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WO2007146163A2 - Protéines de fusion-fc présentant des activités effectrices médiées par fc réduites - Google Patents

Protéines de fusion-fc présentant des activités effectrices médiées par fc réduites Download PDF

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Publication number
WO2007146163A2
WO2007146163A2 PCT/US2007/013567 US2007013567W WO2007146163A2 WO 2007146163 A2 WO2007146163 A2 WO 2007146163A2 US 2007013567 W US2007013567 W US 2007013567W WO 2007146163 A2 WO2007146163 A2 WO 2007146163A2
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WO
WIPO (PCT)
Prior art keywords
fusion protein
fragment
human
isolated
modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2007/013567
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English (en)
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WO2007146163A3 (fr
Inventor
Le Sun
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WELSON PHARMACEUTICALS Inc
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WELSON PHARMACEUTICALS Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by WELSON PHARMACEUTICALS Inc filed Critical WELSON PHARMACEUTICALS Inc
Publication of WO2007146163A2 publication Critical patent/WO2007146163A2/fr
Publication of WO2007146163A3 publication Critical patent/WO2007146163A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to IgG Fc fusion proteins and to related methods of production. More specifically, the present invention relates to IgG Fc fusion proteins with reduced or no Fc-mediated effector activities and to methods of making such fusion proteins.
  • the Fc-fusion proteins produced in accordance with the present invention are desirable for use in therapeutic treatments of human subjects.
  • Therapeutic proteins for human use are susceptible to quick renal clearance.
  • One of the strategies for extending the circulating half-life of a therapeutic protein is to fuse the protein with the Fc portion of a human IgG molecule.
  • Enbrel is a fusion protein of the extracellular domain of a TNF receptor with an IgGl Fc fragment, which is used in the treatment of rheumatoid arthritis by titrating TNF thereby preventing TNF from binding to its receptor. Fusion of human IgG Fc fragments to therapeutic proteins is also believed to facilitate the solubility and purification of the proteins.
  • the Fc region of human immunoglobulins plays a significant role in immune defense for the elimination of pathogens. Effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) binding to the cell surface Fc receptors (Fc ⁇ Rs) can lead to ingestion of pathogens by phagocytosis or lysis by killer cells via the antibody-dependent cellular cytotoxicity (ADCC) pathway; or (2) binding to the CIq part of the first complement component Cl initiates the complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of pathogens.
  • Fc ⁇ Rs cell surface Fc receptors
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • IgGl and IgG3 are effective in binding to Fc ⁇ R.
  • the binding affinity of IgG4 to Fc ⁇ R is an order of magnitude lower than that of IgGl or IgG3, while binding of IgG2 to Fc ⁇ R is below detection.
  • Human IgGl and IgG3 are also effective in binding to CIq and activating the complement cascade. Human IgG2 fixes complement poorly, and IgG4 appears quite deficient in the ability to activate the complement cascade.
  • Some therapeutic proteins for human use are intended to block either the binding of ligands to their cell surface receptors or the association of cell surface proteins, which in turn, inhibits cell signaling without damaging the target cells.
  • Fc fusion products it is important that the Fc portion of the fusion products does not mediate undesirable effector functions, leading to the lysis or removal of the target cells.
  • TNF- ⁇ is a potent mediator of inflammation and the host response to pathogens.
  • TNF- ⁇ has a key role in the pathogenesis of rheumatoid arthritis and its antagonists, such as etanercept (Enbrel), a TNFR2 immunoglobulin Fc fusion protein, and infliximab (Remicade), an TNF- ⁇ -specific monoclonal antibody, can improve the clinical course of rheumatoid arthritis (Feldman, M. et al., Nat. Med.9: 1245-1250 (2003)). Septic arthritis is a rapidly progressive and highly destructive joint disease induced by bacterial infection in which TNF- ⁇ also has an important role.
  • TNF- ⁇ only binds to CRD2 and CRD3 of the TNFRs (Locksley, R. M. et al., Cell 104: 487-501 (2001)). It has been found that a self-association domain that overlaps CRDl at the amino terminus of TNFRl (P60), TNFR2 (P80) and CD95 was found to be essential for signaling (Chan, F. K. et al., Science 288: 2351-2354 (2000); Siegel, R. M. et al., Science 288: 2354-2357 (2000)).
  • the Fc portion of the fusion protein is an Fc fragment of a human IgG molecule, which has been modified at one or more amino acid positions to reduce or completely abolish the binding of the Fc fragment to its receptor (Fc ⁇ R).
  • the Fc portion of the fusion protein is a modified human IgG4 Fc region, wherein amino acid residues 233-236 of the wild type of human IgG4 Fc are replaced with the corresponding amino acids of the wild type human IgG2 Fc.
  • the Fc portion of the fusion protein is a modified Fc fragment of a human IgG molecule, wherein a wild type human IgG constant region has been modified at its glycosylation site(s) such that the resulting Fc fragment is incapable of being glycosylated.
  • the Fc portion of the fusion proteih contains the CH2 and CH3 domains, but not the CHl. or the hinge region, of a human IgG molecule.
  • the Fc fusion proteins of the present invention include at least one therapeutic protein component, which can be a receptor, a cytokine, or a hormone.
  • IL-2 interleukin-2
  • IL-3 interleukin-3
  • fusion proteins examples include insulin, growth hormone, or glucagon-like peptide 1 (GLP-I)-
  • the fusion proteins produced in accordance with the present invention can be combined with a pharmaceutically acceptable carrier for use in therapeutic treatments of human subjects. Methods for treating various diseases in humans by employing the Fc- fusion proteins of the present invention are also provided.
  • Figure 3 shows inhibition of hTNF- ⁇ induced cell death by the IgG4 Fc -TNFRl PLAD fusion protein in a dose-dependent fashion.
  • the Fc portion of the fusion protein of the present invention is derived from the wild-type human IgG4 constant region (SEQ ID NO: 1).
  • Human IgG4 has much weaker ADCC activity than both IgGl and IgG3, and has no CDC activity.
  • the Fc portion of the fusion protein of the present invention is a modified Fc fragment of a human IgG molecule.
  • a wild type human IgG constant region has been modified at one or more amino acid positions to reduce or completely abolish the binding of the resulting Fc fragment to its receptor
  • the Fc portion of the fusion protein contains the CH2 and CH3 domains, but not the CHl or the hinge region, of a human IgG molecule. Removal of the hinge region prevents dimerization via cysteine.
  • the CHl, hinge, CH2 and CH3 domains of human IgGl encompass amino acids 142-238, 239-259, 260-359, and 365-464 of SEQ ID NO: 2, respectively.
  • the CHl, hinge, CH2 and CH3 domains of human IgG4 encompass amino acids 2-98, 99-116, 117- 216, and 222-318 of SEQ ID NO: 1 , respectively.
  • Examples of preferred receptors include TNFRl, TNFR2, CD40, RANK, DR5, Fas, TrailR, the PLADs (pre-ligand binding assembly domains) of the TNFRs, receptors for interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17 and IL- 18; hematopoietic factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), G-CSF and erythropoietin; EGFR, Her-2, Her-3, Her-4, PDGFRs, NGFRs, FGFRs, and IGFRs.
  • IL-2 interleukin-2
  • IL-3 interleukin-4
  • IL-5 IL-6
  • IL-7 IL-10
  • IL-12 IL-13
  • IL-14 IL-15
  • IL-16
  • the Fc-receptor fusion protein of the present invention displays a specific neutralization activity. That is, the fusion protein of Fc and a receptor protein specifically binds and sequesters either the ligand of the receptor or the endogenous receptor, thereby preventing the ligand from binding to its receptor on the cell surface and inhibiting the ligand-mediated biological activities.
  • a protein containing the extracellular domain of a TNF receptor e.g., amino acids 30-82 of the human TNFRl (SEQ ID NO: 4), is employed to make the Fc fusion protein.
  • the Fc portion and the therapeutic component(s) can be fused together with or without a peptide linker.
  • a linker of 4-25 amino acids in length can be employed, a preferred linker for use in the present invention is the five-amino acid linker, GGGGS (or "G4S", SEQ ID NO: 9).
  • the present invention provides nucleic acid molecules that encode a fusion protein described hereinabove, i.e., a fusion protein of a human IgG Fc portion fused in frame with at lease one therapeutic protein.
  • the nucleic acid molecules can also include a signal sequence, which encodes a signal peptide that directs the secretion of the fusion protein from the host cells.
  • the signal peptide may be cleaved from the fusion protein during the secretion process.
  • the signal sequence is placed at the 5' end of the nucleic acid sequence coding for the fusion protein.
  • the present invention provides therapeutic methods for treating diseases in a human subject based on administration of an Fc-fusion protein to the subject.
  • the Fc-fusion proteins of the present invention have longer circulating half lives and minimal Fc-mediated effector activities, and are therefore especially suitable for providing effective treatment of diseases in humans.
  • the present therapeutic methods are not limited to treating any particular disease. Those skilled in the art can select an appropriate therapeutic protein suitable for treating a disease of interest and generate a fusion protein of Fc with such therapeutic protein in accordance with the teachings of the present invention.
  • the Fc-fusion proteins of the present invention can be combined with a pharmaceutical carrier in any convenient and practical manner into a form suitable for administration.
  • the fusion proteins can be administered to the subject by standard routes, including the oral, ophthalmic nasal, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular) route.
  • the fusion proteins can be introduced into the body, by injection or by surgical implantation or attachment.
  • the Fc component of the Fc-PLAD-P ⁇ O is composed of amino acids 118-322 of human IgG4 constant region (SEQ ID NO: 1), which contains the CH2 and CH3 domains, but not the CHl or hinge region of IgG4.
  • Fc-PLAD- P60 which consists of 271 amino acids (SEQ ID NO: 6), was produced by recombinant DNA technology in an E. coli bacterial expression system.
  • L929 cells were first treated with different doses of the recombinant IgG4 Fc-TNFRl PLAD protein for 30 min, after which TNF-Ol (0.2 ng) was added to the mixture. The cells were incubated at 37 °C and the amount of cell death was determined by an MTT assay 24 hours later ( Figure 3).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des protéines de fusion Fc présentant des activités effectrices médiées par le fragment Fc réduites ou nulles et destinées à être utilisées en thérapie. Les protéines de fusion Fc produites selon la présente invention sont aisément purifiées, présentent des demi-vies de circulation plus longues par comparaison avec les protéines ne comportant pas de fragment Fc, et font cependant preuve d'activités effectrices médiées par le fragment Fc minimales ou nulles.
PCT/US2007/013567 2006-06-09 2007-06-08 Protéines de fusion-fc présentant des activités effectrices médiées par fc réduites Ceased WO2007146163A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81252806P 2006-06-09 2006-06-09
US60/812,528 2006-06-09

Publications (2)

Publication Number Publication Date
WO2007146163A2 true WO2007146163A2 (fr) 2007-12-21
WO2007146163A3 WO2007146163A3 (fr) 2008-04-03

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PCT/US2007/013567 Ceased WO2007146163A2 (fr) 2006-06-09 2007-06-08 Protéines de fusion-fc présentant des activités effectrices médiées par fc réduites

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109879969A (zh) * 2017-12-06 2019-06-14 天士力生物医药股份有限公司 一种hm-3融合蛋白及其应用
EP3732197A1 (fr) * 2017-12-28 2020-11-04 Julius-Maximilians-Universität Würzburg PROTÉINES DE FUSION D'ANTICORPS ACTIVANT LE RÉCEPTEUR DE LA SUPERFAMILLE (TNFRSF) DES RÉCEPTEURS DU FACTEUR DE NÉCROSE TUMORALE (TNF), AYANT UNE ACTIVITÉ AGONISTE INDÉPENDANTE DE FCyR (PROTÉINES DE FUSION D'ANTICORPS ACTIVANT LE RÉCEPTEUR TNFRSF AYANT UNE ACTIVITÉ AGONISTE INDÉPENDANTE DE FCyR ; TRAAFFIAA)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0230203D0 (en) * 2002-12-27 2003-02-05 Domantis Ltd Fc fusion
WO2005007809A2 (fr) * 2003-05-30 2005-01-27 Alexion Pharmaceuticals, Inc. Anticorps et proteines de fusion comprenant des regions constantes modifiees
BRPI0518762A2 (pt) * 2004-12-02 2008-12-09 Domantis Ltd fusço de droga, conjugado de droga, Ácido nucleico recombinante ou isolado, construÇço de Ácido nucleico, cÉlula hospedeira, mÉtodo para produzir a fusço de droga, composiÇço farmacÊutica, mÉtodo para tratamento de um indivÍduo tendo uma doenÇa inflamatària, uso de um conjugado de droga ou fusço de droga, e, composiÇço de droga

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109879969A (zh) * 2017-12-06 2019-06-14 天士力生物医药股份有限公司 一种hm-3融合蛋白及其应用
CN109879969B (zh) * 2017-12-06 2024-04-09 天士力生物医药股份有限公司 一种hm-3融合蛋白及其应用
EP3732197A1 (fr) * 2017-12-28 2020-11-04 Julius-Maximilians-Universität Würzburg PROTÉINES DE FUSION D'ANTICORPS ACTIVANT LE RÉCEPTEUR DE LA SUPERFAMILLE (TNFRSF) DES RÉCEPTEURS DU FACTEUR DE NÉCROSE TUMORALE (TNF), AYANT UNE ACTIVITÉ AGONISTE INDÉPENDANTE DE FCyR (PROTÉINES DE FUSION D'ANTICORPS ACTIVANT LE RÉCEPTEUR TNFRSF AYANT UNE ACTIVITÉ AGONISTE INDÉPENDANTE DE FCyR ; TRAAFFIAA)

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Publication number Publication date
WO2007146163A3 (fr) 2008-04-03

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