WO2022087274A1 - Antibodies that neutralize type-i interferon (ifn) activity - Google Patents

Abstract

Antibodies against type-I interferon receptor subunit 2 (lFNAR2) are provided. The antibodies have been shown to have neutralizing activity against the action of type I interferons, such as interferon-alpha (IFN-alpha), and their biological function(s) and can be a novel technology to be used as a therapeutic treatment in patients with inflammatory or viral disease or other immune-related diseases and disorders.

Description

ANTIBODIES THAT NEUTRALIZE TYPE-1 INTERFERON (IFN) ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is an International Application under the Patent Cooperation Treaty, claiming priority to United States Provisional Patent Application No. 63/094,572, filed 21 October 2020, the contents of which are incorporated herein by reference in their entirety.

GOVERNMENT INTERESTS— NIH GRANT INFO

[0002] This invention was made partly using funds from the following United States Federal agencies: The Intramural Programs of the Division of Intramural Research, NIAID, NIH. The US Federal Government has certain rights to this invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] Pursuant to the EFS-Web legal framework and 37 CFR §§ 1 .821-825 (see MPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled “Sequence_Listing_3000094-013977_ST25.txt” created on 20 October 2021 , and 18,143 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.

BACKGROUND

[0002] 1 . Field

[0003] The present disclosure relates to antibodies against type-l interferon receptor subunit 2 (IFNAR2). The antibodies have neutralizing activity against the action of interferon alpha (IFN-alpha) and its biological function, and can be used as a therapeutic in patients with inflammatory or viral disease.

[0004] 2. Description of Related Art

[0005] Interferons (IFNs) are members of a family of cytokines that have antiviral, antiproliferative, and immunomodulatory properties. There are several types of IFNs, each of which interacts with a type-specific receptor complex. Type I IFNs, which include IFN-a, IFN- , and IFN-co, are ubiquitously expressed in mammals and interact with the IFN-a receptor (IFNAR) subunits 1 and 2. Activated T lymphocytes, monocytes, and natural killer (NK) cells produce a single species of type II IFN (IFN-y), which interacts with the IFN- y receptor (IFNGR) subunits 1 and 2. Nearly every cell type expresses receptors for type I IFNs and IFN- y. Type III IFNs, including IFN-A 1 (IL-29), IFN-A2 (IL-28A), and IFN-A3 (IL-28B), bind to the IFN-A receptor (IFNLR1) and the IL- 10Rp subunit (IL-1OR0).

[0006] Human IFN-a is represented by a group of related subtypes, encoded by a multigene family comprising 14 non-allelic genes. At the protein level, there is 75%-99% identity in the primary structure of human IFN-a species. Individual subtypes show quantitatively distinct spectra of antiviral, antiproliferative and immunomodulatory activities. The existence of the numerous subtypes of IFN-a may provide a fine mechanism of regulating the biological effect of IFN.

[0007] The type I IFNs bind with distinct affinities to the common type I IFN receptor complex (IFNAR), expressed on the surface of target cells in low numbers (100-5000 molecules/cell). Functional human IFNAR is a heterodimeric complex composed of two transmembrane polypeptide chains, IFNAR1 and IRNAR2, with distinct complementary functions, which associate to form a heterodimer upon IFN-binding. The IFNAR1 , with a molecular weight of 110-130 kD, has a very low affinity for IFN-as (KD ~ 10-⁶ M).

IFNAR1 alone binds only one species of IFN-a (a8), but is required for signalling by all type I IFNs. The second component, IFNAR2, has a molecular weight of 95 kD. The protein alone displays a relatively high affinity for binding of IFNs (Kd ~ 10-⁹ M) and is the major ligand-binding receptor subunit. The dimerization of IFNAR2 with IFNAR1 results in a fully functional receptor and its binding affinity increases by approximately 100-fold (Kd ~ 10-¹¹ M).

[0008] Human IFNAR2 (Genbank Accession No. NP_001276054) is a 515 amino acid protein including a signal peptide (amino acid positions 1-26), an extracellular domain (amino acid positions 27-243), a transmembrane domain (amino acid positions 244-264), and an intracellular domain (amino acid positions 265-515). There are three known isoforms (isoforms a, b, and c) and two additional predicted isoforms (isoforms d and e) of human IFNAR2 due to alternative splicing. [0009] Blockade of interferon activity has a wide range of clinical and experimental applications, especially in chronic inflammatory and viral diseases. Worldwide, three of five people die due to chronic inflammatory diseases like stroke, chronic respiratory diseases, heart disorders, cancer, obesity, and diabetes. Viral diseases such as HIV affect nearly 38 million people worldwide according to the Foundation for AIDS Research. Thus, there is a need to develop effective therapeutic agents, such as monoclonal antibodies, that neutralize interferon activity.

[0010] There remains a need in the art for therapeutic agents that block interferon activity.

BRIEF SUMMARY

[0011] The present disclosure provides antibodies (including antigen-binding molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of IFNAR2, compositions, and methods of use thereof. In particular, the disclosure provides antibodies (including antigen-binding molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of human IFNAR2 (SEQ ID NO: 33), preferably the extracellular domain of human IFNAR2. The present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with aberrant IFN or IFN receptor expression or inappropriate function of IFN or IFN receptor in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, use of antibodies (including antigenbinding molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2. Diseases and disorders which can be detected, diagnosed, or prognosed with the disclosed antibodies (including antigen-binding molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’s disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), viral infections (e.g., HIV and SARS-CoV-2), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma). The present disclosure further provides methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant IFN or IFNAR2 receptor expression or inappropriate function of IFN or IFNAR2 receptor in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, administering to said animal an effective amount of one or more antibodies (including antigen-binding molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2. Diseases and disorders which can be prevented, treated or ameliorated by administering an effective amount of an antibody of the present disclosure include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’s disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), viral infections (e.g., HIV and SARS-CoV-2), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

[0012] The present inventors identified antibodies that immunospecifically bind to IFNAR2, in particular, to the extracellular domain of IFNAR2. The antibodies described herein have a general “Y” structure consisting of four polypeptides, two heavy chains and two light chains. Each heavy and light chain consists of a constant region and a variable region, e.g., variable heavy (VH) and variable light (VL). Each heavy and light chain comprises a set of three (3) complementarity-determining regions (CDRs) in the variable regions. Antigen-binding molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those described herein), that immunospecifically bind the extracellular domain of IFNAR2, are also presented by the disclosure, as are nucleic acid molecules that encode these antibodies, vectors and host cells comprising the same, and/or antigen-binding molecules.

[0013] In an embodiment, antibodies may comprise a VH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 18, and a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 20. Antigen-binding molecules comprising, or alternatively consisting of, fragments or variants of these monoclonal antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those described herein), that immunospecifically bind the extracellular domain of IFNAR2, are also provided by the disclosure, as are nucleic acid molecules that encode these antibodies, vectors and host cells comprising the same, and/or antigen-binding molecules.

[0014] In an embodiment, the antibodies of the disclosure comprise a VH domain comprising, or alternatively consisting of, a polypeptide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 18 and a VL domain comprising, or alternatively consisting of, a polypeptide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 20. In an embodiment, antibodies of the present disclosure comprise a VH domain comprising, or alternatively consisting of, the polypeptide of SEQ ID NO: 2 and a VL domain comprising, or alternatively consisting of, the polypeptide of SEQ ID NO: 4. In other embodiments, antibodies of the present disclosure comprise a VH domain comprising, or alternatively consisting of, the polypeptide of SEQ ID NO: 18 and a VL domain comprising, or alternatively consisting of, the polypeptide of SEQ ID NO: 20.

[0015] Molecules comprising, or alternatively consisting of, fragments or variants of VH domains having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 (including VH CDRs), and VL domains having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20 (including VL CDRs), that immunospecifically bind the extracellular domain of IFNAR2, are also provided by the disclosure, as are nucleic acid molecules that encode these antigen-binding molecules.

[0016] In an embodiment, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may immunospecifically bind to a polypeptide or a polypeptide fragment of IFNAR2, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one, two, three or more of the VH complementarity determining regions (“CDRs”) (e.g., VH CDR1 , VH CDR2, or VH CDR3) of a VH domain having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 and/or any one, two, three or more of the VL CDRs (e.g., VL CDR1 , VL CDR2, or VL CDR3) of a VL domain having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20. In an embodiment, antibodies of the present disclosure comprise a polypeptide having the amino acid sequence of any one of the VH CDR1s of a VH domain having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 and/or any one of the VL CDR1 s of a VL domain having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20. In another embodiment, antibodies of the present disclosure comprise a polypeptide having the amino acid sequence of any one of the VH CDR2s of a VH domain having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 and/or any one of the VL CDR2s of a VL domain having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20. In another embodiment, antibodies of the present disclosure comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s of a VH domain having an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18 and/or any one of the VL CDR3s of a VL domain having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20. Molecules comprising, or alternatively consisting of, fragments or variants of the VH domains of SEQ ID NO: 2 or SEQ ID NO: 18, (e.g., VH CDRs), and/or the VL domains of SEQ ID NO: 4 or SEQ ID NO: 20 (e.g., VL CDRs), that immunospecifically bind the extracellular domain of IFNAR2, are also provided by the disclosure, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0017] In another embodiment, antibodies of the present disclosure (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) immunospecifically bind to a polypeptide or polypeptide fragment of IFNAR2, and comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR1 s of the VH domains of SEQ ID NO: 2 or SEQ ID NO: 18, any one of the VH CDR2s of the VH domains of SEQ ID NO: 2 or SEQ ID NO: 18, and/or any one of the VH CDR3s of the VH domains of SEQ ID NO: 2 or SEQ ID NO: 18. In another embodiment, antibodies of the present disclosure comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VL CDR1s of the VL domains of SEQ ID NO: 4 or SEQ ID NO: 20, any one of the VL CDR2s of the VL domains of SEQ ID NO: 4 or SEQ ID NO: 20, and/or any one of the VL CDR3s of the VL domains of SEQ ID NO: 4 or SEQ ID NO: 20. In an embodiment, antibodies of the present disclosure comprise CDR1 , CDR2, and CDR3 of the VH domain of SEQ ID NO: 2 or SEQ ID NO: 18 and/or CDR1 , CDR2, and CDR3 of the VL domain of SEQ ID NO: 4 or SEQ ID NO: 20. In an embodiment, antibodies of the present disclosure comprise CDR1 , CDR2, and CDR3 of the VH domain of SEQ ID NO: 2 and/or CDR1 , CDR2, and CDR3 of the VL domain of SEQ ID NO: 4. In other embodiments, antibodies of the present disclosure comprise CDR1 , CDR2, and CDR3 of the VH domain of SEQ ID NO: 18 and/or CDR1 , CDR2, and CDR3 of the VL domain of SEQ ID NO: 20. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence within any one of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32), that immunospecifically bind the extracellular domain of IFNAR2, are also provided by the disclosure, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0018] In an embodiment, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may immunospecifically bind to the extracellular domain of IFNAR2 (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 1-243 of SEQ ID NO: 33); immunospecifically bind to the membrane-bound form of IFNAR2 (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 1-264 of SEQ ID NO: 33); immunospecifically bind to the extracellular domain of IFNAR2 without the signal peptide (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 27-243 of SEQ ID NO: 33); and/or immunospecifically bind to the membrane-bound from of IFNAR2 (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 27- 264 of SEQ ID NO: 33). In an embodiment, antibodies of the present disclosure immunospecifically bind to the extracellular domain of IFNAR2 (e.g., a polypeptide comprising, or alternative consisting of, amino acids 1-243 or 27-243 of SEQ ID NO: 33) and comprise, or alternatively consist of, a VH domain, VH CDR1 , VH CDR2, VH CDR3, VL domain, VL CDR1 , VL CDR2, and/or VL CDR3 corresponding to one or more VH domains of SEQ ID NO: 2 or SEQ ID NO: 18 and/or one or more VL domains of SEQ ID NO: 4 or SEQ ID NO: 20. In another embodiment, antibodies of the present disclosure immunospecifically bind to the membrane-bound form of IFNAR2 (e.g., a polypeptide comprising, or alternative consisting of, amino acids 1-264 or 27-264 of SEQ ID NO: 33) and comprise, or alternatively consist of, a VH domain, VH CDR1 , VH CDR2, VH CDR3, VL domain, VL CDR1 , VL CDR2, and/or VL CDR3 corresponding to one or more VH domains of SEQ ID NO: 2 or SEQ ID NO: 18 and/or one or more VL domains of SEQ ID NO: 4 or SEQ ID NO: 20.

[0019] A VH domain of an amino acid sequence disclosed herein may be combined with a VL domain of an amino acid sequence disclosed herein, or other VL domains, to provide a VH/VL pairing representing an antigen-binding site of an antibody. Similarly, a VL domain of an amino acid sequence disclosed herein may be combined with a VH domain of an amino acid sequence disclosed herein, or other VH domains. Further, one or more CDRs disclosed herein may be taken from a VH or VL domain and incorporated into a suitable framework as discussed infra.

[0020] In an embodiment, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof (including derivatives)) may comprise, or alternatively consist of, VH domains, VL domains and/or CDRs described herein, which antibodies immunospecifically bind to IFNAR2 (e.g., the extracellular domain of IFNAR2) and can be routinely assayed for immunospecific binding to IFNAR2 using methods known in the art, such as, for example, the immunoassays disclosed herein. Antibodies and antibody fragments or variants (including derivatives) of the present disclosure may include, for example, one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue). These alterations may be made in one or more framework regions and/or one or more CDRs. The antibodies described herein (including antibody fragments, and variants and derivative thereof) can be routinely made by methods known in the art. Molecules comprising, or alternatively consisting of, fragments or variants of any of the VH domains, VH CDRs, VL domains, and VL CDRs whose sequences are specifically disclosed herein may be employed in accordance with the present disclosure. Nucleic acid molecules encoding these antibodies and molecules (including fragments, variants, and derivatives) are also provided by the present disclosure.

[0021] In an embodiment, panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) are described, wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the present disclosure (e.g., whole antibodies, Fabs, F(ab’)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-ld) antibodies, and scFvs). The present disclosure further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the disclosure (e.g., whole antibodies, Fabs, F(ab’)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-ld) antibodies, and scFvs).

[0022] In an embodiment, compositions may comprise or consist of one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present disclosure (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the present disclosure may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, a composition of the present disclosure may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the disclosure.

[0023] In an embodiment, fusion proteins may comprise an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the present disclosure, and a heterologous polypeptide (e.g., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also provided by the present disclosure. A composition of the present disclosure may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the disclosure. Alternatively, a composition of the present disclosure may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the disclosure.

[0024] In an embodiment, a recombinant nucleic acid molecule, generally isolated, encoding an antibody (including molecules which may comprise or consist of an antibody fragment or variant thereof) of the present disclosure is provided. The present disclosure also provides a host cell transformed with a nucleic acid molecule of the present disclosure and progeny thereof. The present disclosure also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the present disclosure. The present disclosure further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the present disclosure from a recombinant nucleic acid molecule. These and other aspects of the present disclosure are described in further detail below.

[0025] In an embodiment, methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant IFN or IFN receptor expression or inappropriate IFN or IFN receptor function in an animal, preferably a mammal, and most preferably a human, may comprise using antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2. Diseases and disorders which can be detected, diagnosed or prognosed with the antibodies described herein include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’s disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), viral infections (e.g., HIV and SARS-CoV-2), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

[0026] In an embodiment, methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant IFN or IFN receptor expression or inappropriate IFN or IFN receptor function in an animal, preferably a mammal, and most preferably a human, may comprise administering to said animal an effective amount of one or more antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2. Diseases and disorders which can be prevented, treated or inhibited by administering an effective amount of one or more antibodies or molecules of the present disclosure include, but are not limited to, immune disorders (e.g., lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’s disease, and immunodeficiency syndrome), inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoid arthritis), viral infections (e.g., HIV and SARS-CoV-2), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).

[0027] In an embodiment, autoimmune disorders, diseases, or conditions that may be detected, diagnosed, prognosed, prevented, treated or ameliorated using the antibodies described herein include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter’s Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (e.g., Hashimoto’s thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture’s syndrome, Pemphigus, Receptor autoimmunities such as, for example, Graves’ Disease, Myasthenia Gravis, and insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison’s disease, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren’s syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post- Ml, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders.

[0028] Immunodeficiencies that may be detected, diagnosed, prognosed, prevented, treated or ameliorated using the antibodies described herein include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton’s disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott- Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.

[0030] FIG. 1A and 1B shows data demonstrating the neutralizing effect of anti- IFNAR2 antibodies of the present disclosure on IFN-a activity. FIG. 1A provides results demonstrating that anti-IFNAR2 antibodies of the present disclosure neutralize IFN-a- induced antiviral activity in human alveolar basal epithelial (A549) cells in response to murine encephalomyelitis virus (ECMV). FIG. 1B provides results demonstrating that anti-IFNAR2 antibodies of the present disclosure neutralize IFN-a-induced antiproliferative activity in Daudi (Burkitt’s lymphoma) cells.

[0031] FIG. 2 shows data demonstrating the neutralizing effect of anti-IFNAR2 antibodies of the present disclosure on antiproliferative activity of IFN-a.

[0032] FIG. 3 shows that anti-IFNAR2 antibodies significantly block the ability of IFN- a to activate signaling pathways.

[0033] FIG. 4 shows the quality of the anti-IFNAR2 antibodies as evaluated in SDS- PAGE using 10-20% Tris-Glycine gel under reducing and non-reducing conditions.

[0034] FIG. 5 shows a comparison of binding abilities of anti-IFNAR2 antibodies of the disclosure (A10 GP and B7 GP) against commercial antibodies.

[0035] FIG. 6 shows basis for a new model to study the inflammatory effects of type I interferon.

[0036] FIG. 7A-7C show that anti-IFNAR2 antibodies A10 GP and B7 GP inhibit the induction of inflammatory cytokines induced by treatment with IFN-p and TNF-a. The effect of A10 GP and B7 GP on MIP1 -p (7A), RANTES (7B), and IL-6 (7C) are shown.

DETAILED DESCRIPTION

[0037] Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0038] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Definitions

[0039] As used herein, the terms “type I interferon” and “human type I interferon,” refer broadly to all species of native human interferon which fall within the human interferon-a, interferon-co and interferon-p classes and which bind to a common cellular receptor. Natural human interferon-a comprises 23 or more closely related proteins encoded by distinct genes with a high degree of structural homology (Weissmann and Weber, Prog. Nucl Acid. Res. Mol Biol., 33: 251 (1986); J. Interferon Res., 13: 443-444 (1993)). The human IFN-a locus comprises two subfamilies. The first subfamily consists of at least 14 functional, non-allelic genes, including genes encoding IFN-aA (IFN-a2), IFN-aB (IFN-a8), IFN-aC (IFN-a10), IFN-aD (IFN-a1), IFN-aE (IFN-a22), IFN-aF (IFN- a21), IFN-aG (IFN-a5), and IFN-aH (IFN-a14), and pseudogenes having at least 80% homology. The second subfamily, an or co, contains at least 5 pseudogenes and 1 functional gene (denoted herein as “IFN-an1” or “IFN-co”) which exhibits 70% homology with the IFN-a genes (Weissmann and Weber (1986)). The human IFN-p is encoded by a single copy gene.

[0040] As used herein, the terms “type-l interferon receptor subunit 2”, “IFN-aR”, “hlFNAR2”, and “IFNAR2,” refer broadly to the 515 amino acid receptor protein cloned by Domanski et al., J. Biol. Chem., 37: 21606-21611 (1995), including an extracellular domain of 217 residues, a transmembrane domain of 21 residues, and an intracellular domain of 250 residues, as shown in FIG. 1 on page 21608 of Domanski et al. Also encompassed by the foregoing terms are fragments of IFNAR2 that contain the extracellular domain (ECD) (or fragments of the ECD) of IFNAR2, and soluble forms of IFNAR2, such as IFNAR2 ECD fused to an immunoglobulin sequence. [0041] As used herein, the term “anti-IFNAR2 antibody,” refers broadly to as an antibody that is capable of binding to IFNAR2.

[0042] As used herein, an anti-IFNAR2 antibody with the property or capability of “blocking the binding of a type I interferon to IFNAR2,” refers broadly to an anti-IFNAR2 antibody capable of binding to IFNAR2 such that the ability of IFNAR2 to bind to one or more type I interferons is impaired or eliminated. An anti-IFNAR2 antibody candidate can be tested for such activity, for example, by adsorbing anti-IFNAR2 antibody to immobilized IFNAR2 followed by subjecting the adsorbed antibody to elution with an excess of a selected type I interferon. If an eluent comprising an excess of the selected type I interferon produces an eluate containing a greater concentration of the candidate antibody than the concentration of candidate antibody present in an eluate produced by a “blank” eluent (the same eluent containing no type I interferon) in a control elution, as determined by, e.g., radioimmunoassays performed on the respective eluates with radiolabelled, soluble IFNAR2, then the candidate antibody competes with the selected type I interferon for binding to IFNAR2. In an embodiment, the anti-IFNAR2 antibody of the present disclosure competes with a selected type I interferon for binding to IFNAR2 and accordingly impairs or eliminates the binding of the selected type I interferon to IFNAR2.

[0043] As used herein, an anti-IFNAR2 antibody with the property or capability of “neutralizing IFN activity,” refers broadly to as an anti-IFNAR2 antibody capable of reducing or inhibiting the activity of one or more type I interferons. An anti-IFNAR2 antibody candidate can be tested for such activity, for example, by measuring suppression of IFN activity in one or more biological assays, such as an antiproliferative or antiviral assay.

[0044] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity.

[0045] “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).

[0046] The term “variable” refers broadly to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0047] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0048] “Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigenbinding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0049] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0050] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (A), based on the amino acid sequences of their constant domains.

[0051] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, I g D, IgE, IgG, and I g M , and several of these can be further divided into subclasses (isotypes), e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, £, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. “Therapeutic Antibody Engineering” (1^st Ed.) Strohl & Strohl Woodhead Publishing (2012).

[0052] The term “antibody” encompasses monoclonal antibodies, including antibody fragment clones.

[0053] “Antibody fragments” comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2, and Fv fragments; diabodies; single-chain antibody molecules, including single-chain Fv (scFv) molecules; and multispecific antibodies formed from antibody fragments. “Human Monoclonal Antibodies: Methods and Protocols” (2^nd Ed.) Steinitz (Ed.) Humana Press (2019).

[0054] The term “monoclonal antibody” as used herein, refers broadly to an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” also include clones of antigen-recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991 ), for example.

[0055] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)); “Antibody Engineering” Volume 2 (2^nd Ed.) Kontermann & Diibel. Springer Press (2010).

[0056] A “human” antibody (also called a “fully human” antibody) is an antibody that includes human framework regions and all of the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and/or light chain amino acid sequence. However, frameworks from one human antibody can be engineered to include CDRs from a different human antibody.

[0057] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (such as mouse, rat or rabbit) or a synthetic sequence (donor antibody), having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In an embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering. See for example, U.S. Patent No. 5,585,089.

[0058] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269- 315 (1994).

[0059] The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0060] An “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0061] The term “variant” as used herein refers broadly to a polypeptide that possesses a similar or identical function as an IFN, an IFNAR2 polypeptide, an anti- IFNAR2 antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of an IFN, an IFNAR2 polypeptide, anti-IFNAR2 or antibody fragment thereof, or possess a similar or identical structure of an IFN, an IFNAR2 polypeptide, an anti-IFNAR2 antibody or antibody fragment thereof. A variant having a similar amino acid identity refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, 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 an IFN, an IFNAR2 polypeptide, anti-IFNAR2 or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those described herein); (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding an IFNAR2 polypeptide or fragment thereof, an anti-IFNAR2 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those described herein), of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, 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 nucleotide sequence encoding an IFN or fragment thereof, an IFNAR2 polypeptide or fragment thereof, an anti-IFNAR2 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence described herein). A polypeptide with similar structure to an IFN or fragment thereof, an IFNAR2 polypeptide or fragment thereof, an anti-IFNAR2 antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of an IFN or fragment thereof, an IFNAR2 polypeptide or fragment thereof, an anti-IFNAR2 antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % identity = number of identical overlapping positions/total number of positions x 100%). In an embodiment, the two sequences are the same length.

[0062] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mai. Biol. 215:403-410(1990) have incorporated such an algorithm. BLAST nucleotide searches can be performed with the BLASTn program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the present disclosure. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3389-3402(1997). Alternatively, PSI- BLAST can be used to perform an iterated search, which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. (See ncbi.nlm.nih.gov).

[0063] Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10:3-5(1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8(1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0064] “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to IFNAR2. For example, a monoclonal antibody that immunospecifically binds IFNAR2 can include at most about 1 , at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and immunospecifically bind an IFNAR2 polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody immunospecifically binds IFNAR2. Non-conservative substitutions are those that reduce an activity or binding to IFNAR2.

[0065] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0066] The term “derivative” as used herein, refers to a variant polypeptide of the present disclosure that comprises, or alternatively consists of, an amino acid sequence of an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, or an antibody of the present disclosure that immunospecifically binds to IFNAR2, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, an antibody that immunospecifically binds to IFNAR2 which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, or an anti-IFNAR2 antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, or an anti-IFNAR2 antibody or fragment thereof, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, or an anti-IFNAR2 antibody or fragment thereof, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as an IFN polypeptide or fragment thereof, an IFNAR2 polypeptide or fragment thereof, or an anti-IFNAR2 antibody or fragment thereof, described herein.

[0067] The term “epitopes” as used herein refers to portions of IFNAR2 having antigenic or immunogenic activity in an animal, preferably a mammal. An epitope having immunogenic activity is a portion of IFNAR2 that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of IFNAR2 to which an antibody immunospecifically binds as determined by any method known in the art, for example, by the immunoassays described herein. Antigenic epitopes need not necessarily be immunogenic.

[0068] The term “fragment” as used herein refers broadly to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues, of the amino acid sequence of an IFN, an IFNAR2, or an anti-IFNAR2 antibody (including molecules such as scFvs, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to IFNAR2.

[0069] The term “fusion protein” as used herein refers broadly to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-IFNAR2 antibody of the present disclosure and an amino acid sequence of a heterologous polypeptide (e.g., a polypeptide unrelated to an antibody or antibody domain).

[0070] The term “host cell” as used herein refers broadly to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0071] “Treatment” refers broadly to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, the term “treating,” refers broadly to treating a disease, arresting, or reducing the development of the disease or its clinical symptoms, and/or relieving the disease, causing regression of the disease or its clinical symptoms. Therapy encompasses prophylaxis, treatment, remedy, reduction, alleviation, and/or providing relief from a disease, signs, and/or symptoms of a disease. Therapy encompasses an alleviation of signs and/or symptoms in patients with ongoing disease signs and/or symptoms. Therapy also encompasses “prophylaxis”. The term “reduced”, for purpose of therapy, refers broadly to the clinical significant reduction in signs and/or symptoms. Therapy includes treating relapses or recurrent signs and/or symptoms. Therapy encompasses but is not limited to precluding the appearance of signs and/or symptoms anytime as well as reducing existing signs and/or symptoms and eliminating existing signs and/or symptoms. Therapy includes treating chronic disease (“maintenance”) and acute disease. For example, treatment includes treating or preventing relapses or the recurrence of signs and/or symptoms.

[0072] “Effective amount,” as used herein, refers broadly to the amount of a compound, antibody, antigen, or cells that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The effective amount may be an amount effective for prophylaxis, and/or an amount effective for prevention. The effective amount may be an amount effective to reduce, an amount effective to prevent the incidence of signs/symptoms, to reduce the severity of the incidence of signs/symptoms, to eliminate the incidence of signs/symptoms, to slow the development of the incidence of signs/symptoms, to prevent the development of the incidence of signs/symptoms, and/or effect prophylaxis of the incidence of signs/symptoms. The “effective amount” may vary depending on the disease and its severity and the age, weight, medical history, susceptibility, and pre-existing conditions, of the patient to be treated. The term “effective amount” is synonymous with “therapeutically effective amount” for purposes of this disclosure.

[0073] “Mammal,” as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Mammals include, but are not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, gerbils, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington D.C. Similarly, the term “subject” or “patient” includes both human and veterinary subjects and/or patients.

Anti-IFNAR2 Antibodies

[0074] Anti-IFNAR2 antibodies may be used in the treatment of immune-mediated diseases or disorders in which a partial or total blockade and/or neutralization of type I interferon activity is desired. In an embodiment, the anti-IFNAR2 antibodies of the present disclosure are used to treat autoimmune disorders, such as type I and type II diabetes, systemic lupus erythematosis (SLE), and rheumatoid arthritis. In another embodiment, the anti-IFNAR2 antibodies provided herein are used to treat graft rejection or graft versus host disease. In yet another embodiment, the anti-IFNAR2 antibodies provided herein are used to treat or prevent viral infection, such as HIV. The unique properties of the anti-IFNAR2 antibodies of the present disclosure make them particularly useful for immunosuppression. The anti-IFNAR2 antibodies provided herein effect broad-spectrum neutralization of type I interferon activity, which can be highly effective in suppressing an undesired immune response. In particular, the anti-IFNAR2 antibodies provided herein neutralize or inhibit one or more species of type I interferon.

[0075] In another aspect, the anti-IFNAR2 antibodies of the present disclosure find utility as reagents for detection and isolation of IFNAR2, such as detection and/or quantification of IFNAR2 expression in various cells and/or tissues. In yet another aspect, the present anti-IFNAR2 antibodies are useful for the development of IFNAR2 antagonists with type I interferon blocking activity patterns similar to those of the subject antibodies. The anti-IFNAR2 antibodies of the present disclosure can be used in IFNAR2 signal transduction assays to screen for antagonists of IFNAR2 that will exhibit similar pharmacological effects.

[0076] In an embodiment, antibodies of the present disclosure immunospecifically bind an IFNAR2 polypeptide having the amino acid sequence of SEQ ID NO: 33 or a polypeptide comprising a portion (e.g., a fragment) of the amino acid sequence of SEQ ID NO: 33. The present disclosure also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that: immunospecifically bind to the extracellular domain of IFNAR2 (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 1-243 of SEQ ID NO:33); that immunospecifically bind to the membrane-bound form of IFNAR2 (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 1 - 264 of SEQ ID NO: 33); that immunospecifically bind to the extracellular domain of IFNAR2 without the signal peptide (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 27-243 of SEQ ID NO: 33); and/or that immunospecifically bind to the membrane-bound from of IFNAR2 without the signal peptide (e.g., a polypeptide comprising, or alternatively consisting of, amino acids 27-264 of SEQ ID NO: 33).

[0077] Moreover, polypeptide fragments that may be bound by antibodies of the present disclosure can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context, “about” means the particularly recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini.

[0078] Additional embodiments of the present disclosure encompass antibodies that bind IFNAR2 polypeptide fragments comprising, or alternatively consisting of, functional regions of polypeptides of the present disclosure, such as the Garnier-Robson alpharegions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, betaregions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Emini surface-forming regions and Jameson-Wolf regions of high antigenic index. In a preferred embodiment, the polypeptide fragments bound by the antibodies described herein are antigenic (e.g., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1 .5, as identified using the default parameters of the Jameson-Wolf program) of a complete (e.g., full-length) IFNAR2 polypeptide (e.g., SEQ ID NO: 33).

[0079] In an embodiment, the antibodies described herein bind a polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide described herein. The epitope of this polypeptide portion may be an immunogenic or antigenic epitope of a polypeptide described herein. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81 :3998- 4002 (1983).

[0080] As to the selection of polypeptides bearing an antigenic epitope (e.g., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) “Antibodies that react with predetermined sites on proteins”, Science, 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (e.g., immunogenic epitopes) nor to the amino or carboxyl terminals. Antigenic epitopebearing peptides and polypeptides described herein are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide described herein. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. [0081] The antibodies described herein may bind antigenic epitope-bearing peptides and polypeptides of IFNAR2 and preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids contained within the amino acid sequence of an IFNAR2 polypeptide. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.

Anti-IFNAR2 Antibody Epitopes

[0082] The present disclosure encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO: 33. Predicted epitopes of the extracellular domain of SEQ ID NO: 33 include, but are not limited to, a polypeptide comprising, or alternative consisting of, one or more of the following: SPDYTDES (SEQ ID NO: 34); LKNHSIV (SEQ ID NO: 35); SKPEDLKWKNCANTTR (SEQ ID NO: 36); TDEWRSTH (SEQ ID NO: 37); GNTTLFSCSH (SEQ ID NO: 38); FWLAIDMSFEPPE (SEQ ID NO: 39); PSIVEEELQFDL (SEQ ID NO: 40); SEGIVKKHKPEIKGNMSGNFTYIIDKLIP (SEQ ID NO: 41 ); and/or HSDEQAVIKSPLKCTLLPPGQESESAES (SEQ ID NO: 42).

[0083] The term “epitopes,” as used herein, refers broadly to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present disclosure encompasses antibodies that bind a polypeptide comprising an epitope. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81 :3998- 4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include, but are not limited to, the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0084] Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittie et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes of IFNAR2 may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

[0085] IFNAR2 polypeptide fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131- 5135 (1985) and U.S. Patent No. 4,631 ,211).

[0086] Epitope-bearing IFNAR2 polypeptides may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittie et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, antipeptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund’s adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

Anti-IFNAR2 Antibody Fusion Proteins

[0087] The antibodies described herein may bind polypeptides comprising an immunogenic or antigenic epitope fused to other polypeptide sequences. For example, the IFNAR2 polypeptides may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1 , CH2, CH3, or any combination thereof and portions thereof), or albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, herein incorporated by reference in their entirety), resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331 :84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fe fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991 , Proc. Natl. Acad. Sci. USA 88:8972- 897): In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Nl²⁺ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

[0088] In another embodiment, the antibodies described herein bind IFNAR2 polypeptides and/or the epitope-bearing fragments thereof that are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid). In specific embodiments, the heterologous antigen is an immunogen.

Anti-IFNAR2 Antibody Specificity

[0089] The binding specificity of antibodies described herein to IFNAR2 polypeptides, or fragments or variants thereof can be determined by any suitable means. Examples of suitable assays to measure binding specificity include, but are not limited to, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay (ELISA). Other means, such as, surface plasmon resonance may also be used.

[0090] The binding affinity of antibodies can, for example, be determined by the Scatchard analysis described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In another embodiment, antibody affinity is measured by flow cytometry.

[0091] An antibody that “specifically binds” or “immunospecifically binds” an antigen (such as IFNAR2 or fragments or variants thereof) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0092] The antibodies described herein may bind an IFNAR2 polypeptide or fragment thereof (such as soluble and/or cell-surface IFNAR2) with a dissociation constant (Kd) of about 1 nM or less. In some embodiments, the antibodies bind an IFNAR2 polypeptide or fragment thereof with a binding affinity of about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.15 nM, about 0.1 nM, about 0.05 nM, about 0.04 nM, about 0.03 nM, about 0.02 nM or about 0.01 nM.

[0093] The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence least 80%, 85%, 90% identical and more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33.

[0094] The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 90% identical to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33. The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 95% identical to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33. The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 96% identical to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33.

[0095] The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 97% to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33. The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 98% to an IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33. The antibodies described herein may bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 99% identical to IFNAR2 polypeptide having the amino acid sequence at positions 27-243 of SEQ ID NO: 33.

[0096] The antibodies described herein may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO: 33, such as (i) polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) polypeptides in which one or more of the amino acid residues includes a substituent group, or (iii) polypeptides in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence.

[0097] Amino acids in the IFNAR2 polypeptides that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for functional activity, such ligand binding. Accordingly, antibodies of the present disclosure may bind amino acids in the IFNAR2 polypeptides that are essential for function. The antibodies described herein may bind amino acids in the IFNAR2 polypeptides that are essential for IFN binding. The antibodies described herein may bind amino acids in the IFNAR2 polypeptides that inhibit or reduce IFN signalling and function. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labelling (Smith et al., J. Mai. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

[0100] Anti-IFNAR2 antibodies described herein may possess the unique properties described herein, which can be identified by screening anti-IFNAR2 antibodies, fragments, or variants thereof for the desired properties by any convenient method. For example, if an anti-IFNAR2 antibody that blocks or does not block the binding of certain type I interferons to IFNAR2 is desired, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, wherein plate wells are coated with IFNAR2, and a solution of antibody in an excess of the type I interferon of interest is layered onto the coated plates, and bound antibody is detected enzymatically, e.g., contacting the bound antibody with HRP-conjugated anti-lg antibody or biotinylated anti-lg antibody and developing the HRP color reaction., e.g., by developing plates with streptavidin-HRP and/or hydrogen peroxide and detecting the HRP color reaction by spectrophotometry at 490 nm with an ELISA plate reader.

Anti-IFNAR2 Antibody Activity [0101] The anti-IFNAR2 antibodies described herein inhibit the antiviral activity of one or more type I interferon(s) and, optionally, do not inhibit the antiviral activity of other type I interferon(s). Any convenient type I interferon viral infectivity inhibition assay is suitable for use herein. Such assays are well known in the art, and include, for example, type I interferon-induced inhibition of vesicular stomatitis virus (VSV) infectivity in Daudi cells as described in Schmeisser et al., J. Virol. (October 2010), p. 10671- 10680, or type I interferon-induced inhibition of encephalomyocarditis virus (EMC) infectivity in A549 cells as described in Current Protocols in Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-lnterscience, (1992), vol. 2, unit 6.9.1. Generally, cells are seeded in attached cell culture plates, grown for 1 day, and then incubated for an additional day in the presence of a predetermined number of units of a selected type I interferon plus various concentrations of the candidate anti-IFNAR2 antibody. Culture supernatants are then removed and cells are challenged with virus and incubated for an additional day. The candidate anti-IFNAR2 antibody that inhibits the antiviral activity of a selected type I interferon will inhibit more antiviral activity than the baseline level of antiviral activity inhibition measured in the presence of an equivalent concentration of control antibody. Optionally, the candidate anti-IFNAR2 antibody that inhibits the antiviral activity of a selected type I interferon will inhibit at least and/or about 30%, or at least and/or about 40%, or at least and/or about 50%, or at least and/or about 60%, or at least and/or about 70%, or at least and/or about 80%, or at least and/or about 90%, or at least and/or about 95%, or at least and/or about 96%, or at least and/or about 97%, or at least and/or about 98%, or at least and/or about 99%, or about 100% of the activity of the type I interferon in the antiviral activity assay as compared to baseline activity measured in the presence of an equivalent concentration of control antibody. The candidate anti-IFNAR2 antibody that does not inhibit the antiviral activity of a selected type I interferon will exhibit similar or approximately the same level of antiviral activity inhibition as a control antibody.

[0102] Each type I interferon species used in the viral infectivity assay may be titrated to a concentration that provides the same level of inhibition of viral activity as that induced by a preselected number of units of an IFN-a standard. This concentration serves to provide the normalized units of the subject type I interferon species. In order to assess the ability of an anti-IFNAR2 antibody to inhibit the antiviral activity of various type I interferons, the effective concentration (EC50) of anti-IFNAR2 antibody for inhibiting 50% of a particular type I interferon’s antiviral activity (at the concentration titrated to provide the normalized units of activity) is determined for each type I interferon to be tested. In another embodiment, each type I interferon to be tested is normalized to at least about 1 unit/ml, or from about 1 unit/ml to about 1 ,000 units/ml, or from about 1 unit/ml to about 100 units/ml, of human IFN-a2. In yet another preferred embodiment, each type I interferon to be tested is normalized to 100 units/ml of the NIH reference standard for recombinant human IFN-a2 (IFN-aA).

[0103] In another embodiment, the candidate anti-IFNAR2 antibody that does not inhibit the antiviral activity of a selected type I interferon will exhibit no antiviral effect at a concentration of at least and/or about 1 pg/ml, or at least and/or about 10 pg/ml, or at least and/or about 20 pg/ml, or at least and/or about 30 pg/ml, or at least and/or about 50 pg/ml, or at least and/or about 100 pg/ml, in the inhibition of EMC infectivity in A549 cells assay described in Current Protocols in Immunology, Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds, Greene Publishing Associates and Wiley-lnterscience, (1992), vol. 2, unit 6.9.1 , wherein each type I interferon is normalized to 100 units/ml of NIH reference standard for recombinant human IFN-a2 (IFN-aA).

[0104] If an anti-IFNAR2 monoclonal antibody that binds to a particular IFNAR2 determinant(s) is desired, the candidate antibody can be screened for the presence or absence of differential affinity to wild type IFNAR2 and to mutant IFNAR2 that contains Ala substitution(s) at the determ inant(s) of interest as described above. In one aspect, the candidate antibody can be tested for binding to wild type IFNAR2 and mutant IFNAR2 in an immunoprecipitation or immunoadsorption assay. For example, a capture ELISA can be used wherein plates are coated with a given concentration of wild type IFNAR2 or an equal concentration of mutant IFNAR2, the coated plates are contacted with equal concentrations of the candidate antibody, and the bound antibody is detected enzymatically, e.g. contacting the bound antibody with HRP-conjugated anti-lg antibody and developing the HRP color reaction. The candidate antibody that binds to the particular IFNAR2 determ inant(s) of interest will exhibit binding activity with wild type IFNAR2 that is greater than the candidate antibody’s binding activity with the corresponding Ala-substituted IFNAR2 mutant (e.g., a binding level with wild type IFNAR2 that is above the background binding level with mutant IFNAR2). Optionally, the candidate antibody that binds to the particular IFNAR2 determinant(s) of interest will exhibit binding activity with the corresponding Ala-substituted IFNAR2 mutant that is less than about 50%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 7%, or less than about 6%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1 %, or about 0% of the antibody’s binding activity with wild type IFNAR2, e.g., as determined by dividing the HRP color reaction optical density observed for capture ELISA with IFNAR2 mutant adsorbent by the HRP color reaction optical density observed for capture ELISA with wild type IFNAR2 adsorbent.

[0105] The anti-IFNAR2 antibodies described herein may possess combinations of the type I interferon activity inhibition and the IFNAR2 determinant binding properties described herein. Anti-IFNAR2 antibodies corresponding to these embodiments can be obtained by using combinations of the type I interferon competitive binding and/or activity inhibition assays described herein for selection of antibodies with unique type I interferon inhibiting properties and the immunoprecipitation or immunoadsorption screening procedures described herein for selection of antibodies with unique IFNAR2 determinant binding properties.

[0106] The anti-IFNAR2 antibodies described herein may neutralize one or more type I IFN, such as an IFN-a, an IFN-co and/or an IFN-p. For example, the antibodies described herein may neutralize one or more of the following type I interferons: IFN-aA (IFN-a2), IFN-aB (IFN-a8), IFN-aC (IFN-a10), IFN-aD (IFN-a1), IFN-aE (IFN-a22), IFN- aF (IFN-a21), IFN-aG (IFN-a5), IFN-aH (IFN-a14), IFN-a_N1 (IFN-w”), IFN-p, or variants or fragments thereof. The antibodies described herein may neutralize IFNs derived from any animal, preferably from a mammal. Most preferably, the antibodies described herein may neutralize a human type I IFN.

A10 and B7 Anti-IFNAR2 Antibodies

[0107] Provided herein are antibodies A10 and B7 that immunospecifically bind to IFNAR2 with high affinity and neutralize a broad spectrum of IFNs. These antibodies have been demonstrated, for example, to neutralize antiviral and antiproliferative activities of type I interferons, such as IFN-a2 and IFN-p.

[0108] The nucleotide and amino acid sequences of the VH and VL domains and CDRs of the A10 and B7 antibodies are shown below. [0109] A10 VH Domain Nucleotide Sequence (SEQ ID NO: 1)

[0110] CAGGTCCAGCTGCAGCAGTCTGGAACTGAGCTGGTAAGGCCTGGGACT

TCAGTGAGTCTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTACTTGATAGA

GTGGGTAAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGTGATTAATCCT

GGAAGTGGTAATACTAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAGCA

CTGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTTTATTTCTGT

GCAAGATCCTCCCACTACGGTGCGGGCTTTGACTACTGGGGCCAAGGCACCACTC

TCACAGTCTCCTCA

[0111] A10 VH Domain Amino Acid Sequence (SEQ ID NO: 2)

[0112] QVQLQQSGTELVRPGTSVSLSCKASGYAFTNYLIEWVKQRPGQGLEWIGVI

NPGSGNTKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSSHYGAGFDYWGQGT TLTVSS

[0113] A10 VL Domain Nucleotide Sequence (SEQ ID NO: 3)

[0114] GATGTTGTGATGACCCAGACTCCAGTCACTTTGTCGGTTACCATTGGACA

ACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGGAAG

ACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTA

TCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCA

GGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAAGATTTGGGAGTTTA

TTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAA

[0115] A10 VL Domain Amino Acid Sequence (SEQ ID NO: 4)

[0116] DWMTQTPVTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKR

LIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKL EIK

[0117] Table 1 : CDR sequences for A10 antibody

[0118] B7 VH Domain Nucleotide Sequence (SEQ ID NO: 17)

[0119] GAAGTGAAGCTTGAGGAGTCGGGAGGAGGCTTGGTGCAACCTGGAGGA

TCCATGAAACTCTCCTGTGTTGTTTCTGGATTCACTTTCAATAACTACTGGATGAAC

TGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTCAAATTAGATTGA

AATCTGATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCAT

CTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGGGCTG

AAGACACTGGAATTTATTACTGCACAGGCGGGGGAGTACCTGCCTGGTTTGTTTAC

TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0120] B7 VH Domain Amino Acid Sequence (SEQ ID NO: 18)

[0121] EVKLEESGGGLVQPGGSMKLSCWSGFTFNNYWMNWVRQSPEKGLEWVA

QIRLKSDNYATHYAESVKGR FTI S R DDS KSSVYLQM N N LRAEDTGIYYCTGGGVPAW FVYWGQGTLVTVSA

[0122] B7 VL Domain Nucleotide Sequence (SEQ ID NO: 19)

[0123] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGA

TCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTACATAGTAATGGAAACA

CCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC

AAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAG

GGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTA

TTACTGCTTTCAAGGTTCACATGTTCCTCGGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAA

[0124] B7 VL Domain Amino Acid Sequence (SEQ ID NO: 20) [0125] DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGNTYLEWYLQKPGQSPKLL

IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEI K

[0126] Table 2: CDR sequences for B7 antibody

[0127] In an embodiment, the antibody or fragment thereof described herein may comprise a VH domain of SEQ ID NO: 2.

[0128] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VH domain of SEQ ID NO: 2. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 8, a CDR3 of SEQ ID NO: 10, or any combination thereof.

[0129] In an embodiment, the antibody or fragment thereof described herein may comprise a VL domain of SEQ ID NO: 4.

[0130] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VL domain of SEQ ID NO: 4. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 12, a CDR2 of SEQ ID NO: 14, a CDR3 of SEQ ID NO: 16, or any combination thereof. [0131] In an embodiment, the antibody or fragment thereof comprises a VH domain of SEQ ID NO: 2 and a VL domain of SEQ ID NO: 4.

[0132] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VH domain of SEQ ID NO: 2 and one, two, or all three CDRs of the VL domain of SEQ ID NO: 4. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 6, a CDR2 of SEQ ID NO: 8, a CDR3 of SEQ ID NO: 10, or any combination thereof and a CDR1 of SEQ ID NO: 12, a CDR2 of SEQ ID NO: 14, a CDR3 of SEQ ID NO: 16, or any combination thereof.

[0133] In an embodiment, the antibody or fragment thereof comprises all three CDRs of the VH domain of SEQ ID NO: 2 and all three CDRs of the VL domain of SEQ ID NO: 4. For example, the antibody or fragment thereof comprises a VHCDR1 of SEQ ID NO: 6, a VHCDR2 of SEQ ID NO: 8, a VHCDR3 of SEQ ID NO: 10, a VLCDR1 of SEQ ID NO: 12, a VLCDR2 of SEQ ID NO: 14, and a VLCDR3 of SEQ ID NO: 16.

[0134] In an embodiment, the antibody or fragment thereof comprises a VH domain encoded by a nucleotide sequence of SEQ ID NO: 1 .

[0135] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VH domain encoded by SEQ ID NO: 1 . For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 5, a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 7, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 9, or any combination thereof.

[0136] In an embodiment, the antibody or fragment thereof comprises a VL domain encoded by a nucleotide sequence of SEQ ID NO: 3.

[0137] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VL domain encoded by SEQ ID NO: 3. For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 11 , a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 13, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 15, or any combination thereof.

[0138] In an embodiment, the antibody or fragment thereof comprises a VH domain encoded by the nucleotide sequence of SEQ ID NO: 1 and a VL domain encoded by the nucleotide sequence of SEQ ID NO: 3. [0139] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VH domain encoded by the nucleotide sequence of SEQ ID NO: 1 and one, two, or all three CDRs of the VL domain encoded by the nucleotide sequence of SEQ ID NO: 3. For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded SEQ ID NO: 5, a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 7, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 9, or any combination thereof and a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 11 , a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 13, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 15, or any combination thereof.

[0140] In an embodiment, the antibody or fragment thereof comprises all three CDRs of the VH domain encoded by the nucleotide sequence of SEQ ID NO: 1 and all three CDRs of the VL domain encoded by the nucleotide sequence of SEQ ID NO: 3. For example, the antibody or fragment thereof may comprise a VHCDR1 comprising an amino acid sequence encoded by SEQ ID NO: 5, a VHCDR2 comprising an amino acid sequence encoded by SEQ ID NO: 7, a VHCDR3 comprising an amino acid sequence encoded by SEQ ID NO: 9, a VLCDR1 comprising an amino acid sequence encoded by SEQ ID NO: 11 , a VLCDR2 comprising an amino acid sequence encoded SEQ ID NO: 13, and a VLCDR3 comprising an amino acid sequence encoded SEQ ID NO: 15.

[0141] In an embodiment, the antibody or fragment thereof described herein may comprise a VH domain of SEQ ID NO: 18.

[0142] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VH domain of SEQ ID NO: 18. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 22, a CDR2 of SEQ ID NO: 24, a CDR3 of SEQ ID NO: 26, or any combination thereof.

[0143] In an embodiment, the antibody or fragment thereof described herein may comprise a VL domain of SEQ ID NO: 20.

[0144] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VL domain of SEQ ID NO: 20. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 30, a CDR3 of SEQ ID NO: 32, or any combination thereof. [0145] In an embodiment, the antibody or fragment thereof comprises a VH domain of SEQ ID NO: 18 and a VL domain of SEQ ID NO: 20.

[0146] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VH domain of SEQ ID NO: 18 and one, two, or all three CDRs of the VL domain of SEQ ID NO: 20. For example, the antibody or fragment thereof may comprise a CDR1 of SEQ ID NO: 22, a CDR2 of SEQ ID NO: 24, a CDR3 of SEQ ID NO: 26, or any combination thereof and a CDR1 comprising SEQ ID NO: 28, a CDR2 comprising SEQ ID NO: 30, a CDR3 of SEQ ID NO: 32, or any combination thereof.

[0147] In an embodiment, the antibody or fragment thereof comprises all three CDRs of the VH domain of SEQ ID NO: 18 and all three CDRs of the VL domain of SEQ ID NO: 20. For example, the antibody or fragment thereof may comprise a VHCDR1 of SEQ ID NO: 22, a VHCDR2 of SEQ ID NO: 24, a VHCDR3 of SEQ ID NO: 26, a VLCDR1 of SEQ ID NO: 28, a VLCDR2 of SEQ ID NO: 30, and a VLCDR3 of SEQ ID NO: 32.

[0148] In an embodiment, the antibody or fragment thereof comprises a VH domain encoded by a nucleotide sequence of SEQ ID NO: 17.

[0149] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VH domain encoded by SEQ ID NO: 17. For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 21 , a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 23, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 25, or any combination thereof.

[0150] In an embodiment, the antibody or fragment thereof comprises a VL domain encoded by a nucleotide sequence of SEQ ID NO: 19.

[0151] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRS of the VL domain encoded by SEQ ID NO: 19. For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 27, a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 29, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 31 , or any combination thereof. [0152] In an embodiment, the antibody or fragment thereof comprises a VH domain encoded by the nucleotide sequence of SEQ ID NO: 17 and a VL domain encoded by the nucleotide sequence of SEQ ID NO: 19.

[0153] In an embodiment, the antibody or fragment thereof comprises one, two, or all three CDRs of the VH domain encoded by the nucleotide sequence of SEQ ID NO: 17 and one, two, or all three CDRs of the VL domain encoded by the nucleotide sequence of SEQ ID NO: 19. For example, the antibody or fragment thereof may comprise a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 21 , a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 23, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 25, or any combination thereof and a CDR1 comprising an amino acid sequence encoded by SEQ ID NO: 27, a CDR2 comprising an amino acid sequence encoded by SEQ ID NO: 29, a CDR3 comprising an amino acid sequence encoded by SEQ ID NO: 31 , or any combination thereof.

[0154] In an embodiment, the antibody or fragment thereof comprises all three CDRs of the VH domain encoded by the nucleotide sequence of SEQ ID NO: 17 and all three CDRs of the VL domain encoded by the nucleotide sequence of SEQ ID NO: 19. For example, the antibody or fragment thereof comprises a VHCDR1 comprising an amino acid sequence encoded by SEQ ID NO: 21 , a VHCDR2 comprising an amino acid sequence encoded by SEQ ID NO: 23, a VHCDR3 comprising an amino acid sequence encoded by SEQ ID NO: 25, a VLCDR1 comprising an amino acid sequence encoded by SEQ ID NO: 27, a VLCDR2 comprising an amino acid sequence encoded by SEQ ID NO: 29, and a VLCDR3 comprising an amino acid sequence encoded by SEQ ID NO: 31.

Anti-IFNAR2 Antibody Variants

[0155] The antibodies described herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. In an embodiment, the antibodies are human antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries and xenomice or other organisms that have been genetically engineered to produce human antibodies. For a detailed discussion of a few of the technologies for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661 ,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 ; and 5,939,598; and Lenberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), which are incorporated by reference herein in their entirety. Human antibodies or “humanized” chimeric monoclonal antibodies can be produced using techniques described herein or otherwise known in the art. For example, methods for producing chimeric antibodies are known in the art. See, for review the following references which are hereby incorporated in their entirety: Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671 ; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

Chimeric Anti-IFNAR2 Antibodies

[0156] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a human antibody and a non-human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non-human immunoglobulin molecule (e.g., framework regions from a canine or feline immunoglobulin molecule) can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332). In an embodiment, chimeric antibodies comprise a CDR3 having an amino acid sequence of any one of the VH CDR3s within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 10 or SEQ ID NO: 26 or VL CDR3s within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 16 or SEQ ID NO: 32, or a variant thereof, and non-human framework regions or human framework regions. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well 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. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties).

Anti-IFNAR2 Antibody Mimics

[0157] Further, the antibodies of the described herein can, in turn, be utilized to generate antibodies that “mimic” IFN polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1993); and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies described herein which bind to IFNAR2 and competitively inhibit the binding of IFN to its receptor (as determined by assays well known in the art such as, for example, that disclosed, infra) can be used to generate anti-idiotypes that “mimic” a IFN ligand/receptor-binding domain and, as a consequence, bind to and neutralize IFN. Such neutralizing antiidiotypes (including molecules comprising, or alternatively consisting of, antibody fragments or variants, such as Fab fragments of such anti-idiotypes) can be used in therapeutic regimens to neutralize IFN. For example, such anti-idiotypic antibodies can be used to bind IFN ligands/receptors, and thereby block IFN-mediated biological activity.

[0158] Alternatively, anti-idiotypes that “mimic” an IFN binding domain may bind to IFNAR2 and induce IFN receptor mediated signaling. Such agonistic anti-idiotypes (including agonistic Fab fragments of these anti-idiotypes) can be used in therapeutic regimens to induce or enhance IFN receptor mediated signalling. For example, such anti-idiotypic antibodies can be used to bind IFN ligands/receptors, and thereby stimulate IFN- mediated biological activity (such as antiviral, antiproliferative and immunomodulatory effects in response to viral or bacterial infection).

Monoclonal Anti-IFNAR2 Antibodies

[0159] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgG 1 or an lgG2. The class of an antibody that immunospecifically binds IFNAR2 can be switched with another (for example, IgG can be switched to IgM), according to well- known procedures. Class switching can also be used to convert one IgG subclass to another, such as from lgG1 to lgG2.

[0160] The antibodies described herein may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).

[0161] The antibodies described herein may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of an IFNAR2 polypeptide, or fragment thereof, or may be specific for both an IFNAR2 polypeptide, or fragment thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681 ; 4,925,648; 5,573,920; 5,601 ,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Anti-IFNAR2 Antibody Cross-reactivity

[0162] Antibodies of the present disclosure may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present disclosure are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present disclosure are also included in the present disclosure.

[0163] Antibodies of the present disclosure may cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present disclosure are also included in the present disclosure. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present disclosure are antibodies that bind polypeptides encoded by polynucleotides that hybridize to a polynucleotide of the present disclosure under hybridization conditions (as described herein).

[0164] In an embodiment, the antibodies described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), immunospecifically bind to IFNAR2 and do not cross-react with any other antigens.

Anti-IFNAR2 Variants and Derivatives

[0165] The present disclosure also provides antibodies that comprise, or alternatively consist of, variants (including derivatives) of the VH domains, VH CDRs, VL domains, and VL CDRs described herein, which antibodies immunospecifically bind to IFNAR2. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the present disclosure, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1 , VHCDR2, VHCDR3, VL domain, VLCDR1 , VLCDR2, or VLCDR3. In an embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind IFNAR2). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind IFNAR2) can be determined using techniques described herein or by routinely modifying techniques known in the art.

[0166] The antibodies described herein include derivatives (e.g., variants) that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not affect the ability of the antibody to immunospecifically bind to IFNAR2. For example, but not by way of limitation, derivatives of the present disclosure include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

Anti-IFNAR2 Antibodies Sequences and Structure

[0167] The antibodies described herein (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically bind IFNAR2, may comprise, or alternatively consist of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH orVL domains disclosed herein under stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC/0.1 % SDS at about 50-65° C, under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68° C, or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDRs or VL CDRs disclosed herein under stringent conditions, e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDR3s disclosed herein under stringent conditions e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. Nucleic acid molecules encoding these antibodies are also provided by the present disclosure.

[0168] The antibodies described herein (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically bind to IFNAR2 may comprise, or alternatively consist of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VH domains of SEQ ID NO: 2 or SEQ ID NO: 18. In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2 comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VH CDRs within SEQ ID NO: 2 or SEQ ID NO: 18, such as one or more of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NQ:10, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26. In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2 comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VH CDR3s within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 10 or SEQ ID NO: 26. Nucleic acid molecules encoding these antibodies are also provided by the present disclosure.

[0169] The antibodies described herein (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically bind to IFNAR2 may comprise, or alternatively consist of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VL domains of SEQ ID NO: 4 or SEQ ID NO: 20. In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2 comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VL CDRs within SEQ ID NO: 4 or SEQ ID NO: 20, such as one or more of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32. In another embodiment, an antibody of the present disclosure that immunospecifically binds to IFNAR2 comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, 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 any one of the VL CDR3s within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO:16 or SEQ ID NO: 32. Nucleic acid molecules encoding these antibodies are also provided by the present disclosure.

[0170] The antibodies described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may have one or more of the same biological characteristics as one or more of the antibodies described herein. By “biological characteristics” is meant, the in vitro or in vivo activities or properties of the antibodies, such as, for example, the ability to bind to IFNAR2 (e.g., the soluble form of IFNAR2, the membrane-bound form of IFNAR2, the soluble form and membrane-bound form of IFNAR2), and/or an antigenic and/or epitope region of IFNAR2), the ability to substantially block IFN/IFNAR2 binding, or the ability to block IFN-mediated biological activity. Optionally, the antibodies described herein will bind to the same epitope as at least one of the antibodies specifically referred to herein. Such epitope binding can be routinely determined using assays known in the art.

[0171] The present disclosure also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that neutralize IFN or a fragment thereof, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, or VL CDR3) having an amino acid sequence contained within SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32, or having an amino acid sequence contained within the polypeptide encoded by SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31 , or a fragment or variant thereof. By an antibody that “neutralizes IFN or a fragment thereof’ is meant an antibody that diminishes or abolishes the ability of an IFN to bind to IFNAR2 and/or to stimulate the IFNAR2 receptor signaling cascade and/or to inhibit or reduce one or more biological activities of IFN. In an embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH domain contained within SEQ ID NO:2 or SEQ ID NO: 18, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained within SEQ ID NO: 4 or SEQ ID NO: 20, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR domain contained within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26, or a fragment or variant thereof. In an embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 10 or SEQ ID NO: 26, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR domain contained within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes IFN or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 16 or SEQ ID NO: 32, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also provided by the present disclosure.

[0172] The antibodies described herein may competitively inhibit binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide. In preferred embodiments, the present disclosure provides antibodies which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by between 1 % and 10% in a competitive inhibition assay.

[0173] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 10% and up to 20% in a competitive inhibition assay.

[0174] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 20% and up to 30% in a competitive inhibition assay.

[0175] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 30% and up to 40% in a competitive inhibition assay.

[0176] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 40% and up to 50% in a competitive inhibition assay. [0177] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 50% and up to 60% in a competitive inhibition assay.

[0178] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 60% and up to 70% in a competitive inhibition assay.

[0179] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 70% and up to 80% in a competitive inhibition assay.

[0180] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, or VLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 80% and up to 90% in a competitive inhibition assay.

[0181] The antibodies described herein may reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1 , VHCDR2, VHCDR3, VLCDR1 , VLCDR2, orVLCDR3) described herein or variant thereof to an IFNAR2 polypeptide by at least 90% and up to 100% in a competitive inhibition assay.

[0182] The present disclosure also provides for mixtures of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2, wherein the mixture has at least one, two, three, four, five or more different antibodies of the present disclosure. In particular, the present disclosure provides for mixtures of different antibodies that immunospecifically bind to the soluble form of IFNAR2, the membrane-bound form of IFNAR2, and/or both the membrane-bound form and soluble form of IFNAR2. In an embodiment, the present disclosure provides mixtures of at least 2, preferably at least 4, at least 6, at least 8, at least 10, at least 12, at least 15, at least 20, or at least 25 different antibodies that immunospecifically bind to IFNAR2, wherein at least 1 , at least 2, at least 4, at least 6, or at least 10, antibodies of the mixture is an antibody of the present disclosure. In an embodiment, each antibody of the mixture is an antibody of the present disclosure.

Panels of Antibodies

[0183] The present disclosure also provides for panels of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to IFNAR2, wherein the panel has at least one, two, three, four, five or more different antibodies of the present disclosure. In particular, the present disclosure provides for panels of different antibodies that immunospecifically bind to the soluble form of IFNAR2, the membrane-bound form of IFNAR2, and/or both the membrane-bound form and soluble form of IFNAR2. In an embodiment, the present disclosure provides for panels of antibodies that have different affinities for IFNAR2, different specificities for IFNAR2, or different dissociation rates. The present disclosure provides panels of at least 10, preferably at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000, antibodies. Panels of antibodies can be used, for example, in 96 well plates for assays such as ELISAs.

Compositions

[0184] The present disclosure further provides for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the present disclosure). In an embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained within SEQ ID NO: 2 or SEQ ID NO: 18 or a variant thereof. In another embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 6 or SEQ ID NO: 22 or a variant thereof. In another embodiment, a composition of the present disclosure comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 8 or SEQ ID NO: 24, or a variant thereof. In a preferred embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any- one or more of the VH CDR3s contained within SEQ ID NO: 2 or SEQ ID NO: 18, such as SEQ ID NO: 10 or SEQ ID NO: 26, or a variant thereof.

[0185] In an embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternative consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained within SEQ ID NO: 4 or SEQ ID NO: 20, or a variant thereof. In another embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1 s contained within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 12 or SEQ ID NO: 28, or a variant thereof. In another embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s contained within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 14 or SEQ ID NO: 30, or a variant thereof. In an embodiment, a composition of the present disclosure comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained within SEQ ID NO: 4 or SEQ ID NO: 20, such as SEQ ID NO: 16 or SEQ ID NO: 32, or a variant thereof.

[0186] As discussed in more detail below, a composition described herein may be used either alone or in combination with other compositions. The antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants described herein) may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies described herein may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.

[0187] The composition described herein may be a pharmaceutical composition. The composition, including pharmaceutical compositions, may comprise an antibody or antigen-binding fragment described herein and an adjuvant, carrier, buffers, antioxidants, wetting agents, lubricating agents, gelling agents, thickening agents, binding agents, disintegrating agents, humectants, preservatives, diluent, stabilizer, filler, excipient, or a combination thereof.

[0188] Antibodies described herein (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants described herein) may be used, for example, but not limited to, to purify and detect IFNAR2, and to target the polypeptides described herein to cells expressing membrane-bound IFNAR2, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of IFNAR2 in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

Nucleic Acids, Vectors, and Host Cells

[0189] The present disclosure also provides for an isolated nucleic acid molecule encoding an antibody described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof).

[0190] Nucleic acid molecules that encode the anti-IFNAR2 antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid may be isolated by purification away from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis and others well known in the art. See Ausubel, et al. (2011 ) Current Protocols in Molecular Biology John Wiley & Sons, Inc. A nucleic acid described herein may be, for example, DNA or RNA and may or may not contain intronic sequences. The nucleic acid may be a cDNA molecule. Nucleic acids described herein may be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma may be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody may be recovered from the library. Specifically, degenerate codon substitutions may be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues. Batzer, et al. (1991) Nucleic Acid Res. 19: 5081 ; Ohtsuka, et al. (1985) J. Biol. Chem. 260: 2605-08; Rossolini, et al. (1994) Mol. Cell. Probes 8: 91-98.

Methods for Producing Antibodies

[0191] The antibodies described herein (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants described herein) can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[0192] Single chain Fvs (scFvs) that immunospecifically bind IFNR2 may be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANT AB 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 an antigen of interest (e.g., IFNAR2 or a fragment or variant thereof) 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 described herein include, but are not limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401 ; W097/13844; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821 ,047; 5,571 ,698;

5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0193] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human or humanized 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 PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

[0194] To generate whole antibodies, 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. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin 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. [0195] Once an antibody molecule described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) has been chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, 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. Further, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

[0196] The present disclosure also provides methods for recombinantly producing the anti-IFNAR2 antibodies described herein. Methods of producing the antibodies described herein are well known to those of ordinary skill in the art. The anti-IFNAR2 antibodies described herein may also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing an operon and a DNA sequence encoding the anti-IFNAR2 antibodies described herein. Furthermore, the present disclosure relates to vectors, especially plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-mentioned nucleic acid molecules described herein. The nucleic acid molecules contained in the vectors may be linked to regulatory elements that ensure the transcription in prokaryotic and eukaryotic cells.

[0197] Vectors contain elements that facilitate manipulation for the expression of a foreign protein within the target host cell. Conveniently, manipulation of sequences and production of DNA for transformation is first performed in a bacterial host (e.g., E. coli) and usually vectors will include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media. Exemplary vectors and methods for transformation of yeast are described in the art. See, e.g., Burke, et al. (2000) Methods in Yeast Genetics Cold Spring Harbor Laboratory Press.

[0198] The polynucleotide coding for the anti-IFNAR2 antibodies may be operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in yeast cells. These vector components may include, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide may also be included (e.g., a signal sequence).

[0199] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" refers broadly to contiguous linked DNA sequences, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.

[0200] Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (e.g., that increase levels of transcription in response to absence of a repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to some change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature.)

[0201] The expression vectors are transfected into a host cell by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said anti-IFNAR2 antibodies.

[0202] The host cells used to express the anti-IFNAR2 antibodies may be either a bacterial cell such as E.coli, yeast (e.g., S. cerevisiae), or a eukaryotic cell (e.g., a mammalian cell line). A mammalian cell of a well-defined type for this purpose, such as a myeloma cell, 3T3, HeLa, C6A2780, Vero, MOCK II, a Chinese hamster ovary (CHO), Sf9, Sf21 , COS, NSO, or HEK293 cell line may be used.

[0203] The general methods by which the vectors may be constructed, transfection methods required to produce the host cell and culturing methods required to produce the antibodies, and fragments thereof, from said host cells all include conventional techniques. Although preferably the cell line used to produce the anti-IFNAR2 antibodies is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. coli-de ved bacterial strain, or a yeast cell line, may be used.

[0204] Similarly, once produced the anti-IFNAR2 antibodies may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, and affinity column chromatography.

Diagnostic Uses ofAnti-IFNAR2 Antibodies

[0205] Labeled antibodies described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to IFNAR2 can be used for diagnostic purposes to detect, diagnose, prognose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of IFN or IFNAR2. The present disclosure provides for the detection of aberrant expression of IFNAR2 comprising: (a) assaying the expression of IFNAR2 in a biological sample from a subject using one or more antibodies described herein that immunospecifically binds to IFNAR2; and (b) comparing the level of IFNAR2 with a standard level of IFNAR2, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of IFNAR2 compared to the standard level of IFNAR2 is indicative of aberrant expression.

[0206] By “biological sample” is intended any fluids and/or cells obtained from a subject, body fluid, body tissue, body cell, cell line, tissue culture, or other source that may contain IFNAR2 protein or mRNA. Body fluids include, but are not limited to, sera, plasma, urine, synovial fluid, spinal fluid, saliva, and mucous. Tissues samples may be taken from virtually any tissue in the body. Tissue samples may also be obtained from autopsy material. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source. [0207] The anti-IFNAR2 antibodies described herein are unique research reagents which provide anti-type I interferon activity templates for use in chemical library screening, wherein the practitioner can use a signal transduction assay as an initial, high volume screen for agents that exhibit an anti-type I interferon activity pattern that is similar to the anti-type I interferon activity pattern of an anti-IFNAR2 antibody described herein. In this way, candidate agents likely to exhibit a desired type I interferon activity inhibition profile can be obtained with ease, avoiding prohibitively expensive and logistically impossible numbers of type I interferon induced viral inhibition assays or cell proliferation inhibition assays on large chemical libraries.

[0208] In an embodiment, the anti-IFNAR2 antibodies described herein are used to screen chemical libraries in a Kinase Receptor Activation (KIRA) Assay as described in WO 95/14930. The KIRA assay is suitable for use herein because ligand binding to the type I interferon receptor complex in situ in on the surface of host cells expressing the receptor induces a rapid increase in the phosphorylation of tyrosine residues in the intracellular domains of both IFNAR1 and IFNAR2 components of the receptor as taught in Platanias and Colamonici, J. Biol. Chem., 269: 17761-17764 (1994). The level of tyrosine phosphorylation can be used as a measure of signal transduction. The effect of an anti-IFNAR2 antibody described herein on the levels of tyrosine phosphorylation induced by various type I interferons in the KIRA assay can be used as a benchmark activity pattern for comparison to the activity patterns generated by the library compounds in the assay.

[0209] The KIRA assay suitable for use herein employs a host cell that expresses the type I interferon receptor (both IFNAR1 and IFNAR2 components of the receptor) and the particular series of type I interferons which define the inhibitor profile of interest. Cells that naturally express the human type I interferon receptor, such as the human Daudi cells and U-266 human myeloma cells described in Colamonici and Domanski, J. Biol. Chem. 268: 10895-10899 (1993), can be used. In addition, cells which are transfected with the IFNAR1 and IFNAR2 components and contain intracellular signaling proteins necessary for type I interferon signal transduction, such as mouse L-929 cells as described in Domanski et al., J. Biol. Chem., 270: 21606-21611 (1995), can be used. In the KIRA assay, the candidate antagonist is incubated with each type I interferon ligand to be tested, and each incubation mixture is contacted with the type I interferon receptor-expressing host cells. The treated cells are lysed, and IFNAR2 protein in the cell lysate is immobilized by capture with solid phase anti-IFNAR2 antibody. Signal transduction is assayed by measuring the amount of tyrosine phosphorylation that exists in the intracellular domain (ICD) of captured IFNAR2 and the amount of tyrosine phosphorylation that exists in the intracellular domain of any co-captured IFNAR1 . Alternatively, cell lysis and immunoprecipitation can be performed under denaturing conditions in order to avoid co-capture of IFNAR1 and permit measurement of IFNAR2 tyrosine phosphorylation alone, e.g. as described in Platanias et al., J. Biol. Chem., 271 : 23630-23633 (1996). The level of tyrosine phosphorylation can be accurately measured with labeled anti-phosphotyrosine antibody which identifies phosphorylated tyrosine residues.

[0210] In another embodiment, a host cell co-expressing IFNAR1 and a chimeric construct containing IFNAR2 fused at its carboxy terminus to an affinity-handle polypeptide is used in the KIRA assay. The chimeric IFNAR2 construct permits capture of the construct from cell lysate by use of a solid phase capture agent (in place of an anti-IFNAR2 antibody) specific for the affinity handle polypeptide. In a preferred embodiment, the affinity-handle polypeptide is Herpes simplex virus glycoprotein D (gD) and the capture agent is an anti-gD monoclonal antibody as described in Examples 2 and 3 of WO 95/14930.

[0211] In this system, the anti-IFNAR2 antibody described herein that possesses the type I interferon inhibition activity profile of interest is used as a standard for analysis of the tyrosine phosphorylation patterns generated by the members of the chemical library that is screened. The IFNAR2 ICD tyrosine phosphorylation pattern generated by the anti-IFNAR2 antibody standard is compared to the tyrosine phosphorylation patterns produced in the library screen, and patterns found to match that of the anti-IFNAR2 antibody standard identify candidate agents that are likely to have a type I interferon activity inhibition profile similar to that of the anti-IFNAR2 antibody standard. Accordingly, the anti-IFNAR2 antibody described herein provides a useful means to quickly and efficiently screen large chemical libraries for compounds likely to exhibit the particular type I interferon activity inhibition profile of the antibody.

[0212] In addition, the anti-IFNAR2 antibodies described herein are useful in diagnostic assays for IFNAR2 expression in specific cells or tissues wherein the antibodies are labeled as described below and/or are immobilized on an insoluble matrix. Anti-IFNAR2 antibodies also are useful for the affinity purification of IFNAR2 from recombinant cell culture or natural sources.

[0213] Anti-IFNAR2 antibodies can be used for the detection of IFNAR2 in any one of a number of well-known diagnostic assay methods. For example, a biological sample may be assayed for IFNAR2 by obtaining the sample from a desired source, admixing the sample with anti-IFNAR2 antibody to allow the antibody to form antibody/IFNAR2 complex with any IFNAR2 present in the mixture, and detecting any antibody/IFNAR2 complex present in the mixture. The biological sample may be prepared for assay by methods known in the art that are suitable for the particular sample. The methods of admixing the sample with antibodies and the methods of detecting antibody/IFNAR2 complex are chosen according to the type of assay used. Such assays include competitive and sandwich assays, and steric inhibition assays. Competitive and sandwich methods employ a phase-separation step as an integral part of the method while steric inhibition assays are conducted in a single reaction mixture.

[0214] Analytical methods for IFNAR2 all use one or more of the following reagents: labeled IFNAR2 analogue, immobilized IFNAR2 analogue, labeled anti-IFNAR2 antibody, immobilized anti-IFNAR2 antibody and steric conjugates. The labeled reagents also are known as “tracers.”

[0215] The label used is any detectable functionality that does not interfere with the binding of IFNAR2 and anti-IFNAR2 antibody. Numerous labels are known for use in immunoassay, examples including moieties that may be detected directly, such as fluorochrome, chemiluminescent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes ³²P, ¹⁴C, ¹²⁵l, ³H, and ¹³¹l, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, p-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like. [0216] Conventional methods are available to bind these labels covalently to proteins or polypeptides. For instance, coupling agents such as dialdehydes, carboduimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., Nature, 144: 945 (1962); David eta al., Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods, 40: 219-230 (1981 ); and Nygren, J.

Histochem. and Cytochem., 30: 407-412 (1982). Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase.

[0217] The conjugation of such label, including the enzymes, to the antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, for example, O’Sullivan et al., “Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.

[0218] Immobilization of reagents is required for certain assay methods. Immobilization entails separating the anti-IFNAR2 antibody from any IFNAR2 that remains free in solution. This conventionally is accomplished by either insolubilizing the anti-IFNAR2 antibody or IFNAR2 analogue before the assay procedure, as by adsorption to a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.

3,720,760), by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the anti-IFNAR2 antibody or IFNAR2 analogue afterward, e.g., by immunoprecipitation.

[0219] Other assay methods, known as competitive or sandwich assays, are well established and widely used in the commercial diagnostics industry.

[0220] Competitive assays rely on the ability of a tracer IFNAR2 analogue to compete with the test sample IFNAR2 for a limited number of anti-IFNAR2 antibody antigen-binding sites. The anti-IFNAR2 antibody generally is insolubilized before or after the competition and then the tracer and IFNAR2 bound to the anti-IFNAR2 antibody are separated from the unbound tracer and IFNAR2. This separation is accomplished by decanting (where the binding partner was preinsolubilized) or by centrifuging (where the binding partner was precipitated after the competitive reaction). The amount of test sample IFNAR2 is inversely proportional to the amount of bound tracer as measured by the amount of marker substance. Dose-response curves with known amounts of IFNAR2 are prepared and compared with the test results to quantitatively determine the amount of IFNAR2 present in the test sample. These assays are called ELISA systems when enzymes are used as the detectable markers.

[0221] Another species of competitive assay, called a “homogeneous” assay, does not require a phase separation. Here, a conjugate of an enzyme with the IFNAR2 is prepared and used such that when anti-IFNAR2 antibody binds to the IFNAR2 the presence of the anti-IFNAR2 antibody modifies the enzyme activity. In this case, the IFNAR2 or its immunologically active fragments are conjugated with a bifunctional organic bridge to an enzyme such as peroxidase. Conjugates are selected for use with anti-IFNAR2 antibody so that binding of the anti-IFNAR2 antibody inhibits or potentiates the enzyme activity of the label. This method per se is widely practiced under the name of EMIT.

[0222] Steric conjugates are used in steric hindrance methods for homogeneous assay. These conjugates are synthesized by covalently linking a low-molecular-weight hapten to a small IFNAR2 fragment so that antibody to hapten is substantially unable to bind the conjugate at the same time as anti-IFNAR2 antibody. Under this assay procedure the IFNAR2 present in the test sample will bind anti-IFNAR2 antibody, thereby allowing anti-hapten to bind the conjugate, resulting in a change in the character of the conjugate hapten, e.g., a change in fluorescence when the hapten is a fluorophore.

[0223] Sandwich assays particularly are useful for the determination of IFNAR2 or anti-IFNAR2 antibodies. In sequential sandwich assays an immobilized anti-IFNAR2 antibody is used to adsorb test sample IFNAR2, the test sample is removed as by washing, the bound IFNAR2 is used to adsorb a second, labeled anti-IFNAR2 antibody and bound material is then separated from residual tracer. The amount of bound tracer is directly proportional to test sample IFNAR2. In “simultaneous” sandwich assays, the test sample is not separated before adding the labeled anti-IFNAR2. A sequential sandwich assay using an anti-IFNAR2 monoclonal antibody as one antibody and a polyclonal anti-IFNAR2 antibody as the other is useful in testing samples for IFNAR2. [0224] The foregoing are merely exemplary diagnostic assays for IFNAR2. Other methods now or hereafter developed that use anti-IFNAR2 antibody for the determination of IFNAR2 are included within the scope hereof, including the bioassays described above.

Therapeutic Methods

[0225] Antibodies described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to IFNAR2 can be used for detection of soluble IFNAR2 in serum or other body fluids of patients with autoimmune diseases producing IFN naturally, such as, e.g., SLE (systemic lupus erythematosus), or in diseases where type I IFN is used for treatment, such as, e.g., MS (multiple sclerosis) or some viral infections, such as, e.g., HCV (hepatitis C).

[0226] Antibodies described herein (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to IFNAR2 can be used for diagnostic purposes to detect, diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies, and/or diseases or conditions associated therewith.

[0227] Autoimmune disorders, diseases, or conditions that may be detected, diagnosed, prognosed, or monitored using the antibodies described herein may include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, glutensensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter’s Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, diabetes mellitus (e.g. Type I diabetes mellitus or insulin dependent diabetes mellitus), juvenile onset diabetes, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (e.g., Hashimoto’s thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture’s syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves’ Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia (Addison’s disease), idiopathic Addison’s disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren’s syndrome, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders and other disorders such as inflammatory skin diseases including psoriasis and sclerosis, responses associated with inflammatory bowel disease (such as Crohn’s disease and ulcerative colitis), respiratory distress syndrome (including adult respiratory distress syndrome, ARDS), meningitis, encephalitis, colitis, allergic conditions such as eczema and other conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, leukocyte adhesion deficiency, Reynaud’s syndrome, and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, granulomatosis and diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorder, multiple organ injury syndrome, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, Lambert-Eaton myasthenic syndrome, Beheet disease, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or autoimmune thrombocytopenia etc.

[0228] The anti-IFNAR2 antibodies described herein may be used in methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiencies). In other embodiments, the present disclosure encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).

[0229] Immunodeficiencies that may be detected, diagnosed, prognosed, or monitored using the antibodies described herein include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton’s disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott- Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

[0230] The antibodies described herein which specifically bind to IFNAR2, can be used for diagnostic purposes to detect, diagnose, prognose, or monitor viral infection, such as HIV infection or conditions associated therewith (e.g. AIDS).

Therapeutic Compositions and Administration of Anti-IFNAR2 Antibodies

[0231] Therapeutic formulations of the anti-IFNAR2 antibodies described herein are prepared for storage by mixing antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, Pa.: 1995)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

[0232] The anti-IFNAR2 antibody to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The anti-IFNAR2 antibody ordinarily will be stored in lyophilized form or in solution.

[0233] Therapeutic anti-IFNAR2 antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0234] The route of anti-IFNAR2 antibody administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intraocular, intraarterial, intracerebrospinal, or intralesional routes, or by sustained release systems as noted below. Preferably, the antibody is given systemically.

[0235] Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481 ), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release anti-IFNAR2 antibody compositions also include liposomally entrapped antibody. Liposomes containing antibody are prepared by methods known per se: DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 ; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal antibody therapy.

[0236] Anti-IFNAR2 antibody can also be administered by inhalation. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, anti-IFNAR2 antibody can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0237] An “effective amount” of anti-IFNAR2 antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, the type of anti-IFNAR2 antibody employed, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the anti-IFNAR2 antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

[0238] Anti-IFNAR2 antibodies described herein can be used to neutralize naturally- produced type I IFN in inflammatory diseases such as, for example, rheumatoid arthritis (RA); in autoimmune diseases such as, for example, systemic lupus erythematosus (SLE); and in chronic infectious diseases (such as, e.g., HCV, HIV, or HCV/HIV coinfection and COVID-19), where production of type I IFN can have negative impact on course of disease.

[0239] The patients to be treated with the anti-IFNAR2 antibody described herein include preclinical patients or those with recent onset of immune-mediated disorders, and particularly autoimmune disorders. Patients are candidates for therapy in accord with this invention until such point as no healthy tissue remains to be protected from immune-mediated destruction. For example, a patient suffering from insulin-dependent diabetes mellitus (IDDM) can benefit from therapy with an anti-IFNAR2 antibody described herein until the patient’s pancreatic islet cells are no longer viable. It is desirable to administer an anti-IFNAR2 antibody as early as possible in the development of the immune-mediated or autoimmune disorder, and to continue treatment for as long as is necessary for the protection of healthy tissue from destruction by the patient’s immune system. For example, the IDDM patient is treated until insulin monitoring demonstrates adequate islet response and other indicia of islet necrosis diminish (e.g. reduction in anti-islet antibody titers), after which the patient can be withdrawn from anti- IFNAR2 antibody treatment for a trial period during which insulin response and the level of anti-islet antibodies are monitored for relapse. [0240] In the treatment, prevention, and/or amelioration of an immune-mediated or autoimmune disorder by an anti-IFNAR2 antibody, the antibody composition comprising the anti-IFNAR2 antibodies described herein may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “therapeutically effective amount” of antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder, including treating chronic autoimmune conditions and immunosuppression maintenance in transplant recipients. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.

[0241] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may administered to an animal to treat, prevent or ameliorate immune disorders. Immune disorders include, but are not limited to, autoimmune disorders (e.g., arthritis, graft rejection, Hashimoto’s thyroiditis, insulindependent diabetes, lupus, idiopathic thrombocytopenic purpura, systemic lupus erythrematosus and multiple sclerosis), elective IgA deficiency, ataxia-telangiectasia, common variable immunodeficiency (CVID), X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, idiopathic hypereosinophilic syndrome, monocytic leukemoid reaction, monocytic leukocytosis, monocytic leukopenia, monocytopenia, monocytosis, and graft or transplant rejection.

[0242] The anti-IFNAR2 antibodies and antibody compositions described herein, may be used to treat, prevent, ameliorate, diagnose or prognose various immune system- related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of antibody and antibody compositions described herein that can inhibit an immune response may be an effective therapy in treating and/or preventing autoimmune disorders. Thus, in preferred embodiments, antibodies and antibody compositions described herein are used to treat, prevent, ameliorate, diagnose and/or prognose an autoimmune disorder, or condition(s) associated with such disorder.

[0243] Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter’s Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disease.

[0244] Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may include, but are not limited to, autoimmune thyroiditis, hypothyroidism (e.g., Hashimoto’s thyroiditis) (often characterized, e.g., by cell-mediated and humeral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), discoid lupus, Goodpasture’s syndrome (often characterized, e.g., by antibasement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), Receptor autoimmunities such as, for example, (a) Graves’ Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody- sensitized platelets. [0245] Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), scleroderma with anticollagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison’s disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren’s syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies), chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies, and many other inflammatory, granulomatous, degenerative, and atrophic disorders. [0246] Therapeutic and pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to treat, prevent, ameliorate, diagnose or prognose, HIV infection and/or medical conditions associated therewith (e.g. AIDS).

[0247] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be administered to an animal to treat, prevent or ameliorate an IgE-mediated allergic reaction or histamine-mediated allergic reaction. Examples of allergic reactions include, but are not limited to, asthma, rhinitis, eczema, chronic urticaria, and atopic dermatitis. In another embodiment, therapeutic or pharmaceutical compositions described herein are administered to an animal to treat, prevent, or ameliorate anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. In another embodiment, therapeutic or pharmaceutical compositions described herein are administered to an animal to treat, prevent or ameliorate or modulate inflammation or an inflammatory disorder. Examples of chronic and acute inflammatory disorders that may be treated prevented or ameliorated with the therapeutic and pharmaceutical compositions described herein include, but are not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, Crohn’s disease, inflammatory bowel disease, chronic and acute inflammatory pulmonary diseases, bacterial infection, psoriasis, septicemia, cerebral malaria, arthritis, gastroenteritis, and glomerular nephritis.

[0248] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be administered to an animal to treat, prevent or ameliorate ischemia and arteriosclerosis. Examples of such disorders include, but are not limited to, reperfusion damage (e.g., in the heart and/or brain) and cardiac hypertrophy.

[0249] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may also be administered to modulate blood clotting and to treat or prevent blood clotting disorders, such as, for example, antibody-mediated thrombosis (e.g., antiphospholipid antibody syndrome (APS)). For example, therapeutic or pharmaceutical compositions described herein may inhibit the proliferation and differentiation of cells involved in producing anticardiolipin antibodies. These compositions described herein can be used to treat, prevent, ameliorate, diagnose, and/or prognose thrombotic related events including, but not limited to, stroke (and recurrent stroke), heart attack, deep vein thrombosis, pulmonary embolism, myocardial infarction, coronary artery disease (e.g., antibody-mediated coronary artery disease), thrombosis, graft reocclusion following cardiovascular surgery (e.g., coronary arterial bypass grafts, recurrent fetal loss, and recurrent cardiovascular thromboembolic events.

[0250] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may also be administered to treat, prevent, or ameliorate organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of antibodies described herein, that inhibit an immune response, may be an effective therapy in preventing organ rejection or GVHD.

[0251] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to treat or prevent a disorder characterized by deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, therapeutic or pharmaceutical compositions described herein may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), COVID-19, and/or pneumocystis carnii.

[0252] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immunodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic 13- cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura. de [0253] Additionally, therapeutic or pharmaceutical compositions comprising the anti- IFNAR2 antibodies described herein may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune- mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

[0254] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to enhance antiviral immune responses. Antiviral immune responses that may be enhanced using the compositions described herein, include, but are not limited to, virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions described herein are used to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBY, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions described herein are used to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/ AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever. In another specific embodiment, the compositions described herein are used to enhance an immune response to the HIV gp120 antigen.

[0255] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions described herein include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions described herein are used to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B. In another specific embodiment, the compositions described herein are used to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).

[0256] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be used to enhance anti-parasitic immune responses. Antiparasitic immune responses that may be enhanced using the compositions described herein include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions described herein are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions described herein are used as an adjuvant to enhance an immune response to Plasmodium (malaria).

[0257] Antibody polypeptides or polynucleotides comprising the anti-IFNAR2 antibodies described herein may be used to treat, prevent, and/or diagnose diseases and disorders of the pulmonary system (e.g., bronchi such as, for example, sinopulmonary and bronchial infections and conditions associated with such diseases and disorders and other respiratory diseases and disorders. In specific embodiments, such diseases and disorders include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical bronchiectasis, and saccular bronchiectasis ), bronchiolar adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans, and proliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani’s bronchitis, chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, obliterative bronchitis, plastic bronchitis, pseudomembranous bronchitis, putrid bronchitis, and verminous bronchitis), bronchocentric granulomatosis, bronchoedema, bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g., bronchopulmonary aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm, bronchostaxis, bronchostenosis, Biot’s respiration, bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration, respiratory acidosis, respiratory alkalosis, respiratory distress syndrome of the newborn, respiratory insufficiency, respiratory scleroma, respiratory syncytial virus, and the like.

[0258] Antibody polypeptides or polynucleotides described herein may be used to treat, prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).

[0259] Antibody polypeptides or polynucleotides described herein may be used to treat, prevent, and/or diagnose fibroses and conditions associated with fibroses, including, but not limited to, cystic fibrosis (including such fibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestem fibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers’ clay pipestem fibrosis.

[0260] Therapeutic or pharmaceutical compositions comprising the anti-IFNAR2 antibodies described herein may be administered to an animal to treat, prevent or ameliorate infectious diseases. Infectious diseases include diseases associated with yeast, fungal, viral and bacterial infections. Viruses causing viral infections which can be treated or prevented in accordance with this invention include, but are not limited to, retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arenaviruses (e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus virus, human respiratory syncytial virus, mumps, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), coronaviruses (e.g., SARS-CoV-1 , MERS-CoV, SARS-CoV-2 (COVID-19), HCoV- OC43, HCoV-HKLH , HCoV-NL63, HCoV-229E), filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses (e.g., papillomaviruses), picomaviruses (e.g., rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses, reoviruses (e.g., rotaviruses), togaviruses (e.g., rubella virus), rhabdoviruses (e.g., rabies virus). Microbial pathogens causing bacterial infections include, but are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella jlexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carnii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., and Helicobacter pylori.

[0261] As a general proposition, the initial pharmaceutically effective amount of the antibody administered parenterally will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day. The desired dosage can be delivered by a single bolus administration, by multiple bolus administrations, or by continuous infusion administration of antibody, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve.

[0262] The antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the immune-mediated or autoimmune disorder in question. For example, in rheumatoid arthritis, the antibody may be given in conjunction with a glucocorticosteroid. The effective amount of such other agents depends on the amount of anti-IFNAR2 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.

Kits

[0263] A pharmaceutical pack or kit may comprise one or more containers filled with one or more of the ingredients of the pharmaceutical compositions comprising the anti- IFNAR2 antibodies described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0264] Kits are provided that can be used in the methods described herein. A kit may comprise an antibody described herein, preferably a purified antibody, in one or more containers. In an alternative embodiment, a kit may comprise an antibody fragment that immunospecifically binds to IFNAR2. Kits may contain a substantially isolated IFNAR2 polypeptide as a control.

[0265] Kits may further comprise a control antibody that does not react with IFNAR2. In another specific embodiment, the kits described herein contain a means for detecting the binding of an antibody to IFNAR2 (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized IFNAR2. The IFNAR2 provided in the kit may also be attached to a solid support. In a more specific embodiment, the detecting means of the above-described kit includes a solid support to which IFNAR2 is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to IFNAR2 can be detected by binding of the said reporter-labeled antibody.

[0266] A diagnostic kit for use in screening a biological sample containing antigens of the polypeptide described herein. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with IFNAR2, and means for detecting the binding of IFNAR2 to the antibody. In an embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

[0267] In one diagnostic configuration, a biological sample is reacted with a solid phase reagent having a surface-bound IFNAR2 obtained by the methods described herein. After IFNAR2 binds to a specific antibody, the unbound serum components are removed by washing, reporter-labeled anti-human antibody is added, unbound antihuman antibody is removed by washing, and a reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-IFNAR2 antibody on the solid support. Typically, the reporter is an enzyme, which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate.

[0268] The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

[0269] Thus, the present disclosure provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant IFNAR2, and a reporter-labeled anti-human antibody for detecting surfacebound anti-IFNAR2 antibody.

[0270] Further details described herein can be found in the following examples, which further defines the scope of the present disclosure. All references cited throughout the specification, and the references cited therein, are hereby expressly incorporated by reference in their entirety.

EXAMPLES

[0271] EXAMPLE 1

[0272] Development of monoclonal antibodies to the extracellular domain of type-l IFN receptor subunit II that neutralize IFN-alpha.

[0273] The immunogen (purified recombinant extracellular domain of type-l IFN receptor subunit II) was expressed and purified. The purified immunogen was used to immunize BALB/c mice. 200 individual hybridoma clones were screened and more than 20 clones were identified as positive in ELISA against the target protein (type-l IFN receptor subunit II) and expanded for future use. Antiproliferative and antiviral assays identified several clones with suppressive biological activity against IFN-alpha, among which 2 clones (A10 and B7) were the most effective.

[0274] In particular, both A10 and B7 antibodies neutralized IFN-a induced antiviral activity in an antiviral assay. Human alveolar basal epithelial cells (A549) were seeded at 3x10⁴ cells per well in 96-well plates and incubated with IFN-a2 (0.036 ng/mL) in the presence or absence of supernatants containing A10 or B7 antibodies 24 hours before infection with murine encephalomyelitis virus (EMCV). Upon observation of cytopathic effect, cells were fixed and stained with crystal violet. OD was measured using a suitable plate reader. As shown in FIG. 1A, exposure of A549 cells to EMCV significantly decreased antiviral protection which was restored by treatment with IFN-a2. Addition of either A10 or B7 antibodies completely blocked the effect of IFN-a2.

[0275] Furthermore, both A10 and B7 antibodies neutralized IFN-a induced antiproliferative activity in an antiproliferation assay. Daudi (Burkitt’s lymphoma) cells were seeded at 3x10⁴ cells per well in 96-well plates and incubated with IFN-a2 (0.036 ng/mL) in the presence or absence of supernatants containing A10 or B7 antibodies for 48 hours. To measure inhibition of cell proliferation, a standard MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed as previously described in Schmeisser et al. (J. Virol. Oct 2010, p. 10671-10680). OD was measured using a suitable plate reader. As shown in FIG. 1 B, treatment of Daudi cells with IFN-a2 significantly reduced inhibition of proliferation. Addition of either A10 or B7 antibodies neutralized this effect.

[0276] Recent antibody gene sequencing performed by Genscript documented that A10 (VH Domain: SEQ ID NO: 2 (aa) and SEQ ID NO: 1 (nt); VL Domain: SEQ ID NO: 4 (aa) and SEQ ID NO: 3 (nt)) and B7 (VH Domain: SEQ ID NO: 18 (aa) and SEQ ID NO: 17 (nt); VL Domain: SEQ ID NO: 20 (aa) and SEQ ID NO: 19 (nt)) are very distinct antibodies differing in both the heavy and light chains.

[0277] EXAMPLE 2

[0278] Neutralization of antiproliferative activity of IFN-a

[0279] Daudi (Burkitt’s lymphoma) cells were seeded at 3x10⁴ cells per well in 96- well plates and incubated with IFN-a2 (5 ng/mL and 10-fold dilutions thereof) in the presence or absence of antibodies A10 or B7 for 48 hours. T o measure inhibition of cell proliferation, a standard MTT assay was performed as previously described in Schmeisser et al., JVI, Oct 2010, p. 10671-10680. Optical density (OD) was measured using a plate reader.

[0280] Data are presented in FIG. 2, further confirming that both A10 and B7 antibodies neutralized IFN-a induced antiproliferative activity.

[0281] EXAMPLE 3

[0282] Comparison of the effect of the neutralizing activity of anti-INFAR2 antibodies on signalling pathways activated by IFN-a

[0283] Human PBMC were seeded at 1 .5 x 10⁶/mL in 6-well plates (5 mL/well) and incubated with monoclonal antibodies (A10 or B7) at a concentration of 1 pg/mL for 1 hour, and then treated with IFN-a (5 ng/mL) for 1 hour. Cell pellets were harvested and activation of signaling pathways was probed in Western blot using specific commercially antibodies to proteins in signaling pathways.

[0284] Data are presented in FIG. 3, demonstrating that the anti-IFNAR2 antibodies significantly block the ability of IFN-a to activate signalling pathways.

[0285] EXAMPLE 4

[0286] Antibody quality evaluation by SDS-PAGE

[0287] The variable domains of B7 and A10 antibodies were sequenced and subcloned by GenScript as described above.

[0288] Recently, new batches of mAbs B7 and A10 (further referred to here as B7 GP and A10 GP) were produced as follows: Equal amounts of plasmids expressing the heavy chain and the light chain were transfected into HEK293FS cells using Freestyle™ MAX Reagent (Thermo Fisher Scientific; 16447500) as per the manufacturer’s protocol. After a 4-day incubation at 37°C, the supernatants were collected by centrifugation and filtered through a 0.22-pm membrane. The supernatants were then loaded onto protein A column and washed using PBS. The bound proteins were eluted with low-pH IgG elution buffer (Thermo Fisher Scientific; 21009) and neutralized with 100mM Tris-HCI pH 9.0. The eluted antibodies were concentrated and buffer-exchanged into PBS. The quality of the antibodies was evaluated in SDS-PAGE using 10-20% Tris-Glycine gel under reducing and non-reducing conditions (FIG. 4). Both the subcloned A10-GP and B7-GP monoclonal antibodies and the original A10-1 (batch #1 ), A10-2 (batch #2), and B7 showed high purity and excellent quality in SDS- PAGE.

[0289] The binding activity of the antibodies were tested in ELISA and neutralizing activities were evaluated in neutralization assays and compared with commercial antibodies against IFNAR2.

[0290] EXAMPLE 5

[0291] Comparison of binding of anti-IFNAR2 antibodies using ELISA

[0292] The binding abilities of monoclonal antibodies (A10 GP, B7 GP) and two commercial antibodies (PBL cat # 21385-1 , R&D cat # MAB 4016) were compared in ELISA. ELISA plates were coated with soluble IFNAR2-EC overnight at 4°C (1 pg/mL, 50 pl/well in PBS) and then specific anti-soluble IFNAR2 antibodies were added.

[0293] After incubation for 1 hour at 37°C, the plates were washed, and secondary peroxidase-labeled goat anti-mouse IgG (H+L) antibodies were added (3 pg/mL and 10 fold dilutions thereof), followed by incubation for 1 hour at 37°C. Positive reactions were visualized and absorbance at 450 nm was measured in an ELISA microplate reader.

[0294] Data is presented in FIG. 5 demonstrating that the A10 GP and B7 GP antibodies have superior binding activity compared to commercial anti-IFNAR2 antibodies (PBL cat # 21385-1 , R&D cat # MAB 4016).

[0295] EXAMPLE 6

[0296] Neutralizing activities of anti-IFNAR2 antibodies on inflammatory effect of type I IFN

[0297] Type I IFNs have pleiotropic effects that include the induction of antiviral, antiproliferative, and immunomodulatory activities. However, Type I IFNs could also have an inflammatory effect, especially in the presence of TNF-a. The inflammatory effect of Type I IFN has been described in autoimmune diseases like SLE, chronic viral diseases like HIV and HCV, and recently also in COVID-19.

[0298] Cross-regulation of TNF-a and Type I IFN has an important clinical relevance. This cross-regulation was recently described in SARS-CoV-2 infection (Lee et al., Science Immunology, 2020). An additional benefit of inhibition of CCR5 in terminally ill COVID-19 patients receiving leronlimab has been published (Peterson et al., International Journal of Infectious Diseases, 2020).

[0299] We developed a new experimental model to investigate the pro-inflammatory activity of type I IFN, whereby IFN-a is greatly potentiated by the addition of TNF-a in human PBMC cultures, leading to the production of high levels of inflammatory cytokines and chemokines (FIG. 6).

[0300] To test if anti-IFNAR2 mAbs could inhibit induction of inflammatory chemokines induced by treatments with IFN- and TNF-a, human PBMC were seeded at 1.5 x 10⁶/mL in 24-well plates, incubated with anti-IFNAR2 antibodies A10 GP, B7 GP, or two commercial antibodies (PBL cat # 21385-1 , R&D cat # MAB 4016; 3pg/mL of each) for 1 hour, and then treated with either TNF-a or IFN- alone or a combination of the two (1 pg/mL and 10-fold dilutions thereof).

[0301] Cells were cultured for 72 hours. Supernatants were harvested and levels of cytokines were tested in ELISA using commercially available kits from R&D.

[0302] A strong neutralizing effect of anti-IFNAR2 antibodies A10 GP and B7 GP on the induction of inflammatory chemokines was observed as shown in FIGS. 7A-7C.

[0303] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0304] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

Atty Docket No. 3000094-013977 CLAIMS What is claimed is:

1 . An isolated anti-IFNAR2 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable (VL) region that specifically binds a type-l interferon receptor subunit 2 (IFNAR2) polypeptide.

2. The antibody, or antigen binding fragment thereof, of claim 1 , wherein the heavy chain variable region comprises an amino acid sequence that share at least about 80% sequence homology with the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 18.

3. The antibody, or antigen binding fragment thereof, of claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence that share at least about 80% sequence homology with the amino acid sequences of SEQ ID NO: 4 or SEQ ID NO: 20.

4. The antibody, or antigen binding fragment thereof, of any one of claims 1-3, wherein the heavy chain variable region comprises complementary determining region 1 (CDR1 ) comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 22, CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 24, and/or CDR3 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 26.

5. The antibody, or antigen binding fragment thereof, of any one of claims 1-4, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 28, CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 30, and/or CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 32.

- 87 - Atty Docket No. 3000094-013977 The antibody, or antigen binding fragment thereof, of any one of claims 1-5, wherein the antibody specifically binds to the IFNAR2 comprising the amino acid sequence of SEQ ID NO: 33. The antibody, or antigen binding fragment thereof, of any one of claims 1-6, wherein the antibody specifically binds to an epitope on IFNAR2 comprising the amino acid sequence of SEQ ID NO: 33. The antibody, or antigen binding fragment thereof, of any one of claims 1-7, wherein the heavy chain is encoded by a nucleic acid sequence with at least about 80% sequence homology to the nucleic acid sequence of SEQ ID NO: 1 . The antibody, or antigen binding fragment thereof, of any one of claims 1-8, wherein the heavy chain is encoded by a nucleic acid sequence with at least about 80% sequence homology to the nucleic acid sequence of SEQ ID NO: 17. The antibody, or antigen binding fragment thereof, of any one of claims 1 -9, wherein the light chain is encoded by a nucleic acid sequence with at least about 80% sequence homology to the nucleic acid sequence of SEQ ID NO: 3. The antibody, or antigen binding fragment thereof, of any one of claims 1 -10, wherein the light chain is encoded by a nucleic acid sequence with at least about 80% sequence homology to the nucleic acid sequence of SEQ ID NO: 19. The antibody, or antigen binding fragment thereof, of any one of claims 1-11 , wherein the sequence homology is at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology. An anti-IFNAR2 antibody that competes for binding to IFNAR2 with the antibody of any one of claims 1-12.

- 88 - Atty Docket No. 3000094-013977 The antibody, or antigen binding fragment thereof, of any one of claims 1-13, wherein the antigen-binding fragment is an Fab, Fab’, or F(ab’)2. The antibody, or antigen binding fragment thereof, of any one of claims 1-14, wherein the antibody has neutralizing activity against IFN-a. The antibody, or antigen binding fragment thereof, of any one of claims 1-15, wherein the antibody is monoclonal. The antibody, or antigen binding fragment thereof, of any one of claims 1-16, wherein the antibody is a humanized antibody. The antibody, or antigen binding fragment thereof, of any one of claims 1-16, wherein the antibody is a chimeric antibody. The antibody, or antigen binding fragment thereof, of any one of claims 1 -18, wherein the antibody is conjugated to a label, cytotoxic agent, and/or immunosuppressive agent. The antibody, or antigen binding fragment thereof, of claim 19, wherein the label is a detectable label. The antibody, or antigen binding fragment thereof, of claim 20, wherein the detectable label is a fluorescent label, luminescent label, bioluminescent label, radioactive label, chemiluminescent label, colorimetric label, fluorogenic label, enzymatic label, or a combination thereof. A composition comprising the antibody, or antigen binding fragment thereof, of any one of claims 1-21 .

- 89 - Atty Docket No. 3000094-013977 The composition of claim 22, wherein the composition is a pharmaceutical composition further comprising a pharmaceutical excipient, carrier, diluent, adjuvant, or a combination thereof. An isolated nucleotide comprising a nucleic acid sequence comprising at least about 80% sequence homology with the nucleic acid sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, or 31. The isolated nucleotide of claim 24, wherein the sequence homology is at least about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology. A vector comprising the isolated nucleotide of claim 24 or 25. A host cell comprising the vector of claim 26. A composition comprising the isolated nucleotide of claim 24 or 25. A composition comprising the vector of claim 26. A composition comprising the host cells of claim 27. The composition of any one of claims 28-30, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, diluent, adjuvant, vehicle, or a combination thereof. A method for detecting a type-l interferon receptor subunit 2 (IFNAR2) polypeptide comprising contacting a sample with the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 . The method of claim 32, wherein the antibody, or antigen binding fragment thereof, attached to a solid phase support.

- 90 - Atty Docket No. 3000094-013977 The method of claim 33, wherein said solid phase support is a bead, plate, matrix, polymer, test tube, sheet, culture dish, or test strip. A method for treating an immune disorder comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1- 21. The method of claim 35, wherein the immune disorder is lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’ s disease, and immunodeficiency syndrome, or a combination thereof. A method for treating an inflammatory disorder comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1- 21. The method of claim 37, wherein the inflammatory disorder is asthma, an allergic disorder, rheumatoid arthritis, or a combination thereof. A method for treating a viral infection comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1-21 . The method of claim 39, wherein the viral infection is HIV, coronavirus, influenza, herpes, or a combination thereof. The method of claim 39 or 40, wherein the virus is a coronavirus, preferably SARS- CoV-1 , MERS-CoV, SARS-CoV-2 (COVID-19), HCoV-OC43, HCoV-HKLH , HCoV- NL63, HCoV-229E or a combination thereof. A method for treating a proliferative disorder comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1- 21.

- 91 - Atty Docket No. 3000094-013977 The method of claim 42, wherein the proliferative disorder is leukemia, carcinoma, lymphoma, or a combination thereof. A method for treating a disorder associated with aberrant IFN or IFNAR2 receptor expression or inappropriate function of IFN or IFNAR2 receptor comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 . A method for treating a chronic inflammatory disease comprising administering an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 . The method of claim 45, wherein the chronic inflammatory disease is stroke, chronic respiratory disease, heart disorders, cancer, obesity, diabetes, or a combination thereof. The method of any one of claims 35-46, wherein the antibody, or antigen binding fragment thereof is administered as part of a pharmaceutical composition. The method of any one of claims 35-46, wherein the effective amount is between about 1 ng and 1 ,000 ng. The method of any one of claims 35-46, wherein the effective amount is between about 1 pg and 1 ,000 pg. The method of any one of claims 35-46, wherein the effective amount is between about 1 mg and 1 ,000 mg. The method of any one of claims 35-46, wherein the effective amount is between about 1 g and 1 ,000 g.

- 92 - Atty Docket No. 3000094-013977 The method of any one of claims 35-51 , wherein the antibody or antigen binding fragment thereof is administered intravenously, subcutaneously, via infusion, orally, intrathecally , intraperitoneally, parenterally, or a combination thereof. The method of any one of claims 35-52, wherein the antibody or antigen binding fragment thereof is administered at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The method of any one of claims 35-53, wherein the antibody or antigen binding fragment thereof is administered over the course of 1 , 2, 3, 4, 5, 6, or 7 days. The method of any one of claims 35-53, wherein the antibody or antigen binding fragment thereof is administered over the course of 1 , 2, 3, or 4 weeks. A kit comprising the antibody, or antigen binding fragment thereof, of any one of claims 1-21 . The kit of claim 56, wherein the antibody, or antigen binding fragment thereof, attached to a solid phase support. The kit of claim 57, wherein said solid phase support is a bead, plate, matrix, polymer, test tube, sheet, culture dish, or test strip. The kit of claim 58, wherein said solid support is an array. Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating an immune disorder. The use of claim 60, wherein the immune disorder is lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’ s disease, and immunodeficiency syndrome, or a combination thereof.

- 93 - Atty Docket No. 3000094-013977 Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating an inflammatory disorder. The use of claim 62, wherein the inflammatory disorder is asthma, an allergic disorder, rheumatoid arthritis, or a combination thereof. Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating a viral infection. The use of claim 64, wherein the viral infection is HIV, coronavirus, influenza, herpes, or a combination thereof. The use of claim 64 or 65, wherein the virus is a coronavirus, preferably SARS-CoV-1 , MERS-CoV, SARS-CoV-2 (COVID-19), HCoV-OC43, HCoV-HKU1 , HCoV-NL63, HCoV-229E or a combination thereof. Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating a proliferative disorder. The use of claim 67, wherein the proliferative disorder is leukemia, carcinoma, lymphoma, or a combination thereof. Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating a disorder associated with aberrant IFN or IFNAR2 receptor expression or inappropriate function of IFN or IFNAR2 receptor. Use of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 for treating a chronic inflammatory disease comprising. The use of claim 70, wherein the chronic inflammatory disease is stroke, chronic respiratory disease, heart disorders, cancer, obesity, diabetes, or a combination thereof.

- 94 - Atty Docket No. 3000094-013977 The use of any one of claims 60-71 , wherein the antibody or antigen binding fragment thereof is formulated for intravenous, subcutaneous, infusion, oral, intrathecal, intraperitoneal, parenteral administration, or a combination thereof. A composition for treating an immune disorder comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 . The composition of claim 73, wherein the immune disorder is lupus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, Hashimoto’s disease, and immunodeficiency syndrome, or a combination thereof. A composition for treating an inflammatory disorder comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1-21 . The composition of claim 75, wherein the inflammatory disorder is asthma, an allergic disorder, rheumatoid arthritis, or a combination thereof. A composition for treating a viral infection comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1 -21 . The composition of claim 77, wherein the viral infection is HIV, coronavirus, influenza, herpes, or a combination thereof. The composition of claim 77 or 78, wherein the virus is a coronavirus, preferably SARS-CoV-1 , MERS-CoV, SARS-CoV-2 (COVID-19), HCoV-OC43, HCoV-HKLH , HCoV-NL63, HCoV-229E or a combination thereof. A composition for treating a proliferative disorder comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1-21 . Atty Docket No. 3000094-013977 The composition of claim 80, wherein the proliferative disorder is leukemia, carcinoma, lymphoma, or a combination thereof. A composition for treating a disorder associated with aberrant IFN or IFNAR2 receptor expression or inappropriate function of IFN or IFNAR2 receptor comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1-21 . A composition for treating a chronic inflammatory disease comprising an effective amount of the antibody, or antigen binding fragment thereof, of any one of claims 1- 21. The composition of claim 83, wherein the chronic inflammatory disease is stroke, chronic respiratory disease, heart disorders, cancer, obesity, diabetes, or a combination thereof. The composition of any one of claims 73-84, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, diluent, vehicle, adjuvant, or a combination thereof. The composition of any one of claims 73-85, wherein the effective amount is between about 1 ng and 1 ,000 ng. The composition of any one of claims 73-85, wherein the effective amount is between about 1 pg and 1 ,000 pg. The composition of any one of claims 73-85, wherein the effective amount is between about 1 mg and 1 ,000 mg. The composition of any one of claims 73-85, wherein the effective amount is between about 1 g and 1 ,000 g. Atty Docket No. 3000094-013977 The composition of any one of claims 73-89, wherein the antibody or antigen binding fragment thereof is formulated for intravenous, subcutaneous, infusion, oral, intrathecal, intraperitoneal, parenteral administration, or a combination thereof.

- 97 -