US20110059078A1 - Anti-ifnar1 antibodies with reduced fc ligand affinity - Google Patents

Anti-ifnar1 antibodies with reduced fc ligand affinity Download PDF

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US20110059078A1
US20110059078A1 US12/866,579 US86657909A US2011059078A1 US 20110059078 A1 US20110059078 A1 US 20110059078A1 US 86657909 A US86657909 A US 86657909A US 2011059078 A1 US2011059078 A1 US 2011059078A1
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antibody
antibodies
ifnar1
amino acid
seq
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Anthony Coyle
Peter Kiener
Herren Wu
Ricardo Cibotti
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MedImmune LLC
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Definitions

  • the present invention relates to isolated antibodies and compositions specific for the interferon alpha receptor 1 (IFNAR1) with reduced affinity for Fc ligands.
  • the invention also comprises nucleic acids encoding such antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic compositions, formulations, administrations and devices.
  • Type I interferons (IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ ) are a family of structurally related cytokines having antiviral, antitumor and immunomodulatory effects (Hardy et al. (2001) Blood 97:473; Cutrone and Langer (2001) J. Biol. Chem. 276:17140).
  • the human IFN ⁇ locus includes two subfamilies. The first subfamily consists of 14 non-allelic genes and 4 pseudogenes having at least 80% homology. The second subfamily, ⁇ II or omega ( ⁇ )), contains 5 pseudogenes and 1 functional gene which exhibits 70% homology with the IFN ⁇ genes (Weissmann and Weber (1986) Prog. Nucl.
  • IFN ⁇ Interferon alpha subtypes have been identified with the following nomenclature: IFN ⁇ 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • the interferon ⁇ (IFN ⁇ ) is encoded by a single gene, which has approximately 50% homology with the IFN ⁇ genes.
  • Interferon ⁇ which is produced by activated lymphocytes, does not possess any homology with the alpha/beta interferons and it does not react with their receptor.
  • IFN alpha receptor IFN alpha receptor
  • IFNAR1 IFN alpha receptor 1
  • IFNAR2 IFNAR2
  • IFNAR1 is essential for high affinity binding and differential specificity of the IFNAR complex (Cutrone et al. 2001 J. Bio Chem 276(20):17140-8) While functional differences for each of the type I IFN subtypes have not been identified, it is thought that each may exhibit different interactions with the IFNAR receptor components leading to potentially diverse signaling outcomes (Cook et al. (1996) J. Biol. Chem. 271:13448).
  • type I IFNs have been shown to facilitate differentiation of na ⁇ ve T cells along the Th1 pathway (Brinkmann et al. (1993) J. Exp. Med. 178:1655), to enhance antibody production (Finkelman et al. (1991) J. Exp. Med. 174:1179) and to support the functional activity and survival of memory T cells (Santini et al. (2000) J. Exp. Med. 191:1777; Tough et al. (1996) Science 272:1947).
  • IDM insulin-dependent diabetes mellitus
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • interferon ⁇ has been reported to exacerbate underlying disease in patients with psoriasis and multiple sclerosis and to induce an SLE like syndrome in patients without a previous history of autoimmune disease.
  • Interferon ⁇ has also been shown to induce glomerulonephritis in normal mice and to accelerate the onset of the spontaneous autoimmune disease of NZB/W mice.
  • IFN ⁇ therapy has been shown in some cases to lead to undesired side effects, including fever and neurological disorders. Hence there are pathological situations in which inhibition of Type I IFN activity may be beneficial to the patient and a need exists for agents effective in inhibiting Type I IFN activity.
  • the Fc region of an antibody interacts with a number of ligands (also referred herein as “Fc ligands” which include but are not limited to agents that specifically bind to the Fc region of antibodies, such as Fc receptors and Clq) including Fc receptors and Clq, imparting an array of important functional capabilities referred to as effector functions.
  • Fc ligands also referred herein as “Fc ligands” which include but are not limited to agents that specifically bind to the Fc region of antibodies, such as Fc receptors and Clq
  • the Fc receptors mediate communication between antibodies and the cellular arm of the immune system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu Rev Immunol 19:275-290).
  • this protein family includes Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIA, Fc ⁇ RIB, and Fc ⁇ RIC; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIA, Fc ⁇ RIIB, and Fc ⁇ RIIC; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIA and Fc ⁇ RIIB (Jefferis et al., 2002, Immunol Lett 82:57-65). These receptors typically have an extracellular domain that mediates binding to Fc, a membrane spanning region, and an intracellular domain that may mediate some signaling event within the cell.
  • Fc/Fc ⁇ R complex recruits these effector cells to sites of bound antigen, typically resulting in signaling events within the cells and important subsequent immune responses such as release of inflammation mediators, B cell activation, endocytosis, phagocytosis, and cytotoxic attack.
  • the ability to mediate cytotoxic and phagocytic effector functions is a potential mechanism by which antibodies destroy targeted cells.
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause lysis of the target cell is referred to as antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290).
  • the cell-mediated reaction wherein nonspecific cytotoxic cells that express Fc ⁇ Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell is referred to as antibody dependent cell-mediated phagocytosis (ADCP).
  • ADCP antibody dependent cell-mediated phagocytosis
  • an overlapping site on the Fc region of the molecule also controls the activation of a cell independent cytotoxic function mediated by complement, otherwise known as complement dependent cytotoxicity (CDC).
  • Fc ⁇ RI Human Fc ⁇ Rs are divided into three distinct classes: Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16).
  • Fc ⁇ RI is a high affinity receptor (K a : 10 ⁇ 8 -10 ⁇ 9 M ⁇ 1 ) and binds both immune complexes and monomeric IgG molecules while the Fc receptors Fc ⁇ RII and Fc ⁇ RIII exhibit lower affinities ( ⁇ 10 ⁇ 7 M ⁇ 1 and 2-3 ⁇ 10 ⁇ 7 respectively) (Gessner J. E. et al., 1998, Ann Hematology 76:231-48).
  • Fc ⁇ Rs Signaling through the Fc ⁇ Rs is either through an immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibitory motif (ITIM) for all the transmembrane receptors (Presta 2006, Adv Drug Deli Rev 58:640-656).
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • the 72 kDa extracellular glycoprotein Fc ⁇ RI is mainly expressed on myeloid cells such as monocytes, macrophages CD4+ progenitor cells and may elicit the ADCC, endocytosis, and phagocytosis responses (Siberil et al. 2006, J Immunol Lett 106:111-118).
  • the 40 kDa Fc ⁇ RII group of receptors exhibit extracellular domains but do not contain active signal transduction domains. These receptors propagate signals through phosphorylation of a cytoplasmic tail domain (Amigorena S. et al., 1992 Science. 256:1808-12).
  • the Fc ⁇ RIIA is mainly expressed on monocytes, macrophages, neutrophils, and platelets whereas the Fc ⁇ RIIC receptor has only been identified on NK cells. These two receptors have been shown to initiate ADCC, endocytosis, phagocytosis and inflammatory mediator release (Cassel et al. 1993. Mol Immunol 30:451-60).
  • Fc ⁇ RIIB B1 and B2 types
  • B1 and B2 types Fc ⁇ RIIB receptors
  • B1 and B2 types Fc ⁇ RIIB receptors are expressed on B cells, Mast cells, basophils, monocytes, macrophages and dendritic cells and has been shown to downregulate the immune response triggered by the A and C isoforms.
  • the 50 kDa Fc ⁇ RIIIA expressed on NK cells, monocytes, macrophages and a subset of T lymphocytes where it activates ADCC, phagocytosis, endocytosis and cytokine release (Gessner et al.).
  • the Fc ⁇ RIIIB isoforms is a glycosyl-phosphatidylinositol (GPI) anchored peripheral membrane protein involved in the degranulation and the production of reactive oxygen intermediates (Salmon J. E. et al. 1995 J Clin Inves 95:2877-85).
  • IgG molecules also exhibit differential isotype specificity for Fc ⁇ Rs.
  • IgG3 molecules bind strongly to all Fc ⁇ R isoforms.
  • IgG1 the most prevalent isoforms in the blood binds to all Fc ⁇ Rs albeit with a lower affinity for the Fc ⁇ RIIA/ ⁇ isoforms.
  • IgG4 is an intermediate binder to Fc ⁇ RI and a weak binder to Fc ⁇ RIIB.
  • IgG2 binds only weakly to one allelic form of Fc ⁇ RIIA (Fc ⁇ RIIA-H131) (Siberil et al. 2006, J Immunol Lett 106:111-118).
  • the complement inflammatory cascade is a part of the innate immune response and is crucial to the ability for an individual to ward off infection.
  • Another important Fc ligand is the complement protein Clq. Fc binding to Clq mediates a process called complement dependent cytotoxicity (CDC) (reviewed in Ward et al., 1995 , Ther Immunol 2:77-94). Clq is capable of binding six antibodies, although binding to two IgGs is sufficient to activate the complement cascade. Clq forms a complex with the Clr and Cls serine proteases to form the C1 complex of the complement pathway.
  • the binding and stimulation of effector functions mediated by the Fc region of immunoglobulins is highly beneficial, however, in certain instances it may be more advantageous to decrease or eliminate effector function.
  • a drug e.g., toxins and isotopes
  • the Fc/Fc ⁇ R mediated effector functions bring healthy immune cells into the proximity of the deadly payload, resulting in depletion of normal lymphoid tissue along with the target cells
  • FIG. 1A Nucleic acid (SEQ ID No:7) and amino acid (SEQ ID No:8) sequence alignment of 3F11 VH with the CDR regions are indicated by the overline.
  • FIG. 1B Nucleic acid (SEQ ID No:9) and amino acid (SEQ ID No:10) sequence alignment of 3F11 VK with the CDR regions outlined are indicated by the overline.
  • FIG. 2A Nucleic acid (SEQ ID No:17) and amino acid (SEQ ID No:18) sequence alignment of 4G5 VH with the CDR regions outlined are indicated by the overline.
  • FIG. 2B Nucleic acid (SEQ ID No:19) and amino acid (SEQ ID No:20) sequence alignment of 4G5 VK with the CDR regions outlined are indicated by the overline.
  • FIG. 3A Nucleic acid (SEQ ID No:27) and amino acid (SEQ ID No:28) sequence alignment of 11E2 VH with the CDR regions outlined are indicated by the overline.
  • FIG. 3B Nucleic acid (SEQ ID No:29) and amino acid (SEQ ID No:30) sequence alignment of 11E2 VK with the CDR regions outlined are indicated by the overline.
  • FIG. 4A Nucleic acid (SEQ ID No:37) and amino acid (SEQ ID No:38) sequence alignment of 9D4 VH with the CDR regions outlined are indicated by the overline.
  • FIG. 4B Nucleic acid (SEQ ID No:39) and amino acid (SEQ ID No:40) sequence alignment of 9D4 VK with the CDR regions outlined are indicated by the overline.
  • FIG. 5 Amino acid sequence alignment of heavy chain constant regions for 9D4. Arrows indicate amino acid substitutions (unmodified to modified) to increase stability and reduce affinity to at least one Fc ligand.
  • FIG. 6A Immunohistochemical staining profile of human cerebrum tissue treated with various anti-IFNAR1 antibodies.
  • the 9D4 antibody exhibits a lower staining profile when incubated with human cerebrum tissue compared to 4G5 and MDX-1333 antibodies.
  • FIG. 6B Immunohistochemical staining profile of human monocytes treated with various anti-IFNAR1 antibodies. As a positive control, various anti-IFNAR1 antibodies were tested for reactivity to human monocytes.
  • FIG. 7 The anti-IFNAR1 antibody 9D4 inhibits IFN ⁇ signaling in a cell based STAT activation assay. Treatment with antibody 9D4 inhibits STAT1/3/4 tyrosine phosphorylation in response to stimulation with interferon alpha as determined by Western Blot analysis with commercially available STAT antibodies.
  • FIG. 8 Anti-IFNAR1 antibodies block signaling of various concentrations of pDC Cell derived Type I IFNs. Presented are the IC50 values for antibody 9D4 blocking IFN signaling in a luciferase reporter assay utilizing type I IFN supernatants purified from 3 independent donors. Included are the relative amounts of IFN ⁇ , IFN ⁇ , and IFN ⁇ in each purified type I interferon supernatant.
  • FIG. 9 A, B, C Anti-IFNAR1 antibodies 9D4, 9D4-DM (Double Mutant), and 9D4-TM (Triple Mutant) exhibit similar binding characteristics.
  • 9D4-DM Double Mutant
  • 9D4-TM Triple Mutant
  • the modified antibodies exhibit similar IFNAR1 binding characteristics to the unmodified antibody.
  • FIG. 10A The anti-IFNAR1 antibody 9D4 binds soluble interferon alpha receptor (sIFN ⁇ R1). Presented are equilibrium binding data that demonstrate dose dependent binding of 9D4 to soluble interferon alpha receptor.
  • FIG. 10B Determination of the Kd of 9D4 on human PBMCs. Presented is the dissociation constant determination of 9D4 measured by binding to human PBMCs.
  • FIG. 11 Anti-IFNAR1 antibodies inhibit IFN ⁇ induced signaling in a luciferase reporter assay.
  • Anti-IFNAR1 antibodies including unmodified and modified antibodies demonstrate similar IC50 values for blocking Leukocyte IFN signaling in a luciferase reporter assay system.
  • FIG. 12A Determination of the isoelectric point of 9D4 (unmodified) and modified 9D4 antibodies. Presented is an IEF gel documenting the relative pI values for the 9D4 WT (unmodified), 9D4-DM, and 9D4-TM antibodies.
  • FIG. 12B Determination of the thermal melting temperatures of 9D4 (unmodified) and modified 9D4 antibodies. Presented here is a melt cure documenting the relative melting temperatures (Tm) for the 9D4, 9D4-DM, and 9D4-TM antibodies.
  • FIG. 13 Prophylactic treatment with anti-IFNAR antibodies blocks Adv-IFN ⁇ induced proteinuria. Mice treated with control vector, Adv-IFN ⁇ , Adv-IFN ⁇ +isotype control pretreatment, and Adv-IFN ⁇ +anti-IFNAR pretreatment were analyzed for proteinuria over 9 weeks. Mice pretreated with anti-IFNAR did not exhibit proteinuria after IFN ⁇ challenge.
  • FIG. 14 Prophylactic treatment with anti-IFNAR antibodies blocks the upregulation of IFN ⁇ responsive genes (IFIT1, IF144, CXCL11, IFI202b, CXCL19, CXCL9) in blood. Mice pre-treated with anti-IFNAR antibodies did not exhibit not upregulated selected IFN ⁇ responsive genes upon challenge with adenovirus encoded IFN alpha as compared to mice pretreated with control virus, PBS, or isotype IgG controls. Presented are the relative expression of six genes known to be responsive to IFN ⁇ in blood samples taken from mice 3 weeks post IFN ⁇ induction by infection with Adv-IFN ⁇ .
  • FIG. 15 A, B Prophylactic treatment with anti-IFNAR antibodies blocks IFN ⁇ induced autoantibody production. Mice pre-treated with anti-IFNAR antibodies did not exhibit elevated autoantibody production upon challenge with adenovirus encoded IFN ⁇ as compared to mice pretreated with control virus, PBS or isotype IgG controls. Presented are the concentrations of anti-dsDNA and anti-SSA/Ro in blood samples taken from mice 6 weeks post IFN ⁇ induction by infection with Adv-IFN ⁇ .
  • FIG. 16 A, B Prophylactic treatment with anti-IFNAR antibodies blocks the upregulation of cytokines in the kidney. Mice pretreated with anti-IFNAR antibodies did not exhibit upregulated cytokines in the kidney upon challenge with adenovirus encoded IFN ⁇ 5 as compared to mice pretreated with, control virus, PBS or isotype IgG controls. Presented are the measurement of IP-10, and IL-18 levels in kidney samples taken from mice 6 weeks post IFN ⁇ induction by infection with Adv-IFN ⁇ 5.
  • FIG. 17 Prophylactic treatment with anti-IFNAR antibodies blocks IFN induced autoantibody production.
  • ANA anti-nuclear antigen
  • FIG. 18 Antibody mediated inhibition of SLE plasma mediated Dendritic cell development. Presented are the results of 5 individual experiments in which IFN derived from SLE patients was incubated in the presence of anti-IFNAR1 antibody 9D4 and subsequently added to human monocytes. The presence of anti-IFNAR1 antibody 9D4 inhibited the ability of IFN derived from SLE patients to induce the dendritic cell markers CD38 and CD123 in differentiating monocytes.
  • FIG. 19 Anti-IFNAR1 antibodies suppress the expression of CD38, CD123 and CD86 in monocytes stimulated with Leukocyte Interferon. As measured by percent suppression of control stimulated expression, anti-IFNAR1 antibodies 9D4, 9D4-DM and 9D4-TM exhibited similar inhibition profiles for the expression of CD38, CD123 and CD86 in differentiating monocytes.
  • FIG. 20 Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor Fc ⁇ RI as compared to unmodified anti-IFNAR1 antibodies.
  • Anti-IFNAR1 antibodies 9D4 (unmodified), 9D4-DM (modified) and 9D4-TM (modified) were analyzed for the ability to bind to plate bound Fc ⁇ RI in an ELISA experiment. As a positive control for Fc receptor binding, an unrelated unmodified antibody was used (control antibody).
  • FIG. 21 , A, B, C Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor Fc ⁇ RIIIA as compared to unmodified anti-IFNAR1 antibodies.
  • Plate bound unmodified anti-IFNAR1 antibody 9D4(A) and modified anti-IFNAR1 antibodies 9D4-DM (B) and 9D4-TM(C) were analyzed for the ability to bind free Fc ⁇ RIIIA in an ELISA experimental format.
  • FIG. 22 , A, B, C Modified anti-IFNAR1 antibodies exhibit decreased binding to the Fc receptor Fc ⁇ RIIIA.
  • Free unmodified anti-IFNAR1 antibody 9D4(A) and modified anti-IFNAR1 antibodies 9D4-DM(B) and 9D4-TM(C) were analyzed for the ability to bind plate bound Fc ⁇ RIIIA in an ELISA experimental format.
  • FIG. 23 A-E Neutralization of IFN subtypes in SLE patient serum.
  • anti-IFNAR1 antibodies MDX-1333, 9D4-WT and 9D4-TM inhibited IFN mediated signaling of ⁇ 10 (A), Leukocyte interferon (B), ⁇ 2b (C), ⁇ (D), and ⁇ (E).
  • FIG. 24 Anti-IFNAR1 antibodies neutralize type I interferon from SLE patients.
  • the anti-IFNAR1 antibody, 9D4 inhibited type I interferon mediated signaling as compared to a control, unrelated antibody.
  • FIG. 25 A-D Anti-IFNAR antibodies suppress the IFN ⁇ induced pDC population in PBMC's. Anti-IFNAR antibodies blocked the elevation of pDC cells measured by cell surface epitope expression, induced by ectopic adenoviral induced expression of interferon alpha in spleen (A), lymph nodes (B), peripheral blood (C) and bone marrow (D).
  • FIG. 26 Binding analysis of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM, and 9D4-TM to the Fc receptor Fc ⁇ RI was determined by BIACore analysis. Briefly, anti-IFNAR1 antibodies were immobilized and free Fc ⁇ RI was added to measure affinity. As demonstrated by the tracing, the modified antibodies, 9D4-DM, and 9D4-TM exhibited lower affinities to the free Fc ⁇ RI as compared to the unmodified 9D4-WT antibody.
  • FIG. 27 A-C Binding analysis of anti-IFNAR1 antibodies 9D4-WT, 9D4-DM, and 9D4-TM to the Fc receptor Fc ⁇ RI was determined by BiaCore analysis. Briefly, free anti-IFNAR1 antibodies were passed over immobilized Fc ⁇ RI to measure affinity. As demonstrated by the tracing, the modified antibodies 9D4-DM (B), and 9D4-TM (C) exhibited lower affinities to the bound Fc ⁇ RI as compared to the unmodified 9D4-WT (A) antibody.
  • FIG. 28 Anti-IFNAR antibodies inhibit IFN ⁇ responsive gene induction in the kidney. Briefly, in the accelerated lupus mouse model, treatment with anti-IFNAR antibodies blocks induction in the kidney of six genes (ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1) mediated by the ectopically expression of IFN ⁇ compared to control mice as measured by a Taqman assay.
  • IAM1, VCAM1, CXCL9, CXCL10, and IFIT1 six genes mediated by the ectopically expression of IFN ⁇ compared to control mice as measured by a Taqman assay.
  • FIG. 29 Anti-IFNAR antibodies inhibit the production of anti-ds DNA antibodies in the accelerated lupus mouse model. Briefly, mice ectopically expressing IFN ⁇ and treated with anti-IFNAR antibodies did not accumulate anti-ds DNA antibodies to the same level as mice similarly infected and treated with an IgG control antibody.
  • FIG. 30 Anti-IFNAR antibodies are able to reduce proteinuria in a therapeutic setting of the accelerated lupus mouse model.
  • mice ectopically expressing IFN ⁇ developed Lupus like symptoms, such as proteinuria.
  • anti-IFNAR antibodies were administered to mice once a threshold proteinuria score was reached.
  • Anti-IFNAR antibodies, PBS, or control IgG were administered semi-weekly over a 5 week time course.
  • the anti-IFNAR antibody treated group exhibited decreased severity of proteinuria during the experiment compared to PBS only or control IgG treated groups.
  • FIG. 31 Anti-IFNAR antibodies are able to increase survival in a therapeutic setting of the accelerated lupus mouse model.
  • mice ectopically expressing IFN ⁇ had a reduced survival rate at about 8 weeks after developing Lupus-like symptoms such as proteinuria.
  • anti-IFNAR antibodies were administered to mice once a threshold proteinuria score was reached.
  • Anti-IFNAR antibodies, PBS, or control IgG were administered semi-weekly over a 5 week time course. After the five weeks, antibody treatment was stopped and the mortality tracked for all three treatment groups. The anti-IFNAR antibody treated group exhibited a much lower rate of mortality than the PBS alone, or control IgG groups, which both exhibited complete mortality by 9 weeks.
  • FIG. 32 Representation of the asymmetric unit contents of the crystals of Fc-TM that comprises L234F/L235E/P331S mutations.
  • the mutation P331 is indicated in red.
  • One zinc ion is chelated by two spatially close Histidine residues.
  • the carbohydrate residues attached to 297 were modeled according to their electron density.
  • FIG. 33 Kinetic images demonstrate 9D4-TM internalization.
  • THP-1 cells were stained with 1 ⁇ M CFSE in a 37° C. CO 2 incubator for 10 min followed by 1 ⁇ g/ml of Alexa647-9D4-TM on ice for 1 hr. After removal of unbound the cells were incubated at 37° C. for the times indicated (0, 15, 30 and 60 minutes) and the images of cells were taken.
  • FIG. 34 The anti-IFNAR1 antibody, 9D4-TM does not exhibit CDC activity in an in vitro assay.
  • a CDC assay to determine the ability of the 9D4-TM antibody to elicit CDC activity.
  • the 9D4-TM antibody did not exhibit any CDC activity as compared to the positive control antibody.
  • CDC activity was also undetectable for an unrelated control antibody, R347. Briefly, cells expressing IFNAR1 antigen were incubated with either the positive control antibody, 9D4-TM, or R347. After a series of washes, freshly prepared human serum was added. Complement dependent cytotoxicity (CDC) was measured using a LDH release assay.
  • CDC complement dependent cytotoxicity
  • interferon alpha IFN alpha proteins encoded by a functional gene of the interferon alpha gene locus with 75% or greater sequence identity to IFN alpha 1 (GenBank accession number NP — 076918 or protein encoded by GenBank accession number NM — 024013).
  • IFN alpha subtypes include IFN alpha 1, alpha 2a, alpha 2b, alpha 4, alpha 4b alpha 5, alpha 6, alpha 7, alpha 8, alpha 10, alpha 13, alpha 14, alpha 16, alpha 17 and alpha 21.
  • IFN alpha interferon alpha
  • IFN ⁇ interferon alpha
  • IFN alpha immunoglobulin alpha
  • naturally occurring preparations that comprise IFN alpha proteins such as leukocyte IFN and lymphoblastoid IFN.
  • Interferon alpha receptor-1 is used interchangeably, and include variants, isoforms, species homologs of human IFNAR-1, and analogs having at least one common epitope with IFNAR-1.
  • human antibodies of the invention may, in certain embodiments, cross-react with IFNAR-1 from species other than human, or other proteins which are structurally related to human IFNAR-1 (e.g., human IFNAR-1 homologs). In other embodiments, the antibodies may be completely specific for human IFNAR-1 and not exhibit species or other types of cross-reactivity.
  • the complete cDNA sequence of human IFNAR-1 has the Genbank accession number NM — 000629.
  • conservative sequence modifications is intended to include amino acid modifications that do not affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide.
  • Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine).
  • Families of amino acid residues having similar side chains have been defined in the art. Several non-limiting examples of families of conservative amino acid substitutions are shown in Table 1.
  • anti-IFNAR1 antibodies with reduced or ablated effector function are desired for the treatment of chronic autoimmune and/or inflammatory diseases.
  • antibodies directed against IFNAR1 were developed with the understanding that effector function would play a role in mediating treatment or at least moderation of a chronic autoimmune and/or inflammatory disease state (see, for example U.S. Publication No. 20060029601 or PCT publication No. WO06002177).
  • anti-IFNAR1 antibodies with reduced or ablated effector function such as ADCC and/or CDC.
  • tissue cross-reactivity studies it was surprisingly found that anti-IFNAR1 antibodies with strong or enhanced effector function displayed a propensity for unwanted toxicity due to the prevalence of staining of anti-IFNAR10n non-target tissues. This toxicity would result from the non-specific activation of ADCC and/or CDC at inappropriate sites.
  • the inventors recognized the need to reduce effector function of polypeptides comprising an Fc region.
  • one aspect of the invention encompasses modified antibodies or other polypeptides comprising the Fc region of an antibody, comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region resulting in reduced or ablated affinity for at least one Fc ligand (referred to herein as “modified antibodies of the invention”, “modified antibodies” or “antibodies of the invention”).
  • modified antibodies of the invention refers to herein as “modified antibodies of the invention”, “modified antibodies” or “antibodies of the invention”.
  • the Fc region interacts with a number of ligands including but not limited to Fc Receptors (e.g., FcRn, Fc ⁇ RIIIa, Fc ⁇ RIIb), the complement protein Clq, and other molecules such as proteins A and G.
  • the modified antibodies of the invention have reduced or ablated affinity for an Fc ligand responsible for facilitating effector function compared to an antibody having the same amino acid sequence as the antibody of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region (also referred to herein as an “unmodified antibody”).
  • antibodies of the invention comprise at least one or more of the following properties: reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc receptors, or reduced or ablated toxicities. More specifically, embodiments of the invention provide anti-IFNAR1 antibodies with reduced affinity for Fc receptors (e.g., FcRn, Fc ⁇ RIIIa, Fc ⁇ RIIb) and/or the complement protein Clq.
  • FcRn FcRn, Fc ⁇ RIIIa, Fc ⁇ RIIb
  • antibodies of the invention comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue selected from the positions consisting of: 234, 235, and 331, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • antibodies of the invention comprise an Fc region comprising at least one amino acid substitution selected from the group consisting of: L234F, L235E, and P331S, wherein the first letter and number represent the unmodified amino acid and its position and the second letter represents the substituted amino acid at said position.
  • antibodies of the invention further comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue that is correlated with increased stability of the antibody.
  • the addition, substitution, or deletion of an amino acid residue is at position 228 of the Fc region, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al.
  • antibodies of the invention comprise an Fc region comprising an amino acid substitution at position 228, wherein the substitution is a serine residue.
  • antibodies of the invention of the IgG4 subtype comprise an amino acid substitution of serine at position 228 of the Fc region.
  • antibodies of the invention already comprise a serine residue at position 228 of the Fc region; in such embodiments, no modification is required. In alternative embodiments, antibodies of the invention do not require modification of residue 228 of the Fc region or already comprise serine at said position.
  • antibodies of the invention may be any of any class (for example, but not limited to IgG, IgM, and IgE).
  • antibodies of the invention are members of the IgG class of antibodies.
  • antibodies of the invention are of the IgG1 subclass.
  • antibodies of the invention are of the IgG1 subclass and comprise the following amino acid substitutions: 234F, 235E and 331S of the Fc region.
  • antibodies of the invention are of the IgG4 subclass.
  • antibodies of the invention are of the IgG4 subclass and comprise the following amino acid substitutions: S228P and L235E of the Fc region.
  • modified antibodies of the present invention may be produced by combining a variable domain, or fragment thereof, with an Fc domain comprising one or more of the amino acid substitutions disclosed herein.
  • modified antibodies of the invention may be produced by modifying an Fc domain-containing antibody by introducing one or more of the amino acid substitutions residues into the Fc domain.
  • antibodies of the invention may have altered (relative to an unmodified antibody) Fc ⁇ R and/or Clq binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (K off and K on respectively), binding affinity and/or avidity) and that certain alterations are more or less desirable.
  • binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (K off and K on respectively), binding affinity and/or avidity) and that certain alterations are more or less desirable.
  • the equilibrium dissociation constant (K D ) is defined as k off /k on .
  • One skilled in the art can determine which kinetic parameter is most important for a given antibody application.
  • a modification that reduces binding to one or more positive regulator (e.g., Fc ⁇ RIIIA) and/or enhanced binding to an inhibitory Fc receptor (e.g., Fc ⁇ RIIB) would be suitable for reducing ADCC activity.
  • the ratio of binding affinities e.g., equilibrium dissociation constants (K D )
  • K D equilibrium dissociation constants
  • a modification that reduces binding to Clq would be suitable for reducing or eliminating CDC activity.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, i.e., specific binding of an Fc region to an Fc ⁇ R including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • ELISA enzyme-linked immunoabsorbent assay
  • RIA radioimmunoassay
  • kinetics e.g., BIACORE® analysis
  • indirect binding assays e
  • These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • antibodies of the invention exhibit reduced binding affinity for one or more Fc receptors including, but not limited to Fc ⁇ RI (CD64) including isoforms Fc ⁇ RIA, Fc ⁇ RIB, and Fc ⁇ RIC; Fc ⁇ RII (CD32 including isoforms Fc ⁇ RIIA, Fc ⁇ RIIB, and Fc ⁇ RIIC); and Fc ⁇ RIII (CD16, including isoforms Fc ⁇ RIIIA and Fc ⁇ RIIB) as compared to an unmodified antibody.
  • Fc ⁇ RI CD64
  • Fc ⁇ RII CD32 including isoforms Fc ⁇ RIIA, Fc ⁇ RIIB, and Fc ⁇ RIIC
  • Fc ⁇ RIII CD16, including isoforms Fc ⁇ RIIIA and Fc ⁇ RIIB
  • antibodies of the invention do not comprise a concomitant increase in binding the Fc ⁇ RIIB receptor as compared to an unmodified (for example, containing a wild type Fc region) antibody.
  • antibodies of the invention exhibit decreased affinities to Fc ⁇ RI relative to an unmodified antibody.
  • antibodies of the invention exhibit affinities for Fc ⁇ RI receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.
  • antibodies of the invention exhibit affinity for Fc ⁇ RI receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.
  • antibodies of the invention exhibit decreased affinity for the Fc ⁇ RIIIA receptor relative to an unmodified antibody.
  • antibodies of the invention exhibit affinities for Fc ⁇ RIIIA receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.
  • antibodies of the invention exhibit affinities for Fc ⁇ RIIIA receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.
  • the F158V allelic variant of the Fc ⁇ RIIIA receptor has altered binding characteristics to antibodies.
  • antibodies of the invention bind with decreased affinities to Fc ⁇ RIIIA (F158V) relative to an unmodified antibody.
  • antibodies of the invention exhibit affinities for Fc ⁇ RIIIA (F158V) receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RIIIA(F158V) receptor that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.
  • antibodies of the invention exhibit increased affinities for the Fc ⁇ RIIB receptor as compared to unmodified antibody.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RIIB receptor that are unchanged or increased by at least at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold than that of an unmodified antibody.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RIIB receptor that are increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% than an unmodified antibody.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RI, Fc ⁇ RIIIA, or Fc ⁇ RIIIA (F158V) receptors that are between about 100 nM to about 100 ⁇ M, or about 100 nM to about 10 ⁇ M, or about 100 nM to about 1 ⁇ M, or about 1 nM to about 100 ⁇ M, or about 10 nM to about 100 ⁇ M, or about 1 ⁇ M to about 100 ⁇ M, or about 10 ⁇ M to about 100 ⁇ M.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RI, Fc ⁇ RIIIA, or Fc ⁇ RIIIA (F158V) receptors that are greater than 1 ⁇ M, greater than 5 ⁇ M, greater than 10 ⁇ M, greater than 25 ⁇ M, greater than 50 ⁇ M, or greater than 100 ⁇ M.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RIIB receptor that are between about 100 nM to about 100 ⁇ M, or about 100 nM to about 10 ⁇ M, or about 100 nM to about 1 ⁇ M, or about 1 nM to about 100 ⁇ M, or about 10 nM to about 100 ⁇ M, or about 1 ⁇ M to about 100 ⁇ M, or about 10 ⁇ M to about 100 ⁇ M.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RI, Fc ⁇ RIIIA, or Fc ⁇ RIIIA (F158V) receptors that are less than 100 ⁇ M, less than 50 ⁇ M, less than 10 ⁇ M, less than 5 ⁇ M, less than 2.5 ⁇ M, less than 1 ⁇ M, or less than 100 nM, or less than 10 nM.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RIIB receptor that are between about 100 nM to about 100 ⁇ M, or about 100 nM to about 10 ⁇ M, or about 100 nM to about 1 ⁇ M, or about 1 nM to about 100 ⁇ M, or about 10 nM to about 100 ⁇ M, or about 1 ⁇ M to about 100 ⁇ M, or about 10 ⁇ M to about 100 ⁇ M.
  • antibodies of the invention exhibit affinities for the Fc ⁇ RI, Fc ⁇ RIIIA, or Fc ⁇ RIIIA (F158V) receptors that are less than 100 ⁇ M, less than 50 ⁇ M, less than 10 ⁇ M, less than 5 ⁇ M, less than 2.5 ⁇ M, less than 1 ⁇ M, or less than 100 nM, or less than 10 nM.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • IgG antibodies directed to the surface of target cells “arm” the cytotoxic cells and are required for such killing.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-mediated lysis
  • an antibody of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al., 1987 , J Exp Med 166:1351-1361; Wilkinson et al., 2001 , J Immunol Methods 258:183-191; Patel et al., 1995 J Immunol Methods 184:29-38.
  • ADCC activity of the antibody of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA 95:652-656.
  • antibodies of the invention are characterized by in vitro functional assays for determining one or more Fc ⁇ R mediated effector cell functions.
  • antibodies of the invention have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude antibodies of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • antibodies of the invention exhibit decreased ADCC activities as compared to an unmodified antibody.
  • antibodies of the invention exhibit ADCC activities that are at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold less than that of an unmodified antibody.
  • antibodies of the invention exhibit ADCC activities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.
  • antibodies of the invention have no detectable ADCC activity.
  • the reduction and/or ablatement of ADCC activity may be attributed to the reduced affinity antibodies of the invention exhibit for Fc ligands and/or receptors.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule, an antibody for example, complexed with a cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., 1996 , J. Immunol. Methods, 202:163, may be performed.
  • antibodies of the invention exhibit decreased affinities to Clq relative to an unmodified antibody.
  • antibodies of the invention exhibit affinities for Clq receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than an unmodified antibody.
  • antibodies of the invention exhibit affinities for Clq that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than an unmodified antibody.
  • antibodies of the invention exhibit affinities for Clq that are between about 100 nM to about 100 ⁇ M, or about 100 nM to about 10 ⁇ M, or about 100 nM to about 1 ⁇ M, or about 1 nM to about 100 ⁇ M, or about 10 nM to about 100 ⁇ M, or about 1 ⁇ M to about 100 ⁇ M, or about 10 ⁇ M to about 100 ⁇ M. In certain embodiments, antibodies of the invention exhibit affinities for Clq that are greater than 1 ⁇ M, greater than 5 ⁇ M, greater than 10 ⁇ M, greater than 25 ⁇ M, greater than 50 ⁇ M, or greater than 100 ⁇ M.
  • antibodies of the invention exhibit decreased CDC activities as compared to an unmodified antibody.
  • antibodies of the invention exhibit CDC activities that are at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold less than that of an unmodified antibody.
  • antibodies of the invention exhibit CDC activities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.
  • antibodies of the invention exhibit no detectable CDC activities.
  • the reduction and/or ablatement of CDC activity may be attributed to the reduced affinity antibodies of the invention exhibit for Fc ligands and/or receptors.
  • antibodies of the invention exhibit reduced staining of non-targeted tissues as compared to an unmodified antibody. In another embodiment, antibodies of the invention exhibit reduced staining of non-targeted tissues that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody.
  • antibodies of the invention exhibit reduced staining of non-targeted tissues that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.
  • antibodies of the invention exhibit a reduced antibody related toxicity as compared to an unmodified antibody.
  • antibodies of the invention exhibit toxicities that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of an unmodified antibody.
  • antibodies of the invention exhibit toxicities that are reduced by at least 10%, or at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or by at least 300%, or by at least 400%, or by at least 500% relative to an unmodified antibody.
  • Antibodies of the invention may bind to cell-surface antigens that may internalize, further carrying the antibodies into the cell. Once inside the cell, the antibodies may be released into the cytoplasm, targeted to a specific compartment, or recycled to the cell surface. In some embodiments, the antibodies of the invention bind to a cell-surface antigen that internalizes. In other embodiments, antibodies of the invention may be targeted to specific organelles or compartments of the cell. In yet other embodiments, the antibodies of the invention may be recycled to the cell surface or periphery after internalization. In a specific embodiment, the antibody of the invention is specific for IFNAR1.
  • the extent of internalization is represented as a percentage of total antibody bound to cells. In other embodiments, the extent of antibody internalization is represented as a comparison to a non-specific control antibody. In other embodiments, the extent of antibody internalization is represented as a comparison to an antibody that binds a cell-surface antigen that does not internalize. In yet other embodiments, the extent of antibody internalization is correlated with the degradation of the antibody. In yet other embodiments, the extent of antibody internalization is represented as a ratio of cytoplasmic versus cell surface staining.
  • the antibodies of the invention once bound, internalize into cells wherein internalization is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 100%, at least about 110%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, or at least about 170% more than a non-specific control antibody.
  • the antibodies of the invention once bound, internalize into cells wherein internalization is 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than a non-specific control antibody.
  • the antibodies of the invention once bound, internalize into cells wherein internalization is 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, 140-150%, 150-160%, 160-170% more than control antibodies as determined by the internalization assay using a secondary antibody.
  • the present invention also provide crystalline forms of a human IgG Fc region, wherein the human Fc region, designated as Fc-TM, comprises amino acid substitutions of L234F, L235E and P331S as numbered by the EU index as set forth in Kabat and exhibits reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc receptors, and/or reduced or ablated toxicities.
  • the crystals are of diffraction quality to permit the determination of the three-dimensional X-ray diffraction structure of the crystalline polypeptide(s) to high resolution, preferably to a resolution of greater than about 3 ⁇ , typically in the range of about 2 ⁇ to about 3 ⁇ .
  • the present invention further provides the high-resolution three-dimensional structures and atomic structure coordinates of the Fc-TM crystals.
  • the specific methods used to obtain crystals and structure coordinates are provided in the examples, infra.
  • Fc-TM The overall three-dimensional structure of Fc-TM was very similar to previously reported structures of unliganded human Fc regions (Deisenhofer, (1981). Biochemistry, 20, 2361-2370; Krapp et al., (2003). J. Mol. Biol. 325, 979-989; Matsumiya et al., (2007). J. Mol. Biol. 368, 767-779; Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press).
  • Fc-TM C H 2 and C H 3 domains showed great structural conservation and rigidity when compared with other unliganded, unmutated human Fc structures.
  • the structure information can be used in a variety of computation or computer-based methods to screen, design or identify anti-IFNAR antibodies that have alter biological properties.
  • the crystals and structure coordinates obtained therefrom can be used to screen, design or identify amino acid additions, substitutions or deletions in Fc region that result in reduced or ablated binding to Fc receptors, reduced or ablated effector (ADCC and/or CDC) function, or reduced or ablated toxicities.
  • ADCC and/or CDC reduced or ablated effector
  • the present invention also encompasses anti-IFNAR1 antibodies that are designed or selected by the use of the structure information of Fc-TM and that exhibit the desired biological activities.
  • such antibodies comprise an Fc region with the mutations of L234F, L235E, and P331S.
  • such antibodies comprise an Fc region with one or more addition, substitution, or deletion of an amino acid residue other than amino acid residues 234, 235, and 331.
  • antibodies of the invention are specific for (i.e. specifically bind) IFNAR1. Such antibodies may also be referred to herein as “anti-IFNAR1 antibodies of the invention.” In another embodiment, antibodies of the invention are specific for human IFNAR1. In another embodiment, the anti-IFNAR1 antibodies of the invention may cross-react with IFNAR1 from species other than human, or other proteins which are structurally related to human IFNAR1(for example, human IFNAR1 homologs). In other embodiments, the anti-IFNAR1 antibodies of the invention may be specific for human IFNAR1 only and not exhibit species or other types of cross-reactivity.
  • the anti-IFNAR1 antibodies of the invention exhibit reduced binding affinities for Fc ligands and have at least one of the following properties: reduced or ablated effector (ADCC and/or CDC) function, reduced or ablated binding to Fc ligands, or reduced or ablated toxicities as compared to an unmodified antibody.
  • ADCC and/or CDC reduced or ablated effector
  • anti-IFNAR1 antibodies of the invention comprise the addition, substitution or deletion of at least one amino acid residue selected from the group consisting of: L234F, L235E, and P331S.
  • the anti-IFNAR1 antibodies of the invention comprise the amino acid substitutions: L234F, L235E, and P331S of the Fc region.
  • an anti-IFNAR1 antibody of the invention is an IgG isotype antibody.
  • anti-IFNAR1 antibodies of the invention are of the IgG4 subclass.
  • anti-IFNAR1 IgG4 antibodies of the invention comprise the amino acid substitution L235E of the Fc region.
  • the anti-IFNAR1 IgG4 antibodies of the invention also comprise an amino acid change that is correlated with increased stability.
  • anti-IFNAR1 IgG4 antibodies of the invention further comprise the amino acid substitution S228P of the Fc region.
  • anti-IFNAR1 antibodies of the invention exhibit reduced or ablated binding affinities for Fc receptors (for example, but not limited to Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIA, Fc ⁇ RIB, and Fc ⁇ RIC; Fc ⁇ RII (CD32), including isoforms Fc ⁇ RIIA, Fc ⁇ RIIB, and Fc ⁇ RIIC; and Fc ⁇ RIII (CD16), including isoforms Fc ⁇ RIIIA and Fc ⁇ RIIB) as compared to an unmodified antibody.
  • the anti-IFNAR 1 antibodies of the invention exhibit decreased affinities to Fc ⁇ RI relative to an unmodified antibody.
  • the anti-IFNAR1 antibodies of the invention exhibit decreased affinities for the Fc ⁇ RIIIA receptor relative to an unmodified antibody. In another specific embodiment, the anti-IFNAR1 antibodies of the invention bind with decreased affinities to the F158V allele of Fc ⁇ RIIIA relative to an unmodified antibody.
  • anti-IFNAR1 antibodies of the invention exhibit reduced or ablated binding affinities for Clq as compared to an unmodified antibody.
  • the anti-IFNAR 1 antibodies of the invention exhibit decreased affinities to Fc ⁇ RI relative to an unmodified antibody.
  • anti-IFNAR1 antibodies of the invention exhibit reduced or ablated effector function. In a specific embodiment, anti-IFNAR1 antibodies of the invention exhibit reduced or ablated ADCC and/or CDC activity. In another specific embodiment, the anti-IFNAR1 antibodies of the invention exhibit reduced or ablated toxicity.
  • the amino acid sequences of the heavy chain variable regions and/or light chain variable regions of the anti-IFNAR1 antibodies of the invention are provided herein as FIGS. 1A , 2 A, 3 A, 4 A and FIGS. 1B , 2 B, 3 B, 4 B respectively.
  • the polynucleotide sequence encoding the heavy chain variable and light chain variable regions of the anti-IFNAR1 antibodies of the invention are provided herein as FIGS. 1A , 2 A, 3 A, 4 A and FIGS. 1B , 2 B, 3 B, 4 B respectively.
  • sequences of anti-IFNAR1 antibodies of the invention can be found in U.S. Pat. No. 5,919,453, U.S. patent application Ser. Nos. 10/831,459, 10/182,058, 11/157,494, and 11/521,102 each of which are incorporated by reference in their entireties for all purposes.
  • the sequences of the anti-IFNAR1 antibodies of the invention do not comprise the sequences found in U.S. Pat. No. 5,919,453, U.S. patent application Ser. Nos. 10/831,459, 10/182,058, 11/157,494, and 11/521,102.
  • antibodies of the invention are disclosed in U.S. Patent Provisional Application Ser. Nos. 60/842,925, filed Sep. 8, 2006, 60/866,917; filed Nov. 22, 2006; 60/911,397, filed Apr. 12, 2007; 60/915,309, filed May 22, 2007; U.S. patent application Ser. No. 11/852,106, filed Sep. 7, 2007; and PCT Application Serial No. US2007/07791, filed Sep. 7, 2007, each of which are incorporated in its entirety for all purposes.
  • anti-IFNAR1 antibodies of the invention also include antibodies that comprise an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of the 3F11, 11E2, 4G5, and 9D4 antibodies (see FIGS. 1-4 for sequences).
  • CDRs residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). Specifically, residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of “spacer” residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al. supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
  • the anti-IFNAR1 antibodies of the invention comprise at least one VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 2. In another embodiment, the anti-IFNAR1 antibodies of the invention comprise at least one VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 2. In other embodiments, the anti-IFNAR1 antibodies of the invention comprise one or more of the VH CDRs and one or more of the VL CDRs listed in Table 2. In still other embodiments, the anti-IFNAR1 antibodies of the invention comprise any combination of the VH CDRs and VL CDRs listed in Table 2.
  • the anti-IFNAR1 antibodies of the invention may comprise at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6 CDRs selected from Table 2.
  • anti-IFNAR1 antibodies of the invention may comprise a VH domain and/or a VL domain each comprising 1, 2 or 3 CDRs.
  • the anti-IFNAR1 antibodies of the invention may comprise a VH further comprising 1, 2, or 3 heavy chain CDRs (CDRH#) listed in Table 2.
  • the anti-IFNAR1 antibodies of the invention may comprise a VL further comprising 1, 2, or 3 light chain CDRs (CDRL#) listed in Table 2.
  • the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 3F11 (see for example Table 2). In another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 4G5 (see for example Table 2). In another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 11E2 (see for example Table 2). In yet another specific embodiment, the anti-IFNAR1 antibodies of the invention comprise the CDRs of antibody 9D4 (see for example Table 2).
  • anti-IFNAR1 antibodies of the invention comprise an amino acid sequence of a variable heavy chain and/or variable light chain that comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 amino acid substitutions, additions, or deletions as compared to the variable heavy chains and/or light chains represented in FIG. 1 , 2 , 3 , or 4 .
  • anti-IFNAR1 antibodies of the invention comprise one or more CDRs with at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid substitutions, deletions, or additions of one or more CDRs listed in Table 2.
  • anti-IFNAR1 antibodies of the invention comprise antibodies encoded by a polynucleotide sequence that hybridizes to the nucleotide sequence represented in FIG. 1 , 2 , 3 , or 4 under stringent conditions.
  • anti-IFNAR1 antibodies of the invention comprise one or more CDRs encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of one or more CDRs listed in FIG. 1 , 2 , 3 , or 4 .
  • Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C.
  • anti-IFNAR1 antibodies of the invention include, but are not limited to antibodies encoded by a polynucleotide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide sequence encoding antibodies 3F11, 11E2, 4G5, or 9D4 (see FIGS. 1-4 ).
  • the anti-IFNAR1 antibodies of the invention exhibit a high binding affinity for IFNAR1.
  • anti-IFNAR1 antibodies of the invention exhibit association rate (k on ) of at least 10 5 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 5 M ⁇ s ⁇ 1 , at least 10 6 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 6 M ⁇ 1 s ⁇ 1 at least 10 7 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 7 M ⁇ 1 s ⁇ 1 , or at least 10 8 M ⁇ 1 s ⁇ 1 .
  • anti-IFNAR1 antibodies of the invention exhibit a k on of at least 2 ⁇ 10 5 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 5 M ⁇ 1 s ⁇ 1 , at least 10 6 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 6 M ⁇ 1 s ⁇ 1 , at least 10 7 M ⁇ 1 s ⁇ 1 , at least 5 ⁇ 10 7 M ⁇ 1 s ⁇ 1 , or at least 10 8 M ⁇ 1 s ⁇ 1 .
  • anti-IFNAR1 antibodies of the invention exhibit a dissociation rate (k off ) of less than 10 ⁇ 1 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 1 s ⁇ 1 , less than 10 ⁇ 2 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 2 s ⁇ 1 , less than 10 ⁇ 3 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 3 s ⁇ 1 , less than 10 ⁇ 4 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 4 s ⁇ 1 , less than 10 ⁇ 5 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 5 s ⁇ 1 , less than 10 ⁇ 6 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 6 s ⁇ 1 , less than 10 ⁇ 7 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 7 s ⁇ 1 , less than 10 ⁇ 8 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 8 s ⁇ 1 , less than 10 ⁇ 1
  • anti-IFNAR1 antibodies of the invention exhibit a k off , of less than 5 ⁇ 10 ⁇ 4 s ⁇ 1 , less than 10 ⁇ 5 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 5 s ⁇ 1 , less than 10 ⁇ 6 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 6 s ⁇ 1 , less than 10 ⁇ 7 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 7 s ⁇ 1 , less than 10 ⁇ 8 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 8 s ⁇ 1 , less than 10 ⁇ 9 s ⁇ 1 , less than 5 ⁇ 10 ⁇ 9 s ⁇ 1 , or less than 10 ⁇ 10 s ⁇ 1 .
  • anti-IFNAR1 antibodies of the invention exhibit an affinity constant or K a (k on /k off ) of at least 10 2 M ⁇ 1 , at least 5 ⁇ 10 2 M ⁇ 1 , at least 10 3 M ⁇ 1 , at least 5 ⁇ 10 3 M ⁇ 1 , at least 10 4 M ⁇ 1 , at least 5 ⁇ 10 4 M ⁇ 1 , at least 10 5 M ⁇ 1 , at least 5 ⁇ 10 5 M ⁇ 1 , at least 10 6 M ⁇ 1 , at least 5 ⁇ 10 6 M ⁇ 1 , at least 10 7 M ⁇ 1 , at least 5 ⁇ 10 7 M ⁇ 1 , at least 10 8 M ⁇ 1 , at least 5 ⁇ 10 8 M ⁇ 1 , at least 10 9 M ⁇ 1 , at least 5 ⁇ 10 9 M ⁇ 1 , at least 10 10 M ⁇ 1 , at least 5 ⁇ 10 1 M ⁇ 1 , at least 10 11 M ⁇ 1 , at least 5 ⁇ 10 11 M ⁇ 1 , at least 10
  • anti-IFNAR1 antibodies of the invention exhibit a dissociation constant or K d (k off /k on ) of less than 10 ⁇ 2 M, less than 5 ⁇ 10 2 M, less than 10 ⁇ 3 M, less than 5 ⁇ 10 ⁇ 3 M, less than 10 ⁇ 4 M, less than 5 ⁇ 10 ⁇ 4 M, less than 10 ⁇ 5 M, less than 5 ⁇ 10 ⁇ 5 M, less than 10 ⁇ 6 M, less than 5 ⁇ 10 ⁇ 6 M, less than 10 ⁇ 7 M, less than 5 ⁇ 10 ⁇ 7 M, less than 10 ⁇ 8 M, less than 5 ⁇ 10 ⁇ 8 M, less than 10 ⁇ 9 M, less than 5 ⁇ 10 ⁇ 9 M, less than 10 ⁇ 10 M, less than 5 ⁇ 10 ⁇ 10 M, less than 10 ⁇ 11 M, less than 5 ⁇ 10 ⁇ 11 M, less than 10 ⁇ 12 M, less than 5 ⁇ 10 ⁇ 12 M, less than 10 ⁇ 13 M, less than 5 ⁇ 10 ⁇ 13 M, less than 10 ⁇ 14
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one or more Type I interferon (IFN) including, but not limited to, IFN ⁇ , IFNI ⁇ , and IFN ⁇ . Binding of IFN ⁇ subtypes can be determined by routine competition assays such as that described in Antibodies: A Laboratory Manual, CSHL.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of, including but not limited to, IFN ⁇ , IFNI ⁇ , and IFN ⁇ .
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one or more subtypes of IFN ⁇ including, but not limited to, IFN ⁇ subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of all subtypes of IFN ⁇ .
  • anti-IFNAR1 antibodies of the invention exhibit the ability to block the binding of and/or neutralize the biological activity of IFN ⁇ subtypes IFN ⁇ 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • anti-IFNAR1 antibodies of the invention do not exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of one or more subtypes of IFN ⁇ including, but not limited to, IFN ⁇ subtypes 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity all IFN ⁇ subtypes except IFN ⁇ 21.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the following IFN ⁇ subtypes: 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • the anti-IFNAR1 antibodies of the invention do not exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the following IFN ⁇ subtypes: 1, 2a, 2b, 4, 4b, 5, 6, 7, 8, 10, 14, 16, 17, and 21.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of non-naturally occurring type I-like interferons.
  • non-naturally occurring type I-like interferons, or hybrid type I-like interferons represent molecules that have been altered from their naturally occurring structures by recombinant or synthetic techniques.
  • Hybrid interferons as described in U.S. Pat. No. 7,232,563, represent a molecular replacement of various segments of a naturally occurring interferon structure to create a molecule that has increased potency and/or reduced toxicity.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of mutated type I interferons. Mutated type I interferons are described in U.S. Pat. Nos. 6,299,870 and 6,300,474 which are incorporated by reference in their entireties.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block binding to IFNAR1 and/or neutralize the biological activity of type I-like interferons derived from other animal species.
  • type I-like interferons are isolated from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species.
  • human type I interferons are isolated from cells derived from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species.
  • human type I interferons entail different glycosylation patterns when derived from chicken, cat, mouse, rat, rabbit, goat, horse or other animal species. Further discussion of interferons from other animal species can be found in WIPO publication No. WO06099451A3 which is hereby incorporated by reference.
  • the ability of anti-IFNAR1 antibodies of the invention to neutralize the activity of IFN ⁇ can be monitored, for example, in a Kinase Receptor Activation (KIRA) Assay as described in WO 95/14930, published Jun. 1, 1995, by measuring the ability of a candidate antibody to reduce tyrosine phosphorylation (resulting from ligand binding) of the IFNAR1/R2 receptor complex.
  • KIRA Kinase Receptor Activation
  • the ability of anti-IFNAR1 antibodies of the invention to neutralize the elicitation of a cellular response by IFN ⁇ may be tested by monitoring the neutralization of the antiviral activity of IFN ⁇ , as described by Kawade, J. Interferon Res. 1:61 70 (1980), or Kawade and Watanabe, J. Interferon Res. 4:571 584 (1984), or Yousefi, et al., Am. J. Clin. Pathol.
  • ISGF3 interferon-stimulated factor 3
  • ISRE interferon-stimulated response element
  • anti-IFNAR1 antibodies of the invention exhibit the ability to inhibit at least one IFN ⁇ mediated function of the IFNAR1 receptor. In one embodiment, the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFN ⁇ or subtypes thereof by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
  • the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFN ⁇ or subtypes thereof as measured by the KIRA assay described above by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
  • the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFN ⁇ or subtypes thereof as measured by the binding of the signaling molecule, interferon-stimulated factor 3 (ISGF3), to an oligonucleotide derived from the interferon-stimulated response element (ISRE), in an electrophoretic mobility shift assay, as described by Kurabayashi et al., Mol. Cell Biol., 15: 6386 (1995) by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
  • ISGF3 interferon-stimulated factor 3
  • ISRE interferon-stimulated response element
  • the anti-IFNAR1 antibodies of the invention inhibit the activity of the IFNAR1 receptor in response to IFN ⁇ or subtypes thereof as measured by an assay known in the art by at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
  • anti-IFNAR1 antibodies of the invention exhibit the ability to neutralize the anti-viral properties of IFN ⁇ or subtypes thereof.
  • the anti-IFNAR1 antibodies of the invention neutralize at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the anti-viral activity of IFN ⁇ or subtypes thereof, as determined by the anti-viral assay of Kawade (1980), or Yousefi (1985).
  • anti-IFNAR1 antibodies of the invention do not neutralize the anti-viral properties of IFN ⁇ or subtypes thereof.
  • the ability of anti-IFNAR1 antibodies of the invention to block the binding of IFN ⁇ or subtypes thereof to IFNAR1 can be determined by a routine competition assay such as that described in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block or inhibit binding of the following IFN ⁇ subtypes: 1, 2, 4, 5, 8, 10, and 21 to IFNAR1.
  • the anti-IFNAR1 antibodies of the invention exhibit the ability to block ore inhibit binding of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of the following IFN ⁇ subtypes: 1, 2, 4, 5, 8, 10, and 21 to IFNAR1.
  • Antibodies of the invention may act on IFNAR to regulate IFN-I responsive genes.
  • IFN-I responsive genes have been identified in US Patent Applications entitled “IFN alpha-induced Pharmacodynamic Markers” with the following serial numbers; 60/873,008, filed Dec. 6, 2006; 60/907,762, filed Apr. 16, 2007; 60/924, 584, filed May 21, 2007; 60/960,187, filed Sep. 19, 2007; 60/966, 176, filed Nov. 5, 2007 and PCT application serial number PCT/US2007/02494, filed Dec. 6, 2007 each of which are incorporated by reference in their entireties.
  • Antibodies of the invention may include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • the terms “antibody” and “antibodies” specifically include the modified antibodies described herein.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
  • Antibodies of the invention can be of any isotype. In one embodiment, antibodies of the invention are of the IgG1, IgG2, IgG3 or IgG4 isotype.
  • Antibodies of the invention can be full-length antibodies comprising variable and constant regions, or they can be antigen-binding fragments thereof, such as a single chain antibody.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IFNAR1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V L , V H , C L and C H1 domains
  • F(ab′) 2 fragment a bivalent fragment comprising two
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody.
  • the invention provides fusion proteins (hereinafter referred to as “fusion proteins of the invention”) comprising a modified Fc region with reduced or ablated affinity for an Fc ligand responsible for facilitating effector function compared to an Fc region having the same amino acid sequence as the fusion protein of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region.
  • fusion proteins of the invention comprising a modified Fc region with reduced or ablated affinity for an Fc ligand responsible for facilitating effector function compared to an Fc region having the same amino acid sequence as the fusion protein of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region.
  • fusion proteins of the invention may comprise a peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or an antibody mimetic fused to a modified Fc region.
  • fusion proteins of the invention comprise a linker region connecting the peptide, polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or an antibody mimetic to the modified Fc region.
  • fusion proteins of the invention comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue selected from the group consisting of: 234, 235, and 331, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • fusion proteins of the invention comprise an Fc region comprising at least one amino acid residue selected from the group consisting of: L234F, L235E, and P331S.
  • fusion proteins of the invention further comprise an Fc region comprising at least one addition, substitution, or deletion of an amino acid residue that is correlated with increased stability of the fusion protein.
  • the addition, substitution, or deletion of an amino acid residue is at position 228 of the Fc region, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (supra).
  • fusion proteins of the invention comprise an Fc region comprising an amino acid substitution at position 228, wherein the substitution is a serine residue.
  • the antibodies or fusion proteins of the present invention comprise one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to a molecule comprising an Fc region.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to reducing effector function.
  • Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed.
  • Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999 , Nat.
  • the present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • moieties including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452, which are incorporated by reference in their entireties.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be modified by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to IFNAR1 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the antibodies or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the “Flag” tag.
  • antibodies of the present invention or fragments, analogs or derivatives thereof conjugated to a diagnostic or detectable agent can be useful for monitoring or prognosing the development or progression of an inflammatory disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine ( 131 I, 125 I, 123 I, 125 I), carbon ( 14 C),
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
  • the therapeutic moiety or drug conjugated to an antibody or fragment thereof that specifically binds to IFNAR1 should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject.
  • a clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to an antibody or fragment thereof that specifically binds to IFNAR1: the nature of the disease, the severity of the disease, and the condition of the subject.
  • the antibodies or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with IFNAR1 and once an immune response is detected, e.g., antibodies specific for IFNAR1 are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • monoclonal antibodies can be generated by culturing a hybridoma cell secreting an antibody of the invention wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with IFNAR1 with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind IFNAR1.
  • Antibody fragments which recognize specific IFNAR1 epitopes may be generated by any technique known to those of skill in the art.
  • Fab and F(ab′) 2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′) 2 fragments).
  • F(ab′) 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues).
  • the DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to the IFNAR1 epitope of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce Fab, Fab′ and F(ab′) 2 fragments can also be employed using methods known in the art such as those disclosed in International Publication No.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • VH constant region e.g. the human gamma 4 constant region
  • VL constant region e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains comprise an EF-1 alpha promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,311,415, which are incorporated herein by reference in their entirety.
  • a humanized antibody is an antibody or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′) 2 , Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD; IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit: cytotoxic activity and the class is typically IgG 1 .
  • the constant domain may be of the IgG 2 class.
  • the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and possibly greater than 95%.
  • FR parental framework region
  • Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter or improve antigen binding.
  • framework substitutions are identified by methods known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323, which are incorporated herein by reference in their entireties.)
  • the invention also encompass polynucleotides that hybridize under high stringency, intermediate or lower stringency hybridization conditions, e.g., as defined above, to polynucleotides that encode an antibody of the invention.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of the antibodies are known, nucleotide sequences encoding these antibodies can be determined using methods known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody or fragment thereof of the invention.
  • Such a polynucleotide encoding the antibody maybe assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmejer et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmejer et al., 1994, BioTechniques 17:242
  • oligonucleotides e.g., as described in Kutmejer et al., 1994, BioTechniques 17:242
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, usually poly A+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be clolified
  • the nucleotide sequence of the antibody may be manipulated using methods known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques.
  • the framework regions may be naturally occurring or consensus framework regions, and in certain instances human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to IFNAR1.
  • one or more amino acid substitutions may be made within the framework regions, and, in certain instances, the amino acid substitutions improve binding of the antibody to its antigen.
  • Such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • antibodies of the invention are encoded by polynucleotide sequences exemplified in FIGS. 1-4 .
  • polynucleotides of the invention encode antibodies comprising light chain and heavy chain constant regions corresponding to SEQ ID Nos: 41 and 42 respectively.
  • polynucleotides of the invention encode antibodies comprising heavy chain constant regions corresponding to SEQ ID No: 42 with an allowance for allelic variation wherein the variation is at least one or more residue selected from the group consisting of positions 214, 221, 356, and 358 as defined by the EU index numbering system.
  • an antibody of the invention e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention
  • an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (but not necessarily containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques known in the art.
  • a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication No. WO 86/05807; International Publication No. WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody maybe cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention (see, e.g., U.S. Pat. No. 5,807,715).
  • host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces and Pichia ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.
  • Ti plasmid containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • bacterial cells such as Escherichia coli
  • eukaryotic cells especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2).
  • CHO Chinese hamster ovary cells
  • the expression of nucleotide sequences encoding antibodies or fragments thereof which specifically bind to IFNAR1 is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • GST glutathione 5-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2 197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • antibodies of the invention may be produced by a scalable process (hereinafter referred to as “scalable process of the invention”).
  • scalable process of the invention antibodies may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the antibodies of the invention in analytical scale bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L, or 50 L bioreactors).
  • the antibodies may be produced by a scalable process of the invention in the research laboratory that may be scaled up to produce the antibodies of the invention in production scale bioreactors (for example, but not limited to 75 L, 100 L, 150 L, 300 L, or 500 L).
  • the scalable process of the invention results in little or no reduction in production efficiency as compared to the production process performed in the research laboratory.
  • the scalable process of the invention produces antibodies at production efficiency of about 10 mg/L, about 20 m/L, about 30 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 250 mg/L, about 300 mg/L or higher.
  • fusion proteins may be produced by scalable processes of the invention.
  • the scalable process of the invention produces antibodies at production efficiency of at least about 10 mg/L, at least about 20 m/L, at least about 30 mg/L, at least about 50 mg/L, at least about 75 mg/L, at least about 100 mg/L, at least about 125 mg/L, at least about 150 mg/L, at least about 175 mg/L, at least about 200 mg/L, at least about 250 mg/L, at least about 300 mg/L or higher.
  • the scalable process of the invention produces antibodies at production efficiency from about 10 mg/L to about 300 mg/L, from about 10 mg/L to about 250 mg/L, from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about 175 mg/L, from about 10 mg/L to about 150 mg/L, from about 10 mg/L to about 100 mg/L, from about 20 mg/L to about 300 mg/L, from about 20 mg/L to about 250 mg/L, from about 20 mg/L to about 200 mg/L, from 20 mg/L to about 175 mg/L, from about 20 mg/L to about 150 mg/L, from about 20 mg/L to about 125 mg/L, from about 20 mg/L to about 100 mg/L, from about 30 mg/L to about 300 mg/L, from about 30 mg/L to about 250 mg/L, from about 30 mg/L to about 200 mg/L, from about 30 mg/L to about 175 mg/L, from about 30 mg/L to about 150 mg/L, from about 10 mg/
  • an Fc hinge region of an antibody of the invention is mutated to decrease the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcal protein A
  • an antibody is modified to increase its biological half life.
  • Various approaches are possible.
  • one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375.
  • one or more of the following mutations can be introduced: M252Y, S254T, T256E, as described in U.S. Pat. No. 7,083,784.
  • the antibody can be modified within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
  • an Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to reduce the effector function(s) of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has reduced affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is reduced can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has reduced Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues within amino acid positions 231 and 239 are modified to thereby reduce the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
  • an Fc region of an antibody of the invention is further modified to decrease the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody for an Fc ⁇ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439.
  • ADCC antibody dependent cellular cytotoxicity
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
  • the structural features of anti-IFNAR1 antibodies are used to create structurally related anti-IFNAR1 antibodies that retain at least one functional property of antibodies of the invention, such as binding to IFNAR1.
  • one or more CDR regions of 3F11, 4G5, 11E2, or 9D4, or mutations thereof can be combined recombinantly with known framework regions and/or other CDRs to create additional, recombinantly-engineered, anti-IFNAR1 antibodies of the invention, as discussed above.
  • Other types of modifications include those described in the previous section.
  • the starting material for the engineering method is one or more of the V H and/or V L sequences provided herein, or one or more CDR regions thereof.
  • To create the engineered antibody it is not necessary to actually prepare (i.e., express as a protein) an antibody having one or more of the V H and/or V L sequences provided herein, or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as the starting material to create a “second generation” sequence(s) derived from the original sequence(s) and then the “second generation” sequence(s) is prepared and expressed as a protein.
  • Such compositions may include one or a combination of (e.g., two or more different) antibodies, fusion proteins, immunoconjugates or bispecific molecules of the invention.
  • such compositions are physiologically tolerable and as such are suitable for administration to a subject (also referred to as a “pharmaceutical composition of the invention.”
  • pharmaceutical compositions of the invention may comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.
  • compositions of the invention may include one or more pharmaceutically acceptable salts.
  • salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • compositions of the invention also may include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxy
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions of the invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions (e.g., liquid formulations) of the invention are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. In another embodiment, endotoxin and pyrogen levels in the composition are less then about 10 EU/mg, or less then about 5 EU/mg, or less then about 1 EU/mg, or less then about 0.1 EU/mg, or less then about 0.01 EU/mg, or less then about 0.001 EU/mg.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, also from about 0.1 percent to about 70 percent, also from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months.
  • Dosage regimens for an anti-IFNAR1 antibody of the invention include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • an antibody of fusion protein may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and usually until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a therapeutically effective dosage of an anti-IFNAR1 antibody of the invention results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a therapeutically effective dose may prevent further deterioration of physical symptoms associated with SLE, such as, for example, pain, fatigue or weakness.
  • a therapeutically effective dose may also prevent or delays onset of SLE, such as may be desired when early or preliminary signs of the disease are present. Likewise it includes delaying chronic progression associated with SLE.
  • Laboratory tests utilized in the diagnosis of SLE include chemistries, hematology, serology and radiology.
  • any clinical or biochemical assay that monitors any of the foregoing may be used to determine whether a particular treatment is a therapeutically effective dose for treating SLE.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • a composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes and/or mode of administration will vary depending upon the desired results.
  • Selected routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems , J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S
  • antibodies of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
  • antibodies of the present invention have in vitro and in vivo diagnostic and therapeutic utilities.
  • these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.
  • antibodies of the invention can be used to detect levels of IFNAR1, or levels of cells that express IFNAR1. This can be achieved, for example, by contacting a sample (such as an in vitro sample) and a control sample with the anti-IFNAR1 antibody under conditions that allow for the formation of a complex between the antibody and IFNAR1. Any complexes formed between the antibody and IFNAR1 are detected and compared in the sample and the control. For example, standard detection methods, well-known in the art, such as ELISA and flow cytometric assays, can be performed using the compositions of the invention.
  • the invention further provides methods for detecting the presence of IFNAR1(e.g., human IFNAR1 antigen) in a sample, or measuring the amount of IFNAR1, comprising contacting the sample, and a control sample, with antibodies of the invention, or an antigen binding portion thereof, which specifically binds to IFNAR1, under conditions that allow for formation of a complex between the antibody or portion thereof and IFNAR1. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of IFNAR1 in the sample.
  • IFNAR1 e.g., human IFNAR1 antigen
  • IFNAR1 is part of the cellular receptor for Type I interferons, and Type I interferons are known to be immunoregulatory cytokines that are involved in T cell differentiation, antibody production and activity and survival of memory T cells. Moreover, increased expression of Type I interferons has been described in numerous autoimmune diseases, in HIV infection, in transplant rejection and in graft versus host disease (GVHD). Accordingly, the anti-IFNAR1 antibodies of the invention or fragments thereof, which inhibit the functional activity of Type I interferons, can be used in a variety of clinical indications involving aberrant or undesired Type I interferon activity.
  • the invention encompasses methods of preventing, treating, maintaining, ameliorating, or inhibiting a Type I interferon-mediated disease or disorder, wherein the methods comprise administering antibodies, or antigen-binding portions thereof, of the invention.
  • autoimmune conditions include, but are not limited to, the following: systemic lupus erythematosus (SLE), insulin dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's Disease, Ulcerative Colitis and Celiac's Disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA) and glomerulonephritis.
  • SLE systemic lupus erythematosus
  • IDDM insulin dependent diabetes mellitus
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • psoriasis autoimmune thyroiditis
  • RA rheumatoid arthritis
  • glomerulonephritis glomerulonephritis.
  • the antibody compositions of the invention can be used for inhibiting or preventing transplant rejection or in the treatment of graft versus host disease (GVHD) or in the treatment of HIV infection/AIDS.
  • anti-IFNAR1 antibodies of the invention can be used in the treatment of SLE by administering the antibody to a subject in need of treatment.
  • IFN ⁇ also has been implicated in the pathology of Type I diabetes.
  • the presence of immunoreactive IFN ⁇ in pancreatic beta cells of Type I diabetes patients has been reported (Foulis et al. (1987) Lancet 2:1423-1427). Prolonged use of IFN ⁇ in anti-viral therapy also has been shown to induce Type I diabetes (Waguri et al. (1994) Diabetes Res. Clin. Pract. 23:33-36).
  • the anti-IFNAR1 antibodies or fragments thereof of the invention can be used in the treatment of Type I diabetes by administering the antibody to a subject in need of treatment.
  • the antibody can be used alone or in combination with other anti-diabetic agents, such as insulin.
  • Antibodies to IFNAR1 have been shown to be effective in an animal model of inflammatory bowel disease (see U.S. Patent Application 60/465,155).
  • the anti-IFNAR1 antibodies or fragments thereof of the invention can be used in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, by administering the antibody to a subject in need of treatment.
  • IBD inflammatory bowel disease
  • anti-IFNAR1 antibodies of the invention can be used in the treatment of autoimmune thyroid disease, including autoimmune primary hypothyroidism, Graves Disease, Hashimoto's thyroiditis and destructive thyroiditis with hypothyroidism, by administering an antibody of the invention to a subject in need of treatment.
  • Antibodies of the invention can be used alone or in combination with other agents or treatments, such as anti-thyroid drugs, radioactive iodine and subtotal thyroidectomy.
  • anti-IFNAR1 antibodies of the invention may be used in the treatment of HIV infection or AIDS by administering the antibody of the invention to a subject in need of treatment.
  • antibodies of the invention can be used alone or in combination with other anti-HIV agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors.
  • Antibodies to IFNAR1 have been demonstrated to be effective in inhibiting allograft rejection and prolonging allograft survival (see e.g., Tovey et al. (1996) J. Leukoc. Biol. 59:512-517; Benizri et al. (1998) J. Interferon Cytokine Res. 18:273-284). Accordingly, the anti-IFNAR1 antibodies of the invention also can be used in transplant recipients to inhibit allograft rejection and/or prolong allograft survival.
  • the invention provides a method of inhibiting transplant rejection by administering anti-IFNAR1 antibodies of the invention to a transplant recipient in need of treatment.
  • tissue transplants that can be treated include, but are not limited to, liver, lung, kidney, heat, small bowel, and pancreatic islet cells, as well as the treatment of graft versus host disease (GVHD).
  • Antibodies of the invention can be used alone or in combination with other agents for inhibiting transplant rejection, such as immunosuppressive agents (e.g., cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate mofetil, sirilimus, rapamycin, tacrolimus), anti-infective agents (e.g., acyclovir, clotrimazole, ganciclovir, nystatin, trimethoprimsulfarnethoxazole), diuretics (e.g., bumetanide, furosemide, metolazone) and ulcer medications (e.g., cimetidine, famotidine, lansoprazole,
  • the invention provides methods of administering and using compositions and antibodies of the invention to treat and prevent a wide range of inflammatory conditions including both chronic and acute conditions, such as, but not limited to, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prosta
  • methods of administration and compositions of antibodies of the invention may be useful in the prevention, treatment, amelioration of symptoms associated with the following conditions or disease states: Graves's disease, Hashimoto's thyroiditis, Crohn's disease, psoriasis, psoriatic arthritis, sympathetic opthalmitis, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome.
  • methods of administration and compositions of antibodies of the invention may be useful in the prevention, treatment, amelioration of symptoms associated with Sjogren's syndrome.
  • Sjögren's syndrome is an autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva. It is named after Swedish ophthalmologist Henrik Sjögren (1899-1986), who first described it. Sjögren's syndrome is also associated with rheumatic disorders such as rheumatoid arthritis, and it is rheumatoid factor positive in 90 percent of cases. The hallmark symptoms of the disorder are dry mouth and dry eyes.
  • Sjögren's syndrome may cause skin, nose, and vaginal dryness, and may affect other organs of the body, including the kidneys, blood vessels, lungs, liver, pancreas, and brain.
  • Sjögren's patients are women and the average age of onset is late 40s, although Sjögren's occurs in all age groups in both women and men. It is estimated to strike as many as 4 million people in the United States alone making it the second most common autoimmune rheumatic disease.
  • Myositis is general condition characterized by inflammation of skeletal muscle or voluntary muscle. Muscle inflammation may be caused by an allergic reaction, exposure to a toxic substance or medicine, another disease such as cancer or rheumatoid conditions, or a virus or other infectious agent.
  • the chronic inflammatory myopathies are idiopathic, meaning they have no known cause. They are understood to be autoimmune disorders, in which the body's white blood cells (that normally fight disease) attack blood vessels, normal muscle fibers, and connective tissue in organs, bones, and joints.
  • Polymyositis affects skeletal muscles (involved with making movement) on both sides of the body. It is rarely seen in persons under age 18; most cases are in patients between the ages of 31 and 60. In addition to symptoms listed above, progressive muscle weakness leads to difficulty swallowing, speaking, rising from a sitting position, climbing stairs, lifting objects, or reaching overhead. Patients with polymyositis may also experience arthritis, shortness of breath, and heart arrhythmias.
  • Dermatomyositis is characterized by a skin rash that precedes or accompanies progressive muscle weakness.
  • the rash looks patchy, with bluish-purple or red discoloration, and characteristically develops on the eyelids and on muscles used to extend or straighten joints, including knuckles, elbows, heels, and toes. Red rashes may also occur on the face, neck, shoulders, upper chest, back, and other locations, and there may be swelling in the affected areas.
  • the rash sometimes occurs without obvious muscle involvement.
  • Adults with dermatomyositis may experience weight loss or a low-grade fever, have inflamed lungs, and be sensitive to light.
  • IBM Inclusion body myositis
  • IBM is similar to polymyositis but has its own distinctive features. The onset of muscle weakness is generally gradual (over months or years) and affects both proximal and distal muscles. Muscle weakness may affect only one side of the body. Small holes called vacuoles are seen in the cells of affected muscle fibers. Falling and tripping are usually the first noticeable symptoms of IBM. For some patients the disorder begins with weakness in the wrists and fingers that causes difficulty with pinching, buttoning, and gripping objects. There may be weakness of the wrist and finger muscles and atrophy (thinning or loss of muscle bulk) of the forearm muscles and quadricep muscles in the legs.
  • Difficulty swallowing occurs in approximately half of IBM cases. Symptoms of the disease usually begin after the age of 50, although the disease can occur earlier. Unlike polymyositis and dermatomyositis, IBM occurs more frequently in men than in women.
  • Juvenile myositis has some similarities to adult dermatomyositis and polymyositis. It typically affects children ages 2 to 15 years, with symptoms that include proximal muscle weakness and inflammation, edema (an abnormal collection of fluids within body tissues that causes swelling), muscle pain, fatigue, skin rashes, abdominal pain, fever, and contractures (chronic shortening of muscles or tendons around joints, caused by inflammation in the muscle tendons, which prevents the joints from moving freely). Children with juvenile myositis may also have difficulty swallowing and breathing, and the heart may be affected. Approximately 20 to 30 percent of children with juvenile dermatomyositis develop calcinosis. Juvenile patients may not show higher than normal levels of the muscle enzyme creatine kinase in their blood but have higher than normal levels of other muscle enzymes.
  • antibodies of the invention may be useful in the prevention, treatment, or amelioration of myositis, inflammatory myositis, idiopathic myositis, polymyositis, dermatomyositis, inclusion body myositis (IBM), juvenile myositis or symptoms associated with these conditions.
  • myositis inflammatory myositis, idiopathic myositis, polymyositis, dermatomyositis, inclusion body myositis (IBM), juvenile myositis or symptoms associated with these conditions.
  • IBM inclusion body myositis
  • antibodies of the invention may be useful in the prevention, treatment, or amelioration of symptoms associated with vasculitis.
  • Antibodies of the invention may be useful for the treatment of scleroderma. Methods of treating Scleroderma are described in a U.S. patent application entitled “Methods Of Treating Scleroderma” with an application Ser. No. 60/996,175, filed on Nov. 5, 2007 and PCT Application No. PCT/US2008/82481 are incorporated by reference in their entireties for all purposes.
  • antibodies of the invention may be useful in the prevention, treatment, or amelioration of symptoms associated with sarcoidosis.
  • Sarcoidosis also called sarcoid or Besnier-Boeck disease
  • granulomas small inflammatory nodules.
  • Virtually any organ can be affected; however, granulomas most often appear in the lungs or the lymph nodes. Symptoms can occasionally appear suddenly but usually appear gradually.
  • sarcoidosis can have the appearance of tuberculosis or lymphoma.
  • kits comprising the compositions (e.g., anti-IFNAR1 antibodies) of the invention and instructions for use.
  • the kit can further contain a least one additional reagent, or one or more additional antibodies of the invention (e.g., an antibody having a complementary activity which binds to an epitope on the target antigen distinct from the first antibody).
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • compositions of the invention also can be administered in combination therapy, such as, combined with other agents.
  • the combination therapy can include an anti-IFNAR1 antibody of the present invention combined with at least one other immunosuppressant.
  • two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • the antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient.
  • dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g/ml and in some methods about 25-300 ⁇ g/ml.
  • an anti-IFNAR1 antibody of the invention can be used in combination with one or more of the following agents: drugs containing mesalamine (including sulfasalazine and other agents containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (e.g., prednisone, hydrocortisone), TNF-inhibitors (including adalimumab (HUMIRA®), etanercept (ENBREL®) and infliximab (REMICADE®)), immunosuppressants (such as 6-mercaptopurine, azathioprine and cyclosporine A), and antibiotics anti-IFN ⁇ antibody, anti-IFN ⁇ receptor antibody, and soluble IFN ⁇ receptor.
  • drugs containing mesalamine including sulfasalazine and other agents containing 5-aminosalicylic
  • compositions of the invention may also include agents useful in the treatment of SLE.
  • agents useful in the treatment of SLE include analgesics, corticosteroids (e.g., prednisone, hydrocortisone), immunosuppressants (such as cyclophosphamide, azathioprine, and methotrexate), antimalarials (such as hydroxychloroquine) and biologic drugs that inhibit the production of dsDNA antibodies (e.g., LJP 394).
  • Immunohistochemistry techniques to study antibody binding characteristics are readily known in the art and for example, could be performed by isolating the desired cells or tissues and preparing them for microscopy by standard fixation and mounting techniques.
  • Mouse Macrophages A cell suspension was spun down to form a loose pellet. The pellet was frozen in OCT freezing medium to form a block. Slide sections were cut to 5 microns thickness, soaked in acetone for 10 minutes and allowed to dry with desiccant overnight. Prior to use, the slides were dipped into 10% neutral buffered formalin for 10 sec and washed 3 ⁇ in buffer (1 ⁇ TBS with 0.01% Tween20).
  • Human Monocytes A cell suspension was smeared/spotted directly onto slides. The slides were allowed to dry overnight and then soaked in Acetone for 10 min and allowed to air dry. Prior to use, slides were dipped into 10% neutral buffered formalin for 10 secs and washed 3 ⁇ in buffer (1 ⁇ TBS with 0.01% Tween20).
  • Human Cerebrum and Cardiac Tissue Tissue samples from donors were frozen in OCT freezing medium to form a block. Slide sections were cut to 5 micron thickness, soaked in acetone for 10 minutes and allowed to dry with desiccant overnight. Prior to use, the slides were dipped into 10% neutral buffered formalin for 10 sec and washed 3 ⁇ in buffer (1 ⁇ TBS with 0.01% Tween 20).
  • Antibodies were conjugated to biotin by the following protocol. Approximately 500 ⁇ g of antibody was mixed with a 20 fold excess of biotin and incubated for 2 hours in the dark at 4° C. After the 2 hour incubation, the antibody/biotin mix was applied to a pre-equilibrated PD10 column with 1 ⁇ PBS. Subsequently, the biotin conjugated antibodies were concentrated to a desired concentration using an YM-30 Centricon concentration tube.
  • Slide staining After washing in buffer, slides were treated to quench endogenous peroxidases by treatment with a solution of Glucose Oxidase (1 U/ml, Sigma G0543), B-D(+) Glucose (10 mM, Sigma G5250), Sodium Azide (1 mM, Sigma, S8032) for 1 hour at room temperature. Slides were then rinsed in wash buffer (1 ⁇ TBS with 0.01% Tween 20). Slides were placed in a protein block solution (1 ⁇ PBS pH7.2, 0.5% casein(N-Z amine, Sigma CO626), 1% BSA (Sigma A7906), 1.5% Normal Goat serum (Jackson Labs #005-000-001) for 30 min at room temperature.
  • Biotinylated antibody (see above) was applied to the slides by dilution into the protein block solution. Incubation of the slides with the biotinylated antibody was performed at room temperature for 2 hours. Slides were rinsed 3 ⁇ in wash buffer (1 ⁇ TBS, 0.01% Tween 20). Antibody detection was performed using a Vectastain Kit (Vector Laboratories). Slides were washed and counterstained with hematoxylin. Slides were dehydrated and mounted with coverslips prior to viewing.
  • results Presented in FIG. 6A are the results of an IHC analysis of Human cerebrum tissue stained with various anti-IFNAR1 and control antibodies.
  • the antibodies MDX-1333 (75 ⁇ g/ml) and 4G5 (50 ⁇ g/ml) exhibited strong staining of the cerebrum tissue as exemplified by the brown/dark staining seen throughout the samples.
  • Antibody 9D4 (50 ⁇ g/ml) did not stain the human cerebrum tissue sample as well as MDX-1333 and 4G5 as demonstrated by the reduced brown/dark staining throughout the sample.
  • An IgG1 isotype control was included to demonstrate that binding specificity of the individual antibodies.
  • FIG. 6B are the results of an IHC analysis of monocytes stained with various anti-IFNAR1 and control antibodies.
  • the antibodies MDX-1333 (50 and 20 ⁇ g/ml), 4G5 (50 ⁇ g/ml) and 9D4 (50 and 20 ⁇ g/ml) all exhibited prominent staining on human monocytes as demonstrated by the brown/dark staining of the samples.
  • the isotype control antibody R3-47 did not exhibit prominent staining on human monocytes.
  • MDX-1333(50 ⁇ g/ml) did not stain purified mouse macrophages.
  • the modified anti-IFNAR1 antibody designated “9D4-TM” was generated through the following procedure;
  • Human ⁇ 1 Fc was cloned and engineered from human PBLs by first isolating total RNA, transcribing cDNA, and PCR amplifying the constant regions with gene-specific primers containing restriction sites Apa I and EcoRI for cloning into the mammalian vector PEE6.
  • the triple mutant (TM) includes three amino acid changes in human IgG to decrease ADCC effector function (L234F, L235E, and P331S).
  • TM was engineered using human IgG1 (KOL) as a template, and utilizing site-directed mutagenesis (QuickChange XL, Stratagene) to encode three residue changes in the Fc. Sequence of the mutagenic primers used to encode the L234F/L235E/P331S changes were as follows:
  • MD1056 5′ cgtgcccagcacctgaaTtcGAggggggaccgtcagtcttc 3′ L234F, L235E forward (SEQ ID NO: 44)
  • MD1057 5′ gaagactgacggtcccccTCgaAttcaggtgctgggcacg 3′ L234F
  • L235E reverse SEQ ID NO: 45
  • MD1058 5′ ccaacaaagccctcccagccTccatcgagaaaaccatctcc 3′ P331S forward (SEQ ID NO: 46)
  • MD1059 5′ ggagatggtttctcgatggAggctgggagggctttgtgg 3′ P331S reverse
  • Clones encoding the 9D4-TM antibody were sequenced to confirm the triple mutations, and resolved on the ABI3100 genetic analyzer.
  • the modified anti-IFNAR1 antibody designated “9D4-DM” was generated through the following procedure;
  • Human ⁇ 4 Fc was cloned and engineered from human PBLs by first isolating total RNA, transcribing cDNA, and PCR amplifying the constant regions with gene-specific primers containing restriction sites Apa I and EcoRI for cloning into the mammalian vector PEE6.
  • the double mutant (DM) consists of two mutations in human IgG4 Fc: S228P and L235E. Mutagenic primers to encode DM include:
  • MD1060 5′ ggtcccccatgcccaCcatgcccagcacctg 3′ hinge S228P forward (SEQ ID NO: 48)
  • MD1061 5′ caggtgctgggcatgGtgggcatgggggacc 3′ hinge S228P reverse (SEQ ID NO: 49)
  • MD1062 5′ ccagcacctgagttcGAggggggaccatcagtc 3′IgG4 L234F, L235E forward (SEQ ID NO: 50)
  • MD1063 5′ gactgatggtcccccTCgaactcaggtgctgg 3′ IgG4 L234F, L235E reverse
  • Clones encoding the 9D4-DM antibody were sequenced to confirm the encoded changes, and resolved on the ABI3100 genetic analyzer.
  • Anti-IFNAR1Antibodies Inhibit IFN Mediated STAT Phosphorylation
  • Peripheral blood mononuclear cells were purified from healthy human donors using LSM media (MP Biomedical, Solon Ohio). PBMCs were quantified and seeded at 10 6 cell per condition per well. Antibodies were added at 10 ⁇ g/mL to appropriate well and incubated at 37° C., 5% CO 2 for 10 minutes. After pre-incubation with antibodies, recombinant human IFN ⁇ 2a (PBL Biomedical, Piscataway N.J.) or human plasmacytoid dendritic cell-derived IFN (see below for generation of PDCs derived type-I IFN supernatants) was added to appropriate wells at 100 or 5001 U/mL for 20 minutes.
  • LSM media MP Biomedical, Solon Ohio
  • Cells were spun at 1200 rpm for 5 minutes and washed with sterile 1 ⁇ PBS. After one additional spin, PBS was removed and cells were lysed using mammalian protein extraction reagent (Pierce, Rockford Ill.) supplemented with 300 ⁇ L of 1 ⁇ phosphatase inhibitor cocktails 1 and 2 (Sigma, St. Louis Mo.) and 1 ⁇ protease inhibitor (Roche Biomedical, Nutley N.J.). Lysates were incubated for 10 minutes on an orbital shaker to ensure complete lysis, transferred to microfuge tubes and spun at 14000 rpm to remove cellular debris. NuPAGE sample buffer (Invitrogen, Carlsbad Calif.) and dTT (Sigma, St.
  • Blocking media was subsequently removed and 0.2 ⁇ g/mL anti-STAT1, anti-STAT1 pY701, or 1:1000 dilution of 13-Actin antibodies (Cell Signaling Technology, Danvers Mass.) were added to appropriate blots and incubated overnight at 4° C. Blots were washed 3 ⁇ in 1 ⁇ TBS with 0.05% Tween20 (Sigma, St. Louis Mo.). 1:2500 diluted, HRP conjugated anti-rabbit secondary antibody was added to blots and incubated for 1 hr at room temperature.
  • Blots were washed as described before and 3 mL of a 1:1 mixture of Pico Supersignal West reagent (Pierce, Rockford Ill.) was added to each blot for 1 minute. Blots were drained, excess reagent was removed and bands were visualized using a Kodak X-omat 1000A Processor.
  • Type I IFN Using purified Type I IFN from pDC cells, a reporter assay was used to test the ability of anti-IFNAR1 antibodies to block Type I IFN signaling.
  • Plasmacytoid dendritic cells were isolated from whole blood of healthy donors using a lymphocyte separation media (MP Biomedical, Solon Ohio) followed by positive selection using CD304 (BDCA-4/Neuropilin-1) MicroBead Kit (Milteny Biotec, Auburn Calif.). Purified PDCs were then cultured at 1 ⁇ 10 6 cells/mL in RPMI 1640 supplemented with 10% FBS (Gibco BRL) and 6 ⁇ g/mL CpGA (InVivogen, San Diego Calif.).
  • HEK293 ATCC, Manassas Va. cells were stably transfected with pHTS-ISRE reported plasmid (Biomyx Technology, San Diego Calif.) and were maintained in DMEM supplemented with 10% FBS, 1 ⁇ NEAA, and 700 ⁇ g/mL G418 (Invitrogen, Carlsbad Calif.). Cells were seeded at a concentration of 80,000 cells per well in Optilix white/clear 96 well plates (VWR, West Chester Pa.).
  • Type I IFN supernatants were harvested from pDC cells derived from three individual donors.
  • incubation of anti-IFNAR1 antibodies inhibited the signaling ability of various concentrations of Type I IFN supernatant ( FIG. 8 ).
  • 200,000 HEK 293F cells were seeded in a round bottom, 96-well plate using 50 ⁇ L RPMI 1640 media supplemented with 10% FBS.
  • Europium-labeled 9D4-TM was prepared under contract by PerkinElmer Life and Analytical Sciences.
  • 25 ⁇ L of 100-fold excess unlabeled, serially diluted anti-IFNAR1 antibodies were added to appropriate wells of the 96 well for 5-10 minutes prior to the addition of labeled 9D4-TM.
  • 25 ⁇ L of europium conjugated, serially diluted antibody was then added to appropriate wells and cells and antibodies were agitated gently at room temperature for 1-2 hours.
  • srIFNAR1 ligand was coated onto UltraLink® Biosupport beads (PIERCE, Rockford, Ill.) at concentrations of 5 ⁇ g/mL and 50 ⁇ g/mL in coating buffer (50 mM sodium carbonate buffer, pH9) for a period of 1-2 days at 4° C. Coated beads were then separated (gentle pulse spin) from unreacted ligand solution, and gently rocked in block buffer (1 mL 1M Tris, pH8, containing BSA at 10 mg/mL) for about 15 min at room temperature (RT). After this, the bead slurry was again spun to remove the blocking solution, and then the block step was repeated for about 2 hrs at RT using a fresh aliquot of block buffer.
  • UltraLink® Biosupport beads PIERCE, Rockford, Ill.
  • the coated beads were stored at 4° C. until used. Prior to use, the srIFNAR1-coated beads were transferred to a bead vial, resuspended in 27 mLs of instrument run buffer (PBS, pH7.4-0.02% NaN3), then attached to the KinExA 3000 instrument.
  • instrument run buffer PBS, pH7.4-0.02% NaN3
  • K D equilibrium binding constants
  • Results Depicted in FIG. 10A are the binding curves for three concentrations of 9D4-TM (1 ⁇ M, 10 ⁇ M, and 50 ⁇ M) with sIFN ⁇ RI. Data obtained from at least three independent experiments were fitted to a software derived binding curve to establish a relative K D for 9D4-TM.
  • the K D of 9D4-TM in this binding assay was determined to be 1.1 pM with a 95% confidence interval of 0.603 pM-1.8 pM. The percentage error of the K D determination of 1.1 pM was 1.96%.
  • the Kon and Koff for 9D4-TM was also determined to be 7 ⁇ 10 6 +/ ⁇ 1.3 ⁇ 10 6 S ⁇ 1 and 7.7 ⁇ 10 ⁇ 6 +/ ⁇ 1.57 ⁇ 10 ⁇ 6 1/Ms respectively (data not shown).
  • Peripheral blood mononuclear cells were purified from healthy human donors using LSM media (MP Biomedical, Solon Ohio). Cells were counted and 200,000 cells were seeded in a round bottom, 96-well plate using 50 ⁇ L RPMI 1640 media supplemented with 10% FBS.
  • Europium-labeled 9D4-TM was prepared under contract by PerkinElmer Life and Analytical Sciences. To measure non-specific europium signal, 25 ⁇ L of 100-fold excess unlabeled, serially diluted 9D4-TM was added to appropriate wells of the 96 well for 5-10 minutes prior to the addition of labeled 9D4-TM.
  • modified anti-IFNAR2 antibodies i.e. anti-IFNAR1 antibodies with reduced Fc ligand affinity
  • the Luciferase Reporter assay system used in this example has been previously described above (See Example 3).
  • Antibodies to IFNAR1 used in this example include 9D4, 9D4-DM, 9D4-TM, MDX-1333. Included is a control antibody R3-47.
  • IC50 values were generated for the various anti-IFNAR1 antibodies described above (See FIG. 11A ).
  • the anti-IFNAR1 antibody 9D4 (0.01 nM) and the modified antibodies, such as 9D4-DM (0.01 nM) and 9D4-TM (0.02 nM) each elicit a similar IC50 value in the reporter assay demonstrating that they exhibit a similar potency.
  • Another anti-IFNAR1 antibody, MDX1333 (0.04 nM) also exhibits a similar potency to the unmodified 9D4 antibody.
  • the isotype control does not inhibit Type I IFN mediated signaling in this Luciferase reporter assay.
  • Modified anti-IFNAR1 antibodies share similar potencies to the unmodified versions as demonstrated by IC50 values generated in a Luciferase Reporter assay system designed to quantify IFN signaling events.
  • Type I Interferon 9D4-TM IC50 (nM) IFN- ⁇ 2b 0.07 +/ ⁇ 0.01 IFN- ⁇ 2a 0.3 +/ ⁇ 0.2 IFN- ⁇ 6 0.04 +/ ⁇ 0.01 IFN- ⁇ 16 0.02 +/ ⁇ 0.03 IFN- ⁇ 8 0.03 +/ ⁇ 0.04 IFN- ⁇ 10 0.01 +/ ⁇ 0.01 Leukocyte Interferon 0.01 +/ ⁇ 0.01 IFN- ⁇ 17 0.04 +/ ⁇ 0.03 IFN- ⁇ 14 0.02 +/ ⁇ 0.01 IFN- ⁇ 1 0.004 +/ ⁇ 0.01 IFN- ⁇ 21 0.01 +/ ⁇ 0.002 IFN- ⁇ 7 0.04 +/ ⁇ 0.01 IFN- ⁇ 4b 0.02 +/ ⁇ 0.01 IFN- ⁇ 1 6.8 +/ ⁇ 9.4 IFN- ⁇ 0.1 +/ ⁇ 0
  • 9D4-TM exhibits IC50 values in the sub-nanomolar range for multiple interferon alpha isoforms, leukocyte interferon, and interferon omega.
  • the modified anti-IFNAR1 antibody 9D4-TM demonstrates the ability to inhibit the signaling attributed to multiple specific interferon alpha subtypes as well as leukocyte interferon alpha in a reporter assay
  • FIG. 12A Depicted in FIG. 12A is the isoelectric point (pI) determination for antibodies 9D4WT, 9D4DM, and 9D4TM.
  • Samples of the antibodies were run according to the methods above and exhibited the following characteristics.
  • the 9D4 WT antibody exhibited prominent protein bands corresponding to 8.2, 8.35 and 8.51.
  • the 9D4 DM antibody exhibited a single prominent protein band corresponding to 7.13.
  • the 9D4 TM antibody exhibited prominent protein bands corresponding to 8.09 and 8.18.
  • the modified antibodies 9D4-DM and 9D4-TM exhibit very similar biophysical characteristics (pI) to the parental unmodified antibody 9D4.
  • T m thermal melting temperatures
  • VP-DSC MicroCal, LLC
  • a filter period of 8 seconds was used along with a 15 minute pre-scan.
  • Samples were prepared by dialysis into 10 mM Histidine-HCl, pH 6 using Pierce dialysis cassettes (3.5 kD).
  • Mab concentrations were 0.14 mg/ml, 0.79 mg/ml, and 0.64 mg/ml as determined by A 280 .
  • Melting temperatures were determined following manufacturer procedures using Origin software supplied with the system. Briefly, multiple baselines were run with buffer in both the sample and reference cell to establish thermal equilibrium. After the baseline was subtracted from the sample thermogram, the data were concentration normalized.
  • the antibodies 9D4, 9D4-DM, 9D4-TM were subjected to differential scanning calorimetry as detailed above with the results presented in FIG. 12B .
  • the modified antibodies 9D4-DM and 9D4-TM exhibit very similar biophysical characteristics (T m ) to the parental unmodified antibody 9D4.
  • mice Female NZB/W Fl mice were purchased from Jackson Labs and housed in pathogen-free barrier facility.
  • the recombinant adenovirus vector containing the mouse IFN ⁇ subtype 5 cDNA under the control of the CMV promoter/enhancer (Adv-mIFN ⁇ 5) was used to induce early lupus in these mice.
  • Mice (8 mice/group) were treated at 8-11 wk of age with a single i.v. injection of 0.3 ⁇ 10 10 Adv-mIFN ⁇ 5 viral particles (vp). Controls received the same amount of control Adv particles.
  • mice were injected with gradual doses of Adv-mIFN ⁇ 5 ranging from 0.01 ⁇ 10 10 to 1.0 ⁇ 10 10 vp/mouse.
  • mice were treated with successive 5 daily i.p. dosing of antibody at 10 mg/kg starting at the time of Adv delivery.
  • proteinuria urine was tested using a dipstick (Chemstrip 2 GP; Roche Diagnostics). Proteinuria scored as 1 for levels of 30 mg/dl, 2 for 100 mg/dl, and 3 for levels ⁇ 500 mg/dl. Mice were considered to have proteinuria if two consecutive urine samples scored 2 or higher.
  • mice infected with Adv-mIFN ⁇ 5 exhibit proteinuria with an onset of about 3 weeks.
  • Infected mice treated with control mouse IgG antibody are not protected from the onset of proteinuria over the course of the experiment as demonstrated by an onset of proteinuria of about 4 weeks.
  • Mice treated with anti-IFNAR antibodies do not show evidence of proteinuria throughout the 8 week time course.
  • Mice treated with an adenovirus control show no proteinuria over the experimental time course.
  • RNA was prepared from tissues using RLT lysis buffer (Qiagen). For solid tissues (kidney, spleen, skin), no more than 50 mg of tissue was used for RNA processing each time. Samples were placed in lysis buffer and lysing matrix A (Qbiogene), and processed for 30 sec at 4.5 m/s using Fastprep24 homogenizer instrument (Thermo Electron Corporation, Waltham, Mass.). For PBMC, whole blood samples were centrifuged and the pellet was lysed in RLT buffer. Upon lysis, samples were snap frozen at ⁇ 80° C. until further processed.
  • RLT lysis buffer Qiagen
  • cDNA was generated from 3 ⁇ g of RNA using SuperScript III reverse transcriptase and oligo d(T) as described in the manufacturer's protocol (Invitrogen, Corp. Carlsbad, Calif.). Samples of cDNA were diluted in nuclease-free water and stored at ⁇ 80° C.
  • Expression levels of selected genes were measured by real-time PCR TaqMan® analysis using the ABI 7900HT Fast Real-time PCR system (Applied Biosystems, Foster City, Calif.). Housekeeping gene ⁇ -actin was used for endogenous control. Reaction mixtures had a final volume of 20 ⁇ A consisting of 1 ⁇ A of cDNA, 2 ⁇ A of 20 ⁇ primers and probes (TaqMan® Gene Expression Assays, Applied Biosystems) and 18 ⁇ A of diluted TaqMan® Fast Universal PCR Master Mix. Amplification conditions were: 20 seconds at 95° C., 50 cycles of 1 second at 95° C. and 20 seconds at 60° C. CT values range from 0 to 50, with the latter number assumed to represent no product formation. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the housekeeping gene.
  • Type I interferon ectopically expressed in mice leads to induction of a number of genes.
  • FIG. 14 are the fold changes of six Type I interferon responsive genes in the different populations of mice used in this experiment. Specifically, genes IFIT1, IF144, IFI202b, CXCL9, CXCL10, and CXCL11 are all induced in the mice ectopically expressing IFN ⁇ and treated with nonspecific Mouse IgG. Mice ectopically expressing IFN ⁇ and treated with anti-IFNAR antibodies do not show any induction of the six Type I interferon responsive genes.
  • mice treated with PBS, or control adenovirus do not show any induction of these 6 genes.
  • Anti-IFNAR antibodies can block the regulation of Type I responsive genes in mouse model of SLE.
  • Anti-IFNAR Antibodies Block the Production of Anti-dsDNA and Anti-SSA/Ro (Anti-Nuclear Antigen) Antibodies Induced by Type I Interferon
  • mice were prepared and treated as described in Example 13. Serum anti-dsDNA autoantibody levels were assessed by ELISA. Briefly, ELISA plates pretreated with poly (L-lysine) (100 ⁇ g/ml) were coated with calf thymus activated DNA (5 ⁇ g/ml in carbonate-bicarbonate buffer) (SIGMA). After overnight incubation at 4° C., plates were blocked with PBS/10% FCS. Sera ( 1/200 dilution) were incubated for 30 minutes at room temperature. Bound IgG was detected with peroxidase-conjugated goat anti-mouse IgG ( 1/4000) (KPL) added to the plates for 30 min.
  • SIGMA carbonate-bicarbonate buffer
  • Binding was measured by adding TMB substrate (KPL) and stop solution (KPL), and the OD was read at 450 nm.
  • KPL TMB substrate
  • KPL stop solution
  • a mouse anti-ds DNA IgG standard in serum was run in serial dilution (from 625 ng/ml) (Alpha Diagnostic) on each plate to allow standardization.
  • Serum anti-SSA/Ro autoantibody levels were measured by ELISA (Alpha Diagnostic) following the manufacturer's instructions.
  • mice Type I interferon ectopically expressed in mice (See Example 13) leads to accumulation of anti-dsDNA and anti-SSA/Ro antibodies.
  • FIG. 15 are the relative quantities of anti-dsDNA (A) and anti-SSA/Ro (B) antibodies in the different populations of mice (control adenovirus, Adv-IFN ⁇ +PBS, Adv-IFN ⁇ +MuIgG, and Adv-IFN ⁇ +Anti-IFNAR) as measured by ELISA. Control adenovirus infected mice show little accumulation of anti-dsDNA or anti-SSA/Ro antibodies in this experiment.
  • mice infected with adenovirus encoding IFN ⁇ and treated with PBS accumulate anti-dsDNA and anti-SSA/Ro antibodies.
  • Adv-IFN ⁇ infected mice treated with anti-IFNAR antibodies acquire less anti-dsDNA and anti-SSA/Ro antibodies than Adv-IFN ⁇ infected mice treated with non-specific IgG.
  • Anti-IFNAR Antibodies Block the Production of IP-10 and IL-18 Induced by Type I Interferon
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. Serum levels of cytokines were measured by ELISA (R&D systems) following the manufacturer's instructions.
  • Type I interferon ectopically expressed in mice leads to accumulation of IP-10 and IL-18 cytokines.
  • FIG. 16 are the relative quantities of IP-10 (A) and IL-18 (B) in the different populations of mice (PBS, control adenovirus, Adv-IFN ⁇ +MuIgG, and Adv-IFN ⁇ +Anti-IFNAR) as measured by ELISA.
  • Type I interferon ectopically expressed in mice leads to accumulation of the cytokines, IP-10 and IL-18. Control adenovirus infected mice show little accumulation of IP-10 (A) or IL-18 (B) cytokines in this experiment.
  • Adv-IFN ⁇ infected mice treated with anti-IFNAR antibodies accumulate less IP-10 and IL-18 cytokines than Adv-IFN ⁇ infected mice treated with non-specific IgG.
  • Anti-IFNAR antibodies are able to block the accumulation of IFN ⁇ induced cytokines in a mouse model of SLE.
  • Anti-IFNAR Antibodies Block the Production of ANA (Anti-Nuclear Antibodies) Induced by Type I Interferon
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example.
  • Antinuclear antibody (ANA) levels were measured by ANA test kit (Antibodies Incorporated) with Hep-2 stabilized substrate and mitotic figures following the manufacturer's instruction. Serum was serially diluted and incubated with the Hep-2 cells on slides and the bound antinuclear antibody was detected by Hi-FITC labeled goat anti-mouse IgG (H+L) (Antibodies Incorporated).
  • the titer of ANA is defined as the serum dilution factor where the ANA is no longer detectable.
  • mice Type I interferon ectopically expressed in mice (See Example 13) leads to accumulation of anti-ANA antibodies.
  • FIG. 17 is the serum titer of anti-ANA antibodies in the different populations of mice (no virus, control adenovirus, Adv-IFN ⁇ +PBS, Adv-IFN ⁇ +MuIgG, and Adv-IFN ⁇ +Anti-IFNAR) as measured by serial dilution staining on HEP2 cells.
  • Control adenovirus infected mice show little accumulation of anti-ANA antibodies in this experiment.
  • Mice infected with adenovirus encoding IFN ⁇ and treated with PBS accumulate anti-ANA antibodies.
  • Adv-IFN ⁇ infected mice treated with anti-IFNAR antibodies acquire less anti-ANA antibodies than Adv-IFN ⁇ infected mice treated with non-specific IgG.
  • Anti-IFNAR antibodies are able to block the accumulation of anti-nuclear antibodies induced by IFN ⁇ in a mouse model of SLE.
  • SLE plasma induces dendritic cell development from normal human monocytes.
  • the purified monoclonal antibody 9D4-TM was tested for the inhibition of dendritic cell development, as assessed by the ability of the antibodies to inhibit the induction of the cell surface markers CD38, and CD123 by SLE plasma.
  • Monocytes were isolated by incubating PBMCs (2.0 ⁇ 10 7 cells/5 ml/25 cm 2 flask) for 1.5 hours at 37° C. in culture media and then washing away non-adherent cells twice.
  • PBMCs 2.0 ⁇ 10 7 cells/5 ml/25 cm 2 flask
  • IgG1 isotype control
  • Each culture was resuspended in staining medium (Hanks's Balanced Salt Solution with 0.2% sodium bicarbonate, 0.01% sodium azide, 0.1 mM EDTA, 20 mM HEPES, and 2% fetal calf serum) and separated equally into six wells of a V bottom 96 well plate.
  • the cells were pulse-spun at 2100 rpm on a Sorvall RTH-750 rotor, and resuspended in 25 ⁇ l of staining media.
  • One microgram of specific phycoerythrin conjugated antibody was added to each well and incubated on ice for 45 minutes.
  • the cells were washed three times, resuspended in 200 ⁇ l of 2% paraformaldehyde in PBS, and analyzed by flow cytometry with the Becton Dickinson FACScalibur. Gates were drawn on the forward v side scatter graph to remove contaminating cells from the analysis.
  • the anti-IFNAR1 antibody 9D4-TM was able to block the ability of IFN ⁇ derived form SLE patients to induce pDC maturation as measured by cell surface marker expression.
  • Anti-IFNAR Antibodies Suppress the Expression of CD38, CD123 and CD86 in Monocytes Stimulated with Leukocyte-IFN
  • the antibodies 9D4, 9D4-DM and 9D4 TM were tested for the inhibition of dendritic cell development, as assessed by the ability of the antibodies to inhibit the induction of the cell surface markers CD38, and CD123 by Leukocyte IFN.
  • Monocytes were isolated from whole blood of healthy donors using a lymphocyte separation media (MP Biomedical, Solon Ohio) followed by positive selection using Monocyte Isolation kit II (Milteny Biotec, Auburn Calif.). Purified monocytes were then cultured at 1 ⁇ 10 6 cells/mL in RPMI 1640 supplemented with 10% FBS (Gibco BRL). Serially diluted antibodies were prepared at final concentrations of 3 ⁇ g/mL-20 pg/mL in media and were added to appropriate wells of cells.
  • the anti-IFNAR antibodies elicited similar suppression curves which were utilized to generate IC50 values.
  • the plate was washed with PBS (pH7.4) containing 0.05% of Tween 20 and 25 ⁇ l of HRP conjugated Avidin was added to each well. After an hour incubation at 37° C., the plates were washed again and 50 ⁇ l/well of substrate—SureBlue TMB peroxidase (KPL cat. 52-00-03) was added. The reaction was stopped with 50 ⁇ l of 0.2 M H 2 SO 4 after 5-10 minutes development. The ELISA signal was read at 450 nM.
  • the Fc Receptor Fc ⁇ RIIIA Exhibits Reduced Binding to the Modified Anti-IFNAR1Antibodies
  • results The results from an ELISA based binding assay between anti-IFNAR1 antibodies (9D4WT, 9D4DM, and 9D4TM) and the high and low affinity Fc receptor Fc ⁇ RIIIA are presented in FIG. 21(A , B, C).
  • 9D4WT antibodies coated on the ELISA plate efficiently bind the high affinity Fc ⁇ RIIIA receptor at concentrations greater than 3 ng/ml while there is limited binding of the low affinity Fc ⁇ RIIIA receptor at all concentrations tested.
  • FIG. 21(B) Modified 9D4DM antibodies coated on the ELISA plate do not efficiently bind the high or low affinity Fc ⁇ RIIIA receptors at any concentrations tested.
  • FIG. 21(C) Modified 9D4TM antibodies coated on the ELISA plate do not efficiently bind the high or low affinity Fc ⁇ RIIIA receptors at any concentrations tested.
  • Fc ⁇ RIIIA variants Fc ⁇ RIIIA-10 158F and Fc ⁇ RIIIA-10 158V
  • Immunlon IV microtiter plate After washing and blocking with 4% milk 1 hr at room temperature biotinylated 9D4, 9D4TM, and 9D4DM antibodies were added to the wells of the blocked plate at 100 ⁇ g/mL. The plate was washed one hour later and incubated with HRP conjugated Avidin. The unbound materials were removed by washing one hr after incubation. The binding signal was detected with the substrate TMB.
  • results The results from an ELISA based binding assay between the high and low affinity Fc receptors Fc ⁇ RIIIA and anti-IFNAR1 antibodies (9D4WT, 9D4DM, and 9D4TM) are presented in FIG. 22(A , B, C).
  • the unmodified anti-IFNAR1 antibody 9D4 at concentrations greater than 3 ng/ml, efficiently binds the high affinity Fc ⁇ RIIIA receptor immobilized on the ELISA plate, whereas the antibody demonstrates limited binding to the immobilized low affinity Fc ⁇ RIIIA receptor at all concentrations tested.
  • FIG. 22(A) the unmodified anti-IFNAR1 antibody 9D4 at concentrations greater than 3 ng/ml, efficiently binds the high affinity Fc ⁇ RIIIA receptor immobilized on the ELISA plate, whereas the antibody demonstrates limited binding to the immobilized low affinity Fc ⁇ RIIIA receptor at all concentrations tested.
  • the modified anti-IFNAR1 antibody 9D4DM does not efficiently bind the immobilized high or low affinity Fc ⁇ RIIIA receptors at any concentrations tested compared to the unmodified 9D4WT anti-IFNAR1 antibody.
  • the modified anti-IFNAR1 antibody 9D4TM does not efficiently bind the immobilized high or low affinity Fc ⁇ RIIIA receptors at any concentrations tested compared to the unmodified 9D4WT anti-IFNAR1 antibody.
  • IFN ⁇ neutralization is measured by a HiL3 based reporter assay.
  • An example of how a IFN ⁇ neutralization assay using HiL3 cells as a reporter is as follows: A human hepatoma cell line HiL3 was transfected with a plasmid containing an IFN ⁇ stimulated response element—luciferase (ISRE-Luc), and a neomycin resistance gene. These cells were kindly provided by Dr Michael Tovey (CNRS, Paris, France).
  • HiL3, 30,000 cells/well was cultured in white reflective 96 well plates (DYNEX Microlite) and grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1 mg/ml G418 (+penicillin/streptomycin/ L -glutamine). After this incubation, various forms of interferon were added and the plates were cultured for 18 hours. The reaction was terminated by adding 10 ml of lysis buffer to luciferase substrate vial (Luc Lite Plus kit, Perkin-Elmer); 100 ⁇ l of this substrate solution was added to each well and read on Top Count for 10 minutes (10 minutes waiting in the dark, then 1 second read/well). The counts per second (cps) at each IFN concentration were determined and the IFN concentration or cps in each sample was calculated from the IFN titration curve using Prism software (San Diego, Calif.) with linear regression parameters.
  • results The neutralization capacity for anti-IFNAR1 antibodies for various IFN species in a HiL3 reporter assay is presented in FIGS. 23 (A-E).
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM inhibit multiple Type I interferon subtypes with similar potency.
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFN ⁇ 10(A) with IC50 values of 0.09880 ⁇ g/ml, 0.008345 ⁇ g/ml, and 0.004287 ⁇ g/ml respectively.
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize Human Leukocyte IFN (B) with IC50 values of 1.121 ⁇ g/ml, 0.02104 ⁇ g/ml, and 0.02120 ⁇ g/ml respectively.
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFN ⁇ 2b (C) with IC50 values of 0.0006462 ⁇ g/ml, 0.002789 ⁇ g/ml, and 0.0008279 ⁇ g/ml respectively.
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFN ⁇ (D) with IC50 values of 5.323 ⁇ g/ml, 0.01015 ⁇ g/ml, and 0.01423 ⁇ g/ml respectively.
  • the anti-IFNAR1 antibodies MDX-1333, 9D4WT and 9D4TM neutralize IFN ⁇ (E) with IC50 values of 18.97 ⁇ g/ml, 0.7403 ⁇ g/ml, and 0.2611 ⁇ g/ml respectively.
  • Anti-IFNAR1Antibodies Neutralize Type I IFN in Plasma from SLE Patients
  • Stably transfected PIL-5 ISRE cells were maintained in RPMI 1640+1 ⁇ Pen-strep-glutamine+ 10% FBS and seeded at 100,000 cells per well in Optilix white/clear 96 well plates (VWR, West Chester Pa.). Antibodies were titrated added to appropriate wells for a final concentration ranging from 90 ⁇ g/mL-60 pg/mL. Type-I interferon positive human SLE patient serum samples were added to each well for a final serum percentage of 50% per well. Cells, IFN, and antibodies were incubated overnight at 37° C., 5% CO 2 .
  • 9D4-TM neutralizes Type I interferons in SLE patient plasma.
  • the results from a neutralization assay of Type I interferons in SLE patient plasma is presented in FIG. 24 .
  • Neutralization of Type I interferon contained in the SLE patient plasma sample is specifically neutralized with 9D4-TM versus an Isotype control at increasing antibody concentrations.
  • 9D4-TM exhibits an IC50 of 0.04 nM for neutralization of Type I interferons in this plasma sample taken from an SLE patient.
  • Anti-IFNAR Antibodies Suppress the IFN ⁇ Induced pDC Population in PBMCs
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example.
  • PBMCs were isolated from Spleen, Lymph Nodes, Bone Marrow and Peripheral Blood using standard isolation techniques and stained for the B220 and Ly6C surface markers. Isolated PBMCs were analyzed by FACS and double positive (B220 and Ly6C) cells were scored as pDC cells and the relative populations are represented in FIG. 25 .
  • ectopic expression of IFN ⁇ triggers an increase in pDC cells within the PBMCs isolated from spleen (A), lymph nodes (B), blood (C), and bone marrow (D) in the presence of PBS or mouse non-specific IgG.
  • Mice treated with anti-IFNAR antibodies do not accumulate pDC cells in response to IFN-alpha.
  • Mice treated with control Adenovirus do not accumulate pDCs in the PBMC population.
  • Fc ⁇ RI was prepared at 20 nM in HBS-EP buffer (BIAcore, Inc., consisting of the following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20. Between Fc ⁇ RI injections, the IgG surface was regenerated with a 1 min. injection of 5 mM HCl. Sensorgram overlays were generated using the BIAevaluation 4.1 software (BIAcore, Inc, Uppsala, Sweden).
  • the anti-IFNAR1 antibody 9D4 and modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM were tested for binding affinity to immobilized Fc ⁇ RI protein in a BIAcore assay format. As depicted in FIG. 26 , the anti-IFNAR1 antibody 9D4 exhibits a high affinity for the immobilized Fc ⁇ RI. The binding of the anti-IFNAR1 antibody 9D4 to Fc ⁇ RI is specific as the similar assay run with ovalbumin exhibits very little affinity for the immobilized receptor.
  • the modified anti-IFNAR1 antibodies 9D4-TM and 9D4-DM exhibit a lower affinity of the immobilized receptor Fc ⁇ RI compared to the unmodified 9D4 anti-IFNAR1 antibody.
  • antibodies were prepared at 333 nM in HBS-EP buffer (BIAcore, Inc., consisting of the following: 10 mM HEPES buffer, pH7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20. Between antibody injections, the Fc ⁇ RI surface was regenerated with a 1 min. injection of 3 M MgCl2. Sensorgram overlays were generated using the BIAevaluation 4.1 software (BIAcore, Inc, Uppsala, Sweden).
  • the anti-IFNAR1 antibodies 9D4, 9D4-TM and 9D4-DM were immobilized and incubated with soluble Fc ⁇ RI. Binding affinity of the soluble Fc ⁇ RI receptor to each of the anti-IFNAR1 antibodies were measured in a BIAcore assay and the resultant tracings are represented in FIG. 27 A, B, C.
  • the Fc ⁇ RI bound the immobilized anti-IFNAR1 antibody 9D4 with a high affinity as represented in FIG. 27A . This interaction was highly specific as soluble ovalbumin did not show any binding to the immobilized anti-IFNAR1 antibody 9D4.
  • the modified antibodies 9D4-TM and 9D4-DM do not bind the Fc ⁇ RI as strongly as the wild type unmodified 9D4 antibody.
  • the modified anti-IFNAR1 antibody 9D4-DM was immobilized and incubated with either soluble Fc ⁇ RI or ovalbumin.
  • the Fc ⁇ RI exhibited a low binding affinity for the immobilized 9D4-DM antibody. This binding affinity is similar to the non-specific interaction seen with soluble ovalbumin.
  • FIG. 27C the modified anti-IFNAR1 antibody 9D4-TM was immobilized and incubated with either soluble Fc ⁇ RI or ovalbumin.
  • the Fc ⁇ RI exhibited a low binding affinity for the immobilized 9D4-TM antibody. This binding affinity is similar to the non-specific interaction seen with soluble ovalbumin.
  • Anti-IFNAR Antibodies Block IFN ⁇ Responsive Gene Induction
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. After 8 weeks into the experiment the mice were sacrificed and kidney tissue was removed. No more than 50 mg of tissue was used for RNA extraction using RLT lysis buffer (Qiagen). Samples were placed in lysis buffer and lysing matrix A (Qbiogene), and processed for 30 sec at 4.5 m/s using Fastprep24 homogenizer instrument (Thermo Electron Corporation, Waltham, Mass.). To isolate RNA, thawed tissue lysates were first processed using Qiashredder spin columns, then equal volumes of 70% ethanol were added to the tissue lysates and RNA was purified using Rneasy mini spin column kits according to the manufacturer's instruction.
  • RLT lysis buffer Qiagen
  • Samples were placed in lysis buffer and lysing matrix A (Qbiogene), and processed for 30 sec at 4.5 m/s using Fastprep24 homogenizer instrument (Thermo Electron Corporation, Waltham, Mass.).
  • cDNA was generated from 3 ⁇ g of RNA using SuperScript III reverse transcriptase and oligo d(T) as described in the manufacturer's protocol (Invitrogen, Corp. Carlsbad, Calif.). Samples of cDNA were diluted in nuclease-free water and stored at ⁇ 80° C.
  • Expression levels of selected genes were measured by real-time PCR TaqMan® analysis using the ABI 7900HT Fast Real-time PCR system (Applied Biosystems, Foster City, Calif.). Housekeeping gene ⁇ -actin was used for endogenous control. Reaction mixtures had a final volume of 20 ⁇ A consisting of 1 ⁇ A of cDNA, 2 ⁇ A of 20 ⁇ primers and probes (TaqMan® Gene Expression Assays, Applied Biosystems) and 18 ⁇ A of diluted TaqMan® Fast Universal PCR Master Mix. Amplification conditions were: 20 seconds at 95° C., 50 cycles of 1 second at 95° C. and 20 seconds at 60° C. CT values range from 0 to 50, with the latter number assumed to represent no product formation. Quantification of gene expression was performed using the comparative CT method (Sequence Detector User Bulletin 2; Applied Biosystems) and reported as the fold difference relative to the housekeeping gene.
  • mice ectopically expressing interferon alpha were treated with mouse IgG or anti-IFNAR antibodies.
  • the mice treated with control IgG demonstrated a high induction of IFN ⁇ responsive genes namely ICAM1, VCAM1, CXCL9, CXCL10, and IFIT1.
  • Mice treated with anti-IFNAR antibodies did not show induction of IFN ⁇ responsive genes after 8 weeks.
  • Anti-IFNAR Antibodies Inhibit Accumulation of Autoantibodies in Serum
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example.
  • Whole blood samples were taken at 1 week intervals from week 2-7 of the regimen.
  • Serum anti-dsDNA autoantibody levels were assessed by ELISA. Briefly, ELISA plates pretreated with poly (L-lysine) (100 ⁇ g/ml) were coated with calf thymus activated DNA (5 ⁇ g/ml in carbonate-bicarbonate buffer) (SIGMA). After overnight incubation at 4° C., plates were blocked with PBS/10% FCS. Sera ( 1/200 dilution) were incubated for 30 minutes at room temperature.
  • Bound IgG was detected with peroxidase-conjugated goat anti-mouse IgG ( 1/4000) (KPL) added to the plates for 30 min. Binding was measured by adding TMB substrate (KPL) and stop solution (KPL), and the OD was read at 450 nm. A mouse anti-ds DNA IgG standard in serum was run in serial dilution (from 625 ng/ml) (Alpha Diagnostic) on each plate to allow standardization.
  • mice ectopically expressing IFN ⁇ were treated with anti-IFNAR antibodies or mouse IgG control antibodies during an 7 week regimen.
  • the mice treated with anti-IFNAR antibodies did not accumulate anti-dsDNA antibodies at the same rate or to the same extent of mice treated with control IgG antibodies.
  • Mice infected with control adenovirus did not develop anti-ds DNA antibodies over the time course.
  • Anti-IFNAR Antibodies Reduce Proteinuria in the Accelerated Lupus Mouse Model
  • mice from the experimental procedures described in Example 13 also provided samples for analysis in this example. However, in a therapeutic approach, mice were allowed to develop proteinuria as a symptom of Lupus before application of the antibodies. Specifically, mice were allowed to develop a proteinuria score of 2.0-2.5 as described previously. Once the threshold level of proteinuria was passed, a treatment regimen of semi-weekly doses of PBS, control IgG or anti-IFNAR antibodies was conducted for 5 additional weeks. At semi-weekly intervals urine samples were tested and given a proteinuria score.
  • mice were allowed to develop proteinuria at which time, the cohort was either given PBS, control IgG or anti-IFNAR antibodies as treatment. As documented within the figure, the proteinuria score decreased for only the group receiving anti-IFNAR antibodies. The mice receiving PBS or control IgG as treatment continued to increase the proteinuria score over time.
  • PBS control IgG
  • Anti-IFNAR Antibodies Reduce Mortality in the Accelerated Lupus Mouse Model
  • mice from the experimental procedures described in Example 30 also provided samples for analysis in this example.
  • mice were allowed to develop proteinuria as a symptom of Lupus before application of the antibodies.
  • mice were allowed to develop a proteinuria score of 2.0-2.5 as described previously.
  • a treatment regimen of semi-weekly doses of PBS, control IgG or anti-IFNAR antibodies was conducted for 5 additional weeks.
  • Overall mortality was tracked for an additional 4 weeks.
  • mice were allowed to develop proteinuria at which time, the cohort was either given PBS, control IgG or anti-IFNAR antibodies as treatment.
  • mice treated with anti-IFNAR antibodies exhibited no mortality at week 5, whereas mice treated with PBS or control IgG exhibited mortality rates of 87.5% and 62.5% respectively.
  • anti-IFNAR treated animals exhibited a high survival rate compared to PBS or control IgG treated animals.
  • 293F target cells were labeled with DiO cell label (Invitrogen, experiments I & II) and combined with unlabeled effectors PBMCs (for 4 h at 37° C., in the absence or presence 10 ⁇ g/ml of 9D4-TM, human IgG1 isotype negative control R3-47, 9D4-WT or anti-EphA2 antibody used as a positive control.
  • DiO cell label Invitrogen, experiments I & II
  • PBMCs for 4 h at 37° C., in the absence or presence 10 ⁇ g/ml of 9D4-TM, human IgG1 isotype negative control R3-47, 9D4-WT or anti-EphA2 antibody used as a positive control.
  • lysis buffer Percent of double positive staining in media alone
  • Fc-TM The human Fc/TM fragment was obtained from the enzymatic cleavage of 9D4-TM. Digestion was carried out using immobilized ficin according to the manufacturer's instructions (Pierce). Purification was first performed on HiTrap Protein A columns according to the manufacturer's instructions (GE Healthcare, Piscataway, N.J.). After dialysis in 50 mM NaOAc/pH 5.2, the protein solution was applied to a HiTrap SP HP column (GE Healthcare) and collected in the flow through. The flow through was loaded onto a HiTrap Q column (GE Healthcare) and eluted in a NaCl gradient to yield a homogenous Fc/TM preparation, as judged by reducing and non-reducing SDS-PAGE.
  • Fc-TM SDS-PAGE profile showed the presence of only one band around 25 kDa or 50 kDa under reducing or non reducing conditions, respectively. This observation clearly demonstrated the presence of at least one interchain disulfide bond at positions C226 and/or C229. Consequently, mutated ‘downstream’ residues F234 and E235 were present in the polypeptide chain comprising the crystal.
  • Crystallization of Fc-TM Purified Fc-TM was concentrated to about 5 mg/ml using a Centricon concentrator (Millipore, Billerica Mass., 30 KDa cutoff). Crystallization conditions were identified using the commercial screens from Hampton Research (Hampton Research, Aliso Viejo, Calif.), Emerald BioSystems (Emerald BioSystems, Inc., Bainbridge Island, Wash.) and Molecular Dimensions (Molecular Dimensions Inc., Apopka, Fla.). Each screen yielded several potentially usable crystallization conditions.
  • diffraction-quality crystals were obtained from 0.2 M Zinc acetate, 0.1 M Imidazole-Malate, pH 8.0, 5% PEG 3350, 5% glycerol at protein concentration of 2.0 mg/ml. Under these conditions, well-shaped crystals with three dimensions ranging from 0.1 to 0.2 mm grew in 2-3 days.
  • Diffraction data were collected from a single crystal at the Center for Advanced Research in Biotechnology (CARB, University of Maryland Biotechnology Institute, Rockville, Md.) using a Rigaku MicroMaxTM 007 rotating anode generator with an R-AXIS IV++ imaging plate (Rigaku/MSC, The Woodlands, Tex.). Prior to cooling, the crystal was kept for a few minutes in its growth solution supplemented with 20% glycerol. The crystal was then cooled to 105 kelvins with an X-stream 2000 Cryogenic cooler (Rigaku/MSC). Diffraction of up to 2.3 ⁇ was achieved after one round of annealing as described (Oganesyan et al., 2007).
  • Diffraction data comprising 234 images were collected using an oscillation range of 0.5°, a crystal/detector distance of 200 mm and an exposure time of 600 s. Data were integrated and scaled using the HKL 2000 software (Otwinowski & Minor, 1997).
  • the C H 2 and C H 3 domains were considered separately to minimize any bias in terms of the domains relative conformation.
  • Data up to 3.0 ⁇ were used for the molecular replacement problem using Phaser (McCoy et al., (2005) Acta Cryst. D61, 458-464).
  • the final LL-gain and the Z-score were 1192 and 31, respectively.
  • Weighted electron density calculated with FWT/PHWT at 3.0 ⁇ showed a good match with the model with the exception of some loops in the C H 2 and C H 3 domains. Strong positive difference electron density calculated with DELFWT/PHDELWT was visible in the expected place of N-linked carbohydrate residues attached to N297.
  • Zinc ions present in the crystallization buffer were detected in the electron density and modeled as such when the coordination sphere and distance permitted. In particular, one zinc ion was found coordinated by H310 and H435. Another was coordinated by H285 and H268 of the symmetry related polypeptide. Two others were bound to E318 and E345. In all cases, water molecules completed the expected tetrahedral coordination sphere of the zinc ions.
  • the carbohydrate moiety was modeled according to its electron density and the final model contained nine sugar residues, essentially as described by us in the context of another human Fc structure (Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press). The final model contained 75 solvent molecules. Crystallographic data and refinement statistics are given in Table 6.
  • Fc-TM The atomic coordinates and experimental structure factors of Fc-TM have been deposited with the Protein Data Bank under accession number 3C2S.
  • Fc-TM The overall three-dimensional structure of Fc-TM was very similar to previously reported structures of unliganded human Fc regions (Deisenhofer, (1981). Biochemistry, 20: 2361-2370; Krapp et al., (2003). J. Mol. Biol. 325, 979-989; Matsumiya et al., (2007). J. Mol. Biol. 368:767-779; Oganesyan et al., (2007) Molecular Immunology, Dec. 11, 2007, in press). More precisely, the human Fc structures corresponding to PDB ID numbers 1H3W (Krapp et al., (2003). J. Mol. Biol.
  • Fc-TM C H 2 and C H 3 domains showed great structural conservation and rigidity when compared with other unliganded, unmutated human Fc structures. For instance, rms coordinate displacements of C ⁇ atoms were 0.6 and 0.4 ⁇ for the C H 2 and C H 3 domains, respectively, when superimposing Fc-TM with chain A of PDB ID number 2DTQ (Matsumiya et al., (2007). J. Mol. Biol. 368, 767-779).
  • Table 7 following below, provides the atomic structure coordinates of Fc-TM. The following abbreviations are used in Table 7
  • Atom Type refers to the element whose coordinates are provided. The first letter in the column defines the element.
  • A.A refers to amino acid.
  • X, Y and Z provide the Cartesian coordinates of the element.
  • B is a thermal factor that measures movement of the atom around its atomic center.
  • OCC refers to occupancy, and represents the percentage of time the atom type occupies the particular coordinate. OCC values range from 0 to 1, with 1 being 100%.
  • REMARK 3 CROSS-VALIDATION METHOD THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.21928 REMARK 3 R VALUE (WORKING SET) : 0.21637 REMARK 3 FREE R VALUE : 0.27541 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.9 REMARK 3 FREE R VALUE TEST SET COUNT : 619 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN.
  • REMARK 3 ALL ATOMS 1867 REMARK 3 REMARK 3 B VALUES.
  • REMARK 3 FROM WILSON PLOT (A**2) NULL REMARK 3 MEAN B VALUE (OVERALL, A**2) : 43.320 REMARK 3 OVERALL ANISOTROPIC B VALUE.
  • REMARK 3 B11 (A**2) : ⁇ 3.83 REMARK 3 B22 (A**2) : 0.96 REMARK 3 B33 (A**2) : 2.88 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 0.00 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR.
  • REFINED ATOMS (A): 85; 0.168; 0.200 REMARK 3 POTENTIAL METAL-ION REFINED ATOMS (A): 1; 0.013; 0.200 REMARK 3 SYMMETRY VDW REFINED ATOMS (A): 45; 0.267; 0.200 REMARK 3 SYMMETRY H-BOND REFINED ATOMS (A): 10; 0.166; 0.200 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS.
  • THP-1 cells were cultured in RPMI-1640 media containing 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum at 37° C. in 5% CO 2 incubator. THP-1 cells were seeded at 2 ⁇ 10 5 cells/ml in fresh growth media one day prior to experiments. At the day of the experiment, cells were washed, counted and resuspended in PBS at 3 ⁇ 10 6 cells/ml. The cells were stained with 1 ⁇ M CFSE in 37° C. CO 2 incubator for 10 min.
  • the fluorescence images of cells were analyzed using an algorithm.
  • the algorithm used CFSE cytosolic dye to identify the boundary of a cell and a membrane region.
  • the algorithm quantified the 9D4-TM associated fluorescence inside cells as well as on membrane. Rate of fluorescence accumulated inside the cells was calculated by model fitting of the data using SAAMII software.
  • HI heat-inactivated
  • Daudi B cells were used as target cells as they express CD20 (target for positive control antibody) and IFNAR1.
  • Target cells were washed and resuspended in either phenol-free RPMI media with 10% non-heat inactivated serum or in phenol-free RPMI media with 10% heat inactivated serum at a final concentration of 0.4 ⁇ 10 6 cells/mL.
  • Antibody solutions were prepared as a 3 ⁇ dilution series with the concentrations ranging from 50 ug/mL-1.3 ⁇ 10 ⁇ 6 ⁇ g/mL. Replicate preparations of antibody dilutions were made in either media with heat-inactivated or non-heat-inactivated human serum.
  • the CDC assay was prepared by adding 50 ⁇ L of NHI or HI media to appropriate wells of a 96 well, round bottom plate. 50 ⁇ L of antibody dilution series were added to the appropriate wells. Subsequently, 50 ⁇ L of the target cell preparation was added to the wells, including extra wells with target cells alone for controls. The plates were incubated for 37° C. for 4 hours in 5% CO 2 . After 3.5 hour incubation, 20 uL lysis buffer was added to appropriate control wells designated for determination of maximum lysis signal. The QuantitateTM LDH release assay was performed using protocols defined in Promega non-radioactive cytotoxicity assay, #G1780. Absorbance was measured at 490 nM and Kd values were generated using GraphPad Prism 4 analysis software.
  • results Presented in FIG. 34 are the results from the CDC performed as described above.
  • the modified anti-IFNAR1 antibody, 9D4-TM exhibited no detectable CDC activity on target Daudi B cells over that observed with the R347 antibody.
  • the positive control antibody, which binds CD20 expressed on Daudi B cells caused a dose-dependent increase in cytotoxicity over background levels.
  • the Modified Anti-IFNAR1Antibody, 9D4-TM Does Not Display Any Adverse Toxicity

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