  • the proteins according to the invention may be administered to human patients or other mammals by any route appropriate to the condition to be treated, suitable routes including oral or parenteral (including intramuscular or intravenous), intradermal). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient.
  • the amount required of the protein or fragment thereof will depend upon a number of factors including the severity and nature of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician.
  • a suitable, effective dose will be in the range 0.1 to 100 ⁇ g per kilogram body weight of recipient per day, preferably in the range 1 to 10 ⁇ g per kilogram body weight per day.
  • the desired dose may if desired be presented as two, three, four or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms.
  • Proteins and fragments thereof of the invention may also be used in in vitro screening methods to identify antagonists or agonists of LIF. Accordingly, the present invention provides a method of screening candidate agonist or antagonist substances of LIF which comprises bringing a candidate substance into contact with a cell responsive to LIF in the presence of LIF;
  • candidate substances which show agonist or antagonist activity By using proteins or fragments thereof of the invention with the ability to bind either LIF-R or gp130 in the above manner, it is possible to select candidate substances which show true agonist or antagonist activity against LIF. This is because stimulation or inhibition of cell growth in the presence of LIF which is blocked or activated by the candidate substance can be compared to the activity of the candidate substance in the presence of a LIF protein variant or fragment thereof of the invention. This allows a distinction to be made between candidate substances which have a general effect on cells and those which interact with or through the LIF/gp130 receptor system.
  • Suitable candidate substances include peptides (e.g. of from 5 to 20 amino acids) based on part of the sequence of LIF or other cytokines, synthetic or naturally occurring pharmaceutical drugs or plant extracts.
  • proteins and fragments thereof of the invention may also be used in in vitro screening methods to identify or characterize new cytokines which also bind to the gp130 receptor and/or LIF receptor.
  • proteins or fragments thereof of the invention can be used to antagonise the binding of cytokines to the gp130 receptor, providing information about the binding profile and mode of action of said cytokine.
  • Screening programmes may also include the use of LIF variants of the invention to bind to soluble forms of the LIF receptor and/or gp 130 in solution.
  • the present invention also provides antibodies capable of binding to proteins and fragments thereof of the invention.
  • Such antibodies desirably bind to the protein or fragments thereof of the invention with an affinity which is at least 10 fold, e.g. 100 fold or 1000 fold higher than their affinity to human and/or murine LIF.
  • the antibodies will also have an affinity for that cytdleine which is at least 10 fold, e.g. 100 fold or 1000 fold lower than their affinity to the protein or fragment thereof of the invention.
  • the affinity of antibodies of the invention to a LIF and/or other cytokines may be determined by routine techniques known in the art per se.
  • An antibody of the invention may be monoclonal or polyclonal.
  • antibody includes fragments of whole antibodies which retain their binding activity for a protein or fragment thereof of the invention. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies.
  • monoclonal antibodies according to the invention may be analyzed (eg. by DNA sequence analysis of the genes expressing such antibodies) and humanized antibody with complementarity determining regions of an antibody according to the invention may be made, for example in accordance with the methods disclosed in EP-A-0239400.
  • Monoclonal antibodies may be prepared by conventional hybridoma technology using the proteins or peptide fragments thereof, as an immunogen or, in the case of modified antibodies or fragments, by recombinant DNA technology, eg by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter.
  • suitable host cells include bacterial (eg. E.coli), yeast, insect and mammalian.
  • Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a protein or fragment thereof of the invention and recovering immune serum.
  • the following examples provide an analysis of LIF function by "homolog-scanning" mutagenesis, and reveal two regions of the LIF molecule involved in receptor interaction and biological function. The first, located within the D-helix comprising residues 161-180, and the second, located between residues 150-160 at the C-terminus of the CD loop - two surface regions that are separated by the AB loop. There are significant differences between these findings and the study of Owczarek et al. (1993, ibid) who also analyzed a series of human/mouse LIF chimeras for interaction with human LIF-R in a similar COS cell transfection system.
  • Owczarek et al identified a secondary effect for binding to LIF-R within the region of LIF residues 103-130.
  • Murine LIF was expressed as a fusion protein with glutathione-S-transferase in E. coli strain JM109.
  • Murine LIF cDNA encoding the mature form of the polypeptide was cloned into the bacterial expression vector pGEX-2T, as described by Mereau et al., 1993 Cell Biol. 122, 713-719.
  • pGEX-2T bacterial expression vector
  • For large- scale protein inductions (30 L) cultures were grown in LB + ampicillin (100 mg/ml) at 37°C, 300 rpm until they reached mid-log phase (A 600 0.6-0.8). IPTG was then added to the culture to a final concentration of 0.1 mM. Cultures were incubated at 37°C for a further 3 hours.
  • Intracellular fusion protein was recovered from cell extracts by affinity binding to a slurry of glutathione sepharose (glutathione sepharose 4B; Pharmacia; 100 ml solution of 50%) in MTPBS (150 mM NaCl, 16 mM Na 2 HPO 4 , 4 mM NaH 2 PO 4 , pH 7.3) for 2 hours at 4°C. This was followed by washing once with 5 bead volumes of 0.5% octyl-b-glucopyranoside in MTPBS, then one wash each, with 50 mM Tris.HCl, pH 8.5, 150 mM NaCl and 50 mM Tris.HCl, pH 8.5, 150 mM NaCl, 2.5 mM CaCl 2 .
  • Isolation of recombinant LIF was achieved by cleavage of the fusion protein with human thrombin (T3010; Sigma) whilst attached to the matrix in 50 mM Tris.HCl, pH 8.5, 150 mM NaCl, 2.5 mM CaCl 2 .
  • Thrombin was added to a final enzyme:protein ratio of 1:100 and digestion was carried out at room temperature for 6 h. After thrombin digestion, the supernatant was separated and combined with five washes of the gel matrix. The supernatant from this reaction was dialysed against two changes of 20 mM MES, pH 6.0, 12 h at 4°C.
  • Cleaved protein was further purified on a Mono S cationic exchange column. Elution was carried out with a linear gradient of 0-1 M NaCl in 20 mM MES, pH 6.0. Positive fractions from a single peak were then pooled and concentrated by ultrafiltration (Amicon membrane; molecular weight cut-off of 3,000 Da) to 10 mg/ml for use in crystallization trials. Time of flight mass spectrometry was performed on purified LIF after HPLC purification, using a Finnigan Lasermat (matrix assisted laser desorption, nitrogen laser at 337 nm). LIF samples ( ⁇ 50 pmol) were analyzed using a sinapinic acid (11 mg/ml) matrix at a sample to matrix molar ratio of 1:5000.
  • LIF is a compact molecule with overall dimensions of approximately 22 ü x 28 ⁇ x 46 ⁇ . As had been predicted (Bazan, 1991, ibid), the LIF structure conforms to the up-up- down-down four helix bundle topology common to the hematopoietic growth factors ( Figure 1).
  • the structure thus comprises 4 main a-helices conventionally labelled A, B, C and D, linked by two long loops (AB and CD) and one short loop BC.
  • This topological motif may be considered in terms of two pairs of antiparallel a-helices B:C and A:D.
  • the B and C helices (29 and 27 residues respectively), are relatively straight and pack in a classic antiparallel manner, tilted to cross approximately half way down their length.
  • These kinks require breaks in the normal a-helix hydrogen bonding pattern, substitute hydrogen bonds are made to the polar sidechains of serines (Ser-36 in helix A and Ser-174 in helix D) and tightly bound water molecules (Figure 1).
  • the compact core is predominantly composed of hydrophobic residues contributed by the four a-helices.
  • the N-terminal region is wrapped around the molecule; the long loops AB (the first part of which contains a fifth short a-helix A' ) and CD are similarly tightly packed against the four helix bundle. Thus these three regions also contribute to the molecular core.
  • the N-terminal region is pinned to the four helix bundle at the bottom of helix C by two disulphide bridges (Cys-12 to Cys- 134 and Cys-18 to Cys-131).
  • the third disulphide bridge (Cys-60 to Cys-163) tethers the first part of the AB loop to the top of helix D.
  • the surface of murine LIF is relatively characteristic of that expected for a small globular protein and shows no pronounced clustering of positive or negative charge.
  • Two proline residues are in the cis conformation; Pro-17 before a disulphide bridge in the N-terminal region and Pro-51 at the start of the AB loop.
  • the helical cytokine structures have been classified (Boulay and Paul, 1993, Curr. Biol. 3, 573-581; Sprang and Bazan, 1993, ibid) in terms of two subgroups; short-chain (Sc) and long-chain (Lc) cytokines.
  • the major variation in the four helices of LIF compared to GH and GCSF occurs in helix A which exhibits a distinctive kink in LIF which is thus far unique to this helical cytokine.
  • the first part of the AB loop in all three structures contains a short helical region and the C-terminal half of this loop superimposes well between LIF and GCSF, however, this loop is markedly shorter in LIF and this is manifested in the acute angle at which the first half of the loop crosses in front of the D helix.
  • the AB loop in LIF overlays the surface of the D helix at a point approximately one third of the way down its length rather than at its N-terminus as in GH.
  • the conformation of the CD loop is similar in all three molecules, but in both GCSF and GH this long loop contains highly flexible regions.
  • the CD loop in LIF has a well defined single mainchain conformation throughout its length, this rigidity appears to be inherent to the molecule since this region is not involved in lattice contacts within the crystal.
  • the N-terminal region in LIF prior to helix A, follows a unique path wrapping around the base of the four helix bundle.
  • the major distinctive features of the LIF structure within the Lc cytokine family are the N-terminal region, the kink in helix A and the position of the AB loop on crossing helix D.
  • Figure 2 includes a sequence alignment between human and murine LIF.
  • the positions of the a-helices and the degree of residue solvent accessibility are indicated based on the murine LIF structure.
  • the two sequences differ for 39 residues with no insertions or deletions. Of these differences none seem likely to perturb the structure greatly.
  • the majority of the changes are at solvent exposed residues which are distributed evenly over the surface of the molecule and cannot, taken in isolation, indicate the structural basis of the species specificity which is observed for binding to human LIF-R (Owczarek, et al., 1993, ibid and below).
  • LIF From sequence alignment LIF was predicted to belong to the helical cytokine family and assigned to a subgroup which also comprises OSM and CNTF (Bazan, 1991), molecules that have subsequently been shown to bind LIF-R and hence are referred to here as the "LIF-R binding" subgroup.
  • the LIF structure is the first to be determined for a member of this subgroup.
  • the alignment does show strong conservation of key structural residues along the lengths of all four helices, most notably in helix D, which supports the assumption of structural equivalence for residues in these regions.
  • gross comparisons may also be made with respect to the rest of the LIF structure.
  • the N-terminal region is truncated and the C-terminal region is extended for both OSM and CNTF.
  • the AB loop in LIF and OSM is tethered by a disulphide bridge to the equivalent point on helix D.
  • the BC loop is lengthened in OSM and, to a lesser extent, in CNTF.
  • Example 3 Expression vectors for human and chimeric LIF Human LIF was cloned into the pGEX-2T expression plasmid in an identical manner to murine LIF as set out in Example 1.
  • Chimeric (human/murine) LIF proteins were constructed by taking advantage of a unique Sma I site located at analogous positions in both species of cDNA (human and murine) and unique Sma I and Eco RI sites located in the parental pGEX-2T vector at the 3' end of the LIF inserts.
  • HM and MH chimeras were constructed with Sma I restriction fragments and the chimeras 161-180 and 150-180 were constructed with Sma I/Eco RI fragments assembled by SOE (splicing by overlap extension; Higiuchi et al., 1988, Nucleic Acids Res. 16, 7351-7367). Subcloning resulted in the production of H-MLIF and M-HLIF plasmids pGEX-2T expression vector plasmids. The fragment and junctions were sequenced by the dideoxy chain termination method using Sequenase (US Biochemical) and Circumvent kits (NE Biolabs).
  • HLIF and species chimeric LIF proteins were produced under similar conditions to those for murine LIF, with the exceptions that protein inductions were carried out at 22°C instead of 37°C, and a reverse phase step replaced the final Mono S column.
  • Recombinant human OSM was purchased from Preprotech Inc.
  • Example 4 Site Directed H-M LIF proteins
  • the LIF mutants and chimeras of the Examples 3 and 4 were tested for their biological activity in two bioassays.
  • the first was a murine bioassay based on the LIF dependant growth of the murine Da- la cell line (Moreau et al., 1988, ibid; Godard et al., 1988, Blood 6, 1618-1623) and the second, a human bioassay, based upon the ability of experimental molecules to support the multiplication of murine Ba/F3 cells co-transfected with human LIF-R and human gp130.
  • Da-1a cells were maintained in RPMI 1640 (ICN Flow Laboratories) supplemented with glutamine (2 mM), penicillin (50 IU/ml), streptomycin (50 mg/ml), 10% FCS (selected batches) and 10 ng/ml recombinant mouse LIF.
  • Ba/F3 [gp130 + LIF-R] cells were maintained in RPMI 1640 medium supplemented with 8% FCS, glutamine (2 mM), penicillin (50 IU/ml), streptomycin (50 mg/ml) and recombinant human LIF (25 ng/ml).
  • WEHI-3 conditioned medium was added at 10% to the RPMI culture medium and used as a source of the murine IL3 (Lee et al., 1982, Immunol. 128, 2393-2398).
  • the pKCSRa eukaryotic expression vector containing the cDNA encoding the human LIF-R was then electroporated into the Ba/F3 [gp130] cells and positive clones were selected in culture medium supplemented with 240 IU/ml of human LIF the source of which was obtained from a transfectant CHO cell line expressing the human LIF protein (Moreau et al., 1988, ibid).
  • the Ba/F3 [LIF-R + gp130] LIF- dependent cell line was isolated.
  • Biological assays were performed using the recombinant factors as previously described (Moreau et al., 1988, ibid; Godard et al., 1988, ibid). In brief, cells were washed 3 times with a large volume of LIF-free medium before being cultured at a density of 10 5 cells/ml in RPMI medium supplemented with glutamine, penicillin, streptomycin and 10% FCS, in the presence of twofold successive dilutions of the factors to be tested. Assays were performed in triplicate.
  • chimeras Two chimeras were tested which comprised either the N-terminal 'half of murine LIF (residues 0-98) and the C-terminus of human LIF (residues 99-180), or the reciprocal substitution of the N-terminal 'half' of human LIF and the C-terminal region of murine LIF (HM-LIF).
  • MH-LIF was similar to human LIF in activity whereas HM-LIF was equivalent to murine LIF in activity.
  • HM-LIF was then subjected to further mutations in which increasing regions of murine C-terminal sequence were substituted for human sequence.
  • a series of point substitotions were made at individual amino acids which differed between human and murine LIF in sequence.
  • the strategy behind this approach was to detect regions of murine LIF sequence in the HM chimera which could be 'activated', by substitotion mutagenesis, into functionality in the human bioassay thereby reconstructing regions of the molecule required for activity mediated by human LIF-R and gp130.
  • HM-LIF was chosen as a template molecule for substitotion mutagenesis to permit the identification of sequences in the region 99-180 whose species-specific activity depended upon contributions from residues 1-98.
  • Human LIF-R was transiently expressed in COS-7 cells by electroporating aliquots of 0.8 X 10 7 cells at 330 mV and 500 ⁇ F in the presence of 30 ⁇ g of human LIF-R cDNA subcloned into the expression vector PXMT2. The surviving cells were plated at a density of 10 5 cells. Seventy two hours later the cells were used in binding assays conducted as described previously (Mereau et al., 1993, ibid) with two modifications.
  • the crystal structure of the GH ligand/receptor complex provides a paradigm for receptor binding by the helical cytokines.
  • the higher affinity site I involves the AB loop and the C-terminal half of helix D; site II is formed by residues from helix A and helix C.
  • the work of Cunningham and Wells, (1993, ibid) indicates that a small number of key residues within the receptor binding sites are critical in stabilizing the ligand/receptor complex.
  • Both sites I and II in GH are binding sites for signal-transducing receptor components; the resultant subunit homodimerization initiates signal transduction.
  • the mutagenesis data implicate residues in the region 161-180 of murine LIF in receptor interaction. This region would correspond in location to Site I of the GH model.
  • the difference in activity between mutants in which the N terminal half is derived from either mouse (Owczarek et al., 1993, ibid) or human sequences also indicates that the activity of this site either includes, or is influenced by, additional sequences in the region 1-98. This could involve the region of the AB loop which crosses the D helix of LIF or regions of helix A which are able to interact with helix D by virtue of physical proximity. This assignment of site I therefore resembles the GH prototype. Homolog-scanning, however, has not revealed the binding site for gp130.
  • a number of distinctive surface features of the LIF structure such as the kink in helix A and Ser-36 (conserved Ser or Asp) and Ala-117 (conserved apolar) on helix C, cluster within site II of the GH receptor binding model.
  • site II may be tentatively assigned as a gp130 binding site in the LIF sub-family of helical cytokines, which, in the absence of any species difference in activity in human/mouse LIF chimeras may be comprised of functionally conserved residues.
  • this classical two site model fails to account for all of the data obtained in the above examples.
  • LIF mutants were made using the pGeX-hLif expression vector system described in Example 1. Mutant DNA sequences were created by PCR overlap (Ho et al., 1989) using pGeX-hLif and oligonucleotides containing an appropriate coding change for each individual mutant. The LIF mutants were expressed as glutathione-S-transferase fusion proteins in E. coli JM109 as described in Example 1.
  • Oncostatin-M amino acids 1-196 was a kind gift from Dr. D Staunton, was also expressed as a glutathione-S-transferase fusion protein and purified as above.
  • the LIF mutants were tested for their ability to bind to LIF-R and to gp130, and for their ability to stimulate the Ba/F3 cell line, which expresses both hLIF-R and hGP130, as described in Example 5.
  • expression plasmids were made. PCR was used to amplify the region coding for amino acids 2-538 of the human LIF-R. This fragment was cloned into the pIG plasmid (Simmons, 1992, Cloning Cell Surface Molecules by Transient Expression in Mammalian Cells in Cellular Interaction in Development.
  • Both the human LIF-R and the gp130 receptor were expressed as fusion proteins with the Fc region (hinge-CH2-CH3) of human IgG-1.
  • the pIG expression plasmids were transfected by the calcium- phosphate technique into a human epithelial kidney cell line 293T, which expresses the large T antigen of SV40 (Dubridge et al., 1987, Molecular and Cellular Biology 7, 379-387). After transfection of 293T cells the media was changed to a serum free media (Ultra-Cho, Biowhittaker) and the Fc-fiision proteins were left to accumulate in the media for six days.
  • Receptor-Fc proteins were purified from clarified supernatants by chromatography on protein-A sepharose (Pharmacia). Elution of the receptor was achieved with 0.1 M citric acid, pH 3.0 and subsequent neutralisation with Tris base. Purity of the receptor-Fc proteins was assessed as >90%.
  • Iodinated LIF had a specific activity of 1-4 x 10 3 cpm/fmol and was equally active in the bioassay as wild type hLIF.
  • Recombinant Oncostatin-M was biotinylated with Biotinamidocaproate N-hydroxysuccinimide ester (Sigma) following the procedure of (Harlow and Lane, 1988, Antibodies: A laboratory manual. New York, Cold Spring Harbor Laboratory).
  • Binding stodies to gp130 was performed in a similar manner to that for LIF-R. These assays differed in that for gp130 competition binding, biotinylated Oncostatin-M was used instead of 125 I-hLIF and the bound Oncotatin-M was detected by incubation with a streptavidin-horseradish peroxidase conjugate (Amersham). Specifically, after washing plates with PBS, 0.05% Tween 20 the wells were rinsed with PBS and then incubated with 100 ml of streptavidin-horseradish peroxidase (1/1000 dilution) in PBS-1 % BSA.
  • Example 7c antagonism of OSM and hLIF.
  • the 04 mutant was assayed for its ability to antagonise the action of OSM and hLIF in the Baf- LIFR/gp130 system described above.
  • Antagonism of hLIF is shown in Figure 4, and of OSM in Figure 5.

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Abstract

L'invention concerne des protéines qui sont des variantes du facteur inhibiteur de la leucémie chez l'homme (hLIF) et qui présentent des affinités modifiées de fixation au récepteur de LIF, ainsi qu'au récepteur de gp130. Ces protéines comprennent hLIF qui a été modifié par substitution au niveau d'une partie de ladite séquence, ou par insertion dans ladite partie, ou par suppression à l'intérieur de ladite partie, de telle manière que l'affinité pour le récepteur de LIF est diminuée par rapport à LIF ou à un fragment de ladite protéine contenant ladite modification. Ces protéines comprennent également hLIF qui a été modifié par substitution au niveau d'une partie de ladite séquence, ou par insertion dans ladite partie, ou par suppression à l'intérieur de ladite partie, de telle manière que l'affinité pour le récepteur de gp130 est diminuée par rapport à LIF ou à un fragment de ladite protéine contenant ladite modification.
PCT/GB1995/001528 1994-07-01 1995-06-30 Variantes du facteur inhibiteur de la leucemie WO1996001319A1 (fr)

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WO1998001149A3 (fr) * 1996-07-10 1998-04-23 Angeletti P Ist Richerche Bio VARIANTS DE FACTEUR NEUROTROPHIQUE CILIAIRE HUMAIN (hCNTF) AYANT UN CHAMP D'ACTION DIFFERENT DE CELUI DE LA MOLECULE DE TYPE SAUVAGE
WO2001064239A1 (fr) * 2000-03-03 2001-09-07 The Walter And Eliza Hall Institute Of Medical Research Methode de traitement
WO2005030803A1 (fr) * 2003-09-29 2005-04-07 The Walter And Eliza Hall Institute Of Medical Research Molecules therapeutiques
US7504096B1 (en) 1998-07-06 2009-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for in vitro fertilization
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
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

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WO1994018236A1 (fr) * 1993-02-03 1994-08-18 Amrad Corporation Limited Determinant de fixation du recepteur tire du facteur d'inhibition de leucemie

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M. J. LAYTON ET AL: "Cross-species receptor binding characteristics of human and mouse Leukemia inhibitory factor suggest a complex binding reaction", JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 269, no. 25, 24 June 1994 (1994-06-24), MD US, pages 17048 - 17055 *
M. VAN DAM ET AL: "Structure-function analysis of interleukin-6 utilizing Human/murine chimeric molecules", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 20, 15 July 1993 (1993-07-15), MD US, pages 15285 - 15290 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001149A3 (fr) * 1996-07-10 1998-04-23 Angeletti P Ist Richerche Bio VARIANTS DE FACTEUR NEUROTROPHIQUE CILIAIRE HUMAIN (hCNTF) AYANT UN CHAMP D'ACTION DIFFERENT DE CELUI DE LA MOLECULE DE TYPE SAUVAGE
US6756357B1 (en) 1996-07-10 2004-06-29 Istituto Di Richerche Di Biologia Molecolare Di Angeletti S.P.A. Variants of human ciliary neurotrophic factor (hCNTF)
US7504096B1 (en) 1998-07-06 2009-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for in vitro fertilization
WO2001064239A1 (fr) * 2000-03-03 2001-09-07 The Walter And Eliza Hall Institute Of Medical Research Methode de traitement
WO2005030803A1 (fr) * 2003-09-29 2005-04-07 The Walter And Eliza Hall Institute Of Medical Research Molecules therapeutiques
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
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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