Antibodies that immunospecifically bind to B lymphocyte stimulator protein

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) based on the following U.S. Provisional Applications: 60/331,469 filed Nov. 16, 2001 and 60/340,817 filed Dec. 19, 2001; and this application is a continuation-in-part of, and claims the benefit of priority under 35 U.S.C. § 120 of U.S. patent application Ser. No. 09/880,748 filed Jun. 15, 2001 which claims benefit under 35 U.S.C. § 119(e) based on the following U.S. Provisional Applications: 60/212,210 filed Jun. 16, 2000; 60/240,816 filed Oct. 17, 2000; 60/276,248 filed Mar. 16, 2001; 60/277,379 filed Mar. 21, 2001; and 60/293,499 filed May 25, 2001. Each of the applications identified above is herein incorporated by reference in their entireties.

[0002] The present invention relates to antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator (BLyS™) protein. The present invention also relates to methods and compositions for detecting, diagnosing, or prognosing a disease or disorder associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, comprising antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to B Lymphocyte Stimulator. The present invention further relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator function or B Lymphocyte Stimulator 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 B Lymphocyte Stimulator.

[0003] B Lymphocyte Stimulator (BLyS™) protein is a member of the tumor necrosis factor (“TNF”) superfamily that induces both in vivo and in vitro B cell proliferation and differentiation (Moore et al., Science 285: 260–263 (1999)). B Lymphocyte Stimulator 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. B Lymphocyte Stimulator expression is not detected on natural killer (“NK”) cells, T cells or B cells, but is restricted to cells of myeloid origin. B Lymphocyte Stimulator expression on resting monocytes is upregulated by interferon-gamma (IFN-gamma). The gene encoding B Lymphocyte Stimulator has been mapped to chromosome 13q34.

[0004] B Lymphocyte Stimulator is expressed as a 285 amino acid type II membrane-bound polypeptide and a soluble 152 amino acid polypeptide (Moore et al., 1999 supra). The membrane-bound form of B Lymphocyte Stimulator has a predicted transmembrane spanning domain between amino acid residues 47 and 73. The NH₂-terminus of the soluble form of B Lymphocyte Stimulator begins at Ala¹³⁴ of the membrane-bound form of B Lymphocyte Stimulator. Soluble recombinant B Lymphocyte Stimulator 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 B Lymphocyte Stimulator administration to mice has been shown to result in an increase in the proportion of CD45R^dull, Ly6D^bright (also known as ThB) B cells and an increase in serum IgM and IgA levels (Moore et al., 1999 supra). Thus, B Lymphocyte Stimulator displays a B cell tropism in both its receptor distribution and biological activity.

[0005] Levels of B Lymphocyte Stimulator protein have been found to be elevated in patients with autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren'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). Furthermore, administration of a soluble form of a B Lymphocyte Stimulator receptor, TACI, has been shown to alleviate the autoimmune phenotype of NZBWF1 and MRL-lpr/lpr mice (Gross et al., Nature, (2000) 404:995–999). Thus, antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator may find medical utility in, for example, the treatment of B cell disorders associated with autoimmunity. In other embodiments, antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator may find medical utility in for example, neoplasia or immunodeficiency syndromes.

[0006] 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 B Lymphocyte Stimulator. In particular, 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 B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (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 B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes), preferably human B Lymphocyte Stimulator. The present invention also encompasses methods and compositions for detecting, diagnosing, or prognosing diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator 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 B Lymphocyte Stimulator. 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). The present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate function of B Lymphocyte Stimulator or B Lymphocyte Stimulator 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 B Lymphocyte Stimulator. 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).

[0007] Using phage display technology, the present inventors have identified single chain antibody molecules (“scFvs”) that immunospecifically bind to B Lymphocyte Stimulator, including scFvs that immunospecifically bind to soluble B Lymphocyte Stimulator, scFvs that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator, and scFvs that immunospecifically bind to both the soluble form and the membrane-bound form of B Lymphocyte Stimulator. Antibodies of the present invention are defined as able to bind the membrane bound and/or soluble forms of B Lymphocyte Stimulator 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 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

[0008] In particular, the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOS: 1–2128, preferably SEQ ID NOS:834–872, 1570–1595, and 1886–1908, and most preferably SEQ ID NOS:1–46, 321–329, 1563–1569, and 1881–1885, as referred to in Table 1 below. In specific embodiments, the present invention relates to scFvs that immunospecifically bind the soluble form of B Lymphocyte Stimulator, 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. In other embodiments, the present invention also relates to scFvs that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator, 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 B Lymphocyte Stimulator, 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 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

[0009] 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 B Lymphocyte Stimulator, 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 (“VL”) domains referred to in Table 1. In a preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH domain contained in SEQ ID NOS:1–46, 321–329, 834–872, 1563–1595, or 1881–1908, as referred to in Table 1 below. In another preferred embodiment, 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 VL domain contained SEQ ID NOS:1–46, 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0010] 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 B Lymphocyte Stimulator, 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. In a preferred embodiment, 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. In another preferred embodiment, antibodies of the present invention, comprise, or alternatively consist of, a VH domain from an scFv of SEQ ID NOS:1–46, 321–329, 834–872, 1563–1595, or 1881–1908, as disclosed in Table 1, and a VL domain from an scFv SEQ ID NOS:1–46, 321–329, 834–872, 1563–1595, or 1881–1908, as disclosed in Table 1. In another preferred embodiment, antibodies of the present invention comprise, or alternatively consist of, the VH and VL domain from a single scFv of SEQ ID NOS:1–46, 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0011] 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 B Lymphocyte Stimulator, 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. In one embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR1s referred to in Table 1 and/or any one of the VL CDR1s referred to in Table 1. In another embodiment, 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. In a preferred embodiment, 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0012] In another embodiment, antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, and comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VH CDR1s 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. In another embodiment, antibodies of the present invention comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one of the VL CDR1s 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. In a preferred embodiment, 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 CDR1, CDR2, and CDR3 of the VL domain correspond to the same scFv or where CDR1, 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0013] 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 B Lymphocyte Stimulator (e.g., a polypeptide consisting of amino acids 134–285 of SEQ ID NO:3228); that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator (e.g., a polypeptide consisting of amino acids 1–285 of SEQ ID NO:3228 or a B Lymphocyte Stimulator polypeptide expressed on the surface of monocytes) and/or that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator. In a preferred embodiment, antibodies of the present invention immunospecifically bind to the soluble form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the soluble form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the present invention immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator. In yet another preferred embodiment, antibodies of the present invention immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator and comprise, or alternatively consist of, a VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, and/or VL CDR3 corresponding to one or more scFvs, that immunospecifically binds to the soluble form and membrane-bound form of B Lymphocyte Stimulator. In another preferred embodiment, 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, or both the soluble form and membrane-bound form of B Lymphocyte Stimulator. Nucleic acid molecules encoding these antibodies are also encompassed by the invention. 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 B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the soluble form and membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0014] The present invention also provides antibodies (including molecules comprising or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to both B Lymphocyte Stimulator and APRIL (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. In a preferred embodiment, 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. In another preferred embodiment, antibodies of the present invention that immunospecifically bind to both B Lymphocyte Stimulator 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. In another preferred embodiment, antibodies of the present invention that immunospecifically bind to both B Lymphocyte Stimulator 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 B Lymphocyte Stimulator and APRIL, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0015] 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 B Lymphocyte Stimulator 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. In a preferred embodiment, the antibodies of the invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator 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. In another preferred embodiment, antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator 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. In another preferred embodiment, antibodies of the present invention that immunospecifically bind heterotrimeric protein comprising at least one B Lymphocyte Stimulator 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 B Lymphocyte Stimulator polypeptide, are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies, and/or molecules.

[0016] A VH domain of an amino acid sequence disclosed herein may be combined with a VL domain of an amino acid sequence disclosed herein, or other VL domains, to provide a VH/VL pairing representing an antigen-binding site of an antibody. Similarly, a VL domain of an amino acid sequence disclosed herein may be combined with a VH domain of an amino acid sequence disclosed herein, or other VH domains. Further, one or more CDRs disclosed herein may be taken from a VH or VL domain and incorporated into a suitable framework as discussed infra.

[0017] 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 B Lymphocyte Stimulator (e.g., soluble B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator) and can be routinely assayed for immunospecific binding to B Lymphocyte Stimulator using methods known in the art, such as, for example, the immunoassays disclosed infra. 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 (including antibody fragments, and variants and derivative thereof) can be routinely made by methods known in the art. Molecules comprising, or alternatively consisting of, fragments or variants of any of the VH domains, VH CDRs, VL domains, and VL CDRs whose sequences are specifically disclosed herein may be employed in accordance with the present invention. Nucleic acid molecules encoding these antibodies and molecules (including fragments, variants, and derivatives) are also encompassed by the invention.

[0018] 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). 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. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

[0019] 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. Alternatively, 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.

[0020] 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. These and other aspects of the invention are described in further detail below.

[0021] The present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator 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 B Lymphocyte Stimulator. 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).

[0022] In specific embodiments, 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).

[0023] The present invention further encompasses methods and compositions for preventing, treating or ameliorating diseases or disorders associated with aberrant B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expression or inappropriate B Lymphocyte Stimulator or B Lymphocyte Stimulator 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 B Lymphocyte Stimulator. 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).

[0024] In specific embodiments, the present invention encompasses methods and compositions (e.g., antagonistic anti-B Lymphocyte Stimulator antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypergammaglobulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiency syndromes). In other specific embodiments, the present invention encompasses methods and compositions (e.g., agonistic anti-B Lymphocyte Stimulator antibodies) for preventing, treating or ameliorating diseases or disorders associated with hypogammaglobulinemia (e.g., an immunodeficiency syndrome).

[0025] 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 encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjögren's syndrome, diabetes mellitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders).

[0026] 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-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

Definitions

[0027] The term “antibody,” as used herein, 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. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. Examples of molecules which are described by the term “antibody” in this application include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody. Antibodies that immunospecifically bind to B Lymphocyte Stimulator may have cross-reactivity with other antigens. Preferably, antibodies that immunospecifically bind to B Lymphocyte Stimulator do not cross-react with other antigens. Antibodies that immunospecifically bind to B Lymphocyte Stimulator 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.

[0028] 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., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule.

[0029] Antibodies of the invention may also include multimeric forms of antibodies. For example, 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 within an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, 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. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509–7514, which is hereby incorporated by reference in its entirety. 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.

[0030] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-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. and Frigerio et al., (2000) Plant Physiology 123:1483–94, both of which are hereby incorporated by reference in their entireties.) 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.

[0031] Unless otherwise defined in the specification, specific binding or immunospecific binding by an anti-B Lymphocyte Stimulator antibody means that the anti-B Lymphocyte Stimulator antibody binds B Lymphocyte Stimulator but does not significantly bind to (i.e., cross react with) proteins other than B Lymphocyte Stimulator, such as other proteins in the same family of proteins, e.g., other TNF family ligands). An antibody that binds B Lymphocyte Stimulator 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 B Lymphocyte Stimulator-specific antibody of the invention preferentially binds B Lymphocyte Stimulator 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). Given that B Lymphocyte Stimulator-specific antibodies bind to epitopes of B Lymphocyte Stimulator, an antibody that specifically binds B Lymphocyte Stimulator may or may not bind fragments of B Lymphocyte Stimulator and/or variants of B Lymphocyte Stimulator (e.g., proteins that are at least 90% identical to B Lymphocyte Stimulator) depending on the presence or absence of the epitope bound by a given B Lymphocyte Stimulator-specific antibody in the B Lymphocyte Stimulator fragment or variant. Likewise, B Lymphocyte Stimulator-specific antibodies of the invention may bind species orthologues of B Lymphocyte Stimulator (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, B Lymphocyte Stimulator-specific antibodies of the invention may bind modified forms of B Lymphocyte Stimulator, for example, B Lymphocyte Stimulator fusion proteins. In such a case when antibodies of the invention bind B Lymphocyte Stimulator fusion proteins, the antibody must make binding contact with the B Lymphocyte Stimulator moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to B Lymphocyte Stimulator 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.

[0032] Furthermore, in the present application certain antibodies may be specific for either the membrane bound form of B Lymphocyte Stimulator, or the soluble form of B Lymphocyte Stimulator (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 B Lymphocyte Stimulator according to the assays described in Examples 1 through 19.

[0033] Preferably, 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.

[0034] An antibody of the invention “which binds the soluble form of B Lymphocyte Stimulator” is one which binds the 152 amino acid soluble form of the B Lymphocyte Stimulator 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 B Lymphocyte Stimulator” does not also bind the membrane-bound or membrane-associated form of B Lymphocyte Stimulator. Assays which measure binding to the soluble form of B Lymphocyte Stimulator include, but are not limited to, receptor binding inhibition assay or capture of soluble B Lymphocyte Stimulator from solution as described in Examples 8 and 9.

[0035] An antibody of the invention “which binds the membrane-bound form of B Lymphocyte Stimulator” is one which binds the membrane-associated (uncleaved) B Lymphocyte Stimulator protein. In specific embodiments of the invention, an antibody of the invention “which binds the membrane-bound form of B Lymphocyte Stimulator” does not also bind the soluble form of B Lymphocyte Stimulator. Binding to HIS-tagged B Lymphocyte Stimulator (as described herein) in an ELISA is an indicator that an antibody binds the membrane-bound form of B Lymphocyte Stimulator, but should not be relied upon as proof of specificity for the membrane-bound form of B Lymphocyte Stimulator. Assays that may be relied upon as proof of an antibody's specificity for membrane-bound B Lymphocyte Stimulator, include, but are not limited to, binding to plasma membranes expressing B Lymphocyte Stimulator as described in Example 2. An antibody of the invention “which binds the both the soluble form and the membrane-bound form of B Lymphocyte Stimulator” is one which binds both the membrane-bound form and the soluble form of B Lymphocyte Stimulator.

[0036] The term “variant” as used herein refers to a polypeptide that possesses a similar or identical function as a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, or possess a similar or identical structure of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator 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 B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator 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 B Lymphocyte Stimulator polypeptide (e.g., SEQ ID NO:3228), a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator 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, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator 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. A polypeptide with similar structure to a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quarternary structure of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an anti-B Lymphocyte Stimulator 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.

[0037] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

[0038] 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 algorithm. BLAST nucleotide searches can be performed with the BLASTn program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3389–3402(1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. (See http://www.ncbi.nlm.nih.gov.)

[0039] Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10:3–5(1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444–8(1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0040] The term “derivative” as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, an antibody that immunospecifically binds to B Lymphocyte Stimulator which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator 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 B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator 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. Further, a derivative of a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a B Lymphocyte Stimulator polypeptide, a fragment of B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody, described herein.

[0041] The term “epitopes” as used herein refers to portions of B Lymphocyte Stimulator having antigenic or immunogenic activity in an animal, preferably a mammal. An epitope having immunogenic activity is a portion of B Lymphocyte Stimulator that elicits an antibody response in an animal. An eptiope having antigenic activity is a portion of B Lymphocyte Stimulator 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.

[0042] The term “fragment” as used herein 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 B Lymphocyte Stimulator, or an anti-B Lymphocyte Stimulator antibody (including molecules such as scFv's, that comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically binds to B Lymphocyte Stimulator.

[0043] The term “fusion protein” as used herein refers to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an anti-B Lymphocyte Stimulator antibody of the invention and an amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain).

[0044] 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.

[0045] By “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.

[0063] The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator or a fragment or variant of B Lymphocyte Stimulator. In particular, the invention provides antibodies such as, for example, single chain Fvs (scFvs) having an amino acid sequence of any one of SEQ ID NOS:1–2128, as referred to in Table 1. In particular, the present invention encompasses antibodies that immunospecifically bind to a polypeptide, a polypeptide fragment or variant, or an epitope of human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or B Lymphocyte Stimulator expressed on human monocytes; murine B Lymphocyte Stimulator (SEQ ID NOS:3230 and/or 3231) or B Lymphocyte Stimulator expressed on murine monocytes; rat B Lymphocyte Stimulator (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 B Lymphocyte Stimulator (e.g., the monkey B Lymphocyte Stimulator polypeptides of SEQ ID NOS:3236 and/or 3237, the soluble form of monkey B Lymphocyte Stimulator, or B Lymphocyte Stimulator expressed on monkey monocytes) (as determined by immunoassays known in the art for assaying specific antibody-antigen binding).

[0064] The polypeptide sequence shown in SEQ ID NO:3228 was obtained by sequencing and translating the cDNA of the HNEDU15 clone which was deposited on Oct. 22, 1996 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110–2209, and assigned ATCC™ Accession No. 97768. The deposited clone is contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). 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.

[0065] The polypeptide sequence shown in SEQ ID NO:3229 was obtained by sequencing and translating the cDNA of the HDPMC52 clone, which was deposited on Dec. 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, Calif.). 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.

[0066] The B Lymphocyte Stimulator 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 B Lymphocyte Stimulator polypeptides of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the antibodies of the invention bind B Lymphocyte Stimulator 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 B Lymphocyte Stimulator.

[0067] Multimeric B Lymphocyte Stimulator bound by the antibodies of the invention may be homomers or heteromers. A B Lymphocyte Stimulator homomer, refers to a multimer containing only B Lymphocyte Stimulator polypeptides (including B Lymphocyte Stimulator fragments, variants, and fusion proteins, as described herein). These homomers may contain B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences. In specific embodiments, the antibodies of the invention bind a B Lymphocyte Stimulator homodimer (e.g., containing two B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences) or a B Lymphocyte Stimulator homotrimer (e.g., containing three B Lymphocyte Stimulator polypeptides having identical or different amino acid sequences). In a preferred embodiment, the antibodies of the invention bind homotrimers of B Lymphocyte Stimulator. In additional embodiments, the antibodies of the invention bind a homomeric B Lymphocyte Stimulator multimer which is at least a homodimer, at least a homotrimer, or at least a homotetramer.

[0068] Heteromeric B Lymphocyte Stimulator refers to a multimer containing heterologous polypeptides (i.e., polypeptides of a different protein) in addition to the B Lymphocyte Stimulator polypeptides of the invention. In a specific embodiment, the antibodies of the invention bind a B Lymphocyte Stimulator heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the antibodies of the invention bind a heteromeric B Lymphocyte Stimulator multimer which is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer. In highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising both B Lymphocyte Stimulator polypeptides and APRIL 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. In other highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising one B Lymphocyte Stimulator polypeptide (including fragments or variants) and two APRIL polypeptides (including fragments or variants). In still other highly preferred embodiments, the antibodies of the invention bind a heterotrimer comprising two B Lymphocyte Stimulator polypeptides (including fragments or variants) and one APRIL polypeptide (including fragments or variants). In a further nonexclusive embodiment, the heteromers bound by the antibodies of the invention contain CD40 ligand polypeptide sequence(s), or biologically active fragment(s) or variant(s) thereof.

[0069] In particularly preferred embodiments, the antibodies of the invention bind homomeric, especially homotrimeric, B Lymphocyte Stimulator polypeptides, wherein the individual protein components of the multimers consist of the mature form of B Lymphocyte Stimulator (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. In other specific embodiments, antibodies of the invention bind heteromeric, especially heterotrimeric, B Lymphocyte Stimulator polypeptides such as a heterotrimer containing two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide or a heterotrimer containing one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides, and wherein the individual protein components of the B Lymphocyte Stimulator heteromer consist of the mature extracellular soluble portion of either B Lymphocyte Stimulator (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.

[0070] In specific embodiments, the antibodies of the invention bind conformational epitopes of a B Lymphocyte Stimulator monomeric protein. In specific embodiments, the antibodies of the invention bind conformational epitopes of a B Lymphocyte Stimulator multimeric, especially trimeric, protein. In other embodiments, antibodies of the invention bind conformational epitopes that arise from the juxtaposition of B Lymphocyte Stimulator with a heterologous polypeptide, such as might be present when B Lymphocyte Stimulator forms heterotrimers (e.g., with APRIL polypeptides (e.g., SEQ ID SEQ ID NO:3239)), or in fusion proteins between B Lymphocyte Stimulator and a heterologous polypeptide.

[0071] In a specific embodiment, antibodies of the invention that specifically bind heterotrimers containing at least one B Lymphocyte Stimulator 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). In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides.

[0072] In a specific embodiment, antibodies of the invention that specifically bind heterotrimers containing at least one B Lymphocyte Stimulator 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 VL domain). The sequences of SEQ ID NOS: 3240–3247 are presented after Table 1 just prior to the claims. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise two B Lymphocyte Stimulator polypeptides and one APRIL polypeptide. In a specific embodiment, the heterotrimers containing at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide comprise one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides.

[0073] B Lymphocyte Stimulator 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. Thus, in one embodiment, B Lymphocyte Stimulator multimers, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, B Lymphocyte Stimulator heteromultimers, such as, for example, B Lymphocyte Stimulator heterotrimers or B Lymphocyte Stimulator heterotetramers, 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. In other embodiments, B Lymphocyte Stimulator multimers are formed by covalent associations with and/or between the B Lymphocyte Stimulator 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). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a B Lymphocyte Stimulator fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a B Lymphocyte Stimulator-Fc fusion protein. In another specific example, 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). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from CD40L, or a soluble fragment thereof. In another embodiment, two or B Lymphocyte Stimulator polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple B Lymphocyte Stimulator polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.

[0074] In one embodiment, antibodies of the invention immunospecifically bind a B Lymphocyte Stimulator 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. In another embodiment, the invention provides an antibody that binds an isolated B Lymphocyte Stimulator 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.

[0075] Antibodies of the invention that bind B Lymphocyte Stimulator polypeptides may bind them in as isolated polypeptides, in their naturally occurring state and/or their native conformation. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced by and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell. Thus, antibodies of the present invention may bind recombinantly produced B Lymphocyte Stimulator polypeptides.

[0076] Antibodies of the present invention may also bind B Lymphocyte Stimulator expressed on the surface of a cell, wherein said B Lymphocyte Stimulator 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. In certain embodiments, said B Lymphocyte Stimulator polypeptide expressed on the surface of a cell is a recombinant B Lymphocyte Stimulator polypeptide. In other embodiments, said B Lymphocyte Stimulator polypeptide expressed on the surface of the cell is a naturally occurring B Lymphocyte Stimulator polypeptide. As a non-limiting example, an antibody of the invention may bind a B Lymphocyte Stimulator expressed on the surface of the cell wherein Lys-132 and/or Arg-133 of the B Lymphocyte Stimulator 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 B Lymphocyte Stimulator from cells expressing B Lymphocyte Stimulator.

[0077] Antibodies of the present invention may also bind B Lymphocyte Stimulator secreted by a cell, wherein said B Lymphocyte Stimulator 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. In certain embodiments, said B Lymphocyte Stimulator polypeptide secreted by a cell is a recombinant B Lymphocyte Stimulator polypeptide. In other embodiments, said B Lymphocyte Stimulator polypeptide secreted by a cell is a naturally occurring B Lymphocyte Stimulator polypeptide.

[0078] 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.

[0079] Additionally, antibodies of the present invention bind polypeptides comprising or alternatively, consisting of, the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCC™ accession No. 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 No. 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.

[0080] In addition, 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.

[0081] In specific embodiments, 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. Representative examples of 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. Moreover, 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.

[0082] In specific embodiments, 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. In a specific embodiment, antibodies of the invention bind an epitope comprising amino acids 165–171 of SEQ ID NO:3228. In another embodiment, the CDRs of antibodies of the invention make contacts with one or more amino acids in the sequence of amino acids 165–171 of SEQ ID NO:3228. In another embodiment, antibodies of the invention whose CDRs make contact with one or more amino acids in the sequence of amino acids 165–171 of SEQ ID NO:3228 disrupt B Lymphocyte Stimulator-B Lymphocyte Stimulator receptor interactions.

[0083] It will be recognized by one of ordinary skill in the art that mutations targeted to regions of a B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 which encompass the nineteen amino acid residue insertion which is not found in the B Lymphocyte Stimulator polypeptide sequence of SEQ ID NO:3229 (i.e., amino acid residues Val-142 through Lys-160 of the sequence of SEQ ID NO:3229) may affect the observed biological activities of the B Lymphocyte Stimulator polypeptide. More specifically, a partial, non-limiting and non-exclusive list of such residues of the B Lymphocyte Stimulator polypeptide sequence which may be targeted for mutation includes the following amino acid residues of the B Lymphocyte Stimulator polypeptide sequence as shown in SEQ ID NO:3228: V-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; I-158; Q-159; and K-160. Thus, in specific embodiments, antibodies of the present invention that bind B Lymphocyte Stimulator polypeptides which have one or more mutations in the region from V-142 through K-160 of SEQ ID NO:3228 are contemplated.

[0084] 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. Representative examples of 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. Additional representative examples of 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. Moreover, 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. In this context, “about” means the particularly recited ranges and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or both the amino- and carboxy-termini.

[0085] Additional preferred embodiments encompass antibodies that bind polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of B Lymphocyte Stimulator (e.g., amino acid residues 1–46 of SEQ ID NO:3228), the predicted transmembrane domain of B Lymphocyte Stimulator (e.g., amino acid residues 47–72 of SEQ ID NO:3228), the predicted extracellular domain of B Lymphocyte Stimulator (e.g., amino acid residues 73–285 of SEQ ID NO:3228), the mature soluble extracellular domain of B Lymphocyte Stimulator (e.g., amino acids residues 134–285 of SEQ ID NO:3228), the predicted TNF conserved domain of B Lymphocyte Stimulator (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 B Lymphocyte Stimulator (amino acid residues 1–46 fused to amino acid residues 73–285 of SEQ ID NO:3228).

[0086] Further additional preferred embodiments encompass polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of B Lymphocyte Stimulator (amino acid residues 1–46 of SEQ ID NO:3229), the predicted transmembrane domain of B Lymphocyte Stimulator (amino acid residues 47–72 of SEQ ID NO:3229), the predicted extracellular domain of B Lymphocyte Stimulator (amino acid residues 73–266 of SEQ ID NO:3229), the predicted TNF conserved domain of B Lymphocyte Stimulator (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 B Lymphocyte Stimulator (amino acid residues 1–46 fused to amino acid residues 73–266 of SEQ ID NO:3229).

[0087] 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 B Lymphocyte Stimulator polypeptides of SEQ ID NO:3228 and SEQ ID NO:3229. These polypeptide fragments comprising the beta-pleated sheets of B Lymphocyte Stimulator 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 Ile-185, Gly-191 to Lys-204, His-210 to Val-219, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to Ile-263 and/or Val-276 to Leu-284 of SEQ ID NO:3228. In another, nonexclusive embodiment, these polypeptide fragments comprising the beta-pleated sheets of B Lymphocyte Stimulator 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.

[0088] 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 [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 [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 Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly-249 to Leu-285] of SEQ ID NO:3228. Other combinations of amino acids sequences that may be bound by the antibodies of the invention may include the 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). Other combinations of amino acids sequences that may be bound by the antibodies of the invention may also include 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-248] of SEQ ID NO:3228 fused to [Gly-249 to Leu-285] of SEQ ID NO:3228).

[0089] 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. Other of 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). Other combinations of amino acid sequences that may be bound by the antibodies of the invention may also include 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] of SEQ ID NO:3229.

[0090] Additional embodiments of the invention encompass antibodies that bind B Lymphocyte Stimulator polypeptide fragments comprising, or alternatively consisting of, functional regions of polypeptides of the invention, such as the Garnier-Robson alpha-regions, beta-regions, turn-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. In a preferred embodiment, 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) B Lymphocyte Stimulator polypeptide (e.g., SEQ ID NOS:3228 and 3229).

[0091] The data representing the structural or functional attributes of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 (Table 9) or the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229 (Table 10), as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. Column I represents the results of a Garnier-Robson analysis of alpha helical regions; Column II represents the results of a Chou-Fasman analysis of alpha helical regions; Column III represents the results of a Garnier Robson analysis of beta sheet regions; Column IV represents the results of a Chou-Fasman analysis of beta sheet regions; Column V represents the results of a Garnier Robson analysis of turn regions; Column VI represents the results of a Chou-Fasman analysis of turn regions; Column VII represents the results of a Garnier Robson analysis of coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column IX represents a Hopp-Woods hydrophobicity plot; Column X represents the results of an Eisenberg analysis of alpha amphipathic regions; Column XI represents the results of an Eisenberg analysis of beta amphipathic regions; Column XII represents the results of a Karplus-Schultz analysis of flexible regions; Column XIII represents the Jameson-Wolf antigenic index score; and Column XIV represents the Emini surface probability plot.

[0092] In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Tables 9 and 10 can be used to determine regions of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228 (Table 9) or the B Lymphocyte Stimulator 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.

[0093] 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 Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-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. Preferably, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides or B Lymphocyte Stimulator polypeptide fragments and variants comprising regions of B Lymphocyte Stimulator that combine several structural 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.

[0094]

TABLE 9
Res	Position	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII	XIV
Met	1	A	.	.	.	.	.	.	0.73	−0.71	.	.	.	0.95	1.39
Asp	2	A	.	.	.	.	T	.	1.12	−0.66	*	.	.	1.15	1.56
Asp	3	A	.	.	.	.	T	.	1.62	−1.09	*	.	.	1.15	2.12
Ser	4	A	.	.	.	.	T	.	2.01	−1.51	.	.	.	1.15	4.19
Thr	5	A	.	.	.	.	T	.	2.40	−2.13	.	.	F	1.30	4.35
Glu	6	A	A	.	.	.	.	.	2.70	−1.73	*	*	F	0.90	4.51
Arg	7	A	A	.	.	.	.	.	2.81	−1.34	*	*	F	0.90	4.51
Glu	8	A	A	.	.	.	.	.	2.00	−1.73	*	*	F	0.90	6.12
Gln	9	A	A	.	.	.	.	.	1.99	−1.53	*	*	F	0.90	2.91
Ser	10	A	.	.	B	.	.	.	2.00	−1.04	*	*	F	0.90	2.15
Arg	11	A	.	.	B	.	.	.	1.33	−0.66	*	*	F	0.90	1.66
Leu	12	A	.	.	B	.	.	.	0.41	−0.09	*	*	F	0.45	0.51
Thr	13	A	.	.	B	.	.	.	0.46	0.20	*	*	F	−0.15	0.32
Ser	14	A	A	.	.	.	.	.	0.50	−0.19	*	*	.	0.30	0.32
Cys	15	A	A	.	.	.	.	.	0.91	−0.19	*	*	.	0.30	0.78
Leu	16	A	A	.	.	.	.	.	0.80	−0.87	*	*	F	0.90	1.06
Lys	17	A	A	.	.	.	.	.	1.61	−1.36	.	*	F	0.90	1.37
Lys	18	A	A	.	.	.	.	.	1.32	−1.74	.	*	F	0.90	4.44
Arg	19	A	A	.	.	.	.	.	1.67	−1.70	.	*	F	0.90	5.33
Glu	20	A	A	.	.	.	.	.	1.52	−2.39	.	*	F	0.90	5.33
Glu	21	A	A	.	.	.	.	.	2.38	−1.70	.	*	F	0.90	2.20
Met	22	A	A	.	.	.	.	.	2.33	−1.70	.	*	F	0.90	2.24
Lys	23	A	A	.	.	.	.	.	1.62	−1.70	*	*	F	0.90	2.24
Leu	24	A	A	.	.	.	.	.	0.66	−1.13	*	*	F	0.75	0.69
Lys	25	A	A	.	.	.	.	.	0.36	−0.49	.	*	F	0.45	0.52
Glu	26	A	A	.	B	.	.	.	−0.53	−0.71	*	*	.	0.60	0.35
Cys	27	A	A	.	B	.	.	.	−0.74	−0.03	*	*	.	0.30	0.30
Val	28	A	A	.	B	.	.	.	−1.00	−0.03	*	*	.	0.30	0.12
Ser	29	A	A	.	B	.	.	.	−0.08	0.40	*	*	.	−0.30	0.11
Ile	30	A	.	.	B	.	.	.	−0.08	0.40	*	*	.	−0.30	0.40
Leu	31	A	.	.	B	.	.	.	−0.08	−0.17	*	.	.	0.45	1.08
Pro	32	.	.	.	B	.	.	C	0.29	−0.81	*	.	F	1.10	1.39
Arg	33	.	.	.	.	T	.	.	0.93	−0.81	.	*	F	1.50	2.66
Lys	34	.	.	.	.	T	.	.	0.93	−1.07	.	.	F	1.84	4.98
Glu	35	.	.	.	.	.	.	C	0.97	−1.37	*	*	F	1.98	4.32
Ser	36	.	.	.	.	.	T	C	1.89	−1.16	*	*	F	2.52	1.64
Pro	37	.	.	.	.	.	T	C	1.80	−1.16	*	*	F	2.86	1.60
Ser	38	.	.	.	.	T	T	.	1.39	−0.77	*	.	F	3.40	1.24
Val	39	A	.	.	.	.	T	.	1.39	−0.39	.	*	F	2.36	1.24
Arg	40	A	.	.	.	.	.	.	1.39	−0.77	*	*	F	2.46	1.60
Ser	41	A	.	.	.	.	.	.	1.34	−1.20	*	*	F	2.46	2.00
Ser	42	.	.	.	.	T	T	.	1.60	−1.16	.	*	F	3.06	2.67
Lys	43	.	.	.	.	T	T	.	1.09	−1.80	.	*	F	3.06	2.72
Asp	44	.	.	.	.	T	T	.	1.13	−1.11	*	*	F	3.40	1.67
Gly	45	A	.	.	.	.	T	.	0.43	−0.81	*	*	F	2.66	1.03
Lys	46	A	A	.	.	.	.	.	0.14	−0.70	.	.	F	1.77	0.52
Leu	47	A	A	.	.	.	.	.	0.13	−0.20	*	.	.	0.98	0.31
Leu	48	A	A	.	.	.	.	.	−0.72	0.29	*	.	.	0.04	0.46
Ala	49	A	A	.	.	.	.	.	−1.53	0.54	.	*	.	−0.60	0.19
Ala	50	A	A	.	.	.	.	.	−2.00	1.23	.	.	.	−0.60	0.19
Thr	51	A	A	.	.	.	.	.	−2.63	1.23	.	.	.	−0.60	0.19
Leu	52	A	A	.	.	.	.	.	−2.63	1.04	.	.	.	−0.60	0.19
Leu	53	A	A	.	.	.	.	.	−2.63	1.23	.	.	.	−0.60	0.15
Leu	54	A	A	.	.	.	.	.	−2.34	1.41	.	.	.	−0.60	0.09
Ala	55	A	A	.	.	.	.	.	−2.42	1.31	.	.	.	−0.60	0.14
Leu	56	A	A	.	.	.	.	.	−2.78	1.20	.	.	.	−0.60	0.09
Leu	57	A	.	.	.	.	T	.	−2.78	1.09	.	.	.	−0.20	0.06
Ser	58	A	.	.	.	.	T	.	−2.28	1.09	.	.	.	−0.20	0.05
Cys	59	A	.	.	.	.	T	.	−2.32	1.07	.	.	.	−0.20	0.09
Cys	60	A	.	.	.	.	T	.	−2.59	1.03	.	.	.	−0.20	0.08
Leu	61	.	.	B	B	.	.	.	−2.08	0.99	.	.	.	−0.60	0.04
Thr	62	.	.	B	B	.	.	.	−1.97	0.99	.	.	.	−0.60	0.11
Val	63	.	.	B	B	.	.	.	−1.91	1.20	.	.	.	−0.60	0.17
Val	64	.	.	B	B	.	.	.	−1.24	1.39	.	.	.	−0.60	0.33
Ser	65	.	.	B	B	.	.	.	−1.43	1.10	.	.	.	−0.60	0.40
Phe	66	A	.	.	B	.	.	.	−1.21	1.26	.	.	.	−0.60	0.40
Tyr	67	A	.	.	B	.	.	.	−1.49	1.11	.	.	.	−0.60	0.54
Gln	68	A	.	.	B	.	.	.	−1.44	0.97	.	.	.	−0.60	0.41
Val	69	A	.	.	B	.	.	.	−0.59	1.27	.	.	.	−0.60	0.39
Ala	70	A	.	.	B	.	.	.	−0.63	0.89	.	.	.	−0.60	0.43
Ala	71	A	.	.	B	.	.	.	0.07	0.56	.	*	.	−0.60	0.25
Leu	72	A	.	.	.	.	T	.	−0.50	0.16	.	*	.	0.10	0.55
Gln	73	A	.	.	.	.	T	.	−1.09	0.20	.	.	F	0.25	0.45
Gly	74	A	.	.	.	.	T	.	−0.53	0.20	.	.	F	0.25	0.45
Asp	75	A	.	.	.	.	T	.	−0.76	0.09	.	*	F	0.25	0.73
Leu	76	A	A	.	.	.	.	.	−0.06	0.09	.	*	F	−0.15	0.35
Ala	77	A	A	.	.	.	.	.	0.17	−0.31	.	*	.	0.30	0.69
Ser	78	A	A	.	.	.	.	.	0.17	−0.24	.	*	.	0.30	0.42
Leu	79	A	A	.	.	.	.	.	−0.30	−0.24	.	*	.	0.30	0.88
Arg	80	A	A	.	.	.	.	.	−0.30	−0.24	.	*	.	0.30	0.72
Ala	81	A	A	.	.	.	.	.	0.17	−0.34	.	*	.	0.30	0.93
Glu	82	A	A	.	.	.	.	.	0.72	−0.30	.	*	.	0.45	1.11
Leu	83	A	A	.	.	.	.	.	0.99	−0.49	.	*	.	0.30	0.77
Gln	84	A	A	.	.	.	.	.	1.21	0.01	.	*	.	−0.15	1.04
Gly	85	A	A	.	.	.	.	.	1.10	0.01	*	*	.	−0.30	0.61
His	86	A	A	.	.	.	.	.	1.73	0.01	*	*	.	−0.15	1.27
His	87	A	A	.	.	.	.	.	0.92	−0.67	.	*	.	0.75	1.47
Ala	88	A	A	.	.	.	.	.	1.52	−0.39	.	*	.	0.45	1.22
Glu	89	A	A	.	.	.	.	.	0.93	−0.39	.	.	.	0.45	1.39
Lys	90	A	A	.	.	.	.	.	0.93	−0.39	*	.	F	0.60	1.03
Leu	91	A	.	.	.	.	T	.	0.38	−0.46	*	.	.	0.85	1.01
Pro	92	A	.	.	.	.	T	.	0.07	−0.46	.	.	.	0.70	0.59
Ala	93	A	.	.	.	.	T	.	0.07	−0.03	.	.	.	0.70	0.29
Gly	94	A	.	.	.	.	T	.	−0.14	0.47	.	.	.	−0.20	0.36
Ala	95	A	.	.	.	.	.	.	−0.14	0.21	.	*	.	−0.10	0.36
Gly	96	A	.	.	.	.	.	.	0.08	−0.21	.	.	F	0.65	0.71
Ala	97	A	.	.	.	.	.	.	−0.06	−0.21	.	.	F	0.65	0.72
Pro	98	A	.	.	.	.	.	.	−0.28	−0.21	.	*	F	0.65	0.71
Lys	99	A	A	.	.	.	.	.	0.07	−0.03	.	.	F	0.45	0.59
Ala	100	A	A	.	.	.	.	.	0.66	−0.46	.	.	F	0.60	1.01
Gly	101	A	A	.	.	.	.	.	0.41	−0.96	.	.	F	0.90	1.13
Leu	102	A	A	.	.	.	.	.	0.79	−0.89	.	.	F	0.75	0.57
Glu	103	A	A	.	.	.	.	.	0.41	−0.46	*	.	F	0.45	0.88
Glu	104	A	A	.	.	.	.	.	−0.49	−0.46	*	.	F	0.45	0.89
Ala	105	A	A	.	.	.	.	.	−0.21	−0.24	.	.	.	0.30	0.81
Pro	106	A	A	.	.	.	.	.	−0.46	−0.44	.	.	.	0.30	0.67
Ala	107	A	A	.	.	.	.	.	0.01	0.06	.	.	.	−0.30	0.39
Val	108	A	A	.	.	.	.	.	−0.80	0.49	.	*	.	−0.60	0.38
Thr	109	A	A	.	.	.	.	.	−0.76	0.67	.	*	.	−0.60	0.20
Ala	110	A	A	.	.	.	.	.	−1.06	0.24	*	*	.	−0.30	0.40
Gly	111	A	A	.	.	.	.	.	−1.54	0.43	*	*	.	−0.60	0.38
Leu	112	A	A	.	.	.	.	.	−0.96	0.57	*	*	.	−0.60	0.23
Lys	113	.	A	B	.	.	.	.	−0.31	0.09	*	*	.	−0.30	0.39
Ile	114	.	A	B	.	.	.	.	−0.21	0.01	*	.	.	−0.30	0.61
Phe	115	.	A	B	.	.	.	.	−0.21	0.01	*	.	.	0.15	1.15
Glu	116	.	A	.	.	.	.	C	−0.08	−0.17	*	.	F	1.25	0.58
Pro	117	.	A	.	.	.	.	C	0.39	0.26	*	*	F	1.10	1.28
Pro	118	.	.	.	.	.	.	C	0.34	−0.00	.	.	F	2.20	1.47
Ala	119	.	.	.	.	.	T	C	0.89	−0.79	.	*	F	3.00	1.47
Pro	120	.	.	.	.	.	T	C	1.59	−0.36	.	*	F	2.25	0.94
Gly	121	.	.	.	.	T	T	.	1.29	−0.39	.	*	F	2.15	0.98
Glu	122	.	.	.	.	T	T	.	1.20	−0.43	.	.	F	2.00	1.30
Gly	123	.	.	.	.	.	.	C	1.41	−0.54	.	.	F	1.60	1.12
Asn	124	.	.	.	.	.	T	C	2.00	−0.57	.	.	F	1.50	1.97
Ser	125	.	.	.	.	.	T	C	1.91	−0.60	.	*	F	1.50	1.82
Ser	126	.	.	.	.	.	T	C	2.37	−0.21	.	*	F	1.54	2.47
Gln	127	.	.	.	.	.	T	C	2.37	−0.64	.	*	F	2.18	3.01
Asn	128	.	.	.	.	.	.	C	2.76	−0.64	.	.	F	2.32	3.61
Ser	129	.	.	.	.	.	T	C	2.87	−1.03	.	.	F	2.86	5.39
Arg	130	.	.	.	.	T	T	.	2.58	−1.41	*	.	F	3.40	6.09
Asn	131	.	.	.	.	T	T	.	2.02	−1.31	*	.	F	3.06	3.83
Lys	132	.	.	.	.	T	T	.	2.02	−1.07	*	.	F	2.72	2.12
Arg	133	.	.	.	.	T	.	.	1.68	−1.06	*	.	F	2.18	1.88
Ala	134	.	.	.	.	.	.	C	1.77	−0.63	*	.	F	1.64	1.15
Val	135	.	.	.	.	.	.	C	1.66	−0.60	*	.	F	1.49	0.89
Gln	136	.	.	.	.	.	.	C	1.66	−0.60	*	.	F	1.83	0.79
Gly	137	.	.	.	.	.	T	C	1.30	−0.60	*	.	F	2.52	1.35
Pro	138	.	.	.	.	.	T	C	0.33	−0.61	*	.	F	2.86	2.63
Glu	139	.	.	.	.	T	T	.	0.61	−0.61	*	.	F	3.40	1.13
Glu	140	A	.	.	.	.	T	.	1.47	−0.53	*	.	F	2.66	1.64
Thr	141	A	.	.	.	.	.	.	1.47	−0.56	.	.	F	2.12	1.84
Val	142	A	.	.	.	.	.	.	1.14	−0.99	.	.	F	1.78	1.77
Thr	143	A	.	.	.	.	T	.	0.54	−0.41	.	.	F	1.19	0.55
Gln	144	A	.	.	.	.	T	.	0.54	0.27	*	.	F	0.25	0.31
Asp	145	A	.	.	.	.	T	.	−0.27	0.19	*	.	F	0.25	0.73
Cys	146	A	.	.	.	.	T	.	−0.84	0.23	*	.	.	0.10	0.42
Leu	147	A	A	.	.	.	.	.	−0.58	0.43	*	.	.	−0.60	0.17
Gln	148	A	A	.	.	.	.	.	−0.27	0.53	*	.	.	−0.60	0.10
Leu	149	A	A	.	.	.	.	.	−0.57	0.53	*	*	.	−0.30	0.32
Ile	150	A	A	.	.	.	.	.	−0.57	0.34	*	.	.	0.30	0.52
Ala	151	.	A	.	.	.	.	C	−0.21	−0.34	.	*	.	1.40	0.52
Asp	152	.	.	.	.	T	T	.	0.39	−0.26	.	*	F	2.45	0.91
Ser	153	.	.	.	.	.	T	C	0.08	−0.51	.	.	F	3.00	2.00
Glu	154	.	.	.	.	.	T	C	−0.00	−0.71	.	.	F	2.70	2.86
Thr	155	.	.	.	.	.	T	C	0.89	−0.53	*	.	F	2.40	1.20
Pro	156	.	.	.	B	.	.	C	1.52	−0.13	*	.	F	1.56	1.55
Thr	157	.	.	.	B	T	.	.	1.18	−0.51	*	.	F	1.92	1.79
Ile	158	A	.	.	B	.	.	.	1.18	−0.09	.	.	F	1.08	1.23
Gln	159	.	.	.	.	T	T	.	0.93	−0.19	.	.	F	2.04	1.07
Lys	160	.	.	.	.	T	T	.	0.93	0.14	*	.	F	1.60	1.16
Gly	161	.	.	.	.	T	T	.	0.44	0.14	*	.	F	1.44	2.38
Ser	162	.	.	.	.	T	T	.	−0.10	0.24	*	.	F	1.28	1.19
Tyr	163	.	.	.	B	T	.	.	0.58	0.49	*	.	.	0.12	0.44
Thr	164	.	.	B	B	.	.	.	0.29	0.91	*	.	.	−0.44	0.69
Phe	165	.	.	B	B	.	.	.	−0.57	1.40	*	.	.	−0.60	0.54
Val	166	.	.	B	B	.	.	.	−1.03	1.70	.	.	.	−0.60	0.29
Pro	167	.	.	B	B	.	.	.	−1.03	1.63	.	.	.	−0.60	0.16
Trp	168	A	.	.	B	.	.	.	−1.49	1.53	.	*	.	−0.60	0.25
Leu	169	A	.	.	B	.	.	.	−1.13	1.53	*	.	.	−0.60	0.29
Leu	170	A	.	.	B	.	.	.	−0.32	0.89	*	.	.	−0.30	0.38
Ter	171	A	.	.	.	.	.	.	0.19	0.46	*	.	.	0.20	0.71
Phe	172	.	.	.	.	T	.	.	0.10	−0.03	*	.	.	1.80	0.85
Lys	173	.	.	.	.	T	T	.	−0.20	−0.33	*	.	F	2.60	1.38
Arg	174	.	.	.	.	.	T	C	−0.20	−0.51	.	.	F	3.00	1.04
Gly	175	.	.	.	.	.	T	C	0.61	−0.21	.	.	F	2.25	0.99
Ser	176	A	.	.	.	.	T	.	0.91	−1.00	*	.	F	2.05	0.86
Ala	177	A	A	.	.	.	.	.	1.66	−1.00	*	.	F	1.35	0.76
Leu	178	A	A	.	.	.	.	.	1.61	−1.00	.	.	F	1.20	1.54
Glu	179	A	A	.	.	.	.	.	1.50	−1.43	.	.	F	0.90	1.98
Glu	180	A	A	.	.	.	.	.	1.89	−1.41	*	.	F	0.90	3.16
Lys	181	A	A	.	.	.	.	.	1.30	−1.91	*	.	F	0.90	7.66
Glu	182	A	A	.	.	.	.	.	1.08	−1.91	.	.	F	0.90	3.10
Asn	183	A	A	.	.	.	.	.	1.03	−1.23	*	*	F	0.90	1.48
Lys	184	A	A	.	.	.	.	.	1.08	−0.59	*	.	F	0.75	0.55
Ile	185	A	A	.	.	.	.	.	1.08	−0.59	*	*	.	0.60	0.63
Leu	186	A	A	.	.	.	.	.	0.72	−0.59	*	*	.	0.60	0.68
Val	187	A	A	.	.	.	.	.	0.38	−0.50	.	*	.	0.30	0.49
Lys	188	A	A	.	.	.	.	.	0.13	−0.07	*	*	F	0.45	0.69
Glu	189	A	.	.	.	.	T	.	−0.61	0.00	*	*	F	0.40	1.32
Thr	190	.	.	.	.	T	T	.	−0.42	0.10	.	*	F	0.80	1.54
Gly	191	.	.	.	.	T	T	.	−0.50	0.24	*	.	F	0.65	0.67
Tyr	192	.	.	.	.	T	T	.	0.11	0.93	*	*	.	0.20	0.27
Phe	193	.	.	B	B	.	.	.	−0.28	1.69	.	.	.	−0.60	0.29
Phe	194	.	.	B	B	.	.	.	−0.28	1.63	.	*	.	−0.60	0.29
Ile	195	.	.	B	B	.	.	.	−0.82	1.60	.	.	.	−0.60	0.32
Tyr	196	.	.	B	B	.	.	.	−1.29	1.49	.	.	.	−0.60	0.28
Gly	197	.	.	.	B	T	.	.	−1.29	1.39	.	.	.	−0.20	0.26
Gln	198	.	.	.	B	T	.	.	−0.90	1.36	.	.	.	−0.20	0.59
Val	199	.	.	.	B	.	.	C	−0.20	1.16	.	.	.	−0.40	0.54
Leu	200	.	.	.	B	.	.	C	0.73	0.40	.	.	.	−0.10	0.92
Tyr	201	.	.	.	.	T	T	.	0.67	−0.03	.	.	.	1.25	1.06
Thr	202	.	.	.	.	T	T	.	0.77	0.06	.	.	F	0.80	2.06
Asp	203	.	.	.	.	T	T	.	0.18	0.17	.	.	F	0.80	3.91
Lys	204	A	.	.	.	.	T	.	0.43	−0.01	.	.	F	1.00	2.52
Thr	205	A	A	.	.	.	.	.	0.90	−0.16	.	.	F	0.60	1.73
Tyr	206	A	A	.	.	.	.	.	1.11	−0.21	.	.	.	0.45	1.03
Ala	207	A	A	.	.	.	.	.	0.61	0.29	.	.	.	−0.30	0.70
Met	208	A	A	.	.	.	.	.	−0.28	0.97	.	.	.	−0.60	0.40
Gly	209	A	A	.	B	.	.	.	−0.32	1.17	*	.	.	−0.60	0.18
His	210	A	A	.	B	.	.	.	0.10	0.81	*	.	.	−0.60	0.31
Leu	211	A	A	.	B	.	.	.	0.39	0.31	.	.	.	−0.30	0.61
Ile	212	A	A	.	B	.	.	.	1.02	−0.30	.	.	.	0.45	1.22
Gln	213	A	A	.	B	.	.	.	0.77	−0.73	.	*	.	0.75	1.80
Arg	214	A	A	.	B	.	.	.	1.08	−0.59	.	*	F	0.90	1.62
Lys	215	A	A	.	B	.	.	.	0.26	−0.77	*	*	F	0.90	3.14
Lys	216	A	A	.	B	.	.	.	0.37	−0.81	.	*	F	0.90	1.35
Val	217	.	A	B	B	.	.	.	0.91	−0.43	*	*	.	0.30	0.60
His	218	.	A	B	B	.	.	.	0.91	−0.00	.	*	.	0.30	0.29
Val	219	.	A	B	B	.	.	.	0.80	−0.00	*	*	.	0.30	0.25
Phe	220	.	.	B	B	.	.	.	−0.06	−0.00	*	.	.	0.30	0.57
Gly	221	A	.	.	B	.	.	.	−0.40	0.04	.	*	.	−0.30	0.35
Asp	222	A	.	.	.	.	.	.	−0.36	−0.07	*	.	.	0.50	0.63
Glu	223	A	.	.	.	.	.	.	−1.18	−0.03	*	.	.	0.50	0.60
Leu	224	A	.	.	B	.	.	.	−0.63	−0.17	.	.	.	0.30	0.45
Ser	225	A	.	.	B	.	.	.	−0.74	−0.11	.	.	.	0.30	0.39
Leu	226	A	.	.	B	.	.	.	−1.10	0.57	.	*	.	−0.60	0.18
Val	227	A	.	.	B	.	.	.	−0.99	1.36	.	*	.	−0.60	0.19
Thr	228	A	.	.	B	.	.	.	−1.66	0.67	*	*	.	−0.60	0.28
Leu	229	A	.	.	B	.	.	.	−1.73	0.86	*	.	.	−0.60	0.18
Phe	230	A	.	.	B	.	.	.	−1.43	0.86	*	.	.	−0.60	0.17
Arg	231	A	.	.	B	.	.	.	−0.62	0.61	*	.	.	−0.60	0.21
Cys	232	.	.	.	B	T	.	.	−0.37	0.53	*	.	.	−0.20	0.41
Ile	233	.	.	.	B	T	.	.	−0.27	0.46	*	.	.	−0.20	0.46
Gln	234	.	.	.	B	T	.	.	0.54	0.10	*	.	.	0.10	0.37
Asn	235	.	.	.	B	.	.	C	0.93	0.10	*	.	.	0.05	1.19
Met	236	.	.	.	B	.	.	C	0.01	0.01	*	.	F	0.20	2.44
Pro	237	.	.	.	B	.	.	C	0.47	0.01	*	.	F	0.44	1.16
Glu	238	.	.	.	.	T	.	.	1.36	0.04	*	.	F	1.08	1.12
Thr	239	.	.	.	.	.	.	C	1.36	0.04	*	.	F	1.12	1.82
Leu	240	.	.	.	.	.	.	C	1.06	−0.17	*	.	F	1.96	1.89
Pro	241	.	.	.	.	T	.	.	0.99	−0.21	.	.	F	2.40	1.46
Asn	242	.	.	.	.	T	.	.	0.96	0.36	.	.	F	1.41	0.54
Asn	243	.	.	.	.	T	T	.	0.66	0.63	.	.	F	1.22	1.03
Ser	244	.	.	.	.	T	T	.	0.38	0.33	.	.	F	1.13	0.89
Cys	245	.	.	.	.	T	T	.	0.84	0.40	.	.	.	0.74	0.56
Tyr	246	.	.	.	.	T	T	.	0.17	0.43	.	.	.	0.20	0.35
Ser	247	A	.	.	.	.	.	.	−0.42	0.71	.	.	.	−0.40	0.18
Ala	248	A	A	.	.	.	.	.	−0.38	0.83	.	.	.	−0.60	0.34
Gly	249	A	A	.	.	.	.	.	−0.89	0.26	.	.	.	−0.30	0.43
Ile	250	A	A	.	.	.	.	.	−0.22	0.19	*	.	.	−0.30	0.27
Ala	251	A	A	.	.	.	.	.	0.02	−0.20	*	.	.	0.30	0.46
Lys	252	A	A	.	.	.	.	.	−0.02	−0.70	.	.	.	0.60	0.80
Leu	253	A	A	.	.	.	.	.	0.57	−0.70	.	.	F	0.90	1.13
Glu	254	A	A	.	.	.	.	.	0.91	−1.39	.	.	F	0.90	1.87
Glu	255	A	A	.	.	.	.	.	0.99	−1.89	.	.	F	0.90	1.62
Gly	256	A	A	.	.	.	.	.	1.58	−1.20	.	*	F	0.90	1.62
Asp	257	A	A	.	.	.	.	.	0.72	−1.49	.	*	F	0.90	1.62
Glu	258	A	A	.	.	.	.	.	0.94	−0.80	*	*	F	0.75	0.77
Leu	259	A	A	.	.	.	.	.	0.06	−0.30	*	*	.	0.30	0.79
Gln	260	A	A	.	.	.	.	.	−0.16	−0.04	*	.	.	0.30	0.33
Leu	261	A	A	.	.	.	.	.	0.30	0.39	*	.	.	−0.30	0.30
Ala	262	A	A	.	.	.	.	.	0.30	0.39	*	.	.	−0.30	0.70
Ile	263	A	A	.	.	.	.	.	0.30	−0.30	.	*	.	0.30	0.70
Pro	264	A	.	.	.	.	T	.	0.52	−0.30	.	*	F	1.00	1.37
Arg	265	A	.	.	.	.	T	.	0.52	−0.49	.	*	F	1.00	1.37
Glu	266	A	.	.	.	.	T	.	0.44	−0.59	*	*	F	1.30	3.38
Asn	267	A	.	.	.	.	T	.	0.73	−0.59	*	*	F	1.30	1.53
Ala	268	A	.	.	.	.	.	.	0.81	−0.63	*	*	.	0.95	1.05
Gln	269	A	.	.	.	.	.	.	1.02	0.06	*	*	.	−0.10	0.50
Ile	270	A	.	.	.	.	.	.	0.57	0.06	.	*	.	0.15	0.52
Ser	271	.	.	.	.	.	.	C	0.57	0.09	.	*	.	0.60	0.51
Leu	272	.	.	.	.	.	.	C	−0.29	−0.41	.	*	F	1.60	0.49
Asp	273	.	.	.	.	T	T	.	−0.01	−0.17	.	*	F	2.25	0.52
Gly	274	.	.	.	.	T	T	.	−0.71	−0.37	.	*	F	2.50	0.56
Asp	275	.	.	.	.	T	T	.	−0.52	0.03	.	*	F	1.65	0.59
Val	276	A	.	.	.	.	T	.	−0.57	0.13	.	*	F	1.00	0.30
Thr	277	A	.	.	B	.	.	.	−0.34	0.56	.	*	.	−0.10	0.30
Phe	278	A	.	.	B	.	.	.	−1.16	0.63	.	*	.	−0.35	0.18
Phe	279	A	.	.	B	.	.	.	−0.77	1.31	.	*	.	−0.60	0.20
Gly	280	A	A	.	.	.	.	.	−1.58	0.67	.	*	.	−0.60	0.28
Ala	281	A	A	.	.	.	.	.	−1.53	0.87	.	*	.	−0.60	0.27
Leu	282	A	A	.	.	.	.	.	−1.61	0.77	*	.	.	−0.60	0.26
Lys	283	A	A	.	.	.	.	.	−1.30	0.41	*	.	.	−0.60	0.33
Leu	284	A	A	.	.	.	.	.	−0.99	0.41	.	.	.	−0.60	0.42
Leu	285	A	A	.	.	.	.	.	−1.03	0.34	*	.	.	−0.30	0.65

[0095]

TABLE 10
Res	Position	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII	XIV
Met	1	A	.	.	.	.	.	.	0.73	−0.71	.	.	.	0.95	1.39
Asp	2	A	.	.	.	.	T	.	1.12	−0.66	*	.	.	1.15	1.56
Asp	3	A	.	.	.	.	T	.	1.62	−1.09	*	.	.	1.15	2.12
Ser	4	A	.	.	.	.	T	.	2.01	−1.51	.	.	.	1.15	4.19
Thr	5	A	.	.	.	.	T	.	2.40	−2.13	.	.	F	1.30	4.35
Glu	6	A	A	.	.	.	.	.	2.70	−1.73	*	*	F	0.90	4.51
Arg	7	A	A	.	.	.	.	.	2.81	−1.34	*	*	F	0.90	4.51
Glu	8	A	A	.	.	.	.	.	2.00	−1.73	*	*	F	0.90	6.12
Gln	9	A	A	.	.	.	.	.	1.99	−1.53	*	*	F	0.90	2.91
Ser	10	A	.	.	B	.	.	.	2.00	−1.04	*	*	F	0.90	2.15
Arg	11	A	.	.	B	.	.	.	1.33	−0.66	*	*	F	0.90	1.66
Leu	12	A	.	.	B	.	.	.	0.41	−0.09	*	*	F	0.45	0.51
Thr	13	A	.	.	B	.	.	.	0.46	0.20	*	*	F	−0.15	0.32
Ser	14	A	A	.	.	.	.	.	0.50	−0.19	*	*	.	0.30	0.32
Cys	15	A	A	.	.	.	.	.	0.91	−0.19	*	*	.	0.30	0.78
Leu	16	A	A	.	.	.	.	.	0.80	−0.87	*	*	F	0.90	1.06
Lys	17	A	A	.	.	.	.	.	1.61	−1.36	.	*	F	0.90	1.37
Lys	18	A	A	.	.	.	.	.	1.32	−1.74	.	*	F	0.90	4.44
Arg	19	A	A	.	.	.	.	.	1.67	−1.70	.	*	F	0.90	5.33
Glu	20	A	A	.	.	.	.	.	1.52	−2.39	.	*	F	0.90	5.33
Glu	21	A	A	.	.	.	.	.	2.38	−1.70	.	*	F	0.90	2.20
Met	22	A	A	.	.	.	.	.	2.33	−1.70	.	*	F	0.90	2.24
Lys	23	A	A	.	.	.	.	.	1.62	−1.70	*	*	F	0.90	2.24
Leu	24	A	A	.	.	.	.	.	0.66	−1.13	*	*	F	0.75	0.69
Lys	25	A	A	.	.	.	.	.	0.36	−0.49	.	*	F	0.45	0.52
Glu	26	A	A	.	B	.	.	.	−0.53	−0.71	*	*	.	0.60	0.35
Cys	27	A	A	.	B	.	.	.	−0.74	−0.03	*	*	.	0.30	0.30
Val	28	A	A	.	B	.	.	.	−1.00	−0.03	*	*	.	0.30	0.12
Ser	29	A	A	.	B	.	.	.	−0.08	0.40	*	*	.	−0.30	0.11
Ile	30	A	.	.	B	.	.	.	−0.08	0.40	*	*	.	−0.30	0.40
Leu	31	A	.	.	B	.	.	.	−0.08	−0.17	*	.	.	0.45	1.08
Pro	32	.	.	.	B	.	.	C	0.29	−0.81	*	.	F	1.10	1.39
Arg	33	.	.	.	.	T	.	.	0.93	−0.81	.	*	F	1.50	2.66
Lys	34	.	.	.	.	T	.	.	0.93	−1.07	.	.	F	1.84	4.98
Glu	35	.	.	.	.	.	.	C	0.97	−1.37	*	*	F	1.98	4.32
Ser	36	.	.	.	.	.	T	C	1.89	−1.16	*	*	F	2.52	1.64
Pro	37	.	.	.	.	.	T	C	1.80	−1.16	*	*	F	2.86	1.60
Ser	38	.	.	.	.	T	T	.	1.39	−0.77	*	.	F	3.40	1.24
Val	39	A	.	.	.	.	T	.	1.39	−0.39	.	*	F	2.36	1.24
Arg	40	A	.	.	.	.	.	.	1.39	−0.77	*	*	F	2.46	1.60
Ser	41	A	.	.	.	.	.	.	1.34	−1.20	*	*	F	2.46	2.00
Ser	42	.	.	.	.	T	T	.	1.60	−1.16	.	*	F	3.06	2.67
Lys	43	.	.	.	.	T	T	.	1.09	−1.80	*	*	F	3.06	2.72
Asp	44	.	.	.	.	T	T	.	1.13	−1.11	*	*	F	3.40	1.67
Gly	45	A	.	.	.	.	T	.	0.43	−0.81	*	*	F	2.66	1.03
Lys	46	A	A	.	.	.	.	.	0.14	−0.70	.	.	F	1.77	0.52
Leu	47	A	A	.	.	.	.	.	0.13	−0.20	*	.	.	0.98	0.31
Leu	48	A	A	.	.	.	.	.	−0.72	0.29	*	.	.	0.04	0.46
Ala	49	A	A	.	.	.	.	.	−1.53	0.54	.	*	.	−0.60	0.19
Ala	50	A	A	.	.	.	.	.	−2.00	1.23	.	.	.	−0.60	0.19
Thr	51	A	A	.	.	.	.	.	−2.63	1.23	.	.	.	−0.60	0.19
Leu	52	A	A	.	.	.	.	.	−2.63	1.04	.	.	.	−0.60	0.19
Leu	53	A	A	.	.	.	.	.	−2.63	1.23	.	.	.	−0.60	0.15
Leu	54	A	A	.	.	.	.	.	−2.34	1.41	.	.	.	−0.60	0.09
Ala	55	A	A	.	.	.	.	.	−2.42	1.31	.	.	.	−0.60	0.14
Leu	56	A	A	.	.	.	.	.	−2.78	1.20	.	.	.	−0.60	0.09
Leu	57	A	.	.	.	.	T	.	−2.78	1.09	.	.	.	−0.20	0.06
Ser	58	A	.	.	.	.	T	.	−2.28	1.09	.	.	.	−0.20	0.05
Cys	59	A	.	.	.	.	T	.	−2.32	1.07	.	.	.	−0.20	0.09
Cys	60	A	.	.	.	.	T	.	−2.59	1.03	.	.	.	−0.20	0.08
Leu	61	.	.	B	B	.	.	.	−2.08	0.99	.	.	.	−0.60	0.04
Thr	62	.	.	B	B	.	.	.	−1.97	0.99	.	.	.	−0.60	0.11
Val	63	.	.	B	B	.	.	.	−1.91	1.20	.	.	.	−0.60	0.17
Val	64	.	.	B	B	.	.	.	−1.24	1.39	.	.	.	−0.60	0.33
Ser	65	.	.	B	B	.	.	.	−1.43	1.10	.	.	.	−0.60	0.40
Phe	66	A	.	.	B	.	.	.	−1.21	1.26	.	.	.	−0.60	0.40
Tyr	67	A	.	.	B	.	.	.	−1.49	1.11	.	.	.	−0.60	0.54
Gln	68	A	.	.	B	.	.	.	−1.44	0.97	.	.	.	−0.60	0.41
Val	69	A	.	.	B	.	.	.	−0.59	1.27	.	.	.	−0.60	0.39
Ala	70	A	.	.	B	.	.	.	−0.63	0.89	.	.	.	−0.60	0.43
Ala	71	A	.	.	B	.	.	.	0.07	0.56	.	*	.	−0.60	0.25
Leu	72	A	.	.	.	.	T	.	−0.50	0.16	.	.	.	0.10	0.55
Gln	73	A	.	.	.	.	T	.	−1.09	0.20	.	.	F	0.25	0.45
Gly	74	A	.	.	.	.	T	.	−0.53	0.20	.	.	F	0.25	0.45
Asp	75	A	.	.	.	.	T	.	−0.76	0.09	.	*	F	0.25	0.73
Leu	76	A	A	.	.	.	.	.	−0.06	0.09	.	*	F	−0.15	0.35
Ala	77	A	A	.	.	.	.	.	0.17	−0.31	.	*	.	0.30	0.69
Ser	78	A	A	.	.	.	.	.	0.17	−0.24	.	*	.	0.30	0.42
Leu	79	A	A	.	.	.	.	.	−0.30	−0.24	.	*	.	0.30	0.88
Arg	80	A	A	.	.	.	.	.	−0.30	−0.24	.	*	.	0.30	0.72
Ala	81	A	A	.	.	.	.	.	0.17	−0.34	.	*	.	0.30	0.93
Glu	82	A	A	.	.	.	.	.	0.72	−0.30	.	*	.	0.45	1.11
Leu	83	A	A	.	.	.	.	.	0.99	−0.49	.	*	.	0.30	0.77
Gln	84	A	A	.	.	.	.	.	1.21	0.01	.	*	.	−0.15	1.04
Gly	85	A	A	.	.	.	.	.	1.10	0.01	*	*	.	−0.30	0.61
His	86	A	A	.	.	.	.	.	1.73	0.01	*	*	.	−0.15	1.27
His	87	A	A	.	.	.	.	.	0.92	−0.67	.	*	.	0.75	1.47
Ala	88	A	A	.	.	.	.	.	1.52	−0.39	.	*	.	0.45	1.22
Glu	89	A	A	.	.	.	.	.	0.93	−0.39	.	.	.	0.45	1.39
Lys	90	A	A	.	.	.	.	.	0.93	−0.39	*	.	F	0.60	1.03
Leu	91	A	.	.	.	.	T	.	0.38	−0.46	*	.	.	0.85	1.01
Pro	92	A	.	.	.	.	T	.	0.07	−0.46	.	.	.	0.70	0.59
Ala	93	A	.	.	.	.	T	.	0.07	−0.03	.	.	.	0.70	0.29
Gly	94	A	.	.	.	.	T	.	−0.14	0.47	.	.	.	−0.20	0.36
Ala	95	A	.	.	.	.	.	.	−0.14	0.21	.	*	.	−0.10	0.36
Gly	96	A	.	.	.	.	.	.	0.08	−0.21	.	.	F	0.65	0.71
Ala	97	A	.	.	.	.	.	.	−0.06	−0.21	.	.	F	0.65	0.72
Pro	98	A	.	.	.	.	.	.	−0.28	−0.21	.	*	F	0.65	0.71
Lys	99	A	A	.	.	.	.	.	0.07	−0.03	.	.	F	0.45	0.59
Ala	100	A	A	.	.	.	.	.	0.66	−0.46	.	.	F	0.60	1.01
Gly	101	A	A	.	.	.	.	.	0.41	−0.96	.	.	F	0.90	1.13
Leu	102	A	A	.	.	.	.	.	0.79	−0.89	.	.	F	0.75	0.57
Glu	103	A	A	.	.	.	.	.	0.41	−0.46	*	.	F	0.45	0.88
Glu	104	A	A	.	.	.	.	.	−0.49	−0.46	*	.	F	0.45	0.89
Ala	105	A	A	.	.	.	.	.	−0.21	−0.24	.	.	.	0.30	0.81
Pro	106	A	A	.	.	.	.	.	−0.46	−0.44	.	.	.	0.30	0.67
Ala	107	A	A	.	.	.	.	.	0.01	0.06	.	.	.	−0.30	0.39
Val	108	A	A	.	.	.	.	.	−0.80	0.49	*	*	.	−0.60	0.38
Thr	109	A	A	.	.	.	.	.	−0.76	0.67	.	*	.	−0.60	0.20
Ala	110	A	A	.	.	.	.	.	−1.06	0.24	*	*	.	−0.30	0.40
Gly	111	A	A	.	.	.	.	.	−1.54	0.43	*	*	.	−0.60	0.38
Leu	112	A	A	.	.	.	.	.	−0.96	0.57	*	*	.	−0.60	0.23
Lys	113	.	A	B	.	.	.	.	−0.31	0.09	*	*	.	−0.30	0.39
Ile	114	.	A	B	.	.	.	.	−0.21	0.01	*	.	.	−0.30	0.61
Phe	115	.	A	B	.	.	.	.	−0.21	0.01	*	.	.	0.15	1.15
Glu	116	.	A	.	.	.	.	C	−0.08	−0.17	*	.	F	1.25	0.58
Pro	117	.	A	.	.	.	.	C	0.39	0.26	*	*	F	1.10	1.28
Pro	118	.	.	.	.	.	.	C	0.34	0.00	*	.	F	2.20	1.47
Ala	119	.	.	.	.	.	T	C	0.89	−0.79	.	*	F	3.00	1.47
Pro	120	.	.	.	.	.	T	C	1.59	−0.36	.	*	F	2.25	0.94
Gly	121	.	.	.	.	T	T	.	1.29	−0.39	.	*	F	2.15	0.98
Glu	122	.	.	.	.	T	T	.	1.20	−0.43	.	.	F	2.00	1.30
Gly	123	.	.	.	.	.	.	C	1.41	−0.54	.	.	F	1.60	1.12
Asn	124	.	.	.	.	.	T	C	2.00	−0.57	.	.	F	1.50	1.97
Ser	125	.	.	.	.	.	T	C	1.91	−0.60	.	*	F	1.50	1.82
Ser	126	.	.	.	.	.	T	C	2.37	−0.21	.	*	F	1.54	2.47
Gln	127	.	.	.	.	.	T	C	2.37	−0.64	.	*	F	2.18	3.01
Asn	128	.	.	.	.	.	.	C	2.76	−0.64	.	.	F	2.32	3.61
Ser	129	.	.	.	.	.	T	C	2.87	−1.03	.	.	F	2.86	5.39
Arg	130	.	.	.	.	T	T	.	2.58	−1.41	*	.	F	3.40	6.09
Asn	131	.	.	.	.	T	T	.	2.02	−1.31	*	.	F	3.06	3.83
Lys	132	.	.	.	.	T	T	.	2.02	−1.07	*	.	F	2.72	2.12
Arg	133	.	.	.	.	T	.	.	1.68	−1.06	*	.	F	2.18	1.88
Ala	134	.	.	.	.	.	.	C	1.77	−0.63	*	.	F	1.64	1.15
Val	135	.	.	.	.	.	.	C	1.66	−0.60	*	.	F	1.15	0.89
Gln	136	.	.	.	.	.	.	C	1.66	−0.60	*	.	F	1.49	0.79
Gly	137	.	.	.	.	.	T	C	1.30	−0.60	*	.	F	2.18	1.35
Pro	138	.	.	.	.	.	T	C	0.84	−0.61	*	.	F	2.52	2.63
Glu	139	.	.	.	.	.	T	C	1.13	−0.83	*	.	F	2.86	1.50
Glu	140	.	.	.	.	T	T	.	1.74	−0.84	.	.	F	3.40	2.03
Thr	141	.	.	.	.	T	.	.	1.43	−0.51	.	.	F	2.86	2.06
Gly	142	.	.	.	.	T	T	.	1.08	−0.46	.	.	F	2.42	1.72
Ser	143	.	.	.	.	T	T	.	0.43	0.33	.	.	F	1.33	0.86
Tyr	144	.	.	.	.	T	T	.	0.22	0.97	.	.	.	0.54	0.44
Thr	145	.	.	.	.	T	T	.	−0.07	0.91	.	.	.	0.20	0.69
Phe	146	.	.	B	B	.	.	.	−0.57	1.40	.	.	.	−0.60	0.54
Val	147	.	.	B	B	.	.	.	−1.03	1.70	.	.	.	−0.60	0.29
Pro	148	.	.	B	B	.	.	.	−1.03	1.63	.	.	.	−0.60	0.16
Trp	149	A	.	.	B	.	.	.	−1.49	1.53	.	*	.	−0.60	0.25
Leu	150	A	.	.	B	.	.	.	−1.13	1.53	*	.	.	−0.60	0.29
Leu	151	A	.	.	B	.	.	.	−0.32	0.89	*	.	.	−0.30	0.38
Ser	152	A	.	.	.	.	.	.	0.19	0.46	*	.	.	0.20	0.71
Phe	153	.	.	.	.	T	.	.	0.10	−0.03	*	.	.	1.80	0.85
Lys	154	.	.	.	.	T	T	.	−0.20	−0.33	*	.	F	2.60	1.38
Arg	155	.	.	.	.	.	T	C	−0.20	−0.51	.	.	F	3.00	1.04
Gly	156	.	.	.	.	.	T	C	0.61	−0.21	.	.	F	2.25	0.99
Ser	157	A	.	.	.	.	T	.	0.91	−1.00	*	.	F	2.05	0.86
Ala	158	A	A	.	.	.	.	.	1.66	−1.00	*	.	F	1.35	0.76
Leu	159	A	A	.	.	.	.	.	1.61	−1.00	.	.	F	1.20	1.54
Glu	160	A	A	.	.	.	.	.	1.50	−1.43	.	.	F	0.90	1.98
Glu	161	A	A	.	.	.	.	.	1.89	−1.41	*	.	F	0.90	3.16
Lys	162	A	A	.	.	.	.	.	1.30	−1.91	*	.	F	0.90	7.66
Glu	163	A	A	.	.	.	.	.	1.08	−1.91	.	.	F	0.90	3.10
Asn	164	A	A	.	.	.	.	.	1.03	−1.23	*	*	F	0.90	1.48
Lys	165	A	A	.	.	.	.	.	1.08	−0.59	*	.	F	0.75	0.55
Ile	166	A	A	.	.	.	.	.	1.08	−0.59	*	*	.	0.60	0.63
Leu	167	A	A	.	.	.	.	.	0.72	−0.59	*	*	.	0.76	0.68
Val	168	A	A	.	.	.	.	.	0.38	−0.50	.	*	.	0.92	0.49
Lys	169	A	A	.	.	.	.	.	0.13	−0.07	*	*	F	0.93	0.69
Glu	170	A	.	.	.	.	T	.	−0.61	0.00	*	*	F	1.64	1.32
Thr	171	.	.	.	.	T	T	.	−0.42	0.10	.	*	F	1.60	1.54
Gly	172	.	.	.	.	T	T	.	−0.50	0.24	*	.	F	1.29	0.67
Tyr	173	.	.	.	.	T	T	.	0.11	0.93	*	*	.	0.68	0.27
Phe	174	.	.	B	B	.	.	.	−0.28	1.69	.	.	.	−0.28	0.29
Phe	175	.	.	B	B	.	.	.	−0.28	1.63	.	*	.	−0.44	0.29
Ile	176	.	.	B	B	.	.	.	−0.82	1.60	.	.	.	−0.60	0.32
Tyr	177	.	.	B	B	.	.	.	−1.29	1.49	.	.	.	−0.60	0.28
Gly	178	.	.	.	B	T	.	.	−1.29	1.39	.	.	.	−0.20	0.26
Gln	179	.	.	.	B	T	.	.	−0.90	1.36	.	.	.	−0.20	0.59
Val	180	.	.	.	B	.	.	C	−0.20	1.16	.	.	.	−0.40	0.54
Leu	181	.	.	.	B	.	.	C	0.73	0.40	.	.	.	−0.10	0.92
Tyr	182	.	.	.	.	T	T	.	0.67	−0.03	.	.	.	1.25	1.06
Thr	183	.	.	.	.	T	T	.	0.77	0.06	.	.	F	0.80	2.06
Asp	184	.	.	.	.	T	T	.	0.18	0.17	.	.	F	0.80	3.91
Lys	185	A	.	.	.	.	T	.	0.43	−0.01	.	.	F	1.00	2.52
Thr	186	A	A	.	.	.	.	.	0.90	−0.16	.	.	F	0.60	1.73
Tyr	187	A	A	.	.	.	.	.	1.11	−0.21	.	.	.	0.45	1.03
Ala	188	A	A	.	.	.	.	.	0.61	0.29	.	.	.	−0.30	0.70
Met	189	A	A	.	.	.	.	.	−0.28	0.97	.	.	.	−0.60	0.40
Gly	190	A	A	.	B	.	.	.	−0.32	1.17	*	.	.	−0.60	0.18
His	191	A	A	.	B	.	.	.	0.10	0.81	*	.	.	−0.60	0.31
Leu	192	A	A	.	B	.	.	.	0.39	0.31	.	.	.	−0.30	0.61
Ile	193	A	A	.	B	.	.	.	1.02	−0.30	.	.	.	0.45	1.22
Gln	194	A	A	.	B	.	.	.	0.77	−0.73	.	*	.	0.75	1.80
Arg	195	A	A	.	B	.	.	.	1.08	−0.59	*	*	F	0.90	1.62
Lys	196	A	A	.	B	.	.	.	0.26	−0.77	*	*	F	0.90	3.14
Lys	197	A	A	.	B	.	.	.	0.37	−0.81	.	*	F	0.90	1.35
Val	198	.	A	B	B	.	.	.	0.91	−0.43	*	*	.	0.30	0.60
His	199	.	A	B	B	.	.	.	0.91	0.00	*	*	.	0.30	0.29
Val	200	.	A	B	B	.	.	.	0.80	0.00	*	*	.	0.30	0.25
Phe	201	.	.	B	B	.	.	.	−0.06	0.00	*	.	.	0.30	0.57
Gly	202	A	.	.	B	.	.	.	−0.40	0.04	.	*	.	−0.30	0.35
Asp	203	A	.	.	.	.	.	.	−0.36	−0.07	*	.	.	0.50	0.63
Glu	204	A	.	.	.	.	.	.	−1.18	−0.03	*	.	.	0.50	0.60
Leu	205	A	.	.	B	.	.	.	−0.63	−0.17	.	.	.	0.30	0.45
Ser	206	A	.	.	B	.	.	.	−0.74	−0.11	.	.	.	0.30	0.39
Leu	207	A	.	.	B	.	.	.	−1.10	0.57	.	*	.	−0.60	0.18
Val	208	A	.	.	B	.	.	.	−0.99	1.36	.	*	.	−0.60	0.19
Thr	209	A	.	.	B	.	.	.	−1.66	0.67	*	*	.	−0.60	0.28
Leu	210	A	.	.	B	.	.	.	−1.73	0.86	*	.	.	−0.60	0.18
Phe	211	A	.	.	B	.	.	.	−1.43	0.86	*	.	.	−0.60	0.17
Arg	212	A	.	.	B	.	.	.	−0.62	0.61	*	.	.	−0.60	0.21
Cys	213	.	.	.	B	T	.	.	−0.37	0.53	*	.	.	−0.20	0.41
Ile	214	.	.	.	B	T	.	.	−0.27	0.46	*	.	.	−0.20	0.46
Gln	215	.	.	.	B	T	.	.	0.54	0.10	*	.	.	0.10	0.37
Asn	216	.	.	.	B	.	.	C	0.93	0.10	*	.	.	0.05	1.19
Met	217	.	.	.	B	.	.	C	0.01	0.01	*	.	F	0.20	2.44
Pro	218	.	.	.	B	.	.	C	0.47	0.01	*	.	F	0.44	1.16
Glu	219	.	.	.	.	T	.	.	1.36	0.04	*	.	F	1.08	1.12
Thr	220	.	.	.	.	.	.	C	1.36	0.04	*	.	F	1.12	1.82
Leu	221	.	.	.	.	.	.	C	1.06	−0.17	*	.	F	1.96	1.89
Pro	222	.	.	.	.	T	.	.	0.99	−0.21	.	.	F	2.40	1.46
Asn	223	.	.	.	.	T	.	.	0.96	0.36	.	.	F	1.41	0.54
Asn	224	.	.	.	.	T	T	.	0.66	0.63	.	.	F	1.22	1.03
Ser	225	.	.	.	.	T	T	.	0.38	0.33	.	.	F	1.13	0.89
Cys	226	.	.	.	.	T	T	.	0.84	0.40	.	.	.	0.74	0.56
Tyr	227	.	.	.	.	T	T	.	0.17	0.43	.	.	.	0.20	0.35
Ser	228	A	.	.	.	.	.	.	−0.42	0.71	.	.	.	−0.40	0.18
Ala	229	A	A	.	.	.	.	.	−0.38	0.83	.	.	.	−0.60	0.34
Gly	230	A	A	.	.	.	.	.	−0.89	0.26	.	.	.	−0.30	0.43
Ile	231	A	A	.	.	.	.	.	−0.22	0.19	*	.	.	−0.30	0.27
Ala	232	A	A	.	.	.	.	.	0.02	−0.20	*	.	.	0.30	0.46
Lys	233	A	A	.	.	.	.	.	−0.02	−0.70	.	.	.	0.60	0.80
Leu	234	A	A	.	.	.	.	.	0.57	−0.70	.	.	F	0.90	1.13
Glu	235	A	A	.	.	.	.	.	0.91	−1.39	.	.	F	0.90	1.87
Glu	236	A	A	.	.	.	.	.	0.99	−1.89	.	.	F	0.90	1.62
Gly	237	A	A	.	.	.	.	.	1.58	−1.20	.	*	F	0.90	1.62
Asp	238	A	A	.	.	.	.	.	0.72	−1.49	.	*	F	0.90	1.62
Glu	239	A	A	.	.	.	.	.	0.94	−0.80	*	*	F	0.75	0.77
Leu	240	A	A	.	.	.	.	.	0.06	−0.30	*	*	.	0.30	0.79
Gln	241	A	A	.	.	.	.	.	−0.16	−0.04	*	.	.	0.30	0.33
Leu	242	A	A	.	.	.	.	.	0.30	0.39	*	.	.	−0.30	0.30
Ala	243	A	A	.	.	.	.	.	0.30	0.39	*	.	.	−0.30	0.70
Ile	244	A	A	.	.	.	.	.	0.30	−0.30	—	*	.	0.30	0.70
Pro	245	A	.	.	.	.	T	.	0.52	−0.30	—	*	F	1.00	1.37
Arg	246	A	.	.	.	.	T	.	0.52	−0.49	.	*	F	1.00	1.37
Glu	247	A	.	.	.	.	T	.	0.44	−0.59	*	*	F	1.30	3.38
Asn	248	A	.	.	.	.	T	.	0.73	−0.59	*	*	F	1.30	1.53
Ala	249	A	.	.	.	.	.	.	0.81	−0.63	*	*	.	0.95	1.05
Gln	250	A	.	.	.	.	.	.	1.02	0.06	*	*	.	−0.10	0.50
Ile	251	A	.	.	.	.	.	.	0.57	0.06	*	*	.	0.15	0.52
Ser	252	.	.	.	.	.	.	C	0.57	0.09	.	*	.	0.60	0.51
Leu	253	.	.	.	.	.	.	C	−0.29	−0.41	.	*	F	1.60	0.49
Asp	254	.	.	.	.	T	T	.	−0.01	−0.17	.	*	F	2.25	0.52
Gly	255	.	.	.	.	T	T	.	−0.71	−0.37	.	*	F	2.50	0.56
Asp	256	.	.	.	.	T	T	.	−0.52	0.03	.	*	F	1.65	0.59
Val	257	A	.	.	.	.	T	.	−0.57	0.13	.	*	F	1.00	0.30
Thr	258	A	.	.	B	.	.	.	−0.34	0.56	.	*	.	−0.10	0.30
Phe	259	A	.	.	B	.	.	.	−1.16	0.63	.	*	.	−0.35	0.18
Phe	260	A	.	.	B	.	.	.	−0.77	1.31	.	*	.	−0.60	0.20
Gly	261	A	A	.	.	.	.	.	−1.58	0.67	.	*	.	−0.60	0.28
Ala	262	A	A	.	.	.	.	.	−1.53	0.87	.	*	.	−0.60	0.27
Leu	263	A	A	.	.	.	.	.	−1.61	0.77	*	.	.	−0.60	0.26
Lys	264	A	A	.	.	.	.	.	−1.30	0.41	*	.	.	−0.60	0.33
Leu	265	A	A	.	.	.	.	.	−0.99	0.41	.	.	.	−0.60	0.42
Leu	266	A	A	.	.	.	.	.	−1.03	0.34	*	.	.	−0.30	0.65