WO2007047829A2 - Nouvelles protéines hétérodimères et utilisations de celles-ci - Google Patents

Nouvelles protéines hétérodimères et utilisations de celles-ci Download PDF

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WO2007047829A2
WO2007047829A2 PCT/US2006/040860 US2006040860W WO2007047829A2 WO 2007047829 A2 WO2007047829 A2 WO 2007047829A2 US 2006040860 W US2006040860 W US 2006040860W WO 2007047829 A2 WO2007047829 A2 WO 2007047829A2
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fragment
subunit
amino acid
acid sequence
hybrid protein
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WO2007047829A3 (fr
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Sean D. Mckenna
Robert K. Campbell
Xuliang Jiang
Giampiero De Luca
Meijia Yang
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Laboratoires Serono S.A.
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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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]
    • C07K14/5412—IL-6
    • 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
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • 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
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • Cytokines are small secreted proteins which mediate and regulate immunity, inflammation, growth and hematopoeisis, among other cellular and developmental processes. Generally, these molecules bind to specific membrane receptors, which then signal the cell via second messengers to alter cell activation, for example, by altering gene expression. Responses to cytokines include, but are not limited to, increasing or decreasing expression of membrane proteins, proliferation and secretion of effector molecules.
  • Cytokines may either act on cells that secrete them (autocrine action), or on cells that are nearby (paracrine action), or in some instances on distant cells (endocrine action).
  • Cytokines can be redundant in their activities. For example, the same cellular activities can be stimulated by different cytokines. Cytokines can also act either synergistically or antagonistically. [0004] Various cytokines have been implicated in different disease states. For example, it has been reported that interleukin-6 (IL-6) promotes the proliferation of advanced stage melanoma cells in vitro. In addition, high IL-6 levels have been associated with transverse myelitis (TM), a disease related to multiple sclerosis.
  • IL-6 interleukin-6
  • TM transverse myelitis
  • VEGF vascular endothelial growth factor
  • IFN- ⁇ interferon-alpha
  • EDDM insulin-dependent diabetes mellitus
  • TNF- ⁇ is known to be a major cause for the symptoms of septic shock.
  • TNF- ⁇ has been reported to play a role in congestive heart failure, rheumatoid arthritis, cachexia syndrome, Crohn's disease, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, and psoriasis.
  • increased expression of the human growth hormone (hGH) has been associated with acromegaly and gigantism.
  • This invention is based, at least in part, on the discovery that non- immunoglobulin proteins such as heterodimeric proteinaceous hormones, can be used for making cytokine antagonists.
  • the subject cytokine antagonists employ the ⁇ and ⁇ chains of a heterodimeric hormone or a portion thereof as a scaffold to which a cytokine or a receptor binding portion thereof is linked.
  • An example of a heterodimeric proteinaceous hormone is the human chorionic gonadotropin (hCG) hormone, a stable secreted protein with a long half-life.
  • hCG human chorionic gonadotropin
  • Examples of hybrid proteins employing hCG can be found in U.S. Patent No. 6,194,177, to Campbell et ah, the entire content of which is incorporated by reference herein.
  • this invention relates to hybrid proteins including at least two polypeptide chains, where each polypeptide chain includes at least one cytokine or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone, or a fragment thereof.
  • the hybrid proteins of the invention have an antagonist activity, for example, an activity that is opposite to the activity of the cytokine, and are useful in the treatment of the various diseases where a particular antagonist activity is desirable.
  • the invention comprises a hybrid protein having antagonist activity, comprising at least two polypeptide chains, with each polypeptide ⁇ ( chain comprising a cytokine or a fragment thereof, which retains the ability to bind its 1 receptor, linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, where the subunit or fragment thereof is capable of forming a heterodimer with ⁇ another subunit of the heterodimeric hormone.
  • a hybrid protein comprises two polypeptide chains, with one chain comprising interleukin-6 (IL-6) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising IL-6 or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone, wherein the hybrid protein has IL-6 antagonist activity.
  • IL-6 interleukin-6
  • the hybrid protein comprises two polypeptide chains, with one chain comprising vascular endothelial growth factor (VEGF) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising VEGF or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone, where the hybrid protein has VEGF antagonist activity.
  • VEGF vascular endothelial growth factor
  • the hybrid protein comprises two polypeptide chains, with one chain comprising interferon- ⁇ (IFN- ⁇ ) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising IFN- ⁇ or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone, wherein the hybrid protein has IFN- ⁇ antagonist activity.
  • a hybrid protein comprises two polypeptide chains, with one chain comprising one or more subunits (i.e., alpha, beta and/or gamma) of tumor necrosis factor (TNF) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a
  • TNF tumor necrosis factor
  • the hybrid protein includes two polypeptide chains, i with one chain comprising human growth hormone (hGH) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising hGH or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone, wherein the hybrid protein has hGH antagonist activity.
  • hGH human growth hormone
  • the hybrid protein includes two polypeptide chains, i with one chain comprising human growth hormone (hGH) or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising hGH or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone, wherein the hybrid protein has hGH antagonist activity.
  • the hybrid protein comprises one polypeptide chain comprising a first cytokine or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and a second polypeptide chain comprising a second cytokine or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone or a fragment thereof.
  • the first cytokine is chosen from the group comprising but not limited to IL-6, VEGF, IFN- ⁇ , TNF and hGH
  • a second cytokine is chosen from the group comprising, but not limited to IL-6, VEGF, IFN- ⁇ , TNF and hGH, wherein the first and the second cytokines are different cytokines.
  • the hybrid protein comprises more than one cytokine or a receptor binding fragment thereof on the same polypeptide chain, wherein the cytokine is chosen from the group comprising, but not limited to IL-6, VEGF, IFN- ⁇ , TNF- ⁇ and hGH.
  • hybrid proteins having antagonist activity described herein comprise at least two polypeptide chains, with each chain comprising: (a) an amino acid sequence of a cytokine or a fragment thereof; and (b) an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof ⁇ wherein the cytokine or a fragment thereof has receptor binding activity and wherein the subunit of the heterodimeric proteinaceous hormone or a fragment thereof is capable of dimerizing with another subunit of the hormone or a fragment thereof.
  • hybrid proteins comprise the amino acid sequence of a cytokine or a fragment thereof and the amino" acid sequence of a subunit of the heterodimeric proteinaceous hormone joined by a linker peptide.
  • cytokines for use in this invention include but are not limited to IL-6, VEGF, IFN- ⁇ , TNF- ⁇ and hGH.
  • proteinaceous heterodimeric hormones for use in this invention include but are not limited to FSH, inhibin, TSH, hCG, and LH.
  • At least one subunit of the heterodimeric proteinaceous hormone includes one or more alterations to reduce or eliminate the biological activity of the hormone, while preserving its ability to form heterodimers.
  • the hybrid proteins disclosed herein can have the amino acid sequence of a cytokine or a fragment thereof linked either to the amino-terminus or the carboxy- terminus of the amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof.
  • multiple amino acid sequences of one or more cytokines can be linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, thereby resulting in a hybrid protein with an antagonist activity toward one or more cytokines.
  • an isolated nucleic acid molecule encoding a fusion protein comprises (a) a first nucleotide sequence encoding a cytokine or a fragment thereof, wherein the cytokine or fragment thereof has receptor-binding activity, and (b) a second nucleotide sequence encoding a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, wherein the subunit or fragment thereof is capable of forming a heterodimer with another subunit of the heterodimeric hormone and wherein the nucleic acid molecule is a DNA molecule.
  • a fusion protein comprises (a) a first amino acid sequence of a cytokine or a fragment thereof, wherein the cytokine or fragment has receptor- binding activity; and (b) a second amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, wherein the subunit or fragment thereof is capable of forming a heterodimer with another subunit of the hormone.
  • Exemplary cytokines for use in the invention include but are not limited to IL-6, VEGF, IFN- ⁇ , TNF- ⁇ and hGH and exemplary heterodimeric proteinaceous hormones for use in the invention include but are not limited to hCG, FSH, LH, TSH and inhibin.
  • Fusion proteins of the invention include, but are not limited to, fusion proteins comprising: (a) IL-6 or a receptor binding fragment thereof that has a biological activity of IL-6, linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; (b) VEGF or a receptor binding fragment thereof that has a biological activity of VEGF, linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; (c) IFN- ⁇ or a receptor binding fragment thereof that has a biological activity of IFN- ⁇ , linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; (d) TNF or a receptor binding fragment thereof that has a biological activity of TNF, linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; and (e) hGH or a receptor binding fragment thereof that has a biological activity of hGH, linked to a subunit of a heterodimeric proteinaceous hormone or
  • the fusion proteins of the invention include a linker peptide located between the first and the second amino acid sequences.
  • the linker peptide can be enzymatically cleavable, for example, to include a thrombin cleavage site. Fusion proteins can also be secreted.
  • hybrid proteins having antagonist activity comprise: (a) a first fusion protein comprising an amino acid sequence of a cytokine or a fragment thereof linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; and (b) a second fusion protein comprising an amino acid sequence of a cytokine or a fragment thereof linked to an amino acid sequence of another subunit of the heterodimeric proteinaceous hormone or a fragment thereof, wherein the cytokine or fragment thereof has receptor-binding activity and wherein the subunits of the heterodimeric proteinaceous hormone are capable of dimerizing with each other.
  • cytokines for use in the invention include, but are not limited to, IL- 6, VEGF, hGH, BFN- ⁇ and TNF, and examples of heterodimeric proteinaceous hormones for use in the invention include, but are not limited to LH, FSH, TSH, inhibin andhCG.
  • the amino acid sequence, of a cytokine or a fragment thereof is linked to a subunit of a heterodimeric proteinaceous hormone via a peptide linker.
  • the peptide linked may be enzymatically cleavable or degradable.
  • the invention further comprises expression vectors which comprise nucleic acid molecules which encode the fusion proteins or hybrid proteins described herein.
  • the expression vector comprises a nucleic acid molecule encoding a fusion protein wherein the fusion protein comprises an amino acid sequence of a cytokine or a fragment thereof linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof.
  • the expression vector comprises one or more nucleic acid molecules which encode two fusion proteins, with each fusion protein comprising an amino acid sequence of a cytokine or a fragment thereof linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and wherein the two fusion proteins are capable of forming a hybrid protein.
  • the invention further comprises host cells comprising one or more expression vectors described herein.
  • Host cells can either be co-transfected with two expression vectors, each comprising a nucleotide sequence encoding a polypeptide chain or a fusion protein which form a hybrid protein, or host cells can be transfected sequentially with the two expression vectors. Alternatively, host cells can be transfected with one expression vector which comprises a nucleotide sequence encoding two fusion proteins or polypeptide chains forming a hybrid protein. [0025] The invention further comprises methods of treating disorders where antagonism of a cytokine activity is desirable.
  • the methods of the invention include for example, disorders which can be treated by antagonism of one or more cytokines including, but not limited to, hGH, EL-6, VEGF, IFN- ⁇ and TNF.
  • the invention comprises a method of treating melanoma comprising administering to a subject in need thereof, a hybrid protein comprising two polypeptide chains, with each polypeptide chain comprising an amino acid sequence set forth in SEQ TD NO:1 or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has IL-6 antagonist activity.
  • the hybrid protein of the invention can be used in a method for treating melanoma wherein the hybrid protein comprises two polypeptide chains, with one chain comprising an amino acid sequence of IL-6 linked to the alpha subunit of hCG (SEQ ID NO:2), and the second chain comprising an amino acid sequence of IL-6 linked to the beta subunit of hCG (SEQ ID NO:3), wherein the hybrid protein has IL-6 antagonist activity.
  • a method of treating cancer comprises administering to a subject in need thereof, a hybrid protein of the invention, said hybrid protein of the invention comprising two polypeptide chains, with each polypeptide chain comprising an amino acid sequence set forth in SEQ ID NO:4 or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has VEGF antagonist activity
  • a hybrid protein of the invention used in a method for treating cancer comprises two polypeptide chains, with one chain comprising an amino acid sequence of VEGF linked to the alpha subunit of hCG (SEQ E) NO:5), and the second chain comprising an amino acid sequence of VEGF linked to the beta subunit of hCG (SEQ ID NO:6), wherein the hybrid protein has VEGF antagonist activity.
  • methods of the invention wherein IFN-alpha antagonism is desirable comprise administration of a hybrid protein of the invention comprising two polypeptide chains, with each polypeptide chain comprising an amino acid sequence set forth in SEQ ID NO:7 or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has IFN-alpha antagonist activity.
  • IFN- alpha antagonism disorders where IFN- alpha antagonism is desirable include but are not limited to lupus, rheumatoid arthritis and insulin-dependent diabetes mellitus and can be treated by administration of a hybrid protein comprising two polypeptide chains, with one chain comprising the amino acid sequence of IFN-alpha linked to the alpha subunit of hCG (SEQ ID NO:8), and the second chain comprising an amino acid sequence of IFN-alpha linked to the beta subunit of hCG (SEQ ID NO:9), wherein the hybrid protein has IFN-alpha antagonist activity.
  • a hybrid protein comprising two polypeptide chains, with one chain comprising the amino acid sequence of IFN-alpha linked to the alpha subunit of hCG (SEQ ID NO:8), and the second chain comprising an amino acid sequence of IFN-alpha linked to the beta subunit of hCG (SEQ ID NO:9), wherein the hybrid protein has IFN-alpha antagonist activity.
  • methods of the invention wherein hGH antagonism is desirable comprise administration of a hybrid protein of the invention comprising two polypeptide chains, with each polypeptide chain comprising an amino acid sequence set forth in SEQ ID NO: 10 or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has hGH antagonist activity.
  • ThaGH antagonism is desirable include but are not limited to acromegaly or gigantism and can be treated by administration of a hybrid protein comprising two polypeptide chains, with one chain comprising the amino acid sequence of hGH linked to the alpha subunit of hCG (SEQ ID NO:11), and the second chain comprising an amino acid sequence of hGH linked to the beta subunit of hCG (SEQ ID NO: 12), wherein the hybrid protein has hGH antagonist activity.
  • TNF antagonism is .
  • a hybrid protein of the invention comprising two polypeptide chains, with each polypeptide chain comprising an amino acid sequence set forth in SEQ ID NO: 13 or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has TNF antagonist activity.
  • TNF ⁇ antagonism disorders where TNF ⁇ antagonism is desirable include but are not limited to septic shock, congestive heart failure, rheumatoid arthritis, cachexia syndrome, Crohn's disease, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease and psoriasis and can be treated by administration of a hybrid protein comprising two polypeptide chains, with one chain comprising the amino acid sequence of TNF linked to the alpha subunit of hCG (SEQ ID NO: 14), and the second chain comprising an amino acid sequence of hGH linked to the beta subunit of hCG (SEQ ID NO: 15), wherein the hybrid protein has TNF antagonist activity.
  • one of the subunits of the heterodimeric proteinaceous hormone in the hybrid protein comprises one or more alterations which reduce or eliminate the biological activity of the hormone, while preserving the ability of the altered subunit to dimerize with another subunit of the hormone
  • an altered subunit is an alpha subunit of hCG which comprises a deletion of amino acids 88-92, thereby rendering the hCG biologically inactive; however, preserving the ability of the alpha subunit to dimerize with the beta subunit of hCG.
  • an altered subunit is an alpha subunit which comprises substitution of a cysteine residue at amino acid position 26 with an alanine (C26A).
  • an altered subunit is an alpha subunit comprising a deletion of amino acids 88-92 (del 88-92) and substitution of a cysteine residue at amino acid position 26 with an alanine (C26A).
  • an altered subunit is a beta subunit comprising a deletion of amino acids 104-145 (del 104-145).
  • the hybrid proteins of the invention may comprise: a) an altered alpha subunit and an unaltered beta subunit; b) an altered alpha subunit and an altered beta subunit; c) an unaltered alpha subunit and an altered beta subunit; or d) an unaltered alpha subunit and an unaltered beta subunit.
  • the invention comprises pharmaceutical compositions comprising an effective amount of one or more hybrid proteins described herein in combination with a pharmaceutically acceptable carrier.
  • fusion proteins and hybrid proteins of the invention have a prolonged half-life in vivo relative to the half-life of the naturally occurring cytokine or fragment thereof, which forms a part of the fusion or the hybrid protein.
  • some fusion proteins and hybrid proteins of the invention have a prolonged shelf-life in vitro relative to the shelf-life of the naturally occurring cytokine or fragment thereof, which forms a part of the fusion or the hybrid protein.
  • a method of making a hybrid protein comprises (a) transfecting a cell with two vectors, wherein each vector comprises a nucleic acid molecule encoding a fusion protein comprising an amino acid sequence of a cytokine or a receptor binding fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof; and (b) culturing the cell under suitable conditions, thereby to produce the hybrid protein.
  • a method may further comprise the step of testing the hybrid protein for antagonist activity.
  • Figure 1 shows an SDS-PAGE gel stained with Coomassie of a cellular extract from CHO cells cotransfected with the ⁇ and ⁇ polypeptide chains of hGH-hCG. The protein was purified from culture supernatants. The purity and composition of the resultant protein was tested by SDS-PAGE. Lane 1 illustrates the heterodimer run under non-reducing conditions; lane 2 represents the heterodimer run under reducing conditions; and lane 3 contains molecular weight markers. As shown in the gel, about 96% of the recovered protein is heterodimeric.
  • Figures 2A-2C show a Western blot analysis of a cellular extract from CHO cells cotransfected with the ⁇ and ⁇ polypeptide chains of hGH-hCG.
  • Panel A was probed with anti-growth hormone antiserum
  • panel B with anti-hCG ⁇
  • panel C with anti- hCG ⁇ .
  • lane 1 contains non-reduced hCG ⁇ -hCG ⁇ heterodimer
  • lane 3 contains molecular weight standards
  • lane 4 contains reduced hCG ⁇ -hCG ⁇ heterodimer
  • land 5 contains reduced hCG.
  • Figure 3 is a graph depicting the in vitro potency of the hGH-hCG hybrid protein (AS902333/1) as compared to recombinant growth hormone (rGH), as measured by cell proliferation assays in Nb2 cells.
  • the hGH-hCG protein has about a 10-fold lower specific-activity than rGH.
  • Figure 4 is a graph depicting the pharmacokinetic profile of hGH-hCG relative to rGH.
  • the hGH-hCG hybrid protein has a prolonged half live in vivo as measured by the serum concentration relative to the rGH.
  • Figure 5 is a graph depicting the hi vivo activity of hGH-hCG in rats measuring tibial growth plate depth. The hGH-hCG hybrid protein was significantly reduced in vivo relative to rGH.
  • the invention is based, at least in part, on the discovery that non- immunoglobulin proteins such as heterodimeric proteinaceous hormones can be used for making therapeutic proteins and more specifically therapeutic proteins with cytokine antagonist activity.
  • a heterodimeric proteinaceous hormone is hCG.
  • hCG a heterodimeric proteinaceous hormone
  • hybrid protein refers to a protein which comprises two or more polypeptide chains which are capable of dimerizing to form the hybrid proteins of the invention, hi some embodiments, two or more polypeptide chains dimerize using non-covalent interactions between the two polypeptide chains, such as, for example, between ⁇ and ⁇ subunits of a heterodimeric proteinaceous hormone such as, hCG.
  • heterodimeric proteinaceous hormones include, but are not limited to, FSH, LH inhibin, and TSH.
  • fusion protein refers to a polypeptide which comprises two or more proteins or fragments thereof linked in frame to each other.
  • the two or more proteins or fragments thereof may either be linked via a peptide linker or they can be linked directly.
  • peptide linker refers to one or more peptides used to couple two proteins together (for example, a cytokine and a subunit of a heterodimeric proteinaceous hormone), hi one embodiment, the peptide linker comprises a stretch of about 10 to 15 amino acids, for example, in certain embodiments, glycine and/or serine.
  • a linker peptide of the invention comprises the sequence Ser
  • receptor refers to a membrane protein, whose binding with its cognate ligand, e.g., a cytokine, triggers secondary cellular responses that result in the activation or inhibition of intracellular process:
  • receptor binding fragment and "receptor binding portion,” as used herein, refer to that portion of the cytokines of the invention which retains the ability to bind to its receptor.
  • heterodimeric and “heterodimer formation” refer to the stable association of two or more different proteins or polypeptide chains either through covalent or non-covalent interaction.
  • An example of a covalent interaction is disulphide bonding.
  • the hybrid proteins described herein comprise two polypeptide chains, one comprising an alpha chain of a heterodimeric hormone and the second comprising a beta chain of a heterodimeric hormone.
  • the terms “treat,” “treating,” and “treatment” refer to a reduction (partial or complete) in at least one symptom associated with a disease or disorder based on antagonism of a cytokine activity.
  • IL-6 antagonism can be used for but is not limited to treating melanoma or to inhibit proliferation of melanoma cells in a patient.
  • antagonists of IL-6 activity may be found in US 5,795,965.
  • VEGF antagonism can be used for but is not limited for inhibiting vascularization of tumors to treat cancer. Examples of antagonists of VEGF activity may be found in WO 94/10202.
  • IFN- ⁇ antagonism can be used for but is not limited for treating rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus (IDDM) and transplant rejection. Examples of antagonists of IFN- ⁇ activity may be found in US
  • TNF- ⁇ antagonism can be used for but is not limited to reducing symptoms associated with septic shock and also for treating congestive heart failure, rheumatoid arthritis, cachexia syndrome, Crohn's disease, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease, and psoriasis.
  • antagonists of TNF- ⁇ activity may be found in US 5,981,701.
  • hGH antagonism can be used for but is not limited for treating acromegaly and gigantism. Examples of antagonists of hGH activity may be found in US 5,350,836.
  • the term "pharmaceutically acceptable carrier” comprises compounds that are compatible with the other ingredients in a pharmaceutical formulation and not injurious to a subject when administered in a therapeutically effective amount.
  • salts refers to salts that are physiologically tolerated by a subject. Such salts are typically prepared from an inorganic and/or organic acid.
  • suitable inorganic acids include, but are not limited to, hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, and phosphoric acid.
  • Organic acids may be aliphatic, aromatic, carboxylic, and/or sulfonic acids.
  • Suitable organic acids include, but are not limited to, formic, acetic, propionic, succinic, camphorsulfonic, citric, fumaric, gluconic, lactic, malic, mucic, tartaric, para- toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, pamoic, methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic (besylate), stearic, sulfanilic, alginic, galacturonic, and the like.
  • therapeutically effective amount refers to that amount of a hybrid protein which is sufficient to effect treatment, as described herein, when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can be readily determined by one of ordinary skill in the art.
  • the dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, , about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 ⁇ g to about 3,500 mg, about 5 ⁇ g to about 3,000 mg, about 10 ⁇ g to about 2,600 mg, about 20 ⁇ g to about 2,575 mg, about 30 ⁇ g to about 2,550 mg, about 40 ⁇ g to about 2,500 mg, about 50 ⁇ g to about 2,475 mg, about 100 ⁇ g to about 2,450 mg, about 200 ⁇ g to about 2,425 mg, about 300 ⁇ g to about 2,000, about 400 ⁇ g to about 1,
  • cytokine antagonist refers to a compound that competes with a cytokine for binding to a cytokine receptor and which does not transduce a signal via the receptor or results in a lower level of signaling than the naturally occurring cytokine.
  • antagonist activity refers to an activity of a compound which acts as a cytokine antagonist.
  • Exemplary antagonists of the invention and described herein comprise the various hybrid proteins comprising a cytokine or receptor binding domain thereof linked to a heterodimeric proteinaceous hormone scaffold.
  • such cytokine antagonists have one or more of IL-6 antagonist activity, VEGF antagonist activity, IFN- ⁇ antagonist activity, TNF antagonist activity and hGH antagonist activity.
  • a hybrid protein having an antagonist activity, as described herein will generally have a specific activity which is at least 5-fold, or 10- fold, or 20-fold, or 30-fold, or 40-fold, or 50-fold, or 60-fold, or 70-fold, or 80-fold, or 90-fold, or 100-fold, or greater than 100-fold, less than the specific activity of the corresponding naturally occurring cytokine or recombinant cytokine which is not linked to a subunit of a heterodimeric proteinaceous hormone, when measured in one or more assays described herein or those routinely used by one of ordinary skill in the art.
  • IL-6 antagonist activity refers to the ability of a molecule, for example, an IL-6 containing hybrid protein described herein, to interfere with the normal functioning of IL-6, as determined by one or more assays described herein or those that would be well-known to one of ordinary skill in the art.
  • a hybrid protein having IL-6 antagonist activity inhibits induction of myeloma or plasmacytoma growth by IL-6 and/or inhibits IL-6 induced immunoglobulin synthesis by human B cells.
  • VEGF antagonist activity refers to the ability of a molecule, for example, a VEGF containing hybrid protein described herein, to interfere with the normal functioning of VEGF, as determined by one or more assays described herein or those that would be well-known to one of ordinary skill in the art.
  • a hybrid protein having VEGF antagonist activity inhibits endothelial cell growth.
  • IFN-alpha antagonist activity refers to the ability of a molecule, for example, an IFN-alpha containing hybrid protein described herein, to interfere with the normal functioning of IFN-alpha, as determined by one or more assays described herein or those that would be well-known to one of ordinary skill in the art.
  • a hybrid protein having IFN-antagonist activity induces replication of Newcastle disease virus, as described by Park et ah, J Virol.
  • TNF-antagonist activity refers to the ability of a molecule, for example, a TNF containing hybrid protein described herein, to interfere with the normal functioning of TNF, as determined by one or more assays described herein or those that would be well-known to one of ordinary skill in the art.
  • hGH antagonist activity refers to the ability of a molecule, for example, an hGH containing hybrid protein of the invention and described herein, to interfere with the normal functioning of hGH, as determined by one or more assays described herein or those that would be well-known to one of ordinary skill in the art.
  • a hybrid protein having hGH antagonist activity has lower specific activity than the naturally occurring growth hormone or recombinant growth hormone, as measured by cell proliferation assays described herein.
  • Cytokines are molecules that have pleiotropic effects on the proliferation, differentiation, and activation of cells.
  • Various cytokines, or receptor binding portions thereof, can be utilized in the fusion proteins and hybrid proteins described herein.
  • Exemplary cytokines include the interleukins ⁇ e.g. IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-10, IL-Il, IL-12, IL-13, and IL-18), the colony stimulating factors (CSFs) ⁇ e.g.
  • a polypeptide comprising the complete amino acid sequence of a cytokine may be included in a fusion or a hybrid protein.
  • a polypeptide comprising the receptor binding portion of a cytokine may be included in a fusion protein or a hybrid protein described herein.
  • a fusion protein of the invention comprises an amino acid sequence from a human cytokine.
  • cytokine sequences from non-human mammals may also be employed.
  • sequences and receptor binding portions of these cytokines are known in the art.
  • an exemplary nucleotide sequence of human IL-6 can be found in the GenBank database (Accession number AF372214; gi 14278708), and exemplary amino acid sequence of human IL-6 can also be found in the Genbank database (Accession number AAK48987; gi:13899131).
  • the amphipathical alpha-helix structure in the COOH terminus of IL-6 has been shown to be responsible for receptor binding.
  • the region between Ser 178 and Arg 183 has been identified as being important in receptor binding.
  • a hybrid protein can comprise a polypeptide comprising the COOH terminus or the region between Ser 178 and Arg 183, inclusive, of the molecule.
  • An exemplary nucleotide and amino acid sequence of human VEGF can be found in the GenBank database (Accession number M32977; gi: 181970).
  • VEGF is a homodimeric glycosylated protein that exists in five isoforms of 121, 145, 165, 189, and 206 amino acids.
  • VEGF, placental growth factor, VEGF-B, and VEGF-C form a family of related growth factors having structural homology to platelet-derived growth factor (PDGF).
  • PDGF platelet-derived growth factor
  • the crystal structure of VEGF was recently solved and receptor binding has been associated with loop/turn II and loop/turn III. Because of the symmetry, the VEGF dimer contains two receptor binding interfaces lying on each pole of the molecule.
  • a fusion protein of the invention may comprise these portions of the molecule. (See, for example, Sieffle et al., Proc. Natl. Acad. Sci.
  • IFN-alpha interferon-alpha
  • the 29-35 and 123-140 domains are responsible for IFN-alpha receptor binding interactions and constitute receptor recognition sites in IFN-alpha, with residues 29-35 influencing the active configuration of IFN-alpha most significantly. (See, for example, Fish, J Interferon Res., 12:257-66 (1992)).
  • TNF-alpha An exemplary nucleotide and amino acid sequence of human TNF-alpha can be found in the GenBank database (Accession number X02910; gi:37209). With respect to portions of the molecule that can be used, the receptor binding domain includes the region close to the N-terminus of the TNF- ⁇ molecule. (See, for example, Tchorzewski et al., Cytokine, 5(2): 125-32 (1993))
  • GenBank database accesion number V00520; gi:31906
  • site 1 and site 2 which bind two identical receptor molecules.
  • the cytokines or receptor binding portions thereof for use in the claimed compositions and methods bind to their cognate receptors.
  • the cytokine or portion thereof incorporated into a fusion protein or a hybrid protein is naturally occurring, i.e., has not been mutated to alter receptor binding.
  • heterodimeric proteinaceous hormones include FSH, inhibin, TSH, hCG, and LH.
  • nucleotide and amino acid sequence of the alpha and beta subunits of hCG can be found in the GenBank database at Accession number JOOl 17; gi:180436 and Accession number CAA23777; gi:31869, respectively. Also, see, for example, Morgan et al., J Biol Chem., 250(13):5247-58 (1975) and Fiddes et al., Nature, 281(5730): 351-6 (1979).
  • amino acids 20-161 of the alpha subunit of hCG and amino acids 20-161 of the beta subunit of hCG can be included in a hybrid protein described herein.
  • Heterodimeric hormones hCG, TSH, FSH and LH share the same alpha subunit, which heterodimerizes with the respective beta subunit.
  • An exemplary nucleotide and amino acid sequence of the beta subunit of human FSH can be found in the GenBank database (Accession number NM_000510; gi:66528900).
  • human FSH coding region was derived from the DdeI-Sau3Al subfragment of the 15B genomic clone described by Watkins, P.C. et al., DNA 6:205-212 (1987).
  • amino acids 1-111 of FSH may be incorporated into a hybrid protein described herein.
  • An exemplary nucleotide and amino acid sequence of the beta subunit of human TSH can be found in the GenBank database (Accession number NM_000549; gi:42490754).
  • An exemplary nucleotide and amino acid sequence of the beta subunit of human LH can be found in the GenBank database (Accession number X00264; gi:34351).
  • An exemplary nucleotide and amino acid sequence of the alpha chain of human inhibin can be found in the GenBank database (Accession number M13981; gi:186410), and the nucleotide and amino acid sequence of the beta chain of human inhibin can be found in the GenBank database (Accession number M31669; gi:186419).
  • one or more of the subunits of the heterodimeric proteinaceous hormone in the hybrid protein includes one or more alterations which reduce or eliminate the biological activity of the hormone, while preserving the ability of the altered subunit to dimerize with another subunit of the hormone to form a heterodimer.
  • an altered subunit is an alpha subunit of hCG which includes a deletion of amino acids 88- 92 (del 88-92), thereby rendering the hCG biologically inactive; however, preserving the ability of the alpha subunit to dimerize with the beta subunit of hCG, thereby to generate a hybrid protein
  • an altered subunit is an alpha subunit which comprises a substitution of a cysteine residue at amino acid position 26 with an alanine (C26A).
  • an altered subunit is an alpha subunit comprising a deletion of amino acids 88-92 (del 88-92) and substitution of a cysteine residue at amino acid position 26 with an alanine (C26A).
  • an altered subunit is a beta subunit comprising a deletion of amino acids 104-145 (del 101-145).
  • the hybrid proteins of the invention may comprise: a) an altered alpha subunit and an unaltered beta subunit; b) an altered alpha subunit and an altered beta subunit; c) an unaltered alpha subunit and an altered beta subunit; or d) an unaltered alpha subunit and an unaltered beta subunit.
  • the invention comprises homologs of the various cytokine and heterodimeric proteinaceous hormone genes and proteins encoded by those genes.
  • a "homolog,” in reference to a gene refers to a nucleotide sequence that is substantially identical over at least part of the gene or to its complementary strand or a part thereof, provided that the nucleotide sequence encodes a protein that has substantially the same activity/function as the protein encoded by the gene which it is a homolog of.
  • Homologs of the genes described herein can be identified by percent identity between amino acid or nucleotide sequences for putative homologs and the sequences for the genes or proteins encoded by them (e.g., nucleotide sequences for genes encoding IL-6, VEGF, ESfF- ⁇ , TNF and hGH, or their complementary strands, and also nucleotide sequences for genes encoding LH, hCG, TSH, inhibin and FSH or their complementary strands). Percent identity may be determined, for example, by visual inspection or by using various computer programs known in the art or as described herein.
  • percent identity of two nucleotide sequences can be determined by comparing sequence information using the GAP computer program described by Devereux et al. (1984) Nucl. Acids. Res., 12:387 and available from the University of Wisconsin Genetics Computer Group (UWGCG). Percent identity can also be determined by aligning two nucleotide sequences using the Basic Local Alignment Search Tool (BLASTTM) program (as described by Tatusova et al. (1999) FEMS Microbiol. Lett., 174:247).
  • BLASTTM Basic Local Alignment Search Tool
  • the terms "homology” and “homologous” are not limited to designate proteins having a theoretical common genetic ancestor, but includes proteins which may be genetically unrelated that have, nonetheless, evolved to perform similar functions and/or have similar structures. Functional homology to the various proteins described herein also encompasses proteins that have an activity of the corresponding protein of which it is a homolog.
  • proteins to have functional homology it is not required that they have significant identity in their amino acid sequences, but, rather, proteins having functional homology are so defined by having similar or identical activities, for example, binding to a cytokine receptor.
  • proteins with structural homology are defined as having analogous tertiary (or quaternary) structure and do not necessarily require amino acid identity or nucleic acid identity for the genes encoding them.
  • structural homologs may include proteins which maintain structural homology only at the active site or binding site of the protein.
  • the present invention further encompasses proteins having amino acid identity to the various cytokine and heterodimeric proteinaceous hormone amino acid sequences described herein that may be used in the hybrid proteins described herein.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the amino acid sequence of one protein for optimal alignment with the amino acid sequence of another protein).
  • the amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other, then the molecules are identical at that position.
  • nucleic acid and amino acid sequences of molecules described herein comprise a nucleotide sequence or amino acid sequence which hybridizes to or is at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleic acid or amino acid sequence described herein.
  • nucleic acid molecules appropriate for use in the fusion proteins of the invention comprise a nucleotide sequence which hybridizes under stringent conditions to the complement of a nucleic acid molecule encoding a cytokine or heterodimeric proteinaceous hormone.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC)
  • a fusion protein comprises a polypeptide chain comprising receptor binding domain(s) of a cytokine, e.g., a naturally occurring cytokine which binds to its cognate receptor, with a chain of a heterodimeric proteinaceous hormone.
  • the subject fusion proteins can be made using methods known in the art.
  • the fusion proteins of the invention may be constructed as described in U.S. Patent 6,194,177.
  • the subject fusion proteins can be made employing methods used to make chimeric antibodies in which a variable domain from an antibody of one species is substituted for the variable domain of another species.
  • nucleic acid molecule encoding a hormone ⁇ or ⁇ chain.
  • nucleic acid molecule encoding the fusion protein is subsequently transfected into a host cell for expression.
  • the sequence of the final construct can be confirmed by sequencing.
  • a nucleic acid molecule encoding the binding domain of the ligand will be fused in frame C-terminally to nucleic acid molecule encoding the N terminus of the hormone. N-terminal fusions are also possible in which the ligand is fused to the N- terminus of the hormone.
  • the precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the molecule.
  • the signal sequence of the proteinaceous hormone is excluded prior to incorporation of the hormone amino acid sequence into a fusion protein, of the invention.
  • the signal sequence for the cytokine moiety of the fusion protein may be included, however, such sequences may also be omitted and replaced with the signal sequence for a different protein if secretion of the fusion protein is desired.
  • the pharmaceutical compositions of the invention will comprise the mature form of a cytokine.
  • introns can be excluded from either one or both the cytokine moiety and the hormone moiety prior to incorporation into a fusion.
  • the amino acid sequence of a cytokine or a fragment thereof is linked to a subunit of a heterodimeric proteinaceous hormone via a peptide linker.
  • Exemplary peptide linkers are well known in the art and generally comprise several GIy and several Ser residues, e.g., such as GlyGlyGlySerSerGlyGlyGlySerGly.
  • a peptide linker for use in a fusion protein of the invention acts as a flexible hinge.
  • a peptide linker for use in a fusion protein of the invention is cleavable in vivo (e.g., by an enzyme) to limit the activity of the cytokine antagonist.
  • cleavable linkers include linkers comprising a thrombin cleavage site.
  • the linker is degradable by natural factors found in the circulation.
  • cytokine moiety may be linked via a peptide linker to alpha and beta subunits of hCG starting at residues Cys7 in the alpha subunit or Pro7 in the beta subunit, respectively.
  • V. HYBRID PROTEINS [0085] The fusion proteins of the invention are assembled as multimers, particularly as heterodimers.
  • heterodimeric hybrid proteins described herein typically comprise two polypeptide chains, with one chain comprising a cytokine or a receptor binding fragment thereof or a polypeptide chain of a cytokine linked to a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, and the second chain comprising the same cytokine, another polypeptide chain of the cytokine, a different cytokine, or a receptor binding fragment thereof linked to another subunit of a heterodimeric proteinaceous hormone or fragment thereof.
  • the two subunits of the heterodimeric proteinaceous hormone are capable of dimerizing to form the hybrid protein.
  • modifications can be made, such as chemical or protease cleavage of the protein backbone, or chemical or enzymatic modification of certain amino acid side chains, to reduce the activity of or inactivate one or more molecules which form part of the hybrid proteins.
  • modifications can also be accomplished through the use of recombinant DNA techniques, for example, by altering the coding sequence for one or more molecules which form a part of a hybrid protein, thereby resulting in reducing the activity of or inactivating a molecule which forms a part of the hybrid protein.
  • such a modification can render hybrid protein more amenable to subsequent chemical or enzymatic modification.
  • Hybrid proteins of the invention can either be monofunctional, bifunctional or multifunctional, depending on the cytokines that are used.
  • each polypeptide chain of a hybrid protein includes an amino acid sequence of a different cytokine, thereby, rendering more than one antagonist activity to the hybrid protein.
  • a hybrid protein comprises at least two polypeptide chains, with each polypeptide chain comprising the same cytokine or a fragment thereof linked to a subunit of a heterodimeric proteinaceous hormone.
  • each polypeptide chain which forms a part of a hybrid protein comprises a different chain of a dimeric cytokine.
  • each polypeptide chain which forms a part of a hybrid protein comprises a different cytokine
  • one or more polypeptide chains which form a part of the hybrid protein comprise more than one cytokine or a fragment thereof
  • hi hybrid proteins of the invention, a cytokine or a fragment thereof can be linked either to the amino terminus or to the carboxy terminus of a subunit of a heterodimeric proteinaceous hormone, or in some instances, to both termini.
  • hybrid proteins of the invention are formed by non- covalent linkage between at least two polypeptide chains which form the hybrid protein.
  • One or more covalent bonds can also be added between the two subunits of a heterodimeric proteinaceous hormone to enhance the stability of the resulting hybrid protein. This can be achieved by, for example, adding one or more non-native interchain disulfide bonds.
  • One skilled in the art can easily identify appropriate sites for such cross-links, for example, based on the known structures of heterodimeric hormones.
  • cysteine residues can be incorporated into an hCG molecule at Lys45 in the ⁇ subunit and Glu21 in the ⁇ subunit, thereby replacing a salt bridge (non-covalent bond) with a disulfide bond (covalent bond).
  • Methods for insertion of cysteine residues are well known in the art.
  • Other forms of modifications include PEGylation and other types of chemical modifications of the hybrid proteins.
  • the invention comprises isolated nucleic acid molecules which encode, for example, a polypeptide chain of a hybrid protein.
  • Two isolated nucleic acid molecules, each comprising a nucleotide sequence encoding a cytokine or a receptor binding fragment thereof linked to a nucleotide sequence encoding a subunit of a heterodimeric proteinaceous hormone can either be co-expressed or they may be expressed separately.
  • VI. EXPRESSION OF FUSION PROTEINS AND HYBRID PROTEINS [0092] hi order to express the fusion or hybrid proteins of the invention, DNA molecules obtained by any of the methods described herein or those that are known in the art, can be inserted into appropriate expression vectors by techniques well known in the art.
  • a double stranded cDNA can be cloned into a suitable vector by homopolymeric tailing or by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation.
  • DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.
  • the invention comprises vectors (e.g., recombinant plasmids and bacteriophages) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules comprising genes) as described herein.
  • the term "recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived.
  • a recombinant vector includes a cytokine gene or a receptor binding fragment thereof operably linked to regulatory sequences, for example, promoter sequences, terminator sequences and/or artificial ribosome binding sites
  • a "recombinant vector” includes a nucleic acid molecule encoding a subunit of a heterodimeric proteinaceous hormone or a fragment thereof operably linked to regulatory sequences known in the art and those described herein. Further, a “recombinant vector” includes a vector which includes a nucleic acid molecule including a cytokine gene or a receptor binding fragment thereof linked to a nucleic acid molecule encoding a subunit of a heterodimeric proteinaceous hormone or a fragment thereof, operably linked to regulatory sequences.
  • Recombinant vectors which allow for expression of the genes or nucleic acids included in them are referred to as "expression vectors.”
  • expression vectors For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples include, but are not limited to, the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • one or more DNA molecules comprising a nucleotide sequence encoding one or more polypeptide chains of a hybrid protein are operably linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a host cell.
  • Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • a selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use, if necessary.
  • Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • the vector(s) may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
  • Host cells may either be prokaryotic or eukaryotic.
  • eukaryotic host cells include, for example, mammalian cells, such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells. Such cells facilitate post-translational modifications of proteins, including, for example, correct folding or glycosylation.
  • yeast cells can also be used to express hybrid proteins of the invention. Like most mammalian cells, yeast cells also enable post-translational modifications of proteins, including, for example, glycosylation.
  • Yeast transcription and translation machinery can recognize leader sequences on cloned mammalian gene products, thereby enabling the secretion of peptides bearing leader sequences (i.e., pre- peptides).
  • a particularly preferred method of high-yield production of the hybrid proteins of the invention is through the use of dihydrofolate reductase (DHFR) amplification in DHFR-def ⁇ cient CHO cells, by the use of successively increasing levels of methotrexate as described in US 4,889,803.
  • the polypeptide obtained may be in a glycosylated form.
  • host cells After the introduction of one or more vector(s), host cells are usually grown in a selective medium, which selects for the growth of vector-containing cells.
  • Purification of the recombinant proteins can be carried out by any of the methods known in the art, for example, any conventional procedures involving extraction, precipitation, chromatography and electrophoresis.
  • a further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein.
  • crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized.
  • the protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.
  • Antagonism of receptor binding by various cytokines can be achieved by hybrid proteins described herein.
  • Cytokine receptors are generally divided into several families based on their structure and activities.
  • the Hematopoietin family receptors are dimers or trimers with conserved cysteines in their extracellular domains and a conserved Trp-Ser-X-Trp-Ser sequence. Examples include receptors for IL-2 through IL-7 and GM-CSF.
  • the Interferon family receptors have the conserved cysteine residues but not the Trp-Ser-X-Trp-Ser sequence, and include the receptors for IFN ⁇ , IFN ⁇ , and IFN ⁇ .
  • the Tumor Necrosis Factor family receptors have four extracellular domains; they include receptors for soluble TNF ⁇ and TNF ⁇ as well as membrane- bound CD40 (important for B cell and macrophage activation) and Fas (which signals the cell to undergo apoptosis).
  • the Chemokine family receptors have seven transmembrane helices and interact with G protein. Exemplary receptors in this family include receptors for IL-8, MIP-I and RANTES.
  • antagonism of receptor binding can be detected by contacting cells expressing a cytokine receptor with a labeled cytokine (for example, radio-activ ⁇ label) and increasing concentrations of an unlabeled hybrid protein, which competes for binding to the same receptor are added. The cells are subsequently washed and labeled cytokine is measured. A decrease in the amount of the labeled cytokine to its receptor in the presence of the unlabeled hybrid protein is indicative of antagonism of receptor binding by the hybrid protein.
  • a labeled cytokine for example, radio-activ ⁇ label
  • antagonism of biological activity can also be measured using one or more assays known to those of skill in the art.
  • an assay for detecting antagonism of biological activity involves contacting cells which express a particular cytokine receptor with a hybrid protein, assaying a cellular response such as, for example, cell proliferation, and comparing the cellular response in presence of the naturally occurring or recombinant form of the cytokine; where a decreased cellular response by the hybrid protein relative to the response elicited by the cytokine indicates that the hybrid protein has antagonist activity. Also, a decrease in a downstream signaling event from the receptor can also be measured as an indicia of antagonist activity.
  • the invention also pertains to pharmaceutical compositions comprising one or more hybrid proteins described herein and a pharmaceutically acceptable diluent or carrier.
  • Such pharmaceutical compositions may be included in a kit or container.
  • kit or container may be packaged with instructions pertaining to the extended in vivo half-life or the in vitro shelf life of the hybrid proteins.
  • Such compositions may be used in methods of treating, preventing, or ameliorating a disease or a disease symptom in a patient, preferably a mammal, and most preferably a human, by administering the pharmaceutical composition to the patient.
  • a therapeutically effective amount of a pharmaceutical composition of the invention would be from about 0.0001 mg/Kg to 0.001 mg/Kg; 0.001 mg/kg to about 10 mg/kg body weight or from about 0.02 mg/kg to about 5 mg/kg body weight, hi some embodiments, a therapeutically effective amount of a hybrid protein is from about 0.001 mg to about 0.01 mg, about 0.01 mg to about 100 mg, or from about 100 mg to about 1000 mg, for example.
  • Hybrid proteins of the invention for use in the methods or compositions described herein can be formulated for the most effective route of administration, including for example, oral, transdermal, sublingual, buccal, parenteral, rectal, intranasal, intrabronchial or intrapulmonary administration.
  • Hybrid proteins of the invention and described herein can either by administered alone or in combination of other therapeutic agents known to be useful in the treatment of the disease being treated.
  • the invention includes methods of treating disorders wherein antagonism of a cytokine activity is desirable.
  • methods of the invention include, but are not limited to disorders which can be treated by antagonism of one or more cytokines.
  • Exemplary antagonists of the invention antagonize at least one biological activity of a cytokine selected from the group consisting of IL-6, VEGF, IFN- ⁇ and TNF.
  • a method of treating a subject suffering from a disorder that would benefit from antagonism of ⁇ L- ⁇ comprises administering to a subject in need thereof, a hybrid protein having IL-6 antagonist activity.
  • a hybrid protein of the invention comprises two polypeptide chains, with each polypeptide chain comprising an amino acid sequence of human IL-6, or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has IL-6 antagonist activity.
  • the disorder that would benefit from IL-6 antagonism includes but is not limited to cancer or precancerous condition.
  • the cancer is melanoma.
  • the disorder that would benefit from IL-6 antagonism includes but is not limited to disorders of the nervous system.
  • a hybrid protein used in a method for treating melanoma includes two polypeptide chains, with one chain comprising an amino acid sequence of IL-6 linked to the alpha subunit of hCG (SEQ ID NO:2), and the second chain comprising an amino acid sequence of IL-6 linked to the beta subunit of hCG (SEQ ID NO: 3), wherein the hybrid protein has IL-6 antagonist activity.
  • a method of treating a subj ect suffering from a disorder that would benefit from antagonism of VEGF comprises administering to a subject in need thereof, a hybrid protein having VEGF antagonist activity.
  • a hybrid protein of the invention comprises two polypeptide chains, with each polypeptide chain comprising an amino acid sequence of human VEGF, or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has VEGF antagonist activity.
  • the disorder that would benefit from VEGF antagonism includes but is not limited to cancer or precancerous condition.
  • the disorder involves other unwanted proliferation of blood vessels, e.g., as occurs in diabetic retinopathy.
  • a hybrid protein used in a method for treating cancer includes two polypeptide chains, with one chain comprising an amino acid sequence of VEGF linked to the alpha subunit of hCG (SEQ ID NO:5), and the second chain comprising an amino acid sequence of VEGF linked to the beta subunit of hCG (SEQ ID NO:6), where the hybrid protein has VEGF antagonist activity.
  • a method of treating a subject suffering from a disorder that would benefit from antagonism of IFN- ⁇ comprises administering to a subject in need thereof, a hybrid protein having IFN- ⁇ antagonist activity.
  • a hybrid protein of the invention comprises two polypeptide chains, with each polypeptide chain comprising an amino acid sequence of human IFN- ⁇ , or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has
  • the disorder that would benefit from IFN- ⁇ antagonism is rheumatoid arthritis.
  • a condition that would benefit from IFN- ⁇ antagonism includes but is not limited to insulin-dependent diabetes mellitus.
  • a condition that would benefit from IFN- ⁇ antagonism includes but is not limited to systemic lupus erythematosus. In some embodiments, such a condition includes but is not limited to immune rejection of transplanted cells or tissue.
  • a hybrid protein used in a method for treating a condition that would benefit from IFN- ⁇ antagonism comprises two polypeptide chains, with one chain comprising an amino acid sequence of IFN- ⁇ linked to the alpha subunit of hCG (SEQ ID NO: 8), and the second chain comprising an amino acid sequence of IFN- ⁇ linked to the beta subunit of hCG (SEQ ID NO:9), where the hybrid protein has IFN- ⁇ antagonist activity.
  • a method of treating a subject suffering from a disorder that would benefit from antagonism of TNF- ⁇ comprises administering to a subject in need thereof, a hybrid protein having TNF- ⁇ antagonist activity.
  • a hybrid protein used in such a method comprises two polypeptide chains, with each polypeptide chain comprising an amino acid sequence of human TNF- ⁇ , or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has TNF- ⁇ antagonist activity.
  • TNF- ⁇ antagonism examples include but are not limited to septic shock, congestive heart failure, rheumatoid arthritis, cachexia syndrome, Crohn's disease, ankylosing spondylitis, juvenile chronic arthritis, adult Still's disease and psoriasis.
  • a hybrid protein used in a method for treating a disorder that would benefit from antagonism of TNF- ⁇ activity comprises two polypeptide chains, with one chain comprising an amino acid sequence of TNF- ⁇ linked to the alpha subunit of hCG (SEQ ID NO: 14), and the second chain comprising an amino acid sequence of TNF- ⁇ linked to the beta subunit of hCG (SEQ ID NO:15), where the hybrid protein has TNF- ⁇ antagonist activity.
  • a method of treating a subject suffering from a disorder that would benefit from antagonism of hGH comprises administering to a subject in need thereof, a hybrid protein having hGH antagonist activity.
  • a hybrid protein of the invention comprises two polypeptide chains, with each polypeptide chain comprising an amino acid sequence of human GH, or a receptor binding fragment thereof, linked to an amino acid sequence of a subunit of a heterodimeric proteinaceous hormone chosen from the group including but not limited to hCG, FSH, LH, TSH, inhibin, or a fragment thereof, wherein the hybrid protein has hGH antagonist activity.
  • the disorder that would benefit from hGH antagonism includes but is not limited to acromegaly or gigantism.
  • a hybrid protein used in a method for treating a disorder that would benefit from hGH antagonism comprises two polypeptide chains, with one chain comprising an amino acid sequence of hGH linked to the alpha subunit of hCG (SEQ ID NO:11), and the second chain comprising an amino acid sequence of hGH linked to the beta subunit of hCG (SEQ K) NO:12), wherein the hybrid protein has hGH antagonist activity.
  • hGH human growth hormone
  • One of the subunits of hCG i.e., the alpha subunit
  • the nucleic acid sequence of the hGH linked to the altered alpha subunit of hCG is set forth in SEQ ID NO:16 and the nucleic acid sequence of the hCG linked to the beta subunit of hCG is set forth in SEQ ID NO: 17.
  • Example 2 The hGH-hCG hybrid protein exhibits a prolonged half-life and lower activity in vivo compared to rGH
  • hCG-hCG in vivo activity was significantly reduced as compared to the activity of recombinant growth hormone (rGH).
  • rGH recombinant growth hormone
  • Days 2-16 0.5 ml solution administered twice (interspaced by 6 hrs).
  • the compound AS902333 in Figure 5 represents the hGH- hCG hybrid protein.

Abstract

La présente invention concerne des protéines hybrides comprenant au moins deux chaînes polypeptidiques, chaque chaîne polypeptidique comprenant une séquence d'acides aminés d'une cytokine ou d'un fragment de celle-ci liée à une séquence d'acides aminés d'une sous-unité d'une hormone protéique hétérodimère. L'invention concerne également des procédés de fabrication et d'utilisation de telles protéines hybrides dans le traitement de différentes maladies.
PCT/US2006/040860 2005-10-19 2006-10-17 Nouvelles protéines hétérodimères et utilisations de celles-ci WO2007047829A2 (fr)

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US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9493578B2 (en) 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6194177B1 (en) * 1996-02-20 2001-02-27 Applied Research Systems Ars Holding N.V. DNA encoding a hybrid heterodimeric protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6194177B1 (en) * 1996-02-20 2001-02-27 Applied Research Systems Ars Holding N.V. DNA encoding a hybrid heterodimeric protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN ET AL.: 'The Carboxy-Terminal Region of the Glycoprotein Hormone Alpha-Subunit: Contribution to Receptor Binding and Signaling in Human Chorionic Gonadotropin' MOL. ENDOC. vol. 6, no. 6, 1992, pages 914 - 919, XP002445836 *

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US7820165B2 (en) 2005-11-21 2010-10-26 Merck Serono, S.A. Compositions and methods of producing hybrid antigen binding molecules and uses thereof
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