WO2003055979A2 - Anticorps se liant à blys selon un mode immunospécifique - Google Patents

Anticorps se liant à blys selon un mode immunospécifique Download PDF

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Publication number
WO2003055979A2
WO2003055979A2 PCT/US2002/036496 US0236496W WO03055979A2 WO 2003055979 A2 WO2003055979 A2 WO 2003055979A2 US 0236496 W US0236496 W US 0236496W WO 03055979 A2 WO03055979 A2 WO 03055979A2
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replaced
amino acid
blys
antibody
seq
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PCT/US2002/036496
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WO2003055979A3 (fr
Inventor
Steven M. Ruben
Steven C. Barash
Gil H. Choi
Tristan J. Vaughan
David Hilbert
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Human Genome Sciences, Inc.
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Priority to CA002467521A priority Critical patent/CA2467521A1/fr
Priority to AU2002364954A priority patent/AU2002364954A1/en
Priority to EP02802570A priority patent/EP1456347A4/fr
Publication of WO2003055979A2 publication Critical patent/WO2003055979A2/fr
Publication of WO2003055979A3 publication Critical patent/WO2003055979A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to antibodies and related molecules that immunospecifically bind to BLyS.
  • the present invention also relates to methods and compositions for detecting, diagnosing, or prognosing a disease or disorder associated with aberrant BLyS or BLyS receptor expression or inappropriate function of BLyS or BLyS receptor, comprising antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to BLyS.
  • the present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS function or BLyS receptor function, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to BLyS.
  • B lymphocyte stimulator is a member of the tumor necrosis factor (“TNF”) superf amily that induces both in vivo and in vitro B cell proliferation and differentiation (Moore et al, Science 285: 260-263 (1999)).
  • BLyS is distinguishable from other B cell growth and differentiation factors such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15, CD40L, or CD27L (CD70) by its monocyte-specific gene and protein expression pattern and its specific receptor distribution and biological activity on B lymphocytes.
  • BLyS expression is not detected on natural killer (“NK”) cells, T cells or B cells, but is restricted to cells of myeloid origin.
  • BLyS expression on resting monocytes is upregulated by interferon-gamma (IFN-gamma).
  • IFN-gamma interferon-gamma
  • BLyS is expressed as a 285 amino acid type ⁇ membrane-bound polypeptide and a soluble 152 amino acid polypeptide (Moore et al., 1999 supra).
  • the membrane- bound form of BLyS has a predicted transmembrane spanning domain between amino acid residues 47 and 73.
  • the NH 2 -terminus of the soluble form of BLyS begins at Ala 134 of the membrane-bound form of BLyS.
  • Soluble recombinant BLyS has been shown to induce in vitro proliferation of murine splenic B cells and to bind to a cell-surface receptor on these cells (Moore et al., 1999 supra).
  • Soluble BLyS administration to mice has been shown to result in an increase in the proportion of CD45R duU , Ly6D ⁇ gh (also known as ThB) B cells and an increase in serum IgM and IgA levels (Moore et al, 1999 supra).
  • BLyS displays a B cell tropism in both its receptor distribution and biological activity.
  • Levels of BLyS protein have been found to be elevated in patients with autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjogren's syndrome (Zhang et al., The Journal of Immunology, (2001) 166:6-10; Cheema et al., Arthritis and Rheumatism (2001) 44:1313-1319; and Groom et al., Journal of Clinical Investigation (2002) 109:59-68).
  • SLE systemic lupus erythematosus
  • Sjogren's syndrome Zhang et al., The Journal of Immunology, (2001) 166:6-10
  • Cheema et al., Arthritis and Rheumatism (2001) 44:1313-1319 and Groom et al., Journal of Clinical Investigation (2002) 109:59-68.
  • antibodies and related molecules that immunospecifically bind to BLyS may find medical utility in, for example, the treatment of B cell disorders associated with autoimmunity.
  • antibodies and related molecules that immunospecifically bind to BLyS may find medical utility in for example, neoplasia or immunodeficiency syndromes.
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of BLyS.
  • the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of human BLyS (SEQ ID NOS:3228 and/or 3229) or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOS:3230 and/or 3231) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NO
  • the present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate function of BLyS or BLyS receptor in an animal, preferably a mammal, and most preferably a human, comprising, or alternatively consisting of, use of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to BLyS.
  • Diseases and disorders which can be detected, diagnosed, or prognosed with the antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention 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), infectious diseases ⁇ e.g., AIDS), and proliferative disorders ⁇ e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases ⁇ e.g
  • the present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate function of BLyS or BLyS 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 molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to BLyS.
  • Diseases and disorders which can be prevented, treated or ameliorated by administering an effective amount of an antibody of the invention 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), infectious diseases ⁇ e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases ⁇ e.g., AIDS
  • proliferative disorders e.g., leukemia
  • scFvs single chain antibody molecules
  • Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of BLyS according to the assays described in Examples 1 through 19.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including NH domains, NH CDRs, NL domains, or NL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.
  • the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID ⁇ OS: 1 - 2128, preferably SEQ ID ⁇ OS:834 - 872, 1570 - 1595, and 1886 - 1908, and most preferably SEQ ID NOS:l - 46, 321 - 329, 1563 - 1569, and 1881 - 1885, as referred to in Table 1 below.
  • the present invention relates to scFvs that immunospecifically bind the soluble form of BLyS, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1563 - 1880, preferably SEQ ID NOS:1570 - 1595, and most preferably SEQ ID NOS: 1563 - 1569, as referred to in Table 1, below.
  • the present invention also relates to scFvs that immunospecifically bind the membrane-bound form of BLyS, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1881 - 2128, preferably SEQ ID NOS:1886 - 1908, and most preferably SEQ ID NOS: 1881 - 1885, as referred to in Table 1 below.
  • the present invention further relates to scFvs that immunospecifically bind both the membrane-bound form and soluble form of BLyS, said scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1 - 1562, preferably SEQ ID NOS: 834 - 872, and most preferably SEQ ID NOS: 1 - 46, and 321 - 329, as referred to in Table 1 below.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including NH domains, NH CDRs, NL domains, or NL CDRs having an amino acid sequence of any one of those referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.
  • the present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the variable heavy ("VH") domains referred to in Table 1, below, or any one of the variable light (“NL”) domains referred to in Table 1.
  • VH variable heavy
  • NL variable light domains referred to in Table 1
  • antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a NH domain contained in SEQ ID ⁇ OS:l
  • antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a NL domain contained SEQ ID ⁇ OS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908, as referred to in Table 1 below.
  • 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 referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • the present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS, said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1 , below, and any one of the VL domains referred to in Table 1.
  • the antibodies of the invention comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1.
  • antibodies of the present invention comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:l - 46, 321 - 329, 834
  • antibodies of the present invention comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908, as disclosed in Table 1.
  • 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 referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • the present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS, 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") ⁇ i.e., VH CDR1, VH CDR2, or VH CDR3) referred to in Table 1 and/or any one, two, three or more of the VL CDRs ⁇ i.e., VL CDR1, VL CDR2, or VL CDR3) referred to in Table 1.
  • CDRs VH complementarity determining regions
  • antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDRls referred to in Table 1 and/or any one of the VL CDRls referred to in Table 1.
  • antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR2s referred to in Table 1 and/or any one of the VL CDR2s referred to in Table 1.
  • antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR3s referred to in Table 1 and/or any one of the VL CDR3s referred to in Table 1.
  • 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 referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • antibodies of the present invention immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, and comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDRls referred to in Table 1, any one of the VH CDR2s referred to in Table 1, and/or any one of the VH CDR3s referred to in Table 1.
  • antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VL CDRls referred to in Table 1, any one of the VL CDR2s referred to in Table 1, and/or any one of the VL CDR3s referred to in Table 1.
  • antibodies of the present invention comprise, or alternatively consist of, at least one, two, three, four, five, six, or more CDRs that correspond to the same scFv referred to in Table 1, more preferably where CDRl, CDR2, and CDR3 of the VL domain correspond to the same scFv or where CDRl, CDR2, and CDR3 of the VH domain correspond to the same scFv, and most preferably where all six CDRs correspond to the same scFv referred to in Table 1.
  • 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 referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • the present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that: immunospecifically bind to the soluble form of BLyS (e.g., a polypeptide consisting of amino acids 134 - 285 of SEQ ID NO:3228); that immunospecifically bind to the membrane-bound form of BLyS (e.g., a polypeptide consisting of amino acids 1 - 285 of SEQ ID NO:3228 or a BLyS polypeptide expressed on the surface of monocytes) and/or that immunospecifically bind to both the soluble form and membrane-bound form of BLyS.
  • BLyS e.g., a polypeptide consisting of amino acids 134 - 285 of SEQ ID NO:3228
  • the membrane-bound form of BLyS e.g., a polypeptide consisting of amino acids 1 - 285 of SEQ ID NO:3228 or a BLyS polypeptide expressed
  • antibodies of the present invention immunospecifically bind to the soluble form of BLyS and comprise, or alternatively consist of, a VH domain, VH CDRl, VH CDR2, VH CDR3, VL domain, VL CDRl, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the soluble form of BLyS.
  • antibodies of the present invention immunospecifically bind to the membrane-bound form of BLyS and comprise, or alternatively consist of, a VH domain, VH CDRl, VH CDR2, VH CDR3, VL domain, VL CDRl, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the membrane-bound form of BLyS.
  • antibodies of the present invention immunospecifically bind to the soluble form and membrane-bound form of BLyS and comprise, or alternatively consist of, a VH domain, VH CDRl, VH CDR2, VH CDR3, VL domain, VL CDRl, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically binds to the soluble form and membrane-bound form of BLyS.
  • antibodies of the present invention comprise, or alternatively consist of, a VH domain and a VL domain corresponding to the same scFv disclosed in Table 1, which antibodies immunospecifically bind to the soluble form of BLyS, the membrane-bound form of BLyS, or both the soluble form and membrane-bound form of BLyS.
  • Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
  • Molecules comprising, or altematively 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 referred to in Table 1), that immunospecifically bind the soluble form of BLyS, the membrane-bound form of BLyS, and/or both the soluble form and membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • the present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to both BLyS and APR-CL (preferably to the soluble forms of each of these molecules), said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, below, and any one of the VL domains referred to in Table 1.
  • the antibodies of the invention comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1.
  • antibodies of the present invention that immunospecifically bind to both BLyS and APRIL comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:3240-3247 as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:3240-3247, as disclosed in Table 1.
  • antibodies of the present invention that immunospecifically bind to both BLyS and APRIL comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS: SEQ ID NOS:3240-3247, as disclosed in Table 1.
  • 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 referred to in Table 1), that immunospecifically bind both BLyS and APRIL, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • the present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a heterotrimeric protein comprising at least one BLyS polypeptide (preferably amino acids 134-285 of SEQ ID NO:3228), said antibodies comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, below, and any one of the VL domains referred to in Table 1.
  • antibodies of the invention that immunospecifically bind heterotrimeric protein comprising at least one BLyS polypeptide comprise or alternatively consist of, a polypeptide having the amino acid sequence of a VH and VL domain contained in the same scFv referred to in Table 1.
  • antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one BLyS polypeptide comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:3240- 3247 as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:3240-3247, as disclosed in Table 1.
  • antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one BLyS polypeptide comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS:3240-3247, as disclosed in Table 1.
  • 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 referred to in Table 1), that immunospecifically bind a heterotrimeric protein comprising at least one BLyS polypeptide, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.
  • 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/NL pairing representing an antigen-binding site of an antibody.
  • 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.
  • one or more CDRs disclosed herein may be taken from a VH or VL domain and incorporated into a suitable framework as discussed infra.
  • the present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof (including derivatives)) comprising, or alternatively consisting of, of VH domains, VL domains and/or CDRs described herein, which antibodies, immunospecifically bind to BLyS (e.g., soluble BLyS and membrane-bound BLyS) and can be routinely assayed for immunospecific binding to BLyS using methods known in the art, such as, for example, the immunoassays disclosed infra.
  • BLyS e.g., soluble BLyS and membrane-bound BLyS
  • Antibodies and antibody fragments or variants (including derivatives) of the invention 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 CDR's.
  • the antibodies of the invention 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 invention. Nucleic acid molecules encoding these antibodies and molecules (including fragments, variants, and derivatives) are also encompassed by the invention.
  • the present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab') 2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs).
  • antibodies including molecules comprising, or alternatively consisting of, antibody fragments or variants
  • the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab') 2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs).
  • the present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab') 2 fragments, Fd fragments, disulfide- linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies, and scFvs)).
  • the present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof).
  • a composition of the invention 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.
  • a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.
  • the present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide ⁇ i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
  • a composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.
  • a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.
  • the present invention also provides for a nucleic acid molecule, generally isolated, encoding an antibody (including molecules such as scFvs, which comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention.
  • the present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof.
  • the present invention 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 invention.
  • the present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule.
  • the present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an animal, preferably a mammal, and most preferably a human, comprising using antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to BLyS.
  • Diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention 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), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases e.g., AIDS
  • proliferative disorders e.g., leukemia, carcinoma, and lymphoma
  • the present invention encompasses 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 specific embodiments, the present invention encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency).
  • hypergammaglobulinemia e.g., AIDS, autoimmune diseases, and some immunodeficiencies
  • hypogammaglobulinemia e.g., an immunodeficiency
  • the present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an animal, preferably a mammal, and most preferably a human, comprising 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 BLyS.
  • antibodies including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof
  • Diseases and disorders which can be prevented, treated or inhibited by administering an effective amount of one or more antibodies or molecules of the invention 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), infectious diseases (e.g., AIDS), and proliferative disorders (e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases e.g., AIDS
  • the present invention encompasses methods and compositions (e.g., antagonistic anti-BLyS antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiency syndromes).
  • the present invention encompasses methods and compositions (e.g., agonistic anti-BLyS antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency syndrome).
  • Autoimmune disorders, diseases, or conditions that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention 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 encephalomyehtis, 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 glomerul
  • Immunodeficiencies that may be detected, diagnosed, prognosed, or monitored using the antibodies of the invention 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-
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
  • antibody examples include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab') , disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • scFvs single chain Fvs
  • Fab fragments fragments
  • Fab' fragments F(ab')
  • sdFvs disulfide linked Fvs
  • Fvs fragments comprising or alternatively consisting of, either a VL or a VH domain.
  • Antibodies that immunospecifically bind to BLyS may have cross- reactivity with other antigens.
  • antibodies that immunospecifically bind to BLyS do not cross-react with other antigens.
  • Antibodies that immunospecifically bind to BLyS can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.
  • Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • Antibodies of the invention may also include multimeric forms of antibodies.
  • antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab') 2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent.
  • Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers.
  • individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art.
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-l-carboxylate] and S ATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, IL)) can be used to form antibody multimers.
  • antibody homodimers can be converted to Fab'2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.
  • antibodies can be made to multimerize through recombinant D ⁇ A techniques.
  • IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide.
  • ⁇ on-IgA or non-IgM molecules such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules, (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31.
  • ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety.
  • Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.
  • specific binding or immunospecifc binding by an anti-BLyS antibody means that the anti-BLyS antibody binds BLyS but does not significantly bind to (i.e., cross react with) proteins other than BLyS, such as other proteins in the same family of proteins, e.g., other TNF family ligands).
  • an antibody that binds BLyS protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the BLyS -specific antibody of the invention preferentially binds BLyS compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope.
  • An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein).
  • an antibody that specifically binds BLyS may or may not bind fragments of BLyS and/or variants of BLyS (e.g., proteins that are at least 90% identical to BLyS) depending on the presence or absence of the epitope bound by a given BLyS-specific antibody in the BLyS fragment or variant.
  • BLyS-specific antibodies of the invention may bind species orthologues of BLyS (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue.
  • BLyS-specific antibodies of the invention may bind modified forms of BLyS, for example, BLyS fusion proteins, hi such a case when antibodies of the invention bind BLyS fusion proteins, the antibody must make binding contact with the BLyS moiety of the fusion protein in order for the binding to be specific.
  • Antibodies that specifically bind to BLyS can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.
  • antibodies may be specific for either the membrane bound form of BLyS, or the soluble form of BLyS (i.e., 134-285 of SEQ ID NO:2, preferably trimers of proteins consisting of amino acids 134-285 of SEQ ID NO:2), or both.
  • Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of BLyS according to the assays described in Examples 1 through 19.
  • an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof.
  • An antibody of the invention "which binds the soluble form of BLyS" is one which binds the 152 amino acid soluble form of the BLyS protein (amino acids 134- 285 of SEQ ID NO:3228). In specific embodiments of the invention, an antibody of the invention "which binds the soluble form of BLyS" does not also bind the membrane- bound or membrane-associated form of BLyS.
  • Assays which measure binding to the soluble form of BLyS include, but are not limited to, receptor binding inhibition assay or capture of soluble BLyS from solution as described in Examples 8 and 9.
  • An antibody of the invention "which binds the membrane-bound form of BLyS" is one which binds the membrane-associated (uncleaved) BLyS protein. In specific embodiments of the invention, an antibody of the invention "which binds the membrane-bound form of BLyS" does not also bind the soluble form of BLyS.
  • Binding to HIS-tagged BLyS (as described herein) in an ELISA is an indicator that an antibody binds the membrane-bound form of BLyS, but should not be relied upon as proof of specificity for the membrane-bound form of BLyS.
  • Assays that may be relied upon as proof of an antibody's specificity for membrane-bound BLyS include, but are not limited to, binding to plasma membranes expressing BLyS as described in Example 2.
  • An antibody of the invention "which binds the both the soluble form and the membrane-bound form of BLyS" is one which binds both the membrane-bound form and the soluble form of BLyS.
  • variant refers to a polypeptide that possesses a similar or identical function as a BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment thereof, or possess a similar or identical stracture of a BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment thereof.
  • a variant having a similar amino acid 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 a BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding a BLyS polypeptide (e.
  • a polypeptide with similar stracture to a BLyS polypeptide, a fragment of BLyS, an anti-BLyS antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quartemary stracture of a BLyS polypeptide, a fragment of BLyS, an anti-BLyS 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.
  • 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 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. Mol. Biol 215:403-410(1990) have incorporated such an alogrithm.
  • Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3389-3402(1997).
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules ⁇ Id.).
  • ktup is a control option that sets the sensitivity and speed of the search.
  • derivative refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a BLyS polypeptide, a fragment of BLyS, or an antibody of the invention that immunospecifically binds to BLyS, which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
  • derivative as used herein also refers to a BLyS polypeptide, a fragment of BLyS, an antibody that immunospecifically binds to BLyS which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide.
  • a BLyS polypeptide, a fragment of BLyS, or an anti-BLyS 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 a BLyS polypeptide, a fragment of BLyS, or an anti-BLyS antibody 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.
  • a derivative of a BLyS polypeptide, a fragment of BLyS, or an anti-BLyS antibody may contain one or more non-classical amino acids.
  • a polypeptide derivative possesses a similar or identical function as a BLyS polypeptide, a fragment of BLyS, or an anti-BLyS antibody, described herein.
  • epitopes refers to portions of BLyS having antigenic or immunogenic activity in an animal, preferably a mammal.
  • An epitope having immunogenic activity is a portion of BLyS that elicits an antibody response in an animal.
  • An eptiope having antigenic activity is a portion of BLyS 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.
  • fragment refers 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 BLyS, or an anti-BLyS antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to BLyS.
  • an anti-BLyS antibody including molecules such as
  • fusion protein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-BLyS antibody of the invention and an amino acid sequence of a heterologous polypeptide ⁇ i.e., a polypeptide unrelated to an antibody or antibody domain).
  • the term "host cell” as used herein refers 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.
  • isolated antibody is intended an antibody removed from its native environment. Thus, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • Figure 1 ELISA results for three scFvs, I006E07, 1008D05 and I016F04, that immunospecifically bind to U937 membranes, but not to bind to or cross-react with TNF- alpha or BSA.
  • Figure 2 The results for three scFvs, I016H07, 1001C09 and I018D07, in a receptor inhibition assay.
  • Figure 3 ELISA results for two scFvs (I022D01 and 1031F02) demonstrating their ability to bind to human BLyS and to cross-react with mouse BLyS, but not to bind to or cross-react with other antigens of the TNF ligand family.
  • Figure 4. ELISA results for three scFvs (1031F09, 1050A12, and 1051C04) binding to U937 plasma membranes when either BLyS or TNF-alpha is used as a competitor.
  • FIG. 1 Kinetic analysis of scFv antibody I003C02. A dilution series of I003C02 from 3nM to 825nM is shown. Association and dissociation curves were generated using a BIAcore 2000 and BIAevaluation 3.0 software. [0050] Figure 6. Typical titration curves for two scFv antibodies (I007F11 and I050A07) are shown in Figure 6. Unlabelled BLyS competed for binding to its receptor with an IC 50 value of 0.8 nM. The IC 50 values for I007F11 and I050A07 are 7.9 nM and 17.1 nM , respectively. The assay was performed in triplicate and standard error bars are shown.
  • FIG. 7 ELISA results for three scFvs clones (I074B12, 1075F12 and 1075 A02) that immunospecifically bind to immobilized BLyS, but not to U937 plasma membranes, TNF-alpha or BSA. As a control, a phage antibody that recognizes TNF ⁇ , is also shown in Figure 7.
  • Figure 8 The results for two scFvs (I025B09 and I026C04) in a receptor inhibition assay.
  • Figure 9 ELISA results for two scFvs clones (I067F05 and I078D02) demonstrating their ability to bind to immobilized human BLyS and to cross-react with immobilized mouse BLyS, but not to bind to or cross-react with other antigens of the TNF ligand family.
  • Figure 11 Typical titration curves for two scFvs, I0068C06 and I074B 12, are shown in Figure 11. Unlabelled BLyS competed for binding to its receptor with an inhibitory constant 50 (IC 50 ) value of 0.66 nM. The IC 50 values for I0068C06 and I074B12 are 61 nM and 13 nM , respectively. The assay was performed in triplicate and standard error bars are shown.
  • FIG. 12 ELISA results for three clones (I079C01, 1081C10 and I082A02) demonstrating their ability to bind histidine-tagged BLyS, U937 plasma membranes, but not to bind immobilized biotinylated BLyS.
  • BLyS is used as a competitor.
  • Figure 14 An example of the dissociation section of a typical sensorgram for 8 scFvs is shown in Figure 14. An anti-TNFo. antibody that does not recognize BLyS was included as a control. Of the 8 scFvs exemplified, I079F06 was identified for further study due to the relatively high numbers of RU's bound to the surface.
  • Figure 15 A typical example of the binding curves generated for the scFv antibody I082C03 is shown in Figure 15. The off-rate for this clone was calculated as
  • BLyS is used as a competitor.
  • the present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to BLyS or a fragment or variant of BLyS.
  • the invention provides antibodies such as, for example, single chain Fvs (scFvs) having an amino acid sequence of any one of SEQ ID NOS:l - 2128, as referred to in Table 1.
  • the present invention encompasses antibodies that immunospecifically bind to a polypeptide, a polypeptide fragment or variant, or an epitope of human BLyS (SEQ ID NOS:3228 and or 3229) or BLyS expressed on human monocytes; murine BLyS (SEQ ID NOS:3230 and/or 3231) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ID NOS:3232, 3233, 3234 and/or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes) (as determined by immunoassays known in the art for assaying specific antibody-antigen binding).
  • human BLyS SEQ ID
  • polypeptide sequence shown in SEQ ID NO:3228 was obtained by sequencing and translating the cDNA of the HNEDU15 clone which was deposited on October 22, 1996 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, and assigned ATCC Accession No. 97768.
  • the deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).
  • the ATCC deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
  • polypeptide sequence shown in SEQ ID NO:3229 was obtained by sequencing and translating the cDNA of the HDPMC52 clone, which was deposited on. December 10, 1998 at the American Type Culture Collection, and assigned ATCC Accession No. 203518.
  • the deposited clone is contained in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).
  • the ATCC deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
  • the BLyS polypeptides bound by the antibodies of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to antibodies that bind monomers and multimers of the BLyS polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the antibodies of the invention bind BLyS monomers, dimers, trimers or tetramers. In additional embodiments, the antibodies of the invention bind at least dimers, at least trimers, or at least tetramers of BLyS.
  • Multimeric BLyS bound by the antibodies of the invention may be homomers or heteromers.
  • a BLyS homomer refers to a multimer containing only BLyS polypeptides (including BLyS fragments, variants, and fusion proteins, as described herein). These homomers may contain BLyS polypeptides having identical or different amino acid sequences.
  • the antibodies of the invention bind a BLyS homodimer (e.g., containing two BLyS polypeptides having identical or different amino acid sequences) or a BLyS homotrimer (e.g., containing three BLyS polypeptides having identical or different amino acid sequences).
  • the antibodies of the invention bind homotrimers of BLyS. In additional embodiments, the antibodies of the invention bind a homomeric BLyS multimer which is at least a homodimer, at least a homotrimer, or at least a homotetramer.
  • Heteromeric BLyS refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the BLyS polypeptides of the invention.
  • the antibodies of the invention bind a BLyS heterodimer, a heterotrimer, or a heterotetramer.
  • the antibodies of the invention bind a heteromeric BLyS multimer which is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
  • the antibodies of the invention bind a heterotrimer comprising both BLyS polypeptides and APR-1L polypeptides (SEQ ID NO:3239; GenBank Accession No. AF046888; PCT International Publication Number WO97/33902; J. Exp. Med. 188(6):1185-1190) or fragments or variants thereof.
  • the antibodies of the invention bind a heterotrimer comprising one BLyS polypeptide (including fragments or variants) and two APRIL polypeptides (including fragments or variants).
  • the antibodies of the invention bind a heterotrimer comprising two BLyS polypeptides (including fragments or variants) and one APRIL polypeptide (including fragments or variants).
  • the heteromers bound by the antibodies of the invention contain CD40 ligand polypeptide sequence(s), or biologically active fragment(s) or variant(s) thereof.
  • the antibodies of the invention bind homomeric, especially homotrimeric, BLyS polypeptides, wherein the individual protein components of the multimers consist of the mature form of BLyS (e.g., amino acids residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID NO:3229) or fragments or variants thereof.
  • antibodies of the invention bind heteromeric, especially heterotrimeric, BLyS polypeptides such as a heterotrimer containing two BLyS polypeptides and one APRE polypeptide or a heterotrimer containing one BLyS polypeptide and two APR.1L polypeptides, and wherein the individual protein components of the BLyS heteromer consist of the mature extracellular soluble portion of either BLyS (e.g., amino acids residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID NO:3229) or fragments or variants thereof, or the mature extracellular soluble portion APRIL (e.g., amino acid residues 105-250 of SEQ ID NO:3239) or fragments or variants thereof.
  • BLyS e.g., amino acids residues 134-285 of SEQ ID NO:3228, or amino acids residues 134-266 of SEQ ID NO:3229
  • APRIL e.g.,
  • the antibodies of the invention bind conformational epitopes of a BLyS monomeric protein. In specific embodiments, the antibodies of the invention bind conformational epitopes of a BLyS multimeric, especially trimeric, protein. In other embodiments, antibodies of the invention bind conformational epitopes that arise from the juxtaposition of BLyS with a heterologous polypeptide, such as might be present when BLyS forms heterotrimers (e.g., with APRIL polypeptides (e.g., SEQ ID SEQ ID NO:3239)), or in fusion proteins between BLyS and a heterologous polypeptide.
  • APRIL polypeptides e.g., SEQ ID SEQ ID NO:3239
  • antibodies of the invention that specifically bind heterotrimers containing at least one BLyS polypeptide and at least one APRIL polypeptide comprise all or a portion of SEQ ID NOS: 1881 or 1884 (e.g., one or more CDR regions, a VH domain or a VL domain).
  • the heterotrimers containing at least one BLyS polypeptide and at least one APRIL polypeptide comprise two BLyS polypeptides and one APR-tL polypeptide.
  • the heterotrimers containing at least one BLyS polypeptide and at least one APR-D polypeptide comprise one BLyS polypeptide and two APRIL polypeptides.
  • antibodies of the invention that specifically bind heterotrimers containing at least one BLyS polypeptide and at least one APRIL polypeptide comprise all or a portion of any one of SEQ ID NOS: 3240-3247 (e.g., one or more CDR regions, a VH domain or a NL domain).
  • SEQ ID NOS: 3240-3247 e.g., one or more CDR regions, a VH domain or a NL domain.
  • the sequences of SEQ ID ⁇ OS: 3240-3247 are presented after Table 1 just prior to the claims.
  • the heterotrimers containing at least one BLyS polypeptide and at least one APR-tL polypeptide comprise two BLyS polypeptides and one APRIL polypeptide.
  • the heterotrimers containing at least one BLyS polypeptide and at least one APRIL polypeptide comprise one BLyS polypeptide and two APRIL polypeptides.
  • BLyS multimers bound by the antibodies of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • BLyS multimers such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.
  • BLyS heteromultimers such as, for example, BLyS heterotrimers or BLyS heterotetramer s, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution
  • BLyS multimers are formed by covalent associations with and or between the BLyS polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:3228 or SEQ ID NO:3229).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a BLyS fusion protein.
  • covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in a BLyS-Fc fusion protein.
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from CD40L, or a soluble fragment thereof.
  • two or BLyS polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers).
  • antibodies of the invention immunospecifically bind a BLyS polypeptide having the amino acid sequence of SEQ ID NO:3228 or as encoded by the cDNA clone contained in ATCC No. 97768, or a polypeptide comprising a portion (i.e., a fragment) of the above polypeptides.
  • the invention provides an antibody that binds an jsolated BLyS polypeptide having the amino acid sequence of SEQ ID NO: 3229 or the amino acid sequence encoded by the cDNA clone contained in ATCC No. 203518, or an antibody that binds polypeptide comprising a portion (i.e, fragment) of the above polypeptides.
  • Antibodies of the invention that bind BLyS polypeptides may bind them in as isolated polypeptides, in their naturally occurring state and/or their native conformation.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced by and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • antibodies of the present invention may bind recombinantly produced BLyS polypeptides.
  • Antibodies of the present invention may also bind BLyS expressed on the surface of a cell, wherein said BLyS polypeptide is encoded by a polynucleotide encoding amino acids 1 to 285 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide.
  • said BLyS polypeptide expressed on the surface of a cell is a recombinant BLyS polypeptide.
  • said BLyS polypeptide expressed on the surface of the cell is a naturally occurring BLyS polypeptide.
  • an antibody of the invention may bind a BLyS expressed on the surface of the cell wherein Lys-132 and/or Arg-133 of the BLyS sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, thereby preventing or diminishing release of the soluble form of BLyS from cells expressing BLyS.
  • Antibodies of the present invention may also bind BLyS secreted by a cell, wherein said BLyS polypeptide is encoded by a polynucleotide encoding amino acids 1 to 285 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide.
  • said BLyS polypeptide secreted by a cell is a recombinant BLyS polypeptide.
  • said BLyS polypeptide secreted by a cell is a naturally occurring BLyS polypeptide.
  • Antibodies of the present invention immunospecifically bind to polypeptides comprising or alternatively, consisting of, the amino acid sequence of SEQ ID NO:3228, encoded by the cDNA contained in the plasmid having ATCC accession number 97768, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
  • Antibodies of the present invention also bind to fragments of the amino acid sequence of SEQ ID NO:3228, encoded by the cDNA contained in the plasmid having ATCC accession number 97768, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
  • antibodies of the present invention bind polypeptides comprising or altematively, consisting of, the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCC accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
  • Antibodies of the present invention also bind to fragments of the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCC accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
  • antibodies of the invention bind polypeptides or polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NOS: 3230 through 3237.
  • the antibodies of the present invention immunospecifically bind polypeptide fragments including polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:3228, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
  • Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • polypeptide fragments that may be bound by the antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, and/or 251 to 285 of SEQ ID NO:3228.
  • polypeptide fragments 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.
  • antibodies of the present invention bind polypeptide fragments comprising, or alternatively consisting of, amino acid residues: 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152, 166-181, 185-209, 210-221, 226-237, 244-249, 253-265, and/or 277-285 of SEQ ID NO:3228.
  • antibodies of the invention bind an epitope comprising amino acids 165-171 of SEQ ID NO:3228.
  • the CDRs of antibodies of the invention make contacts with one or more amino acids in the the sequence of amino acids 165-171 of SEQ ID NO:3228.
  • antibodies of the invention whose CDRs make contact with one or more amino acids in the the sequence of amino acids 165-171 of SEQ ID NO:3228 disrupt BLyS- BLyS receptor interactions.
  • a partial, non-limiting and non-exclusive list of such residues of the BLyS polypeptide sequence which may be targeted for mutation includes the following amino acid residues of the BLyS polypeptide sequence as shown in SEQ ID NO:3228: N-142; T-143; Q-144; D-145; C-146; L-147; Q-148; L-149; 1-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157; 1-158; Q-159; and K-160.
  • antibodies of the present invention that bind BLyS polypeptides which have one or more mutations in the region from N-142 through K-160 of SEQ ID ⁇ O:3228 are contemplated.
  • Polypeptide fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • polypeptide fragments that may be bound by antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 15, 16-30, 31-46, 47-55, 56- 72, 73-104, 105-163, 163-188, 186-210 and 210-284 of the amino acid sequence disclosed in SEQ ID NO:3228.
  • polypeptide fragments that may be bound by antibodies of the present invention, include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 143, 1-150, 47- 143, 47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150, 140-200, 140-225, and 140-266 of the amino acid sequence disclosed in SEQ ID NO:3229.
  • polypeptide fragments that may be bound by antibodies of the present invention 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.
  • "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.
  • Additional preferred embodiments encompass antibodies that bind polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of BLyS (e.g., amino acid residues 1-46 of SEQ ID NO:3228), the predicted transmembrane domain of BLyS (e.g., amino acid residues 47-72 of SEQ ID NO:3228), the predicted extracellular domain of BLyS (e.g., amino acid residues 73-285 of SEQ ID NO:3228), the mature soluble extracellular domain of BLyS (e.g., amino acids residues 134-285 of SEQ ID NO:3228), the predicted TNF conserved domain of BLyS (e.g., amino acids 191 to 284 of SEQ ID NO:3228), and a polypeptide comprising, or alternatively, consisting of the predicted intracellular domain fused to the predicted extracellular domain of BLyS (amino acid residues 1-46 fused to amino acid residues 73-285 of SEQ ID NO:32
  • polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of BLyS (amino acid residues 1-46 of SEQ ID NO:3229), the predicted transmembrane domain of BLyS (amino acid residues 47-72 of SEQ ID NO:3229), the predicted extracellular domain of BLyS (amino acid residues 73-266 of SEQ ID NO:3229), the predicted TNF conserved domain of BLyS (amino acids 172 to 265 of SEQ ID NO:3229), and a polypeptide comprising, or alternatively, consisting of the predicted intracellular domain fused to the predicted extracellular domain of BLyS (amino acid residues 1-46 fused to amino acid residues 73-266 of SEQ ID NO:3229).
  • Certain additional embodiments of the invention encompass antibodies that bind polypeptide fragments comprising, or alternatively consisting of, the predicted beta- pleated sheet regions of the BLyS polypeptides of SEQ ID NO:3228 and SEQ ID NO: 3229.
  • polypeptide fragments comprising the beta-pleated sheets of BLyS comprise, or alternatively consist of, amino acid residues Gln-144 to Ala-151, Phe-172 to Lys-173, Ala-177 to Glu-179, Asn-183 to He-185, Gly-191 to Lys-204, His-210 to Val- 219, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to He-263 and/or Val-276 to Leu- 284 of SEQ TD NO:3228.
  • these polypeptide fragments comprising the beta-pleated sheets of BLyS comprise, or alternatively consist of, amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160, Asn-164 to Ile-166, Gly- 172 to Lys-185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile-244 and/or Val-257 to Leu-265 of SEQ ID NO:3229.
  • a partial, non-limiting, and exemplary list of polypeptides that may be bound by the antibodies of the invention includes polypeptides that comprise, or alternatively consist of, combinations of amino acid sequences of the invention includes, for example, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Nal-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID ⁇ O:3228; or [Met-1 to Lys-113] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Ly
  • polypeptide fragments in an order other than that recited above (e.g., [Leu-114 to Thr-141] fused to [Val-199 to Ala-248] fused to [Gly-249 to Leu-285] fused to [Val-142 to Lys-160] of (SEQ ID NO:3228).
  • heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the present invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Val-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly- 249 to Leu-285] of SEQ ID NO:3228 fused to a FLAG tag ; or [Met-1 to Lys-113] of SEQ ID NO:3228 fused to [Leu-114 to Thr-141] of SEQ ID NO:3228 fused to [Glu- 135 to Asn-165] of SEQ ID NO:39 fused to [Val-142 to Lys-160] of SEQ ID NO:3228 fused to [Gly-161 to Gln-198] of SEQ ID NO:3228 fused to [Val-199 to Ala
  • a partial, non-limiting, and exemplary list of polypeptides that may be bound by the antibodies of the invention includes polypeptides that comprise, or alternatively consist of, combinations of amino acid sequences includes, for example, [Met-1 to Lys- 113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229; [Met-1 to Lys-113] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229; or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229.
  • amino acids sequences that may be bound by the antibodies of the invention combinations may include the polypeptide fragments in an order other than that recited above (e.g., [Leu-114 to Thr- 141] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] fused to [Gly-142 to Gln-179] of SEQ ID NO:3229).
  • heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the present invention (e.g., [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly- 142 to Gln-179] fused to [Gly-230 to Leu-266] of SEQ ID NO:3229 fused to a FLAG tag (SEQ ID NO:3238) or, [Met-1 to Lys-113] of SEQ ID NO:3229 fused to [Leu-114 to Thr- 141] of SEQ ID NO:3229 fused to [Glu-135 to Asn-165] of SEQ ID NO:39 fused to [Gly- 142 to Gln-179] of SEQ ID NO:3229 fused to [Val-180 to Ala-229] of SEQ ID NO:3229 fused to [Gly-230 to Leu-266]
  • heterologous polypeptide fragments as described herein and/or other poly
  • Additional embodiments of the invention encompass antibodies that bind BLyS polypeptide fragments comprising, or alternatively consisting of, functional regions of polypeptides of the invention, such as the Garnier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, 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 set out in Tables 9 and 10 and as described herein.
  • functional regions of polypeptides of the invention such as the Garnier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic
  • the polypeptide fragments bound by the antibodies of the invention are antigenic (i.e., 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 (i.e., full-length) BLyS polypeptide (e.g., SEQ ID NOS:3228 and 3229).
  • the data presented in columns NHL, IX, XIII, and XIV of Tables 9 and 10 can be used to determine regions of the BLyS polypeptide of SEQ ID NO:3228 (Table 9) or the BLyS polypeptide of SEQ ID NO:3229 (Table 10) which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
  • the above-mentioned preferred regions set out in Tables 9 and 10 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in Tables 9 and 10, such preferred regions include Gamier-Robson alpha-regions, beta-regions, tum-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and tum-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson- Wolf regions of high antigenic index and Emini surface-forming regions.
  • antibodies of the present invention bind BLyS polypeptides or BLyS polypeptide fragments and variants comprising regions of BLyS that combine several stractural features, such as several (e.g., 1, 2, 3 , or 4) of the same or different region features set out above and in Tables 9 and 10.
  • Val 108 A A -0.80 0.49 * -0.60 0.38
  • Lys 184 A A 1.08 -0.59 ⁇ F 0.75 0.55 lie 185 A A 1.08 -0.59 i * 0.60 0.63
  • Tyr 206 A A 1.11 -0.21 0.45 1.03
  • Lys 283 A A -1.30 0.41 * -0.60 0.33
  • Val 108 A A -0.80 0.49 -0.60 0.38
  • Val 168 A A 0.38 -0.50 ⁇ 0.92 0.49
  • the invention provides antibodies that bind a polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide of the invention.
  • the epitope of this polypeptide portion may be an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • 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.
  • polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiseram 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 rales, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al, Cell 37:767-778 (1984) at 777.
  • antibodies of the present invention bind antigenic epitope-bearing peptides and polypeptides of BLyS 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 a BLyS 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.
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate BLyS-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-115 to about Leu-147 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about lie- 150 to about Tyr-163 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-171 to about Phe-194 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-223 to about Tyr-246 in SEQ ID NO:3228; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-271 to about Phe-278 in Figures 1 A and IB (SEQ ID NO:3228).
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate BLyS-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-32 to about Leu-47 in SEQ ED NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-116 to about Ser-143 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-153 to about Tyr-173 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-218 to about Tyr-227 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ala-232 to about Gln-241 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of,
  • BLyS epitope-bearing peptides and polypeptides may be produced by any conventional means. See, e.g., Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl Acad. Sci. USA 52:5131-5135; this "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U. S. Patent No. 4,631,211 to Houghten et al. (1986).
  • SMPS Simultaneous Multiple Peptide Synthesis
  • the present invention encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3228, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 97768, or encoded by a polynucleotide that hybridizes to cDNA sequence contained in ATCC deposit No. 97768 (e.g., under hybridization conditions described herein).
  • the present invention also encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3229, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. 203518, or encoded by a polynucleotide that hybridizes to the cDNA sequence contained in ATCC deposit No. 203518 (e.g., under hybridization conditions described herein).
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • the present invention 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)).
  • antigenic epitope 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.
  • BLyS polypeptide fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antibodies of the present invention bind antigenic epitopes preferably containing 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.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes that may be bound by antibodies of the present invention 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. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include 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)).
  • 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 Bittle 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 BLyS 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.
  • a carrier protein such as an albumin
  • 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).
  • Epitope-bearing BLyS 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 Bittle et al., J. Gen. Nirol., 66:2347-2354 (1985).
  • animals may be immunized with free peptide; however, anti- peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- ⁇ -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.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which 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.
  • the antibodies of the present invention may bind polypeptides comprising an immunogenic or antigenic epitope fused to other polypeptide sequences.
  • the BLyS polypeptides may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, 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, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
  • chimeric polypeptides 5,766,883, issued June 16, 1998, 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).
  • antigens e.g., insulin
  • FcRn binding partner such as IgG or Fc fragments
  • IgG Fusion proteins that have a disulfide-linked dimeric stracture 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.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • HA hemagglutinin
  • 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).
  • 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 Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • the antibodies of the present invention bind BLyS polypeptides and/or the epitope-bearing fragments thereof that are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid).
  • a heterologous antigen e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid.
  • the heterologous antigen is an immunogen.
  • the heterologous antigen is the gpl20 protein of HIN, or a fragment thereof.
  • antibodies of the present invention bind BLyS polypeptides and/or the epitope-bearing fragments thereof that are fused with polypeptide sequences of another T ⁇ F ligand family member (or biologically active fragments or variants thereof).
  • the antibodies of the present invention bind BLyS polypeptides of the present invention are fused with a CD40L polypeptide sequence.
  • the CD40L polypeptide sequence is soluble.
  • antibodies of the present invention bind mutant BLyS polypeptides that have been generated by random mutagenesis of a polynucleotide encoding the BLyS polypeptide, by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • antibodies of the present invention bind one or more components, motifs, sections, parts, domains, fragments, etc., of BLyS recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM- ⁇ (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185- 1190), endokine-alpha (International Publication No.
  • WO 98/07880 OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TRIO (International Publication No. WO 98/54202),312C2 (International Publication No.
  • heterologous molecules are any member of the TNF family.
  • antibodies of the present invention bind BLyS polypeptides of the invention (including biologically active fragments or variants thereof), that are fused with soluble APRIL polypeptides (e.g., amino acid residues 105 through 250 of SEQ ID NO:3239), or biologically active fragments or variants thereof.
  • protein engineering may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron et al., J. Biol.
  • antibodies of the present invention may bind BLyS polypeptide mutants or variants generated by protein engineering.
  • deletions of N-terminal amino acids up to the Gly (G) residue at position 191 in SEQ ID NO:3228 may retain some biological activity such as, for example, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and cytotoxicity to appropriate target cells.
  • Polypeptides having further N-terminal deletions including the Gly (G) residue would not be expected to retain biological activities because it is known that this residue in TNF-related polypeptides is in the beginning of the conserved domain required for biological activities.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the BLyS of SEQ ID NO:3228, up to the glycine residue at position 191 (Gly-191 residue from the amino terminus).
  • the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n ! -285 of SEQ ID NO:3228, where n 1 is an integer in the range of the amino acid position of amino acid residues 2-190 of the amino acid sequence in SEQ ID NO:3228.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide may retain some biological activity such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities.
  • lymphocyte e.g., B cell
  • the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. [0116] Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of BLyS shown in SEQ ID NO:3228, up to the glycine residue at position number 280.
  • the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 2 -285 of SEQ ID NO:3228, where n 2 is an integer in the range of the amino acid position of amino acid residues 73-280 in SEQ ID NO:3228, and 73 is the position of the first residue from the N-terminus of the predicted extracellular domain of the BLyS polypeptide (disclosed in SEQ ID NO:3228).
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • Highly preferred embodiments of the invention are directed to antibodies that 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 BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
  • Preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 90% identical to a BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 95% identical to a BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
  • More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 96% identical to a BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ED NO:3228.
  • more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 97% to a BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 98% to a BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
  • more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 99% identical to BLyS polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
  • antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, one of the following N-terminally deleted polypeptide fragments of BLyS: amino acid residues Ala-71 through Leu-285, amino acid residues Ala-81 through Leu-285, amino acid residues Leu-112 through Leu-285, amino acid residues Ala-134 through Leu-285, amino acid residues Leu-147 through Leu-285, and amino acid residues Gly-161 through Leu-285 of SEQ ID NO:3228.
  • amino acid residues Ala-71 through Leu-285 amino acid residues Ala-81 through Leu-285, amino acid residues Leu-112 through Leu-285, amino acid residues Ala-134 through Leu-285, amino acid residues Leu-147 through Leu-285, and amino acid residues Gly-161 through Leu-285 of SEQ ID NO:3228.
  • Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (D ⁇ beli et al., J. Biotechnology 7:199-216 (1988). Since the present protein is a member of the TNF polypeptide family, deletions of C-terminal amino acids up to the leucine residue at position 284 are expected to retain most if not all biological activity such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication.
  • lymphocyte e.g., B cell
  • Polypeptides having deletions of up to about 10 additional C-terminal residues also may retain some activity such as receptor binding, although such polypeptides would lack a portion of the conserved TNF domain which extends to about Leu-284 of SEQ ED NO:3228.
  • deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained.
  • the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the BLyS polypeptide of SEQ ED NO: 3228, up to the glycine residue at position 274 (Gly-274).
  • the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m 1 of the amino acid sequence in SEQ ID NO:3228, where m 1 is any integer in the range of the amino acid position of amino acid residues 274-284 in SEQ ED NO:3228.
  • the invention provides antibodies that bind BLyS polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283 and 1-284 of SEQ ED NO:3228.
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • antibodies that bind BLyS polypeptides comprising, or alternatively consisting of, BLyS polypeptides with one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n ⁇ m 1 of SEQ ED NO:3228, where n 1 and m 1 are integers as defined above. Also included are antibodies that bind a polypeptide comprising, or alternatively consisting of, a portion of the complete BLyS amino acid sequence encoded by the deposited cDNA clone contained in ATCC Accession No.
  • deletions of C-terminal amino acid residues of the predicted extracellular domain of BLyS up to the leucine residue at position 79 of SEQ ED NO:3228 may retain some biological activity, such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities.
  • Polypeptides having further C-terminal deletions including Leu-79 of SEQ ED NO:3228 would not be expected to retain biological activities.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of BLyS polypeptide shown in SEQ ED NO:3228, up to the leucine residue at position 79 of SEQ ID NO:3228.
  • the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 73-m of the amino acid sequence in SEQ ID NO:3228, where m 2 is any integer in the range of the amino acid position of amino acid residues 79 to 285 in the amino acid sequence in SEQ ED NO:3228, and residue 78 is the position of the first residue at the C- terminus of the predicted extracellular domain of the BLyS polypeptide (disclosed in SEQ ED NO:3228).
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to Leu-285; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to N-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to 1-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to ⁇ -267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to 1-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%), 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • the invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of BLyS, which may be described generally as having residues n 2 -m 2 of SEQ ID NO:3228 where n 2 and m 2 are integers as defined above. [0128] In another embodiment, antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the BLyS amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCC accession no.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the BLyS shown in SEQ ID NO:3228, up to the glycine residue at position number 280 of the sequence shown SEQ ED NO:3228 and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n 3 -285 of the sequence shown in SEQ ED NO:3228, where n 3 is an integer in the range of the amino acid position of amino acid residues 1 to 280 of the amino acid sequence in SEQ ID NO:3228.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-285; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-ll to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; N-28 to L-285; S-29 to L-285; 1-30 to L-285; L-31
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • functional activities e.g., biological activity
  • a shortened BLyS mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a BLyS mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., biological or immunogenic) activities.
  • the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the BLyS shown in SEQ ID NO:3228, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m 3 of SEQ ED NO:3228, where m is an integer in the range of the amino acid position of amino acid residues 6-284 of the amino acid sequence in SEQ ID NO:3228.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-l to L-284; M-l to K-283; M-l to L-282; M-l to A-281; M-l to G-280; M-l to F-279; M-l to F-278; M-l to T-277; M-l to V-276; M-l to D-275; M-l to G-274; M-l to D-273; M-l to L-272; M-l to S-271; M-l to 1-270; M-l to Q-269; M-l to A-268; M-l to N-267; M-l to E-266; M-l to R-265; M-l to P-264; M-l to 1-263; M-l to A-262; M-l to L-261; M-l to Q-260; M-l to L-259; M-l to E-25
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • the invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a BLyS polypeptide, which may be described generally as having residues n 3 -m 3 of SEQ ED NO:3228, where n 3 and m 3 are integers as defined above.
  • deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, to stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity.
  • functional activity e.g., biological activity
  • deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, to stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity.
  • lymphocyte e.g., B cell
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of BLyS shown in SEQ ED NO:3229, up to the glycine residue at position number 261.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n 4 -266 of SEQ ID NO:3229, where n 4 is an integer in the range of the amino acid position of amino acid residues 73-261 of the amino acid sequence in SEQ ED NO:3229, and 261 is the position of the first residue from the N-terminus of the predicted extracellular domain BLyS polypeptide (shown in SEQ ID NO:3229).
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266;
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • deletions of C-terminal amino acid residues of the predicted extracellular domain of BLyS up to the leucine residue at position 79 of SEQ rD NO:3229 may retain some functional activity, such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity.
  • Polypeptides having further C-terminal deletions including Leu-79 of SEQ ED NO: 3229 would not be expected to retain biological activities.
  • the present invention further provides antibodies that bind polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of BLyS shown in SEQ ED NO:3229, up to the leucine residue at position 79 of SEQ ED NO:3229.
  • the present invention provides antibodies that bind polypeptides having the amino acid sequence of residues 73-m 4 of the amino acid sequence in SEQ -ED NO:3229, where m 4 is any integer in the range of the amino acid position of amino acid residues 79-265 of the amino acid sequence in SEQ ID NO:3229.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to L-265; Q-73 to K-264; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to 1-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-245; Q-73 to 1-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240;
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of BLyS, which may be described generally as having residues n 4 -m 4 of SEQ ED NO:3229 where n 4 and m are integers as defined above.
  • antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the BLyS amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC Accession No. 203518, where this portion excludes from 1 to about 260 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCC Accession No. 203518, or from 1 to about 187 amino acids from the carboxy terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCC Accession No. 203518, or any combination of the above amino terminal and carboxy terminal deletions, of the entire extracellular domain of the amino acid sequence encoded by the cDNA clone contained in the deposit having ATCC Accession No. 203518.
  • the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the BLyS polypeptide shown in SEQ ID NO: 3229, up to the glycine residue at position number 261 of the sequence shown SEQ ID NO: 3229 and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n 5 -266 of the sequence shown in SEQ ID NO:3229, where n 5 is an integer in the range of the amino acid position of amino acid residues 1 to 261 of the amino acid sequence in SEQ ED NO:3229.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-266; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-ll to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; N-28 to L-266; S-29 to L-266; 1-30 to L-266; L-31
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%), 96%, 97%), 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%), 96%, 97%), 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • functional activities e.g., biological activities
  • a shortened BLyS mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus.
  • Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a BLyS mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., immunogenic) activities. In fact, peptides composed of as few as six BLyS amino acid residues may often evoke an immune response.
  • the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the BLyS shown in SEQ ID NO:3229, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m 5 of SEQ ID NO:3229, where m 5 is an integer in the range of the amino acid position of amino acid residues 6 to 265 in the amino acid sequence of SEQ ED NO:3229.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-l to L-265; M-l to K-264; M-l to L-263; M-l to A-262; M-l to G-261; M-l to F-260; M-l to F-259; M-l to T-258; M-l to V-257; M-l to D-256; M-l to G-255; M-l to D-254; M-l to L-253; M-l to S-252; M-l to 1-251; M-l to Q-250; M-l to A-249; M-l to N-248; M-l to E-247; M-l to R-246; M-l to P-245; M-l to 1-244 M-l to A-243; M-l to L-242; M-l to Q-241; M-l to L-240; M-l to E-2
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • the invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a BLyS polypeptide, which may be described generally as having residues n 5 -m 5 of SEQ ID NO:3229, where n 5 and m 5 are integers as defined above.
  • the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 134-m 6 of SEQ ED NO:3228, where m 6 is an integer from 140 to 285, corresponding to the position of the amino acid residue in SEQ ED NO:3228.
  • the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues A-134 to Leu-285; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to 1-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to 1-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-
  • T-228 A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224 A-134 to
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%,
  • antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-l to C-15; D-2 to L-16; D-3 to K-17; S-
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%), 96%), 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-l to C-15; D-2 to L-16; D-3 to K-17; S- 4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-ll to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to 1-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K- 43; 1-30 to D-44; L-31
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-l to F-15; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-ll to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to 1-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to 1-42; P-29 to C- 43; 1-30 to R-44; T-31
  • the present invention is also directed to antibodies that bind BLyS polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of BLyS polypeptides described above.
  • the invention further includes antibodies that bind variations of BLyS polypeptides which show BLyS polypeptide functional activity (e.g., biological activity) or which include regions of BLyS polypeptide such as the polypeptide fragments described herein.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rales known in the art so as have little effect on activity.
  • antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ED NO:3228, or that encoded by the deposited cDNA plasmid, 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
  • Antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO:3229, or that encoded by the deposited cDNA plasmid, 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, a soluble biologically active fragment of another TNF ligand family member (e.g.,
  • the antibodies of the invention may bind BLyS polypeptides that include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 13).
  • antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a BLyS polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions, hi one embodiment of the invention, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a BLyS polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • site directed changes at the amino acid level of BLyS can be made by replacing a particular amino acid with a conservative substitution.
  • Antibodies of the present invention may bind BLyS amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ED NO:3228 including: Ml replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; Rll replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or
  • T202 replaced with A, G, I, L, S, M, or V
  • D203 replaced with E
  • K204 replaced with H, or R
  • T205 replaced with A, G, I, L, S, M, or V
  • Y206 replaced with F, or W
  • A207 replaced with G, I, L, S, T, M, or V
  • M208 replaced with A, G, I, L, S, T, or V
  • G209 replaced with A, I, L, S, T, M, or V
  • H210 replaced with K, or R
  • L211 replaced with A, G, I, S, T, M, or V
  • 1212 replaced with A, G, L, S, T, M, or V
  • Q213 replaced with ⁇
  • R214 replaced with H, or K
  • K215 replaced with H, or R
  • K216 replaced with H, or R
  • V217 replaced with A, G, I, L, S, T, or M
  • H218 replaced with K, or R
  • V219
  • G, I, S, T, M, or V G, I, S, T, M, or V
  • A262 replaced with G, I, L, S, T, M, or V
  • 1263 replaced with A, G, L, S, T, M, or N
  • R265 replaced with H, or K
  • E266 replaced with D
  • ⁇ 267 replaced with Q
  • A268 replaced with G, I, L, S, T, M, or V
  • S271 replaced with A, G, I, L, T, M, or V
  • L272 replaced with A, G, I, S, T, M, or V
  • D273 replaced with E
  • G274 replaced with A, I, L, S, T, M, or V
  • D275 replaced with E
  • V276 replaced with A, G, I, L, S, T, or M
  • T277 replaced with A, G, I, L,
  • site directed changes at the amino acid level of BLyS can be made by replacing a particular amino acid with a conservative substitution.
  • Antibodies of the present invention may bind BLyS amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ID NO:3229 including: Ml replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; Rll replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or
  • site directed changes at the amino acid level of BLyS can be made by replacing a particular amino acid with a conservative substitution.
  • Antibodies of the present invention may bind BLyS amino acid sequences containing conservative substitution mutations of the polypeptide of any one of SEQ ID ⁇ OS:3230- 3237.
  • Amino acids in the BLyS 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 and the ability to stimulate lymphocyte (e.g., B cell) as, for example, proliferation, differentiation, and/or activation. Accordingly, antibodies of the present invention may bind amino acids in the BLyS polypeptides that are essential for function.
  • antibodies of the present invention bind amino acids in the BLyS polypeptides that are essential for function and inhibit BLyS polypeptide function. In other preferred embodiments, antibodies of the present invention bind amino acids in the BLyS polypeptides that are essential for function and enhance BLyS polypeptide function.
  • substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation. Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic (Pinckard et al, Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al, Crit. Rev. ⁇ xerapeutic Drug Carrier Systems i0:307-377 (1993).
  • the invention provides for antibodies that bind polypeptides having amino acid sequences containing non-conservative substitutions of the amino acid sequence provided in SEQ ID NO:3228.
  • non-conservative substitutions of the BLyS protein sequence provided in SEQ ID NO:3228 include: Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced
  • E, H, K, R, ⁇ , Q, F, W, Y, P, or C T51 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; L52 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R
  • E, H, K, R, ⁇ , Q, F, W, Y, P, or C AllO replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; Gl 11 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; LI 12 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V, ⁇ , Q, F, W, Y, P, or C; 1114 replaced with D, E, H, K, R, ⁇ , Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, ⁇ , Q, A, G, I, L, S, T, M, V, P, or C; El 16 replaced with H, K, R, A, G, I, L, S, T, M,
  • E, H, K, R, N, Q, F, W, Y, P, or C V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A,
  • E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • E140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • V142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • T143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • Q144 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C
  • D145 replaced with H, K, R, A, G, I, L, S, T, M, V, F, W, Y
  • E, H, K, R, N, Q, F, W, Y, P, or C L272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D273 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G274 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D275 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T277 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F278 replaced with D, E, H, K, R, N, Q, A, G
  • antibodies of the present invention bind BLyS polypeptides comprising, or alternatively consisting of, a BLyS amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative).
  • more than one amino acid e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50
  • antibodies of the present invention bind BLyS polypeptides with non-conservative substitutions of the sequence provided in SEQ ID NO:3229 including: Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E
  • D, E, H, K, R, N, Q, F, W, Y, P, or C H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E
  • E, H, K, R, N, Q, F, W, Y, P, or C A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; AlOO replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; GlOl replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V
  • V200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • F201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C
  • G202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • E204 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
  • L205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • S206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • N224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C
  • S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • C226 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P
  • Y227 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C
  • S228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • A229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • G230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
  • T258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
  • F259 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C
  • F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C
  • G261 replaced with D,
  • E, H, K, R, N, Q, F, W, Y, P, or C A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K264 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
  • site directed changes at the amino acid level of BLyS can be made by replacing a particular amino acid with a non-conservative substitution.
  • Antibodies of the present invention may bind BLyS amino acid sequences containing non- conservative substitution mutations of the polypeptide of any one of SEQ ID NOS:3230- 3237.
  • antibodies of the present invention bind BLyS polypeptides comprising, or alternatively consisting of, a BLyS amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative) .
  • more than one amino acid e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50
  • Replacement of amino acids can also change the selectivity of the binding of a ligand to cell surface receptors.
  • Ostade et al Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Since BLyS is a member of the TNF polypeptide family, mutations similar to those in TNF-alpha are likely to have similar effects in BLyS polypeptides.
  • Sites that are critical for ligand-receptor binding can also be determined by stractural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol Biol. 224:899-904 (1992) and de Vos et al Science 255:306-312 (1992)).
  • BLyS is a member of the TNF-related protein family
  • mutations may be made in sequences encoding amino acids in the TNF conserved domain, e.g., in positions Gly-191 through Leu-284 of SEQ ID NO:3228 or in positions Gly-172 through Leu-265 of SEQ ED NO:3229, may modulate rather than completely eliminate functional activities (e.g., biological activities) of BLyS polypeptides or fragments or variants thereof.
  • antibodies of the present invention may bind BLyS polypeptides that have mutations in the TNF conserved domain.
  • antibodies of the present invention may bind BLyS polypeptides that have mutations in the TNF conserved domain and act as antagonists of BLyS. In other preferred embodiments, antibodies of the present invention may bind BLyS polypeptides that have mutations in the TNF conserved domain and act as agonists of BLyS.
  • Recombinant DNA technology known to those skilled in the art (see, for instance, DNA shuffling supra) can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • the invention also encompasses antibodies that bind BLyS derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate BLyS polypeptides, e.g., that are better suited for expression, scale up, etc., in the host cells.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
  • mutation of the serine at position 244 to alanine either singly or in combination with mutation of the asparagine at position 242 to glutamine abolishes glycosylation of the mature soluble form of BLyS (e.g., amino acids 134-285 of SEQ ID NO:3228) when expressed in the yeast Pichea pastoris.
  • the glycosylation event may be due to the activation or unmasking of an O-linked glyscosylation site at serine 244.
  • BLyS polypeptide of SEQ ED NO:3229 Similar mutations affecting glycosylation could also be made in the BLyS polypeptide of SEQ ED NO:3229, i.e., aspargine-223 to glutamine and/or serine-224 to alanine of SEQ ID NO:3229.
  • one or more of the amino acid residues of the polypeptides of the invention e.g., arginine and lysine residues
  • proteases such as, for example, furins or kexins.
  • antibodies of the invention may bind BLyS derivatives and analogs that have one or more amino acid residues deleted, added, or substituted. In other embodiments, antibodies of the invention may bind BLyS derivatives, variants or analogs that are unable to be cleaved from the cell surface.
  • antibodies of the invention bind BLyS polypeptides in which Lys-132 and/or Arg-133 of the BLyS sequence shown in SEQ ED NO:3228 is mutated to another amino acid residue, or deleted altogether, to prevent or diminish release of the soluble form of BLyS from cells expressing BLyS.
  • antibodies of the invention bind BLyS polypeptides in which Lys-132 of the BLyS sequence shown in SEQ TD NO:3228 is mutated to Ala-132.
  • antibodies of the invention bind BLyS polypeptides in which Arg-133 of the BLyS sequence shown in SEQ ID NO:3228 is mutated to Ala-133.
  • mutated proteins have uses such as, for example, in ex vivo therapy or gene therapy, to engineer cells expressing a BLyS polypeptide that is retained on the surface of the engineered cells.
  • antibodies of the invention bind BLyS polypeptides in which Cys-146 of the BLyS sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant BLyS polypeptide when expressed in an expression system.
  • antibodies of the invention bind BLyS polypeptides in which Cys-146 is replaced with a serine amino acid residue.
  • antibodies of the invention bind BLyS polypeptides in which Cys-232 of the BLyS sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant BLyS polypeptide when expressed in an expression system.
  • antibodies of the invention bind BLyS polypeptides in which Cys-232 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.
  • antibodies of the invention bind BLyS polypeptides in which Cys-245 of the BLyS sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant BLyS polypeptide when expressed in an expression system.
  • antibodies of the invention bind BLyS polypeptides in which Cys-245 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.
  • the polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of the BLyS polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • the antibodies of the present invention bind BLyS polypeptides including the complete polypeptide encoded by the deposited cDNA (ATCC Deposit No. 97768) including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ED NO:3228, the mature soluble polypeptide of SEQ ID NO:3228, e.g., amino acids 134-285 of SEQ ID NO:3228, the extracellular domain of SEQ -ID NO:3228, amino acid residues 73-285 of SEQ ID NO:3228 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%
  • polypeptides encoding these polypeptides are also encompassed by the invention.
  • the antibodies of the present invention bind BLyS polypeptides including the complete polypeptide encoded by the deposited cDNA including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA (ATCC Deposit No.
  • the mature soluble polypeptide encoded by the deposited cDNA the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ED NO:3229, the mature soluble of SEQ ID NO:3229, e.g., amino acid residues 134-266 of SEQ ID NO:3229, the extracellular domain of SEQ ID NO:3229, e.g., amino acid residues 73-266 of SEQ ID NO:3229 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • polypeptides of the present invention bind polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC Deposit No. 97768) or to the polypeptide of SEQ ID NO:3228, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC Deposit No. 203518) or to the polypeptide of SEQ ED NO:3229, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • % similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a BLyS polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the BLyS polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of SEQ ED NO:3228, the amino acid sequence encoded by the deposited cDNA clone HNEDU15 (ATCC Accession No.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Bratlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. h another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • the present invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to BLyS polypeptides, which antibodies comprise, or alternatively consist of, all or a portion of a heavy and/or light chain variable domain of the scFvs referred to in Table 1.
  • the present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an animal, preferably a mammal, and most preferably a human, comprising using antibodies (including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to BLyS.
  • Diseases and disorders which can be detected, diagnosed or prognosed with the antibodies of the invention 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), infectious diseases ⁇ e.g., AIDS), and proliferative disorders ⁇ e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases ⁇ e.g., AIDS
  • proliferative disorders ⁇ e.g., leukemia,
  • the present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant BLyS or BLyS receptor expression or inappropriate BLyS or BLyS receptor function in an animal, preferably a mammal, and most preferably a human, comprising 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 BLyS.
  • antibodies including molecules which comprise, or alternatively consist of, antibody fragments or variants thereof
  • Diseases and disorders which can be prevented, treated or inhibited by administering an effective amount of one or more antibodies or molecules of the invention 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), infectious diseases ⁇ e.g., AIDS), and proliferative disorders ⁇ e.g., leukemia, carcinoma, and lymphoma).
  • 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
  • infectious diseases ⁇ e.g., AIDS
  • proliferative disorders
  • Anti-BLvS Antibodies [0194] The antibodies of the present invention were discovered, in part, using phage display technology. Single chain antibody molecules (“scFvs”) displayed on the surface of phage particles were screened to identify those scFvs that immunospecifically bind to BLyS, including the membrane-bound form and soluble form of BLyS.
  • scFvs Single chain antibody molecules
  • the present invention encompasses the scFvs and portions thereof that were identified to immunospecifically bind to BLyS, including scFvs that immunospecifically bind to the soluble form of BLyS, scFvs that immunospecifically bind to the membrane-bound form of BLyS, and scFvs that immunospecifically bind to both the soluble form and membrane- bound form of BLyS.
  • the present invention encompasses scFvs comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NOS: 1 - 2128, as referred to in Table 1.
  • the scFvs of the present invention comprise, or alternatively consist of, the amino acid sequence of SEQ ID NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908.
  • the scFvs include scFvs that bind to soluble BLyS (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ED NOS: 1563 - 1880), scFvs that bind to the membrane-bound form of BLyS (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ED NOS: 1881 - 2128), and scFvs that bind to both the soluble form and the membrane-bound form of BLyS (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1 - 1562).
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • scFvs that immunospecifically bind to BLyS comprise a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1 and/or any one of the VL domains referred to in Table 1.
  • scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to BLyS comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs referred to in Table 1 and/or any one, two, three, or more of the VL CDRs referred to in Table 1.
  • scFvs of the present invention comprise the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1.
  • scFvs of the present invention comprise the amino acid sequence of a NH CDR and NL CDR from different scFvs referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, antibody fragments or variants of the scFvs referred to in Table 1 that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • an scFv that immunospecifically binds to a soluble form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ID ⁇ OS1563 - 1880 as referred to in Table 1.
  • an scFv that immunospecifically binds to a soluble form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ED NOS: 1570 - 1595.
  • an scFv that immunospecifically binds to a soluble form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1563 - 1569.
  • an scFv that immunospecifically binds to a membrane-bound form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ED NOS:1881 - 2128 as referred to in Table 1.
  • an scFv that immunospecifically binds to a membrane- bound form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ED NOS: 1886 - 1908.
  • an scFv that immunospecifically binds to a membrane-bound form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 1881 - 1885.
  • an scFv that immunospecifically binds to both the soluble form and membrane-bound form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ED NOS:l - 1562 as referred to in Table 1.
  • an scFv that immunospecifically binds to both the soluble form and membrane-bound form of BLyS comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 834 - 872.
  • an scFv that immunospecifically binds to both the soluble form and membrane-bound form of BLyS comprises, or alternatively consists of, any one of the amino acids sequences of SEQ ED NOS:l - 46 or 321 - 329.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to the soluble form of BLyS and/or the membrane-bound form of BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • scFvs that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS: 1563 - 1880 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ED NOS1563 - 1880 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs SEQ ED NOS:1563 - 1880 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in contained SEQ ID NOS:1563 - 1880, as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the of the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS: 1563 - 1880 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ED NOS: 1563 - 1880 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to BLyS, preferably the soluble form of BLyS, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • scFvs that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ED NOS: 1881 - 2128 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS: 1881 - 2128 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ED NOS: 1881 - 2128 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1881 - 2128 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ED NOS: 1881 - 2128 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ED NOS: 1881 - 2128 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to BLyS, preferably the membrane-bound form of BLyS, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • scFvs that immunospecifically bind to the soluble form and membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:l - 1562 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ED NOS:l - 1562 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form and membrane- bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the soluble form and membrane- bound form of BLyS comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ED NOS:l - 1562 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS:l - 1562 as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble form and membrane-bound form of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of BLyS comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1.
  • scFvs that immunospecifically bind to the soluble and membrane-bound forms of BLyS comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS:l - 1562 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ED NOS:l - 1562, as disclosed in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of BLyS comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1.
  • scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of BLyS, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs or molecules, that immunospecifically bind to BLyS, preferably the soluble and membrane-bound forms of BLyS, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
  • the present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS.
  • the invention provides antibodies corresponding to the scFvs referred to in Table 1, such scFvs may routinely be "converted" to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 20, infra.
  • the invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one of the VH domains contained in the sequences referred to in Table 1.
  • the present invention also provides antibodies that immunospecifically bind to a polypeptide, or polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VH CDRs contained in the sequences referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that immunospecifically bind to BLyS or a BLyS fragment are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants.
  • antibodies that immunospecifically bind BLyS, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR referred to in Table 1.
  • the invention provides antibodies that immunospecifically bind BLyS, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VH CDRl contained in SEQ ED NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1.
  • antibodies that immunospecifically bind BLyS comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR2 contained in SEQ ED NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1.
  • antibodies that immunospecifically bind BLyS comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ED NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1.
  • antibodies that immunospecifically bind BLyS comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDRl contained in SEQ DD NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table 1; a VH CDR2 contained in SEQ ID NOS: SEQ ID NOS: SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908; and/or a VH CDR3 contained in SEQ ID NOS: SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table 1.
  • antibodies of the invention comprise, or alternatively consist of, VH CDRs that are derived from the same scFv as disclosed in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • the present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide, or polypeptide fragment of BLyS.
  • the invention provides antibodies wherein said antibodies comprise, or alternatively consist of, a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1.
  • the present invention also provides antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, a VL CDR having an amino acid sequence of any one, two, three, or more of the VL CDRs contained in the sequences referred to in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • antibodies that immunospecifically bind BLyS, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR referred to in Table 1.
  • the invention provides antibodies that immunospecifically bind BLyS, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VL CDRl contained in SEQ ID NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1.
  • antibodies that immunospecifically bind BLyS comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR2 contained in SEQ ED NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 as disclosed in Table 1.
  • antibodies comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ED NOS: in SEQ ID NOS:l - 46, 321 - 329, 1563 - 1569, or 1881 - 1885 disclosed in Table 1.
  • antibodies that immnospecifically bind BLyS comprise, or alternatively consist of: a polypeptide having the amino acid sequence of a VL CDRl contained in SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table 1; a VL CDR2 SEQ ID NOS :834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table 1; and a VL CDR3 contained SEQ ID NOS:834 - 872, 1570 - 1595, or 1886 - 1908 as disclosed in Table 1.
  • antibodies of the invention comprise, or alternatively consist of, VL CDRs that are derived from the same scFv as disclosed in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • the present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, a VH domain of one of the scFvs referred to in Table 1 combined with a VL domain of one of the scFvs referred to in Table 1, or other VL domain.
  • the present invention further provides antibodies (including molecules comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, a VL domain of one of the scFvs referred to in Table 1 combined with a VH domain of one of the scFvs referred to in Table 1, or other VH domain.
  • antibodies that immunospecifically bind to a polypeptide or a polypeptide fragment of BLyS comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH domain contained SEQ ED NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1 and a VL domain contained in contained SEQ ED NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1.
  • the antibodies of the invention comprise, or alternatively consist of, a VH and a VL domain from the same scFv as disclosed in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • the present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as referred to in Table 1.
  • the invention provides for antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of BLyS, wherein said antibodies comprise, or alternatively consist of, a VH CDRl and a VL CDRl, a VH CDRl and a VL CDR2, a VH CDRl and a VL CDR3, a VH CDR2 and a VL CDRl, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDRl, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VL CDRs referred to in Table 1.
  • one or more of these combinations are from the same scFv as disclosed in Table 1.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to BLyS are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • antibody encompasses not only whole antibody molecules, but also antibody fragments, as well as variants (including derivatives) of antibodies and antibody fragments.
  • Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, single chain Fvs (scFvs), Fab fragments, F(ab') 2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • the immunoglobulin molecules of the invention can be of any type ⁇ e.g., IgG, IgE, IgM, IgD, IgA and IgY), class ⁇ e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • the antibodies of the present invention also include molecules comprising, or alternatively consisting of, a polypeptide having an amino acid sequence of a portion of an amino acid sequence contained SEQ ED NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908.
  • an antibody of the invention comprises, or alternatively consists of, a polypeptide having an amino acid sequence of a VH domain, VH CDR, VL domain, or VL CDR of any one those contained in the sequences referred to in Table 1.
  • Antibodies of the invention also include molecules comprising, or alternatively consisting of, fragments or variants of the above antibodies that immunospecifically bind BLyS.
  • the antibodies of the present invention are whole antibodies or antibody fragments that immunospecifically bind human BLyS.
  • Antibody fragments of the invention that immunospecifically bind human BLyS include, but are not limited to, Fab, Fab' and F(ab')2, Fd fragments, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFvs), fragments comprising, or alternatively consisting of, either a VL or VH domain, and epitope binding fragments of any of the above.
  • BLyS-binding antibody fragments, including single-chain antibodies may comprise, or alternatively consist of, the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention comprise, or alternatively consist of, a polypeptide that immunospecifically binds to BLyS, said polypeptides comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs referred to in Table 1, preferably a polypeptide having an amino acid sequence of a VH CDR3 and/or a VL CDR3 of contained SEQ ID NOS:l - 46, 321 - 329, 834 - 872, 1563 - 1595, or 1881 - 1908 as disclosed in Table 1.
  • antibodies of the invention comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs from the same scFv, as referred to in Table 1.
  • the antibodies of the invention may be from any animal origin, including birds and mammals.
  • the antibodies are human, murine ⁇ e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.
  • the antibodies are human antibodies.
  • "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.
  • 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.
  • the antibodies of the present invention may be monovalent, bivalent, trivalent or multivalent.
  • 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.).
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a BLyS polypeptide, or fragment thereof, or may be specific for both a BLyS polypeptide, or fragment thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • a heterologous epitope such as a heterologous polypeptide or solid support material.
  • the antibodies of the invention may bind immunospecifically to murine BLyS (e.g., a polypeptide having the amino acid sequence of human BLyS (SEQ ID NOS:3228 and/or 3229) or BLyS expressed on human monocytes; murine BLyS (SEQ ED NOS:3230 and/or 3231) or BLyS expressed on murine monocytes; rat BLyS (either the soluble forms as given in SEQ ED NOS:3232, 3233, 3234 and or 3235 or in a membrane associated form, e.g., on the surface of rat monocytes); or monkey BLyS (e.g., the monkey BLyS polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey BLyS, or BLyS expressed on monkey monocytes), preferably the antibodies of the invention bind
  • murine BLyS e.g., a polypeptide having the amino acid sequence
  • the antibodies of the invention bind immunospecifically to human and monkey BLyS. Also preferably, the antibodies of the invention bind immunospecifically to human BLyS and murine BLyS. More preferably, antibodies of the invention, bind immunospecifically and with higher affinity to human BLyS than to murine BLyS.
  • Antibodies of the present invention 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 invention 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 invention are also included in the present invention. In a specific embodiment, antibodies of the present invention cross react with APRIL (SEQ ED NO:3239; GenBank Accession No. AF046888; J. Exp.
  • antibodies of the present invention 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 invention are also included in the present invention.
  • 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.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under hybridization conditions as described herein.
  • antibodies of the present invention cross react with APRIL (SEQ ED NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902).
  • APRIL SEQ ED NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902.
  • antibodies that immunospecifically bind both BLyS and APRIL comprise all or a portion the BAB2001, BAB2080, BAB2015, BAB2019, BAB2087, BAB2016, BAB2034 or BAB2065 scFVs (SEQ ID NOS:3240-3247).
  • scFvs were isolated by panning a phage scFv library comprising VH and VL domains obtained from human bone marrow B cells (BM library). Phage from the BM phage library were first selected for binding to soluble BLyS (amino acids 134-285 of SEQ ID NO:3228). A second round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the BLyS binding phage selected in round one. A third round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the phage selected in round two.
  • a final (fourth) round of selection for binding to the soluble form of BLyS (amino acids 134-285 of. SEQ ID NO:3228) was then performed on the phage selected in round three.
  • Phage clones that bound BLyS in the fourth round of selection were eluted with either 0.1M triethylamine (TEA) or with a TACI-Fc fusion protein (e.g., the extracellular domain of TACI (amino acids 31 to 159 of Genbank Accession No. AAC51790) fused to Fc).
  • Eluted Phage were collected and sequenced (SEQ ED NOS:3240-3247). Of 79 sequences, there were 8 unique sequences (SEQ ID NOS:3240-3247).

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Abstract

la présente invention concerne des anticorps et molécules connexes qui se lient à BLyS selon un mode immunospécifique. Elle concerne également des méthodes et des compositions permettant de détecter ou de diagnostiquer une pathologie ou des troubles en rapport avec une expression aberrante de BLyS, ou avec une fonction inappropriée de BLyS, Ces compositions renferment des anticorps ou des fragments ou des variants de BLyS, ou bien des molécules connexes qui se lient à BLyS selon un mode immunospécifique. Cette invention concerne en outre des méthodes ou des compositions permettant de prévenir, de traiter ou d'atténuer une pathologie ou des troubles en rapport avec une expression aberrante de BLyS, ou avec une fonction inappropriée de BLyS, par administration à un animal d'une dose efficace d'un ou plusieurs anticorps ou fragments d'anticorps, ou bien de molécules connexes se liant à BLyS selon un mode immunospécifique.
PCT/US2002/036496 2001-11-16 2002-11-14 Anticorps se liant à blys selon un mode immunospécifique WO2003055979A2 (fr)

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AU2002364954A AU2002364954A1 (en) 2001-11-16 2002-11-14 ANTIBODIES THAT IMMUNOSPECIFICALLY BIND TO BLyS
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US7083785B2 (en) 1999-08-17 2006-08-01 Biogen Idcc MA Inc. Methods of treatment by administering an anti-BCMA antibody
US7112421B2 (en) 2000-09-18 2006-09-26 Biogen Idec Ma Inc. Nucleic acids encoding BAFF receptor, chimeric proteins and methods and compositions related thereto
US7276241B2 (en) 1999-10-06 2007-10-02 Biogen Idec Ma Inc. Methods of treating a tumor that expresses APRIL by administering BCMA
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US7700317B2 (en) 2003-03-28 2010-04-20 Biogen Idec Ma Inc. Truncated baff receptors
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US9545086B2 (en) 1999-01-25 2017-01-17 Biogen Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response and treatment of autoimmune disorders
US9718883B2 (en) 2003-09-10 2017-08-01 Amgen Fremont Inc. Antibodies to M-CSF
US9726673B2 (en) 2005-11-23 2017-08-08 Genentech, Inc. Methods and compositions related to B cell assays
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US8231873B2 (en) 1996-10-25 2012-07-31 Human Genome Sciences, Inc. Methods of treatment using antibodies to Neutrokine-alpha
US8303951B2 (en) 1996-10-25 2012-11-06 Human Genome Sciences, Inc. Neutrokine-alpha antibodies and methods of use thereof
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US6869605B2 (en) 1999-01-25 2005-03-22 Biogen Idec Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response
US8212004B2 (en) 1999-03-02 2012-07-03 Human Genome Sciences, Inc. Neutrokine-alpha fusion proteins
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US7276241B2 (en) 1999-10-06 2007-10-02 Biogen Idec Ma Inc. Methods of treating a tumor that expresses APRIL by administering BCMA
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WO2005012531A2 (fr) * 2003-08-01 2005-02-10 Genentech, Inc. Polypeptides de liaison avec sequences de diversite restreinte
WO2005012531A3 (fr) * 2003-08-01 2005-10-27 Genentech Inc Polypeptides de liaison avec sequences de diversite restreinte
US10280219B2 (en) 2003-09-10 2019-05-07 Amgen Fremont Inc. Antibodies to M-CSF
US9718883B2 (en) 2003-09-10 2017-08-01 Amgen Fremont Inc. Antibodies to M-CSF
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WO2005095461A3 (fr) * 2004-03-31 2006-12-14 Canon Kk Protéine de liaison or et utilisation en conséquence
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US7871614B2 (en) 2004-03-31 2011-01-18 Canon Kabushiki Kaisha Gold-binding protein and use thereof
US7833731B2 (en) 2004-03-31 2010-11-16 Canon Kabushiki Kaisha Gold-binding protein and use thereof
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US7807391B2 (en) 2004-03-31 2010-10-05 Canon Kabushiki Kaisha Gold-binding protein and use thereof
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US8246953B2 (en) 2007-10-12 2012-08-21 Novartis Ag Compositions and methods for use for antibodies against sclerostin
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WO2011109280A1 (fr) 2010-03-05 2011-09-09 Lerner Research Institute Procédés et compositions permettant de traiter les troubles d'origines immunes
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WO2014139425A1 (fr) * 2013-03-13 2014-09-18 江苏先声药物研究有限公司 Anticorps monoclonal anti-blys et composition pharmaceutique contenant l'anticorps
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US11203636B2 (en) 2017-02-01 2021-12-21 Yale University Treatment of existing left ventricular heart failure
US11384143B2 (en) 2018-01-05 2022-07-12 Novo Nordisk A/S Methods for treating IL-6 mediated inflammation without immunosuppression

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CA2467521A1 (fr) 2003-07-10
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AU2002364954A1 (en) 2003-07-15
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