US20060134100A1 - Dc-sign blockers and their use for preventing or treating viral infections - Google Patents

Dc-sign blockers and their use for preventing or treating viral infections Download PDF

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US20060134100A1
US20060134100A1 US10/533,924 US53392405A US2006134100A1 US 20060134100 A1 US20060134100 A1 US 20060134100A1 US 53392405 A US53392405 A US 53392405A US 2006134100 A1 US2006134100 A1 US 2006134100A1
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sign
binding
effector molecule
blocker
viral
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Ali Amara
Fernando Arenzana-Seisdedos
Philippe Despres
Jean-Louis Virelizier
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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Assigned to INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE, INSTITUT PASTEUR reassignment INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DESPRES, PHILIPPE, AMARA, ALI, ARENZANA-SEISDEDOS, FERNANDO, VIRELIZIER, JEAN-LOUIS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to methods, uses and compositions for preventing or treating diseases of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding or interaction of an effector molecule to a DC-SIGN receptor of the mammal to be treated.
  • the effector molecule may be a molecule of a foreign organism.
  • the foreign organism may be a virus.
  • the invention also relates to compositions, and to methods of identifying compositions, wherein the compositions are useful for treating mammalian diseases for which at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated.
  • Dengue is an acute febrile tropical disease and the virus which causes it is an arbovirus which is transmitted by mosquitoes.
  • the vectors of the disease are mosquitoes of the Aedes genus, in particular Aedes aegypti, which most commonly leave their larvae in domestic and peridomestic areas.
  • the responsible virus isolated in 1951, has been classified into four different antigenic types (DEN1, DEN2, DEN3 and DEN4). It belongs to the Flaviviridae family, genus flavivirus.
  • DHF hemorrhagic- syndrome
  • vascular permeability characterized by increased vascular permeability and deregulation of hemostasis.
  • DSS hypovolemic shock
  • the infectious agent is the dengue virus which belongs to the Flaviviridae family, to which the yellow fever virus and the Japanese encephalitis virus also belong (T. P. Monath et al., (1996) Flaviviruses in B. N. Fields, D. M. Knipe, P. M. Howly et al. (eds.) “Fields Virology” Philadelphia: Lippincott Raven Press Publishers). These viruses have a single-strand RNA with positive polarity which comprises 11000 nucleotides and which encodes a poly-protein of approximately 3400 amino acids.
  • NS1 protein In mammalian cells, but not in insect cells, a portion of the NS1 protein is released into the extracellular medium, either primarily in the form of a soluble protein, or secondarily in a microparticulate form.
  • the protein exists in the form of an oligomer, in particular of a pentamer or of a hexamer (Crooks A. J. et al. J. Chrom. (1990), 502, 59-68 and J. Gen. Virol. (1994), 75, 3453-3460 and Glamand et al., J. Virol. (1999), 73, 6106-6110).
  • DEN virus infection was not enhanced by specific antibody (Marovich et al., JID Symp.Proc. 6:219, 2001).
  • Immature DCs can undergo maturation in response to DEN virus infection. Upregulation of surface markers B7-1, B7-2, HLA-DR CD11b and DC83 and cytokine production were observed following infection of DCs. There is growing evidence that DEN infection can induce functional maturity in DC. Such infection causes upregulation of surface markers B7-1, B7-2, HLA-DR CD11b and DC83 and stimulates cytokine production (Ho et al., Immunology 166: 1499, 2001). Immature DCs exposed to DEN virus produce TNF- ⁇ , which may perturb endothelial cell function.
  • DC Dendritic cells
  • APC Antigen Presenting Cells
  • Myeloid DC are distributed throughout the body in an immature state, exhibiting a high capacity for antigen uptake and processing. Once activated by inflammatory stimuli or infectious agents, DC undergo a maturation process, migrate to lymphoid organs, and acquire their capacity to activate naive T lymphocytes.
  • DC-specific molecules DEN virus uses as a receptor for entry.
  • the human DC-specific adhesion receptor DC-SIGN (ICAM-grabbing non integrin or CD-209), a type 11 integral protein, is of particular interest because its expression is largely restricted to immature DCs.
  • DC-SIGN has been shown to be the ligand of ICAM-3, which enables transient DC-T cell interactions, thus facilitating primary immune response (Geijtenbeek et al., Nature 1: 353, 2000).
  • DC-SIGN appeared to be a critical mediator of the migratory and T cell-interacting capabilities exhibited by maturing immature myeloid monocyte-derived DCs.
  • DC-SIGN expression is ILK dependent and is negatively regulated by IFN- ⁇ , IFN- ⁇ , TGF- ⁇ , and anti-inflammatory agents (Relloso et al. J.Immunol. 168: 2634, 2002). DC-SIGN polymorphisms might also explain why some patients mount protective immunity whereas other do not.
  • DC-SIGN a C-type lectin
  • DC-SIGN has a single carbohydrate recognition domain that interacts with proteins with either mannose or galactose side chains in a calcium-dependent manner (Drichunt, Curr.Opin.Immunol. 13: 585, 1999).
  • DC-SIGN is now believed to bind to high-mannose oligosaccharides that are present on the viral glycoproteins and thereby may capture enveloped viruses (Feinberg et al., Science 294: 2163, 2001).
  • DC-SIGN binds to the HIV envelope glycoprotein gp120 (Geijtenbeek et al., Cell, 100: 587, 2000) and thereby mediates rapid internalization of intact HIV into a nonlysosomal compartment (Kwon et al., Immunity, 16: 135, 2002).
  • DC-SIGN receptor for example on the dendritic cells of mammals.
  • Such methods and compositions are needed, for example, to prevent and treat diseases such as viral infections; for example Dengue virus infections.
  • diseases such as viral infections; for example Dengue virus infections.
  • methods and compositions are needed that allow the specific targeting of cells expressing DC-SIGN receptor, such as dendritic cells or alveolar macrophages, to aid in therapy or diagnosis.
  • this invention identifies DC-SIGN as a receptor involved in the binding of viruses other than HIV to dendritic cells.
  • the invention further provides a number of novel methods, uses and compositions for treating diseases of mammals, including viral infections.
  • a first object of the invention is to provide a method of preventing or treating a disease of a mammal, where at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated, and where the method comprises administering to the mammal an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of the effector molecule to the DC-SIGN receptor to thereby prevent or treat the disease.
  • Another object of the invention is to provide a method of preventing or treating a disease of a mammal, where at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated, and where the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the effector molecule to the DC-SIGN receptor to thereby prevent or treat the disease.
  • the DC-SIGN blocker is a blocking derivative of the effector molecule. In other embodiments the DC-SIGN blocker is an antibody.
  • DC-SIGN blocker is an antibody
  • embodiments where the antibody specifically binds DC-SIGN and embodiments where the antibody specifically binds the effector molecule are included embodiments where the antibody specifically binds DC-SIGN and embodiments where the antibody specifically binds the effector molecule.
  • the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor.
  • the mannosylated molecule may be mannan.
  • a further object of the invention is to provide a method of preventing or treating a viral infection of a mammal, where the viral infection is mediated at least in part by the binding of a viral effector molecule to a DC-SIGN receptor of the mammal to be treated, where the method comprises administering to the mammal an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of the viral effector molecule to the DC-SIGN receptor to thereby prevent or treat the viral infection.
  • Another object of the invention is to provide a method of preventing or treating a viral infection of a mammal, where the viral infection is mediated at least in part by the binding of a viral effector molecule to a DC-SIGN receptor of the mammal to be treated, where the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the viral effector molecule to the DC-SIGN receptor to thereby prevent or treat the viral infection.
  • the DC-SIGN blocker comprises a binding moiety of the viral effector molecule. In other embodiments the DC-SIGN blocker comprises a binding moiety of a viral envelope glycoprotein. In other embodiments the DC-SIGN blocker is an antibody. The antibody may specifically bind DC-SIGN or specifically bind the viral effector molecule. In additional embodiments the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor. The mannosylated molecule may be mannan.
  • the DC-SIGN blocker is an antibody
  • the antibody is a monoclonal antibody
  • the mammal is a human and the antibody is a monoclonal antibody that is humanized
  • the antibody specifically binds DC-SIGN
  • the monoclonal antibody is Mab 1B10.2.6
  • the antibody specifically binds the viral effector molecule
  • the antibody specifically binds the binding moiety of the viral effector molecule.
  • the viral effector molecule is a molecular constituent of the viral envelope.
  • the molecular constituent of the viral envelope is an envelope glycoprotein.
  • the DC-SIGN blocker comprises a binding moiety of the viral effector molecule.
  • the DC-SIGN blocker that is used comprises a binding moiety of the envelope glycoprotein.
  • the viral infection is a Flaviviridae infection and the viral effector molecule is a Flaviviridae effector molecule.
  • the viral infection is a Dengue virus infection and the viral effector molecule is a Dengue virus effector molecule.
  • the mammal is a human.
  • the Dengue virus effector molecule is a molecular constituent of the Dengue virus envelope.
  • the molecular constituent of the Dengue virus envelope is a Dengue virus envelope glycoprotein.
  • the Dengue virus envelope glycoprotein is Dengue virus E glycoprotein.
  • the viral infection is a Dengue virus infection and the viral effector molecule is a Dengue virus effector molecule are embodiments where the DC-SIGN blocker comprises a binding moiety of the Dengue virus effector molecule; the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein; the DC-SIGN blocker is a recombinantly produced protein; and the DC-SIGN blocker is an antibody.
  • the DC-SIGN blocker is an antibody
  • the antibody is a monoclonal antibody
  • the mammal is a human and the monoclonal antibody is humanized
  • the antibody specifically binds DC-SIGN
  • the monoclonal antibody is Mab 1B10.2.6
  • the antibody specifically binds the Dengue virus effector molecule.
  • the antibody specifically binds the Dengue virus effector molecule are embodiments where the Dengue virus effector molecule is Dengue virus E glycoprotein.
  • the invention provides a method of preventing or treating an HIV or SIV infection of a human or a simian, where the method comprises administering to the human or simian an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of HIV or .SIV to the DC-SIGN receptor present on dendritic cells of the human or simian to thereby prevent or treat the HIV or SIV infection.
  • the invention provides a method of preventing or treating an HIV or SIV infection of a human or a simian, where the method comprises administering to the human or simian an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of HIV or SIV to the DC-SIGN receptor present on dendritic cells of the human or simian to thereby prevent or treat the HIV or SIV infection.
  • the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein.
  • an HIV infection of a human is prevented or treated.
  • the invention provides a method of preventing or treating inflammation in a mammal caused by specific binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal, wherein the method comprises administering to the mammal an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal to thereby prevent or treat inflammation.
  • the invention provides a method of preventing or treating inflammation in a mammal caused by specific binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal, wherein the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal to thereby prevent or treat inflammation.
  • the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein.
  • the mammal is a human.
  • a further object of the invention is the use of:
  • an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of an effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a disease of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of an effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a disease of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • a DC-SIGN modulator sufficient to substantially modulate the binding of a viral effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a viral infection of a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of a viral effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a viral infection of a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • a DC-SIGN modulator sufficient to substantially modulate the binding of HIV or SIV to a DC-SIGN receptor present on dendritic cells of a human or a simian for the preparation of a medicament for preventing or treating an HIV or SIV infection of said human or said simian.
  • an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of HIV or SIV to a DC-SIGN receptor present on dendritic cells of a human or a simian for the preparation of a medicament for preventing or treating an HIV or SIV infection of a human or a simian.
  • an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of ICAM-3 present on T cells of a mammal with DC-SIGN receptor present on dendritic cells of the mammal to prepare a drug for preventing or treating inflammation in said mammal caused by specific binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal.
  • DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein.
  • mammal is a human.
  • composition comprising:
  • DC-SIGN modulator is present in the composition at an achievable therapeutic concentration.
  • the invention provides a pharmaceutical composition comprising:
  • DC-SIGN blocker is present in the composition at an achievable therapeutic concentration.
  • the DC-SIGN blocker is a derivative of a viral effector molecule.
  • DC-SIGN blocker comprises the binding moiety of a Dengue virus effector molecule.
  • Dengue virus effector molecule is Dengue virus E glycoprotein.
  • the DC-SIGN blocker is an antibody.
  • Embodiments where the DC-SIGN blocker is an antibody include embodiments where the antibody is a monoclonal antibody; the monoclonal antibody is humanized; the antibody specifically binds DC-SIGN; the monoclonal antibody is Mab 1B10.2.6; the antibody specifically binds the viral effector molecule; or the antibody specifically binds the binding moiety of the viral effector molecule.
  • the invention provides a method of identifying a DC-SIGN modulator, wherein the method comprises:
  • test substance binding inhibition value representing an about 95% modulation of binding of the viral effector molecule to dendritic cells by the test substance, indicates that the test substance is a substance that substantially modulates the binding of a viral effector molecule to the DC-SIGN receptor.
  • the invention provides a method of identifying a DC-SIGN blocker, wherein the method comprises:
  • test substance binding inhibition value representing an about 95% inhibition of binding of the viral effector molecule to dendritic cells by the test substance, indicates that the test substance is a substance that substantially inhibits the binding of a viral effector molecule to the DC-SIGN receptor.
  • the method of identifying a DC-SIGN blocker includes embodiments where the cultured cells are DC; the cultured cells are THP-1 cells; the viral effector molecule is a Dengue virus effector molecule; and the Dengue virus effector molecule is Dengue virus E glycoprotein.
  • the invention provides an isolated DC-SIGN blocker identified by the above method of identifying a DC-SIGN blocker.
  • the invention provides a method of targeting a subject molecule to a cell expressing a DC-SIGN receptor by exposing the cell to a targeting complex, where the targeting complex comprises a subject molecule and a DC-SIGN blocker, and where the exposure is under conditions which allow the DC-SIGN blocker to bind to DC-SIGN on the cell expressing the DC-SIGN receptor, thereby targeting the subject molecule to the cell expressing a DC-SIGN receptor.
  • the method of targeting a subject molecule to a cell expressing a DC-SIGN receptor includes embodiments where the DC-SIGN blocker is an antibody; the DC-SIGN blocker is a monoclonal antibody; the subject molecule is a protein; the subject molecule is an antibody; the subject molecule is labeled; the exposure occurs in vivo; and the exposure occurs in vitro.
  • FIG. 1 depicts DEN-1 virus infection of human DCs ex-vivo.
  • DCs infected with DEN-1 virus strain FGA/NA dld (5 Ap6l FFU/cell) were fixed with 3% PFA in PBS 40 h post-infection and permeabilized with 0.1% Triton X-100 in PBS.
  • Intracellular DEN proteins were visualized with anti-DEN-1 virus HMAF by indirect immunofluorescence and nuclei were stained with Hoechst 33258.
  • DEN virus-infected DCs viral antigens
  • chromatin condensation apoptotic nuclei
  • FIG. 2 depicts apoptotic DNA fragmentation in DCs infected with DEN-1 virus.
  • Infected DCs were assayed simultaneously for the presence of DEN antigens by indirect indirect immunofluorescence (Viral antigens) as described in the legend of FIG. 1 and for apoptosis by the TUNEL assay (TUNEL).
  • TUNEL-positive cells were observed by fluorescence. TUNEL-positive cells are indicated (arrows). Low (A) or high (B) magnification.
  • FIG. 3 shows that Anti-DC-SIGN Mab 1B10.2.6 blocks DEN-1 virus infection of human DCs.
  • DCs Prior to infection, DCs were incubated with anti-DC-SIGN Mab 110 (20 ⁇ g/ml) or anti-DEN E Mab 9D12 (dilution 1:50) (Desprbs et al. Virology, 196: 209-219, 1993) for 20 min.
  • Antibody-treated DCs were infected with DEN-1 virus strain FGA/NA did in the presence of Mab for 2 hrs.
  • Viral antigens were detected by indirect immunofluorescence as described in the legend of FIG. 1 . The percentages of infected DCs 42 h post-infection are indicated.
  • FIG. 4 depicts Flavivirus infections of THP-1 and THP-1l/DC-SIGN cells.
  • Cells infected with DEN-1 virus strain FGA/NA did (5 AP61FFU/cell), YF virus strain 17D-204 (50 VEROFFU/cell), or WN virus strain IS-98-ST1 (5 AP61FFU/cell) were assayed 40 h post-infection for the presence of viral antigens by indirect immunofluorescence as described in the legend of FIG. 1 .
  • Viral antigens were visualized with either anti-DEN-1 virus HMAF (AB ⁇ -DEN-1), anti-YF virus HMAF (AB ⁇ -YF), or anti-WN virus HMAF (Ab ⁇ -WN).
  • THP-1 cells mock-infected (top), or infected with flavivirus (bottom) and infected THP-1/DC cells (bottom) 40 post-infection (m.o.i., multiplicity of infection).
  • B The percentages of infected cells are indicated. Values represent the mean of triplicate assays ⁇ SD.
  • FIG. 5 shows that Mannan, EDTA and antibody specific DC-SIGN block DEN-1 virus infection of THP-1/DC/SIGN cells.
  • THP-1/DC/SIGN cells Prior to infection, THP-1/DC/SIGN cells were incubated with either Mab 9D12 (dilution 1:50), Mab BD12.5 (20 ⁇ g/ml), Mab 1B10.2.6 (20 ⁇ g/ml), EDTA (5 mM), mannan (20 ⁇ g/ml), or mock-treated (control).
  • Treated cells were infected with DEN-1 virus strain FGA/NA dld (5 AP61FFU/cell) in the presence of reagents for 2 hrs. Viral antigens were detected by indirect immunofluorescence as described in the legend of FIG. 1 . The percentages of infected cells 48 h post-infection are indicated. Values represent the mean of triplicate assays ⁇ SD.
  • FIG. 6 depicts DEN-1 virus infection of THP-1 cell clone expressing mutant form of DC-SIGN.
  • FIG. 7 depicts the comparative DEN-1, DEN-2, DEN-3and DENA virus infection of THP-1 cell clone expressing DC-SIGN (THP/DC-SIGN cells) vs THP-1 cells.
  • This invention relates to a method of preventing or treating a disease of a mammal, where at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated.
  • the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the effector molecule to the DC-SIGN receptor to thereby prevent or treat the disease.
  • “Mammal” for purposes of the invention refers to any animal classified within the class mammalia.
  • mammals include: humans and simians; pet animals, such as dogs, cats, ferrets, and guinea pigs; farm animals, such as pigs, cows, horses, sheep, goats, and llamas; and zoo animals, such as bears, zebras, elephants, and water buffalo.
  • the mammal is preferably human.
  • a “disease” is any pathological condition of a mammal, which results, for example, from infection, genetic defect, or exposure to a substance in the environment.
  • the methods and compositions of the invention are useful for preventing or treating diseases that are characterized in that at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to the DC-SIGN receptor present on cells such as dendritic cells or alveolar macrophages of the mammal.
  • diseases include viral infection.
  • a specific examples of viral infections that can be treated by the method is Dengue virus infection of a human.
  • DC-SIGN receptor refers generically to DC-SIGN (described in Curtis et al., 1992) and/or DC-SIGNR (described in Pohlmann et al., 2001.), and/or a homologue of DC-SIGN or DC-SIGNR.
  • DC-SIGN receptor refers generically to DC-SIGN (described in Curtis et al., 1992) and/or DC-SIGNR (described in Pohlmann et al., 2001.), and/or a homologue of DC-SIGN or DC-SIGNR.
  • DC-SIGN receptor refers generically to DC-SIGN (described in Curtis et al., 1992) and/or DC-SIGNR (described in Pohlmann et al., 2001.), and/or a homologue of DC-SIGN or DC-SIGNR.
  • human DC-SIGN can be purified from human dendritic cells which are obtained from an in vivo source, such as human blood, or purified from an in vitro source, such as human dendritic cells produced in tissue culture from human dendritic cell precursor cells. It is also possible to express human DC-SIGN using a recombinant system, using either cultured dendritic cell as a host or a suitable heterologous cell type, such as COS-7 or HeLa cells, or bacteria such as E. coli.
  • DC-SIGN receptor refers to homologues of a human DC-SIGN receptor.
  • proteins may be identified in any of a number of different ways. These include expression cloning, polymerase chain reaction using degenerate oligonucleotide primers, and low stringency screening of a bacterial or bacteriophage library.
  • Dendritic cells are a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. Dendritic cells function as antigen-presenting cells that efficiently capture antigens in the peripheral tissues and process them to form MHC-peptide complexes. Dendritic cells are also involved in the early activation of non-MHC-restricted ⁇ and CDI-restricted T cells specific for various mycobacterial glycolipids, including CAM (Kaufmann, 2001 and Moody, et al., 2000). After antigen uptake, these immature dendritic cells acquire the unique capacity to migrate from the periphery to the T cell areas of the secondary lymphoid organs.
  • Dendritic cells convert antigens from foreign cells and infectious microorganisms into short peptides that are bound to membrane proteins of the major histo-compatibility complex (MHC). These MHC-peptide complexes are formed intracellularly but are ultimately presented on the plasma membrane where they serve as ligands for antigen-specific T cell receptors (TCR). In addition to TCR ligand formation, dendritic cells carry out many other functions, which allow them to control immunity at several points (Steinman, 2000).
  • MHC-peptide complexes are formed intracellularly but are ultimately presented on the plasma membrane where they serve as ligands for antigen-specific T cell receptors (TCR).
  • TCR antigen-specific T cell receptors
  • dendritic cells carry out many other functions, which allow them to control immunity at several points (Steinman, 2000).
  • Alveolar macrophages and dendritic cells are examples of cells expressing a DC-SIGN receptor.
  • Endothelial cells are an example of cells expressing DC-SIGNR.
  • dendritic cells may be obtained from an in vivo source, such as the blood of a mammal, or grown in vitro, by culturing dendritic cell precursor cells under appropriate conditions.
  • Dendritic cell precursor cells include monocytes prepared according to Example 2.
  • effector molecule is any molecule that specifically binds to the DC-SIGN receptor present on cells of a mammal, such as the dendritic cells or the alveolar macrophages of a mammal, and thereby mediates a symptom that is associated with a disease of that mammal.
  • effector molecules are ligands present on viruses that bind to receptors on cells of a mammal and thereby facilitate the entry of the virus into a cell of the mammal.
  • the effector molecules can be referred to as “viral effector molecules.”
  • the effector molecules include gp129 of HIV and glycoprotein E of Dengue virus, which bind with the DC-SIGN receptor present on cells such as dendritic cells or alveolar macrophages of a human to facilitate, in the case of Dengue virus, virus entry into DC-SIGN expressing cells.
  • Dengue virus E glycoprotein is thus a “Dengue virus effector molecule.”
  • Other types of effector molecules are ligands that are endogenous to the mammal. This type of ligand includes both ligands that are bound to the surface of other cells of the mammal and soluble ligands, which may be localized to the extracellular space of a particular tissue or circulating systemically.
  • a “symptom” is any pathological manifestation of the disease to be treated.
  • a symptom is caused at least in part by the binding of an effector molecule to the DC-SIGN receptor present on the dendritic cells of the mammal to be treated if a modulation (a reduction or an increase) in the binding of the effector molecule to the DC-SIGN receptor causes a determinable reduction in the occurrence or severity of the symptom, or both.
  • the symptom is no longer present or is prevented from occurring following the reduction in the binding of the effector molecule to the DC-SIGN receptor.
  • An effector molecule is said to “specifically bind” to the DC-SIGN receptor present on cells such as the dendritic cells or the alveolar macrophages of the mammal to be treated if such binding is not competitively inhibited by the presence of unrelated molecules (e.g., fetal calf serum), but is inhibited by antibodies to DC-SIGN (e.g., 1B10.2.6) and/or additional effector molecule.
  • unrelated molecules e.g., fetal calf serum
  • An example of an effector molecule that specifically binds to the DC-SIGN receptor present on cells such as the dendritic cells or the alveolar macrophages of a mammal to be treated is Dengue virus E glycoprotein.
  • the binding of E glycoprotein present on the surface of Dengue virus to DC-SIGN is not inhibited by 0.2% bovine serum albumin as shown in FIGS. 3 and 5 .
  • binding is inhibited by either antibody specific to DC-SIGN as shown in FIGS. 3 and 5 , or soluble mannan added to the media as shown in FIG. 5 , or EDTA added to the media as shown in FIG. 5 .
  • binding Once an effector molecule is known to specifically bind to the DC-SIGN receptor the binding of the effector molecule to DC-SIGN can be referred to simply as “binding.” It will be understood by one of skill in the art that such binding is specific. In this regard, the “modulation” of binding may be discussed. Modulation can include “inhibition” or “enhancement”.
  • Modulation means the act of regulating. It includes the act of inducing variations of a property of a molecule.
  • modulation means the act of regulating and varying the binding of effector molecules to their receptors. This modulation may serve to either inhibit or enhance binding, or to impose other regulatory controls.
  • “inhibition” of binding means a reduction in the total amount of effector molecule that binds to DC-SIGN over a fixed period of time. Inhibition of binding of the effector molecule is achieved by providing a DC-SIGN blocker.
  • a “DC-SIGN blocker” is any molecule that substantially inhibits the binding of a given effector molecule at a concentration at which the effector molecule specifically binds to DC-SIGN.
  • the DC-SIGN blocker used is a monoclonal antibody that specifically binds DC-SIGN.
  • the DC-SIGN blocker used comprises a binding moiety of the Dengue virus E glycoprotein.
  • “enhancement” of binding means an increase in the total amount of effector molecule that binds to DC-SIGN over a fixed period of time. Enhancement of binding of the effector molecule is achieved by providing a DC-SIGN enhancer.
  • a “DC-SIGN enhancer” is any molecule that substantially enhances the binding of a given effector molecule at a concentration at which the effector molecule specifically binds to DC-SIGN.
  • a “binding moiety” is that portion of a molecule that substantially retains the ability to bind to a second molecule when other portions of the molecule are removed or modified or when the binding moiety is placed into a heterologous context.
  • a binding moiety of the effector molecule can be defined.
  • a binding moiety of an effector molecule is that portion of the effector molecule that substantially retains the ability to bind to DC-SIGN when other portions of the molecule are removed or modified or when the binding moiety is placed into a heterologous context.
  • substantially retains can be defined by one of skill based on the specific properties of the binding moiety that are sought.
  • substantially inhibit means greater than 80% inhibition, greater than 90% inhibition, greater than 95% inhibition, or greater than 99% inhibition. In a preferred embodiment of the present invention about 90% binding inhibition is obtained.
  • “Inhibition” is measured by comparing the extent of effector molecule binding to DC-SIGN in the presence of a DC-SIGN blocker with the extent of effector molecule binding to DC-SIGN in the absence of a DC-SIGN blocker. The ratio of extent of binding in the presence of the DC-SIGN blocker compared to the extent of binding in the absence of the DC-SING blocker is then determined. The percent inhibition is then the proportional reduction in the amount of binding. For example, a ratio of 0.1 represents a 90% reduction in binding.
  • treat refers to the administration of therapy to an individual who already manifests at least one symptom of a disease. Such an individual includes an individual who is diagnosed as having a known disease.
  • prevent refers to the administration of therapy on a prophylactic or preventative basis to an individual who may ultimately acquire the disease but who has not yet done so (i.e., those needing preventative measures). Such individuals may be identified on the basis of risk factors that are known to correlate with the subsequent occurrence of the disease.
  • therapeutic benefit refers to an improvement of at least one symptom of a disease, a slowing of the progression of a disease, as manifested by a slowing in the increase in severity of at least one symptom of a disease, or a cessation in the progression of at least one symptom of a disease.
  • the therapeutic benefit is determined by comparing a symptom of a disease before and after a DC-SIGN blocker is administered.
  • antibody refers to any antibody that can be made by any technique known in the art. Suitable antibodies are obtained by immunizing a host animal with peptides comprising all or a portion of the target protein. Suitable host animals include mouse, rat sheep, goat, hamster, rabbit, etc. The origin of the protein immunogen may be mouse, human, rat, monkey, or microorganism such as a bacteria or virus etc. The host animal will generally be a different species than the immunogen, e.g. human protein used to immunize mice, etc.
  • the immunogen may comprise the complete protein, or fragments and derivatives thereof.
  • Preferred immunogens comprise all or a part of one of the subject proteins, where these residues contain the post-translation modifications, such as glycosylation, found on the native target protein.
  • Immunogens comprising the extracellular domain are produced in a variety of ways known in the art, e.g. expression of cloned genes using conventional recombinant methods, isolation from tumor cell culture supernatants, etc.
  • the first step is immunization of the host animal with the target protein, where the target protein will preferably be in substantially pure form, comprising less than about 1% contaminant.
  • the immunogen may comprise the complete target protein, fragments or derivatives thereof.
  • the target protein may be combined with an adjuvant, where suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like.
  • suitable adjuvants include alum, dextran, sulfate, large polymeric anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, and the like.
  • the target protein may also be conjugated to synthetic carrier proteins or synthetic antigens.
  • a variety of hosts may be immunized to produce the polyclonal antibodies.
  • Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and the like.
  • the target protein is administered to the host, usually intradermally, with an initial dosage followed by one or more, usually at least two, additional booster dosages.
  • the blood from the host will be collected, followed by separation of the serum from the blood cells.
  • the Ig present in the resultant antiserum may be further fractionated using known methods, such as ammonium salt fractionation, DEAE chromatography, and the like.
  • Monoclonal antibodies are produced by conventional techniques.
  • the spleen and/or lymph nodes of an immunized host animal provide a source of plasma cells.
  • the plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells.
  • Culture supernatant from individual hybridomas is screened using standard techniques to identify those producing antibodies with the desired specificity.
  • Suitable animals for production of monoclonal antibodies to the human protein include mouse, rat, hamster, etc.
  • the animal will generally be a hamster, guinea pig, rabbit, etc.
  • the antibody may be purified from the hybridoma cell supernatants or ascites fluid by conventional techniques, e.g. affinity chromatography using protein according to the subject invention bound to an insoluble support, protein A sepharose, etc.
  • the antibody may be produced as a single chain, instead of the normal multimeric structure.
  • Single chain antibodies are described in Jost et al. (1994) J.B.C. 269:26267-73, and others.
  • DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are ligated to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and/or serine.
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.
  • artificial antibodies e.g., antibodies and antibody fragments produced and selected in vitro.
  • such antibodies are displayed on the surface of a bacteriophage or other viral particle.
  • such artificial antibodies are present as fusion proteins with a viral or bacteriophage structural protein, including, but not limited to, M13 gene III protein.
  • Methods of producing such artificial anti-bodies are well known in the art. See, e.g., U.S. Pat. Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988; 5,498,538; 5,403,484; 5,571,698; and 5,625,033.
  • the humanized antibody may be the product of an animal having transgenic human immunoglobulin constant region genes (see for example International Patent Applications WO 90/10077 and WO 90/04036).
  • the antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190).
  • Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521).
  • mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA.
  • the cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).
  • a library is made and screened to isolate the sequence of interest.
  • the DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences.
  • the sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.
  • the antibodies may be fully human antibodies.
  • xenogeneic antibodies which are identical to human antibodies may be employed.
  • xenogenic human antibodies is meant anti-bodies that are the same has human antibodies, i.e. they are fully human antibodies, with exception that they are produced using a non-human host which has been genetically engineered to express human antibodies. See e.g. WO 98/50433; WO 98,24893 and WO 99/53049, the disclosures of which are herein incorporated by reference.
  • Antibody fragments such as Fv, F(ab′) 2 and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage.
  • a truncated gene is designed.
  • a chimeric gene encoding a portion of the F(ab′) 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.
  • Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments.
  • C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.
  • Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like.
  • a convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed.
  • splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions.
  • the resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g.
  • SV40 early promoter (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Ig promoters, etc
  • An example of a disease that can be prevented or treated utilizing the present invention is Dengue virus infection.
  • the results presented in the examples demonstrate for the first time a role for DC-SIGN in Dengue virus binding to human dendritic cells.
  • the results described herein show that highly purified DEN-1 virus bearing mosquito N-linked oligosaccharides is able to replicate in human DC and produce progeny virus. Apoptotic cell death was also observed among DEN-1 virus-infected DCs.
  • the C-lectin molecule, DC-SIGN is expressed at the surface of DCs.
  • the experiments described herein sought to determine whether the DC-specific adhesion receptor DC-SIGN has the ability to promote DEN virus infection of human DC cells.
  • the results showed that DC-SIGN specific antibody has a blocking action at the level of DEN-1 virus infection.
  • a novel function of DC-SIGN has been therefore identified as DEN virus binding protein possibly through interaction with the E glycoprotein.
  • the process of DC-SIGN-mediated DEN virus infectivity to DCs provides a new mechanism for targeting the design of anti-viral compounds.
  • the invention provides a method of preventing or treating a disease of a mammal, where at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor of the mammal to be treated, and where the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the effector molecule to the DC-SIGN receptor to thereby prevent or treat the disease.
  • the DC-SIGN blocker is a blocking derivative of the effector molecule. In other embodiments the DC-SIGN blocker is an antibody.
  • DC-SIGN blocker is an antibody
  • embodiments where the antibody specifically binds DC-SIGN and embodiments where the antibody specifically binds the effector molecule are included embodiments where the antibody specifically binds DC-SIGN and embodiments where the antibody specifically binds the effector molecule.
  • the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor.
  • the mannosylated molecule may be mannan.
  • the invention also provides a method of preventing or treating a viral infection of a mammal, where the viral infection is mediated at least in part by the binding of a viral effector molecule to a DC-SIGN receptor of the mammal to be treated, where the method comprises administering to the mammal an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of the viral effector molecule to the DC-SIGN receptor to thereby prevent or treat the viral infection.
  • the DC-SIGN blocker comprises a binding moiety of the viral effector molecule. In other embodiments the DC-SIGN blocker comprises a binding moiety of a viral envelope glycoprotein. In other embodiments the DC-SIGN blocker is an antibody. The antibody may specifically bind DC-SIGN or specifically bind the viral effector molecule. In additional embodiments the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor. The mannosylated molecule may be mannan.
  • the DC-SIGN blocker is an antibody
  • the antibody is a monoclonal antibody
  • the mammal is a human and the antibody is a monoclonal antibody that is humanized
  • the antibody specifically binds DC-SIGN
  • the monoclonal antibody is Mab 1B10.2.6
  • the antibody specifically binds the viral effector molecule
  • the antibody specifically binds the binding moiety of the viral effector molecule.
  • the viral effector molecule is a molecular constituent of the viral envelope.
  • the molecular constituent of the viral envelope is an envelope glycoprotein.
  • the DC-SIGN blocker comprises a binding moiety of the viral effector molecule.
  • the DC-SIGN blocker that is used comprises a binding moiety of the envelope glycoprotein.
  • the viral infection is a Flaviviridae infection and the viral effector molecule is a Flaviviridae effector molecule.
  • the viral infection is a Dengue virus infection and the viral effector molecule is a Dengue virus effector molecule.
  • the mammal is a human.
  • the Dengue virus effector molecule is a molecular constituent of the Dengue virus envelope.
  • the molecular constituent of the Dengue virus envelope is a Dengue virus envelope glycoprotein.
  • the Dengue virus envelope glycoprotein is Dengue virus E glycoprotein.
  • the viral infection is a Dengue virus infection and the viral effector molecule is a Dengue virus effector molecule are embodiments where the DC-SIGN blocker comprises a binding moiety of the Dengue virus effector molecule; the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein; the DC-SIGN blocker is a recombinantly produced protein; and the DC-SIGN blocker is an antibody.
  • the DC-SIGN blocker is an antibody
  • the antibody is a monoclonal antibody
  • the mammal is a human and the monoclonal antibody is humanized
  • the antibody specifically binds DC-SIGN
  • the monoclonal antibody is Mab 1B10.2.6
  • the antibody specifically binds the Dengue virus effector molecule.
  • the antibody specifically binds the Dengue virus effector molecule are embodiments where the Dengue virus effector molecule is Dengue virus E glycoprotein.
  • the effector molecule and the DC-SIGN blocker are the same. In a second preferred embodiment the effector molecule and the DC-SIGN blocker are different.
  • the invention thus provides a method for the prevention and treatment of HIV infection. Specifically, it is an object of the invention to provide a method of preventing or treating an HIV or SIV infection of a human or a simian.
  • the method comprises administering to the human or simian an amount of a DC-SIGN blocker that is sufficient to inhibit the interaction of HIV or SIV with DC-SIGN receptor present on dendritic cells of the human or simian to thereby prevent or treat the HIV or SIV infection.
  • DC-SIGN is also believed to have a critical role in mediating the known loose adhesion that takes place between dendritic cells, and T cells in the apparent absence of foreign antigen. This adhesion is thought to be necessary to provide an opportunity for the TCR to scan the dendritic cell surface and identify the very small amounts of TCR ligand which are present, and in turn to become activated by this ligand. For this reason, the interaction between DC-SIGN on dendritic cells, and ICAM-3 on T cells, is likely to be critically important for the process of T cell activation and stimulation. This model suggests that the DC-SIGN-ICAM-3 interaction may have a role in mediating and/or potentiating other stimulatory effects of dendritic cells on T cells.
  • DC-SIGN blockers may be potent anti-inflammatory agents, by blocking the interaction of the ICAM-3 effector molecule with DC-SIGN.
  • the invention also provides a method of preventing or treating inflammation in a mammal caused by interaction of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal.
  • the method comprises administering to the mammal an amount of a DC-SIGN blocker that is sufficient to inhibit the interaction of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal to thereby prevent or treat inflammation.
  • the invention also provides the use of an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of an effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a disease of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • the invention also provides use of an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of an effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a disease of a mammal, wherein at least one symptom of the disease is mediated at least in part by the binding of the effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • the DC-SIGN blocker is a blocking derivative of the effector molecule.
  • the DC-SIGN blocker is an antibody.
  • the antibody specifically binds DC-SIGN.
  • the antibody specifically binds the effector molecule.
  • the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor; the mannosylated molecule being preferably mannan.
  • the invention also provides the use of an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of a viral effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a viral infection of a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • the invention also provides the use of an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding of a viral effector molecule to a DC-SIGN receptor for the preparation of a medicament for preventing or treating a viral infection of a mammal, wherein the viral infection is mediated at least in part by the binding of the viral effector molecule to the DC-SIGN receptor of the mammal to be treated.
  • the viral effector molecule is a molecular constituent of the viral envelope.
  • the molecular constituent of the viral envelope is an envelope glycoprotein.
  • the DC-SIGN blocker comprises a binding moiety of the viral effector molecule. In other embodiments the DC-SIGN blocker comprises a binding moiety of the envelope glycoprotein. In other embodiments the DC-SIGN blocker is an antibody. The antibody is a monoclonal antibody. In other embodiments the mammal is a human and the monoclonal antibody is humanized. The antibody may specifically bind DC-SIGN or specifically binds the viral effector molecule. In additional embodiments the antibody specifically binds the binding moiety of the viral effector molecule. In additional the DC-SIGN blocker is a mannosylated molecule that binds to a DC-SIGN receptor. The mannosylated molecule may be mannan.
  • the viral infection is a Flaviviridae virus infection and the viral effector molecule is a Flaviviridae effector molecule.
  • the Flaviviridae viral infection is a Dengue virus infection and the Flaviviridae effector molecule is a Dengue effector molecule.
  • the mammal is human.
  • the Dengue virus effector molecule is a molecular constituent of the Dengue virus envelope.
  • the molecular constituent of the Dengue virus envelope is a Dengue virus envelope glycoprotein.
  • the Dengue virus envelope glycoprotein is Dengue virus E glycoprotein.
  • the viral infection is a Dengue virus infection and the viral effector molecule is a Dengue virus effector molecule are embodiments where the DC-SIGN blocker comprises a binding moiety of the Dengue virus effector molecule; the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein; the DC-SIGN blocker is a recombinantly produced protein; and the DC-SIGN blocker is an antibody.
  • the DC-SIGN blocker is an antibody
  • the antibody is a monoclonal antibody
  • the mammal is a human and the monoclonal antibody is humanized
  • the antibody specifically binds DC-SIGN
  • the monoclonal antibody is Mab 1B10.2.6
  • the antibody specifically binds the Dengue virus effector molecule.
  • the antibody specifically binds the Dengue virus effector molecule are embodiments where the Dengue virus effector molecule is Dengue virus E glycoprotein.
  • the invention also provides the use of an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of HIV or SIV to a DC-SIGN receptor present on dendritic cells of a human or a simian for the preparation of a medicament for preventing or treating an HIV or a SIV infection of said human or said simian.
  • the invention also provides the use of an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding or interaction of HIV or SIV to a DC-SIGN receptor present on dendritic cells of a human or a simian for the preparation of a medicament for preventing or treating an HIV or a SIV infection of said human or said simian.
  • the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein.
  • an HIV infection of a human is prevented or treated.
  • the invention also provides the use of an amount of a DC-SIGN modulator sufficient to substantially modulate the binding of ICAM-3 present on T cells of a mammal with DC-SIGN receptor present on dendritic cells of the mammal for the preparation of a medicament for preventing or treating inflammation in said mammal caused by specific binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal.
  • the invention also provides the use of an amount of a DC-SIGN blocker sufficient to substantially inhibit the binding or interaction of ICAM-3 present on T cells of a mammal with DC-SIGN receptor present on dendritic cells of the mammal for the preparation of a medicament for preventing or treating inflammation in said mammal caused by specific binding of ICAM-3 present on T cells of the mammal with DC-SIGN receptor present on dendritic cells of the mammal.
  • the DC-SIGN blocker comprises a binding moiety of the Dengue virus E glycoprotein.
  • the mammal is a human.
  • the invention also provides pharmaceutical compositions comprising a DC-SIGN blocker.
  • Such compositions may be suitable for pharmaceutical use and administration to patients.
  • the compositions typically contain a purified DC-SIGN blocker at a therapeutically achievable concentration and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions can also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the pharmaceutical compositions can also be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art.
  • the administration may, for example, be intravenous, intramuscular, subcutaneous, or via inhalation.
  • Solutions or suspensions used for subcutaneous application typically include one or more of the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetra acetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Such preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of micro-organisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the DC-SIGN blocker containing compositions are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • a purified DC-SIGN blocker is prepared with carriers that will protect it against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions containing LAM can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • Therapeutically useful agents such as growth factors (e.g., BMPs, TGF- ⁇ , FGF, IGF), cytokines (e.g., interleukins and CDFs), antibiotics, and any other therapeutic agent beneficial for the condition being treated can optionally be included in or administered simultaneously or sequentially with the DC-SIGN blocker.
  • growth factors e.g., BMPs, TGF- ⁇ , FGF, IGF
  • cytokines e.g., interleukins and CDFs
  • antibiotics e.g., antibiotics, and any other therapeutic agent beneficial for the condition being treated can optionally be included in or administered simultaneously or sequentially with the DC-SIGN blocker.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of compositions comprising a DC-SIGN blocker can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • DC-SIGN blockers which exhibit large therapeutic indices are preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test DC-SIGN blocker which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay.
  • a targeting complex of the present invention comprises at least one DC-SIGN blocker molecule covalently attached to at least one subject molecule.
  • a single DC-SIGN blocker molecule is covalently linked to a single subject molecule.
  • more than one DC-SIGN blocker molecule can be covalently linked to a single subject molecule.
  • the multiple DC-SIGN blocker molecules can each be independently covalently linked to the subject molecule; alternatively, one or more of the more than one DC-SIGN blocker molecules can be covalently linked only to one or more other DC-SIGN blocker molecules, at least one of which is itself covalently linked to the subject molecule.
  • multiple subject molecules are covalently linked to a single DC-SIGN blocker molecule.
  • the multiple subject molecules can each be independently covalently linked to the DC-SIGN blocker molecule; alternatively, one or more of the more than one subject molecules can be covalently linked only to one or more other subject molecules, at least one of which is itself covalently linked to the DC-SIGN blocker molecule.
  • compositions of more than one of the various types of DC-SIGN blockers described immediately above there is no limit to the diversity of such compositions which can be used.
  • One of skill in the art will appreciate that the composition to be used for a particular application will be dictated by many factors and that a suitable composition can thus be appropriately chosen for each application of the invention.
  • PC-SIGN blockers of the invention are well known and widely practiced by those of skill in the biochemistry art, and thus need not be detailed here. However, one of skill in the art will recognize that any suitable technique which results in the formation of a covalent bond between a subject molecule and a DC-SIGN blocker molecule can be used.
  • Subject molecules can be any molecule of interest.
  • Nonlimiting examples include: small organic molecules, proteins, nucleic acids, carbohydrates, and lipids.
  • One of ordinary skill in the art will appreciate that any known derivatives and composites of one or more of these classes of molecules can also be used.
  • the subject molecule in the case in which the subject molecule is a protein, nucleic acid, carbohydrate, or lipid, the subject molecule can be obtained from a natural source, i.e., purified from an organism, which comprises the molecule.
  • the subject molecule can be obtained from a recombinant source, i.e., from a recombinant organism, which has been engineered to produce a subject molecule of choice.
  • the recombinant organism that is used to produce the recombinant subject molecule is one that comprises the subject molecule, as the organism occurs in nature, in nonrecombinant form. In other cases, the subject molecule is one that does not naturally occur in the recombinant organism.
  • the subject molecules of the invention also include derivatives of small organic molecules, proteins, nucleic acids, carbohydrates, and lipids.
  • a derivative is a form of small organic molecule, protein, nucleic acid, carbohydrate, or lipid that is modified from its natural state by adding, subtracting, or altering one or more chemically reactive sites present on the small organic molecule, protein, nucleic acid, carbohydrate, or lipid.
  • Techniques for making derivatives of small organic molecules, proteins, nucleic acids, carbohydrates, and lipids are well known and widely practiced by those of skill in the biochemistry art, and thus need not be detailed here.
  • the subject molecule is an antibody.
  • the subject molecule can also be a molecule that is antigenic.
  • a molecule is antigenic when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a molecule can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier molecule for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • the targeting complex of the invention can be exposed to a cell expressing DC-SIGN, such as a dendritic cell either in vivo or in vitro.
  • a cell expressing DC-SIGN such as a dendritic cell either in vivo or in vitro.
  • In vivo exposure is achieved by administering the targeting complex in a pharmaceutical composition as described herein or in any suitable equivalent formulation known in the art.
  • the targeting complex will bind to DC-SIGN on the surface of dendritic cells in vivo.
  • In vitro exposure occurs when dendritic cells grown in vitro are exposed to the targeting complex.
  • DEN type-1 virus strain FGA/NA d1d (GenBank accession number AF226686) (Duarte dos Santos et al. Virology, 274: 292, 2000) and West Nile (WN) virus strain IS-98-ST1 (GenBank accession number AF481864) (Mashimo et al., PNAS, 99: 11311, 2002) from mosquito Aedes pseudoscutellaris AP61 cell monolayers and virus titration on AP61 cells by focus immunodetection assay (FIA) were performed as previously described (DesNase et al., Virology, 196: 209, 1993).
  • Yellow fever (YF) virus vaccine strain 17D-204 (STAMARIL, Pasteur Vaccins, Lot E113) (GenBank accession number: X07755) was propagated twice in African green monkey kidney VERO cell monolayers, purified in sucrose gradients and titrated on VERO cells. Infectivity titers were expressed as focus forming units (FFU).
  • FFU focus forming units
  • FGA/NA d1d E glycoprotein has two N-linked glycosylation sites at positions Asn 67 and Asn 153 . Both N-glycosylation sites of the DEN-1 glycoprotein E appear to be utilized during the N-glycosylation process (Courageot et al., J.Virol., 74: 564-572). IS-98-ST1 E glycoprotein has a single N-linked glycosylation site that appeared to be utilized (Deshunt, personal communication). Whereas 17D-204 E protein is not N-glycosylated. The flavivirus M protein is not glycosylated.
  • FCS heat-inactivated fetal calf serum
  • THP-1 and THP/DC-SIGN cells were adhered to poly-L-lysine-treated glass Lab-tek chambers or polylysine-treated 12-well flasks (5 ⁇ 10 4 cells per cm 2 ).
  • RPMI 1640 Cells were washed once with RPMI 1640, incubated with highly purified virus in RPMI 1640 supplemented with 0.2% bovine serum albumine (BSA, pH 7.5) (Sigma) for 2 hrs at 37° C. and placed into fresh media supplemented with 2% FCS, 2 mM L-glutamine and antibiotics Peni/Strepto at 37° C. for 40 hrs.
  • BSA bovine serum albumine
  • HMAF hyperimmune mouse ascites fluid
  • WN virus-specific HMAF 0801 strain IS-98-ST1
  • YF virus-specific HMAF 9803 strain FNV
  • Adherent cells were incubated either with EDTA (5 mM), mannan (20 ⁇ g/ml), anti-DC-SIGN Mab 1B10.2.6 (20 ⁇ g/mi), anti-LMCV Mab 12.5 (isotype control, 20 ⁇ g/ml), or DEN E-specific Mab 9D12 (dilution 1:50) in RPMI 1640 supplemented with 0.2% BSA for 20 min at room temperature before binding the virus for 2 hrs. Two hours after infection, cells were washed with RPMI 1640 and incubated with RMPI 1640 2% FCS for 40 hrs. Anti-DC-SIGN Mab 1B10.2.6 and anti-LMCV Mab 12.5 were obtained from Ali Amara.
  • TUNEL deoxyterminal transferase-mediated dUTP nick-end labeling
  • FIG. 3 indicates that 20 ⁇ g/ml of Mab 1B10.2.6 blocked FGA/NA d1d infection of DC cells as examined by IF assay.
  • anti-E Mab 9D12 reduced the infectivity of DEN-1 virus by 70%.
  • the production of infectious particles in FGA/NA did-infected DCs treated with Mab 1B10.2.6 was very low ( ⁇ 5 AP 61 FFU/ml).
  • THP/DC-SIGN To determine whether DC-SIGN confers DEN-1 virus infectivity at THP-1, we tested the DC-SIGN-expressing human monocytic THP cell line, THP/DC-SIGN (Kwon et al., Immunity, 16: 135, 2002). At an m.o.i. of 5 AP61FFU/cell, more than 50% of THP/DC-SIGN cells were positive for FGA/NA d1d antigens after 48 h of infection ( FIG. 4 ). Mortality of THP/DC-SIGN cells in which DEN-1 virus was replicating occurred after a 96-h period of infection.
  • YF virus vaccine strain 17D-204 did not replicate in THP/DC-SIGN cells at an m.o.i. as high as 50 VERO FFU/cell. It is also of interest that DC-SIGN could mediate enhancement of WN virus infection by monocytic cells ( FIG. 4 ).
  • THP/DC-SIGN cells were infected with DEN virus [DEN-1 virus strain FGA/NA did (French Guiana); DEN-2 virus strain Jam (Jamaica); DEN-3 virus strain PaH 881 (Thailand); DEN-4 virus strain 63632 (Birmanie)] at the multiplicities of infection (MOI) from 0.1 to 10 AP 61 FFU/cell as specified in example 3.
  • the percentage (%) of DEN antigen-positive THP cells expressing DC-SIGN was determined by indirect immunofluorescence using specific anti-DEN HMAF at 40 h post-infection ( FIG. 7 ). No DEN antigen was detected in THP cells infected with each DEN virus strain.
  • Hela DC-SIGN Flap The Hela cell line denoted “Hela DC-SIGN Flap” was deposited at the C.N.C.M., 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on Oct. 30, 2002, under the accession number 1-2949.
  • DC-SIGN human clone2 The DC-SIGN clone denoted “DC-SIGN human clone2” was deposited at the C.N.C.M., 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on Oct. 30, 2002, under the accession number 1-2950.
  • the hybridoma denoted “1B10.2.6” was deposited at the C.N.C.M., 28 rue du Dondel Roux, 75724 PARIS Cedex 15, France, on Nov. 7, 2002, under the accession number 1-2951.
  • DC-SIGN a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587-97.
  • DC-SIGN A Guide to Some Mysteries of Dendritic Cells. Cell 100, 491-94.

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US20080038286A1 (en) * 2005-09-22 2008-02-14 Prosci Inc. Glycosylated polypeptides produced in yeast mutants and methods of use thereof
US20080292644A1 (en) * 2005-08-31 2008-11-27 Shie-Liang Hsieh Compositions and methods for identifying response targets and treating flavivirus infection responses
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US7427469B2 (en) * 2002-11-05 2008-09-23 Institut Pasteur Method of treating cytomegalovirus with DC-SIGN blockers
DE60327237D1 (de) * 2002-11-05 2009-05-28 Pasteur Institut Dc-sign blockern und deren verwendung zur vorbeugung oder behandlung von viraleninfektionen
AT503387B1 (de) * 2006-03-27 2008-05-15 Univ Wien Pharmazeutische zusammensetzung zur vorbeugung von infektionen
EP2478359A4 (fr) * 2009-09-14 2013-03-20 Univ Rockefeller Procédés permettant d'identifier des composés anti-inflammatoires
CN104459146B (zh) * 2013-09-16 2016-06-22 大连医科大学 Dc-signr的一种新用途

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