WO2013059299A1 - Anticorps dirigés contre divers sous-types d'interféron, complexe ternaire interféron/récepteur d'interféron et utilisations associées - Google Patents

Anticorps dirigés contre divers sous-types d'interféron, complexe ternaire interféron/récepteur d'interféron et utilisations associées Download PDF

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WO2013059299A1
WO2013059299A1 PCT/US2012/060589 US2012060589W WO2013059299A1 WO 2013059299 A1 WO2013059299 A1 WO 2013059299A1 US 2012060589 W US2012060589 W US 2012060589W WO 2013059299 A1 WO2013059299 A1 WO 2013059299A1
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interferon
antibody
fragment
receptor
ifn
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PCT/US2012/060589
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Mark Walter
Sachdev Sidhu
Shane MIERSCH
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The Uab Research Foundation
The University Of Toronto
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    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249—Interferons
    • 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
    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the interferons are a family of inducible alpha-helical cytokines expressed in response to viral and bacterial exposure.
  • the two major classes of interferons (Type I and II) are synthesized by leukocytes and fibroblasts, respectively, and bind functionally distinct receptors expressed on virtually every nucleated cell.
  • Type I interferons ( ⁇ , ⁇ , or ⁇ ) induce hetero- dimerization of a pair of transmembranouse sub-units (interferon- ⁇ / ⁇ receptor 1 (IFNAR1) and interferon- ⁇ / ⁇ receptor 2 (IFNAR2)) resulting in the formation of a ternary complex that transduces a variety of intracellular signals invoking anti-viral responses.
  • IFNAR1 interferon- ⁇ / ⁇ receptor 1
  • IFNAR2 interferon- ⁇ / ⁇ receptor 2
  • type II interferon (the single ⁇ isoform) interacts with interferon- ⁇ receptors 1 and 2 (IFNGR1 and IFNGR2) eliciting immunomodulatory responses meant to confer protection against viral and microbial infection.
  • IFNGR1 and IFNGR2 interferon- ⁇ receptors 1 and 2
  • the interferons modulate the expression of up to 300 gene products involved in regulating critical cellular processes including pathogenic defense, cellular proliferation, inflammation, angiogenesis, and apoptosis.
  • antibodies or fragments thereof that specifically bind an interferon- interferon receptor complex.
  • antibodies or fragments thereof that specifically bind an interferon receptor, wherein binding of the antibody forms a ternary complex between the antibody, interferon receptor 1 and interferon receptor 2.
  • antibodies or fragments thereof that specifically bind one or two type I interferons selected from the group consisting of interferon-al, interferon-a2, interferon- a4, interferon-a5, interferon-a6, interferon-a7, interferon-a8, interferon-alO, interferon-al4, interferon-al 6, interferon-a 17, interferon-a21, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ .
  • methods of detecting an interferon-a subtype in a biological sample from a subject comprise contacting the biological sample with an antibody or fragment thereof specific for the interferon- ⁇ subtype and detecting the bound antibody.
  • the methods comprise administering an effective amount of any of the antibodies or fragments described herein to the subject, wherein the effective amount of the antibody or fragment thereof treats or prevents the autoimmune disease, viral infection, or cancer.
  • FC fragments comprising an interferon ligand and two interferon receptors selected from the group consisting of IFNAR1/IFNAR2, iFN l/iL-10R2, and IFNGR 1 /IFNGR2.
  • Figure 1 shows sequence data for FAB 7 CDRH1 (SEQ ID NO: 19), CDRH2 (SEQ ID NO:20), and CDRH3 (SEQ ID NO:21).
  • Figure 2 is a bar graph showing FAB7 binding (OD 450nm) to IFNa2, IFNa2-post, IFNAR1, IFNAR2, IFNAR1/IFNAR2 heterodimer, IFNAR 1 /IFNAR2/IFNa2 ternary complex, and Fc.
  • Figures 3 A to 3 C are graphs showing FAB7 affinity by surface plasmon resonance (SPR) for IFNAR 1 (Fig. 3A), IFNAR 1 /IFN AR2/IFN ⁇ ternary complex (Fig. 3B), and
  • Figures 4A and 4B show chromatographic analysis of equimolar IFNAR1, IFNAR2, and IFN- ⁇ with (Fig. 4B) or without (Fig. 4A) FAB7.
  • Figure 5 shows immunostaining of fixed HLl 16 cells with FAB7.
  • Figure 6 is a graph showing flow cytometric detection of live cells immunostained for Fab7 in the presence and absence of IFNa2a.
  • Figures 7A and 7B are dose response curves showing bioactivity (luciferase reporter assay) in HLl 16 cells treated with 50nM FAB7 (Fig. 7A) or with Fab7, Fab 8, or Fab 9 in the presence of constant physiological levels of IFN-a2a (1 pM, 10 pM, and 100 pM) (Fig. 7B).
  • Figure 8 shows FAB7 inhibition of IFN-a2a anti-viral activity.
  • Figure 8A is a graph showing anti-viral activity (% cell viability) of IFN-a2a with or without Fab7 as a function of IFN or Fab concentration (M).
  • Figure 8B is a dose response curve showing virus cytotoxicity (OD 490nm) of cells incubated with serial dilutions of IFNa2a with constant 200nM Fab7.
  • Figure 9 shows sequence data for FAB 10 CDRHl (SEQ ID NO:22), CDRH2 (SEQ ID NO:23), and CDRH3 (SEQ ID NO:24); and FABl 1 CDRHl (SEQ ID NO:25), CDRH2 (SEQ ID NO:26), and CDRH3 (SEQ ID NO:27).
  • Figures 10A to 10B are bar graphs showing binding of FAB10 (Fig. 10A) and FABl 1 (Fig. 10B) to IFNa2, IFNa2-post, IFNAR1, IFNAR2, IFNAR1/IFNAR2 heterodimer,
  • Figures 11A to 1 IB are plots showing estimates of FAB10 (Fig. 11A) and FABl 1 (Fig. 1 IB) affinity for IFNAR2 by multi-point competitive ELISA.
  • Figures 12A to 12D are graphs showing FAB10 (Figs. 12A and 12B) and FABl 1 (Figs. 12C and 12D) affinity by SPR for IFNAR2 (Figs. 12A and 12C) or IFNAR l/IFNAR2/IFNa2 ternary complex (Fig. 12B and 12D).
  • Figures 13 A to 13C shows chromatographic analysis of equimolar IFNAR1, IFNAR2, and IFN- ⁇ without (Fig. 13A) or with FAB 10 (Fig. 13B) or FABl 1 (Fig. 13C).
  • Figures 14A and 14B show FAB 10 cell-binding by flow cytometry or
  • Figure 15 is a graph showing IFN-induced luciferase activity after treatment with IFNa2A or treatment with FAB 10, FABl 1, or FAB 12 in combination with ⁇ , ⁇ , or ⁇ IFN.
  • Figure 16 is a graph showing anti-viral activity (% cell viability) of IFN-a2a with or without FAB 10 as a function of IFN or Fab concentration (M).
  • Figure 17 shows sequence data for FABS A2-1 CDRHl (SEQ ID NO:28), CDRH2 (SEQ ID NO:29), and CDRH3 (SEQ ID NO:31); A2-2 CDRHl (SEQ ID NO:31), CDRH2 (SEQ ID NO:32), and CDRH3 (SEQ ID NO:33); and A2-3 CDRHl (SEQ ID NO:34), CDRH2 (SEQ ID NO:35), and CDRH3 (SEQ ID NO:36).
  • Figures 18A to 18C are bar graphs showing binding of FABS A2-1 (Fig. 18A), A2-2
  • Figure 19 is a bar graph showing binding of FABS A2-1, A2-2, and A2-3 to IFN-alpha subtypes al, a2a, a2b, a4, a5, al4, or to BSA or Fc.
  • Figures 20A to 20C are plots showing affinity of FABS A2-1 (Fig. 20A), A2-2 (Fig. 20B), and A2-3 (Fig. 20C) for IFN-a2a by competitive ELISA.
  • Figures 21A to 21C are graphs showing affinity of FABS A2-1 (Fig. 22A), A2-2 (Fig. 22B), and A2-3 (Fig. 22C) for IFN-a2a by SPR.
  • Figure 22 shows ligand-targeted antagonists inhibited IFN-induced signals.
  • Figure 22A is a graph showing bioactivity (luciferase reporter assay) of IFN-a2 in the presence and absence of 50 nM FABS A2-1 or A2-3.
  • Figures 22B to 22E are graphs showing bioactivity of IFN-a2 in the presence of FABS and IFNa subtypes.
  • Figure 23 is a bar graph showing binding of Fabs A2-4, A2-5, and A2-6 to IFN-a2a, IFNa2 post-block, IFNARl/IFNa2 post-block, and Fc .
  • Figure 24 is a bar graph showing binding of FABS A2-4, A2-5, and A2-6 to IFN-alpha subtypes al, a2a, a2b, a4, a5, al4, or to BSA or Fc.
  • Figure 25 is a graph showing affinity of FABS A2-4, A2-5, and A2-6 for IFNa2a determined by multi-point competitive ELISA.
  • Figures 26A to 26C are graphs showing affinity of FABS A2-4 (Fig. 26A), A2-5 (Fig. 26B), and A2-6 (Fig. 26C) for IFNa2a determined by SPR.
  • Figure 27 shows sequence data for FAB1 CDRHl (SEQ ID NO: 1), CDRH2 (SEQ ID NO: 1), and
  • Figure 28 is a bar graph showing binding of FABS 1-6 to IFNa2, IFNa2 -post-block, IFNARl, IFNAR2, IFNAR1/IFNAR2 heterodimer, IFNARl /IFNAR2/IFNa2 ternary complex, and Fc.
  • Figure 29 is a graph showing an SPR protocol for determining affinity for the ternary complex.
  • Figure 30 is a series of graphs showing affinity of FABS 1-6 for IFNa2a ternary-complex by SPR analysis.
  • Figure 31 is a graph showing affinity of FABS 1-6 for ⁇ ternary-complex by SPR analysis.
  • Figure 32 is a graph showing chromatographic analysis of the effects of FABS 1-6 on stability of the ternary complex.
  • Figure 33 shows enhancement of IFNa2 induced gene expression (luciferase activity) and anti-viral activity by Fab5 compared to Fab6.
  • Figure 33A shows dose response curves of bioactivity (luciferase reporter assay) in cells treated with Fabl, Fab2, Fab3, Fab4, Fab5, or Fab6 in the presence of constant physiological levels of IFN-a2a (1 pM or 10 pM).
  • Figure 33B is a graph showing anti-viral activity (% cell viability) of IFN-a2a, or of Fab5 or Fab 6 with or without ⁇ IFN-a2a.
  • Figure 33C is a dose response curve showing virus cytotoxicity (OD 490nm) of cells incubated with serial dilutions of IFNa2a with constant 200nM Fab5 or Fab6.
  • Figure 34 is a graph showing binding of IFN-a2a with IFNAR2 in the presence and absence of Fabl, Fab2, Fab3, Fab4, Fab5, or Fab6 by SPR analysis.
  • Figure 35 is a graph showing binding of IFN-a2a with Fab 4, but not with Fabl, Fab2, Fab3, Fab5, or Fab6 by SPR analysis.
  • Figures 36A and 36B are immunomicroscopy images showing cellular binding of FAB4 with (Fig. 36B) or without (Fig. 36A) IFN-a2a.
  • Figure 37 is a graph showing FAB4 immunostaining of live cells with and without IFN- a2a by flow cytometry.
  • Figure 38 is a graph showing that stabilization of ligand IFNAR2 interactions by FAB5 is pan-IFN-a-subtype specific.
  • Figures 39A and 39B are immunomicroscopy images showing binding of FAB5 with (Fig. 39B) or without (Fig. 39A) IFN-a2a to HL116 cells .
  • Figure 40 is a set of graphs showing FAB5 (left) and FAB6 (right) immunostaining of live cells with and without IFN-a2a by flow cytometry.
  • Figure 41 is a table of the properties of selected Fabs to components of the functional IFN-receptor complex with Type I Interferons.
  • Figure 42 is a bar graph showing binding of A6-1, A6-2, A8-1 and A8-2 antibodies to IFNa6 (bar 1), IFNa8 (bar 3), BSA (bar 2) or blank control (bar 4) by ELISA.
  • Figures 43A and 43B are graphs showing the bioactivity (luciferase units as a function of IFN or Fab concentration (M)) of IFNa6 (Fig. 43 A) or IFNa8 (Fig. 43 B) with and without the addition of the IF a6-specific Fabs A6-1 or A6-2 (Fig. 43 A) or the IFNa8-specific Fabs A8-1 or A8-2 (Fig. 43B).
  • Figures 44A and 44B are graphs showing results of bioassays to determine the subtype specificity of IFNa6 Fabs.
  • Figure 44A is a graph showing bioactivity (luciferase units as a function of IFN or Fab concentration (M)) of IFNa2a, or A6-1 in combination with IFNala, IFNa2a, IFNa2b, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNalO, IFNal4, IFNal6, IFNal7, or IFNa21.
  • Figure 44B is a graph showing bioactivity (luciferase units as a function of IFN or Fab concentration (M)) of IFNa6, or A6-2 in combination with IFNa6, IFNal6, IFNa8, IFNalO, IFNal4, IFNa7, IFNal7, IFNa21, IFNa2a, IFNa2b, IFNa4, IFNa5, or IFNala.
  • Figures 45 A and 45B are graphs showing results of bioassays to determine the subtype specificity of IFNa8 Fabs.
  • Figure 45A is a graph showing bioactivity (luciferase units as a function of IFN or Fab concentration (M)) of IFNa8, or A8-1 in combination with IFNala, IFNa2a, IFNa2b, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNalO, IFNal4, IFNal6, IFNal7, or IFNa21.
  • Figure 45B is a graph showing bioactivity (luciferase units as a function of IFN or Fab concentration (M)) of IFNa8, or A8-2 in combination with IFNala, IFNa2a, IFNa2b, IFNa4, IFNa5, IFNa6, IFNa7, IFNa8, IFNalO, IFNal4, IFNal6, IFNal7, or IFNa21.
  • Figure 46 shows IFNal selective antibody clone sequence and binding affinity.
  • Figure 46A shows sequence data for anti-IFNal CDRL3 (SEQ ID NO: 37), CDRHl (SEQ ID NO: 38), CDRH2 (SEQ ID NO:39), and CDRH3 (SEQ ID NO:40).
  • Figure 46B is a bar graph showing binding (OD 450nm) of IFNal selective antibody to IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 46C is a graph showing affinity of IFNal selective antibody to IFNal (OD450 nM as a function of IFNal concentration ( ⁇ )).
  • Figure 47 shows IFNa2ab selective antibody clone sequence and binding affinity.
  • Figure 47A shows sequence data for anti-IFNa2ab (A2-1) CDRL3 (SEQ ID NO:41), CDRHl (SEQ ID NO:28), CDRH2 (SEQ ID NO:29), and CDRH3 (SEQ ID NO:30).
  • Figure 47B is a bar graph showing binding (OD 450nm) of IFNa2ab selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or blanks.
  • Figure 47C is a graph showing affinity of IFNa2ab selective antibody to IFNa2a (OD450 nM as a function of IFNa2a concentration ( ⁇ )).
  • Figure 48 is an image of a Western blot showing that anti-IFNa2ab antibody clone A2-1 neutralizes cellular IFNa2ab STAT1 phosphorylation.
  • 10 5 Daudi cells were serum starved for 48 hours then stimulated with 0 ng/ml (col. 1-2), 0.1 ng/ml (col. 3-4), 1.0 ng/ml (col. 5-6), 10 ng/ml (col. 7-8), or 100 ng/ml (col. 9-10) IFNa2ab and with (col. 1, 3, 5, 7, 9) or without (col. 2, 4, 6, 8, 10) 10 ⁇ g/ml Fab A2-1.
  • Western blots were immunostained for STAT1 (bottom row) or pSTATl (Tyr701).
  • Figure 49 shows IFNa4ab selective antibody clone sequence and binding affinity.
  • Figure 49A shows sequence data for anti-IFNa4ab clone 1 CDRL3 (SEQ ID NO:42), CDRHl (SEQ ID NO:43), CDRH2 (SEQ ID NO:44), and CDRH3 (SEQ ID NO:45); clone 12 CDRL3 (SEQ ID NO:46), CDRHl (SEQ ID NO:47), CDRH2 (SEQ ID NO:48), and CDRH3 (SEQ ID NO:49); and clone 26 CDRL3 (SEQ ID NO:50), CDRHl (SEQ ID NO:51), CDRH2 (SEQ ID NO:52), and CDRH3 (SEQ ID NO:53).
  • Figures 48B-48D are graphs showing affinity of IFNa4ab selective antibody clone 1 (Fig. 49B), 12 (Fig. 49C), or 26 (Fig. 49D) to IFNa4ab (OD450 nM as a function of IFNa4ab concentration ( ⁇ )).
  • Figure 49E is a bar graph showing binding (OD 450nm) of IFNa4ab selective antibody clone 1, 12, or 26 to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 50 shows IFNa5 selective antibody clone sequence and binding affinity.
  • Figure 50A shows sequence data for anti-IFNa5 CDRL3 (SEQ ID NO:54), CDRHl (SEQ ID NO:55), CDRH2 (SEQ ID NO:56), and CDRH3 (SEQ ID NO:57).
  • Figure 50B is a bar graph showing binding (OD 450nm) of IFNa5 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 50C is a graph showing affinity of IFNa5 selective antibody to IFNa5 (OD450 nM as a function of IFNa5 concentration ( ⁇ )).
  • Figure 51 shows IFNa6 selective antibody clone sequence and binding affinity.
  • 51A shows sequence data for anti-IFNa6 clone GOl CDRL3 (SEQ ID NO:58), CDRHl (SEQ ID NO:58),
  • Figure 5 IB is a bar graph showing binding
  • FIG. 51C is a graph showing affinity of IFNa6 selective antibody clone GOl (Fig. 51C), G10 (Fig. 5 ID), or Hl l
  • FIG. 5 IE to IFNa6 (OD450 nM as a function of IFNa6 concentration ( ⁇ )).
  • Figure 52 shows IFNa7 selective antibody clone sequence and binding affinity.
  • Figure 52A shows sequence data for anti-IFNa7 CDRL3 (SEQ ID NO:70), CDRH1 (SEQ ID NO:71), CDRH2 (SEQ ID NO:72), and CDRH3 (SEQ ID NO:73).
  • Figure 52B is a bar graph showing binding (OD 450nm) of IFNa7 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 52C is a graph showing affinity of IFNa7 selective antibody to IFNa7 (OD450 nM as a function of IFNa7 concentration ( ⁇ )).
  • Figure 53 shows IFNa8 selective antibody clone sequence and binding affinity.
  • Figure 53A shows sequence data for anti-IFNa8 CDRL3 (SEQ ID NO:74), CDRH1 (SEQ ID NO:75), CDRH2 (SEQ ID NO:76), and CDRH3 (SEQ ID NO:77).
  • Figure 53B is a bar graph showing binding (OD 450nm) of IFNa8 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 53C is a graph showing affinity of IFNa8 selective antibody to IFNa8 (OD450 nM as a function of IFNa8 concentration ( ⁇ )).
  • Figure 54 shows IFNalO selective antibody clone sequence and binding affinity.
  • Figure 54A shows sequence data for anti-IFNalO clone D01 CDRL3 (SEQ ID NO:78), CDRH1 (SEQ ID NO:79), CDRH2 (SEQ ID NO:80), and CDRH3 (SEQ ID NO:81); and clone D05 CDRL3 (SEQ ID NO: 82), CDRH1 (SEQ ID NO: 83), CDRH2 (SEQ ID NO: 84), and CDRH3 (SEQ ID NO:85).
  • Figure 54B is a bar graph showing binding (OD 450nm) of IFNalO selective antibody clone D01 or D05 to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 55 shows IFNal4 selective antibody clone sequence and binding affinity.
  • Figure 55A shows sequence data for anti-IFNal4 clone 1 CDRL3 (SEQ ID NO:86), CDRH1 (SEQ ID NO:87), CDRH2 (SEQ ID NO:88), and CDRH3 (SEQ ID NO:89); and clone 30 CDRL3 (SEQ ID NO:90), CDRH1 (SEQ ID NO:91), CDRH2 (SEQ ID NO:92), and CDRH3 (SEQ ID NO:93).
  • Figure 55B is a bar graph showing binding (OD 450nm) of IFNal 4 selective antibody clone 1 or clone 30 to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or blanks.
  • Figure 53C is a graph showing affinity of IFNal4 selective antibody clone 1 (Fig. 55C) or clone 30 (Fig. 55D) to IFNal4 (OD450 nM as a function of IFNal4 concentration ( ⁇ )).
  • Figure 56 shows affinity matured IFNal4 selective antibody clone binding affinity.
  • Figure 56B is a bar graph showing binding (OD 450nm) of affinity matured IFNal4 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 56B is a graph showing affinity of affinity matured IFNal4 selective antibody to IFNal 4 (OD450 nM as a function of IFNal 4 concentration ( ⁇ )).
  • Figure 57 shows IFNal 6 selective antibody clone sequence and binding affinity.
  • Figure 57A shows sequence data for anti-IFNal6 CDRL3 (SEQ ID NO:94), CDRH1 (SEQ ID NO:95), CDRH2 (SEQ ID NO:96), and CDRH3 (SEQ ID NO:97).
  • Figure 57B is a bar graph showing binding (OD 450nm) of IFNal6 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • Figure 57C is a graph showing affinity of IFNal 6 selective antibody to IFNal 6 (OD450 nM as a function of IFNal 6 concentration ( ⁇ )).
  • Figure 58 shows IFNa21 selective antibody clone sequence and binding affinity.
  • Figure 58A shows sequence data for anti-IFNa21 CDRL3 (SEQ ID NO:98), CDRH1 (SEQ ID NO:99), CDRH2 (SEQ ID NO: 100), and CDRH3 (SEQ ID NO: 101).
  • Figure 58B is a bar graph showing binding (OD 450nm) of IFNa21 selective antibody to (from left to right) IFNal, a2a, a2b, a4, a6, a7, a8, alO, al4, al6, al7, a21, BSA, or a blanks.
  • the ternary complex can, for example, comprise a type I interferon, interferon- ⁇ / ⁇ receptor 1 (IFNAR1), and interferon- ⁇ / ⁇ receptor 2 (IFNAR2).
  • the ternary complex comprises an interferon- ⁇ , an interferon- ⁇ receptor 1 (IFNGR1), and an interferon- ⁇ receptor 2 (IFGNR2).
  • the ternary complex comprises an interferon- ⁇ , an interferon- ⁇ receptor 1 (IFN Rl), and an interleukin-10 receptor 2 (IL-10R2).
  • a type I interferon can, for example, be selected from the group consisting of interferon- al, interferon-a2, interferon-a4, interferon-a5, interferon-a6, interferon-a7, interferon-a8, interferon-alO, interferon-al4, interferon-al6, interferon-al7, interferon-a21, interferon- ⁇ , interferon-K, interferon- ⁇ , and interferon- ⁇ .
  • An interferon- ⁇ can be selected from the group consisting of interferon- ⁇ , interferon- 2, and interferon- 3.
  • the antibody or fragment thereof does not bind an uncomplexed interferon or interferon receptor.
  • the antibody or fragment thereof does not bind an interferon alone or an interferon receptor alone, and can only bind an interferon-interferon receptor ternary complex.
  • antibodies or fragments thereof that specifically bind interferon receptors, wherein the binding of the antibody forms a ternary complex between the antibody, interferon receptor 1, and interferon receptor 2.
  • the binding of the antibody activates the interferon signaling cascade.
  • the ternary complex can, for example, comprise the antibody or fragment thereof, IFNAR1, and IFNAR2; the antibody or fragment thereof, IFNGR1, and IFNGR2; and the antibody or fragment thereof, IFN Rl, and IL-10R2.
  • antibodies or fragments thereof that specifically bind one or two type I interferons selected from the group consisting of interferon-al, interferon-a2, interferon- a4, interferon-a5, interferon-a6, interferon-a7, interferon-a8, interferon-alO, interferon-al4, interferon-al 6, interferon-a 17, interferon-a21, interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , and interferon- ⁇ .
  • the antibodies or fragments provided herein are humanized.
  • the antibody fragments provided herein are Fab fragments.
  • the antibodies provided herein are monoclonal antibodies.
  • the antibodies or fragments provided herein are neutralizing, antagonistic, or agonistic antibodies or fragments.
  • a neutralizing antibody can bind an interferon subtype in the serum and prevent the interferon subtype from binding the intended receptor.
  • an antagonistic antibody can bind an interferon/interferon receptor ternary complex and inhibit/reduce the interferon signaling cascade.
  • an agonistic antibody can bind an interferon/interferon receptor ternary complex and activate/enhance the interferon signaling cascade.
  • antibody encompasses, but is not limited to, whole
  • immunoglobulin i.e., an intact antibody of any class.
  • Native antibodies are usually used to construct immunoglobulin (i.e., an intact antibody) of any class.
  • Native antibodies are usually used to construct immunoglobulin (i.e., an intact antibody) of any class.
  • heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains.
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • variable is used herein to describe certain portions of the antibody domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • epitope is meant to include any determinant capable of specific interaction with the provided antibodies or fragments.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Identification of the epitope that the antibody or fragment recognizes is performed as follows. First, various partial structures of the target molecule that the antibody or fragment recognizes are prepared. The partial structures are prepared by preparing partial peptides of the molecule. Such peptides are prepared by, for example, known oligopeptide synthesis technique or by incorporating DNA encoding the desired partial polypeptide in a suitable expression plasmid. The expression plasmid is delivered to a suitable host, such as E.
  • a series of polypeptides having appropriately reduced lengths, working from the C- or N-terminus of the target molecule, can be prepared by established genetic engineering techniques. By establishing which fragments react with the antibody or fragment, the epitope region is identified. The epitope is more closely identified by synthesizing a variety of smaller peptides or mutants of the peptides using established oligopeptide synthesis techniques. The smaller peptides are used, for example, in a competitive inhibition assay to determine whether a specific peptide interferes with binding of the antibody to the target molecule. If so, the peptide is the epitope to which the antibody binds.
  • kits such as the SPOTs Kit (Genosys Biotechnologies, Inc., The Woodlands, TX) and a series of multipin peptide synthesis kits based on the multipin synthesis method (Chiron Corporation, Emeryvile, CA) may be used to obtain a large variety of oligopeptides.
  • antibody or fragments thereof can also encompass chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • fragments of antibodies which maintain interferon, interferon receptor, or interferon/interferon receptor ternary complex binding activity are included within the meaning of the term antibody or fragment thereof.
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane.
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Patent No. 4,704,692, the contents of which are hereby incorporated by reference in their entirety.
  • the antibody is a monoclonal antibody.
  • monoclonal antibody refers to an antibody from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) or Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York (1988). In a hybridoma method, a mouse or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent can be an interferon, interferon receptor, or interferon/interferon receptor ternary complex or an immunogenic fragment thereof.
  • peripheral blood lymphocytes are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).
  • Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium”) substances that prevent the growth of HGPRT-deficient cells.
  • Immortalized cell lines useful here are those that fuse efficiently, support stable high level expression of antibody by the selected antibody -producing cells, and are sensitive to a medium such as HAT medium.
  • Immortalized cell lines include murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center; San Diego, Calif, and the American Type Culture Collection; Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the interferon, interferon receptor, or interferon/interferon receptor ternary complex or selected epitopes thereof.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells can serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, plasmacytoma cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody provided herein, or can be substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for the interferon, interferon receptor, or interferon/interferon receptor ternary complex and another antigen-combining site having specificity for a different antigen.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348, U.S. Patent No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988).
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the Fab fragments produced in the antibody digestion can also contain the constant domains of the light chain and the first constant domain of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region.
  • the F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • One method of producing proteins comprising the provided antibodies or polypeptides is to link two or more peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert- butyloxycarbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA).
  • Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert- butyloxycarbonoyl) chemistry Applied Biosystems, Inc.; Foster City, CA.
  • a peptide or polypeptide corresponding to the antibody provided herein can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the other fragment.
  • peptide condensation reactions By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof.
  • Grant GA Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis. Springer Verlag Inc., NY).
  • the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or fragment thereof via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al, Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776 779 (1994)).
  • the first step is the chemos elective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • IL-8 human interleukin 8
  • unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non peptide) bond (Schnolzer et al., Science 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et al, Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
  • the provided polypeptide fragments can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as a bacterial, adenovirus or baculovirus expression system.
  • an expression system capable of producing the polypeptide fragments thereof, such as a bacterial, adenovirus or baculovirus expression system.
  • amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity.
  • the provided fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or epitope. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio longevity, to alter its secretory characteristics, and the like.
  • the fragment can possess a bioactive property, such as binding activity, regulation of binding at the binding domain, and the like.
  • Functional or active regions may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site specific mutagenesis of the nucleic acid encoding the antigen. (Zoller et al, Nucl. Acids Res. 10:6487- 500 (1982)).
  • the antibody modulates the activity of the interferon, interferon receptor, or interferon/interferon receptor ternary complex by neutralizing, antagonizing, or agonizing the interferon, interferon receptor, or interferon/interferon receptor ternary complex.
  • the humanized or human antibody comprises at least one complementarity determining region (CDR) of an antibody having the same epitope specificity as an antibody produced by a disclosed hybridoma cell line.
  • the antibody can comprise all CDRs of an antibody having the same epitope specificity as an antibody produced by the hybridoma cell line.
  • the humanized or human antibody can comprise at least one residue of the framework region of the monoclonal antibody produced by a disclosed hybridoma cell line.
  • Humanized and human antibodies can be made using methods known to a skilled artisan; for example, the human antibody can be produced using a germ-line mutant animal or by a phage display library.
  • Antibodies can also be generated in other species and humanized for administration to humans.
  • fully human antibodies can also be made by immunizing a mouse or other species capable of making a fully human antibody (e.g., mice genetically modified to produce human antibodies) and screening clones that bind the interferon, interferon receptor, or interferon/interferon receptor ternary complex. See, e.g., Lonberg and Huszar, Int. Rev.
  • humanized and human in relation to antibodies relate to any antibody which is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject.
  • the terms include fully humanized or fully human as well as partially humanized or partially human.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen- binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all or at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature, 321 :522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the methods described in Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); or Verhoeyen et al, Science 239: 1534- 1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • the nucleotide sequences encoding the provided antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). These nucleotide sequences can also be modified, or humanized, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see, e.g., U.S. Patent No. 4,816,567).
  • the nucleotide sequences encoding any of the provided antibodies can be expressed in appropriate host cells. These include prokaryotic host cells including, but not limited to, E.
  • Eukaryotic host cells can also be utilized. These include, but are not limited to, yeast cells (for example, Saccharomyces cerevisiae and Pichia pastoris), and mammalian cells such as VERO cells, HeLa cells, Chinese hamster ovary (CHO) cells, W138 cells, BHK cells, COS-7 cells, 293T cells and MDCK cells.
  • yeast cells for example, Saccharomyces cerevisiae and Pichia pastoris
  • mammalian cells such as VERO cells, HeLa cells, Chinese hamster ovary (CHO) cells, W138 cells, BHK cells, COS-7 cells, 293T cells and MDCK cells.
  • the antibodies produced by these cells can be purified from the culture medium and assayed for binding, activity, specificity or any other property of the monoclonal antibodies by utilizing the methods set forth herein and standard in the art.
  • Transgenic animals e.g., mice
  • J(H) antibody heavy chain joining region
  • chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci.
  • Human antibodies can also be produced in phage display libraries (Hoogenboom et al, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, ed., p. 77 (1985); Boerner et al, J.
  • the provided antibody or fragment can be labeled or fused with another polypeptide or fragment thereof.
  • the provided antibodies or fragments thereof can be fused with a therapeutic agent.
  • an antibody or fragment thereof that binds to the interferon, interferon receptor, or interferon/interferon receptor ternary complex may be linked to a therapeutic agent.
  • the linkage can be covalent or noncovalent (e.g., ionic).
  • Therapeutic agents include but are not limited to toxins, including but not limited to plant and bacterial toxins, small molecules, peptides, polypeptides and proteins.
  • a target cell or target cells are interferon, interferon receptor, or interferon/interferon receptor ternary complex positive cells.
  • therapeutic agents include chemotherapeutic agents, a radiotherapeutic agent, and immunotherapeutic agent, as well as combinations thereof.
  • the antibody complex delivered to the subject can be multifunctional, in that it exerts one therapeutic effect by binding to the interferon, interferon receptor, or interferon/interferon receptor ternary complex and a second therapeutic by delivering a supplemental therapeutic agent.
  • the therapeutic agent can act extracellularly, for example by initiating or affecting an immune response, or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling.
  • the therapeutic agent is optionally cleavable from the antibody or fragment. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.
  • the antibody or fragments thereof can also act extracellularly, for example by initiating, affecting, enhancing or reducing an immune response without being linked in a molecular complex with a therapeutic agent.
  • Such an antibody is known in the art as an unconjugated antibody.
  • An unconjugated antibody can directly induce negative growth signal or apoptosis or indirectly activate a subject's defense mechanism to mediate anti-tumor activity or anti-viral activity.
  • toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof.
  • An antibody or antibody fragment may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e. g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. , dactinomycin
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytar
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. Pat. No. 4,676, 980.
  • an interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody a humanized interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody; heavy and light chain immunoglobulins of an interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody; CDRs of an interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody; and certain truncations of these antibodies or immunoglobulins that perform the functions of the full length antibody or immunoglobulin.
  • nucleic acid sequence coding for the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies can be altered.
  • nucleic acids that encode the polypeptide sequences, variants, and fragments of thereof are disclosed. These sequences include all degenerate sequences related to a specific protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences.
  • the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies and fragments provided herein have a desired function.
  • the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody or fragment binds a specific epitope of the interferon, interferon receptor, or interferon/interferon receptor ternary complex. Binding of the epitope can, for example, neutralize, antagonize, or agonize the biological activity of the interferon, interferon receptor, or interferon/interferon receptor ternary complex.
  • interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies or fragments described herein can be further modified and varied so long as the desired function is maintained. It is understood that one way to define any known modifications and derivatives or those that might arise, of the disclosed nucleic acid sequences and proteins herein is through defining the modifications and derivatives in terms of identity to specific known sequences.
  • polypeptides which have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies or fragments provided herein.
  • the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
  • Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Adv. Appl. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized
  • Protein modifications include amino acid sequence modifications. Modifications in amino acid sequence may arise naturally as allelic variations (e.g., due to genetic
  • polymorphism may arise due to environmental influence (e.g., exposure to ultraviolet light), or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion, and substitution mutants. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional modifications. Insertions include amino and/or terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e., a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • substitutional modifications are those in which at least one residue has been removed and a different residues inserted in its place. Such substitutions generally are made in accordance with the following Table 1 and are referred to as conservative substitutions.
  • Modifications including the specific amino acid substitutions, are made by known methods.
  • modifications are made by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the
  • the methods comprise administering an effective amount of any of the antibodies or fragments described herein to the subject, wherein the effective amount of the antibodies or fragments thereof treat or prevent the autoimmune disease, viral infection, or cancer.
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, Sjorgren's syndrome, systemic lupus erythematosus, and Type I diabetes.
  • the viral infection is selected from hepatitis B or hepatitis C.
  • the cancer is selected from the group consisting of hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, lymphoma, Kaposi's sarcoma, renal cell carcinoma, and melanoma.
  • compositions comprising the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies or fragments described herein.
  • the herein provided compositions are suitable for administration in vitro or in vivo.
  • the compositions comprising the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies can further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
  • the carrier is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5.
  • Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides.
  • Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the composition, e.g., the polypeptides described herein and the adenovirus encoding an antigen to humans or other subjects.
  • compositions are administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • the compositions are administered via any of several routes of administration, including topically, orally, parenterally,
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.
  • compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.
  • a nucleic acid molecule encoding the interferon, interferon receptor, or interferon/interferon receptor ternary complex specific antibody or fragment is administered by a vector comprising the nucleic acid molecule.
  • compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non- viral based deliver systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding expression of the nucleic acid molecule and/or adapter polypeptide in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other R A viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al, Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them.
  • replication-defective adenoviruses has been described (Berkner et al, J. Virology 61 : 1213-20 (1987); Massie et al, Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al, J. Virology 57:267-74 (1986); Davidson et al, J. Virology 61 : 1226-39 (1987); Zhang et al, BioTechniques 15:868-72 (1993)).
  • the benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles.
  • Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites.
  • Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
  • VLPs Virus like particles
  • Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
  • interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies can be delivered by subviral dense bodies (DBs).
  • DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al, Gene Therapy 10:278-84 (2003).
  • interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
  • Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules encoding the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibodies, wherein the nucleic acids are operably linked to an expression control sequence.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions.
  • Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably
  • viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably
  • CMV cytomegalovirus
  • heterologous mammalian promoters e.g., ⁇ -actin promoter or EFla promoter
  • hybrid or chimeric promoters e.g., CMV promoter fused to the ⁇ -actin promoter. Promoters from the host cell or related species are also useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' or 3 ' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pair (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the promoter and/or the enhancer can be inducible (e.g., chemically or physically regulated).
  • a chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal.
  • a physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed.
  • the promoter and/or enhancer region can be active in a cell type specific manner.
  • the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type.
  • Preferred promoters of this type are the CMV promoter, the SV40 promoter, the ⁇ -actin promoter, the EF 1 a promoter, and the retroviral long terminal repeat (LTR).
  • the vectors also can include, for example, origins of replication and/or markers.
  • a marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell.
  • the marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E.
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
  • Tag sequences such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GFP glutathione S-transferase
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • FLAGTM tag FLAGTM tag
  • the methods comprise contacting the biological sample with an antibody or fragment thereof, as described herein, and detecting the bound antibody.
  • the loss of biological activity of the interferon-a subtype is detected as the antibody neutralizes or antagonizes the biological activity of the interferon-a subtype.
  • the bound antibody is detected by an ELISA assay, a Western blot, magnetic beads, or by flow cytometry. Methods of detecting antibody-ligand complexes are known in the art.
  • FC fragments comprising an interferon ligand and two interferon receptors selected from the group consisting of IFNAR1/IFNAR2, IFNGR1/IFNGR2, and IF R1/IL-10R2.
  • the methods comprise producing a phage display library and identifying one or more Fab fragments, wherein the Fab fragment binds an
  • interferon/receptor interface or an interferon-receptor/interferon-receptor interface.
  • peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
  • subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal.
  • a mammal e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
  • patient or subject may be used interchangeably and can refer to a subject with a disease or disorder (e.g., autoimmune disease, viral infection, or cancer).
  • patient or subject includes human and veterinary subjects.
  • a subject at risk of developing a disease or disorder can be genetically predisposed to the disease or disorder, e.g., have a family history or have a mutation in a gene that causes the disease or disorder, or show early signs or symptoms of the disease or disorder.
  • a subject currently with a disease or disorder has one or more than one symptom of the disease or disorder and may have been diagnosed with the disease or disorder.
  • a therapeutically effective amount of the agents described herein are administered to a subject prior to onset (e.g., before obvious signs of autoimmune disease, viral infection, or cancer) or during early onset (e.g., upon initial signs and symptoms of autoimmune disease, viral infection, or cancer or upon exposure to a virus).
  • Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of cancer.
  • Prophylactic administration can be used, for example, in the preventative treatment of subjects diagnosed with a genetic predisposition to cancer.
  • Therapeutic treatment involves administering to a subject a therapeutically effective amount of the agents described herein after diagnosis or development of cancer.
  • the subject is administered an effective amount of the interferon, interferon receptor, or interferon/interferon receptor ternary complex antibody or fragment.
  • effective amount and effective dosage are used interchangeably.
  • the term effective amount is defined as any amount necessary to produce a desired physiologic response (e.g., reduction of fatigue or fever, maintenance of body's ability to fight disease or infection).
  • Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed).
  • the dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of
  • treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of a therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.
  • references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
  • any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus , if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
  • IFNARl is the 65 kDa monomeric partner that binds ligand bound to 25 kDa IFNAR2 to form a functional, ternary signaling complex. It has affinities for alpha IFNs that range of 0.5-5 ⁇ and is thought to bind to ligand following ligand interaction with IFNAR2.
  • IFNARl was targeted using purified protein isolated from a C-terminal Fc-fusion expression construct. For selection, IFNARl was panned against by immobilizing 5 ⁇ g/ml of protein in the wells of the microplate. In an equal number of wells, the same concentration of expressed Fc-tag alone was immobilized.
  • Purified library F (diversity of 1 x 10 10 at a concentration of 10 13 virus particles) was first exposed to immobilized Fc-tag to remove tag-specific binders. The supernatant was then transferred to target protein (IFNARl), allowed to equilibrate, washed and eluted using 100 mM HC1. Eluted phage were used to infect XL-1 Blue eels (OD 0.8-1.0) and plated on carbenicillin agar plates to isolate individual clones that bound specifically to IFNARl .
  • target protein IFNARl
  • Eluted phage were used to infect XL-1 Blue eels (OD 0.8-1.0) and plated on carbenicillin agar plates to isolate individual clones that bound specifically to IFNARl .
  • FAB7 Specificity of FAB7 was determined by target binding of the isolated Fab-phage in an ELISA assay.
  • 2 ⁇ of IFNARl, IFNAR2, IFN-a2a, and IFNAR1/IFNAR2 heterodimer were prepared as separate solutions in IX PBS. Proteins were immobilized by passive adsorption in wells of a microplate by overnight incubation at 4°C. The next day, the plate was blocked with a solution of 0.2% BSA/1X PBS (blocking solution) for 1 hour before washing 4 times with IX PBS with 0.05% Tween (PT) buffer.
  • Fab bioactivity was next assessed in cells. Bioactivity was assessed by a luciferase reporter assay in a cell line (HL116) derived from the HT1080 human fibrosarcoma line transfected with luciferase reporter cDNA driven by the immediate early IFN inducible 6-16 promoter, specific for Type I interferons.
  • HL116 human fibrosarcoma line transfected with luciferase reporter cDNA driven by the immediate early IFN inducible 6-16 promoter, specific for Type I interferons.
  • an anti-viral assay of IFN-a2 was performed in the presence and absence of a range of concentrations of Fab.
  • the earliest noted activity of interferons was their ability to induce an anti-viral state in live cells in culture, limiting viral replication and minimizing cytotoxicity induced by infection. Protocols were designed and used to quantify cellular anti-viral activity and to determine whether the selected Fabs modulate IFN-induced virus inhibiting signals in a manner consistent with in vitro, biophysical observations.
  • the anti-viral assays were performed over a range of IFNa2a concentration ( Figure 8A, diamond trace) and a range of concentrations of Fabs in the presence of ⁇ IFNa2a ( Figure 8A, circle trace).
  • Cells were incubated with serial dilutions of IFNa2a with a constant 200nM Fab shifted the dose response curve to the right indicating inhibition of IFN activity by Fab7 ( Figure 8B).
  • IFN or IFN-Fab mixture was added to Huh-7 cells, which were seeded 24 hours prior at a density of 2 x 10 4 in fresh media, and incubated overnight.
  • % cell viability equals A4 90 treated/(A49o untreated minus A490 background media).
  • the selection strategy was also devised to target Fabs to IFNAR2.
  • the interaction between IFNAR2 and IFN-a is the higher affinity and more stable of the two receptor interactions that most likely establishes first contact with ligand and subsequently recruits IFNAR1.
  • IFNAR2 was targeted using purified protein isolated from a C-terminal Fc-fusion expression construct. For selection, IFNAR2 was panned against by immobilizing 5 ⁇ g/ml of protein in the wells of the microplate. In an equal number of wells, the same concentration of expressed Fc-tag alone was immobilized. Purified Library F was first exposed to immobilized Fc-tag to remove tag-specific binders.
  • the supernatant was then transferred to target protein (IFNAR2), allowed to equilibrate, washed and eluted using 100 mM HC1.
  • Eluted phage were used to infect XL-1 Blue cells and plated on carbenicillin agar plates to isolate individual clones that bound specifically to IFNAR2. Individual colonies were picked and used to inoculate media to generate isolated Fab-phage clones for sequencing and ELISA assays for specificity.
  • the affinity of IFNAR2 -binding FABS 10 and 1 1 were determined first by multi-point competitive ELISA, by pre-incubating Fabs with a range of soluble IFNAR2 concentrations and then assessing binding to plate-immobilized IFNAR2. Half-maximal binding was observed at concentrations of 5-10 ⁇ suggesting affinity within the same range ( Figure 1 1). More accurate confirmation of these estimates was obtained by SPR analysis of the kinetics of binding (See Table 4 and Figure 12). Using the same methodology as described above, except immobilizing IFNAR2, and it was shown that both FABS 10 and 11 bind to IFNAR2 in a pre-assembled ternary complex with virtually identical on- and off-rates and similar affinities (Table 4). This suggested that complex assembly does not impede access to FAB 10 or FAB1 1 epitopes and must be considered in any model of Fab activity.
  • Binding studies were also conducted by separating mixtures of FAB 10 or FAB1 1 with solution-assembled receptor-ligand complex (IFNARl/IFNAR2/IFN-a2a) by size exclusion chromatography, as described above, to acquire insight into target binding and the effects on stability of the receptor complex. Chromatograms revealed that FAB 10 appeared to stabilize the ternary complex, inferred from the increase in the size of ⁇ + FAB 10 peak at the expense of IFNAR2/IFNP peak ( Figure 13).
  • FAB1 1 appeared to have a less dramatic effect on complex stability, perhaps causing a mild destabilization, inferred from the shoulder of the earlier eluting portion of the INFPTC + FAB1 1 peak and the growth in the IFNAR2/IFNP peak ( Figure 13). Importantly, the effect of Fab binding on stability of the complex would have to be considered and brought into accord with any model of Fab activity.
  • HL116 cells by flow cytometric detection of cells stained with Fab and fluoro-labeled with
  • Alexa488 conjugated anti-FLAG secondary antibodies Plots of cell count versus fluorescence intensity showed a population of cells that stained positively with FAB 10 in both the presence or absence of IFN-a2a that is distinct from cells without primary Fab or cells stained with an equivalent concentration of non-cell binding Fab ( Figure 14A). Binding of Fab 10 to cellular IFNAR2 was confirmed by immunofluorescence microscopy. Cells fixed with formaldehyde were immunostained with ⁇ ⁇ FablO for lh, washed with PBS and stained with 1 : 1000 dilution of Alexa 488-labeled anti-FLAG antibody. Cells stained with FablO showed intense staining of the cell surface confirming the cell binding activity of the Fab ( Figure 14B).
  • Example 3 Ligand directed antagonists.
  • the IFN-alpha cytokine is represented by up to 13 different subtypes that bear unique affinities for the receptors IFNAR1 and IFNAR2. These cytokines are related by both a conserved 5-helix structure and significant sequence identity ranging from 30-99%.
  • Fab-phage libraries In order to demonstrate the utility of Fab-phage libraries in selecting Fab molecules that specifically bind and modulate the activity of IFN-alpha subtypes, a selection against IFN-a2a was conducted. This selection was conducted with a simple, one step pre-selection against non-specific protein (BSA) and identified numerous unique IFN-a2a-binding affinity clones (Figure 17). These Fabs, described below, bind and inhibit the activity of alpha interferon subtypes, providing a proof of principle.
  • IFN-a2a binding clones were initially assessed by immobilized plate ELISA in a similar manner as previously described to (1) ensure specificity for the alpha interferon versus nonspecific protein and to (2) determine whether clones that bind free IFN-alpha also bind target when complexed with the TNFAR1/IFNAR2 heterodimer. Experiments confirmed that these Fabs are capable of binding both free ligand and receptor-bound ligand in a similar manner ( Figure 18). Isoform specificity was also evaluated for individual IFN-a2a binding clones using the purified IFN-alpha shown in Figure 19 by ELISA.
  • FAB A2-1 was specific for only IFN-a2a and IFN-a2b, which incidentally differ in primary sequence at only a single residue.
  • FAB A2-2 and A2-3 exhibited similar subtype specificity, binding primarily to the IFN-a2a, IFN-a2b, IFN-a4, IFN-a5, and IFN-al4 isoforms.
  • these Fabs exhibit differential biological responses.
  • Varying concentrations of FAB A2-1, A2-2, and A2-3 in the mobile phase were exposed to immobilized IFN-a2a resulting in kinetic traces that could be fit precisely with a 1 : 1 binding model and indicated high affinity for target, as seen in Figure 21.
  • the luciferase-induced activity of the cytokine was assessed in the presence and absence of 50 nM Fab. This cellular assay was conducted, as described above. For each Fab, a shift right was observed in the dose-response curve, such that at certain concentrations, an order of magnitude higher concentration is required to achieve the same level of activity and that at physiological concentrations of IFN-a2 (pM levels), a 20-80% inhibition of activity could be observed (Figure 22A).
  • the A2-3 Fab exhibited a different specificity pattern where IFNa5 was antagonized to a greater extent than IFNa2a/b.
  • the A2-3 Fab neutralized IFNa5 > IFNa2 IFNal6 > IFNa6.
  • IFNa6 and IFNa8 specific Fabs that bind and modulate the activity of IFN subtypes
  • the screen identified several unique IFNa6 and IFNa8 binding affinity clones (Table 7).
  • ELISA studies to determine the binding affinities revealed 25-50nM affinity of the Fabs to their subtypes (Table 8).
  • the specificity of isolated clones was initially assessed by ELISA as described earlier, which confirmed selective binding to the selected IFN alpha subtype
  • A8- 1 (E7) CDRH1 NIYSYYIH SEQ ID NO 102
  • A8- 1 (E7) CDRH2 ASISSYYGSTYYA SEQ ID NO 103
  • A8- 1 (E7) CDRH3 CARTVRGSKKPYFSGWAMD SEQ ID NO 104
  • IFNa6 and IFNa8 binding Fabs To evaluate the bioactivity of IFNa6 and IFNa8 binding Fabs, IFN induced luciferase activity was determined by the addition of a range of Fab concentrations starting from 1 ⁇ and 8pM IFNa6 for the IFNa6-specific Fabs and 5pM IFNa8 for the IFNa8-specific Fabs. Results revealed that the IFNa6-specific Fabs inhibited IFNa6 at the concentrations above ⁇ and complete inhibition of IFN activity was observed at higher concentrations ( ⁇ ⁇ ) ( Figure 43A). However, A8-1 and A8-2 Fab inhibited the IFNa8 activity starting from ⁇ with complete neutralization observed at ⁇ Fab concentration ( Figure 43 B).
  • Example 4 Anti-IFN FAB A2-4, A2-5 and A2-6.
  • Fabs A2-4, A2-5, and A2-6 were obtained by affinity maturation of a clone (Figure 23) obtained from the third round of selections against IFN-a2a.
  • This clone was specifically chosen as the highest apparent affinity ( ⁇ 50nM) by multi-point competitive ELISA of four clones that specifically bound free IFN-a2a, but could not bind ligand in a pre-assembled ternary complex with IFNAR1/IFNAR2 ( Figure 23). For this reason, this specific clone was chosen for affinity maturation.
  • a maturation library was generated by soft-randomization of the CDRH3 sequence determined for the clone, and re-randomizing the remaining L3, HI, and H2 regions, then repeating selections against IFN-a2a. Additional rounds of selection yielded 10 unique clones, of which three were determined to have affinities ⁇ 10 nM by multi-point competitive ELISA ( Figure 25). Following affinity maturation, the Fabs were purified and the absence of binding to the ternary complex confirmed by clonal ELISA as previously performed; however, using purified Fab rather than Fab-phage.
  • affinity was determined by both multi-potent competitive ELISA and subsequently confirmed by SPR.
  • Example 5 IFN-agonizing FABS.
  • IFNAR1/IFNAR2 surface receptor reduces IC50 up to 4 fold over interferon alone.
  • these Fabs have been termed conditional ternary complex-directed agonists. Biophysical characterization suggests that stabilization of the IFN-a2-IFNAR2 interaction is likely responsible for cytokine-boosting activity and that, in several instances, specificity is exclusive to the a-subtype, and does not affect IFN-P-signaling.
  • the clones obtained from in vitro selections against pre-formed IFNAR1/IFNAR2/ IFN- a2 ternary complex were first characterized for specificity by ELISA.
  • the clones showed predominant binding to the ternary complex and limited binding to the monomeric components or heterodimer receptor without the ligand (Figure 28).
  • the IFNAR1/IFNAR2 was immobilized as a monolayer on a GLC sensor chip
  • Sensograms of Fab binding to the IFNAR1/IFNAR2/ IFN-a2 complex were fit to a 1 : 1 binding model and from the measured kinetic constants revealed in all cases low to sub- nanomolar affinity (Table 9 and Figures 29 and 30).
  • Fab interaction with receptor alone was, except for single Fab (FAB4), either undetectable (FABS 1 and 2) or bound with more than a thousand times less affinity (FABS 3, 5, and 6).
  • the Fabs are interacting with a newly formed epitope that arises strictly from the interaction of all three components.
  • FABS 4, 5, and 6 appear to be indiscriminate with respect to ligand-subtype interacting with ternary complex comprised of either the a2a subtype or the ⁇ subtype, although binding of FAB5 appeared to be compromised in the presence of IFN- ⁇ . It is also important to note that since the Fabs selected do not interact with IFN-a2a , the differential recognition observed here may arise from the receptor conformational changes induced by cytokine subtypes.
  • the EC5 0 for IFN-a2a was determined to be 7.3 + 0.9 pM.
  • FAB4 represents an anomalous mode of binding insofar as it (1) appeared to bind to the IFNAR1/IFNAR2 heterodimer with high affinity ( ⁇ 10 nM) even in the absence of ligand (Figure 30), (2) binds to the ternary complex assembled with IFN- ⁇ essentially as was observed for IFN- a2a ( Figure 31), and (3) appeared to offer only modest activation of IFN-signaling in comparison to FAB1 and FAB5 ( Figure 33/Table 9). Despite the Fab-phage clones selected from the screens were tested by ELISA for specificity against the monomeric components of the receptor complex, specificity was confirmed by SPR analysis.
  • FAB4 exhibits unique binding specificity in comparison to other Fabs obtained.
  • Figure 35 it is observed that FAB4 uniquely binds to IFNAR2 alone. Importantly, despite its apparent specificity for monomeric IFNAR2, binding did not inhibit IFN-signaling in contrast to FAB 10 and FAB 11 obtained from selections against IFNAR2. Thus, FAB4 is valuable for labeling and tracking receptor localization and dynamics without interfering with receptor activation.
  • HL116 cells were immunostained with FAB4 and visualized suing fluoro-labeled anti-FLAG antibodies (specific for the FLAG tag on the C-terminus of the light chain constant domain) by

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Abstract

La présente invention concerne des anticorps ou des fragments d'anticorps qui se lient spécifiquement à un complexe ternaire interféron-récepteur d'interféron. L'invention concerne également des anticorps ou des fragments d'anticorps qui se lient spécifiquement à un ou deux interférons de type I. L'invention concerne par ailleurs des méthodes de traitement ou de prévention d'une maladie auto-immune, d'une infection virale ou d'un cancer chez un sujet, lesdites méthodes comprenant l'administration audit sujet d'une quantité efficace de l'un quelconque des anticorps décrits ici.
PCT/US2012/060589 2011-10-17 2012-10-17 Anticorps dirigés contre divers sous-types d'interféron, complexe ternaire interféron/récepteur d'interféron et utilisations associées WO2013059299A1 (fr)

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CN112358548A (zh) * 2013-07-03 2021-02-12 因美诺克股份公司 人抗IFN-α抗体、IFN-α结合片段、多核苷酸、组合物、试剂盒及应用和制备方法
US10947295B2 (en) 2017-08-22 2021-03-16 Sanabio, Llc Heterodimers of soluble interferon receptors and uses thereof
WO2022032025A1 (fr) * 2020-08-05 2022-02-10 Synthekine, Inc. Cytokines synthétiques de liaison à l'ifngr et méthodes d'utilisation

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