# Patent

## Title
Antibodies that immunospecifically bind to B lymphocyte stimulator

## Abstract

The present invention relates to antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator. The present invention also relates to methods and compositions for detecting or diagnosing a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or inappropriate function of B Lymphocyte Stimulator 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 expression or inappropriate B Lymphocyte Stimulator function comprising administering to an animal an effective amount of one or more antibodies or fragments or variants thereof or related molecules that immunospecifically bind to B Lymphocyte Stimulator.

## Background
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/543,296 filed Feb. 11, 2004 and U.S. Provisional Application No. 60/580,347 filed Jun. 18, 2004. This application is also a continuation in-part of U.S. patent application Ser. No. 10/293,418 filed Nov. 14, 2002 which claims the benefit of U.S. Provisional Application No. 60/331,469 filed Nov. 16, 2001 and U.S. Provisional Application No. 60/340,817 filed Dec. 19, 2001. U.S. patent application Ser. No. 10/293,418 filed Nov. 14, 2002 is a continuation-in-part of U.S. patent application Ser. No. 09/880,748 filed Jun. 15, 2001 which claims benefit of U.S. Provisional Application No. 60/212,210 filed Jun. 16, 2000, U.S. Provisional Application No. 60/240,816 filed Oct. 17, 2000, U.S. Provisional Application No. 60/276,248 filed Mar. 16, 2001, U.S. Provisional Application No. 60/277,379 filed Mar. 21, 2001 and U.S. Provisional Application No. 60/293,499 filed May 25, 2001. This application is a continuation in-part of U.S. patent application Ser. No. 09/880,748 filed Jun. 15, 2001 which claims benefit of U.S. Provisional Application No. 60/212,210 filed Jun. 16, 2000, U.S. Provisional Application No. 60/240,816 filed Oct. 17, 2000, U.S. Provisional Application No. 60/276,248 filed Mar. 16, 2001, U.S. Provisional Application No. 60/277,379 filed Mar. 21, 2001 and U.S. Provisional Application No. 60/293,499 filed May 25, 2001. Each of the applications identified above is herein incorporated by reference in their entireties.


REFERENCE TO SEQUENCE LISTING ON COMPACT DISC
This application refers to a “Sequence Listing” listed below, which is provided as an electronic document on two identical compact discs (CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each contain the file “PF523P3 Seq List.txt” (4,924,816 bytes, created on Jan. 18, 2005), which is incorporated by reference in its entirety.
INTRODUCTION
The present invention relates to antibodies and related molecules that immunospecifically bind to B Lymphocyte Stimulator (BLySTM) 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.
BACKGROUND OF THE INVENTION
B Lymphocyte Stimulator (BLySTM) 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.
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 CD45Rdull, Ly6Dbright (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.
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.
SUMMARY OF THE INVENTION
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 alliteratively consisting of, fragments or variants of any of the VH domains, VH CDRs, VL domains, and VL CDRs whose sequences are specifically disclosed herein may be employed in accordance with the present invention. Nucleic acid molecules encoding these antibodies and molecules (including fragments, variants, and derivatives) are also encompassed by the invention.
The present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (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.
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.
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.
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).
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).
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).
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).
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 erythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, 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).
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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. 215:403410(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.)
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.
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.
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.
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.
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).
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.
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.



DESCRIPTION OF THE FIGURES
FIG. 1. ELISA results for three scFvs, I006E07, I008D05 and I016F04, that immunospecifically bind to U937 membranes, but not to bind to or cross-react with TNF-alpha or BSA.
FIG. 2. The results for three scFvs, I016H07, I001C09 and I018D07, in a receptor inhibition assay.
FIG. 3. ELISA results for two scFvs (I022D01 and I031F02) demonstrating their ability to bind to human B Lymphocyte Stimulator and to cross-react with mouse B Lymphocyte Stimulator, but not to bind to or cross-react with other antigens of the TNF ligand family.
FIG. 4. ELISA results for three scFvs (I031F09, I050A12, and I051C04) binding to U937 plasma membranes when either B Lymphocyte Stimulator or TNF-alpha is used as a competitor.
FIG. 5. Kinetic analysis of scFv antibody I003C02. A dilution series of I003C02 from 3 nM to 825 nM is shown. Association and dissociation curves were generated using a BIAcore 2000 and BIAevaluation 3.0 software.
FIG. 6. Typical titration curves for two scFv antibodies (I007F11 and I050A07) are shown in FIG. 6. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an IC50 value of 0.8 nM. The IC50 values for I007F11 and I050A07 are 7.9 nM and 17.1 nM, respectively. The assay was performed in triplicate and standard error bars are shown.
FIG. 7. ELISA results for three scFvs clones (I074B12, I075F12 and I075A02) that immunospecifically bind to immobilized B Lymphocyte Stimulator, but not to U937 plasma membranes, TNF-alpha or BSA. As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7.
FIG. 8. The results for two scFvs (I025B09 and I026C04) in a receptor inhibition assay.
FIG. 9. ELISA results for two scFvs clones (I067F05 and I078D02) demonstrating their ability to bind to immobilized human B Lymphocyte Stimulator and to cross-react with immobilized mouse B Lymphocyte Stimulator, but not to bind to or cross-react with other antigens of the TNF ligand family.
As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7.
FIG. 10. Kinetic analysis of scFV antibody I002A01. A dilution series of I002A01 from 3 nM to 1650 nM is shown. Association and dissociation curves were generated using a BIAcore 2000 and BIAevaluation 3.0 software.
FIG. 11. Typical titration curves for two scFvs, I0068C06 and I074B12, are shown in FIG. 11. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an inhibitory constant 50 (IC50) value of 0.66 nM. The IC50 values for I0068C06 and I074B12 are 61 nM and 13 nM respectively. The assay was performed in triplicate and standard error bars are shown.
FIG. 12. ELISA results for three clones (I079C01, I081C10 and I082A02) demonstrating their ability to bind histidine-tagged B Lymphocyte Stimulator, U937 plasma membranes, but not to bind immobilized biotinylated B Lymphocyte Stimulator.
FIG. 13. ELISA results for three scFvs (I079B04, I079F08, and I080B01) binding to U937 plasma membranes when either histidine-tagged B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator is used as a competitor.
FIG. 14. An example of the dissociation section of a typical sensorgram for 8 scFvs is shown in FIG. 14. An anti-TNFα antibody that does not recognize B Lymphocyte Stimulator was included as a control. Of the 8 scFvs exemplified, I079F06 was identified for further study due to the relatively high numbers of RU's bound to the surface.
FIG. 15. A typical example of the binding curves generated for the scFv antibody I082C03 is shown in FIG. 15. The off-rate for this clone was calculated as 2×10−3 s−1. The affinity of I082C03 was calculated as 20 nM, assuming 100% activity of the scFv.
FIG. 16. ELISA results for three scFvs (I079B04, I079F08, and I080B01) binding to P388 plasma membranes when either histidine-tagged B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator is used as a competitor.



DETAILED DESCRIPTION OF THE INVENTION
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).
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 ATCCTM Accession No. 97768. The deposited clone is contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). The ATCCTM deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
The 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 ATCCTM Accession No. 203518. The deposited clone is contained in the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). The ATCCTM deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
The 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.
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.
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.
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.
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.
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.
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.
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, osteoprotegerin (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.
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 ATCCTM 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 ATCCTM No. 203518, or an antibody that binds polypeptide comprising a portion (i.e, fragment) of the above polypeptides.
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.
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.
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.
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 ATCCTM 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 ATCCTM 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.
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 ATCCTM accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone. Antibodies of the present invention also bind to fragments of the amino acid sequence of SEQ ID NO:3229, encoded by the cDNA contained in the plasmid having ATCCTM accession number 203518, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone.
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.
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.
In specific embodiments, antibodies of the present invention bind polypeptide fragments comprising, or alternatively consisting of, amino acid residues: 146, 3144, 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.
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; I-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.
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.
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 146 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).
Further additional preferred embodiments encompass polypeptide fragments comprising, or alternatively consisting of, the predicted intracellular domain of B Lymphocyte Stimulator (amino acid residues 146 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).
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.
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-13] 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).
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.
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 Gamier-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).
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 Gamier-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 Gamier 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 Gamier 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 Gamier 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.
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.
The above-mentioned preferred regions set out in Tables 9 and 10 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in Tables 9 and 10, such preferred regions include Gamier-Robson alpha-regions, beta-regions, 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.






















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































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









In another embodiment, the invention provides antibodies that bind a polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion may be an immunogenic or antigenic epitope of a polypeptide of the invention. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other band, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
As to the selection of polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) “Antibodies that react with predetermined sites on proteins”, Science, 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.
In specific embodiments, antibodies of the present invention bind antigenic epitope-bearing peptides and polypeptides of B Lymphocyte Stimulator and preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids contained within the amino acid sequence of a B Lymphocyte Stimulator polypeptide. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
Non-limiting examples of antigenic polypeptides or peptides that can be used to generate B Lymphocyte Stimulator-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-115 to about Leu-147 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ile-150 to about Tyr-163 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-171 to about Phe-194 in SEQ ID NO:3228; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-223 to about Tyr-246 in SEQ ID NO:3228; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-271 to about Phe-278 in FIGS. 1A and 1B (SEQ ID NO:3228). 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. These polypeptide fragments have been determined to bear antigenic epitopes of the B Lymphocyte Stimulator polypeptide by the analysis of the Jameson-Wolf antigenic index, as disclosed Table 9, above.
Non-limiting examples of antigenic polypeptides or peptides that can be used to generate B Lymphocyte Stimulator-specific antibodies and which may be bound by the antibodies of the invention include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-32 to about Leu-47 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Glu-116 to about Ser-143 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Phe-153 to about Tyr-173 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-218 to about Tyr-227 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ala-232 to about Gln-241 in SEQ ID NO:3229; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ile-244 to about Ala-249 in SEQ ID NO:3229; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-252 to about Val-257 in SEQ ID NO:3229. 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. These polypeptide fragments have been determined to bear antigenic epitopes of the B Lymphocyte Stimulator polypeptide by the analysis of the Jameson-Wolf antigenic index, as disclosed in Table 10 generated by the Protean component of the DNA*STAR computer program (as set forth above).
B Lymphocyte Stimulator epitope-bearing peptides and polypeptides may be produced by any conventional means. See, e.g., Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135; this “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
The present invention encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3228, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCCTM deposit No. 97768, or encoded by a polynucleotide that hybridizes to cDNA sequence contained in ATCCTM deposit No. 97768 (e.g., under hybridization conditions described herein).
The present invention also encompasses antibodies that bind polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:3229, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCCTM deposit No. 203518, or encoded by a polynucleotide that hybridizes to the cDNA sequence contained in ATCCTM deposit No. 203518 (e.g., under hybridization conditions described herein).
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses antibodies that bind a polypeptide comprising an epitope. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
B Lymphocyte Stimulator polypeptide fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).
In the present invention, antibodies of the present invention bind antigenic epitopes preferably containing a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes that may be bound by antibodies of the present invention are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes of B Lymphocyte Stimulator may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing B Lymphocyte Stimulator polypeptides may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the antibodies of the present invention may bind polypeptides comprising an immunogenic or antigenic epitope fused to other polypeptide sequences. For example, the B Lymphocyte Stimulator polypeptides may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof), or albumin (including but not limited to recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
In another embodiment, the antibodies of the present invention bind B Lymphocyte Stimulator polypeptides and/or the epitope-bearing fragments thereof that are fused with a heterologous antigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleic acid). In specific embodiments, the heterologous antigen is an immunogen.
In a more specific embodiment, the heterologous antigen is the gp120 protein of HIV, or a fragment thereof.
In another embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides and/or the epitope-bearing fragments thereof that are fused with polypeptide sequences of another TNF ligand family member (or biologically active fragments or variants thereof). In a specific embodiment, the antibodies of the present invention bind B Lymphocyte Stimulator polypeptides of the present invention are fused with a CD40L polypeptide sequence. In a preferred embodiment, the CD40L polypeptide sequence is soluble.
In another embodiment, antibodies of the present invention bind mutant B Lymphocyte Stimulator polypeptides that have been generated by random mutagenesis of a polynucleotide encoding the B Lymphocyte Stimulator polypeptide, by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, antibodies of the present invention bind one or more components, motifs, sections, parts, domains, fragments, etc., of B Lymphocyte Stimulator recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are, for example, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1 BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (International Publication No. WO 98/07880), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR12, CAD, and v-FLIP. In further embodiments, the heterologous molecules are any member of the TNF family.
In another preferred embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides of the invention (including biologically active fragments or variants thereof), that are fused with soluble APRIL polypeptides (e.g., amino acid residues 105 through 250 of SEQ ID NO:3239), or biologically active fragments or variants thereof.
To improve or alter the characteristics of B Lymphocyte Stimulator polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. For instance, for many proteins, including the extracellular domain or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino-terminal amino acid residues were missing. Accordingly, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptide mutants or variants generated by protein engineering.
In the present case, since the protein of the invention is a member of the TNF polypeptide family, deletions of N-terminal amino acids up to the Gly (G) residue at position 191 in SEQ ID NO:3228 may retain some biological activity such as, for example, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and cytotoxicity to appropriate target cells. Polypeptides having further N-terminal deletions including the Gly (G) residue would not be expected to retain biological activities because it is known that this residue in TNF-related polypeptides is in the beginning of the conserved domain required for biological activities. However, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or extracellular domain of the protein generally will be retained when less than the majority of the residues of the complete or extracellular domain of the protein are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the B Lymphocyte Stimulator of SEQ ID NO:3228, up to the glycine residue at position 191 (Gly-191 residue from the amino terminus). In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n1-285 of SEQ ID NO:3228, where n1 is an integer in the range of the amino acid position of amino acid residues 2-190 of the amino acid sequence in SEQ ID NO:3228. More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285, 102-285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-285, 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-285, 155-285, 156-285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285, 169-285, 170-285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-285, 182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, and 190-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Furthermore, since the predicted extracellular domain of the B Lymphocyte Stimulator polypeptides of the invention may itself elicit biological activity, deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide (spanning positions Gln-73 to Leu-285 of SEQ ID NO:3228) may retain some biological activity such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities. However, even if deletion of one or more amino acids from the N-terminus of the predicted extracellular domain of a B Lymphocyte Stimulator polypeptide results in modification or loss of one or more biological functions of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glycine residue at position number 280. In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n2-285 of SEQ ID NO:3228, where n2 is an integer in the range of the amino acid position of amino acid residues 73-280 in SEQ ID NO:3228, and 73 is the position of the first residue from the N-terminus of the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide (disclosed in SEQ ID NO:3228). More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Highly preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence least 80%, 85%, 90% identical and more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
Preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 90% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 95% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. More preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 96% identical to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 97% to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 98% to a B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228. Additionally, more preferred embodiments of the invention are directed to antibodies that bind polypeptides comprising, or alternatively consisting of, a polypeptide having an amino acid sequence at least 99% identical to B Lymphocyte Stimulator polypeptide having the amino acid sequence at positions 134-285 of SEQ ID NO:3228.
In specific embodiments, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, one of the following N-terminally deleted polypeptide fragments of B Lymphocyte Stimulator: amino acid residues Ala-71 through Leu-285, amino acid residues Ala-81 through Leu-285, amino acid residues Leu-112 through Leu-285, amino acid residues Ala-34 through Leu-285, amino acid residues Leu-147 through Leu-285, and amino acid residues Gly-161 through Leu-285 of SEQ ID NO:3228.
Similarly, many examples of biologically functional C-terminal deletion polypeptides are known. For instance, Interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Döbeli et al., J. Biotechnology 7:199-216 (1988). Since the present protein is a member of the TNF polypeptide family, deletions of C-terminal amino acids up to the leucine residue at position 284 are expected to retain most if not all biological activity such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication. Polypeptides having deletions of up to about 10 additional C-terminal residues (i.e., up to the glycine residue at position 274) also may retain some activity such as receptor binding, although such polypeptides would lack a portion of the conserved TNF domain which extends to about Leu-284 of SEQ ID NO:3228. However, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities may still be retained. Thus, the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature protein generally will be retained when less than the majority of the residues of the complete or mature protein are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3228, up to the glycine residue at position 274 (Gly-274). In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m1 of the amino acid sequence in SEQ ID NO:3228, where m1 is any integer in the range of the amino acid position of amino acid residues 274-284 in SEQ ID NO:3228. More in particular, the invention provides antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283 and 1-284 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Also provided are antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, B Lymphocyte Stimulator polypeptides with one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n1-m1 of SEQ ID NO:3228, where n1 and m1 are integers as defined above. Also included are antibodies that bind a polypeptide comprising, or alternatively consisting of, a portion of the complete B Lymphocyte Stimulator amino acid sequence encoded by the deposited cDNA clone contained in ATCCTM Accession No. 97768 where this portion excludes from 1 to 190 amino acids from the amino terminus or from 1 to 11 amino acids from the C-terminus of the complete amino acid sequence (or any combination of these N-terminal and C-terminal deletions) encoded by the cDNA clone in the deposited plasmid.
Similarly, deletions of C-terminal amino acid residues of the predicted extracellular domain of B Lymphocyte Stimulator up to the leucine residue at position 79 of SEQ ID NO:3228 may retain some biological activity, such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, and modulation of cell replication or modulation of target cell activities. Polypeptides having further C-terminal deletions including Leu-79 of SEQ ID NO:3228 would not be expected to retain biological activities.
However, even if deletion of one or more amino acids from the C-terminus of a polypeptide results in modification or loss of one or more biological functions of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete, mature or extracellular forms of the polypeptide generally will be retained when less than the majority of the residues of the complete, mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of the predicted extracellular domain retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of B Lymphocyte Stimulator polypeptide shown in SEQ ID NO:3228, up to the leucine residue at position 79 of SEQ ID NO:3228. In particular, the present invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 73-m2 of the amino acid sequence in SEQ ID NO:3228, where m2 is any integer in the range of the amino acid position of amino acid residues 79 to 285 in the amino acid sequence in SEQ ID NO:3228, and residue 78 is the position of the first residue at the C-terminus of the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide (disclosed in SEQ ID NO:3228). More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to Leu-285; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to S-247; Q-73 to Y-246; Q-73 to C-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to C-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-73 to I-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-13 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; and Q-73 to L-79 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of B Lymphocyte Stimulator, which may be described generally as having residues n2-m2 of SEQ ID NO:3228 where n2 and m2 are integers as defined above.
In another embodiment, antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the B Lymphocyte Stimulator amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCCTM accession no. 97768, where this portion excludes from 1 to about 206 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCCTM accession no. 97768, or from 1 to about 206 amino acids from the carboxy terminus of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCCTM accession no. 97768, or any combination of the above amino terminal and carboxy terminal deletions, of the entire extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit having ATCCTM accession no. 97768.
As mentioned above, even if deletion of one or more amino acids from the N-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functions or biological activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the full-length or mature forms or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the full-length or mature or extracellular domain of the polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted N-terminal amino acid residues may retain some functional (e.g., biological or immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glycine residue at position number 280 of the sequence shown SEQ ID NO:3228 and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n3-285 of the sequence shown in SEQ ID NO:3228, where n3 is an integer in the range of the amino acid position of amino acid residues 1 to 280 of the amino acid sequence in SEQ ID NO:3228.
More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-285; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K-43 to L-285; D44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; C-60 to L-285; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-15 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285; and G-280 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more functional activities (e.g., biological activity) of the protein, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., biological or immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3228, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m3 of SEQ ID NO:3228, where m3 is an integer in the range of the amino acid position of amino acid residues 6-284 of the amino acid sequence in SEQ ID NO:3228.
More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-1 to L-284; M-1 to K-283; M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to C-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1 to K-215; M-1 to R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183; M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-65; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a B Lymphocyte Stimulator polypeptide, which may be described generally as having residues n3-m3 of SEQ ID NO:3228, where n3 and m3 are integers as defined above.
Furthermore, since the predicted extracellular domain of the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229 may itself elicit functional activity (e.g., biological activity), deletions of N- and C-terminal amino acid residues from the predicted extracellular region of the polypeptide at positions Gln-73 to Leu-266 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, to stimulation of lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity. However, even if deletion of one or more amino acids from the N-terminus of the predicted extracellular domain of a B Lymphocyte Stimulator polypeptide results in modification or loss of one or more functional activities of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptides to induce and/or bind to antibodies which recognize the complete or mature or extracellular domains of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature or extracellular domains of the polypeptides are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the glycine residue at position number 261. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n4-266 of SEQ ID NO:3229, where n4 is an integer in the range of the amino acid position of amino acid residues 73-261 of the amino acid sequence in SEQ ID NO:3229, and 261 is the position of the first residue from the N-terminus of the predicted extracellular domain B Lymphocyte Stimulator polypeptide (shown in SEQ ID NO:3229).
More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Similarly, deletions of C-terminal amino acid residues of the predicted extracellular domain of B Lymphocyte Stimulator up to the leucine residue at position 79 of SEQ ID NO:3229 may retain some functional activity, such as, for example, ligand binding, the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation, modulation of cell replication, modulation of target cell activities and/or immunogenicity. Polypeptides having further C-terminal deletions including Leu-79 of SEQ ID NO:3229 would not be expected to retain biological activities.
However, even if deletion of one or more amino acids from the C-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functional activities may still be retained. Thus, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete, mature or extracellular forms of the polypeptide generally will be retained when less than the majority of the residues of the complete, mature or extracellular forms of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of the predicted extracellular domain retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of the predicted extracellular domain of B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the leucine residue at position 79 of SEQ ID NO:3229. In particular, the present invention provides antibodies that bind polypeptides having the amino acid sequence of residues 73-m4 of the amino acid sequence in SEQ ID NO:3229, where m4 is any integer in the range of the amino acid position of amino acid residues 79-265 of the amino acid sequence in SEQ ID NO:3229.
More in particular, in certain embodiments, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues Q-73 to L-265; Q-73 to K-264; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; Q-73 to Q-7389; Q-73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79; and Q-73 to S-78 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of the predicted extracellular domain of B Lymphocyte Stimulator, which may be described generally as having residues n4-m4 of SEQ ID NO:3229 where n4 and m4 are integers as defined above.
In another embodiment, antibodies of the present invention bind polypeptides consisting of a portion of the extracellular domain of the B Lymphocyte Stimulator amino acid sequence encoded by the cDNA clone contained in the deposit having ATCCTM Accession No. 203518, where this portion excludes from 1 to about 260 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCCTM Accession No. 203518, or from 1 to about 187 amino acids from the carboxy terminus of the extracellular domain of the amino acid sequence encoded by cDNA clone contained in the deposit having ATCCTM Accession No. 203518, or any combination of the above amino terminal and carboxy terminal deletions, of the entire extracellular domain of the amino acid sequence encoded by the cDNA clone contained in the deposit having ATCCTM Accession No. 203518.
As mentioned above, even if deletion of one or more amino acids from the N-terminus of a polypeptide results in modification or loss of one or more functional activities (e.g., biological activity) of the polypeptide, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator polypeptide to induce and/or bind to antibodies which recognize the full-length or mature forms or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the full-length or mature or extracellular domain of the polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted N-terminal amino acid residues may retain functional (e.g., immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the predicted full-length amino acid sequence of the B Lymphocyte Stimulator polypeptide shown in SEQ ID NO:3229, up to the glycine residue at position number 261 of the sequence shown SEQ ID NO:3229 and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n5-266 of the sequence shown in SEQ ID NO:3229, where n5 is an integer in the range of the amino acid position of amino acid residues 1 to 261 of the amino acid sequence in SEQ ID NO:3229.
More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues of D-2 to L-266; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266; S-42 to L-266; K-43 to L-266; D44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266; and G-261 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more functional activities (e.g., biological activities) of the protein, other functional activities may still be retained. Thus, the ability of a shortened B Lymphocyte Stimulator mutein to induce and/or bind to antibodies which recognize the complete or mature form or the extracellular domain of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature form or the extracellular domain of the polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a B Lymphocyte Stimulator mutein with a large number of deleted C-terminal amino acid residues may retain some functional (e.g., immunogenic) activities. In fact, peptides composed of as few as six B Lymphocyte Stimulator amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides in another embodiment, antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the B Lymphocyte Stimulator shown in SEQ ID NO:3229, up to the glutamic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 1-m5 of SEQ ID NO:3229, where m5 is an integer in the range of the amino acid position of amino acid residues 6 to 265 in the amino acid sequence of SEQ ID NO:3229.
More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues M-1 to L-265; M-1 to K-264; M-1 to L-263; M-1 to A-262; M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234; M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-215; M-1 to I-214; M-1 to C-213; M-1 to R-212; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-1 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to I-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7; and M-1 to E-6 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a B Lymphocyte Stimulator polypeptide, which may be described generally as having residues n5-m5 of SEQ ID NO:3229, where n5 and m5 are integers as defined above.
In additional embodiments, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 134-m6 of SEQ ID NO:3228, where m6 is an integer from 140 to 285, corresponding to the position of the amino acid residue in SEQ ID NO:3228. For example, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues A-134 to Leu-285; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A-134 to V-142; A-134 to T-141; and A-134 to E-140 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-1 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; K-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R-174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-171 to I-185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to I-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G-249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274; L-261 to D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284; and S-271 to L-285 of SEQ ID NO:3228. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to C-15; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; S-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; A49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to G-142; S-129 to S-143; R-130 to Y-144; N-131 to T-145; K-132 to F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; 1-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-215; G-202 to N-216; D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-248; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265; and S-252 to L-266 of SEQ ID NO:3229. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
In additional embodiments, antibodies of the present invention may bind polypeptide fragments comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of residues: M-1 to F-15; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-1 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F40 to L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q-74; S-61 to A-75; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109; A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to N-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-174; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-168 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to V-190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-240 to I-254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to E-258; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-256 to E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to A-285; A-272 to L-286; Q-273 to K-287; I-274 to L-288; and S-275 to L-289 of SEQ ID NO:38. The present invention is also directed to antibodies that bind B Lymphocyte Stimulator polypeptides comprising, or alternatively, consisting of, a contiguous sequence of amino acid residues at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of B Lymphocyte Stimulator polypeptides described above.
It will be recognized by one of ordinary skill in the art that some amino acid sequences of the B Lymphocyte Stimulator polypeptides can be varied without significant effect of the structure or function of the polypeptide. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the polypeptide which determine activity.
Thus, the invention further includes antibodies that bind variations of B Lymphocyte Stimulator polypeptides which show B Lymphocyte Stimulator polypeptide functional activity (e.g., biological activity) or which include regions of B Lymphocyte Stimulator polypeptide such as the polypeptide fragments described herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.
As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J. U. et al., supra, and the references cited therein. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
Thus, antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO:3228, or that encoded by the deposited cDNA plasmid, such as (i) polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) polypeptides in which one or more of the amino acid residues includes a substituent group, or (iii) polypeptides in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence.
Antibodies of the present invention may bind fragments, derivatives or analogs of the polypeptide of SEQ ID NO:3229, or that encoded by the deposited cDNA plasmid, such as (i) polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) polypeptides in which one or more of the amino acid residues includes a substituent group, or (iii) polypeptides in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) polypeptides in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as, a soluble biologically active fragment of another TNF ligand family member (e.g., CD40 Ligand), an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Thus, the antibodies of the invention may bind B Lymphocyte Stimulator polypeptides that include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 13).







TABLE 13





Conservative Amino Acid Substitutions.


















Aromatic
Phenylalanine




Tryptophan




Tyrosine



Hydrophobic
Leucine




Isoleucine




Valine



Polar
Glutamine




Asparagine



Basic
Arginine




Lysine




Histidine



Acidic
Aspartic Acid




Glutamic Acid



Small
Alanine




Serine




Threonine




Methionine




Glycine










In one embodiment of the invention, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a B Lymphocyte Stimulator polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, even more preferably, not more than 40 conservative amino acid substitutions, still more preferably, not more than 30 conservative amino acid substitutions, and still even more preferably, not more than 20 conservative amino acid substitutions. In one embodiment of the invention, antibodies of the present invention bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of a B Lymphocyte Stimulator polypeptide having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
For example, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ID NO:3228 including: M1 replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or V; L16 replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced with H, or R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22 replaced with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A, G, I, S, T, M, or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G, I, L, S, T, or M; S29 replaced with A, G, I, L, T, M, or V; 130 replaced with A, G, L, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V; P33 replaced with H, or K; K34 replaced with H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38 replaced with A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced with H, or K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T, M, or V; K43 replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T, M, or V; K46 replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced with A, G, I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I, S, T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or V; L61 replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced with F, or W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83 replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91 replaced with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R; A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I, L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105 replaced with G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108 replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I, S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L, S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L, T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced with Q; S129 replaced with A, G, I, L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q; K132 replaced with H, or R; R133 replaced with H, or K; A134 replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced with D; T141 replaced with A, G, I, L, S, M, or V; V142 replaced with A, G, I, L, S, T, or M; T143 replaced with A, G, I, L, S, M, or V; Q144 replaced with N; D145 replaced with E; L147 replaced with A, G61, S, T, M, or V; Q148 replaced with N; L149 replaced with A, G, I, S, T, M, or V; I150 replaced with A, G, L, S, T, M, or V; A151 replaced with G, I, L, S, T, M, or V; D152 replaced with E; S153 replaced with A, G, I, L, T, M, or V; E154 replaced with D; T155 replaced with A, G, I, L, S, M, or V; T157 replaced with A, G, I, L, S, M, or V; I158 replaced with A, G, L, S, T, M, or V; Q159 replaced with N; K160 replaced with H, or R; G161 replaced with A, I, L, S, T, M, or V; S162 replaced with A, G, I, L, T, M, or V; Y163 replaced with F, or W; T164 replaced with A, G, I, L, S, M, or V; F165 replaced with W, or Y; V166 replaced with A, G, I, L, S, T, or M; W168 replaced with F, or Y; L169 replaced with A, G, I, S, T, M, or V; L170 replaced with A, G, I, S, T, M, or V; S171 replaced with A, G, I, L, T, M, or V; F172 replaced with W, or Y; K173 replaced with H, or R; R174 replaced with H, or K; G175 replaced with A, I, L, S, T, M, or V; S176 replaced with A, G, I, L, T, M, or V; A177 replaced with G, I, L, S, T, M, or V; L178 replaced with A, G, I, S, T, M, or V; E179 replaced with D; E180 replaced with D; K181 replaced with H, or R; E182 replaced with D; N183 replaced with Q; K184 replaced with H, or R; I185 replaced with A, G, L, S, T, M, or V; L186 replaced with A, G, I, S, T, M, or V; V187 replaced with A, G, I, L, S, T, or M; K188 replaced with H, or R; E189 replaced with D; T190 replaced with A, G, I, L, S, M, or V; G191 replaced with A, I, L, S, T, M, or V; Y192 replaced with F, or W; F193 replaced with W, or Y; F194 replaced with W, or Y; I195 replaced with A, G, L, S, T, M, or V; Y196 replaced with F, or W; G197 replaced with A, I, L, S, T, M, or V; Q198 replaced with N; V199 replaced with A, G, I, L, S, T, or M; L200 replaced with A, G, I, S, T, M, or V; Y201 replaced with F, or W; T202 replaced with A, G, I, L, S, M, or V; D203 replaced with E; K204 replaced with H, or R; T205 replaced with A, G, I, L, S, M, or V; Y206 replaced with F, or W; A207 replaced with G, I, L, S, T, M, or V; M208 replaced with A, G, I, L, S, T, or V; G209 replaced with A, I, L, S, T, M, or V; H210 replaced with K, or R; L211 replaced with A, G, I, S, T, M, or V; I212 replaced with A, G, L, S, T, M, or V; Q213 replaced with N; R214 replaced with H, or K; K215 replaced with H, or R; K216 replaced with H, or R; V217 replaced with A, G, I, L, S, T, or M; H218 replaced with K, or R; V219 replaced with A, G, I, L, S, T, or M; F220 replaced with W, or Y; G221 replaced with A, I, L, S, T, M, or V; D222 replaced with E; E223 replaced with D; L224 replaced with A, G, I, S, T, M, or V; S225 replaced with A, G, I, L, T, M, or V; L226 replaced with A, G, I, S, T, M, or V; V227 replaced with A, G, I, L, S, T, or M; T228 replaced with A, G, I, L, S, M, or V; L229 replaced with A, G, I, S, T, M, or V; F230 replaced with W, or Y; R231 replaced with H, or K; I233 replaced with A, G, L, S, T, M, or V; Q234 replaced with N; N235 replaced with Q; M236 replaced with A, G, I, L, S, T, or V; E238 replaced with D; T239 replaced with A, G, I, L, S, M, or V; L240 replaced with A, G, I, S, T, M, or V; N242 replaced with Q; N243 replaced with Q; S244 replaced with A, G, I, L, T, M, or V; Y246 replaced with F, or W; S247 replaced with A, G, I, L, T, M, or V; A248 replaced with G, I, L, S, T, M, or V; G249 replaced with A, I, L, S, T, M, or V; 1250 replaced with A, G, L, S, T, M, or V; A251 replaced with G, I, L, S, T, M, or V; K252 replaced with H, or R; L253 replaced with A, G, I, S, T, M, or V; E254 replaced with D; E255 replaced with D; G256 replaced with A, I, L, S, T, M, or V; D257 replaced with E; E258 replaced with D; L259 replaced with A, G, I, S, T, M, or V; Q260 replaced with N; L261 replaced with A, G, I, S, T, M, or V; A262 replaced with G, I, L, S, T, M, or V; I263 replaced with A, G, L, S, T, M, or V; R265 replaced with H, or K; E266 replaced with D; N267 replaced with Q; A268 replaced with G, I, L, S, T, M, or V; Q269 replaced with N; I270 replaced with A, G, L, S, T, M, or V; S271 replaced with A, G, I, L, T, M, or V; L272 replaced with A, G, I, S, T, M, or V; D273 replaced with E; G274 replaced with A, I, L, S, T, M, or V; D275 replaced with E; V276 replaced with A, G, I, L, S, T, or M; T277 replaced with A, G, I, L, S, M, or V; F278 replaced with W, or Y; F279 replaced with W, or Y; G280 replaced with A, I, L, S, T, M, or V; A281 replaced with G, I, L, S, T, M, or V; L282 replaced with A, G, I, S, T, M, or V; K283 replaced with H, or R; L284 replaced with A, G, I, S, T, M, or V; and/or L285 replaced with A, G, I, S, T, M, or V.
In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of SEQ ID NO:3229 including: M1 replaced with A, G, I, L, S, T, or V; D2 replaced with E; D3 replaced with E; S4 replaced with A, G, I, L, T, M, or V; T5 replaced with A, G, I, L, S, M, or V; E6 replaced with D; R7 replaced with H, or K; E8 replaced with D; Q9 replaced with N; S10 replaced with A, G, I, L, T, M, or V; R11 replaced with H, or K; L12 replaced with A, G, I, S, T, M, or V; T13 replaced with A, G, I, L, S, M, or V; S14 replaced with A, G, I, L, T, M, or V; L16 replaced with A, G, I, S, T, M, or V; K17 replaced with H, or R; K18 replaced with H, or R; R19 replaced with H, or K; E20 replaced with D; E21 replaced with D; M22 replaced with A, G, I, L, S, T, or V; K23 replaced with H, or R; L24 replaced with A, G, I, S, T, M, or V; K25 replaced with H, or R; E26 replaced with D; V28 replaced with A, G, I, L, S, T, or M; S29 replaced with A, G, I, L, T, M, or V; 130 replaced with A, G, L, S, T, M, or V; L31 replaced with A, G, I, S, T, M, or V; R33 replaced with H, or K; K34 replaced with H, or R; E35 replaced with D; S36 replaced with A, G, I, L, T, M, or V; S38 replaced with A, G, I, L, T, M, or V; V39 replaced with A, G, I, L, S, T, or M; R40 replaced with H, or K; S41 replaced with A, G, I, L, T, M, or V; S42 replaced with A, G, I, L, T, M, or V; K43 replaced with H, or R; D44 replaced with E; G45 replaced with A, I, L, S, T, M, or V; K46 replaced with H, or R; L47 replaced with A, G, I, S, T, M, or V; L48 replaced with A, G, I, S, T, M, or V; A49 replaced with G, I, L, S, T, M, or V; A50 replaced with G, I, L, S, T, M, or V; T51 replaced with A, G, I, L, S, M, or V; L52 replaced with A, G, I, S, T, M, or V; L53 replaced with A, G, I, S, T, M, or V; L54 replaced with A, G, I, S, T, M, or V; A55 replaced with G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V; L57 replaced with A, G, I, S, T, M, or V; S58 replaced with A, G, I, L, T, M, or V; L61 replaced with A, G, I, S, T, M, or V; T62 replaced with A, G, I, L, S, M, or V; V63 replaced with A, G, I, L, S, T, or M; V64 replaced with A, G, I, L, S, T, or M; S65 replaced with A, G, I, L, T, M, or V; F66 replaced with W, or Y; Y67 replaced with F, or W; Q68 replaced with N; V69 replaced with A, G, I, L, S, T, or M; A70 replaced with G, I, L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72 replaced with A, G, I, S, T, M, or V; Q73 replaced with N; G74 replaced with A, I, L, S, T, M, or V; D75 replaced with E; L76 replaced with A, G, I, S, T, M, or V; A77 replaced with G, I, L, S, T, M, or V; S78 replaced with A, G, I, L, T, M, or V; L79 replaced with A, G, I, S, T, M, or V; R80 replaced with H, or K; A81 replaced with G, I, L, S, T, M, or V; E82 replaced with D; L83 replaced with A, G, I, S, T, M, or V; Q84 replaced with N; G85 replaced with A, I, L, S, T, M, or V; H86 replaced with K, or R; H87 replaced with K, or R; A88 replaced with G, I, L, S, T, M, or V; E89 replaced with D; K90 replaced with H, or R; L91 replaced with A, G, I, S, T, M, or V; A93 replaced with G, I, L, S, T, M, or V; G94 replaced with A, I, L, S, T, M, or V; A95 replaced with G, I, L, S, T, M, or V; G96 replaced with A, I, L, S, T, M, or V; A97 replaced with G, I, L, S, T, M, or V; K99 replaced with H, or R; A100 replaced with G, I, L, S, T, M, or V; G101 replaced with A, I, L, S, T, M, or V; L102 replaced with A, G, I, S, T, M, or V; E103 replaced with D; E104 replaced with D; A105 replaced with G, I, L, S, T, M, or V; A107 replaced with G, I, L, S, T, M, or V; V108 replaced with A, G, I, L, S, T, or M; T109 replaced with A, G, I, L, S, M, or V; A110 replaced with G, I, L, S, T, M, or V; G111 replaced with A, I, L, S, T, M, or V; L112 replaced with A, G, I, S, T, M, or V; K113 replaced with H, or R; I114 replaced with A, G, L, S, T, M, or V; F115 replaced with W, or Y; E116 replaced with D; A119 replaced with G, I, L, S, T, M, or V; G121 replaced with A, I, L, S, T, M, or V; E122 replaced with D; G123 replaced with A, I, L, S, T, M, or V; N124 replaced with Q; S125 replaced with A, G, I, L, T, M, or V; S126 replaced with A, G, I, L, T, M, or V; Q127 replaced with N; N128 replaced with Q; S129 replaced with A, G, I, L, T, M, or V; R130 replaced with H, or K; N131 replaced with Q; K132 replaced with H, or R; R133 replaced with H, or K; A134 replaced with G, I, L, S, T, M, or V; V135 replaced with A, G, I, L, S, T, or M; Q136 replaced with N; G137 replaced with A, I, L, S, T, M, or V; E139 replaced with D; E140 replaced with D; T141 replaced with A, G, I, L, S, M, or V; G142 replaced with A, I, L, S, T, M, or V; S143 replaced with A, G, I, L, T, M, or V; Y144 replaced with F, or W; T145 replaced with A, G, I, L, S, M, or V; F146 replaced with W, or Y; V147 replaced with A, G, I, L, S, T, or M; W149 replaced with F, or Y; L150 replaced with A, G, I, S, T, M, or V; L151 replaced with A, G, I, S, T, M, or V; S152 replaced with A, G, I, L, T, M, or V; F153 replaced with W, or Y; K154 replaced with H, or R; R155 replaced with H, or K; G156 replaced with A, I, L, S, T, M, or V; S157 replaced with A, G, I, L, T, M, or V; A158 replaced with G, I, L, S, T, M, or V; L159 replaced with A, G, I, S, T, M, or V; E160 replaced with D; E161 replaced with D; K162 replaced with H, or R; E163 replaced with D; N164 replaced with Q; K165 replaced with H, or R; I166 replaced with A, G, L, S, T, M, or V; L167 replaced with A, G, I, S, T, M, or V; V168 replaced with A, G, I, L, S, T, or M; K169 replaced with H, or R; E170 replaced with D; T171 replaced with A, G, I, L, S, M, or V; G172 replaced with A, I, L, S, T, M, or V; Y173 replaced with F, or W; F174 replaced with W, or Y; F175 replaced with W, or Y; I176 replaced with A, G, L, S, T, M, or V; Y177 replaced with F, or W; G178 replaced with A, I, L, S, T, M, or V; Q179 replaced with N; V180 replaced with A, G, I, L, S, T, or M; L181 replaced with A, G, I, S, T, M, or V; Y182 replaced with F, or W; T183 replaced with A, G, I, L, S, M, or V; D184 replaced with E; K185 replaced with H, or R; T186 replaced with A, G, I, L, S, M, or V; Y187 replaced with F, or W; A188 replaced with G, I, L, S, T, M, or V; M189 replaced with A, G, I, L, S, T, or V; G190 replaced with A, I, L, S, T, M, or V; H191 replaced with K, or R; L192 replaced with A, G190, S, T, M, or V; I193 replaced with A, G, L, S, T, M, or V; Q194 replaced with N; R195 replaced with H, or K; K196 replaced with H, or R; K197 replaced with H, or R; V198 replaced with A, G, I, L, S, T, or M; H199 replaced with K, or R; V200 replaced with A, G, I, L, S, T, or M; F201 replaced with W, or Y; G202 replaced with A, I, L, S, T, M, or V; D203 replaced with E; E204 replaced with D; L205 replaced with A, G, I, S, T, M, or V; S206 replaced with A, G, I, L, T, M, or V; L207 replaced with A, G, I, S, T, M, or V; V208 replaced with A, G, I, L, S, T, or M; T209 replaced with A, G, I, L, S, M, or V; L210 replaced with A, G, I, S, T, M, or V; F211 replaced with W, or Y; R212 replaced with H, or K; I214 replaced with A, G, L, S, T, M, or V; Q215 replaced with N; N216 replaced with Q; M217 replaced with A, G, L, S, T, or V; E219 replaced with D; T220 replaced with A, G, I, L, S, M, or V; L221 replaced with A, G, I, S, T, M, or V; N223 replaced with Q; N224 replaced with Q; S225 replaced with A, G, I, L, T, M, or V; Y227 replaced with F, or W; S228 replaced with A, G, I, L, T, M, or V; A229 replaced with G, I, L, S, T, M, or V; G230 replaced with A, I, L, S, T, M, or V; I231 replaced with A, G, L, S, T, M, or V; A232 replaced with G, I, L, S, T, M, or V; K233 replaced with H, or R; L234 replaced with A, G, I, S, T, M, or V; E235 replaced with D; E236 replaced with D; G237 replaced with A, I, L, S, T, M, or V; D238 replaced with E; E239 replaced with D; L240 replaced with A, G, I, S, T, M, or V; Q241 replaced with N; L242 replaced with A, G, I, S, T, M, or V; A243 replaced with G, I, L, S, T, M, or V; I244 replaced with A, G, L, S, T, M, or V; R246 replaced with H, or K; E247 replaced with D; N248 replaced with Q; A249 replaced with G, I, L, S, T, M, or V; Q250 replaced with N; I251 replaced with A, G, L, S, T, M, or V; S252 replaced with A, G, I, L, T, M, or V; L253 replaced with A, G, I, S, T, M, or V; D254 replaced with E; G255 replaced with A, I, L, S, T, M, or V; D256 replaced with E; V257 replaced with A, G, I, L, S, T, or M; T258 replaced with A, G, I, L, S, M, or V; F259 replaced with W, or Y; F260 replaced with W, or Y; G261 replaced with A, I, L, S, T, M, or V; A262 replaced with G, I, L, S, T, M, or V; L263 replaced with A, G, I, S, T, M, or V; K264 replaced with H, or R; L265 replaced with A, G, I, S, T, M, or V; and/or L266 replaced with A, G, I, S, T, M, or V.
In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing conservative substitution mutations of the polypeptide of any one of SEQ ID NOS:3230-3237.
Amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for functional activity, such ligand binding and the ability to stimulate lymphocyte (e.g., B cell) as, for example, proliferation, differentiation, and/or activation. Accordingly, antibodies of the present invention may bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function. In preferred embodiments, antibodies of the present invention bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function and inhibit B Lymphocyte Stimulator polypeptide function. In other preferred embodiments, antibodies of the present invention bind amino acids in the B Lymphocyte Stimulator polypeptides that are essential for function and enhance B Lymphocyte Stimulator polypeptide function.
Of special interest are substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation. Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical formulations, because aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
In another embodiment, the invention provides for antibodies that bind polypeptides having amino acid sequences containing non-conservative substitutions of the amino acid sequence provided in SEQ ID NO:3228. For example, non-conservative substitutions of the B Lymphocyte Stimulator protein sequence provided in SEQ ID NO:3228 include: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C15 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F; W, Y, P, or C; E21 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 130 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85 replaced with D, E, H, N, K, R, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109 replaced with D, E, H, N, K, R, N, Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R133 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q144 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D145 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C146 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L149 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D152 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S153 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E154 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q159 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K160 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G161 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S162 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y163 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T164 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F165 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P167 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W168 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L169 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F172 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K173 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R174 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G175 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E179 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E180 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K181 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K184 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L186 replaced with D, E, H K, R, N, Q, F, W, Y, P, or C; V187 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K188 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E189 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y192 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F193 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F194 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y196 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G197 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q198 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K204 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y206 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H210 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I212 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q213 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P214 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K215 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K216 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H218 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F220 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D222 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E223 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L226 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V227 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T228 replaced with D, F, H, K, R, N, Q, F, W, Y, P, or C; L229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F230 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R231 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C232 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q234 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N235 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M236 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P237 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N242 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N243 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C245 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y246 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S247 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K252 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E254 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E255 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D257 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E258 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L259 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q260 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P264 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R265 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E266 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N267 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q269 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I270 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D273 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G274 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D275 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T277 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F278 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F279 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L282 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K283 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L285 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
In an additional embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, a B Lymphocyte Stimulator amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative).
In another embodiment of the invention, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides with non-conservative substitutions of the sequence provided in SEQ ID NO:3229 including: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D2 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D3 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R7 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E8 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q9 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T13 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C15 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K17 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R19 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E20 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E21 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L24 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K25 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E26 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C27 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 130 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R33 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E35 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R40 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S42 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K43 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T51 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L57 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; C60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S65 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F66 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y67 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q68 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G74 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L79 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R80 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A81 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E82 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K90 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L91 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A95 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K99 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A100 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L102 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E103 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P106 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T109 replaced with D, E, H, N, K, R, N, Q, F, W, Y, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G111 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F115 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E116 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P120 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N128 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S129 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R130 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R133 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q136 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E139 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E140 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y144 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F146 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W149 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F153 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K154 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R155 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G156 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E160 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E161 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K162 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E163 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N164 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K165 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L167 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K169 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E170 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G172 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y173 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F174 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F175 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y177 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q179 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V180 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y182 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T183 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D184 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K185 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y187 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H191 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R195 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K196 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V198 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H199 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F201 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D203 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E204 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L205 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S206 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L210 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F211 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R212 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C213 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q215 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N216 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; M217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P218 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E219 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T220 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L221 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P222 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N223 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C226 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y227 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G230 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I231 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A232 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K233 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E235 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E236 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G237 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D238 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E239 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I244 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P245 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R246 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E247 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N248 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A249 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q250 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I251 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D254 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G255 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D256 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F259 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F260 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A262 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K264 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
In another embodiment, site directed changes at the amino acid level of B Lymphocyte Stimulator can be made by replacing a particular amino acid with a non-conservative substitution. Antibodies of the present invention may bind B Lymphocyte Stimulator amino acid sequences containing non-conservative substitution mutations of the polypeptide of any one of SEQ ID NOS:3230-3237.
In an additional embodiment, antibodies of the present invention bind B Lymphocyte Stimulator polypeptides comprising, or alternatively consisting of, a B Lymphocyte Stimulator amino acid sequence in which more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 and 50) is replaced with the substituted amino acids as described above (either conservative or nonconservative).
Replacement of amino acids can also change the selectivity of the binding of a ligand to cell surface receptors. For example, Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Since B Lymphocyte Stimulator is a member of the TNF polypeptide family, mutations similar to those in TNF-alpha are likely to have similar effects in B Lymphocyte Stimulator polypeptides.
Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
Since B Lymphocyte Stimulator is a member of the TNF-related protein family, mutations may be made in sequences encoding amino acids in the TNF conserved domain, e.g., in positions Gly-191 through Leu-284 of SEQ ID NO:3228 or in positions Gly-172 through Leu-265 of SEQ ID NO:3229, may modulate rather than completely eliminate functional activities (e.g., biological activities) of B Lymphocyte Stimulator polypeptides or fragments or variants thereof. Accordingly, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain. In preferred embodiments, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain and act as antagonists of B Lymphocyte Stimulator. In other preferred embodiments, antibodies of the present invention may bind B Lymphocyte Stimulator polypeptides that have mutations in the TNF conserved domain and act as agonists of B Lymphocyte Stimulator.
Recombinant DNA technology known to those skilled in the art (see, for instance, DNA shuffling supra) can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
Thus, the invention also encompasses antibodies that bind B Lymphocyte Stimulator derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate B Lymphocyte Stimulator polypeptides, e.g., that are better suited for expression, scale up, etc., in the host cells. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognition sequences in the B Lymphocyte Stimulator polypeptides of the invention, and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the B Lymphocyte Stimulator at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197). By way of non-limiting example, mutation of the serine at position 244 to alanine either singly or in combination with mutation of the asparagine at position 242 to glutamine abolishes glycosylation of the mature soluble form of B Lymphocyte Stimulator (e.g., amino acids 134-285 of SEQ ID NO:3228) when expressed in the yeast Pichea pastoris. A mutant B Lymphocyte Stimulator polypeptide in which only the asparagine at position 242 is mutated to glutamine, is still glycosylated when expressed in Pichea pastoris. In this mutant, the glycosylation event may be due to the activation or unmasking of an O-linked glycosylation site at serine 244. Similar mutations affecting glycosylation could also be made in the B Lymphocyte Stimulator polypeptide of SEQ ID NO:3229, i.e., aspargine-223 to glutamine and/or serine-224 to alanine of SEQ ID NO:3229. Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins. One possible result of such a mutation is that B Lymphocyte Stimulator polypeptide of the invention is not cleaved and released from the cell surface. Accordingly, antibodies of the invention may bind B Lymphocyte Stimulator derivatives and analogs that have one or more amino acid residues deleted, added, or substituted. In other embodiments, antibodies of the invention may bind B Lymphocyte Stimulator derivatives, variants or analogs that are unable to be cleaved from the cell surface.
In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which 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, to prevent or diminish release of the soluble form of B Lymphocyte Stimulator from cells expressing B Lymphocyte Stimulator. In a more specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Lys-132 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to Ala-132. In another, nonexclusive specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to Ala-133. These mutated proteins have uses such as, for example, in ex vivo therapy or gene therapy, to engineer cells expressing a B Lymphocyte Stimulator polypeptide that is retained on the surface of the engineered cells.
In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-146 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-146 is replaced with a serine amino acid residue.
In another specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-232 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-232 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.
In yet another specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-245 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228 is mutated to another amino acid residue, or deleted altogether, for example, to aid preventing or diminishing oligomerization of the mutant B Lymphocyte Stimulator polypeptide when expressed in an expression system. In a specific embodiment, antibodies of the invention bind B Lymphocyte Stimulator polypeptides in which Cys-245 is replaced with a serine amino acid residue. Polypeptides encoding these polypeptides are also encompassed by the invention.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of the B Lymphocyte Stimulator polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:3140 (1988).
The antibodies of the present invention bind B Lymphocyte Stimulator polypeptides including the complete polypeptide encoded by the deposited cDNA (ATCCTM Deposit No. 97768) including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ID NO:3228, the mature soluble polypeptide of SEQ ID NO:3228, e.g., amino acids 134-285 of SEQ ID NO:3228, the extracellular domain of SEQ ID NO:3228, amino acid residues 73-285 of SEQ ID NO:3228 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 991/o similarity to those described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The antibodies of the present invention bind B Lymphocyte Stimulator polypeptides including the complete polypeptide encoded by the deposited cDNA including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA (ATCCTM Deposit No. 203518), the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of SEQ ID NO:3229, the mature soluble of SEQ ID NO:3229, e.g., amino acid residues 134-266 of SEQ ID NO:3229, the extracellular domain of SEQ ID NO:3229, e.g., amino acid residues 73-266 of SEQ ID NO:3229 minus the intracellular and transmembrane domains, as well as polypeptides which have at least 80%, 85%, 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Further antibodies of the present invention bind polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCCTM Deposit No. 97768) or to the polypeptide of SEQ ID NO:3228, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
Further antibodies of the present invention bind polypeptides including polypeptides at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCCTM Deposit No. 203518) or to the polypeptide of SEQ ID NO:3229, and also include antibodies that bind portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. Polynucleotides encoding these polypeptides are also encompassed by the invention.
By “% similarity” for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482489, 1981) to find the best segment of similarity between two sequences.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a B Lymphocyte Stimulator polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the B Lymphocyte Stimulator polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of SEQ ID NO:3228, the amino acid sequence encoded by the deposited cDNA clone HNEDU15 (ATCCTM Accession No. 97768), or fragments thereof, or, for instance, to the amino acid sequence of SEQ ID NO:3229, the amino acid sequence encoded by the deposited cDNA clone HDPMC52 (ATCCTM Accession No. 203518), or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and -C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
Antibodies that Immunospecifically Bind B Lymphocyte Stimulator Polypeptides
The present invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator polypeptides, which antibodies comprise, or alternatively consist of, all or a portion of a heavy and/or light chain variable domain of the scFvs referred to in Table 1.
The present invention also encompasses methods and compositions for detecting, diagnosing and/or prognosing diseases or disorders associated with aberrant 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).
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).
Anti-B Lymphocyte Stimulator Antibodies
The antibodies of the present invention were discovered, in part, using phage display technology. Single chain antibody molecules (“scFvs”) displayed on the surface of phage particles were screened to identify those scFvs that immunospecifically bind to B Lymphocyte Stimulator, including the membrane-bound form and soluble form of B Lymphocyte Stimulator. The present invention encompasses the scFvs and portions thereof that were identified to immunospecifically bind to B Lymphocyte Stimulator, including scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, and scFvs that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator. In particular, the present invention encompasses scFvs comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NOS: 1-2128, as referred to in Table 1. Preferably, the scFvs of the present invention comprise, or alternatively consist of, the amino acid sequence of SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908. The scFvs include scFvs that bind to soluble B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1563-1880), scFvs that bind to the membrane-bound form of B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1881-2128), and scFvs that bind to both the soluble form and the membrane-bound form of B Lymphocyte Stimulator (e.g., scFvs comprising, or alternatively consisting of, an amino acid sequence of SEQ ID NOS: 1-1562). Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
In one embodiment of the present invention, scFvs that immunospecifically bind to B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1 and/or any one of the VL domains referred to in Table 1. In preferred embodiments, scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention comprise the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs referred to in Table 1 and/or any one, two, three, or more of the VL CDRs referred to in Table 1. In preferred embodiments, scFvs of the present invention comprise the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention comprise the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, antibody fragments or variants of the scFvs referred to in Table 1 that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
(Table 1 can be found at the end of the specification just prior to the claims.)
In another embodiment of the present invention, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator, comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 1563-1880 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 1570-1595. In an even more preferred embodiment, an scFv that immunospecifically binds to a soluble form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1563-1569.
In another embodiment of the present invention, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1881-2128 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 1886-1908. In an even more preferred embodiment, an scFv that immunospecifically binds to a membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:1881-1885.
In another embodiment of the present invention, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS: 1-1562 as referred to in Table 1. In a preferred embodiment, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, the amino acid sequence of SEQ ID NOS:834-872. In another preferred embodiment, an scFv that immunospecifically binds to both the soluble form and membrane-bound form of B Lymphocyte Stimulator comprises, or alternatively consists of, any one of the amino acids sequences of SEQ ID NOS:1-46 or 321-329. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to the soluble form of B Lymphocyte Stimulator and/or the membrane-bound form of B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
In another embodiment of the present invention, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in contained SEQ ID NOS: 1563-1880, as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the of the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator, preferably the soluble form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
In another embodiment of the present invention, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:1881-2128 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs, that immunospecifically bind to B Lymphocyte Stimulator, preferably the membrane-bound form of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
In another embodiment of the present invention, scFvs that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one of the VL domains contained in SEQ ID NOS:1-1562 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In another embodiment, scFvs that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator comprise a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one, two, three, or more of the VL CDRs contained in SEQ ID NOS:1-1562 as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH domain and VL domain from different scFvs referred to in Table 1. In a preferred embodiment, scFvs that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of any one of the VH CDR3s contained in SEQ ID NOS:1-1562 as disclosed in Table 1 and/or any one of the VL CDR3s contained in SEQ ID NOS:1-1562, as disclosed in Table 1. In preferred embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from the same scFv referred to in Table 1. In alternative embodiments, scFvs of the present invention that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator, comprise a polypeptide having the amino acid sequence of a VH CDR and VL CDR from different scFvs referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs or molecules, that immunospecifically bind to B Lymphocyte Stimulator, preferably the soluble and membrane-bound forms of B Lymphocyte Stimulator, are also encompassed by the invention, as are nucleic acid molecules encoding these scFvs, molecules, fragments and/or variants.
The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1, such scFvs may routinely be “converted” to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 20, infra.
In one embodiment, the invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one of the VH domains contained in the sequences referred to in Table 1. The present invention also provides antibodies that immunospecifically bind to a polypeptide, or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VH CDRs contained in the sequences referred to in Table 1. Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that immunospecifically bind to B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants.
In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR referred to in Table 1. In particular, the invention provides antibodies that immunospecifically bind B Lymphocyte Stimulator, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VH CDR1 contained in SEQ ID NOS:1-46, 321-329, 15-63-1569, or 1881-1885 as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR2 contained in SEQ ID NOS: 1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In yet another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR1 contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1; a VH CDR2 contained in SEQ ID NOS: SEQ ID NOS: SEQ ID NOS:834-872, 1570-1595, or 1886-1908; and/or a VH CDR3 contained in SEQ ID NOS: SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1. Preferably, antibodies of the invention comprise, or alternatively consist of, VH CDRs that are derived from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide, or polypeptide fragment of B Lymphocyte Stimulator. In particular, the invention provides antibodies wherein said antibodies comprise, or alternatively consist of, a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. The present invention also provides antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VL CDR having an amino acid sequence of any one, two, three, or more of the VL CDRs contained in the sequences referred to in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR referred to in Table 1. In particular, the invention provides antibodies that immunospecifically bind B Lymphocyte Stimulator, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VL CDR1 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR2 contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1. In a preferred embodiment, antibodies comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS: in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 disclosed in Table 1. In yet another embodiment, antibodies that immunospecifically bind B Lymphocyte Stimulator comprise, or alternatively consist of: a polypeptide having the amino acid sequence of a VL CDR1 contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1; a VL CDR2 SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1 and a VL CDR3 contained SEQ ID NOS:834-872, 1570-1595, or 1886-1908 as disclosed in Table 1. Preferably, antibodies of the invention comprise, or alternatively consist of, VL CDRs that are derived from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VH domain of one of the scFvs referred to in Table 1 combined with a VL domain of one of the scFvs referred to in Table 1, or other VL domain. The present invention further provides antibodies (including molecules comprise, or alternatively consist of, antibody fragments or variants thereof) that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VL domain of one of the scFvs referred to in Table 1 combined with a VH domain of one of the scFvs referred to in Table 1, or other VH domain. In a preferred embodiment, antibodies that immunospecifically bind to a polypeptide or a polypeptide fragment of B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH domain contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1 and a VL domain contained in contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1. In a further preferred embodiment, the antibodies of the invention comprise, or alternatively consist of, a VH and a VL domain from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as referred to in Table 1. In particular, the invention provides for antibodies that immunospecifically bind to a polypeptide or polypeptide fragment of B Lymphocyte Stimulator, wherein said antibodies comprise, or alternatively consist of, a VH CDR1 and a VL CDR1, a VH CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and a VL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VL CDRs referred to in Table 1. In a preferred embodiment, one or more of these combinations are from the same scFv as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
In a preferred embodiment the invention provides antibodies wherein the VH CDRX (where X=1, 2, or 3) and VL CDRY (where Y=1, 2, or 3) are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator, from scFvs that bind membrane-bound B Lymphocyte Stimulator, or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that immunospecifically bind to B Lymphocyte Stimulator are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants.
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. Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, single chain Fvs (scFvs), Fab fragments, F(ab′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), antiidiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The antibodies of the present invention also include molecules comprising, or alternatively consisting of, a polypeptide having an amino acid sequence of a portion of an amino acid sequence contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908. Preferably, an antibody of the invention comprises, or alternatively consists of, a polypeptide having an amino acid sequence of a VH domain, VH CDR, VL domain, or VL CDR of any one those contained in the sequences referred to in Table 1. Antibodies of the invention also include molecules comprising, or alternatively consisting of, fragments or variants of the above antibodies that immunospecifically bind B Lymphocyte Stimulator.
Most preferably the antibodies of the present invention are whole antibodies or antibody fragments that immunospecifically bind human B Lymphocyte Stimulator. Antibody fragments of the invention that immunospecifically bind human B Lymphocyte Stimulator include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd fragments, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFvs), fragments comprising, or alternatively consisting of, either a VL or VH domain, and epitope binding fragments of any of the above.
B Lymphocyte Stimulator-binding antibody fragments, including single-chain antibodies, may comprise, or alternatively consist of, the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. In a preferred embodiment, the antibodies of the invention comprise, or alternatively consist of, a polypeptide that immunospecifically binds to B Lymphocyte Stimulator, said polypeptides comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs referred to in Table 1, preferably a polypeptide having an amino acid sequence of a VH CDR3 and/or a VL CDR3 of contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1. Most preferably, antibodies of the invention comprise, or alternatively consist of, one, two, three, four, five, six or more CDRs from the same scFv, as referred to in Table 1. The antibodies of the invention may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Most preferably, the antibodies are human antibodies. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries and xenomice or other organisms that have been genetically engineered to produce human antibodies. For a detailed discussion of a few of the technologies for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; and Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), which are incorporated by reference herein in their entirety. Human antibodies or “humanized” chimeric monoclonal antibodies can be produced using techniques described herein or otherwise known in the art. For example, methods for producing chimeric antibodies are known in the art. See, for review the following references which are hereby incorporated in their entirety: Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985). In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
The antibodies of the present invention may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a B Lymphocyte Stimulator polypeptide, or fragment thereof, or may be specific for both a B Lymphocyte Stimulator polypeptide, or fragment thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may bind immunospecifically to murine B Lymphocyte Stimulator (e.g., a polypeptide having the amino acid sequence 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 the antibodies of the invention bind immunospecifically to human B Lymphocyte Stimulator. Preferably, the antibodies of the invention bind immunospecifically to human and monkey B Lymphocyte Stimulator. Also preferably, the antibodies of the invention bind immunospecifically to human B Lymphocyte Stimulator and murine B Lymphocyte Stimulator. More preferably, antibodies of the invention, bind immunospecifically and with higher affinity to human B Lymphocyte Stimulator than to murine B Lymphocyte Stimulator.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, antibodies of the present invention cross react with APRIL (SEQ ID NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902). In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under hybridization conditions (as described herein).
In a specific embodiment, antibodies of the present invention cross react with APRIL (SEQ ID NO:3239; GenBank Accession No. AF046888; J. Exp. Med. 188(6):1185-1190; PCT International Publication WO97/33902). In specific embodiments, antibodies that immunospecifically bind both B Lymphocyte Stimulator and APRIL comprise all or a portion the BAB2001, BAB2080, BAB2015, BAB2019, BAB2087, BAB2016, BAB2034 or BAB2065 scFVs (SEQ ID NOS:3240-3247). These scFvs were isolated by panning a phage scFv library comprising VH and VL domains obtained from human bone marrow B cells (BM library). Phage from the BM phage library were first selected for binding to soluble B Lymphocyte Stimulator (amino acids 134-285 of SEQ ID NO:3228). A second round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the B Lymphocyte Stimulator binding phage selected in round one. A third round of selection for binding to the soluble form of APRIL (amino acids 105-250 of SEQ ID NO:3239) was then performed on the phage selected in round two. A final (fourth) round of selection for binding to the soluble form of B Lymphocyte Stimulator (amino acids 134-285 of SEQ ID NO:3228) was then performed on the phage selected in round three. Phage clones that bound B Lymphocyte Stimulator in the fourth round of selection were eluted with either 0.1M triethylamine (TEA) or with a TACI-Fc fusion protein (e.g., the extracellular domain of TACI (amino acids 31 to 159 of Genbank Accession No. AAC51790) fused to Fc). Eluted Phage were collected and sequenced (SEQ ID NOS: 3240-3247). Of 79 sequences, there were 8 unique sequences (SEQ ID NOS:3240-3247).
Isolated scFv clones (e.g., scFvs corresponding to SEQ ID NOS:3240-3247 or other scFvs described in Table 1) or antibodies comprising at least a portion of said scFV clones may be screened for their ability to bind their ability to bind the soluble form of B Lymphocyte Stimulator and the soluble form of APRIL by ELISA. Isolated scFv clones or antibodies comprising at least a portion of said scFv clones may also be screened for their ability to inhibit binding of a soluble form of B Lymphocyte Stimulator or B Lymphocyte Stimulator heterotrimer to TACI, BCMA or BAFF-R (Genbank Accession Nos. AAC51790, NP—00183, and NP—443177, respectively). Isolated scFv clones or antibodies comprising at least a portion of said scFV clones may also be screened for their ability to inhibit B Lymphocyte Stimulator or B Lymphocyte Stimulator heterotrimer mediated biological activities (e.g. stimulation of B cell proliferation and/or stimulation of immunoglobulin production).
In specific embodiments, antibodies that immunospecifically bind both B Lymphocyte Stimulator and APRIL comprise all or a portion (e.g., VHCDR, VLCDR, VH domain, VL domain) of the BAB2001, BAB2015, BAB2016, BAB2019, BAB2034, BAB2065, or BAB2080 scFVs (SEQ ID NOS:3240-3247).
In preferred embodiments, the antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), immunospecifically bind to B Lymphocyte Stimulator and do not cross-react with any other antigens. In more preferred embodiments, the antibodies of the invention immunospecifically bind to B Lymphocyte Stimulator and do not cross-react with TRAIL, APRIL, Endokine-alpha, TNF-alpha, TNF-beta, Fas-L or LIGHT.
The present invention also provides for a nucleic acid molecule, generally isolated, encoding an antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). In one embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1 having an amino acid sequence of any one of the VH CDR1s referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR2 having an amino acid sequence of any one of the VH CDR2s referred to in Table 1. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR3 having an amino acid sequence of any one of the VH CDR3s referred to in Table 1. Nucleic acid molecules encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VH domains and/or VH CDRs are also encompassed by the invention.
In another embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR1 having amino acid sequence of any one of the VL CDR1s referred to in Table 1. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR2 having an amino acid sequence of any one of the VL CDR2s referred to in Table 1. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR3 having an amino acid sequence of any one of the VL CDR3s referred to in Table 1. Nucleic acid encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VL domains and/or VLCDR(s) are also encompassed by the invention.
In another embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains referred to in Table 1 and a VL domain having an amino acid sequence of any one of the VL domains referred to in Table 1. In another embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3, a VL CDR3, or any combination thereof having an amino acid sequence referred to in Table 1. Nucleic acid encoding antibodies that immunospecifically bind B Lymphocyte Stimulator and comprise, or alternatively consist of, fragments or variants of the VL and/or domains and/or VHCDR(s) and/or VLCDR(s) are also encompassed by the invention.
The present invention also provides antibodies that comprise, or alternatively consist of, variants (including derivatives) of the VH domains, VH CDRs, VL domains, and VL CDRs described herein, which antibodies immunospecifically bind to B Lymphocyte Stimulator. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. In specific embodiments, the variants encode substitutions of VHCDR3. In a preferred embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind B Lymphocyte Stimulator). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind B Lymphocyte Stimulator) can be determined using techniques described herein or by routinely modifying techniques known in the art.
The antibodies of the invention include derivatives (i.e., variants) that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not affect the ability of the antibody to immunospecifically bind to B Lymphocyte Stimulator. For example, but not by way of limitation, derivatives of the invention include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
In a specific embodiment, an antibody of the invention (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH or VL domains referred to in Table 1 under stringent conditions, e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDRs or VL CDRs referred to in Table 1 under stringent conditions, e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CDRs or VL CDRs referred to in Table 1 under stringent conditions e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
In another embodiment, an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH domains referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH CDRs referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the VH CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
In another embodiment, an antibody of the invention (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL domains referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDRs referred to in Table 1. In another embodiment, an antibody of the invention that immunospecifically binds to B Lymphocyte Stimulator comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDR3s referred to in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
Antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may also be described or specified in terms of their binding affinity for to B Lymphocyte Stimulator polypeptides or fragments or variants of B Lymphocyte Stimulator polypeptides (e.g., to the soluble form of B Lymphocyte Stimulator and/or membrane-bound form of B Lymphocyte Stimulator). In specific embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides, or fragments or variants thereof, with a dissociation constant or KD of less than or equal to 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×104 M, 104 M, 5×10−5 M, or 10.5 M. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×104 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−8 M, or 10−8 M. Even more preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×10−9 M, 10−9 M, 5×10−11 M, 10−10 M, 5×10−1 M, 10−11 M, 5×10−12 M, 10−12 M, 5×−13 M, 10−3 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with a dissociation constant or KD that is within any one of the ranges that are between each of the individual recited values.
In specific embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an off rate (koff) of less than or equal to 5×10−2 sec−1, 10−2 sec−1, 5×10−3 sec−1 or 10−3 sec−1. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an off rate (koff) less than or equal to 5×10−4 sec−1, 10−4 sec−1, 5×10−5 sec−1, or 10−5 sec−1, 5×10−4 sec−1, 10−6 sec−1, 5×10−7 sec−1 or 10−7 sec−1. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with an off rate (koff) that is within any one of the ranges that are between each of the individual recited values.
In other embodiments, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an on rate (kon) of greater than or equal to 103 M−1 sec−1, 5×103 M−1 sec−1, 104 M−1 sec−1 or 5×104 M−1 sec−1. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with an on rate (kon) greater than or equal to 105 M−1 sec−1, 5×105 M−1 sec−1, 106 M−1 sec−1, or 5×106 M−1 sec−1 or 107 M−1 sec−1. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with on rate (kon) that is within any one of the ranges that are between each of the individual recited values.
The invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that have one or more of the same biological characteristics as one or more of the antibodies described herein. By “biological characteristics” is meant, the in vitro or in vivo activities or properties of the antibodies, such as, for example, the ability to bind to B Lymphocyte Stimulator (e.g., the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, the soluble form and membrane-bound form of B Lymphocyte Stimulator), and/or an antigenic and/or epitope region of B Lymphocyte Stimulator), the ability to substantially block B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177) binding, or the ability to block B Lymphocyte Stimulator mediated biological activity (e.g., stimulation of B cell proliferation and immunoglobulin production). Optionally, the antibodies of the invention will bind to the same epitope as at least one of the antibodies specifically referred to herein. Such epitope binding can be routinely determined using assays known in the art.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that neutralize B Lymphocyte Stimulator or a fragment thereof, said antibodies comprising, or alternatively consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv referred to in Table 1, more preferably having an amino acid sequence contained in SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and even more preferably having an amino acid sequence contained in SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1, or a fragment or variant thereof. By an antibody that “neutralizes B Lymphocyte Stimulator or a fragment thereof” is meant an antibody that diminishes or abolishes the ability of B Lymphocyte Stimulator to bind to its receptor (e.g., TACI-GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177) to stimulate B cell proliferation, to stimulate immunoglobulin secretion by B cells, and/or to stimulate the B Lymphocyte Stimulator receptor signalling cascade. In one embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR domain in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VLCDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that neutralizes B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated B cell proliferation as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS: 1-46, 321-329, 1563-1569, 188I-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of B cell survival as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 188I-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell survival, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of B cell differentiation as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of B cell differentiation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit (i.e., diminish or abolish) B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells as determined by any method known in the art such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, 1886-1908, and even more preferably having an amino acid sequence SEQ ID NOS:1-46, 321-329, 1563-1569, 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that inhibits B Lymphocyte Stimulator-mediated stimulation of immunoglobulin production by B cells, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that enhance the activity of B Lymphocyte Stimulator or a fragment thereof, said antibodies comprising, or alternatively consisting of, a portion (i.e., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and preferably having an amino acid sequence of SEQ ID NOS: 1-46, 321-329, 1563-1569, or 1881-1885, as disclosed in Table 1, or a fragment or variant thereof. By an antibody that “enhances the activity of B Lymphocyte Stimulator or a fragment thereof” is meant an antibody increases the ability of B Lymphocyte Stimulator to bind to its receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP-443177), to stimulate B cell proliferation, to stimulate immunoglobulin secretion by B cells, and/or to stimulate the B Lymphocyte Stimulator receptor signalling cascade. In one embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that enhances B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that enhances the activity of B Lymphocyte Stimulator or a fragment thereof, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that stimulate B Lymphocyte Stimulator-mediated B cell proliferation as determined by any method known in the art, such as, for example, the assays described in Examples 21 and 22, infra, said antibodies comprising, or alternatively consisting of, a portion (e.g., a VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of an scFv having an amino acid sequence of SEQ ID NOS:834-872, 1570-1595, or 1886-1908, and even more preferably having an amino acid sequence of SEQ ID NOS:1-46, 321-329, 1563-1569, or 1881-1885 as disclosed in Table 1 or a fragment or variant thereof. In one embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists 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 disclosed in Table 1, or a fragment or variant thereof. In another embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL domain contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In a preferred embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VH CDR3 contained in SEQ ID NOS:1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. In another preferred embodiment, an antibody that stimulates B Lymphocyte Stimulator-mediated B cell proliferation, comprises, or alternatively consists of, a polypeptide having the amino acid sequence of a VL CDR3 contained in SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, or 1881-1908 as disclosed in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
The present invention also provides for fusion proteins comprising, or alternatively consisting of, an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that immunospecifically binds to B Lymphocyte Stimulator, and a heterologous polypeptide. Preferably, the heterologous polypeptide to which the antibody is fused to is useful for B-cell function or is useful to target the antibody to B-cells. In an alternative preferred embodiment, the heterologous polypeptide to which the antibody is fused to is useful for monocyte cell function or is useful to target the antibody to a monocyte. In another embodiment, the heterologous polypeptide to which the antibody is fused is albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-x of human serum albumin, where x is an integer from 1 to 585 and the albumin fragment has human serum albumin activity. In another preferred embodiment, antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).
In one embodiment, a fusion protein of the invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one or more of the VH domains referred to in Table 1 or the amino acid sequence of any one or more of the VL domains referred to in Table 1 or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein of the present invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs referred to in Table 1, or the amino acid sequence of any one, two, three, or more of the VL CDRs referred to in Table 1, or fragments or variants thereof, and a heterologous polypeptide sequence. In a preferred embodiment, the fusion protein comprises, or alternatively consists of, a polypeptide having the amino acid sequence of, a VH CDR3 referred to in Table 1, or fragment or variant thereof, and a heterologous polypeptide sequence, which fusion protein immunospecifically binds to B Lymphocyte Stimulator. In another embodiment, a fusion protein comprises, or alternatively consists of a polypeptide having the amino acid sequence of at least one VH domain referred to in Table 1 and the amino acid sequence of at least one VL domain referred to in Table 1 or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL domains of the fusion protein correspond to the same scFv referred to in Table 1. In yet another embodiment, a fusion protein of the invention comprises, or alternatively consists of a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs referred to in Table 1 and the amino acid sequence of any one, two, three or more of the VL CDRs referred to in Table 1, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the VHCDR(s) or VLCDR(s) correspond to the same scFv referred to in Table 1. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
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; antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator; and antibodies that immunospecifically bind to both the soluble form and membrane-bound form of B Lymphocyte Stimulator.
In one embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) (including derivative) thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
In another embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or a fragment or variant thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
In another embodiment of the present invention, antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator, are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a fragment or variant thereof. Preferably, the VH and VL domains of the antibody correspond to the same scFv as disclosed in Table 1. In another embodiment, antibodies that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs contained in: SEQ ID NOS: 1-1562 as disclosed in Table 1 and/or the amino acid sequence of any one, two, three, or more of the VL CDRs contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, two, three, four, five, six or more of the VH and VL CDRs of the antibody correspond to the same scFv as disclosed in Table 1. In a preferred embodiment, antibodies that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator are provided that comprise, or alternatively consist of, a polypeptide having the amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1-1562, disclosed in Table 1 and/or the amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1-1562, disclosed in Table 1, or fragment(s) or variant(s) thereof. Preferably, the VHCDR3 and VLCDR3 of the antibody correspond to the same scFv, as disclosed in Table 1.
The present invention also provides for mixtures of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, wherein the mixture has at least one, two, three, four, five or more different antibodies of the invention. In particular, the invention provides for mixtures of different antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the membrane-bound form and soluble form of B Lymphocyte Stimulator. In specific embodiments, the invention provides mixtures of at least 2, preferably at least 4, at least 6, at least 8, at least 10, at least 12, at least 15, at least 20, or at least 25 different antibodies that immunospecifically bind to B Lymphocyte Stimulator, wherein at least 1, at least 2, at least 4, at least 6, or at least 10, antibodies of the mixture is an antibody of the invention. In a specific embodiment, each antibody of the mixture is an antibody of the invention.
The present invention also provides for panels of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, wherein the panel has at least one, two, three, four, five or more different antibodies of the invention. In particular, the invention provides for panels of different antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator, the membrane-bound form of B Lymphocyte Stimulator, and/or both the membrane-bound form and soluble form of B Lymphocyte Stimulator. In specific embodiments, the invention provides for panels of antibodies that have different affinities for B Lymphocyte Stimulator, different specificities for B Lymphocyte Stimulator, or different dissociation rates. The invention provides panels of at least 10, preferably at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000, antibodies. Panels of antibodies can be used, for example, in 96 well plates for assays such as ELISAs.
The present invention further provides for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS: 1563-1880 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1563-1880, as disclosed in Table 1 or a variant thereof.
The present invention further provides for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1 or a variant thereof.
The present invention further provides for compositions comprising, one or more antibodies (including scFvs, or molecules comprising, or alternatively consisting of antibody fragments or variants of the invention). In one embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR1 s contained in SEQ ID NOS:1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR2s contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH CDR3s contained in SEQ ID NOS: 1-1562 as disclosed in Table 1 or a variant thereof.
Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternative consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VLCDR1s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s contained SEQ ID NOS: 1563-1880 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS:1563-1880 as disclosed in Table 1, or a variant thereof.
Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1s contained in SEQ ID NOS: 1881-2128 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s SEQ ID NOS: 1881-2128 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS:1881-2128 as disclosed in Table 1, or a variant thereof.
Other embodiments of the present invention providing for compositions comprising, one or more antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) are listed below. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL domains contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR1 s contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof. In another embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR2s SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof. In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VL CDR3s contained in SEQ ID NOS: 1-1562 as disclosed in Table 1, or a variant thereof.
In a preferred embodiment, a composition of the present invention comprises, one, two, three, four, five, or more antibodies that comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one or more of the VH domains in disclosed in Table 1, or a variant thereof, and an amino acid sequence of any one or more of the VL domains disclosed in Table 1, or a variant thereof wherein the VH and VL domains are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator (SEQ ID NOS: 1563-1880), from scFvs that bind membrane-bound B Lymphocyte Stimulator (SEQ ID 1881-2128), or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator (SEQ ID NOS:1-1562). In a preferred embodiment the invention provides antibodies wherein the VH CDRX (where X=1, 2, or 3) and VL CDRY (where Y=1, 2, or 3) are from scFvs with the same specificity (i.e., from scFvs that bind soluble B Lymphocyte Stimulator (SEQ ID NOS: 1563-1880), from scFvs that bind membrane-bound B Lymphocyte Stimulator (SEQ ID NOS:1881-2128), or from scFvs that bind both soluble and membrane-bound B Lymphocyte Stimulator (SEQ ID NOS: 1-1562). In yet another embodiment, a composition of the present invention comprises one or more fusion proteins.
As discussed in more detail below, a composition of the invention may be used either alone or in combination with other compositions. The antibodies (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the present invention) may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
Antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the present invention) may be used, for example, but not limited to, to purify and detect B Lymphocyte Stimulator, and to target the polypeptides of the present invention to cells expressing membrane-bound B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of B Lymphocyte Stimulator in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
Methods Producing Antibodies
The antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants of the invention) can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
The single chain Fvs disclosed in Table 1 were generated using phage display methods known in the art. Furthermore, other scFvs that immunospecifically bind B Lymphocyte Stimulator may be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g. p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., B Lymphocyte Stimulator or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; WO97/13844; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
ScFvs that immunospecifically bind to both B Lymphocyte Stimulator and APRIL polypeptides (preferably to the mature soluble forms of each) may be obtained, for example, by sequential rounds of selection for binding to one or the other of B Lymphocyte Stimulator and APRIL polypeptides. Thus in one embodiment, the present invention provides for a method of selecting phage that express scFvs that immunospecifically bind both B Lymphocyte Stimulator polypeptides and APRIL polypeptides, comprising at least one round of phage selection for binding to B Lymphocyte Stimulator polypeptide and at least one round of phage selection for binding to APRIL polypeptide. Selection for B Lymphocyte Stimulator binding may either precede or follow selection for APRIL binding. More than one round of selection for binding to either B Lymphocyte Stimulator or APRIL may be conducted.
ScFvs that immunospecifically bind to a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide may be, for example, obtained by sequential rounds of selection for binding to one or the other of B Lymphocyte Stimulator and APRIL polypeptide. Thus in one embodiment, the present invention provides for a method of selecting phage that express scFvs that immunospecifically bind a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide, comprising at least one round of phage selection for binding to B Lymphocyte Stimulator polypeptide and at least one round of phage selection for binding to APRIL polypeptide. Selection for B Lymphocyte Stimulator binding may either precede or follow selection for APRIL binding. More than one round of selection for binding to either B Lymphocyte Stimulator or APRIL may be conducted.
Alternatively, scFvs that immunospecifically bind to a heterotrimer comprising at least one B Lymphocyte Stimulator polypeptide and at least one APRIL polypeptide may be obtained, for example, by selecting scFVs that bind to a B Lymphocyte Stimulator heterotrimer. A B Lymphocyte Stimulator heterotrimer may contain, for example, one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides (2B Lymphocyte Stimulator: 1APRIL heterotrimer). Other B Lymphocyte Stimulator heterotrimers may contain, for example, one B Lymphocyte Stimulator polypeptide and two APRIL polypeptides (1B Lymphocyte Stimulator:2APRIL heterotrimer). Preferably, the heterotrimers comprising B Lymphocyte Stimulator and APRIL polypeptides contain the mature forms of both the B Lymphocyte Stimulator and APRIL polypeptides. ScFvs may be selected in one or more rounds of selection for binding only to the 2B Lymphocyte Stimulator:1APRIL heterotrimer, or only to the 1B Lymphocyte Stimulator:2APRIL heterotrimer. Alternatively, scFVs that immunospecifically bind heterotrimers comprising B Lymphocyte Stimulator and APRIL polypeptides may be obtained through sequential rounds of screening on individual forms of the B Lymphocyte Stimulator/APRIL heterotrimer in any order and/or on both forms of the heterotrimer simultaneously, or any combination thereof.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
The characteristics of an antibody, such as its on-rate, off-rate and/or overall affinity may be altered using in vitro mutation and selection techniques. This process is well known in the art and is commonly referred to as in vitro affinity maturation of antibodies. Starting with a given antibody that binds a particular antigen with a certain affinity, one of skill in the art can engineer variants of that antibody and test the antibody variants for altered (usually improved) antigen binding characteristics. The amino acid sequence of the VH and VL regions of such antibody variants may be substantially from that of the starting or original antibody; for example, the an antibody variants may comprise the original VH or VL paired with a different (from the original) VL or VH region, respectively. Alternatively, the amino acid sequence of the VH and VL region of such antibody variants may be quite similar to that of the starting or original antibody; for example, an antibody variant may have only a few amino acid changes in the VH and/or VL region. It is common for one to engineer the mutations in CDR regions, and particularly in the VHCDR3 region. Examples of both types of in vitro antibody affinity maturation are described for example, Thompson et al., (1996) The Journal of Molecular Biology 256:77-88. Moreover, a review of phage display antibody technology may be found in Vaughan et al., (1998) Nature Biotechnology 16:535-539; both of these articles are herein incorporated by reference in their entireties. The scFvs of SEQ ID NOS:10-37 are CDR3 mutants derived from the scFv of SEQ ID NO:9 and scFvs of SEQ ID NOS:291-327 are CDR3 mutants derived from the scFv of SEQ ID NO:2.
To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Preferably, the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
Cell lines that express antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (“ATCCTM”) on the dates listed in Table 2 and given the ATCCTM Deposit Numbers identified in Table 2. The ATCCTM is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCCTM deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.











TABLE 2






Corre-
SEQ
ATCC TM




sponding
ID
Deposit
ATCC TM Deposit


Cell Line
scFv
NO:
Number
Date



















NSO-B11-15
I050B11-15
24
PTA-3238
Mar. 27, 2001


NSO-anti-B
I006D08
2
PTA-3239
Mar. 27, 2001


Lymphocyte


Stimulator-


6D08-18


NSO-anti-B
I116A01
327
PTA-3240
Mar. 27, 2001


Lymphocyte


Stimulator-


116A01-60


IO26C04K
I026C04-K
1563
PTA-3241
Mar. 27, 2001


IO50A12
I050A12
12
PTA-3242
Mar. 27, 2001


IO50-B11
I050B11
9
PTA-3243
Mar. 27, 2001









Accordingly, in on embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-B11-15.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-anti-B Lymphocyte Stimulator-6D08-18.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-anti-B Lymphocyte Stimulator-116A01-60.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line IO26C04K.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line IO50A12.
In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO-B11.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide. In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by between 1% and 10% in a competitive inhibition assay. In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by between 1% and 10% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 10% and up to 20% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 20% and up to 30% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 30% and up to 40% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 40% and up to 50% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 50% and up to 60% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 60% and up to 70% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 70% and up to 80% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 80% and up to 90% in a competitive inhibition assay.
In preferred embodiments, the invention provides antibodies that which reduce the binding of an antibody comprising a fragment (e.g., VH domain, VL domain, VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, or VLCDR3) or variant of an scFv referred to in Table 1 to a B Lymphocyte Stimulator polypeptide by at least 90% and up to 100% in a competitive inhibition assay.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3238 to a B Lymphocyte Stimulator polypeptide.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3239 to a B Lymphocyte Stimulator polypeptide.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3240 to a B Lymphocyte Stimulator polypeptide.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3241 to a B Lymphocyte Stimulator polypeptide.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3242 to a B Lymphocyte Stimulator polypeptide.
In other preferred embodiments, the invention provides antibodies that competitively inhibit binding of the antibody produced by the cell line having ATCCTM deposit number PTA-3243 to a B Lymphocyte Stimulator polypeptide.
For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. In a specific embodiment, antibodies of the present invention comprise one or more VH and VL domains corresponding to the human scFvs of the invention and framework regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In a specific embodiment, antibodies of the present invention comprise one or more CDRs corresponding to the human scFvs of the invention and framework regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In other embodiments, an antibody of the present invention comprises one, two, three, four, five, six or more VL CDRs or VH CDRs corresponding to one or more of the human scFvs referred to in Table 1, or fragments or variants thereof, and framework regions (and, optionally CDRs not derived from the scFvs in Table 1) from a human immunoglobulin molecule. In a preferred embodiment, an antibody of the present invention comprises a VH CDR3, VL CDR3, or both, corresponding to the same scFv, or different scFvs referred to in Table 1, or fragments or variants thereof, and framework regions from a human immunoglobulin.
A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a human antibody and a non-human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non-human immunoglobulin molecule (e.g., framework regions from a canine or feline immunoglobulin molecule) can be produced using a variety of techniques known in the art including, for example, CDR grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In a preferred embodiment, chimeric antibodies comprise a human CDR3 having an amino acid sequence of any one of the VH CDR3s or VL CDR3s referred to in Table 1, or a variant thereof, and non-human framework regions or human framework regions different from those of the frameworks in the corresponding scFv disclosed in Table 1. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.)
Further, the antibodies of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” B Lymphocyte Stimulator polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1993); and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies of the invention which bind to B Lymphocyte Stimulator and competitively inhibit the binding of B Lymphocyte Stimulator to its receptor (as determined by assays well known in the art such as, for example, that disclosed, infra) can be used to generate antiidiotypes that “mimic” a B Lymphocyte Stimulator ligand/receptor-binding domain and, as a consequence, bind to and neutralize B Lymphocyte Stimulator receptors (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177). Such neutralizing anti-idiotypes (including molecules comprising, or alternatively consisting of, antibody fragments or variants, such as Fab fragments of such anti-idiotypes) can be used in therapeutic regimens to neutralize B Lymphocyte Stimulator. For example, such anti-idiotypic antibodies can be used to bind B Lymphocyte Stimulator ligands/receptors, and thereby block B Lymphocyte Stimulator mediated biological activity. Alternatively, anti-idiotypes that “mimic” a B Lymphocyte Stimulator binding domain may bind to B Lymphocyte Stimulator receptor(s) and induce B Lymphocyte Stimulator receptor mediated signalling (e.g., activation of nuclear factor of activated T cells (NF-AT), nuclear factor-kappa B (NF-kappa B), and/or AP-1). Such agonistic anti-idiotypes (including agonistic Fab fragments of these anti-idiotypes) can be used in therapeutic regimens to induce or enhance B Lymphocyte Stimulator receptor mediated signalling. For example, such anti-idiotypic antibodies can be used to bind B Lymphocyte Stimulator ligands/receptors, and thereby stimulate B Lymphocyte Stimulator mediated biological activity (e.g., B cell proliferation and/or immunoglobulin production).
Once an antibody molecule of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) has been chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
Polynucleotides Encoding an Antibody
The invention provides polynucleotides comprising, or alternatively consisting of, a nucleotide sequence encoding an antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). The invention also encompasses polynucleotides that hybridize under high stringency, or alternatively, under intermediate or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides complementary to nucleic acids having a polynucleotide sequence that encodes an antibody of the invention or a fragment or variant thereof.
The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of the scFv antibodies and VH domains, VL domains and CDRs thereof, are known (as described in Table 1), nucleotide sequences encoding these antibodies can be determined using methods well known in the art, i.e., the nucleotide codons known to encode the particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody, of the invention. Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence of the antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, one or more of the VH and VL domains referred to in Table 1, or fragments or variants thereof, is inserted within framework regions using recombinant DNA techniques known in the art. In a specific embodiment, one, two, three, four, five, six, or more of the CDRs referred to in Table 1, or fragments or variants thereof, is inserted within framework regions using recombinant DNA techniques known in the art. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457479 (1998) for a listing of human framework regions, the contents of which are hereby incorporated by reference in its entirety). Preferably, the polynucleotides generated by the combination of the framework regions and CDRs encode an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically binds to B Lymphocyte Stimulator. Preferably, as discussed supra, polynucleotides encoding variants of antibodies or antibody fragments having one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules, or antibody fragments or variants, lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and fall within the ordinary skill of the art.
Recombinant Expression of an Antibody
Recombinant expression of an antibody of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof (e.g., a heavy or light chain of an antibody of the invention or a portion thereof or a single chain antibody of the invention)), requires construction of an expression vector(s) containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule (e.g., a whole antibody, a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain)), of the invention has been obtained, the vector(s) for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, a single chain Fv, or fragments or variants thereof), operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464, the contents of each of which are hereby incorporated by reference in its entirety) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.
The expression vector(s) is(are) transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing polynucleotide(s) encoding an antibody of the invention (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, or a fragment or variant thereof), operably linked to a heterologous promoter. In preferred embodiments, for the expression of entire antibody molecules, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 1. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pfN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) may be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, NSO, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7O3O and HsS78Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8 17 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); TIB TECH 11(5):155-2 15 (May, 1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the coding sequence of the antibody, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibodies of the present invention may be glycosylated or may be non-glycosylated. In addition, antibodies of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
Antibodies of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y. and Hunkapiller, M., et al., 1984, Nature 310:105-111). For example, a peptide corresponding to a fragment of an antibody of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses antibodies which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-termini or C-termini ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-termini methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the antibody.
Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as, for example, iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 69Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin.
In specific embodiments, antibodies of the invention may be labeled with Europium. For example, antibodies of the invention may be labelled with Europium using the DELFIA Eu-labeling kit (catalog#1244-302, Perkin Elmer Life Sciences, Boston, Mass.) following manufacturer's instructions.
In specific embodiments, antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 111In, 177Lu, 90Y, 166Ho, and 153Sm, to antibodies. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator attached to antibodies of the invention is 90Y, In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA). In specific embodiments, the macrocyclic chelator is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid. In other specific embodiments, the DOTA is attached to the antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to an antibody are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties.
In one embodiment, antibodies of the invention are labeled with biotin. In other related embodiments, biotinylated antibodies of the invention may be used, for example, as an imaging agent or as a means of identifying one or more B Lymphocyte Stimulator receptor(s) or other coreceptor or ligand molecules.
Also provided by the invention are chemically modified derivatives of antibodies of the invention which may provide additional advantages such as increased solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000,40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the antibody. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include, for example, lysine residues and the N-termini amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-termini amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins, e.g., antibodies, via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the antibody or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the antibody.
One may specifically desire antibodies chemically modified at the N-terminus of either the heavy chain or the light chain or both. Using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective chemical modification at the N-terminus may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular antibody, e.g., a heavy chain or alight chain. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction of antibody with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the antibody. Thus, the invention includes antibody-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethane sulphonyl group.
Polyethylene glycol can also be attached to antibodies using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the antibody by a linker can also be produced by reaction of antibodies with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each antibody of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated antibodies of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-1, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per antibody molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
Antibody Characterization
Antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be characterized in a variety of ways. In particular, antibodies and related molecules of the invention may be assayed for the ability to immunospecifically bind to B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator (e.g., to the soluble form or the membrane-bound form of B Lymphocyte Stimulator) using techniques described herein or routinely modifying techniques known in the art. B Lymphocyte Stimulator or B Lymphocyte Stimulator fragments that may be immunospecifically bound by the compositions of the invention include, but are not limited to, 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) or fragments thereof. Preferably compositions of the invention bind human B Lymphocyte Stimulator (SEQ ID NOS:3228 and/or 3229) or fragments thereof. Assays for the ability of the antibodies of the invention to immunospecifically bind B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator may be performed in solution (e.g., Houghten, Bio/Techniques 13:412421(1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of these references is incorporated herein in its entirety by reference). Antibodies that have been identified to immunospecifically bind to B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator can then be assayed for their specificity and affinity for B Lymphocyte Stimulator or a fragment of B Lymphocyte Stimulator using or routinely modifying techniques described herein or otherwise known in the art.
The antibodies of the invention may be assayed for immunospecific binding to B Lymphocyte Stimulator and cross-reactivity with other antigens by any method known in the art. In particular, the ability of an antibody to immunospecifically bind to the soluble form or membrane-bound form of B Lymphocyte Stimulator and the specificity of the antibody, fragment, or variant for B Lymphocyte Stimulator polypeptide from a particular species (e.g., murine, monkey or human, preferably human) may be determined using or routinely modifying techniques described herein or otherwise known in art.
Immunoassays which can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40 degrees C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound antibodies or non-specifically bound antibodies, and detecting the presence of the antibodies specifically bound to the antigen coating the well. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g. Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.
The binding affinity of an antibody (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of the present invention for B Lymphocyte Stimulator and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, B Lymphocyte Stimulator is incubated with an antibody of the present invention conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second anti-B Lymphocyte Stimulator antibody.
In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) to B Lymphocyte Stimulator, or fragments of B Lymphocyte Stimulator. BIAcore kinetic analysis comprises analyzing the binding and dissociation of B Lymphocyte Stimulator from chips with immobilized antibodies on their surface as described in detail in Examples 6, 12, 17 and 18, infra.
The antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) can also be assayed for their ability to inhibit, increase, or not significantly alter, the binding of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177) using techniques known to those of skill in the art. For example, cells expressing a receptor for B Lymphocyte Stimulator (e.g., IM9, REH, ARH-77 cells, Namalwa, and RPMI-8226 B cell tumor lines as wells as peripheral CD20+ B cells) can be contacted with B Lymphocyte Stimulator in the presence or absence of an antibody, and the ability of the antibody to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to the cells can be measured. B Lymphocyte Stimulator binding to cells can be measured by, for example, flow cytometry or a scintillation assay. B Lymphocyte Stimulator or the antibody can be labeled with a detectable compound such as a radioactive label (e.g., 32P, 35S, and 125I) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between B Lymphocyte Stimulator and a B Lymphocyte Stimulator receptor and/or B Lymphocyte Stimulator and an antibody of the invention. Alternatively, the ability of antibodies of the invention to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor can be determined in cell-free assays. For example, native or recombinant B Lymphocyte Stimulator (e.g., that having the amino acid sequence of amino acids 134-285 of SEQ ID NO:3228) or a fragment thereof can be contacted with an antibody and the ability of the antibody to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator from binding to a B Lymphocyte Stimulator receptor can be determined. Preferably, the antibody is immobilized on a solid support and B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment is labeled with a detectable compound. Alternatively, B Lymphocyte Stimulator or a B Lymphocyte Stimulator fragment is immobilized on a solid support and the antibody is labeled with a detectable compound. B Lymphocyte Stimulator may be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. Further, the B Lymphocyte Stimulator polypeptide may be a fusion protein comprising B Lymphocyte Stimulator or a biologically active portion thereof and a domain such as an Immunoglobulin Fe or glutathionine-S-transferase. For example, amino acid residues 1-154 of TACI (GenBank accession number AAC51790), amino acids 148 of BCMA (GenBank accession number NP—001183) or amino acids 1-81 of BAFF-R (GenBank Accession Number NP—443177) may be fused to the Fe region of an IgG molecule and used in a cell free assay to determine the ability of antibodies of the invention to inhibit, increase, or not significantly alter, B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor. Alternatively, B Lymphocyte Stimulator can be biotinylated using techniques well known to those of skill in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).
The antibodies of the invention (including scFvs or other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), can also be assayed for their ability to inhibit, stimulate, or not significantly alter, B Lymphocyte Stimulator-induced B-cell proliferation using techniques known to those of skill in the art. For example, B-cell proliferation can be assayed by 3H-thymidine incorporation assays and trypan blue cell counts (see, e.g., Moore et al., Science 285: 260-263 (1999)). Further, the antibodies of the invention, or fragments or variants thereof, can be assayed for their ability to block, stimulate, or not significantly alter, B Lymphocyte Stimulator-induced activation of cellular signaling molecules and transcription factors such as calcium-modulator and cyclophilin ligand (“CAML”), calcineurin, nuclear factor of activated T cells transcription factor (“NF-AT”), nuclear factor-kappa B (“NF-kappa B”), and AP-1 using techniques known to those of skill in the art (see, e.g., von Bulow and Bram, Science 278:138-141(1997)). For example, NF-AT activity can be determined by electromobility gel shift assays, by detecting the expression of a protein known to be regulated by NF-AT (e.g., IL-2 expression), by detecting the induction of a reporter gene (e.g., an NF-AT regulatory element operably linked to a nucleic acid encoding a detectable marker such as luciferase, beta-galactosidase or chloramphenicol acetyltransferase (CAT)), or by detecting a cellular response (e.g., cellular differentiation, or cell proliferation).
The antibodies of the invention, or fragments or variants thereof can also be assayed for their ability to neutralize, enhance, or not significantly alter, B Lymphocyte Stimulator activity. For example, antibodies or fragments or variants thereof, may be routinely tested for their ability to inhibit B Lymphocyte Stimulator from binding to cells expressing the receptor for B Lymphocyte Stimulator (see Example 3, infra).
Selection and Screening for Antibodies that Immunospecifically Bind to Soluble B Lymphocyte Stimulator
Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be screened in a variety of assays to identify those antibodies that immunospecifically bind to the soluble form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to the biotinylated soluble form of B Lymphocyte Stimulator in solution are captured on streptavidin coated magnetic beads. This assay may be relatively applied to identify antibodies of the invention that neutralize and/or bind to B Lymphocyte Stimulator. Additionally, antibodies may be assayed in neutralization assays described herein or otherwise known in the art (see Example 3, infra). For example, antibodies may be tested for their ability to inhibit soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) from binding to IM9 cells. In this assay, labeled soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) is incubated with candidate anti-B Lymphocyte Stimulator antibodies to allow for the formation of B Lymphocyte Stimulator-anti-B Lymphocyte Stimulator antibody complexes. Following incubation, an aliquot of the B Lymphocyte Stimulator-anti-B Lymphocyte Stimulator antibody sample is added to IM9 cells. The binding of soluble B Lymphocyte Stimulator may be determined using techniques known in the art. For example, the binding of biotinylated B Lymphocyte Stimulator to IM9 cells may be detected using a fluorimeter following the addition of streptavidin-delfia. Biotinylated B Lymphocyte Stimulator, if it is not bound by antibodies that neutralize B Lymphocyte Stimulator, binds to the cells is detected. Thus, an antibody that decreases the amount of bio-B Lymphocyte Stimulator that binds to IM-9 cells (relative to a control sample in which the B Lymphocyte Stimulator had been preincubated with an irrelevant antibody or no antibody at all) is identified as one that binds to and neutralizes the soluble form of B Lymphocyte Stimulator. In another assay, antibodies are screened using ELISAs for those antibodies that bind to biotinylated soluble B Lymphocyte Stimulator, but do not bind membrane-bound B Lymphocyte Stimulator, such as, for example, B Lymphocyte Stimulator on membranes from U937 cells (see Examples 2 and 9, infra). In these assays, soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) and membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are incubated in separate samples with the same antibodies and those antibodies that bind to the soluble B Lymphocyte Stimulator (biotinylated B Lymphocyte Stimulator), but not membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are captured and identified.
Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be tested to identify those antibodies that do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS (see Example 4, infra). Antibodies may also be tested for their affinity for B Lymphocyte Stimulator using, for example, BIAcore analysis (see Examples 6, 12, 17 and 18 infra). Antibodies may also be tested for their ability to stimulate, inhibit, or not alter, B Lymphocyte Stimulator-induced immunoglobulin production and/or B-cell proliferation using techniques known to those of skill in the art. For example, human B-cells, B Lymphocyte Stimulator and antibodies may be incubated together in 96 well plates and 3H-thymidine incorporation may be measured using a scintillation counter.
Selection and Screening for Antibodies that Immunospecifically Bind to Membrane-bound B Lymphocyte Stimulator
Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be screened in a variety of assays to identify those antibodies that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to B Lymphocyte Stimulator on U937 membranes or immobilized histidine-tagged B Lymphocyte Stimulator are captured. Other cell lines that express B Lymphocyte Stimulator that might be useful for testing antibody binding to membrane-bound form of B Lymphocyte Stimulator include, K562, HL 60 and THP-1 cells. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants) that bind to B Lymphocyte Stimulator on U937 membranes or to histidine-tagged B Lymphocyte Stimulator. In this assay, antibodies are added to 96 well plates coated with U937 membranes or histidine-tagged B Lymphocyte Stimulator and those antibodies or antibody fragments or variants that bind to the U937 membranes or histidine-tagged B Lymphocyte Stimulator are captured. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants thereof) that do not bind to biotinylated B Lymphocyte Stimulator (soluble B Lymphocyte Stimulator) but bind to membrane-bound B Lymphocyte Stimulator, such as, for example, that on membranes from U937 cells (see Example 2, infra). In these assays, soluble B Lymphocyte Stimulator (e.g., biotinylated B Lymphocyte Stimulator) and membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are incubated in separate samples with the same antibodies (or antibody fragments or variants) and those antibodies (or antibody fragments or variants) that do not bind to the soluble B Lymphocyte Stimulator (biotinylated B Lymphocyte Stimulator), but bind the membrane-bound B Lymphocyte Stimulator (e.g., on U937 membranes) are captured and identified. In other assays, antibodies are screened using ELISAs to determine which of the antibodies (or antibody fragments or variants) that bind to histidine-tagged B Lymphocyte Stimulator or membranes from U937 cells do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS (See Example 4, infra). ELISAs can also be used to determine which of the antibodies (or antibody fragments or variants) that bind to histidine-tagged B Lymphocyte Stimulator or membranes from U937 cells bind to B Lymphocyte Stimulator in the presence of TNF-alpha (see Example 4, infra). Antibodies or fragments or variants thereof that immunospecifically bind to the membrane-bound form of B Lymphocyte Stimulator may also be tested for their affinity for histidine-tagged B Lymphocyte Stimulator using high-throughput BIAcore analysis (see Example 14, infra).
Additionally, antibodies of the invention may be screened against cells engineered to express an “uncleavable” form of B Lymphocyte Stimulator in order to determine their specificity for the membrane-bound form of B Lymphocyte Stimulator. Mutations in B Lymphocyte Stimulator which may achieve this result include, but are not limited to, the mutation or deletion of amino acid residues Lys-132 and/or Arg-133 of the B Lymphocyte Stimulator sequence shown in SEQ ID NO:3228. Atypical mutagenesis might include mutation of one or both of residues Lys-132 or Arg-133 to alanine residues. Cells expressing such an “uncleavable” form of B Lymphocyte Stimulator provide a profound reagent to use in assaying the ability of antibodies to bind the membrane-bound form of B Lymphocyte Stimulator.
Selection and Screening for Antibodies that Immunospecifically Bind to Soluble and Membrane-bound B Lymphocyte Stimulator
Antibodies of the invention (including scFvs and other molecules comprising, or alternately consisting of, antibody fragments or variants) may be screened in a variety of assays to identify those antibodies or antibody fragments or variants that immunospecifically bind to the soluble form and membrane-bound form of B Lymphocyte Stimulator. In one particular assay, antibodies that bind to immobilized B Lymphocyte Stimulator are captured. In another assay, antibodies are screened using ELISAs for those antibodies (or antibody fragments or variants) that inhibit the binding of soluble B Lymphocyte Stimulator (e.g. soluble bio-B Lymphocyte Stimulator) to IM-9 cells as described supra. In other assays, antibodies are screened using ELISAs for those antibodies that bind to membranes from U937 cells. Additionally, further ELISA assays may be performed using techniques known in the art to determine which antibodies do not cross-react with APRIL, endokine-alpha, VEGI, TRAIL, TNF-alpha, TNF-beta, Fas-L, LIGHT, and PBS, or those antibodies that bind to B Lymphocyte Stimulator in the presence of TNF-alpha (see Example 4 infra). Antibodies may be assayed in neutralization assays using techniques described herein or otherwise known in the art. Antibodies that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator may also be tested for their affinity for B Lymphocyte Stimulator using high-throughput BIAcore analysis.
Antibody Conjugates
The present invention encompasses antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous polypeptide (or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies of the invention may be used to target heterologous polypeptides to particular cell types (e.g., cells of monocytic lineage and B-cells), either in vitro or in vivo, by fusing or conjugating the heterologous polypeptides to antibodies of the invention that are specific for particular cell surface antigens (e.g., membrane-bound B Lymphocyte Stimulator on cells of monocytic lineage) or which bind antigens that bind particular cell surface receptors (e.g., TACI, BCMA, BAFF-R located on B cells). Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
In one embodiment, a fusion protein comprises a polypeptide having an amino acid sequence of any one of the VH domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR1s referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR2s referred to in Table 1, and a heterologous polypeptide. In a preferred embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH CDR3s referred to in Table 1 (i.e., SEQ ID NOS:2129-3227), and a heterologous polypeptide.
In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR1s referred to in Table 1, and a heterologous polypeptide. In yet another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR2s referred to in Table 1, and a heterologous polypeptide. In a preferred embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VL CDR3s referred to in Table 1, and a heterologous polypeptide.
In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of any one of the VH domains referred to in Table 1, and one or more VL domains referred to in Table 1, and a heterologous polypeptide. In another embodiment, a fusion protein of the present invention comprises a polypeptide having the amino acid sequence of any one of the VH CDRs referred to in Table 1, and any one of the VL CDRs referred to in Table 1, and a heterologous polypeptide.
The present invention further includes compositions comprising, or alternatively consisting of, heterologous polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, or a portion thereof. Methods for fusing or conjugating polypeptides to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), such methods can be used to generate antibodies with altered activity (e.g., antibodies with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, polynucleotides encoding antibodies of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more portions of a polynucleotide encoding an antibody which portions immunospecifically bind to B Lymphocyte Stimulator may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Moreover, the antibodies of the present invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), can be fused to marker sequences, such as a polypeptides to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine polypeptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag (DYKDDDDK, (SEQ ID No: 3238) Stratagene, La Jolla, Calif.).
The present invention further encompasses antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor or prognose the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include, but are not limited to, streptavidinlbiotin and avidin/biotin; examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes, but is not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; and examples of suitable radioactive material include, but are not limited to, iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115mIn, 113mIn, 112In, 111In), and technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (113F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 69Y 51Cr, 54Mn, 75Se, 153Sn, and 117Tin.
Further, an antibody of the invention (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof), may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic of cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. In specific embodiments, antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 181In, 177Lu, 90Y, 166Ho, and 153Sm, to polypeptides. In preferred embodiments, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is 111In. In preferred embodiments, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is 90Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA). In other specific embodiments, the DOTA is attached to the antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety.
A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells and includes such molecules as small molecule toxins and enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof. Examples include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide (VP-16), tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease, RNAse, and puromycin and fragments, variants or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine), improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide trimethylolomelamine, chlomaphazine, cholophosphamide, estramustine, ifosfamide, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, chlorozotocin, fotemustine, nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin, esorubicin, idarubicin, marcellomycin, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, quelamycin, rodorubicin, streptonigrin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSKO, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2′,2′′-trichlorotriethylamine, urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (“Ara-C”), taxoids, e.g. paclitaxel (TAXOL′′, Bristol-Myers Squibb Oncology, Princeton, N.J.) doxetaxel (TAXOTERE®, Rhóne-Poulenc Rorer, Antony, France), gemcitabine, ifosfamide, vinorelbine, navelbine, novantrone, teniposide, aminopterin, xeloda, ibandronate, CPT-I 1, topoisomerase inhibitor RFS 2000, difluoromethylomithine (DMFO), retinoic acid, esperamicins, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T reaction).
The antibodies of the invention which are conjugates can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, but are not limited to, for example, a toxin such as abrin, ricin A, alpha toxin, pseudomonas exotoxin, or diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (see, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.
In specific embodiments, antibodies of the invention are conjugated or fused to a polypeptide cytotoxin. An example of a suitable polypeptide cytotoxin is a ribosome-inactivating protein. Type I ribosome-inactivating proteins are single-chain proteins, while type II ribosome-inactivating proteins consist of two nonidentical subunits (A and B chains) joined by a disulfide bond (for a review, see Soria et al., Targeted Diagn. Ther. 7:193 (1992)). In one embodiment, type I ribosome-inactivating proteins that may be used include, but are not limited to, polypeptides from Saponaria officinalis (e.g., saporin-1, saporin-2, saporin-3, saporin-6), Momordica charantia (e.g, momordin), Byronia dioica (e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g., trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin), Phytolacca americana (e.g., pokeweed antiviral protein, pokeweed antiviral protein-II, pokeweed antiviral protein-S), Phytolacca dodecandra (e.g., dodecandrin, Mirabilis antiviral protein). Ribosome-inactivating proteins are described, for example, by Walsh et al., U.S. Pat. No. 5,635,384. In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.
In another embodiment, type II ribosome-inactivating proteins that may be used according to the invention include, but are not limited to, polypeptides from Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin), Adenia digitata (e.g., modeccin). Since type II ribosome-inactiving proteins include a B chain that binds galactosides and a toxic A chain that depurinates adensoine. Type II ribosome-inactivating protein conjugates should include the A chain. Additional ribosome-inactivating proteins that may be used according to the invention include, but are not limited to, bouganin, clavin, maize ribosome-inactivating proteins, Vaccaria pyramidata ribosome-inactivatng proteins, nigrine b, basic nigrine 1, ebuline, racemosine b, luffin-a, luffin-b, luffin-S, and other ribosome-inactivating proteins known to those of skill in the art. See, for example, Bolognesi and Stirpe, International Publication No. WO98/55623, Colnaghi et al., International Publication No. WO97/49726, Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and Stirpe, International Publication No. WO95/07297, Arias et al., International Publication No. WO94/20540, Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al., Agric. Biol. Chem. 55:229 (1991), and Gao et al., FEBS Lett. 347:257 (1994). In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.
Additional ribosome inactivating proteins (RIPs) that may be conjugated or fused to antibodies of the invention include, but are not limited to, Type I Plant RIPs such as Pokeweed antiviral proteins, Tritin, Gelonin, Momordin, Saporin, Dianthin, and Maize RIP; Type II Plant RIPs such as Ricin, Abrin, Modecin, Viscumin, Volkensin, Cinnamomin, Mistletoe lectin I and Luffangulin (6 kda); Bacterial RIPS such as Shiga toxin, and Shiga-like toxin; and Fungal RIPS such as alpha-sarcin, mitogillin, and restrictocin. In specific embodiments, an antibody of the invention is conjugated or fused to a fragment or variant of the ribosome inactivating proteins described above, particularly when said fragment or variant retains activity.
Ricin A homologues that may be conjugated or fused to antibodies of the invention include, but are not limited to, polypeptides that have the same amino acid sequence as a protein selected from the group consisting of: type 2 ribosome-inactivating protein cinnamomin III precursor, abrin-d precursor from Indian licorice or fragment thereof, RIP precursor of Sambucus nigra, RIP bryodin II precursor, alpha-trichosanthin, ribosome-inactivating protein precursor from Sambucus ebulus, karasurin-B, Trichobakin, Beta-Luffin, Beta-galactoside specific lectin 1 A chain (MLA; ML-I A), lectin chain A isoform 2 from Viscum album subsp. coloratum, curcin precursor from Jatropha curcas, trichoanguina from snake gourd, ribosome-inactivating protein IRAb from Iris hollandica, Momordin, trichoanguin from Trichosanthes cucumerina, momordin II from Momordica balsamina, gelonin from Gelonium multiflorum, ribosome inactivating protein RIPm from Polygonatum muliiflorum, ribosome inactivating protein Euserratin 2 precursor from Euphorbia serrata, alpha-PAP (pokeweed antiviral protein) from Phytolacca americana, ribosome-inactivating protein gynostemmin II from Gynostemma pentaphyllum, trichosanthin, ribosome-inactivating protein 2 from Phytolacca insularis, bouganin from Bougainvillea spectabilis, antiviral protein from Clerodendrum aculeatum, anti-viral protein PAP from Phytolacca acinosa, type I RIP moschatin 1, antiviral ribosome inactivating protein from Chenopodium album, Betavulgin from Beta vulgaris subsp. vulgaris, ribosome-inactivating protein benincasin from Benincasa hispida, ribosome-inactivating protein MEI from Mirabilis expansa, MAP-MIRABILIS—Four-o'clock (Marvel-of-peru), RIP amaranthin, amarandin-S from Amaranthus tricolor, ribosome-inactivating protein from Spinacia oleracea, type I RIP musarmin 3 from Muscari, saporin from Saponaria officinalis, dianthin 30 from Dianthus caryophyllus, and tritin from Triticum aestivum, or biologically active fragments or variants thereof.
Other toxins that may be conjugated or fused to antibodies of the invention include, but are not limited to diptheria toxin, Pseudomonas exotoxin A, Botulinum neurotoxin, Clostridium perfringens epsilon toxin, Conotoxins, and Staphylococcal enterotoxins.
Antibodies of the invention (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating a therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R, Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
Alternatively, an antibody of the invention can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
An antibody of the invention (including an scFv or and other molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
Certain forms of antibodies, particularly, IgG3 and IgM antibodies, as well as IgG1 and IgG2 antibodies to a lesser extent, are able to mediate cellular lysis via the complement cascade. Thus antibodies of the invention may be used to kill cells expressing Neutrokine-alpha on their surface or cells bearing Neutrokine-alpha receptors via complement-mediated killing.
Antibodies of the invention may be used to mediate antibody dependent cell-mediated cytotoxicity (ADCC) by, for example, Natural Killer (NK) cells. ADCC occurs when antibodies of the IgG1 or IgG3 isotype interact with FcγRIII receptor on Natural Killer cells triggering an cytotoxic attack by the NK cell on the antibody coated cells. Thus antibodies of the invention may be used to kill cells expressing Neutrokine-alpha on their surface or cells bearing Neutrokine-alpha receptors via ADCC.
Use of Antibodies for Epitope Mapping
The present invention provides antibodies (including scFvs and other molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that can be used to identify epitopes of B Lymphocyte Stimulator. In particular, the antibodies of the present invention can be used to identify epitopes 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) using techniques described herein or otherwise known in the art. Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211.)
Diagnostic Uses of Antibodies
Labeled antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of aberrant expression.
By “biological sample” is intended any fluids and/or cells obtained from an individual, body fluid, body tissue, body cell, cell line, tissue culture, or other source which may contain B Lymphocyte Stimulator protein or mRNA. Body fluids include, but are not limited to, sera, plasma, urine, synovial fluid, spinal fluid, saliva, and mucous. Tissues samples may be taken from virtually any tissue in the body. Tissue samples may also be obtained from autopsy material. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
The invention also provides for the detection of aberrant expression of B Lymphocyte Stimulator receptor comprising (a) assaying the expression of B Lymphocyte Stimulator receptor in a biological sample from an individual using one or more antibodies or fragments or variants thereof that immunospecifically binds only to soluble B Lymphocyte Stimulator, but does not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding. Such an antibody, by way of an example that is not to be construed as limiting, would be one that is able to capture a biotinylated B Lymphocyte Stimulator from solution (see Example 8), but that would not prevent B Lymphocyte Stimulator from binding to IM-9 cells (see Example 3) and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard level of B Lymphocyte Stimulator receptor, e.g., in normal tissue or cell samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator receptor compared to the standard level of B Lymphocyte Stimulator receptor is indicative of aberrant expression.
Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies, and/or diseases or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of an autoimmune disorder or disease and/or an immunodeficiency. In specific embodiments, an increase in the assayed level of B Lymphocyte Stimulator is indicative of an autoimmune disorder or disease. In other specific embodiments, a decrease in the assayed level of B Lymphocyte Stimulator is indicative of an immunodeficiency.
Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to B Lymphocyte Stimulator but, do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding can be used for diagnostic purposes to detect, diagnose, prognose, or monitor autoimmune disorders and/or immunodeficiencies, and/or diseases or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator receptor comprising: (a) assaying the expression of B Lymphocyte Stimulator receptor in a biological sample from an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard level of B Lymphocyte Stimulator receptor, e.g., in normal biological samples, whereby an increase or decrease in the assayed level of B Lymphocyte Stimulator receptor compared to the standard level of B Lymphocyte Stimulator receptor is indicative of an autoimmune disorder or disease and/or an immunodeficiency. In specific embodiments, an increase in the assayed level of B Lymphocyte Stimulator receptor is indicative of an autoimmune disorder or disease. In other specific embodiments, a decrease in the assayed level of B Lymphocyte Stimulator receptor is indicative of an immunodeficiency.
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, dilated cardiomyopathy, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, diabetes mellitus (e.g. Type I diabetes mellitus or insulin dependent diabetes mellitis), juvenile onset diabetes, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemic lupus erythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia (Addison's disease), idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjögren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders and other disorders such as inflammatory skin diseases including psoriasis and sclerosis, responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), respiratory distress syndrome (including adult respiratory distress syndrome, ARDS), meningitis, encephalitis, colitis, allergic conditions such as eczema and other conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, leukocyte adhesion deficiency, Reynaud's syndrome, and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, granulomatosis and diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorder, multiple organ injury syndrome, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, Lambert-Eaton myasthenic syndrome, Beheet disease, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or autoimmune thrombocytopenia etc.
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).
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.
Elevated levels of soluble B Lymphocyte Stimulator have been observed in the serum of patients with Systemic Lupus Erythematosus (SLE). In comparing the sera of 150 SLE patients with that of 38 control individuals, it was found that most of the SLE patients had more than 5 ng/ml of serum B Lymphocyte Stimulator, more than 30% of SLE patients had levels greater than 10 ng/ml, and approximately 10% of SLE patients had serum B Lymphocyte Stimulator levels greater than 20 ng/ml. In contrast, the majority of normal controls had B Lymphocyte Stimulator levels less than 5 ng/ml, and less than 10% had levels higher than 10 ng/ml. The elevated levels of B Lymphocyte Stimulator protein in sera is present in the soluble form and has biologic activity as assayed by the ability to stimulate anti-IgM treated B cells in vitro. SLE patients with more than 15 ng/ml serum B Lymphocyte Stimulator were also found to have elevated levels of anti-dsDNA antibodies compared to both normal controls and SLE patients with less than 5 ng/ml of serum B Lymphocyte Stimulator unpublished data).
In addition the serum of two subgroups of patients which were positive for anti-nuclear antibodies (ANA+) but did not meet the formal requirements of the American College of Rheumatology (ACR) for classification of SLE were analyzed for B Lymphocyte Stimulator levels. The first subgroup of sera was ANA+ sera that came from patients who did not present with the clinical impression of SLE. This group had only slightly elevated levels of B Lymphocyte Stimulator ( ̃9 ng/ml B Lymphocyte Stimulator). The second subgroup however, which was ANA+ sera from patients who presented with the clinical impression of SLE, had significantly increased B Lymphocyte Stimulator levels ( ̃15 ng/ml). These results suggest that an elevated level of B Lymphocyte Stimulator precedes the formal fulfillment of the ACR criteria. The ACR criteria are described in Tan, E. M., et al, Arthritis and Rheumatism 25:1271-1277 (1982).
Thus in specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Systemic Lupus Erythematosus or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of SLE.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor IgA nephropathy or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of IgA nephropathy.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Sjögren's Syndrome or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Sjögren's Syndrome.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor HIV infection or conditions associated therewith (e.g. AIDS). The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of HIV infection.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Myasthenia Gravis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Myasthenia Gravis.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor idiopathic thrombocytopenic purpura (ITP) or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of idiopathic thrombocytopenic purpura (ITP).
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor hemolytic anemia or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of hemolytic anemia.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor thyroiditis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of thyroiditis.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Goodpasture's syndrome or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Goodpasture's syndrome.
In other specific embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor multiple sclerosis or conditions associated therewith. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of multiple sclerosis.
In additional embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor Rheumatoid Arthritis. The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample (e.g., serum and synovial fluid) of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of Rheumatoid arthritis.
In additional embodiments, antibodies of the invention which specifically bind to B Lymphocyte Stimulator can be used for diagnostic purposes to detect, diagnose, prognose, or monitor an immune-based rheumatologic disease, (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia), Seronegative spondyloarthropathy (SpA), Polymyositis/dermatomyositis, Microscopic polyangiitis, Hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder). The invention provides for the detection of aberrant expression of B Lymphocyte Stimulator comprising: (a) assaying the expression of B Lymphocyte Stimulator in a biological sample (e.g., serum and synovial fluid) of an individual using one or more antibodies of the invention that immunospecifically binds to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard level of B Lymphocyte Stimulator, e.g., in normal biological samples, whereby an increase in the assayed level of B Lymphocyte Stimulator compared to the standard level of B Lymphocyte Stimulator is indicative of monitor an immune-based rheumatologic disease.
It has been observed, that serum B Lymphocyte Stimulator levels inversely correlate with nephrotic range proteinuria (>3 gm proteinuria in a 24 hour urine collection) using a sample of 71 SLE patients (p=0.019). Proteinuria was determined in 71 SLE patients within one month of phlebotomy for serum B Lymphocyte Stimulator determination. Serum B Lymphocyte Stimulator was classified as low, normal, or high based on the 5th through 95th percentiles for normal controls. Nephrotic-range proteinuria was inversely correlated with serum Neutrokine-alpha levels. Thus, in specific embodiments, serum levels of B Lymphocyte Stimulator (determined using one or more antibodies of the present invention) in individuals diagnosed with an immune based rheumatologic disease (e.g., SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia), seronegative spondyloarthropathy (SpA), polymyositis/dermatomyositis, microscopic polyangiits, hepatitis C-associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder) may be used to determine, diagnose, prognose, or monitor the severity of certain aspects or symptoms of the disease, such as nephrotic-range proteinuria.
In another specific embodiment, antibodies of the invention are used to diagnose, prognose, treat, or prevent conditions associated with CVID, including, but not limited to, conditions associated with acute and recurring infections (e.g., pneumonia, bronchitis, sinusitis, otitis media, sepsis, meningitis, septic arthritis, and osteomyelitis), chronic lung disease, autoimmunity, granulomatous disease, lymphoma, cancers (e.g., cancers of the breast, stomach, colon, mouth, prostate, lung, vagina, ovary, skin, and melanin forming cells (i.e. melanoma), inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, and ulcerative proctitis), malabsorption, Hodgkin's disease, and Waldenstrom's macroglobulinemia.
The invention provides a diagnostic assay for diagnosing or prognosing a disease or disorder, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without the disease or disorder, whereby an increase or decrease in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of a particular disease or disorder. With respect to cancer, the presence of a relatively high amount of B Lymphocyte Stimulator in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
In specific embodiments, the presence of a relatively high amount of membrane-bound B Lymphocyte Stimulator in a biological sample is indicative of monocytic cell related leukemias or lymphomas, such as, for example acute myelogenous leukemia and/or the severity thereof.
In other specific embodiments, the presence of a relatively high amount of B Lymphocyte Stimulator receptor in a biological sample (as determined using antibodies of the invention that bind to soluble B Lymphocyte Stimulator, but do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding) is indicative of B cell related leukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease), and/or the severity thereof.
In specific embodiments, the invention provides a diagnostic assay for diagnosing or prognosing Systemic Lupus Erythematosus, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without Systemic Lupus Erythematosus, whereby an increase in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of Systemic Lupus Erythematosus.
In specific embodiments, the invention provides a diagnostic assay for diagnosing or prognosing a Rheumatoid Arthritis, comprising: (a) assaying for the level of B Lymphocyte Stimulator in a biological sample of an individual using one or more antibodies of the invention that immunospecifically bind to B Lymphocyte Stimulator; and (b) comparing the level of B Lymphocyte Stimulator with a standard B Lymphocyte Stimulator level, e.g., in a biological sample from a patient without Rheumatoid Arthritis, whereby an increase or decrease in the assayed B Lymphocyte Stimulator level compared to the standard level of B Lymphocyte Stimulator is indicative of Rheumatoid Arthritis.
The invention provides a diagnostic assay for diagnosing or prognosing a disease or disorder, comprising: (a) assaying for the level of B Lymphocyte Stimulator receptor in cells or a tissue sample of an individual using one or more antibodies of the invention that immunospecifically binds only to soluble B Lymphocyte Stimulator, but does not neutralize B Lymphocyte Stimulator/B Lymphocyte Stimulator receptor binding; and (b) comparing the level of B Lymphocyte Stimulator receptor with a standard B Lymphocyte Stimulator receptor level, e.g., in a tissue sample from a patient without the disease or disorder, whereby an increase or decrease in the assayed B Lymphocyte Stimulator receptor level compared to the standard level of B Lymphocyte Stimulator receptor is indicative of a particular disease or disorder. With respect to cancer, the presence of a relatively high amount of B Lymphocyte Stimulator receptor in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) can be used to assay protein levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, alkaline phosphatase, and horseradish peroxidase; radioisotopes, such as iodine (123I, 123I, 125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In, 112In, 113mIn, 115mIn), technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 116Re, 118Re, 142Pr, 105Rh, and 97Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically binds to B Lymphocyte Stimulator; b) waiting for a time interval following the administering for permitting the labeled antibody to preferentially concentrate at sites in the subject where B Lymphocyte Stimulator is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled antibody in the subject, such that detection of labeled antibody or fragment thereof above the background level and above or below the level observed in a person without the disease or disorder indicates that the subject has a particular disease or disorder associated with aberrant expression of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99Tc. The labeled antibody will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
Immunophenotyping
The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be utilized for immunophenotyping of cell lines and biological samples by their B Lymphocyte Stimulator expression or B Lymphocyte Stimulator receptor expression. Various techniques can be utilized using antibodies, fragments, or variants of the invention to screen for cellular populations (i.e., immune cells, particularly monocytic cells or B-cells) expressing B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (see, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:73749 (1999)).
These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e., minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
In one embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) are used to identify cells of monocytic or B cell origin.
Use of Antibodies to Identify, Purify and/or Target B Lymphocyte Stimulator Protein and/or B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor Expressing Cells
The antibodies of the invention may be used to identify, purify and target B Lymphocyte Stimulator protein and/or B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor expressing cells. For example, the antibodies of the invention when coupled to a detectable label can be used to identify cells that express B Lymphocyte Stimulator. More specifically, the antibodies of the invention can be used in immunohistochemistry or in flow cytometry. Additionally, the antibodies of the invention may be used to purified B Lymphocyte Stimulator proteins or portions thereof from cells expressing B Lymphocyte Stimulator. In more specific embodiments, antibodies of the invention may be conjugated (directly or indirectly) to a detectable label such a fluorescent label and used in fluorescence activated cell sorting (FACS). Alternatively, antibodies of the invention may be conjugated (directly or indirectly) to a magnetic particle or bead and used in magnetic cell sorting (MACS).
Antibodies of the invention may be used to target therapeutic or toxic moieties to B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells (see Antibody Conjugates section above). In a specific embodiment, antibodies of the invention may also be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells. In one embodiment, antibodies of the invention are linked to a cytotoxic moiety which kills B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells that have been contacted with such an antibody conjugate.
Certain forms of antibodies, particularly, IgG3 and IgM antibodies, as well as IgG1 and IgG2 antibodies to a lesser extent, are able to mediate cellular lysis via the complement cascade. Thus antibodies of the invention may be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells via complement-mediated killing.
Antibodies of the invention may be used to mediate antibody dependent cell-mediated cytotocicity (ADCC) by, for example, Natural Killer (NK) cells. ADCC occurs when antibodies of the IgG1 or IgG3 isotype interact with FcγRIII receptor on Natural Killer cells triggering an cytotoxic attack by the NK cell on the antibody coated cells. Thus antibodies of the invention may be used to kill B Lymphocyte Stimulator or B Lymphocyte Stimulator Receptor expressing cells via ADCC.
Therapeutic Uses of Antibodies
The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention and nucleic acids encoding antibodies (and anti-idiotypic antibodies) of the invention as described herein. The antibodies of the invention can be used to treat, ameliorate or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of B Lymphocyte Stimulator or B Lymphocyte Stimulator receptor, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant B Lymphocyte Stimulator expression and/or activity or aberrant B Lymphocyte Stimulator receptor expression and/or activity includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
Antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that function as agonists or antagonists of B Lymphocyte Stimulator, preferably of B Lymphocyte Stimulator-induced signal transduction, can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. For example, antibodies of the invention which disrupt the interaction between B Lymphocyte Stimulator and its receptor may be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive of B Lymphocyte Stimulator receptor function. Antibodies of the invention which do not prevent B Lymphocyte Stimulator from binding its receptor but inhibit or downregulate B Lymphocyte Stimulator-induced signal transduction can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. In particular, antibodies of the present invention which prevent B Lymphocyte Stimulator-induced signal transduction by specifically recognizing the unbound B Lymphocyte Stimulator, receptor-bound B Lymphocyte Stimulator or both unbound and receptor-bound B Lymphocyte Stimulator can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. The ability of an antibody of the invention to inhibit or downregulate B Lymphocyte Stimulator-induced signal transduction may be determined by techniques described herein or otherwise known in the art. For example, B Lymphocyte Stimulator-induced receptor activation and the activation of signaling molecules can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or a signaling molecule by immunoprecipitation followed by western blot analysis (for example, as described herein).
In a specific embodiment, an antibody of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that inhibits or downregulates B Lymphocyte Stimulator activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to B Lymphocyte Stimulator activity in absence of the antibody is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function. In another embodiment, a combination of antibodies, a combination of antibody fragments, a combination of antibody variants, or a combination of antibodies, antibody fragments, and/or variants that inhibit or downregulate B Lymphocyte Stimulator activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to B Lymphocyte Stimulator activity in absence of said antibodies, antibody fragments, and/or antibody variants are administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, excessive B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or excessive B Lymphocyte Stimulator receptor function.
Further, antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which activate B Lymphocyte Stimulator-induced signal transduction can be administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. These antibodies may potentiate or activate either all or a subset of the biological activities of B Lymphocyte Stimulator-mediated receptor activation, for example, by inducing multimerization of B Lymphocyte Stimulator and/or multimerization of the receptor. The antibodies of the invention may be administered with or without being pre-complexed with B Lymphocyte Stimulator. In a specific embodiment, an antibody of the present invention that increases B Lymphocyte Stimulator activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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% relative to B Lymphocyte Stimulator activity in absence of the antibody is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression, lack of B Lymphocyte Stimulator function, aberrant B Lymphocyte Stimulator receptor expression, or lack of B Lymphocyte Stimulator receptor function. In another embodiment, a combination of antibodies, a combination of antibody fragments, a combination of antibody variants, or a combination of antibodies, antibody fragments and/or antibody variants that increase B Lymphocyte Stimulator activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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% relative to B Lymphocyte Stimulator activity in absence of the said antibodies or antibody fragments and/or antibody variants is administered to an animal to treat, prevent or ameliorate a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or lack of B Lymphocyte Stimulator function or aberrant B Lymphocyte Stimulator receptor expression or lack of B Lymphocyte Stimulator receptor function.
One or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator may be used locally or systemically in the body as a therapeutic. The antibodies of this invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may also be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, anti-angiogenesis and anti-inflammatory agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments, or variants, (e.g., derivatives), or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that immunospecifically bind to B Lymphocyte Stimulator, or polynucleotides encoding antibodies that immunospecifically bind to B Lymphocyte Stimulator, for both immunoassays directed to and therapy of disorders related to B Lymphocyte Stimulator polynucleotides or polypeptides, including fragments thereof. Such antibodies will preferably have an affinity for B Lymphocyte Stimulator and/or B Lymphocyte Stimulator fragments. Preferred binding affinities include those with a dissociation constant or KD less than or equal to 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, or 10−5 M. More preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×10−6 M, 10−6 M, 5×10−7 M, 10−7 M, 5×10−5 M, or 10−8 M. Even more preferably, antibodies of the invention bind B Lymphocyte Stimulator polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×10−9 M, 10−9 M, 5×10−11 M, 10−11 M, 5×10−11 M, 10−11 M, 5×10−2 M, 10−12 M, 5×−13 M, 10−14 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M. The invention encompasses antibodies that bind B Lymphocyte Stimulator polypeptides with a dissociation constant or KD that is within any one of the ranges that are between each of the individual recited values.
In a preferred embodiment, antibodies of the invention neutralize B Lymphocyte Stimulator activity. In another preferred embodiment, antibodies of the invention inhibit B cell proliferation.
In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment antibodies of the invention inhibit or reduce B cell proliferation induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment antibodies of the invention inhibit or reduce immunoglobulin production induced by the soluble form of B Lymphocyte Stimulator.
In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of membrane-bound B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment, antibodies of the invention inhibit or reduce B cell proliferation induced by the membrane-bound form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention inhibit or reduce immunoglobulin production induced by the membrane bound form of B Lymphocyte Stimulator.
In a preferred embodiment, antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) inhibit or reduce binding of both the soluble and membrane-bound forms of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor. In another preferred embodiment, antibodies of the invention inhibit or reduce B cell proliferation induced by either or both forms of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention inhibit or reduce immunoglobulin production induced by either or both forms of B Lymphocyte Stimulator.
In one embodiment, the invention provides a method of delivering antibody conjugates of the invention to targeted cells, such as, for example, monocytic cells expressing the membrane-bound form of B Lymphocyte Stimulator, or B cells expressing a B Lymphocyte Stimulator receptor.
In one embodiment, the invention provides a method for the specific delivery of antibodies and antibody conjugates of the invention to cells by administering molecules of the invention that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs). In a specific embodiment, the invention provides a method for the specific destruction of cells of monocytic lineage (e.g., monocytic cell related leukemias or lymphomas, such as, for example acute myelogenous leukemia) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs) that immunospecifically bind the membrane-bound form of B Lymphocyte Stimulator. In another specific embodiment, the invention provides a method for the specific destruction of cells of B cell lineage (e.g., B cell related leukemias or lymphomas (e.g., chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and Hodgkin's disease) by administering antibodies or antibody conjugates of the invention (e.g., antibodies conjugated with radioisotopes, toxins, or cytotoxic prodrugs) that bind soluble B Lymphocyte Stimulator, but do not inhibit B Lymphocyte Stimulator binding to a B Lymphocyte Stimulator receptor on B cells.
In another preferred embodiment antibodies of the invention (including antibody fragments and variants) promote or enhance B cell proliferation induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment, antibodies of the invention (including antibody fragments and variants) promote or enhance B cell proliferation induced by the membrane or soluble form of APRIL. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production induced by the soluble form of B Lymphocyte Stimulator. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production induced by the membrane bound or soluble form of APRIL. In another preferred embodiment antibodies of the invention (including antibody fragments and variants) increase or enhance immunoglobulin production in response to T cell dependent immunogens. In another preferred embodiment antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance immunoglobulin production in response to T cell independent immunogens.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate immune disorders. Immune disorders include, but are not limited to, autoimmune disorders (e.g., arthritis, graft rejection, Hashimoto's thyroiditis, insulin-dependent diabetes, lupus, idiopathic thrombocytopenic purpura, systemic lupus erythrematosus and multiple sclerosis), elective IgA deficiency, ataxia-telangiectasia, common variable immunodeficiency (CVID), X-linked agammaglobulinemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, idiopathic hyper-eosinophilic syndrome, monocytic leukemoid reaction, monocytic leukocytosis, monocytic leukopenia, monocytopenia, monocytosis, and graft or transplant rejection.
As discussed herein, antibodies and antibody compositions of the invention, may be used to treat, prevent, ameliorate, diagnose or prognose various immune system-related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of antibody and antibody compositions of the invention that can inhibit an immune response, particularly the proliferation of B cells and/or the production of immunoglobulins, may be an effective therapy in treating and/or preventing autoimmune disorders. Thus, in preferred embodiments, antibodies and antibody compositions of the invention are used to treat, prevent, ameliorate, diagnose and/or prognose an autoimmune disorder, or condition(s) associated with such disorder.
Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions 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 disease.
Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erythematosus (often characterized, e.g., by circulating and locally generated immune complexes), discoid lupus, Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), Receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody-sensitized platelets.
Additional autoimmune disorders and conditions associated with these disorders that may be treated, prevented, ameliorated, diagnosed and/or prognosed with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjögren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies), chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies, and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
In specific embodiments, antagonistic anti-neutrokine-alpha antibodies are useful in the diagnosis and treatment or prevention of dilated cardiomyopathy.
In a preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, a member of the group: autoimmune hemolytic anemia, as primary glomerulonephritis, IgA glomerulonephritis, Goodpasture's syndrome, idiopathic thrombocytopenia, Multiple Sclerosis, Myasthenia Gravis, Pemphigus, polymyositis/dermatomyositis, relapsing polychondritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, Uveitis, vasculitis, and primary biliary cirrhosis.
In specific embodiments, pharmaceutical compositions of the invention are useful in the diagnosis and treatment or prevention of liver diseases, including but not limited to, primary biliary cirrhosis, primary sclerosing cholangitis, chronic hepatitis C infection, autoimmune hepatitis and alcoholic liver disease. In specific embodiments, antagonists of Neutrokine-alpha, such as an anti-Neutrokine-alpha specific antibody, are useful in the diagnosis and treatment or prevention of liver diseases, including but not limited to, primary biliary cirrhosis, primary sclerosing cholangitis, chronic hepatitis C infection, and alcoholic liver disease.
In another preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, an immune based-rheumatologic disease, such as, for example, SLE, rheumatoid arthritis, CREST syndrome (a variant of scleroderma characterized by calcinosis, Raynaud's phenomenon, esophageal motility disorders, sclerodactyly, and telangiectasia), Seronegative spondyloarthropathy (SpA), polymyositis/dermatomyositis, microscopic polyangiitis, hepatitis C associated arthritis, Takayasu's arteritis, and undifferentiated connective tissue disorder.
In a specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, rheumatoid arthritis and/or medical conditions associated therewith.
For example, an antibody, or antibodies, of the present invention are used to treat patients with clinical diagnosis of rheumatoid arthritis (RA). The patient treated preferably will not have a B cell malignancy. Moreover, the patient is optionally further treated with any one or more agents employed for treating RA such as salicylate; nonsteroidal anti-inflammatory drugs such as indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g. ibuprofen and fenoprofen), naphthalene acetic acids (naproxen), pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac), halogenated anthranilic acid (meclofenamate sodium), piroxicam, zomepirac and diflunisal; antimalarials such as chloroquine; gold salts; penicillamine; or immunosuppressive agents such as methotrexate or corticosteroids in dosages known for such drugs or reduced dosages. Preferably however, the patient is only treated with an antibody, or antibodies, of the present invention. Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. The primary response is determined by the Paulus index (Paulus et al. Athritis Rheum. 33:477-484 (1990)), i.e. improvement in morning stiffness, number of painful and inflamed joints, erythrocyte sedimentation (ESR), and at least a 2-point improvement on a 5-point scale of disease severity assessed by patient and by physician. Administration of an antibody, or antibodies, of the present invention will alleviate one or more of the symptoms of RA in the patient treated as described above.
In a specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, amelioate, diagnose or prognose, lupus and/or medical conditions associated therewith. Lupus-associated conditions that may be treated, prevented, ameliorated, prognosed and/or diagnosed with the antibodies and antibody compositions of the invention include, but are not limited to, hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus (pleurisy)), renal disorders (e.g., nephritis), neurological disorders (e.g., seizures, peripheral neuropathy, CNS related disorders), gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In a preferred embodiment, therapeutic and pharmaceutical compositions of the invention are used to treat, prevent, ameliorate, diagnose, or prognose, renal disorders associated with systemic lupus erythematosus. In a most preferred embodiment, therapeutic and pharmaceutical compositions of the invention are used to treat, prevent, ameliorate, diagnose, or prognose, nephritis associated with systemic lupus erythematosus. In another most preferred embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate lupus or glomerular nephritis.
In a further specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent hemolytic anemia. For example, patients diagnosed with autoimmune hemolytic anemia (AIHA), e.g., cryoglobinemia or Coombs positive anemia, are treated with an antibody, or antibodies, of the present invention. AIHA is an acquired hemolytic anemia due to auto-antibodies that react with the patient's red blood cells. The patient treated preferably will not have a B cell malignancy. Further adjunct therapies (such as glucocorticoids, prednisone, azathioprine, cyclophosphamide, vinca-laden platelets or Danazol) may be combined with the antibody therapy, but preferably the patient is treated with an antibody, or antibodies, of the present invention as a single-agent throughout the course of therapy. Antibodies of the present invention are administered to the hemolytic anemia patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. Overall response rate is determined based upon an improvement in blood counts, decreased requirement for transfusions, improved hemoglobin levels and/or a decrease in the evidence of hemolysis as determined by standard chemical parameters. Administration of an antibody, or antibodies of the present invention will improve any one or more of the symptoms of hemolytic anemia in the patient treated as described above. For example, the patient treated as described above will show an increase in hemoglobin and an improvement in chemical parameters of hemolysis or return to normal as measured by serum lactic dehydrogenase and/or bilirubin.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Sjögren's Syndrome and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, HIV infection and/or medical conditions associated therewith (e.g. AIDS).
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Myasthenia gravis and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, IgA nephropathy and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, hemolytic anemia and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, thyroiditis and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Goodpasture's Syndrome and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, multiple sclerosis and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, chronic lymphocytic leukemia (CLL) and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, multiple myeloma and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Non-Hodgkin's lymphoma and/or medical conditions associated therewith.
In another specific preferred embodiment, therapeutic and pharmaceutical compositions of the invention, are used to treat, prevent, ameliorate, diagnose or prognose, Hodgkin's disease and/or medical conditions associated therewith.
In another specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent adult immune thrombocytopenic purpura. Adult immune thrombocytopenic purpura (ITP) is a relatively rare hematologic disorder that constitutes the most common of the immune-mediated cytopenias. The disease typically presents with severe thrombocytopenia that may be associated with acute hemorrhage in the presence of normal to increased megakaryocytes in the bone marrow. Most patients with ITP have an IgG antibody directed against target antigens on the outer surface of the platelet membrane, resulting in platelet sequestration in the spleen and accelerated reticuloendothelial destruction of platelets (Bussell, J. B. Hematol. Oncol. Clin. North Am. (4):179 (1990)). A number of therapeutic interventions have been shown to be effective in the treatment of ITP, Steroids are generally considered first-line therapy, after which most patients are candidates for intravenous immunoglobulin (IVIG), splenectomy, or other medical therapies including vincristine or immunosuppressive/cytotoxic agents. Up to 80% of patients with ITP initially respond to a course of steroids, but far fewer have complete and lasting remissions. Splenectomy has been recommended as standard second-line therapy for steroid failures, and leads to prolonged remission in nearly 60% of cases yet may result in reduced immunity to infection. Splenectomy is a major surgical procedure that may be associated with substantial morbidity (15%) and mortality (2%). IVIG has also been used as second line medical therapy, although only a small proportion of adult patients with ITP achieve remission. Therapeutic options that would interfere with the production of autoantibodies by activated B cells without the associated morbidities that occur with corticosteroids and/or splenectomy would provide an important treatment approach for a proportion of patients with ITP, Patients with clinical diagnosis of ITP are treated with an antibody, or antibodies of the present invention, optionally in combination with steroid therapy. The patient treated will not have a B cell malignancy. Antibodies of the present invention are administered to the RA patient according to a dosing schedule as described infra, which may be readily determined by one of ordinary skill in the art. Overall patient response rate is determined based upon a platelet count determined on two consecutive occasions two weeks apart following treatments as described above. See, George et al. “Idiopathic Thrombocytopenic Purpura: A Practice Guideline Developed by Explicit Methods for The American Society of Hematology”, Blood 88:3-40 (1996), expressly incorporated herein by reference.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate an IgE-mediated allergic reaction or histamine-mediated allergic reaction. Examples of allergic reactions include, but are not limited to, asthma, rhinitis, eczema, chronic urticaria, and atopic dermatitis. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent, or ameliorate anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate or modulate inflammation or an inflammatory disorder. Examples of chronic and acute inflammatory disorders that may be treated prevented or ameliorated with the therapeutic and pharmaceutical compositions of the invention include, but are not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, Crohn's disease, inflammatory bowel disease, chronic and acute inflammatory pulmonary diseases, bacterial infection, psoriasis, septicemia, cerebral malaria, arthritis, gastroenteritis, and glomerular nephritis.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate ischemia and arteriosclerosis. Examples of such disorders include, but are not limited to, reperfusion damage (e.g., in the heart and/or brain) and cardiac hypertrophy.
Therapeutic or pharmaceutical compositions of the invention, may also be administered to modulate blood clotting and to treat or prevent blood clotting disorders, such as, for example, antibody-mediated thrombosis (i.e., antiphospholipid antibody syndrome (APS)). For example, therapeutic or pharmaceutical compositions of the invention, may inhibit the proliferation and differentiation of cells involved in producing anticardiolipin antibodies. These compositions of the invention can be used to treat, prevent, ameliorate, diagnose, and/or prognose thrombotic related events including, but not limited to, stroke (and recurrent stroke), heart attack, deep vein thrombosis, pulmonary embolism, myocardial infarction, coronary artery disease (e.g., antibody-mediated coronary artery disease), thrombosis, graft reocclusion following cardiovascular surgery (e.g., coronary arterial bypass grafts, recurrent fetal loss, and recurrent cardiovascular thromboembolic events.
Therapeutic or pharmaceutical compositions of the invention, may also be administered to treat, prevent, or ameliorate organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of antibodies of the invention, that inhibit an immune response, may be an effective therapy in preventing organ rejection or GVHD.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate a disease or disorder diseases associated with increased apoptosis including, but not limited to, AIDS, neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration), myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia. In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate bone marrow failure, for example, aplastic anemia and myelodysplastic syndrome.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate growth, progression, and/or metastases of malignancies and proliferative disorders associated with increased cell survival, or the inhibition of apoptosis. Examples of such disorders, include, but are not limited to, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia), neoplasms, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma), heavy chain disease, metastases, or any disease or disorder characterized by uncontrolled cell growth.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used to treat or prevent a disorder characterized by hpergammagloulinemia (e.g., AIDS, autoimmune diseases, and some immunodeficiencies).
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used to treat or prevent a disorder characterized by deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, therapeutic or pharmaceutical compositions of the invention may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-bone infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii.
Therapeutic or pharmaceutical compositions of the invention of the invention thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.
Additionally, therapeutic or pharmaceutical compositions of the invention may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., cytomegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.
Additional preferred embodiments of the invention include, but are not limited to, the use of therapeutic or pharmaceutical compositions of the invention in the following applications:
Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response. In a specific nonexclusive embodiment, therapeutic or pharmaceutical compositions of the invention are administered to boost the immune system to produce increased quantities of IgG, In another specific nonexclusive embodiment, antibodies of the are administered to boost the immune system to produce increased quantities of IgA. In another specific nonexclusive embodiment antibodies of the invention are administered to boost the immune system to produce increased quantities of IgM.
Administration to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741).
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a vaccine adjuvant that enhances immune responsiveness to specific antigen. In a specific embodiment, the vaccine is an antibody described herein. In another specific embodiment, the vaccine adjuvant is a polynucleotide described herein (e.g., an antibody polynucleotide genetic vaccine adjuvant). As discussed herein, therapeutic or pharmaceutical compositions of the invention may be administered using techniques known in the art, including but not limited to, liposomal delivery, recombinant vector delivery, injection of naked DNA, and gene gun delivery.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance tumor-specific immune responses.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include, but are not limited to, virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to the HIV gp120 antigen.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria).
In a specific embodiment, compositions of the invention may be administered to patients as vaccine adjuvants. In a further specific embodiment, compositions of the invention may be administered as vaccine adjuvants to patients suffering from an immune-deficiency. In a further specific embodiment, compositions of the invention may be administered as vaccine adjuvants to patients suffering from HIV.
In a specific embodiment, compositions of the invention may be used to increase or enhance antigen-specific antibody responses to standard and experimental vaccines. In a specific embodiment, compositions of the invention may be used to enhance seroconversion in patients treated with standard and experimental vaccines. In another specific embodiment, compositions of the invention may be used to increase the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination.
In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance antigen-specific antibody responses to standard and experimental vaccines by regulating binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance antigen-specific antibody responses to standard and experimental vaccines by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).
In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance seroconversion in patients treated with standard and experimental vaccines by regulating binding of the soluble form of B Lymphocyte Stimulator to B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance seroconversion in patients treated with standard and experimental vaccines by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).
In a preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination by regulating binding of the soluble form of B Lymphocyte Stimulator to a B Lymphocyte Stimulator receptor (e.g., TACI—GenBank accession number AAC51790; BCMA—GenBank accession number NP—001183; and/or BAFF-R—GenBank accession number NP—443177). In another preferred embodiment, antibodies of the invention (including antibody fragments and variants, and anti-antibody antibodies) increase or enhance the repertoire of antibodies recognizing unique epitopes in response to standard and experimental vaccination by regulating binding of the soluble form of APRIL to an APRIL receptor (e.g., BCMA and TACI).
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a stimulator of B cell responsiveness to pathogens.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to induce higher affinity antibodies.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to increase serum immunoglobulin concentrations.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to accelerate recovery of immunocompromised individuals.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among aged populations.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy. B cell immunodeficiencies that may be ameliorated or treated by administering the antibodies and/or compositions 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.
In a specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate selective IgA deficiency.
In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate ataxia-telangiectasia.
In another specific embodiment antibodies and/or compositions of the invention are administered to treat or ameliorate common variable immunodeficiency.
In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate X-linked ammaglobulinemia.
In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate severe combined immunodeficiency (SCID).
In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate Wiskott-Aldrich syndrome.
In another specific embodiment, antibodies and/or compositions of the invention are administered to treat or ameliorate X-deficiency linked Ig deficiency with hyper IgM.
As an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering antibodies and/or compositions of the invention include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering antibodies and/or compositions of the invention include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a regulator of antigen presentation by monocytes, dendritic cells, T cells and/or B-cells. In one embodiment, antibody polypeptides or polynucleotides enhance antigen presentation or antagonize antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonization of antigen presentation may be useful in anti-tumor treatment or to modulate the immune system.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a mediator of mucosal immune responses. The expression of B Lymphocyte Stimulator on monocytes, the expression of B Lymphocyte Stimulator receptor on B cells, and the responsiveness of B cells to B Lymphocyte Stimulator suggests that it may be involved in exchange of signals between B cells and monocytes or their differentiated progeny. This activity is in many ways analogous to the CD40-CD154 signalling between B cells and T cells. Anti-B Lymphocyte Stimulator antibodies and compositions of the invention may therefore be good regulators of T cell independent immune responses to environmental pathogens. In particular, the unconventional B cell populations (CD5+) that are associated with mucosal sites and responsible for much of the innate immunity in humans may respond to antibodies or compositions of the invention thereby enhancing or inhibiting individual's immune status.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly, their susceptibility profile would likely change.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a monocyte cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a B cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of detecting monocytic cells by virtue of its specificity. This application may require labeling the protein with biotin or other agents (e.g., as described herein) to afford a means of detection.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of detecting B-lineage cells by virtue of its specificity. This application may require labeling the protein with biotin or other agents (e.g., as described herein) to afford a means of detection.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable immunodeficiency.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as part of a monocyte selection device the function of which is to isolate monocytes from a heterogeneous mixture of cell types. Antibodies of the invention could be coupled to a solid support to which monocytes would then specifically bind. Unbound cells would be washed out and the bound cells subsequently eluted. A non-limiting use of this selection would be to allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as part of a B cell selection device the function of which is to isolate B cells from a heterogeneous mixture of cell types. Antibodies of the invention (that do not inhibit B Lymphocyte Stimulator/B Lymphocyte Stimulator Receptor interaction) binding soluble B Lymphocyte Stimulator could be coupled to a solid support to which B cells would then specifically bind. Unbound cells would be washed out and the bound cells subsequently eluted. A non-limiting use of this selection would be to allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence such as observed among SCID patients.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as an antigen for the generation of antibodies to inhibit or enhance B Lymphocyte Stimulator mediated responses.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as pretreatment of bone marrow samples prior to transplant. Such treatment would increase B cell representation and thus accelerate recovery.
In a specific embodiment, therapeutic or pharmaceutical compositions of the invention are used as a means of regulating secreted cytokines that are elicited by B Lymphocyte Stimulator and/or B Lymphocyte Stimulator receptor.
Antibody polypeptides or polynucleotides of the invention may be used to modulate IgE concentrations in vitro or in vivo.
Additionally, antibody polypeptides or polynucleotides of the invention may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.
In a specific embodiment, antibody polypeptides or polynucleotides of the invention, are administered to treat, prevent, diagnose, and/or ameliorate selective IgA deficiency.
In another specific embodiment antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate ataxia-telangiectasia.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate common variable immunodeficiency.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked agammaglobulinemia.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate severe combined immunodeficiency (SCID).
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate Wiskott-Aldrich syndrome.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM. In a specific embodiment antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, and/or diagnose chronic myelogenous leukemia, acute myelogenous leukemia, leukemia, hystiocytic leukemia, monocytic leukemia (e.g., acute monocytic leukemia), leukemic reticulosis, Shilling Type monocytic leukemia, and/or other leukemias derived from monocytes and/or monocytic cells and/or tissues.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate monocytic leukemoid reaction, as seen, for example, with tuberculosis.
In another specific embodiment, antibody polypeptides or polynucleotides of the invention are administered to treat, prevent, diagnose, and/or ameliorate monocytic leukocytosis, monocytic leukopenia, monocytopenia, and/or monocytosis.
In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, detect, and/or diagnose monocyte disorders and/or diseases, and/or conditions associated therewith.
In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, detect, and/or diagnose primary B lymphocyte disorders and/or diseases, and/or conditions associated therewith. In one embodiment, such primary B lymphocyte disorders, diseases, and/or conditions are characterized by a complete or partial loss of humoral immunity. Primary B lymphocyte disorders, diseases, and/or conditions associated therewith that are characterized by a complete or partial loss of humoral immunity and that may be prevented, treated, detected and/or diagnosed with compositions of the invention include, but are not limited to, X-Linked Agammaglobulinemia (XLA), severe combined immunodeficiency disease (SCID), and selective IgA deficiency.
In a preferred embodiment antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with any one or more of the various mucous membranes of the body. Such diseases or disorders include, but are not limited to, for example, mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example, American leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis, and New World leishmaniasis), mucocutaneous lymph node syndrome (for example, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medial degeneration (for example, cystic medial necrosis), mucolipidosis (including, for example, mucolipidosis I, mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysis disorders, mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as, for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome), type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type V mucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VI mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e, mucopolysaccharidosis due to beta-glucuronidase deficiency), and mucosulfatidosis), mucopolysacchariduria, mucopurulent conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virus diarrhea), mucous colitis (such as, for example, mucocolitis and myxomembranous colitis), and mucoviscidosis (such as, for example, cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis). In a highly preferred embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose mucositis, especially as associated with chemotherapy.
In a preferred embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with sinusitis.
An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by antibody polypeptides or polynucleotides of the invention is osteomyelitis.
An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by antibody polypeptides or polynucleotides of the invention is endocarditis.
All of the above described applications as they may apply to veterinary medicine.
Antibody polypeptides or polynucleotides of the invention may be used to treat, prevent, and/or diagnose diseases and disorders of the pulmonary system (e.g., bronchi such as, for example, sinopulmonary and bronchial infections and conditions associated with such diseases and disorders and other respiratory diseases and disorders. In specific embodiments, such diseases and disorders include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolar adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans, and proliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, obliterative bronchitis, plastic bronchitis, pseudomembranous bronchitis, putrid bronchitis, and verminous bronchitis), bronchocentric granulomatosis, bronchoedema, bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g., bronchopulmonary aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration, bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration, respiratory acidosis, respiratory alkalosis, respiratory distress syndrome of the newborn, respiratory insufficiency, respiratory scleroma, respiratory syncytial virus, and the like.
In a specific embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).
In another embodiment, antibody polypeptides or polynucleotides of the invention are used to treat, prevent, and/or diagnose fibroses and conditions associated with fibroses, including, but not limited to, cystic fibrosis (including such fibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestern fibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers' clay pipestem fibrosis.
In another embodiment, therapeutic or pharmaceutical compositions of the invention are administered to an animal to treat, prevent or ameliorate infectious diseases. Infectious diseases include diseases associated with yeast, fungal, viral and bacterial infections. Viruses causing viral infections which can be treated or prevented in accordance with this invention include, but are not limited to, retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpes simplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), arena viruses (e.g., lassa fever virus), paramyxoviruses (e.g., morbillivirus virus, human respiratory syncytial virus, mumps, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus), comaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses (e.g., influenza viruses A, B and C), papovaviruses (e.g., papillomavirues), picornaviruses (e.g., rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses, reoviruses (e.g., rotavirues), togaviruses (e.g. rubella virus), rhabdoviruses (e.g., rabies virus). Microbial pathogens causing bacterial infections include, but are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinji, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., and Helicobacter pylori. 
Gene Therapy
In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of B Lymphocyte Stimulator and/or its receptor, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 1 l (5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
In a preferred aspect, a composition of the invention comprises, or alternatively consists of, nucleic acids encoding an antibody, said nucleic acids being part of an expression vector that expresses the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is an scFv; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments or variants thereof, of an antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06 180; WO 92/22635; WO92/203 16; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435438 (1989)).
In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention or fragments or variants thereof are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:29 1-302 (1994), which describes the use of a retroviral vector to deliver the mdr 1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651(1994); Klein et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication W094/12649; and Wang, et al, Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Clin. Pharma. Ther. 29:69-92m (1985)) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody or fragment thereof are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 7 1:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Utility of a Composition
The compounds of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific antibody or composition of the present invention is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered an antibody or composition of the present invention, and the effect of such an antibody or composition of the present invention upon the tissue sample is observed. In various specific embodiments, in vitro assays can be carried out with representative cells of cell types involved in a patient's disorder, to determine if an antibody or composition of the present invention has a desired effect upon such cell types. Preferably, the antibodies or compositions of the invention are also tested in in vitro assays and animal model systems prior to administration to humans.
Antibodies or compositions of the present invention for use in therapy can be tested for their toxicity in suitable animal model systems, including but not limited to rats, mice, chicken, cows, monkeys, and rabbits. For in vivo testing of an antibody or composition's toxicity any animal model system known in the art may be used.
Efficacy in treating or preventing viral infection may be demonstrated by detecting the ability of an antibody or composition of the invention to inhibit the replication of the virus, to inhibit transmission or prevent the virus from establishing itself in its host, or to prevent, ameliorate or alleviate the symptoms of disease a progression. The treatment is considered therapeutic if there is, for example, a reduction in viral load, amelioration of one or more symptoms, or a decrease in mortality and/or morbidity following administration of an antibody or composition of the invention.
Antibodies or compositions of the invention can be tested for the ability to induce the expression of cytokines such as IFN-γ, by contacting cells, preferably human cells, with an antibody or composition of the invention or a control antibody or control composition and determining the ability of the antibody or composition of the invention to induce one or more cytokines. Techniques known to those of skill in the art can be used to measure the level of expression of cytokines. For example, the level of expression of cytokines can be measured by analyzing the level of RNA of cytokines by, for example, RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by, for example, immunoprecipitation followed by western blot analysis and ELISA. In a preferred embodiment, a compound of the invention is tested for its ability to induce the expression of IFN-γ.
Antibodies or compositions of the invention can be tested for their ability to modulate the biological activity of immune cells by contacting immune cells, preferably human immune cells (e.g., T-cells, B-cells, and Natural Killer cells), with an antibody or composition of the invention or a control compound and determining the ability of the antibody or composition of the invention to modulate (i.e, increase or decrease) the biological activity of immune cells. The ability of an antibody or composition of the invention to modulate the biological activity of immune cells can be assessed by detecting the expression of antigens, detecting the proliferation of immune cells (i.e., B-cell proliferation), detecting the activation of signaling molecules, detecting the effector function of immune cells, or detecting the differentiation of immune cells. Techniques known to those of skill in the art can be used for measuring these activities. For example, cellular proliferation can be assayed by 3H-thymidine incorporation assays and trypan blue cell counts. Antigen expression can be assayed, for example, by immunoassays including, but not limited to, competitive and non-competitive assay systems using techniques such as western blots, immunohistochemistry radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and FACS analysis. The activation of signaling molecules can be assayed, for example, by kinase assays and electrophoretic shift assays (EMSAs). In a preferred embodiment, the ability of an antibody or composition of the invention to induce B-cell proliferation is measured. In another preferred embodiment, the ability of an antibody or composition of the invention to modulate immunoglobulin expression is measured.
Antibodies or compositions of the invention can be tested for their ability to reduce tumor formation in in vitro, ex vivo and in vivo assays. Antibodies or compositions of the invention can also be tested for their ability to inhibit viral replication or reduce viral load in in vitro and in vivo assays. Antibodies or compositions of the invention can also be tested for their ability to reduce bacterial numbers in in vitro and in vivo assays known to those of skill in the art. Antibodies or compositions of the invention can also be tested for their ability to alleviate of one or more symptoms associated with cancer, an immune disorder (e.g., an inflammatory disease), a neurological disorder or an infectious disease. Antibodies or compositions of the invention can also be tested for their ability to decrease the time course of the infectious disease. Further, antibodies or compositions of the invention can be tested for their ability to increase the survival period of animals suffering from disease or disorder, including cancer, an immune disorder or an infectious disease. Techniques known to those of skill in the art can be used to analyze the function of the antibodies or compositions of the invention in vivo.
Therapeutic/Prophylactic Compositions and Administration
The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of antibody (or fragment or variant thereof) or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, an antibody or fragment or variant thereof is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to, animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin as described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer antibody or fragment or variant thereof of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:44294432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a preferred embodiment the antibody of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3). In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, 0.01% polysorbate 80, pH 6.5 (±0.3) for intravenous administration.
In a preferred embodiment the antibody of the invention is formulated in 110 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) (±0.3). In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) for intravenous administration. In another preferred embodiment, the antibody of the invention is formulated in 10 mM sodium citrate, 8% sucrose, 0.04% (w/v) polysorbate 80 (pH 6.5) for subcutaneous administration.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).
In yet another embodiment, the composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:20 1 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:35 1 (1989); Howard et al., J. Neurosurg. 7 1:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
In a specific embodiment where the composition of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g. by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868(1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the composition of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. In preferred embodiments, a dose of 1, 4, 10, or 20 mg/kg is administered intravenously to a patient. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of therapeutic or pharmaceutical compositions of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The antibodies and antibody compositions of the invention may be administered alone or in combination with other adjuvants. Adjuvants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with alum. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with QS21. Further adjuvants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS21, QS18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis, and/or PNEUMOVAX-23TM. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In another specific embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated therewith. In one embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose any Gram positive bacterial infection and/or any disease, disorder, and/or condition associated therewith. In another embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with one or more members of the genus Enterococcus and/or the genus Streptococcus. In another embodiment, antibody and antibody compositions of the invention are used in any combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with one or more members of the Group B streptococci. In another embodiment, antibody and antibody compositions of the invention are used in combination with PNEUMOVAX-23TM to treat, prevent, and/or diagnose infection and/or any disease, disorder, and/or condition associated with Streptococcus pneumoniae. 
The antibody and antibody compositions of the invention may be administered alone or in combination with other therapeutic agents, including but not limited to, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents and cytokines. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
In one embodiment, the antibody and antibody compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-IBBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), TRAIL (also known as AIM-1, International Publication No. WO97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6): 1185-1190 (1998)), endokine-alpha (International Publication No. WO 98/07880), Neutrokine-alpha (International Application Publication No. WO 98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
In another embodiment, the compositions of the invention are administered in combination with Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors (e.g., TACI, BCMA and BAFF-R). In preferred in embodiments the Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors are soluble. In other preferred embodiments the Neutrokine-alpha receptors and/or Neurtokine-alpha SV receptors are fused to the FC region of an immunoglobulin molecule (e.g, amino acid residues 1-154 of TACI (GenBank accession number AAC51790), amino acids 148 of BCMA (GenBank accession number NP—001183, or amino acids 1 to 81 of BAFF-R (GenBank Accession Number NP—443177) fused to the Fc region of an IgG molecule.
In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVRENDTM), biologically active fragments, variants, or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonistic or antagonistic antibodies).
In an additional embodiment, the antibody and antibody compositions of the invention are administered alone or in combination with an anti-angiogenic agent(s). Anti-angiogenic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, 1992); and metalloproteinase inhibitors such as BB94.
Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H, Brem and J. Folkman J. Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A4 (CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
Anti-angiogenic agents that may be administered in combination with the compositions of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, AG3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S, San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S, San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).
In particular embodiments, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
In a particular embodiment, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
In particular embodiments, the use of antibody and antibody compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of strokes.
In another embodiment, antibody and antibody compositions of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, heparin, warfarin, and aspirin. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with warfarin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin. In another specific embodiment, antibody and antibody compositions of the invention are administered in combination with heparin and aspirin.
In another embodiment, antibody and antibody compositions of the invention are administered in combination with an agent that suppresses the production of anticardiolipin antibodies. In specific embodiments, the polynucleotides of the invention are administered in combination with an agent that blocks and/or reduces the ability of anticardiolipin antibodies to bind phospholipid-binding plasma protein beta 2-glycoprotein I (b2GPI).
In certain embodiments, antibody and antibody compositions of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz). Protease inhibitors that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with antibody and antibody compositions of the invention to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or diagnose HIV infection.
In certain embodiments, antibody and antibody compositions of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the compositions of the invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine). NNRTIs that may be administered in combination with the compositions of the invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz). Protease inhibitors that may be administered in combination with the compositions of the invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir), INVIRASETM (saquinavir), and VIRACEPTTM (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat AIDS and/or to prevent or treat HIV infection.
Additional NRTIs include LODENOSINETM (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACILTM (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIRTM (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGENTM (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S acyl-2-thioethyl (SATE)-bearing prodrug forms of b-LFD4C and b-LFddC (WO 98/17281).
Additional NNRTIs include COACTINONTM (Emivirine/MKC442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINETM (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).
Additional protease inhibitors include LOPINAVIRTM (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIRTM (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASETM (aniprenavir; Glaxo Wellcome Inc.).
Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).
Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists such as RANTES, SDF-1, MIP-1a, MIP-1b, etc., may also inhibit fusion.
Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIRTM (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.
Additional antiretroviral agents include hydroxyurea-like compounds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOXTM (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors such as VX-497 (Vertex); and myvopholic acids such as CellCept (mycophenolate mofetil; Roche).
Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1a, MIP-1b, SDF-1a, IL-2, PROLEUKINTM (aldesleukin/L2-7001; Chiron), IL-4, IL-8, IL-10, IL-12, and IL-13; interferons such as IFN-a2a; antagonists of TNFs, NFkB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as RemuneTM (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JRFL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targeted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G-5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as g-L-glutamyl-L-cysteine ethyl ester (g-GCE; WO 99/56764).
In other embodiments, antibody and antibody compositions of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM, KETOCONAZOLETM, ACYCLOVIRTM, FAMCICOLVIRTM, PYRIMETHAMINETM, LEUCOVORINTM, NEUPOGENTM (filgrastim/G-CSF), and LEUKINETM (sargramostim/GM-CSF). In a specific embodiment, antibody and antibody compositions of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat, prevent, and/or diagnose an opportunistic Pneumocystis carinji pneumonia infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or KETOCONAZOLETM to prophylactically treat, prevent, and/or diagnose an opportunistic fungal infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection. In another specific embodiment, antibody and antibody compositions of the invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat, prevent, and/or diagnose an opportunistic bacterial infection.
In a further embodiment, the antibody and antibody compositions of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
In a further embodiment, the antibody and antibody compositions of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
Conventional nonspecific immunosuppressive agents, that may be administered in combination with the antibody and antibody compositions of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents, that may be administered in combination with the compositions of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININTM), brequinar, deoxyspergualin, and azaspirane (SKF 105685).
In specific embodiments, antibody and antibody compositions of the invention are administered in combination with immunosuppressants. Immunosuppressants preparations that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, ORTHOCLONETM (OKT® 3 (muromonab-CD3), SANDIMMUNETM, NEORALTM, SANGDYATM (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURANTM (azathioprine), glucorticosteroids, adrenocortical steroids such as DELTASONETM (prednisone) and HYDELTRASOLTM (prednisolone), FOLEXTM and MEXATETM (methotrexate), OXSORALEN-ULTRATM (methoxsalen) and RAPAMUNETM (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with steroid therapy. Steroids that may be administered in combination with the antibody and antibody compositions of the invention, include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with prednisone. In a further specific embodiment, the antibody and antibody compositions of the invention are administered in combination with prednisone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the antibody and antibody compositions of the invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV. In a another specific embodiment, antibody and antibody compositions of the invention are administered in combination with methylprednisolone. In a further specific embodiment, the antibody and antibody compositions of the invention are administered in combination with methylprednisolone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the antibody and antibody compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial. Antimalarials that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.
In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an NSAID.
In a nonexclusive embodiment, the antibody and antibody compositions of the invention are administered in combination with one, two, three, four, five, ten, or more of the following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNF receptor 1 (synergen; Amgen), IPR-6001 (Institute for Pharmaceutical Research), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics), MK663 (Merck), ST-1482 (Sigma-Tau), and butixocort propionate (Warner Lambert).
In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: Infliximab (also known as RemicadeTM Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as AravaTM from Hoechst Marion Roussel), KineretTM (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.), SCIO-469 (p38 kinase inhibitor from Scios, Inc), Humira® (adalimumab from Abbott Laboratories) and/or ASLERATM (prasterone, dehydroepiandrosterone, GL701) from Genelabs Technologies Inc.
In a preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g., as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti-TNF antibody, UP 394 (La Jolla Pharmaceutical Company, San Diego, Calif.) and prednisolone.
In a more preferred embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial, methotrexate, anti-TNF antibody, ENBRELTM and/or sulfasalazine. In one embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with anti-TNF antibody. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate and anti-TNF antibody. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with sulfasalazine. In another specific embodiment, the antibody and antibody compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and sulfasalazine. In another embodiment, the antibody and antibody compositions of the invention are administered in combination ENBRELTM. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBRELTM and methotrexate. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBRELTM, methotrexate and sulfasalazine. In another embodiment, the antibody and antibody compositions of the invention are administered in combination with ENBRELTM, methotrexate and sulfasalazine. In other embodiments, one or more antimalarials is combined with one of the above-recited combinations. In a specific embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELTM, methotrexate and sulfasalazine. In another specific embodiment, the antibody and antibody compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody, and methotrexate.
In an additional embodiment, antibody and antibody compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the antibody and antibody compositions of the invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, and GAMIMUNETM. In a specific embodiment, antibody and antibody compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
In an additional embodiment, the antibody and antibody compositions of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
In specific embodiments, the compositions of the invention are administered alone or in combination with anti-CD4 antibody. In one embodiment, coadministration of the compositions of the invention with anti-CD4 antibody is envisioned for treatment of rheumatoid arthritis.
In specific embodiments, the compositions of the invention are administered alone or in combination with anti-IL-15 antibody. In one embodiment, coadministration of the compositions of the invention with anti-IL-15 antibody is envisioned for treatment of rheumatoid arthritis.
In specific embodiments, the compositions of the invention are administered alone or in combination with CTLA4-Ig and LEA29Y, In one embodiment, coadministration of the compositions of the invention with CTLA4-Ig and LEA29Y is envisioned for treatment of rheumatoid arthritis.
In specific embodiments, the compositions of the invention are administered alone or in combination with anti-IL-6 Receptor antibody. In one embodiment, coadministration of the compositions of the invention with anti-IL-6 Receptor antibody is envisioned for treatment of rheumatoid arthritis.
In specific embodiments, the compositions of the invention are administered alone or in combination with anti-C5 (complement component) antibody. In one embodiment, coadministration of the compositions of the invention with anti-C5 antibody is envisioned for treatment of rheumatoid arthritis.
In specific embodiments, the compositions of the invention are administered alone or in combination with complement cascade inhibitors. Complement cascade inhibitors include, but are not limited to, anti-properdin antibodies (Gliatech); TP-10, a recombinant soluble type I complement receptor (AVANT Immunotheragenetics Inc.); Pexelizmab, a Complement C5 inhibitor (Alexion Pharmaceuticals Inc.); and 5G 1.1, a monoclonal antibody that prevents cleavage of complement component C5 into its pro-inflammatory components. In one embodiment, coadministration of the compositions of the invention with complement cascade inhibitors are is envisioned for treatment of Inflammation, Rheumatoid arthritis, and/or cardiovascular disorders.
In another embodiment, compostions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP, In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab (anti-CD20 antibody from Coulter Pharmaceuticals, San Francisco, Calif.). In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Tositumomab may optionally be associated with 131I. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
In another specific embodiment, the compositions of the invention are administered in combination ZevalinTM. In a further embodiment, compositions of the invention are administered with ZevalinTM and CHOP, or ZevalinTM and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. ZevalinTM may be associated with one or more radioisotopes. Particularly preferred isotopes are 90Y and 111In.
In additional preferred embodiments, compositions of the invention are administered in combination with Rituximab (RituxanTM) and/or Ibritumomab Tiuxetan (ZevalinTM, e.g., either (In-111) Ibritumomab Tiuxetan or (Y-90) Ibritumomab Tiuxetan). In a specific embodiment, compositions of the invention are administered in combination with Rituximab and/or Ibritumomab Tiuxetan for the treatment of non-Hodgkin's lymphoma.
In additional preferred embodiments, compositions of the invention are administered in combination with imatinib mesylate (Gleevec®: 4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate). In a specific embodiment, compositions of the invention are administered in combination with imatinib mesylate for the treatment of chronic myelogenous leukemia.
In additional preferred embodiments, compositions of the invention are administered in combination with bortezomib (VelcadeTM [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid). In a specific embodiment, compositions of the invention are administered in combination with bortezomib for the treatment of multiple myeloma.
In additional preferred embodiments, compositions of the invention are administered in combination with Alemtuzumab (Campath®). In a specific embodiment, compositions of the invention are administered in combination with Alemtuzumab for the treatment of chronic lymphocytic leukemia.
In additional preferred embodiments, compositions of the invention are administered in combination with fludarabine phosphate (Fludara®: 9H-Purin-6-amine, 2-fluoro-9-(5-O-phosphono-b-D-arabinofuranosyl) (2-fluoro-ara-AMP)). In a specific embodiment, compositions of the invention are administered in combination with fludarabine phosphate for the treatment of chronic lymphocytic leukemia.
In further particular embodiments, compositions of the present invention are used to treat, ameliorate and/or prevent hematological cancers. Compositions of the present invention may be used in combination with one or more surgical and/or radiological procedures and/or therapeutic agents to treat, ameliorate and/or prevent hematological cancers. Hematological cancers which may be treated using compositions of the present invention include, but are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicular center cell lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and intestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and plasma cell neoplasms including multiple myeloma.
In preferred embodiments, compositions of the present invention are used to treat, ameliorate and/or prevent hematological cancers compositions of the present invention may be used in combination with one or more surgical and/or radiological procedures and/or therapeutic agents to treat, ameliorate and/or prevent hematological cancers. Hematological cancers which may be treated using compositions of the present invention include, but are not limited to, non-Hodgkin's lymphoma (e.g., small lymphocytic lymphoma, follicular center cell lymphoma, lymphoplasmacytoid lymphoma, marginal zone lymphoma, mantle cell lymphoma, immunoblastic lymphoma, burkitt's lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma and intestinal T-cell lymphoma), leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia and plasma cell neoplasms including multiple myeloma.
In one preferred embodiment, compositions of the present invention are used to treat plasma cell neoplasms. In a specific embodiment, that plasma cell neoplasm is multiple myeloma.
In another preferred embodiment, compositions of the present invention are used to treat non-Hodgkin's lymphoma.
In another preferred embodiment, compositions of the present invention are used to treat leukemia. In a specific embodiment, that leukemia is acute lymphocytic leukemia. In another specific embodiment, that leukemia is chronic lymphocytic leukemia.
Compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of hematological cancer including, but not limited to, bone marrow transplantation, external beam radiation and total body irradiation.
In preferred embodiments, compositions of the present invention are administered in combination with one or more surgical and/or radiological procedures useful in the treatment of hematological cancer including, but not limited to, bone marrow transplantation, external beam radiation and total body irradiation.
In one preferred embodiment, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of multiple myeloma including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support.
In another preferred embodiment, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of non-Hodgkin's lymphoma including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support.
In further specific embodiments, compositions of the present invention may be administered in combination with one or more surgical and/or radiological procedures useful in the treatment of leukemia including, but not limited to, allogeneic bone marrow transplantation and peripheral stem cell support. In one specific preferred embodiment, compositions of the present invention of the invention are used to treat acute lymphocytic leukemia (ALL). In another specific preferred embodiment, compositions of the present invention are used to treat chronic lymphocytic leukemia (CLL).
Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of multiple myeloma including, but not limited to, Alkylating agents, Anthracyclines, Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Dexamethasone (Decadron®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Melphalan (L-PAM, Alkeran®, Phenylalanine mustard), Prednisone, Thalidomide and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of multiple myeloma.
Preferred combinations of therapeutic agents useful in the treatment of multiple myeloma which may be administered in combination with compositions of the present invention include, but are not limited to, Cyclophosphamide+Prednisone, Melphalan+Prednisone (MP), Vincnistine+Adriamycin®+Dexamethasone (VAD), Vincristine+Carmustine+Melphalan+Cyclophosphamide+Prednisone (VBMCP; the M2 protocol), and Vincristine+Melphalan+Cyclophosphamide+Prednisone alternating with Vincristine+Carmustine+Doxorubicin+Prednisone (VMCP/VBAP).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of multiple myeloma.
Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of non-Hodgkin's lymphoma including, but not limited to, 2-chlorodeoxyadenosine, Amifostine (Ethyol®, Ethiofos®, WR-272), Bexarotene (Targretin®, Targretin Gel®, Targretin Oral®, LGD1069), Bleomycin (Blenoxane®), Busulfan (Busulfex®, Myleran®), Carboplatin (Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®), Chlorambucil (Leukeran®), Cisplatin (Platinol®, CDDP), Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®, Neosarg), CTX), Cytarabine (Cytosar-U®1, ara-C, cytosine arabinoside, DepoCyt®), Dacarbazine (DTIC), Daunorubicin (Daunomycin, DaunoXome®&, Daunorubicin®, Cerubidine®)), Denileukin diftitox (Ontak®), Dexamethasone (Decadron®), Dolasetron mesylate (Anzemet®), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Erythropoietin (EPO®&, Epogen®, Procrit®), Etoposide phosphate (Etopophos®), Etoposide (VP-16, Vepesid®), Fludarabine (Fludara®, FAMP), Granisetron (Kytril®), Hydrocortisone, Idarubicin (Idamycin3, DMDR, IDA), Ifosfamide (IFEX®), Interferon alpha (Alfaferone®, Alpha-IF®), Interferon alpha 2a (Intron A®), Mechlorethamine (Nitrogen Mustard, HN2, Mustargen®), Melphalan (LPAM, Alkeran®, Phenylalanine mustard), Methotrexate® (MTX, Mexate®, Folex®), Methylprednisolone (Solumedrol®), Mitoxantrone (Novantrone®, DHAD), Ondansetron (Zofran®), Pentostatin (Nipent®, 2-deoxycoformycin), Perfosfamide (4-hydroperoxycyclophosphamide, 4-HC), Prednisone, Procarbazine (Matulane®), Rituximab® (Rituxan®D, anti-CD20 MAb), Thiotepa (triethylenethiophosphaoramide, Thioplex®), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Vinblastine (Velban®, VLB), Vincristine (Oncovin®, Onco TCS®, VCR, Leurocristine®) and Vindesine (Eldisine®, Fildesin®).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.
Preferred combinations of therapeutic agents useful in the treatment of non-Hodgkin's lymphoma which may be administered in combination with compositions of the present invention include, but are not limited to, Adriamycin®+Blenoxane+Vinblastine+Dacarbazine (ABVD), Anti-idiotype therapy (BsAb)+Interferon alpha, Anti-idiotype therapy (BsAb)+Chlorambucil, Anti-idiotype therapy (BsAb)+Interleukin-2, BCNU (Carmustine)+Etoposide+Ara-C (Cytarabine)+Melphalen (BEAM), Bleomycin+Etoposide+Adriamycin+Cyclophosphamide+Vincristine+Procarbazine+Prednisone (BEACOPP), Bryostatin+Vincristine, Cyclophosphamide+BCNU (Carmustine)+VP-16 (Etoposide) (CBV), Cyclophosphamide+Vincnistine+Prednisone (CVP), Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine (Oncovorin)+Prednisone (CHOP), Cyclophosphamide+Novantrone® (Mitoxantrone)+Vincristine (Oncovorin)+Prednisone (CNOP), Cyclophosphamide+Doxorubicin+Teniposide+Prednisone, Cyclophosphamide+Adriamycin® (Hydroxyldaunomycin)+Vincristine (Oncovorin)+Prednisone+Rituximab (CHOP+Rituximab), Cyclophosphamide+Doxorubicin+Teniposide+Prednisone+Interferon alpha, Cytarabine+Bleomycin+Vincristine+Methotrexate (CytaBOM), Dexamethasone+Cytarabine+Cisplatin (DHAP), Dexamethasone+Ifosfamide+Cisplatin+Etoposide (DICE), Doxorubicin+Vinblastine+Mechlorethamine+Vincristine+Bleomycin+Etoposide+Prednisone (Stanford V), Etoposide+Vinblastine+Adriamycin (EVA), Etoposide+Methylprednisone+Cytarabine+Cisplatin (ESHAP), Etoposide+Prednisone+Ifosfamide+Cisplatin (EPIC), Fludarabine, Mitoxantrone+Dexamethasone (FMD), Fludarabine, Dexamethasone, Cytarabine (ara-C), +Cisplatin (Platnol®D) (FluDAP), Ifosfamide+Cisplatin+Etoposide (ICE), Mechlorethamine+Oncovin® (Vincristine)+Procarbazine+Prednisone (MOPP), Mesna+Ifosfamide+Idarubicin+Etoposide (MIZE), Methotrexate with leucovorin rescue+Bleomycin+Adriamycin+Cyclophosphamide+Oncovorin+Dexamethasone (m-BACOD), Prednisone+Methotrexate+Adriamycin+Cyclophosphamide+Etoposide (ProMACE), Thiotepa+Busulfan+Cyclophosphamide, Thiotepa+Busulfan+Melphalan, Topotecan+Paclitaxel, and Vincristine (Oncovin®)+Adriamycin®+Dexamethasone (VAD).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.
Further examples of therapeutic agents useful in the treatment of non-Hodgkin's lymphoma which may be administered in combination with compositions of the present invention include, but are not limited to, A007 (44′-dihydroxybenzophenone-2,4-dinitrophenylhydrazone), AG2034 (AG2024, AG2032, GARFT [glycinamide ribonucleoside transfonnylase] inhibitor), Aldesleukin (IL-2, Proleukin®), Alemtuzumab (Campath®), Alitretinoin (Panretin®, LGN-1057), Altretamine (Hexalen®, hexamethylmelamine, Hexastat®), Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Anti-CD19/CD3 MAb (anti-CD19/CD3 scFv, anti-NHL MAb), Anti-idiotype therapy (BsAb), Arabinosylguanine (Ara-G, GW506U78), Arsenic trioxide (Trisenox®, ATO), B43-Genistein (anti-CD19 Ab/genistein conjugate), B7 antibody conjugates, Betathine (Beta-LT), B Lymphocyte Stimulator antagonists, Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555), CHML (Cytotropic Heterogeneous Molecular Lipids), Clofarabine (chloro-fluoro-araA), Daclizumab (Zenapax®), Depsipeptide (FR901228, FK228), Dolastatin-10 (DOLA-10, NSC-376128), Epirubicin (Ellence®, EPI, 4′ epi-doxorubicin), Epratuzumab (Lymphocide®, humanized anti-CD22, HAT), Fly3/flk2 ligand (Mobista®), G3139 (Genasense®, GentaAnticode®, Bc-12 antisense), Hu1D10 (anti-HLA-DR MAb, SMART 1D10), HumaLYM (anti-CD20 MAb), Ibritumomab tiuxetan (Zevalin(D), Interferon gamma (Gamma-interferon, Gamma 100®, Gamma-IF), Irinotecan (Camptosar®, CPT-11, Topotecin®, Capto CPT-1), ISIS2053, ISIS3521 (PKCalpha antisense), Lmb-2 immunotoxin (anti-CD25 recombinant immuno toxin, anti-Tac(Fv)-PE38), Leuvectin® (cytofectin+IL-2 gene, IL-2 gene therapy), Lym-1 (131-1 LYM-1), Lymphoma vaccine (Genitope), Nelarabine (Compound 506, U78), Neugene compounds (Oncomyc-NG®, Resten-NG®, myc antisense), NovoMAb-G2 scFv (NovoMAb-G2 IgM), O6-benzylguanine (BG, Procept®), Oxaliplatin (Eloxatine®, Eloxatin®), Paclitaxel (Paxene®, Taxol®), Paclitaxel-DHA (Taxoprexin®), Peldesine (BCX-34, PNP inhibitor), Rebeccamycin and Rebeccamycin analogues, SCH-66336, Sobuzoxane (MST-16, Perazolin®), SU5416 (Semaxanib®, VEGF inhibitor), TER286, Thalidomide, TNP-470 (AGM-1470), Tositumomab (Bexxar®), Valspodar (PSC 833), Vaxid (B-cell lymphoma DNA vaccine), Vinorelbine (Navelbine®), WF10 (macrophage regulator) and XR-9576 (XR-9351, P-glycoprotein/MDR inhibitor).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of non-Hodgkin's lymphoma.
Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of acute lymphocytic leukemia including, but not limited to, Amsacrine, Carboplatin (Paraplatin®, CBDCA), Carmustine (DTI-015, BCNU, BiCNU, Gliadel Wafer®, Cholecaliferol, Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®), Daunorubicin (Daunomycin, DaunoXome®, Daunorubicin®, Cerubidine®), Dexamethasone (Decadron®D), Doxorubicin (Adriamycin®, Doxil®, Rubex®), Etoposide (VP-16, Vepesid®D), Filgrastam® (Neupogen®, GCSF, Leukine®), Fludarabine (Fludara®, FAMP), Idarubicin (Idamycin®, DMDR, IDA), Ifosfamide (IFEX®), Imatinib mesylate (STI-571, Imatinib®, Glivec®, Gleevec®, Abl tyrosine kinase inhibitor), Interferon gamma (Gamma-interferon, Gamma 100®, Gamma-IF), L-asparaginase (Elspar®, Crastinin®, Asparaginase Medac®, Kidrolase®), Mercaptopurine (6-mercaptopurine, 6-MP), Methotrexate® (MTX, Mexate®, Folex®), Mitoxantrone (Novantrone®, DHAD), Pegaspargase® (Oncospar®), Prednisone, Retinoic acid, Teniposide (VM-26, Vumon®)), Thioguanine (6-thioguanine, 6-TG), Topotecan (Hycamtin®, SK&F-104864, NSC-609699, Evotopin®), Tretinoin (Retin-A®, Atragen®, ATRA, Vesanoid®) and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.
Further examples of therapeutic agents useful in the treatment of acute lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF, Aronex®D), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®), Arsenic trioxide (Trisenox®, ATO, Atrivex®), B43-Genistein (anti-CD19 Ab/genistein conjugate), B43-PAP (anti-CD19 Ab/pokeweed antiviral protein conjugate), Cordycepin, CS-682, Decitabine (5-aza-2′-deoxyytidine), Dolastatin-10 (DOLA-10, NSC-376128), G3139 (Genasense®D, GentaAnticode®, Bcl-2 antisense), Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Quinine, TNP-470 (AGM-1470, Fumagillin), Trimetrexate (Neutrexing), Troxacitabine (BCH-204, BCH-4556, Troxatyl®), UCN-01 (7-hydroxystaurosporine), WHI-P131 and WTI Vaccine.
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.
Preferred combinations of therapeutic agents useful in the treatment of acute lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Carboplatin+Mitoxantrone, Carmustine+Cyclophosphamide+Etoposide, Cytarabine+Daunorubicin, Cytarabine+Doxorubicin, Cytarabine+Idarubicin, Cytarabine+Interferon gamma, Cytarabine+L-asparaginase, Cytarabine+Mitoxantrone, Cytarabine+Fludarabine and Mitoxantrone, Etoposide+Cytarabine, Etoposide+Ifosfamide, Etoposide+Mitoxantrone, Ifosfamide+Etoposide+Mitoxantrone, Ifosfamide+ Teniposide, Methotrexate+Mercaptopurine, Methotrexate+Mercaptopurine+Vincristine+Prednisone, Phenylbutyrate+Cytarabine, Phenylbutyrate+Etoposide, Phenylbutyrate+Topotecan, Phenylbutyrate+Tretinoin, Quinine+Doxorubicin, Quinine+Mitoxantrone+Cytarabine, Thioguanine+Cytarabine+Amsacrine, Thioguanine+Etoposide+Idarubicin, Thioguanine+Retinoic acid+Cholecaliferol, Vincristine+Prednisone, Vincristine+Prednisone and L-asparaginase, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Filgrastim, Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate, and Vincristine+Dexamethasone/Prednisone+Asparaginase+Daunorubicin/Doxorubicin+Cyclophosphamide+Methotrexate+Filgrastim.
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of acute lymphocytic leukemia.
Compositions of the present invention may be administered in combination with one or more therapeutic agents useful in the treatment of chronic lymphocytic leukemia including, but not limited to, Chlorambucil (Leukeran®, Cladribine (2-CdA, Leustatin®), Cyclophosphamide (Cytoxan®, Neosar®, CTX), Cytarabine (Cytosar-U®, ara-C, cytosine arabinoside, DepoCyt®, cytarabine ocfosfate, ara-CMP), Doxorubicin (Adriamycin®, Doxil®, Rubex®, Fludarabine (Fludara®, FAMP), Pentostatin (Nipent®, 2-deoxycoformycin), Prednisone and Vincristine (Oncovorin®, Onco TCS®, VCR, Leurocristine®).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.
Further examples of therapeutic agents useful in the treatment of chronic lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Alemtuzumab (Campath®, Aminocamptothecin (9-AC, 9-Aminocamptothecin, NSC 603071), Aminopterin, Annamycin (AR-522, annamycin LF, Aronex®), Arabinosylguanine (Ara-G, GW506U78, Nelzarabine®, Compound 506U78), Arsenic trioxide (Trisenox®, ATO, Atrivex®), Bryostatin-1 (Bryostatin®, BMY-45618, NSC-339555), CS-682, Dolastatin-10 (DOLA-10, NSC-376128), Filgrastim (Neupogen®, G CSF, Leukine), Flavopiridol (NSC-649890, HMR-1275), G3139 (Genasense®, GentaAnticode®, Bc-12 antisense), Irofulven (MGI-114, Ivofulvan, Acylfulvene analogue), MS-209, Phenylbutyrate, Rituximab® (Rituxan®, anti-CD20 MAb), Thalidomide, Theophylline, TNP-470 (AGM-1470, Fumagillin), UCN-01 (7-hydroxystaurosporine) and WHI-P131.
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agents in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.
Preferred combinations of therapeutic agents useful in the treatment of chronic lymphocytic leukemia which may be administered in combination with compositions of the present invention include, but are not limited to, Fludarabine+Prednisone, and Cyclophosphamide+Doxorubicin+Vincristine+Prednisone (CHOP).
In preferred embodiments, compositions of the present invention are administered in combination with one or more of the above-described therapeutic agent combinations in the treatment, amelioration and/or prevention of chronic lymphocytic leukemia.
In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with cytokines. Cytokines that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, GM-CSF, GCSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In preferred embodiments, antibody and antibody compositions of the invention are administered with B Lymphocyte Stimulator (e.g., amino acids 134-285 of SEQ ID NO:3228). In another embodiment, antibody and antibody compositions of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22. In preferred embodiments, the antibody and antibody compositions of the invention are administered in combination with IL-4 and IL-10.
In one embodiment, the antibody and antibody compositions of the invention are administered in combination with one or more chemokines. In specific embodiments, the antibody and antibody compositions of the invention are administered in combination with an α(C×C) chemokine selected from the group consisting of gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8), platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α, GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocyte chemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1 (SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a β(CC) chemokine selected from the group consisting of: RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma (MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophage inflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4 (MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and I-309; and/or the γ(C) chemokine, lymphotactin.
In another embodiment, the antibody and antibody compositions of the invention are administered with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein-4. In a preferred embodiment, the antibody and antibody compositions of the invention are administered with chemokine beta-8.
In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with an IL-4 antagonist IL-4 antagonists that may be administered with the antibody and antibody compositions of the invention include, but are not limited to: soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor without transducing the biological signal elicited by IL-4, anti-IL-4 antibodies that block binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do not transduce the biological signal elicited by IL-4. Preferably, the antibodies employed according to this method are monoclonal antibodies (including antibody fragments, such as, for example, those described herein).
The invention also encompasses combining the polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) with other proposed or conventional hematopoietic therapies. Thus, for example, the polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) can be combined with compounds that singly exhibit erythropoietic stimulatory effects, such as erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, and triiodothyzonine. Also encompassed are combinations of the antibody and antibody compositions of the invention with compounds generally used to treat aplastic anemia, such as, for example, methenolene, stanozolol, and nandrolone; to treat iron-deficiency anemia, such as, for example, iron preparations; to treat malignant anemia, such as, for example, vitamin B12 and/or folic acid; and to treat hemolytic anemia, such as, for example, adrenocortical steroids, e.g., corticoids. See e.g., Resegotti et al., Panminerva Medica, 23:243-248 (1981); Kurtz, FEBS Letters, 14a:105-108 (1982); McGonigle et al., Kidney Int., 25:437-444 (1984); and Pavlovic-Kantera, Expt. Hematol., 8(supp. 8)283-291(1980), the contents of each of which are hereby incorporated by reference in their entireties.
Compounds that enhance the effects of or synergize with erythropoietin are also useful as adjuvants herein, and include but are not limited to, adrenergic agonists, thyroid hormones, androgens, hepatic erythropoietic factors, erythrotropins, and erythrogenins, See for e.g., Dunn, “Current Concepts in Erythropoiesis”, John Wiley and Sons (Chichester, England, 1983); Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J. Med., 289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325 (1979); Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton et al., Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364, Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec, Jul. 1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol., 20:105-108 (1982). Methods for stimulating hematopoiesis comprise administering a hematopoietically effective amount (i.e., an amount which effects the formation of blood cells) of a pharmaceutical composition containing polynucleotides and/or polypeptides of the invention (and/or agonists or antagonists thereof) to a patient. The polynucleotides and/or polypeptides of the invention and/or agonists or antagonists thereof is administered to the patient by any suitable technique, including but not limited to, parenteral, sublingual, topical, intrapulmonary and intranasal, and those techniques further discussed herein. The pharmaceutical composition optionally contains one or more members of the group consisting of erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, and nandrolone, iron preparations, vitamin B12, folic acid and/or adrenocortical steroids.
In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, LEUKINETM (SARGRAMOSTIMTM) and NEUPOGENTM (FILGRASTIMTM).
In an additional embodiment, the antibody and antibody compositions of the invention are administered in combination with fibroblast growth factors. Fibroblast growth factors that may be administered with the antibody and antibody compositions of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
Additionally, the antibody and antibody compositions of the invention may be administered alone or in combination with other therapeutic regimens, including but not limited to, radiation therapy. Such combinatorial therapy may be administered sequentially and/or concomitantly.
Kits
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In an alternative embodiment, a kit comprises an antibody fragment that immunospecifically binds to B Lymphocyte Stimulator. In a specific embodiment, the kits of the present invention contain a substantially isolated B Lymphocyte Stimulator polypeptide as a control. Preferably, the kits of the present invention further comprise a control antibody which does not react with B Lymphocyte Stimulator. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to B Lymphocyte Stimulator (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized B Lymphocyte Stimulator. The B Lymphocyte Stimulator provided in the kit may also be attached to a solid support. In a more specific embodiment the detecting means of the above-described kit includes a solid support to which B Lymphocyte Stimulator is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to B Lymphocyte Stimulator can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with B Lymphocyte Stimulator, and means for detecting the binding of B Lymphocyte Stimulator to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound B Lymphocyte Stimulator obtained by the methods of the present invention. After B Lymphocyte Stimulator binds to a specific antibody, the unbound serum components are removed by washing, reporter-labeled anti-human antibody is added, unbound anti-human antibody is removed by washing, and a reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-B Lymphocyte Stimulator antibody on the solid support. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate.
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific absorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant B Lymphocyte Stimulator, and a reporter-labeled anti-human antibody for detecting surface-bound anti-B Lymphocyte Stimulator antibody.
In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.
In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880.
In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128.
In specific embodiments, the present invention encompasses a single chain Fv (scFv) having an amino acid sequence of one of SEQ ID NOS: 1 to 1562.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, and in which said antibody or fragment thereof immunospecifically binds to the membrane-bound form of B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, and in which said antibody or fragment thereof immunospecifically binds to the soluble form of B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, and in which said antibody or fragment thereof immunospecifically binds to the membrane-bound form of B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, and in which said antibody or fragment thereof immunospecifically binds to the soluble form of B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and in which said VL and said VH domains are derived from the same scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses an antibody or fragment thereof comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.
In specific embodiments, the antibody or fragment thereof of the invention is a whole immunoglobulin molecule.
In specific embodiments, the antibody or fragment thereof of the invention is a Fab fragment.
In specific embodiments, the antibody or fragment thereof of the invention is a Fv fragment.
In specific embodiments, the present invention encompasses a chimeric protein comprising the antibody or fragment thereof of the invention covalently linked to a heterologous polypeptide.
In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and wherein each type of antibody or fragment thereof further comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses a composition comprising two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 3129 to 3227.
In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128.
In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128.
In specific embodiments, the present invention encompasses a panel of two or more types of antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VL domain from a different scFv having an amino acid sequence of one of SEQ ID NO: 1 to 2128 and wherein each type of antibody or fragment further comprises a VH domain from a different scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention encompasses a panel of two or more antibodies or fragments or variants thereof, each of which type immunospecifically binds to B Lymphocyte Stimulator, and each of which type of antibody or fragment thereof comprises a VHCDR3 from a different scFv having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.
In specific embodiments, the antibodies or fragments thereof of the antibody panel of the invention, are each in a well of a 96 well plate.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 1908, wherein the antibody of fragment thereof immunospecifically binds the membrane-bound form of B Lymphocyte Stimulator.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1569, wherein said antibody of fragment thereof immunospecifically binds the soluble form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 46, 321 to 329, 1563 to 1595, and 1881 to 1908, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1881 to 2128, wherein the antibody of fragment thereof immunospecifically binds the membrane-bound form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1563 to 1880, wherein said antibody of fragment thereof immunospecifically binds the soluble form of B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and in which said VL domain and said VH domain are derived from the same scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof comprising a VHCDR3 from an scFv having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator. The present invention also encompasses vectors comprising the isolated nucleic acid molecule described above, including vectors comprising a nucleotide sequence which regulates the expression of the antibody or fragment thereof encoded by the above-described nucleic acid molecule. Additionally the present invention also encompasses host cells, including mammalian host cells, comprising the above-described nucleic acid molecule which is operably linked to a heterologous promoter, as well as host cells, including mammalian host cells, comprising the above-described vectors. Additionally, the present invention also provides a method for producing an antibody or fragment thereof comprising culturing the above-described host cells under conditions in which the nucleic acid molecule is expressed.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH domain encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH domain encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a CDR from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a CDR encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a CDR from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH CDR3 encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.
In specific embodiments, the present invention provides an antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator, said antibody or fragment thereof comprising an amino acid sequence of a VH CDR3 encoded by a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence encoding a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.
In specific embodiments, the present invention provides a method for detecting of aberrant expression of B Lymphocyte Stimulator, comprising:
assaying the level of B Lymphocyte Stimulator expression in cells or a tissue sample of an individual using one or more antibodies or fragments or variants thereof that immunospecifically bind B Lymphocyte Stimulator; and
comparing the level of B Lymphocyte Stimulator assayed in the cells or a tissue sample with a standard level of B Lymphocyte Stimulator or a level of B Lymphocyte Stimulator in cells or a tissue sample from an individual without aberrant B Lymphocyte Stimulator expression, wherein an increase or decrease in the assayed level of B Lymphocyte Stimulator or level in cells or a tissue sample from an individual without aberrant B Lymphocyte Stimulator expression compared to the standard level of B Lymphocyte Stimulator is indicative of aberrant expression.
In specific embodiments, the present invention provides a method for diagnosing a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising:
administering to a subject an effective amount of a labeled antibody or fragment thereof that immunospecifically binds to B Lymphocyte Stimulator;
waiting for a time interval following the administering for permitting the labeled antibody or fragment thereof to preferentially concentrate at sites in the subject where B Lymphocyte Stimulator is expressed;
determining background level; and
detecting the labeled antibody or fragment thereof in the subject, such that detection of labeled antibody or fragment thereof above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of B Lymphocyte Stimulator.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above comprises a VH CDR3 having an amino acid sequence of one of SEQ ID NOS: 2129 to 3227.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is 251I, 131I, 111In, 90Y or 99Tc.
In specific embodiments, the antibody or fragment thereof utilized in the two methods described immediately above is conjugated to a diagnostic agent wherein the diagnostic agent is luciferase, luciferin or aequorin.
A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising at least one antibody or fragment thereof of comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier.
A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.
A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.
A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition comprising at least one antibody or fragment thereof of comprising a VL domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128, wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and which also comprises a VH domain from an scFv having an amino acid sequence of one of SEQ ID NOS: 1 to 2128 and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.
A method of treating, preventing or ameliorating a disease or disorder associated with aberrant B Lymphocyte Stimulator expression or activity, comprising administering to an animal in need thereof the pharmaceutical composition of comprising at least one antibody or fragment thereof of comprising an amino acid sequence of one of SEQ ID NOS: 2129 to 3227 wherein said antibody or fragment thereof immunospecifically binds B Lymphocyte Stimulator and a pharmaceutically acceptable carrier in an amount effective to treat, prevent or ameliorate the disease or disorder. This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.
This method may be used to treat an infectious disorder, cancer, and/or an autoimmune disease such as lupus or glomerular nephritis.
EXAMPLES
Abbreviations


    0.2 M Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose (TES)
    1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
    2TY supplemented with 100 μg/ml ampicillin and 2% glucose (2TYAG)
    2TY supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin (2TYAK)
    3,3′,5,5′-Tetramethyl Benzidine (TMB)
    50% inhibitory concentration (IC50)
    6×PBS containing 18% Marvel blocking solution (6×MPBS)
    Absorbance (A)
    Bovine serum albumin (BSA)
    Enzyme linked immunosorbent assay (ELISA)
    Foetal calf serum (FCS)
    Heavy chain variable (VH)
    Hepes buffered saline (HBS)
    Horseradish peroxidase (HRP)
    Immobilised Metal Affinity Chromatography (IMAC)
    Isopropyl β-D-thiogalactopyranoside (IPTG)
    Light chain variable (VL)
    Multiplicity of infection (MOI)
    N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (Hepes)
    Nanomolar (nM)
    N-Hydroxysuccinimide (NHS)
    PBS containing 3% Marvel (MPBS)
    Phosphate Buffered Saline (PBS)
    Phosphate Buffered Saline+0.1% (v/v) Tween 20 (PBST)
    Picomolar (pM)
    Single chain fragment variable (scFv)
    Tumour Necrosis Factor-alpha (TNF-α)
    Tumour Necrosis Factor-beta (TNF-β)
    TNF-related apoptosis inducing ligand (TRAIL)


DEFINITIONS
In the following section “immobilized B Lymphocyte Stimulator” refers to a soluble form of B Lymphocyte Stimulator or biotinylated B Lymphocyte Stimulator coated on a plastic assay plate (e.g., a 96 well plate), but does not refer to histidine tagged B Lymphocyte Stimulator coated on a plastic assay plate; “biotinylated B Lymphocyte Stimulator” is a soluble form of B Lymphocyte Stimulator except when used to coat an ELISA plate, in which case it would be “immobilized B Lymphocyte Stimulator.” Membrane bound forms of B Lymphocyte Stimulator include, but are not limited to, U937 and P388 plasma membranes. 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.
Example 1
Antibodies Immunospecifically Binding to Soluble And Membrane-Bound B Lymphocyte Stimulator
A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator. Phage displaying scFvs that bound to immobilized B Lymphocyte Stimulator were identified after panning on immobilized B Lymphocyte Stimulator and assessment by ELISA for binding to immobilized B Lymphocyte Stimulator. The B Lymphocyte Stimulator that was immobilized on plates for these assays was purified from supernatants of Sf9 cells infected with a baculovirus expression construct as described in Moore et al., Science 285:260-263 which is hereby incorporated by reference in its entirety. Each of the identified scFvs were then sequenced. Certain sequences were isolated multiple times, thus a panel (panel 1) containing one member of each unique sequences was generated and further characterized for their ability to immunospecifically bind to the soluble and membrane-bound forms of B Lymphocyte Stimulator.
The derived amino acid sequences of these scFvs are shown in Table 1 above. The individual VH and VL segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.
Example 2
Specificity of scFvs for B Lymphocyte Stimulator and Membrane-Bound B Lymphocyte Stimulator
The specificity of each of the scFvs for both B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator was determined by phage ELISA. B Lymphocyte Stimulator was immobilised onto plastic as a purified soluble form of the protein or as a membrane-bound form present on plasma membrane preparations from the human macrophage-like cell line, U937.
Maintenance of U937 Cells
U937 cells are a human monocyte-like, histiocytic lymphoma cell line known to express B Lymphocyte Stimulator on their plasma membranes. They were maintained in RPMI-L 640 supplemented with 4 mM L-glutamine, 10% FCS, 10 U penicillin, 100 g/ml streptomycin (all reagents from Sigma). The cells were thawed from frozen stock and are either used for plasma membrane preparation, or split 1:5, after 2 days in culture when the cell density reaches 1×106/ml.
Preparation of U937 Plasma Membranes
To prepare plasma membranes, 1×109 U937 cells were harvested from their culture medium by centrifugation at 1000 rpm at 4° C. for 5 minutes in a benchtop centrifuge. The cells were resuspended in 40 ml 12 mM Tris, pH 7.5, 250 mM sucrose and placed on ice. The cells are then lysed using a hand-held electric homogenizer (Labortechnik IKA Ultra-Turrax) for four, one minute, bursts. To check that cell lysis had occurred, 10 μl cell lysate was added to 10 μl Trypan blue and the cell lysate was examined under a microscope. After confirming lysis, the homogenate was centrifuged at 270×g, for 10 minutes at 4° C. to pellet the nuclear fraction and the supernatant was retained. The supernatant was centrifuged at 8000×g, 10 mins, 4° C., to pellet the mitochondrial and lysosomal fractions and the supernatant was retained. The supernatant was then centrifuged at 100000×g, 60 mins, 4° C. to pellet the plasma membrane enriched fraction. The supernatant was discarded and the plasma membrane pellet was resuspended in 1 ml PBS and stored at −70° C. The protein concentration of the plasma membrane fraction was determined using a protein quantification kit (Biorad). Typical yields were between 5 and 10 mg of plasma membranes.
Phage ELISA
To determine the specificity of each of the unique scFvs, a phage ELISA was performed for each scFv against human B Lymphocyte Stimulator, U937 plasma membranes, TNFα (R&D Systems, Minneapolis, Minn.), BSA and uncoated well. Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 1 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Plates were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each well to a MOI of 10 and the plates were incubated for a further 1 hour at 37° C. The plates were centrifuged in a benchtop centrifuge at 2000 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 100 μl 2TYAK and incubated at 30° C. overnight, shaking. The next day, plates were centrifuged at 2000 rpm for 10 min and the 100 μl phage-containing supernatant from each well carefully transferred into a fresh 96-well plate. Twenty μl of 6×MPBS was added to each well, and incubated at room temperature for 1 hour to pre-block the phage prior to ELISA.
Flexible 96-well plates (Falcon) were coated overnight at 4° C. with human B Lymphocyte Stimulator (1 μg/ml) in PBS, U937 plasma membranes (10 μg/ml) in PBS, TNFα (1 μg/ml) in PBS, BSA (1 μg/ml) in PBS, or PBS, After coating, the solutions were removed from the wells, and the plates were blocked for 1 hour at room temperature in MPBS, The plates were washed 3 times with PBS and then 50 μl of pre-blocked phage was added to each well. The plates were incubated at room temperature for 1 hour and then washed with 3 changes of PBST followed by 3 changes of PBS. To each well, 50 μl of an anti-gene VIII-HRP conjugate (Pharmacia) at a 1 to 5000 dilution in MPBS was added and the plates incubated at room temperature for 1 hour. Each plate was washed three times with PBST followed by three times with PBS, Then 50 μl of an HRP-labelled anti-mouse polymer (DAKO EnVision) diluted 1/50 in 3% MPBS was added and incubated for 1 hour at room temperature. Each plate was then washed three times with PBST followed by three times with PBS, Fifty μl of TMB substrate was then added to each well, and incubated at room temperature for 30 minutes or until colour development. The reaction was stopped by the addition of 25 μl of 0.5 M H2SO4. The signal generated was measured by reading the absorbance at 450 nm (A450) using a microtiter plate reader (Bio-Rad 3550).
The results for 3 clones (I006E07, I008D05 and I016F04) are shown in FIG. 1. All 3 scFvs recognize immobilized B Lymphocyte Stimulator and U937 plasma membranes but do not recognize TNFα, BSA or an uncoated well (PBS only). These results indicate that these scFvs specifically recognize immobilized B Lymphocyte Stimulator and membrane-bound B Lymphocyte Stimulator.
Example 3
Inhibition in an In Vitro Receptor Binding Assay by Phage ScFvs
All of the unique phage scFvs in panel 1 were assessed for their ability to inhibit soluble B Lymphocyte Stimulator binding to its cognate receptor on IM9 cells.
Biotinylation of B Lymphocyte Stimulator
One hundred μg of either human or mouse B Lymphocyte Stimulator was dialysed overnight at 4° C. against 50 mM sodium bicarbonate (sodium hydrogen carbonate) pH8.5 using a slide-a-lyzer cassette (Pierce). The next day, NHS-biotin (Pierce) was dissolved in DMSO to 13.3 mg/ml. This was then added to the B Lymphocyte Stimulator at a molar ratio of 20:1 biotin:B Lymphocyte Stimulator, mixed and incubated on ice for 2 hours. The biotinylated B Lymphocyte Stimulator was then dialysed back into sterile PBS (Sigma) using a slide-a-lyzer cassette overnight at 4° C. The biological activity of the biotinylated B Lymphocyte Stimulator was confirmed using the receptor binding inhibition assay (see below).
Maintenance of IM9 cells
IM9 cells are a human B lymphocyte cell line. They were maintained in RPMI-1640 supplemented with 4 mM L-glutamine, 10% FCS, 10 U penicillin, 100 g/ml streptomycin (all reagents from Sigma). The cells are thawed from frozen stock and can be used in assays after 5 days in culture when they reach a density of 4-8×105/ml.
Receptor Binding Inhibition Assay
Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 1 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Plates were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each well to a MOI of 10 and the plates were incubated for a further 1 hour at 37° C. The plates were centrifuged in a benchtop centrifuge at 2000 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 100 μl 2TYAK and incubated at 30° C. overnight, shaking. The next day, plates were centrifuged at 2000 rpm for 10 min and the 100 μl phage-containing supernatant from each well carefully transferred into a fresh 96-well plate. Phage were diluted 1 in 2 in MPBS prior to use.
Flat-bottomed 96-well plates (Costar) were coated with 100 μl per well of a 1:10 dilution of poly-L-lysine (Sigma) in PBS for 1 hour at room temperature. The plates were then washed twice with water, allowed to air-dry and placed at 4° C. overnight. One hundred μl of IM9 cells (at 106/ml in RPMI-1640 culture medium) were then added to each well. Plates were then centrifuged at 3200 rpm for 5 mins to pellet the cells. The media was carefully aspirated and 200 μl of MPBS added to each well. The plates were then allowed to block for 1 hour at room temperature.
To a separate 96-well plate 10 μl of biotinylated B Lymphocyte Stimulator (at 162.5 ng/ml) in MPBS was added to each well to give a final concentration of 25 ng/ml. Fifty-five μl of each appropriate phage supernatant was added to each well and the final volume in each well was 65 μl. Plates were then incubated at room temperature for 30 minutes.
The IM9 coated plates were washed twice in PBS, tapped dry and immediately 50 μl of the phage/biotinylated-B Lymphocyte Stimulator mix was added and incubated at room temperature for 1 hour. Plates were washed three times in PBST and three times in PBS, tapped dry and 50 μl of streptavidin-Delfia (Wallac) was added to each well at 1:1000 dilution in the Manufacturer's assay buffer. The plates were then incubated at room temperature for I hour and washed six times in Delfia wash solution (Wallac). After tapping the plates dry, 100 μl per well of Delfia enhancement solution (Wallac) was added. The plates were gently tapped to encourage micelle formation, incubated at room temperature for 10 minutes, and fluorescence read on a Wallac 1420 workstation at 6520 nM.
Results for 3 phage scFvs (I001C09, I018D07 and I016H07) that inhibited the binding of biotinylated B Lymphocyte Stimulator are shown in FIG. 2. Maximal binding of biotinylated B Lymphocyte Stimulator to its receptor (bio-B Lymphocyte Stimulator only), the background signal in the absence of biotinylated B Lymphocyte Stimulator (no bio-B Lymphocyte Stimulator), and results with an irrelevant (i.e., does not recognize B Lymphocyte Stimulator) phage antibody are also shown. All 3 phage scFvs inhibited biotinylated B Lymphocyte Stimulator binding to its receptor on IM9 cells, identifying these scFvs as scFvs that bind the soluble form of B Lymphocyte Stimulator. These scFvs also bind to U937 membranes, thus they also bind the membrane bound form of B Lymphocyte Stimulator.
Forty-eight of the scFvs from panel I that demonstrated the greatest inhibition as phage particles in this assay were chosen for further study. These 48 scFvs are listed in Table 3.







TABLE 3







scFvs that Inhibit the Binding of Biotinylated-B


Lymphocyte Stimulator to its Receptor











Antibody
Antibody
Antibody
Antibody
Antibody





I008C02
I029D07
I008C03
I008C12
I028A06


I022E02
I061E07
I007H08
I061H01
I031C03


I018C02
I006D07
I008A11
I006D08
I031F02


I008B01
I017D10
I061D02
I026E03
I031F09


I016F04
I007B03
I008A09
I027A07
I031G11


I016E05
I018C10
I007F11
I016H07
I050A07


I018H08
I001C09
I037E07
I021B05
1050A12


I018H09
I018D07
I037E12
I031G10
I050B11



I029F11
I016F02
I031G08
I051C04



I022D01

I031C07
I003F12





I012A06









Example 4
Specificity of Anti-B Lymphocyte Stimulator Antibodies
The specificity of the 48 scFvs listed in Table 3 for human and murine B Lymphocyte Stimulator was determined using phage ELISA.
Phage ELISA
To determine the specificity of the 48 scFvs, a phage ELISA was performed against human and mouse B Lymphocyte Stimulator, and a panel of related and unrelated human antigens: Fas ligand, TRAIL, TNFα, TNFβ, and PBS, The: Fas ligand, TRAIL, TNFα, and TNFβ antigens were obtained from R&D Systems, Minneapolis, Minn., Individual E. coli colonies containing phagemid were inoculated into 5 ml 2YTAG and incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage (Pharmacia) was added to each tube to a MOI of 10 and incubated for 30 minutes at 37° C. for 1 hour, the first 30 minutes static and the final 30 minutes with gentle shaking. Cells were pelleted by centrifugation at 3,500 rpm for 10 minutes and the supernatant discarded. Cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight with shaking. The next day, the cells were pelleted by centrifugation at 3,500 rpm for 10 minutes. The phage-containing supernatant (5 ml) was carefully transferred to a fresh tube, 1 ml of 6MPBS was added, and the tube was incubated at room temperature for 1 hour to pre-block the phage prior to ELISA.
All antigens were coated at 1 μg/ml. ELISAs were performed essentially as described in Example 2. The only exception to this being the detection of phage antibody binding to mouse B Lymphocyte Stimulator where the step involving incubation with the HRP-labelled anti-mouse polymer was omitted. Binding to mouse B Lymphocyte Stimulator was detected with TMB as in Example 2.
All 48 scFvs are specific for immobilized human B Lymphocyte Stimulator and 43 out of the 48 scFvs cross-react with immobilized mouse B Lymphocyte Stimulator but not with any other unrelated or related antigen tested. I008C03, I007F11, I037E07, I037E12, and I016H07 did not bind murine B Lymphocyte Stimulator. Results for two scFvs, I022D01 and I031 F02, are shown in FIG. 3. Both these scFvs specifically recognize human and mouse B Lymphocyte Stimulator but not any other unrelated or related antigen tested.
Example 5
Specificity for the Membrane-Bound Form of B Lymphocyte Stimulator
The specificity of 48 scFvs for membrane-bound B Lymphocyte Stimulator was determined by the phage ELISA described in Example 2. B Lymphocyte Stimulator was immobilised onto plastic as a membrane-bound form present on plasma membranes preparations from the human macrophage-like cell line, U937. This cell line is known to express the membrane-bound form of human B Lymphocyte Stimulator.
To demonstrate that this binding is specific for membrane-bound B Lymphocyte Stimulator, a competition ELISA was developed to determine if the ELISA signal for an individual antibody on U937's could be competed out by pre-incubation with either B Lymphocyte Stimulator or TNFα. An anti-B Lymphocyte Stimulator antibody that also recognizes membrane-bound B Lymphocyte Stimulator would be expected to demonstrate a signal reduction with free B Lymphocyte Stimulator but not free TNFα.
Competition ELISA
Individual E. coil colonies containing phagemid for each of the 48 scFvs listed in Table 3 were inoculated into 5 ml 2YTAG and incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage (Pharmacia) was added to each tube to a MOI of 10 and incubated for 30 minutes at 37° C. for 1 hour, the first 30 minutes static and the final 30 minutes with gentle shaking. Cells were pelleted by centrifugation at 3,500 rpm for 10 minutes and the supernatant discarded. Cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight with shaking. The next day, the cells were pelleted by centrifugation at 3,500 rpm for 10 minutes. The phage-containing supernatants (5 ml) were carefully transferred to a fresh tube.
For each of the 48 scFvs listed in Table 3, two aliquots of 20 μl 6×MPBS were pipetted into separate wells of a 96-well plate (Greiner). The first aliquot was supplemented with B Lymphocyte Stimulator to a final concentration of 0.5 μg/ml. The second aliquot was supplemented with TNF-α to a final concentration of 0.5 μg/ml. Each experiment was performed in triplicate. One hundred μl of each phage supernatant was then added to each aliquot and mixed by pipetting up and down. The phage were incubated (±competing antigen) at room temperature for 1 hour.
Flexible 96-well plates (Falcon) were coated overnight at 4° C. with 50 μl of 10 μg/ml U937 plasma membranes. After coating, the plates were washed 3 times with PBS and blocked for 1 hour at room temperature with 200 μl MPBS, The plates were washed 3 times with PBS and 50 μl of phage (±competing antigen) was added to each appropriate well. The plates were incubated at room temperature for 1 hour and then washed with 3 changes of PBST followed by 3 changes of PBS, To each well, 50 μl of a mouse anti-gene VIII-HRP conjugate (Pharmacia) at a 1:5000 dilution in MPBS was added and the plates incubated at room temperature for 1 hour. Each plate was washed three times with PBST followed by three times with PBS, Then 50 μl of an HRP-labelled anti-mouse polymer (DAKO EnVision) diluted 1:50 in 3% MPBS was added and incubated for 1 hour at room temperature. Each plate was then washed three times with PBST followed by three times with PBS, Fifty μl of TMB substrate was then added to each well, and incubated at room temperature for 30 to 60 minutes or until color development. The reaction was stopped by the addition of 25 μl of 0.5 M H2SO4. The signal generated was measured by reading the absorbance at 450 nm (A450) using a microtiter plate reader (Bio-Rad 3550).
All 48 scFvs bind to U937 plasma membrane preparations. This signal could be competed out by pre-incubation of the phage antibody with B Lymphocyte Stimulator but not by pre-incubation with TNF-A. This indicates that the 48 scFvs specifically recognize membrane-bound B Lymphocyte Stimulator as well as soluble B Lymphocyte Stimulator. Typical results are exemplified by scFvs 1031F09, 1050A12 and 1051C04 and are shown in FIG. 4. All 3 scFvs demonstrate binding to U937 plasma membranes. This binding was specifically competed out with B Lymphocyte Stimulator but did not compete with TNF-α, demonstrating specific recognition of membrane-bound B Lymphocyte Stimulator.
Example 6
scFv Off-Rate Determinations
All off-rate determinations were performed on BIAcore 2000 machines, using the BIAcore 2000 Control Software and evaluated using the BIAevaluation 3.0 software.
Preparation of a Low Density B Lymphocyte Stimulator Surface
A 500RU surface was prepared for kinetic studies with purified scFvs. A low density B Lymphocyte Stimulator surface (500 RU B Lymphocyte Stimulator coupled) was prepared in flow cell 2 by amine coupling to a CM5 chip. A new CM5 chip was inserted into the BIAcore and a sensorgram initiated with HBS buffer at a flow rate of 5 μl/min. The NHS and EDC coupling solutions (BIAcore) were mixed according to manufacturer's instructions and 30 μl injected over the CM5 surface. Fifty μl of B Lymphocyte Stimulator at 1 μg/ml in 10 mM sodium acetate buffer, pH4, was then injected followed by 30 μl of ethanolamine-HCl solution (BIAcore). The flow rate was then adjusted to 20 μl/min and 10 μl of 4M guanidine hydrochloride in HBS injected over the surface. This strips the surface of non-covalently bound B Lymphocyte Stimulator.
Measurement of scFv Off-rate Kinetics on the Low Density Surfaces
The chip containing the low density B Lymphocyte Stimulator surface was inserted in to the BIAcore. A dilution series of purified scFvs was prepared in HBS, typically 50 μg/ml doubling dilutions down to 1.5 μg/ml. The dilution series was then injected sequentially over the low density B Lymphocyte Stimulator surface (and blank control) using the following program:















MAIN



FLOWCELL 1,2,3,4












APROG
genab
r1d1
ab1



APROG
genab
r1d2
ab2



APROG
genab
r1d3
ab3



APROG
genab
r1d4
ab4



APROG
genab
r1d5
ab5



APROG
genab
r1d6
ab6









APPEND CONTINUE



END



DEFINE APROG genab



PARAM %Abpos %AbId










FLOW
20









KINJECT %Abpos 200 80










INJECT
r1c6 10!guanidine hydrochloride regeneration step









EXTRACLEAN



END










Bound scFvs were removed by injecting 10 μl 4M GuHCl in HBS over the surface between scFv samples.
The binding curves for individual scFvs were analyzed using the BIAevaluation software to determine antibody off-rates. Kinetic analysis for a typical scFv antibody, I003C02, is shown in FIG. 5. I003C02 has a Koff=6×10−3s−1.
Example 7
Inhibition in an In Vitro Receptor Binding Assay by scFv Antibodies
The 48 scFvs listed in Table 3 were purified and assessed for their ability to inhibit B Lymphocyte Stimulator binding to its receptor on IM9 cells.
Purification of scFv
To determine the inhibitory potency of anti-B Lymphocyte Stimulator scFv, scFv's were first prepared by IMAC. 2TYAG (5 ml) was inoculated with a single colony and grown overnight at 30° C., shaking. This overnight culture was then used to inoculate 500 ml of 2TY containing 100 μg/ml ampicillin and 0.1% Glucose, and grown at 30° C., shaking, until an A600 of 1.0 was attained. IPTG was added to 1 mM and the culture was grown for a further 3.5 hours at 30° C.
Cells were harvested by centrifugation at 5,000 rpm, and resuspended in 10 ml of TES, A further 15 ml of a 1:5 dilution (in water) of TES was added, and the cell suspension incubated on a turning wheel at 4° C. for 30 minutes. This causes osmotic shock and yields a periplasmic extract containing the scFv. Residual cells and debris were pelleted by centrifugation at 9,000 rpm for 20 minutes at 4° C. The supernatant was transferred to a new tube, and 50 μl of 1 M MgCl2 added. Two ml of a Ni-NTA agarose (Qiagen), pre-washed with buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl) together with a protease inhibitor tablet (Boehringer Mannheim) were then added to the periplasmic extract. The preparation was incubated, rotating, overnight at 4° C. The Ni-NTA was pelleted by centrifugation at 2,000 rpm for 5 minutes, and the supernatant was aspirated. The agarose beads were washed 3 times with 50 ml wash buffer, centrifuging to collect the agarose in between each wash. Ten ml of wash buffer was added after the final wash, and the slurry was loaded on to a polyprep column (BioRad). Two ml elution buffer (50 mM NaPi (sodium phosphate), pH 8, 300 mM NaCl, 250 mM imidazole) was added to the drained agarose, and the eluate was collected. IMAC purified scFv was buffer exchanged in to PBS by use of a Nap 5 column (Phamiacia) according to the manufacturer's instructions. The A280 was read and the protein concentration determined using a molar extinction coefficient of 1 mg/ml protein=A280 1.4. Purified scFv was stored in 500 μl aliquots at −70° C.
Receptor Binding Inhibition Assay
Flat-bottomed 96-well plates (Costar) were coated with 100 μl per well of a 1:10 dilution of poly-L-lysine (Sigma) in PBS for 1 hour at room temperature. The plates were then washed twice with water, allowed to air-dry and placed at 4° C. overnight. One hundred μl of IM9 cells (at 106/ml in RPMI-1640) were then added to each well. Plates were then centrifuged at 3200 rpm for 5 mins to pellet the cells. The media was carefully aspirated and 200 μl of MPBS added to each well. The plates were then left to block for 1 hour at room temperature.
To a separate 96-well plate, titrate test scFvs in MPBS, in triplicate, over a concentration range from 10 μg/ml down to 0.001 μg/ml were added. The final volume of test scFv in each well was 55 μl. Competition with unlabelled B Lymphocyte Stimulator was also included in every assay as a control. Unlabelled B Lymphocyte Stimulator, in MPBS, was typically titrated in triplicate, over a concentration range from 1 μg/ml down to 0.001 μg/ml. 10 μl of biotinylated-B Lymphocyte Stimulator (at 162.5 ng/ml) in MPBS was added to each well to give a final concentration of 25 ng/ml. Plates were then incubated at room temperature for 30 minutes.
The IM9 coated plates was washed twice in PBS, tapped dry and immediately 50 μl of the scFv/biotinylated-B Lymphocyte Stimulator mix was added and incubated at room temperature for 1 hour. Plates were washed three times in PBST and three times in PBS, tapped dry and 50 μl per well added of streptavidin-Delfia (Wallac) at 1:1000 dilution in the Manufacturer's assay buffer. The plates were then incubated at room temperature for 1 hour and washed six times in Delfia wash solution (Wallac). After tapping the plates dry, 100 μl per well of Delfia enhancement solution (Wallac) was added. The plates were gently tapped to encourage micelle formation, incubated at room temperature for 10 minutes, and fluorescence read on a Wallac 1420 workstation at 6520 nM.
Typical titration curves for two scFv antibodies, I007F11 and I050A07, are shown in FIG. 6. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an IC50 value of 0.8 nM. The IC50 values for I007F11 and I050A07 are 7.9 nM and 17.1 nM, respectively. The assay was performed in triplicate and standard error bars are shown. The 9 scFvs that demonstrated the greatest inhibition as scFv are listed in Table 4. This data also confirms that these 9 scFvs recognize the soluble form of B Lymphocyte Stimulator.







TABLE 4







9 ScFvs that demonstrated greatest potency in B Lymphocyte


Stimulator Receptor Binding Inhibition Assay









ScFv Antibody







I017D10



I022D01



I008A11



I006D08



I031F02



I050A12



I050B11



I051C04



I003F12S










Example 8
Antibodies Recognizing a Soluble Form of B Lymphocyte Stimulator
A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the soluble but not the membrane-bound forms of B Lymphocyte Stimulator.
A phage library was screened for the ability to bind to biotinylated B Lymphocyte Stimulator. The phage were exposed to biotinylated B Lymphocyte Stimulator, allowed an interval of time to bind the biotinylated B Lymphocyte Stimulator. Phage binding bio-B Lymphocyte Stimulator were then isolated by capture on streptavidin coated magnetic beads.
The phage identified in the screen above (capture of Bio-B Lymphocyte Stimulator from solution) were then screened by ELISA for their ability to bind immobilized B Lymphocyte Stimulator. The scFv expressed by phage that bound immobilized B Lymphocyte Stimulator were then cloned and sequenced. Again, several sequences were identified multiple times, thus a panel (panel 2) consisting of on example of each phage expressing a unique scFv was then characterized further.
The derived amino acid sequences of these scFvs are shown in Table 1 above. The individual VH and VL segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.
Example 9
Specificity For Soluble B Lymphocyte Stimulator
The scFvs were isolated from a library of phage based on their ability to bind a soluble form of B Lymphocyte Stimulator. Briefly, phage were preincubated with biotinylated B Lymphocyte Stimulator in solution. Phage that bound to this biotinylated B Lymphocyte Stimulator were then isolated using streptavidin coated magnetic beads.
The specificity of each of the unique scFvs for B Lymphocyte Stimulator and for the membrane-bound form of B Lymphocyte Stimulator, was determined by phage ELISA. B Lymphocyte Stimulator was immobilised onto plastic as a purified soluble form of the protein or as a membrane-bound form present on plasma membrane preparations from the human macrophage-like cell line, U937. Maintenance of U937 cells and plasma membrane preparations were performed as detailed in Example 2.
Phage ELISA
To determine the specificity of each of the scFvs, a phage ELISA was performed for each antibody against human B Lymphocyte Stimulator, U937 plasma membranes, TNFα, BSA and an uncoated well. Antigen coating conditions were as described in Example 2, apart from human B Lymphocyte Stimulator. B Lymphocyte Stimulator was first biotinylated (as described in Example 3) and coated at 1 μg/ml onto streptavidin coated plates (Reacti-Bind, Pierce) for 30 mins at room temperature. The plates were then washed, blocked and the phage ELISA performed as detailed in Example 2.
The results for 3 clones (I074B12, I075F12 and I075A02) that bind the soluble but not the membrane-bound form of B Lymphocyte Stimulator are shown in FIG. 7. As a control, a phage antibody that recognizes TNFα, is also shown in FIG. 7. There is a small non-specific background signal on the U937 plasma membranes that is evident with both the anti-B Lymphocyte Stimulator scFvs as well as the anti-TNFα control. All 3 anti-B Lymphocyte Stimulator scFvs recognize B Lymphocyte Stimulator but not U937 plasma membranes, TNFα, BSA or an uncoated well (PBS only). This indicates that the scFvs do not bind the membrane-bound form of B Lymphocyte Stimulator. Further, The fact that these scFvs were isolated on the basis of their ability to bind soluble biotinylated B Lymphocyte Stimulator indicates that they bind the soluble form of B Lymphocyte Stimulator. Further confirmation of these scFvs′ specificity for B Lymphocyte Stimulator is provided in Example 10.
Example 10
Inhibition in an in vitro Receptor Binding Assay by Phage scFvs
All of the unique phage scFvs from panel 2 were assessed for their ability to inhibit B Lymphocyte Stimulator binding to its cognate receptor on IM9 cells. The biotinylation of B Lymphocyte Stimulator, maintenance of IM9 cells and receptor binding inhibition assay were performed as described in Example 3.
Results for two phage scFvs, I025B09 and I026C04 are shown in FIG. 8. Maximal binding of biotinylated B Lymphocyte Stimulator to its receptor (bio-B Lymphocyte Stimulator only), the background signal in the absence of biotinylated B Lymphocyte Stimulator (no bio-B Lymphocyte Stimulator), and results with an irrelevant (i.e. does not recognize B Lymphocyte Stimulator) phage antibody are also shown. Both phage scFvs inhibited biotinylated B Lymphocyte Stimulator binding to its receptor on IM9 cells. 33 of the unique scFvs from panel 2 were identified for further study. These 33 scFvs demonstrated the greatest inhibition as phage particles in this assay and are listed in Table 5.







TABLE 5







Identification of 33 phage scFvs to free B Lymphocyte Stimulator


that demonstrate the most significant inhibition of biotinylated-B


Lymphocyte Stimulator binding to its receptor












Antibody
Antibody
Antibody
Antibody







I026C04
I074B12
I073F04
I065D04



I003C06
I075A02
I078D08
I068C08



I025B09
I068B08
I078D02
I068F03



I027B12
I068B04
I075G01
I069B07



I025B06
I068C06
I071B03



I030A10
I075F12
I072B09



I002A01R
I065D08
I078H08



I002A01K
I065F08
I064C04



I026C04R
I067B10
I064C07



I026C04K
I067F05










Example 11
Specificity of Anti-B Lymphocyte Stimulator scFvs
The specificity of the 33 scFvs (listed in Table 5) for immobilized human and murine B Lymphocyte Stimulator was determined using phage ELISA.
Phage ELISA
To determine the specificity of the 33 scFvs, a phage ELISA was performed as described in Example 4 against human and mouse B Lymphocyte Stimulator, and a panel of related human antigens: TRAIL, LIGHT, TNFα, TNFβ, and an uncoated well (PBS only).
Typical results for two scFvs, I067F05 and I078D02 are shown in FIG. 9. A control antibody that specifically recognizes TNFα is also shown. Both anti-B Lymphocyte Stimulator scFvs specifically recognize immobilized human and mouse B Lymphocyte Stimulator but not any other antigen tested.
All 33 scFvs are specific for human B Lymphocyte Stimulator. 14/33 cross-react with mouse B Lymphocyte Stimulator but not with any other unrelated or related antigen tested.
Example 12
scFv Off-Rate Determinations
Off-rate determinations, preparation of a low density B Lymphocyte Stimulator surface and kinetic measurements were as detailed in Example 6.
The binding curves for individual scFvs were analysed using the BIAevaluation software to determine antibody off-rates. Kinetic analysis for a typical scFv antibody, I002A01, is shown in FIG. 10. I002A01 has a Koff=9×10−4s−1.
Example 13
Inhibition in an In Vitro Receptor Binding Assay by scFv Antibodies
The 33 scFvs identified in Table 5 were prepared as purified scFvs and assessed for their ability to inhibit B Lymphocyte Stimulator binding to its receptor on IM9 cells. The scFvs were purified and analysed in the receptor binding inhibition assay as described in Example 6.1.8.
Typical titration curves for two scFvs, I0068C06 and I074B12, are shown in FIG. 11. Unlabelled B Lymphocyte Stimulator competed for binding to its receptor with an inhibitory constant 50 (IC50) value of 0.66 nM. The IC50 values for I0068C06 and I074B 12 are 61 nM and 13 nM, respectively. The assay was performed in triplicate and standard error bars are shown. The 7 scFvs that demonstrated the greatest inhibition as scFv are listed in Table 6.







TABLE 6







Identification of 7 scFvs to free B Lymphocyte Stimulator that


demonstrate the most significant inhibition of biotinylated-B


Lymphocyte Stimulator binding to its receptor as purified scFv's.









Antibody







I002A01-R



I002A01-K



I026C04-R



I026C04-K



I068C06



I075F12



I067B10










Example 14
ScFvs Recognizing Membrane-Bound B Lymphocyte Stimulator
A library of phage was screened in an assay to identify those phage displaying scFvs that immunospecifically bind to the membrane-bound but not the soluble form of B Lymphocyte Stimulator.
As a starting point, a library of phage expressing scFv antibodies were panned on immobilized HIS-tagged B Lymphocyte Stimulator. Phage isolated by panning were then screened for the ability to bind to HIS-tagged B Lymphocyte Stimulator. HIS-tagged B Lymphocyte Stimulator was obtained by expressing amino acids 71-285 of SEQ ID NO:3228 using the pQE9 vector (Qiagen Inc., Valencia, Calif.) in E. coli and purifying the expressed protein. This phage clones identified by this screen were then sequenced. After sequencing, A panel (panel 3) of phage each expressing a unique scFv that bound HIS-tagged B Lymphocyte Stimulator was generated and further characterized.
The derived amino acid sequences of the unique scFvs from panel 3 are shown in Table 1 above. The individual VH and VL segments of the scFvs were aligned to the known human germline sequences in V-BASE (Tomlinson et al, which can accessed on the United Kingdom Medical Research Council (MRC) Centre for Protein Engineering website) and the closest germline identified.
Example 15
Recognition of Membrane-bound B Lymphocyte Stimulator
The specificity of each of the unique scFvs for both the membrane-bound form of B Lymphocyte Stimulator as well as for the soluble form of B Lymphocyte Stimulator, was determined by phage ELISA.
B Lymphocyte Stimulator was immobilised onto plastic either directly as a purified soluble form of the protein or biotinylated and coated on a streptavidin plate as in Example 9. Binding to HIS-tagged B Lymphocyte Stimulator was used as a primary screen for scFv's that would bind the membrane-bound form of B Lymphocyte Stimulator (see below). The membrane-bound form of B Lymphocyte Stimulator was presented as plasma membranes preparations from the human macrophage-like cell line, U937 or the murine cell line P388.
Mouse monoclonal antibodies have been raised against His-tagged B Lymphocyte Stimulator according to standard procedures. Characterization of these mouse monoclonal antibodies revealed that they specifically recognized both His-tagged B Lymphocyte Stimulator and the membrane-bound form of B Lymphocyte Stimulator on U937 cells, but not soluble B Lymphocyte Stimulator. Therefore, specific recognition of His-tagged B Lymphocyte Stimulator was used as supporting evidence for the recognition of the membrane-bound form of B Lymphocyte Stimulator by phage and scFv antibodies.
Phage ELISA
To determine the specificity of each of the scFvs, a phage ELISA was performed for each antibody against His-tagged human B Lymphocyte Stimulator, U937 plasma membranes, TNFα, BSA and an uncoated well. Antigen coating conditions were as described in 2. apart from human B Lymphocyte Stimulator. B Lymphocyte Stimulator was first biotinylated (as described in Example 3) and coated at 1 μg/ml onto streptavidin coated plates (Reacti-Bind, Pierce) for 30 mins at room temperature. The plates were then washed, blocked and the phage ELISA performed as detailed in Example 2.
The results for 3 clones, I079C01, I081C10 and I082A02, and a control phage antibody that recognizes TNFα, are shown in FIG. 12. All 3 scFvs recognize U937 plasma membranes (U937) and His-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not, biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator) or an uncoated well (PBS). This indicates that the scFvs recognize the membrane-bound form of B Lymphocyte Stimulator.
Example 16
Specificity for Membrane-Bound B Lymphocyte Stimulator
The specificity of the scFvs for only the membrane-bound form of B Lymphocyte Stimulator, and not for the soluble form, was confirmed using a competition ELISA. This assay assesses the ability of test phage scFvs to bind to the membrane-bound form of B Lymphocyte Stimulator on U937 plasma membranes in the presence of different forms of competing B Lymphocyte Stimulator. Competing B Lymphocyte Stimulator was either the His-tagged form of B Lymphocyte Stimulator or soluble B Lymphocyte Stimulator. ScFvs specific for the membrane-bound B Lymphocyte Stimulator would be expected to be competed out by pre-incubation with His-tagged B Lymphocyte Stimulator but not by pre-incubation with soluble B Lymphocyte Stimulator.
Maintenance of U937 cells and plasma membrane preparations were performed as detailed in Example 2.
Competition ELISA
U937 plasma membranes (50 μl per well) were coated at 10 μg/ml in PBS onto Falcon 96-well plates overnight at 4° C.
Individual E. coli colonies containing a phagemid representing one of the unique scFvs from the panel 3 were inoculated into 50 ml tubes (Falcon) containing 5 ml 2TYAG medium. Tubes were incubated at 37° C. for 4 hours, shaking. M13KO7 helper phage was added to each tube to an MOI of 10 and the tubes were incubated for a further 1 hour at 37° C. The tubes were centrifuged in a benchtop centrifuge at 3500 rpm for 10 minutes. The supernatant was removed and cell pellets were resuspended in 5 ml 2TYAK and incubated at 30° C. overnight, shaking. The next day, tubes were centrifuged at 3500 rpm for 10 min and the phage-containing supernatant carefully transferred into a fresh tube.
For each test phage antibody, 3 aliquots of 20 μl 18% marvel/6×PBS were transferred into separate wells of a 96-well plate. The first aliquot was supplemented with His-tagged B Lymphocyte Stimulator to a final concentration of 60 μg/ml. The second aliquot was supplemented with soluble B Lymphocyte Stimulator to a final concentration of 60 μg/ml. The third aliquot was not supplemented with any competing antigen. One hundred μl of phage supernatant was then added to each aliquot and left to block at room temperature for 1 hour.
The antigen-coated plates were washed once with PBS before the addition of 200 μl/well 3% marvel/PBS, These plates were left to block at 37° C. for 1 hour and were then washed once with PBS, Duplicate samples of 50 μl pre-blocked phage (above) were added to the antigen-coated plates and left at room temperature for 1 hour. Plates were washed 3× with PBS/0.1% Tween 20, then 3× with PBS, Fifty μl/well mouse anti-M13 HRP (Pharmacia) at 1/5000 in 3% Marvel/PBS was added and left for 1 hour at room temperature. Plates were washed 3 times with PBS/0.1% Tween 20, then 3 times with PBS, Fifty μl/well HRP-labelled anti-mouse Envision polymer (DAKO) at 1/50 in 3% marvel/PBS was added and left for I hour at RT, Plates were washed 3 times with PBS/0.1% Tween 20, then 3 times with PBS, Next, 50 μl/well of TMB (Sigma) was added and plates left to develop for 30 to 60 minutes. When sufficient color has developed, 25 μl/well 0.5M H2SO4 was added to stop the reaction. The plates were read at 450 nm on a microtiter plate reader (Bio-Rad 3550).
The results for 3 clones, I079B04, I079F08 and I080B01, and a control phage antibody that recognizes TNFα, are shown in FIG. 13. All 3 scFvs recognize U937 plasma membranes (U937). This binding is competed out to background levels (i.e. comparable to the signal observed with the anti-TNFα phage antibody) in the presence of His-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator). This confirms that the scFvs specifically recognize the membrane-bound form but not the soluble form of B Lymphocyte Stimulator.
Example 17
High Throughput BIAcore Screen to Identify High Affinity scFvs
This is a 96-well screen where the test samples (scFvs) are derived from 1 ml periplasmic extracts of individual antibody expressing clones. Potentially higher affinity scFvs are then identified principally as those giving a large number of total RU's bound to a HIS-B Lymphocyte Stimulator surface in BIAcore. This method of ranking does assume approximately equal yields of scFv from each clone. Since this is not always the case, some scFvs may also be identified that simply express high levels of scFv. These can be discriminated from those of higher affinity by further characterization of the scFvs (see Example 18).
Preparation of ScFv from 1 ml E. coli Cultures
Individual E. coli colonies containing a phagemid representing one of the unique scFvs from panel 3 were inoculated into 96-well plates containing 100 μl 2TYAG medium per well. Eight wells on each plate were reserved for positive and negative control samples. The plate was grown overnight at 30° C. with shaking at 120 rpm.
Next day, 1 ml of 2TYAG+345 mM sucrose was added to each well of an autoclaved 96 deep well plate (Beckman). Twenty μl of each overnight culture was resuspended and transferred to the appropriate well of the deep well plate. The plate was grown for approximately 3.5 hours at 30° C. with shaking at 250 rpm (or until the OD600=0.6). Fifty μl of 1M IPTG was added to 5 ml 2TY and 10 μl of this was added to each well. The plate was grown overnight at 30° C. with shaking at 250 μm.
Plates were kept at 4° C. for the remainder of the procedure. The overnight plate (above) was centrifuged at 3500 rpm for 10 minutes at 4° C. to pellet the cells. The supernatant was decanted and each pellet resuspended in 100 μl TES (0.2M Tris HCl pH8.0, 0.5 mM EDTA, 0.5M sucrose) and transferred to a fresh 96 well plate. This plate was incubated on ice for 30 minutes and then centrifuged for 10 minutes at 3500 rpm at 4° C. to pellet the cell debris. During centrifugation, 15 μl of freshly made protease inhibitors cocktail (Roche, 1 tablet dissolved in 1.5 ml water) was added to each well of a fresh 96 well plate. Supernatants from the centrifuged plate were then transferred to the plate containing the protease inhibitors. The plate was centrifuged at 3500 rpm for 10 minutes at 4° C. and the supernatant was transferred to a further 96-well plate. This step was repeated at least once more or until there was no sign of any cell debris following centrifugation. Finally, the plate was covered in foil to prevent evaporation of samples during the BIAcore run.
Generation of a High Density HIS-B Lymphocyte Stimulator Surface
All BIAcore analysis was performed on BIAcore 2000 machines, using the BIAcore 2000 control software and evaluated using the BIAevaluation 3.0 software. A high density His-tagged B Lymphocyte Stimulator surface (>1000 RU HIS-B Lymphocyte Stimulator coupled) was prepared in flow cell 2 by amine coupling to a CM5 chip. A new CM5 chip was inserted into the BIAcore and a sensorgram started over flow cell 2 with HBS buffer at a flow rate of 5 μl/min. The NHS and EDC solution were mixed 1:1 before injecting 30 μl over the CM5 surface. Fifty μl HIS-B Lymphocyte Stimulator (at 10 μg/ml in Sodium acetate buffer, pH4) was injected and allowed to couple to the surface. Thirty μl of ethanolamine-HCl solution was then injected to block free NHS esters. Prior to using the chip, 10 μl of 4M Guanidine hydrochloride in HBS was injected over the surface to strip the surface of non-covalently bound B Lymphocyte Stimulator. A blank surface (no HIS-B Lymphocyte Stimulator) was also prepared over flow cell 1 so that non-specific binding effects can be subtracted from the HIS-B Lymphocyte Stimulator binding curves.
Typically, a 5000 RU His-tagged B Lymphocyte Stimulator surface was generated in this way and used for 96-well analysis of scFvs isolated from the periplasm of E. coli. 
BIAcore Analysis
The 96-well plate containing periplasmic scFvs was secured inside the BIAcore. Two ml of 4M Guanidine hydrochloride in HBS was placed in a rack inside the BIAcore for regeneration of the HIS-B Lymphocyte Stimulator surface between samples. The sensorgram was run over flow cells 1 and 2 at a flow rate of 20 μl minute. The following method was run:

    MAIN
    FLOWCELL 1,2,3,4
    LOOP cycle STEP
    APROG inj % pos
    ENDLOOP
    APPEND CONTINUE
    END
    DEFINE LOOP cycle
    LPARAM % pos
    r1a1
    r1b1
    r1c1
    r1d1
    r1e1
    r1f1 etc (all wells listed until r1h12)
    END
    DEFINE APROG inj
    PARAM % pos
    FLOW 20
    KINJECT % pos 35 30 IscFv injection
    QUICKINJECT r2f3 10 Iregeneration
    EXTRACLEAN
    END


When the run had finished, the sensorgram data for flow cell 1 was subtracted from the data for flow cell 2 for each sample using the BIAevaluation software. The clones were compared with one another principally by overall RU change as the scFv dissociates from the surface. In addition a few scFvs were identified as having potentially slower off-rates. An example of the dissociation section of a typical sensorgram for 8 scFvs is shown in FIG. 14. An anti-TNFα antibody that does not recognize B Lymphocyte Stimulator was included as a control. Of the 8 scFvs exemplified, I079F06 was identified for further study due to the relatively high numbers of RU's bound to the surface.
ScFvs were identified principally if they demonstrated a RU change of over 1200, a few were also identified as having potentially slower than typical off-rates. A total of 28 clones were chosen on these criteria and are listed in Table 7.







TABLE 7







Identification of 28 antibodies to membrane-bound


B Lymphocyte Stimulator that demonstrate the


most significant RU changes by BIAcore










Antibody
Antibody







I079C01
I084C04



I082H08
I080E05



I079E02
I083B12



I079B05
I082G01



I079F06
I082G02



I079F08
I082C03



I079F11
I082A05



I079B12
I082D07



I080B01
I082B08



I080G09
I084A01



I099D03
I084B02



I080D03
I080A08



I080A03
I084C11



I083G03



I080G07










Example 18
scFv Affinity Determinations
The affinity (KD) of the 28 scFvs was determined using the BIAcore.
Low Density HIS-B Lymphocyte Stimulator Surface for Kinetic Studies
500RU surfaces were used for kinetic studies of purified scFv binding to HIS-B Lymphocyte Stimulator. The method to prepare these surfaces was identical to the method described in Example 17, only smaller volumes of HIS-B Lymphocyte Stimulator were injected.
Measurement of scFv Binding Kinetics
The chip containing the low density HIS-B Lymphocyte Stimulator surface was inserted into the BIAcore. A dilution series for each of the 28 purified scFvs (prepared as in Example 6) were diluted in HBS (typically starting with 50 μg/ml scFv and double diluting down to 1.5 μg/ml). The dilution series was then injected sequentially over the blank control (flow cell 1) and low density HIS B Lymphocyte Stimulator surface (flow cell 2) using the following program:















MAIN



FLOWCELL 1,2,3,4












APROG
genab
r1d1
ab1



APROG
genab
r1d2
ab2



APROG
genab
r1d3
ab3



APROG
genab
r1d4
ab4



APROG
genab
r1d5
ab5



APROG
genab
r1d6
ab6









APPEND CONTINUE



END



DEFINE APROG genab



PARAM %Abpos %AbId










FLOW
20









KINJECT %Abpos 200 80










INJECT
r2f3 10









EXTRACLEAN



END










Bound scFv were removed by injecting 10 μl of 4M Guanidine hydrochloride in HBS (location r2f3 in the above program), over the surface between samples. Binding curves for individual scFv were analysed using the BIAevaluation software to determine antibody on- and off-rates.
A typical example of the binding curves generated for the scFv antibody I082C03 is shown in FIG. 15. The off-rate for this clone was calculated as 2×10−3s−1. The affinity of I082C03 was calculated as 20 nM, assuming 100% activity of the scFv. The 5 scFvs with the highest affinities as scFvs are given in Table 8.







TABLE 8







Identification of 5 antibodies to membrane-bound B Lymphocyte


Stimulator that have the highest affinities as scFvs











Affinity



Antibody
(KD)















I079F11
5
nM



I079E02
10
nM



I082G02
6
nM



I082H08
1
nM



I099D03
4
nM










Example 19
Recognition of Mouse Membrane-Bound B Lymphocyte Stimulator
The ability of the 5 scFvs listed in Table 8 to also recognize murine membrane-bound B Lymphocyte Stimulator was determined using a competition ELISA. This assay assesses the ability of test phage scFvs to bind to the membrane-bound form of B Lymphocyte Stimulator on the murine cell line, P388, plasma membranes in the presence of different forms of competing human B Lymphocyte Stimulator. Competing B Lymphocyte Stimulator was either presented as the His-tagged form of B Lymphocyte Stimulator, or soluble B Lymphocyte Stimulator. ScFvs that recognize mouse membrane-bound B Lymphocyte Stimulator would give an ELISA signal on the P388 plasma membranes that is competed out by pre-incubation with HIS-tagged B Lymphocyte Stimulator but not by pre-incubation with soluble B Lymphocyte Stimulator.
Maintenance of P388.D1 Cells and Preparation of Plasma Membranes
P388.D1 cells are a mouse monocyte-macrophage like cell line. They were cultured in L15 medium supplemented with 2 mM L-glutamine, 10% CS, 10U penicillin, 100 g/ml streptomycin (all reagents from Sigma). Cells were split 1:4 every 3-4 days to maintain a cell density of 2−8×105 per ml. A fresh aliquot of cells was thawed from liquid nitrogen every 6 weeks. Plasma membrane fractions were prepared as described in Example 2.
Competition ELISA
P388 plasma membranes (501 μl per well) were coated at 10 μg/ml in PBS onto Falcon 96-well plates overnight at 4° C. The method is otherwise essentially as described Example 16.
The results for 3 clones, I079E02, I082H08 and I099D03 are shown in FIG. 16. All 3 scFvs recognize P388 plasma membranes. This binding is competed out in the presence of HIS-tagged B Lymphocyte Stimulator (HIS-B Lymphocyte Stimulator) but not in the presence of biotinylated B Lymphocyte Stimulator (bio-B Lymphocyte Stimulator). This confirms that these scFvs also recognize the membrane-bound form but not the soluble form of mouse B Lymphocyte Stimulator.
Example 20
Conversion of scFvs to IgG1 Format
The VH domain and the VL domains of scFvs that we wished to convert into IgG molecules were cloned into vectors containing the nucleotide sequences of the appropriate heavy (human IgG1) or light chain (human kappa or human lambda) constant regions such that a complete heavy or light chain molecule could be expressed from these vectors when transfected into an appropriate host cell. Further, when cloned heavy and light chains are both expressed in one cell line (from either one or two vectors), they can assemble into a complete functional antibody molecule that is secreted into the cell culture medium. Methods for converting scFvs into conventional antibody molecules are well known within the art.
Generation of NS0 Cell Lines Expressing Anti-B Lymphocyte Stimulator Antibodies (IgG1)
Plasmids containing the heavy and light chains were separately linearized using the Pvu I restriction enzyme. The linearized DNAs were purified by phenol-chloroform extraction followed by ethanol precipitation and then resuspended in H2O. NS0 cells (107) from a growing culture were electroporated (0.25 kV and 975 μF) in PBS with 12.5 μg linearized heavy chain plasmid DNA and 37.5 μg linearized light chain DNA. The cells were washed in 20 ml non-selective medium (10% FCS in DMEM supplemented with 6 mM glutamine, amino acids and penicillin/streptomycin) and then transferred in 12.5 ml medium into a T75 cm2 flask and incubated overnight at 37° C., 5% CO2/air. The day after transfection the cells were resuspended in selective medium containing 1 mg/ml geneticin and dispensed into 5×96-well plates at 200 μl/well. After 18 days at 37° C. (5% CO2/air) the colony supernatants were screened by an ELISA that detects assembled human IgG in order to identify colonies expressing IgG. Approximately twenty positive colonies were expanded and adapted to growth in serum-free, selective medium. Duplicate T25 cm2 flasks were set up. Cells from one flask were frozen down as a stock and cells in the second flask were grown to saturation. The productivity of the saturated cultures was assessed by ELISA. The highest producing cell lines were then selected for large-scale antibody production.
The above procedure is exemplified for the I006D08 anti-B Lymphocyte Stimulator antibody constructs. Following electroporation and selection of NSO cells, supernatants from ninety-three wells each containing a single colony were screened by ELISA to detect assembled IgG1, antibody. Twenty-seven of the supernatants were identified as containing IgG. The colonies from 24 of the positive wells were transferred to 1 ml selective medium in a 24-well plate and allowed to grow for 2 days. The 1 ml cultures of cells were then added to 4 ml selective medium containing reduced serum (0.5% FCS) in a T25 cm2 flask. When the cultures reached confluency 1 ml cells were diluted in 4 ml selective, serum-free medium in a T25 cm2 flask. At confluency this subculture regime was repeated again. Finally 1 ml cells from the culture containing 0.1% FCS was diluted with 9 ml serum-free, selective medium and divided into 2×T25 cm2 to form the saturated and stock cultures. The stock cultures were frozen down and stored in liquid nitrogen once the cultures were confluent. The saturation culture was grown until the viability of the culture was <10%. Twenty-three out of the 24 colonies originally expanded were successfully adapted to growth in serum-free medium. The productivity of these serum-free adapted cell lines ranged from 0.3 to 17 μg/ml by ELISA quantification of the saturated, 5 ml serum-free cultures. The I006D08-32 cell line produced 17 μg/ml.
Large-scale IgG Production
The highest-producing cell lines were revived from frozen stocks and then expanded to 400 ml in selective, serum-free medium in 2 liter roller bottles. The cells were grown at 37° C. and rolled at 4 rpm with the headspace being re-equilibrated with 5% CO2/air every 2-3 days. Finally the culture was expanded to a 4 liter volume by the addition of serum-free medium without selection (400 ml per 2 liter roller bottle). The cultures were then grown to saturation.
This procedure is exemplified by the production of I006D08 antibody from the I006D08-32 cell line. The frozen stock of I006D08-32 was revived into a T25 cm2 containing 5 ml serum-free medium containing 1 mg/ml geneticin and grown at 37° C. in 5% CO2/air incubator. After two days growth the culture was diluted with 7.5 ml fresh medium and transferred to a T75 cm2 flask. After a further three days in the incubator the cells were transferred to 130 ml selective medium and transferred to a 2 liter roller bottle. After three days growth the cells were diluted with 500 ml selective medium and split into 2×2 liter roller bottles. After another 2 days 100 ml fresh selective medium was added to each roller. Finally the next day the culture was expanded to a total volume of 4 liters with non-selective medium and divided into 10×2 liter roller bottles. After three days the medium was supplemented with 6 mM glutamine. The cells were grown for 17 days from the final subculture into a 4 liter volume. The cells grew up to 3×106 cells/ml before viability declined to <0.2×106 cells/ml. At this low viability the culture supernatants were harvested. ELISA analysis indicated that the culture supernatant contained 33 μg/ml IgG, Hence, the 4 liter culture contained 132 mg IgG,
IgG Purification
The purification of the IgG from the fermentation broth is performed using a combination of conventional techniques commonly used for antibody production. Typically the culture harvest is clarified to remove cells and cellular debris prior to starting the purification scheme. This would normally be achieved using either centrifugation or filtration of the harvest. Following clarification, the antibody would typically be captured and significantly purified using affinity chromatography on Protein A Sepharose. The antibody is bound to Protein A Sepharose at basic pH and, following washing of the matrix, is eluted by a reduction of the pH, Further purification of the antibody is then achieved by gel filtration. As well as removing components with different molecular weights from the antibody this step can also be used to buffer exchange into the desired final formulation buffer.
Purification of I006D08 IgG1
The harvest was clarified by sequential filtration through 0.5 μm and 0.22 μm filters. Clarified harvest was then applied to a column of recombinant Protein A Sepharose equilibrated at pH 8.0 and washed with the equilibration buffer. I006D08 antibody was eluted from the Protein A Sepharose by application of a buffer at pH3.5. The collected antibody containing eluate was then neutralized to pH 7.4 by the addition of pH 8.0 buffer. The neutralized eluate was concentrated by ultrafiltration using a 30 KDa cut off membrane. Concentrated material was then purified by Sephacryl S300HR gel filtration using phosphate buffered saline as the mobile phase. The final monomeric IgG1 fraction from the gel filtration column was then concentrated to the desired formulation concentration by ultrafiltration using a 30 KDa cut off membrane. The final product was filtered through a 0.22 μm filter.
Example 21
Antibody Neutralization of Murine Splenocyte Proliferation as Measured by 3HdT Incorporation
To determine if an antibody inhibited B Lymphocyte Stimulator mediated B cell proliferation, a splenocyte proliferation assay was performed Briefly, murine splenocytes were isolated by flushing spleen using a 25 g needle and 10 ml of complete medium (RPMI 1640 with 10% FBS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 5×10−5M P-mercaptoethanol). The cells were passed through a 100 micron nylon filter to remove cell clumps. The cell suspension was then ficolled at 400×g for 25 minutes at room temperature (one 15 ml conical tube/spleen; 3 ml ficol, 10 ml cell suspension/spleen; Ficol 1083 from Sigma). The recovered cells were washed 3 times in complete medium and counted. Recovered cells were then diluted to a concentration of 3×106/ml in complete medium containing a 3× concentration of SAC (3X=1:33,333 dilution of stock; stock is a 10% suspension of Staph. aureus (Cowan 1 strain) available from Calbiochem).
For each antibody, 50 microliters of antibody dilutions at 30 μg/ml, 3.0 μg/ml, and 0.3 μg/ml concentrations were aliquotted into individual wells of a 96 well plate in triplicate. Suitable positive controls, such as, for example monoclonal antibody 15C10, were also used. Antibody 15C10 is described in Moore et al., (1999), Science 285: 260-263 and WO0050597; 15C10 has also been deposited with the ATCCTM on Feb. 1, 2000 as ATCCTM Deposit No. PTA-1158. Medium containing no antibody (and human isotype controls (purchased commercially) when necessary) were used as negative controls.
B Lymphocyte Stimulator protein was diluted in complete medium to concentrations of 300 ng/ml, 90 ng/ml and 30 ng/ml. 50 microliters of each of the B Lymphocyte Stimulator dilutions were then added to the antibody dilution series in the plates. The plate containing the antibody and B Lymphocyte Stimulator dilutions are then incubated for 30 minutes at 37° C., 5% CO2, after which 50 microliters of the splenocyte cell suspension containing SAC was added to all wells. The plates were then incubated for 72 hours (37° C., 5% CO2).
After 72 hours, each well was supplemented with 50 μl of complete medium containing 0.5 μCi of 3H-thymidine (6.7 Ci/mM; Amersham) and cells were incubated for an additional 20-24 hours at (37° C., 5% CO2). Following incubation cells were harvested using a Tomtec Cell Harvester and filters counted in a TopCount Scintillation counter (Packard).
Example 22
Human B Cell Proliferation Assay for In Vitro Screening of B Lymphocyte Stimulator Antagonist Molecules
The bioassay for assessing the effects of putative B Lymphocyte Stimulator antagonists was performed in triplicate in 96 well format by mixing equal volumes of B Lymphocyte Stimulator, responder cells, and putative antagonist each of which is prepared as a 3× stock reagent.
B-lymphocytes were purified from human tonsil by MACS (anti-CD3 depletion), washed, and resuspended in complete medium (CM) (RPMI 1640 with 10% FBS containing 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM glutamine, 5×10E-5 M beta-mercaptoethanol) at a concentration of 3×10e6 cells/mL. Staphylococcus aureus, Cowan I (SAC, CalBiochem) was added to cells at 3× concentration (3×=1:33,333 dilution of stock
Meanwhile, eight serial dilutions (3-fold) of potential antagonist were prepared in CM such that the diluted antagonists are at 3× the final concentrations to be tested in the assay. Antibodies are routinely tested starting at a final concentration of 10 μg/mL and going down to about 1.5 ng/mL.
Human rB Lymphocyte Stimulator was prepared in CM to 3× concentration (3×=300 ng/μL, 30 ng/mL, and 3 ng/mL) in CM. Potential inhibitors were routinely tested at several concentrations of B Lymphocyte Stimulator to avoid false negatives due to unexpectedly low affinity or antagonist concentration.
Fifty microliters of diluted antagonist and 5 uL of diluted B Lymphocyte Stimulator were added to the putative antagonist dilution series.
Cells were then incubated for 72 hours (37° C., 5% CO2) in a fully humidified chamber. After 72 hrs., the cells were supplemented with 0.5 μCi/well 3H-thymidine (6.7 μCi/mmol) and incubated for an additional 24 hours. Plates were harvested using a Tomtec Cell Harvester and filters counted in a TopCount Scintillation counter (Packard).
Example 23
Characterization of I006D08 and I116A01
I116A01 (SEQ ID NO:327) is a CDR3 mutant of the I006D08 (SEQ ID NO:2). The I116A0 I scFv was subsequently isolated from a single chain phage display library containing a repertoire of I006D08 mutations generated by randomization of the last 6 amino acids of the heavy chain CDR3 region of I006D08. The I116A0 I scFv contains 7 amino acid differences from the parental I006D08 sequence, I change in the VH framework 3 region, 5 changes in the VH CDR3, and I change in the VLCDR3. Each of the assays described herein were performed using whole IgG1 molecules comprising the VH and VL regions of I006D08 and I116A01.
Binding studies of I006D08 and I116A01
Equilibrium dissociation constants (Kd) for I116A01 and I006D08 binding to B Lymphocyte Stimulator were assessed using BIAcore technology (BIAcore, Uppsala, Sweden). Briefly, antibodies were captured on low-density Protein A surfaces and B Lymphocyte Stimulator was passed over at various flow rates. The binding of B Lymphocyte Stimulator to immobilized antibody was detected by changes in surface charge that were proportional to the amount of bound B Lymphocyte Stimulator. Binding values were calculated using software provided by the manufacturer.
I116A01 was determined to have an equilibrium dissociation constant, Kd, of 267±70 μM (mean±SD, n=3). The Kd value of I006D08, the parental antibody, was determined to be 498±151 pM (mean±SD, n=3). This was approximately 2-fold higher than the Kd obtained for I116A01. The difference in affinity can be attributed to a 2-fold higher off rate (kd, 1/s) of I006D08 compared with I116A01, the on rates (ka, 1/Ms) being essentially equal (Table 9).







TABLE 9







Kinetic parameters for I116A01 and I006D08


binding to B Lymphocyte Stimulator











ka, 1/Ms
kd, 1/s
Kd, pM


Mab
Mean ± SD
Mean ± SD
Mean ± SD





I116A01
1.26 ± 0.14 × 106
3.32 ± 0.63 × 10−4
267 ± 70


I006D08
1.43 ± 0.13 × 106
 5.8 ± 0.24 × 10−4
498 ± 151









The Kd values obtained for the B Lymphocyte Stimulator antibodies indicate that these antibodies bind to B Lymphocyte Stimulator with high affinity and possess a slow dissociation or off rate.
The binding characteristics of I116A01 and I006D08 were investigated further using a competitive binding ELISA in which B Lymphocyte Stimulator was immobilized directly on the surface of the well to compare epitopes. Biotinylated I006D08 binding was inhibited in a dose-dependent manner by the addition of unlabeled I006D08 or I116A01 suggesting that these antibodies recognize overlapping or identical epitopes on B Lymphocyte Stimulator.
Ability of I006D08 and I116A01 to Inhibit B Lymphocyte Stimulator-Induced Splenocyte Proliferation
The ability of I006D08 and I116A01 to inhibit B Lymphocyte Stimulator-induced splenocyte proliferation was tested using the assay described in Example 21 with slight modifications. In particular, the assay was modified such that the final concentration of B Lymphocyte Stimulator in the assay was 3 ng/ml and the final dilution factor of SAC was 1:100,000. Using this assay it was shown that both antibodies I006D08 and I116A01 were each found to inhibit murine splenocyte proliferation induced by both human (aa134-285 of SEQ ID NO:2) and murine soluble B Lymphocyte Stimulator. The IC50 values for the inhibition of human B Lymphocyte Stimulator induced-proliferation of murine B cells was 0.09 nM±0.01 nM and 0.06 nM±0.01 nM, respectively.
Additional Characterization of I006D08 and I116A01
I006D08 and I116A01 were identified herein as antibodies that bind both the membrane bound and soluble forms of B Lymphocyte Stimulator. The ability to bind the membrane bound form of B Lymphocyte Stimulator was determined according to the assays described above, e.g., ability to bind HIS-tagged B Lymphocyte Stimulator and ability to bind U937 and/or P388 membrane preparations in an ELISA assay. Further characterization of antibodies I006D08 and I116A01 reveals that these antibodies bind recombinant B Lymphocyte Stimulator polypeptides expressed on the surface of a 293T cell. On the other hand, these antibodies are not able to bind naturally occurring B Lymphocyte Stimulator (non-recombinant) expressed on the surface of a cell, e.g., K562 cells (ATCCTM #CCL-243) or U-937 cells (ATCCTM #CRL-1593.2). Thus, I006D08 and I116A01 bind the soluble form of B Lymphocyte Stimulator. I006D08 and I116A01 also bind the membrane bound form of B Lymphocyte Stimulator (as determined by their ability to bind HIS-tagged B Lymphocyte Stimulator and their ability to bind U937 and/or P388 membrane preparations in an ELISA assay). I006D08 and I116A01 also bind recombinant B Lymphocyte Stimulator expressed on the surface of a 293T transfected fibroblast cell line, but do not bind naturally occurring B Lymphocyte Stimulator protein on the surface of a K-562 or U-937 cell.
Example 24
In Vivo Studies of Anti-B Lymphocyte Stimulator Antibodies
Overexpression of B Lymphocyte Stimulator in transgenic animals results in an autoimmune phenotype including increased numbers of mature B cells (leading to enlarged spleens), increased levels of serum immunoglobulins, as well as increased levels of autoantibodies (e.g., anti-dsDNA antibodies) (McKay et al., (1999), The Journal of Experimental Medicine 190:1697-1710 and Gross et al., (2000) Nature 404:995-999, both of which are hereby incorporated by reference in their entireties.) Similar effects are observed when recombinant B Lymphocyte Stimulator is administered to animals such as mice and monkeys. The ability of anti-B Lymphocyte Stimulator antibodies of the invention to reduce or prevent these B Lymphocyte Stimulator induced effects may be tested through either co-administration or sequential administration of soluble B Lymphocyte Stimulator and anti-B Lymphocyte Stimulator antibodies of the invention in an animal. An anti-B Lymphocyte Stimulator antibody of the invention would be considered neutralizing if it prevented or reduced B Lymphocyte Stimulator-induced increases in B cell numbers (resulting in lack of, or less, enlargement of spleen), and serum immunoglobulins, and in particular serum autoantibodies.
Example 25
Antibody Production and Purification
The following example describes a large scale antibody production and purification methods that can be used to make antibodies of the present invention. One of skill in the art will be aware of routine modifications to the protocol described below, for example, as regards column choice, column, loading, wash, and elution buffers, and pH,
Cell Culture Scale-Up and Antibody Production
A serum-free and animal source-free growth medium is used from thawing cells through scale-up to the production bioreactor. The growth medium is prepared by adding 1 kilogram AGT powder granules preformulated with GS supplement to water for injection (WFI) quality water making a total of 51 kilograms CD hybridoma medium. To the CD hybridoma medium, 1 mL/kg cholesterol (synthetic) lipid concentrate (1000×). The AGT powder granules preformulated with GS supplement for preparing the CD hybridoma medium and the synthetic cholesterol lipid concentrate and are available from Invitrogen Corp., Carlsbad, Calif. The medium, hereinafter referred to as INV-CDH medium, is stored at 2-8° C. until use.
Thawing Cells from into Spinner Flask
Approximately 48×106 NS0 cells engineered to express an antibody of the invention, preferably in whole IgG1 format, are thawed at 37° C. in a water bath. The cells are transferred into a 250 mL spinner flask to yield approximately a 150 mL working volume with an inoculation density of approximately 3.2×105 cells/mL. The spinner flask(s) is then placed on magnetic stirrers in a humidified CO2 incubator at 37° C. with 5% CO2 for 4 days. The agitation rate for the spinner flask is 80 rpm.
First Expansion(s) of Culture in Spinner Flask
The culture is aseptically expanded into a 1000 mL spinner flask to give approximately 600 mL working volume, at an inoculation cell density of approximately 2.5×105 cells/mL. The spinner flask is then placed on magnetic stirrers in a humidified CO2 incubator at 37° C. with 5% CO2 for 3 days. The agitation rate for the spinner flask is 80 rpm.
The culture is again expanded aseptically into two 3000 mL spinner flasks to give approximately 2×2000 mL working volume, at an inoculation cell density of approximately 3.0×105 cells/mL. The spinner flask is then placed on magnetic stirrers in a humidified CO2 incubator at 37° C. with 5% CO2 for 3 days. The agitation rate for the spinner flasks is 80 rpm. At the same time, a reserve culture is created in a fresh 250 mL spinner flask. The reserve culture may be used as the seed for another lot as needed.
Seed Culture (25 Liters)
The 25 liter seed bioreactor is equipped with one impeller for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. The first step of the cell cultivation process is the addition of INV-CDH medium into the bioreactor. After the medium temperature reaches 37±0.5° C., the dissolved oxygen (DO) and pH levels are stabilized by addition of N2 and CO2 to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 70 rpm. The pooled cell culture is transferred aseptically to the 25 liter seed bioreactor containing 21 liters of sterile INV-CDH medium. During the cultivation process the temperature is maintained via a plant stem and chilled glycol, which regulate the temperature of the circulating jacket. The oxygen concentration is maintained via oxygen and nitrogen sparging and surface aeration, and pH is controlled by addition of CO2 gas to lower the pH, The cultivation period is 4 days. The bioreactor air/gas inlets and vent lines are protected by hydrophobic 0.2 μm filters.
Seed Culture (200 Liters)
The 200 L seed bioreactor is equipped with 1 impeller for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. The first step of the cell cultivation process is the addition of INV-CDH medium into the bioreactor. After the medium temperature reaches 37±0.5° C., the DO and pH levels are stabilized by addition of N2 and CO2 to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 35 rpm. The 25 L seed culture is transferred aseptically to the 200 L seed bioreactor containing 175 L sterile medium. During the cultivation process the temperature is maintained via plant steam and chilled glycol which regulate the temperature of the circulating jacket. The oxygen concentration is maintained via oxygen and nitrogen sparging and surface aeration, and pH is controlled by addition of CO2 gas to lower the pH, The cultivation period is 4 days. The bioreactor air/gas inlet and vent lines are protected by hydrophobic 0.2 μm filters.
Production Culture (1600 Liters)
The production bioreactor is equipped with 2 impellers for mixing, a dissolved oxygen probe, a temperature probe, a pH probe, aseptic sampling and additional systems. 600 L of INV-CDH medium is aseptically transferred into the 1600 L production bioreactor. After the medium temperature reaches 37±0.5° C., the DO and pH levels are stabilized by addition of N2 and CO2 to decrease dissolved oxygen concentration to 25 to 75% air saturation, and obtain a pH of 7.20±0.2. The agitation rate is 17 rpm. The 200 L seed culture is aseptically transferred into the production bioreactor. During the cultivation process the temperature is maintained via plant steam and chilled glycol which regulate the temperature of the circulating jacket, the oxygen concentration is maintained via oxygen and nitrogen sparging and surface aeration, and pH is controlled by addition of CO2 gas to lower the pH, The bioreactor air/gas inlet and vent lines are protected by hydrophobic 0.2 μm filters. After 48 hours, 800 Liters of INV-CDH medium is transferred into the production bioreactor in order to obtain the final volume. The culture is cultivated in the production bioreactor 43 additional days.
Recovery and Purification
Harvest of Cell Supernatant
Cell supernatant, (e.g., culture supernatant from NS0 cells expressing antibodies of the invention) is harvested on day 5 or 6 post final feeding in the final production bioreactor. Cell culture broth temperature is cooled down to 15° C. in the recovery vessel at the time of harvest and maintained at that temperature during the recovery. Centrifugation followed by depth filtration is used for cell removal and antibody recovery. The filtration process train consists of 0.1 μm nominal rated depth filter and 0.2 μm membrane filters connected in series. A constant flow rate of 13±1 L/min is maintained during the operation. The 0.2 μm filtered culture supernatant is collected in a process vessel, warned to 20° C. and transferred to purification. The purification process is conducted at 22 to 26° C.
Chromatography on rProtein A Sepharose FF Column
The culture supernatant is loaded onto rProtein-A Sepharose FF column (affinity chromatography, Amersham Phamnacia) or equivalent column that is equilibrated in 50 mM Tris-HCl, 0.5 M NaCl, pH 7.5. The rProtein-A Sepharose FF column is washed with 0.05 M sodium citrate, 0.15 M sodium chloride, pH 5.2 and eluted with 0.05 M sodium citrate, 0.15 M sodium chloride, pH 3.2. The elution is monitored by ultraviolet (UV) absorbance at 280 nm. The elution peak is collected, and analyzed by A280 and SDS-PAGE.
Virus Inactivation
The eluate from the rProtein-A Sepharose FF column is adjusted with 1 M citric acid to pH 3.4±0.2 and allowed to stand for 45-60 minutes for viral inactivation. The solution is then readjusted to pH 5.4 with 1 M Tris base. The pH adjusted material is 0.2 μm filtered and stored at 20° C. up to 20 hours.
Chromatography on SP Sepharose FF Column
The inactivated material from the rProtein-A Sepharose FF column is diluted with water for injection (WFI) to a conductivity of 7 mS, and loaded onto a SP Sepharose FF column (cation exchanger, Amersham-Pharmacia), or equivalent column equilibrated with 50 mM sodium acetate, 30 mM sodium chloride, pH 5.4. The antibody is eluted from the SP column with 50 mM sodium acetate, 100 mM sodium chloride, pH 6.0. The elution is monitored by ultraviolet (UV) absorbance at 280 nm. The elution peak is collected and analyzed by A280 and SDS-PAGE.
Virus Removal Filtration, Diafiltration and Concentration
The eluate from the SP Sepharose FF column is filtered through a sequentially connected 0.2 μm filter and a virus removal filter (Pall DV50 Ultipor® VF Filter System). The DV50 filtrate is stored at 20° C. up to 8 hours.
The DV50 filtrate is placed into a 30 kD molecular weight cut-off membrane device (Millipore Pellicon) to concentrate to a target concentration of 35-40 mg/mL. The concentrated material is diafiltered against 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, pH 6.5. The diafiltered material is monitored by A280. The diafiltered bulk is 0.2 μm filtered and stored at 20° C. up to 8 hours.
Chromatography on Q Sepharose FF Column
The diafiltered antibody solution is passed over a Q Sepharose FF column (anion exchanger, Amersham-Pharmacia) or equivalent column equilibrated with 10 mM sodium citrate, 1.9% glycine, 0.5% sucrose, pH 6.5. The antibody is collected in the flow-through and monitored by A280. The elution peak is collected and analyzed by A280 
Bulk Formulation, Filtration and Bulk Drug Substance Fill
Polysorbate 80 (1% stock solution in 10 mM sodium citrate, 1.9% glycine and 0.5% sucrose) is pre-filtered through a 0.2 μm filter and added to the antibody solution to a final concentration of 0.01%. The purified antibody is aseptically filtered under a laminar flow hood through a 0.2 μm filter and filled into polypropylene containers.
Storage of Bulk Drug Substance
The bulk drug substance is stored at 2-8° C. (short-term storage) or at or below −65° C. (long-term storage).
In Process Testing
In-process testing of bioreactor culture at harvest for each batch and in-process testing during the purification process are performed. The bioreactor is sampled aseptically and the culture is tested at various times throughout cultivation for cell density, viability and nutrient determination to ensure consistency of material being supplied for purification. The purification process is monitored at each step. Appearance is checked by visual inspection. The protein concentration is determined by Absorbance at 280 nm. The pH of the material is checked. Purity is checked, for example, by SDS-PAGE and size exclusion chromatography. An ELISA may be performed to check the ability of the antibody to bind its antigen. The biological activity of the antibody is also monitored. Residual DNA content, Endotoxin levels, and the bioburden (the number of viable organisms present in the antibody preparation) are all monitored and kept at or below standard acceptable levels. Additionally the oligosaccharide content may be analyzed; the peptide sequence of the antibody chains may also be analyzed using N-termini sequencing and peptide mapping. Short and long-term studies of antibody stability may also be performed.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in this application is incorporated in their entireties herein by reference. Further, the sequences disclosed herein are also disclosed in the Sequence Listings of U.S. Provisional Application 60/212,210 filed Jun. 16, 2000 and U.S. Non-Provisional application Ser. Nos. 10/293,418 filed Nov. 14, 2002 and 09/880,748 filed Jun. 15, 2001 the contents of which are herein incorporated in their entireties by reference.
The entire disclosure (including the specification, sequence listing, and drawings) of each of the following U.S. Provisional and Non-Provisional Patent Applications and International Patent Applications are herein incorporated by reference in their entireties: U.S. Provisional Application Ser. Nos. 60/543, 296 Filed Feb. 11, 2004; 60/580,347 filed Jun. 18, 2004; 60/331,469 filed Nov. 16, 2001; 60/340,817 filed Dec. 19, 2001; 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; 60/293,499 filed May 25, 2001; U.S. Nonprovisional application Ser. Nos. 10/293,418 filed Nov. 14, 2002 and 09/880,748 filed Jun. 15, 2001; and International Patent Application Serial Nos. PCT/US02/36496 filed Nov. 14, 2002 and PCT/US01/19110 filed Jun. 15, 2001.







TABLE 1







scFvs that Immunospecifically Bind to B Lymphocyte Stimulator



















scFv SEQ

AAs of
AAs of
AAs of

AAs of
AAs of
AAs of




Clone ID
ID NO
AAs of VL
VL CDR1
VL CDR2
VL CDR3
AAs of VH
VH CDR1
VH CDR2
VH CDR3
VH CDR3 Sequence
(SEQ ID NO)





















I003F12S
1
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
HDDDVLTGYYFES
(SEQ ID NO: 2130)


I006D08
2
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I008A11
3
144-254
166-179
195-201
234-243
1-128
26-37
52-69
102-117
DRYDILTGYYYYGMDV
(SEQ ID NO: 2129)


I017D10
4
148-255
169-179
195-201
234-244
1-132
26-35
50-66
99-121
VQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 2132)


I022D01
5
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I031F02
6
138-251
160-173
189-195
228-240
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I050A12
7
142-250
164-174
190-196
229-239
1-124
26-35
50-66
99-113
APYDLLTHYFHYFDY
(SEQ ID NO: 2134)


I051C04
8
146-256
168-181
197-203
236-245
1-129
26-35
50-66
99-118
AATTSQKHNKYAYYFYGMDV
(SEQ ID NO: 2131)


I050B11
9
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I050B11-01
10
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQVWVA
(SEQ ID NO: 2143)


I050B11-02
11
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQVWVA
(SEQ ID NO: 2143)


I050B11-03
12
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I050B11-04
13
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQYFDH
(SEQ ID NO: 2141)


I050B11-05
14
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQVWVA
(SEQ ID NO: 2142)


I050B11-06
15
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQVWVA
(SEQ ID NO: 2140)


I050B11-07
16
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I050B11-08
17
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQYFDH
(SEQ ID NO: 2141)


I050B11-09
18
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQVWVA
(SEQ ID NO: 2142)


I050B11-10
19
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQVWVA
(SEQ ID NO: 2142)


I050B11-11
20
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQVWVA
(SEQ ID NO: 2140)


I050B11-12
21
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQVWVA
(SEQ ID NO: 2140)


I050B11-13
22
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I050B11-14
23
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I050B11-15
24
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQVWVA
(SEQ ID NO: 2143)


I050B11-16
25
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQVWVA
(SEQ ID NO: 2143)


I050B11-17
26
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I050B11-18
27
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I050B11-19
28
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTSYVFQYFDH
(SEQ ID NO: 2139)


I050B11-20
29
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTSYVFQYFDH
(SEQ ID NO: 2139)


I050B11-21
30
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTRYVFQYFDH
(SEQ ID NO: 2138)


I050B11-22
31
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTRYVFQYFDH
(SEQ ID NO: 2138)


I050B11-23
32
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTRYVFQYFDH
(SEQ ID NO: 2138)


I050B11-24
33
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTSYVFQYFDH
(SEQ ID NO: 2139)


I050B11-25
34
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I050B11-26
35
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTSYVFQYFDH
(SEQ ID NO: 2139)


I050B11-27
36
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTRYVFQYFDH
(SEQ ID NO: 2138)


I050B11-28
37
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093D03
38
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLGYYLS
(SEQ ID NO: 2145)


I093D09
39
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093G08
40
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQVWVA
(SEQ ID NO: 2143)


I097D11
41
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDILTSYVFQYFDH
(SEQ ID NO: 2139)


I101A04
42
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101B01
43
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102A02
44
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102E01
45
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTRYVFQYFDH
(SEQ ID NO: 2144)


I102G06
46
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTGYVFQYFDH
(SEQ ID NO: 2141)


I087A07
47
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPRVIP
(SEQ ID NO: 2227)


I087A08
48
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVCRPHF
(SEQ ID NO: 2238)


I087A09
49
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVRCPYV
(SEQ ID NO: 2272)


I087B02
50
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVFRPDL
(SEQ ID NO: 2281)


I087B03
51
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVKSMPT
(SEQ ID NO: 2305)


I087B04
52
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPFLYC
(SEQ ID NO: 2292)


I087B05
53
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPVPST
(SEQ ID NO: 2270)


I087B06
54
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVGIHGL
(SEQ ID NO: 2282)


I087B08
55
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVPCSPPR
(SEQ ID NO: 2261)


I087B09
56
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVCYPPA
(SEQ ID NO: 2240)


I087C02
57
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLPLLS
(SEQ ID NO: 2224)


I087C05
58
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVALYRL
(SEQ ID NO: 2234)


I087C06
59
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVRASFS
(SEQ ID NO: 2271)


I087C07
60
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVCTPVP
(SEQ ID NO: 2319)


I087C08
61
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVWPSFFS
(SEQ ID NO: 2277)


I087D01
62
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVTPRGY
(SEQ ID NO: 2275)


I087D02
63
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVSSLLS
(SEQ ID NO: 2213)


I087D03
64
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVPLLPLC
(SEQ ID NO: 2263)


I087D05
65
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVPPPSFL
(SEQ ID NO: 2266)


I087D07
66
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPTSIT
(SEQ ID NO: 2269)


I087D09
67
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVISCSWA
(SEQ ID NO: 2299)


I087E04
68
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVSALPPP
(SEQ ID NO: 2274)


I087E05
69
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVCRHLF
(SEQ ID NO: 2236)


I087E10
70
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVVSFPSL
(SEQ ID NO: 2307)


I087F02
71
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMGVTPS
(SEQ ID NO: 2322)


I087F04
72
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLFRPVL
(SEQ ID NO: 2326)


I087F05
73
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPSVGG
(SEQ ID NO: 2267)


I087F07
74
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPPTRH
(SEQ ID NO: 2286)


I087F08
75
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLRSRD
(SEQ ID NO: 2243)


I087F09
76
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVPLLPP
(SEQ ID NO: 2310)


I087G05
77
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLRCVL
(SEQ ID NO: 2239)


I087G06
78
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVHPSRS
(SEQ ID NO: 2285)


I087G07
79
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLRLPPQ
(SEQ ID NO: 2241)


I087G09
80
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVGPYGT
(SEQ ID NO: 2284)


I087G10
81
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVTTPCT
(SEQ ID NO: 2276)


I087H02
82
137-244
160-170
186-192
225-233
1-121
26-35
50-66
99-110
ASYLSTSSSLDN
(SEQ ID NO: 2265)


I088A01
83
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088A03
84
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIPFLPL
(SEQ ID NO: 2290)


I088A04
85
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHIYPH
(SEQ ID NO: 2335)


I088A08
86
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTNYVFEYYAS
(SEQ ID NO: 2323)


I088A09
87
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVILYYLH
(SEQ ID NO: 2295)


I088A10
88
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088A11
89
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLMYFPH
(SEQ ID NO: 2220)


I088A12
90
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLFFYPL
(SEQ ID NO: 2325)


I088B01
91
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088B02
92
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFDYYAS
(SEQ ID NO: 2244)


I088B03
93
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIPFLPL
(SEQ ID NO: 2290)


I088B05
94
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088B06
95
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFEYYSL
(SEQ ID NO: 2324)


I088B07
96
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088B08
97
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088B09
98
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEFYLL
(SEQ ID NO: 2303)


I088B10
99
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088B12
100
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLPLDS
(SEQ ID NO: 2223)


I088C01
101
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLYFYPS
(SEQ ID NO: 2317)


I088C03
102
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088C09
103
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088C12
104
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088D01
105
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088D03
106
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYAL
(SEQ ID NO: 2215)


I088D04
107
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLPPSV
(SEQ ID NO: 2225)


I088D07
108
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088D08
109
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088D11
110
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E01
111
140-248
163-174
190-196
229-237
1-122
23-32
47-63
96-111
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E02
112
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYLY
(SEQ ID NO: 2216)


I088E03
113
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E04
114
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E08
115
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E10
116
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088E11
117
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088F07
118
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088G02
119
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088G03
120
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088G07
121
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFHYYPL
(SEQ ID NO: 2260)


I088G09
122
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFPVYYL
(SEQ ID NO: 2264)


I088G10
123
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHFIDH
(SEQ ID NO: 2301)


I088H05
124
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I088H07
125
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092A03
126
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092A05
127
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFHYYDV
(SEQ ID NO: 2258)


I092A06
128
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092A08
129
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVHEFFSL
(SEQ ID NO: 2283)


I092A10
130
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092A11
131
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B01
132
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B02
133
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B04
134
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B05
135
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B10
136
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092B12
137
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092C01
138
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092C02
139
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092C07
140
142-250
165-176
192-198
231-239
1-124
26-35
50-66
99-113
PFYDTLTSYVLALDL
(SEQ ID NO: 2328)


I092C08
141
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFGYYSL
(SEQ ID NO: 2254)


I092C12
142
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092D01
143
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLKYYTD
(SEQ ID NO: 2226)


I092D07
144
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092D09
145
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMHAYPL
(SEQ ID NO: 2255)


I092D10
146
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFHYLPV
(SEQ ID NO: 2256)


I092D11
147
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092E01
148
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092E03
149
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYAFQYFDH
(SEQ ID NO: 2230)


I092E04
150
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFEYFSV
(SEQ ID NO: 2248)


I092E07
151
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092E10
152
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLFYYPL
(SEQ ID NO: 2327)


I092E11
153
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F01
154
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F02
155
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F05
156
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F07
157
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F08
158
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F11
159
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092F12
160
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLAYYPD
(SEQ ID NO: 2306)


I092G01
161
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092G05
162
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092G10
163
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I092H01
164
137-244
160-170
186-192
225-233
1-121
26-35
50-66
99-110
ASYLSTSSSLDN
(SEQ ID NO: 2265)


I093A06
165
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPVYDH
(SEQ ID NO: 2334)


I093A09
166
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFAH
(SEQ ID NO: 2268)


I093A11
167
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093A12
168
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093B02
169
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093B05
170
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIFYYPT
(SEQ ID NO: 2289)


I093B06
171
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093B09
172
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEVYHP
(SEQ ID NO: 2318)


I093B12
173
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFAPLVT
(SEQ ID NO: 2242)


I093C02
174
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHAYAF
(SEQ ID NO: 2332)


I093C03
175
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093C05
176
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVILYYLH
(SEQ ID NO: 2295)


I093D05
177
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFEFLPL
(SEQ ID NO: 2245)


I093D08
178
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVRPFYAH
(SEQ ID NO: 2273)


I093D10
179
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093D12
180
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHFYRV
(SEQ ID NO: 2302)


I093E01
181
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093E02
182
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIQYFDH
(SEQ ID NO: 2297)


I093E05
183
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVHEFFSL
(SEQ ID NO: 2283)


I093E08
184
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMQFFPT
(SEQ ID NO: 2321)


I093E10
185
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLSFYPV
(SEQ ID NO: 2246)


I093F01
186
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLYYYAF
(SEQ ID NO: 2251)


I093F03
187
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093F05
188
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093F08
189
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093F11
190
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHFYPL
(SEQ ID NO: 2333)


I093G07
191
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLQYYVL
(SEQ ID NO: 2237)


I093G11
192
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093012
193
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I093H06
194
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094A08
195
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDY
(SEQ ID NO: 2280)


I094B07
196
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPVWVS
(SEQ ID NO: 2228)


I094B08
197
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094B12
198
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094C11
199
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094C12
200
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094D06
201
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIEYYPV
(SEQ ID NO: 2288)


I094D07
202
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094D08
203
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYLPL
(SEQ ID NO: 2314)


I094D09
204
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094D10
205
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094D11
206
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFHFYPV
(SEQ ID NO: 2218)


I094E04
207
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094E08
208
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEAFSL
(SEQ ID NO: 2311)


I094F04
209
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFGFYPF
(SEQ ID NO: 2252)


I094F05
210
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094F10
211
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PIYDTLTSYVFQYFDH
(SEQ ID NO: 2278)


I094F11
212
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLWYYQD
(SEQ ID NO: 2249)


I094F12
213
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIPFYPL
(SEQ ID NO: 2296)


I094G06
214
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I094G10
215
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095A04
216
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095A12
217
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEYFPL
(SEQ ID NO: 2320)


I095B04
218
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEFFPA
(SEQ ID NO: 2312)


I095B09
219
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIEYLPL
(SEQ ID NO: 2287)


I095B10
220
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYSA
(SEQ ID NO: 2217)


I095C02
221
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLFYYTA
(SEQ ID NO: 2331)


I095C05
222
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYLPV
(SEQ ID NO: 2337)


I095C07
223
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095C08
224
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095C09
225
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMHYYPT
(SEQ ID NO: 2259)


I095D01
226
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095D02
227
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLQYFRY
(SEQ ID NO: 2235)


I095D03
228
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLQVFDT
(SEQ ID NO: 2233)


I095D05
229
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095D09
230
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095E01
231
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLDYYSS
(SEQ ID NO: 2309)


I095E05
232
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDALTSYVFQYFDH
(SEQ ID NO: 2221)


I095E12
233
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095F06
234
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFPFYPH
(SEQ ID NO: 2262)


I095F09
235
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIGFYPV
(SEQ ID NO: 2291)


I095G06
236
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I095G09
237
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMDFYSV
(SEQ ID NO: 2253)


I095G11
238
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096A01
239
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096A10
240
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPFYAL
(SEQ ID NO: 2222)


I096B01
241
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096B03
242
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096C01
243
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096C06
244
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPYLTH
(SEQ ID NO: 2229)


I096C09
245
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096D01
246
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096D02
247
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096D05
248
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096D06
249
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096D09
250
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096E02
251
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLGFYPV
(SEQ ID NO: 2329)


I096E06
252
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYHTH
(SEQ ID NO: 2336)


I096E11
253
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096F02
254
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIHFLPL
(SEQ ID NO: 2330)


I096G01
255
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIPFLPL
(SEQ ID NO: 2290)


I096G02
256
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096G05
257
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096G07
258
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I096G09
259
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVMHYLPV
(SEQ ID NO: 2257)


I096012
260
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEFFSH
(SEQ ID NO: 2315)


I096H01
261
137-244
160-170
186-192
225-233
1-121
26-35
50-66
99-110
ASYLSTSSSLDN
(SEQ ID NO: 2265)


I097A04
262
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIHYLVT
(SEQ ID NO: 2294)


I097A06
263
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLPYYTL
(SEQ ID NO: 2231)


I097A09
264
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYPI
(SEQ ID NO: 2298)


I097B02
265
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLWFYPL
(SEQ ID NO: 2247)


I097B09
266
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097B10
267
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097B11
268
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097C05
269
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYTH
(SEQ ID NO: 2219)


I097C09
270
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYAY
(SEQ ID NO: 2316)


I097C11
271
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097D05
272
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIHFYSL
(SEQ ID NO: 2293)


I097D06
273
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFGFFPH
(SEQ ID NO: 2300)


I097E01
274
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097E04
275
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097E08
276
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097E09
277
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097F09
278
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I097G10
279
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFAH
(SEQ ID NO: 2268)


I097H02
280
137-244
160-170
186-192
225-233
1-121
26-35
50-66
99-110
ASYLSTSSSLDN
(SEQ ID NO: 2265)


I098A04
281
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I098A05
282
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I098B08
283
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLDFYSV
(SEQ ID NO: 2308)


I098C01
284
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PIYDTLTSYVFQYFDH
(SEQ ID NO: 2278)


I098C04
285
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLYYYAF
(SEQ ID NO: 2251)


I098F11
286
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I098F12
287
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFFFYPF
(SEQ ID NO: 2250)


I098G02
288
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I098G12
289
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I098H05
290
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101A01
291
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101B04
292
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101B06
293
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVIPFLTH
(SEQ ID NO: 2304)


I101D04
294
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLEFFPD
(SEQ ID NO: 2313)


I101D07
295
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101E09
296
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDR
(SEQ ID NO: 2279)


I101E12
297
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101G02
298
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I101G11
299
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102C03
300
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102E09
301
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102F02
302
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102G08
303
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I102G09
304
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVLHYYAH
(SEQ ID NO: 2214)


I106A09
305
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106B02
306
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106B06
307
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106C07
308
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106E05
309
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106E12
310
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106G01
311
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I106G03
312
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I109B06
313
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I109D12
314
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I109E12
315
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I109G06
316
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I109H04
317
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I110B03
318
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I112D09
319
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYGFQYFDH
(SEQ ID NO: 2232)


I112F10
320
143-251
166-177
193-199
232-240
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I089F12
321
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHGLDS
(SEQ ID NO: 2146)


I105E12
322
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I108D08
323
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLAPLYP
(SEQ ID NO: 2148)


I108E06
324
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHGLDV
(SEQ ID NO: 2151)


I113E07
325
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSLDL
(SEQ ID NO: 2152)


I114G05
326
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I116A01
327
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHALSP
(SEQ ID NO: 2149)


I116A09
328
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHHSFDL
(SEQ ID NO: 2150)


I116C11
329
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I085A01
330
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDHLLF
(SEQ ID NO: 2602)


I085A02
331
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDPLGF
(SEQ ID NO: 2639)


I085A03
332
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTHPLSF
(SEQ ID NO: 2561)


I085A04
333
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLAPLFF
(SEQ ID NO: 2550)


I085A05
334
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDPLSL
(SEQ ID NO: 2659)


I085A06
335
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLSF
(SEQ ID NO: 2611)


I085A07
336
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLSF
(SEQ ID NO: 2390)


I085A09
337
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPNDALS
(SEQ ID NO: 2632)


I085A10
338
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLRF
(SEQ ID NO: 2609)


I085A11
339
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHDPLE
(SEQ ID NO: 2363)


I085B01
340
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQSPLYP
(SEQ ID NO: 2466)


I085B02
341
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSSLVF
(SEQ ID NO: 2392)


I085B03
342
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYDPLLF
(SEQ ID NO: 2638)


I085B04
343
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLYF
(SEQ ID NO: 2589)


I085B05
344
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLSP
(SEQ ID NO: 2573)


I085B06
345
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLSPLSF
(SEQ ID NO: 2574)


I085B07
346
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDFPMAP
(SEQ ID NO: 2433)


I085B10
347
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHSPLY
(SEQ ID NO: 2470)


I085B12
348
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQDPLSP
(SEQ ID NO: 2372)


I085C02
349
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDDPLLS
(SEQ ID NO: 2430)


I085C03
350
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHGPLLI
(SEQ ID NO: 2400)


I085C05
351
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGSPLLF
(SEQ ID NO: 2491)


I085C06
352
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAALSF
(SEQ ID NO: 2341)


I085C07
353
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTPLRF
(SEQ ID NO: 2375)


I085C09
354
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHSPLT
(SEQ ID NO: 2468)


I085C10
355
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFSPLLF
(SEQ ID NO: 2471)


I085C12
356
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHPLFF
(SEQ ID NO: 2680)


I085D01
357
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRPLLLF
(SEQ ID NO: 2548)


I085D02
358
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSQYLDF
(SEQ ID NO: 2523)


I085D03
359
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLLF
(SEQ ID NO: 2713)


I085004
360
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYFPLVF
(SEQ ID NO: 2646)


I085006
361
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-113
SRDLLLFPGSPLLD
(SEQ ID NO: 2488)


I085007
362
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLLF
(SEQ ID NO: 2694)


I085D08
363
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSYLSP
(SEQ ID NO: 2477)


I085D09
364
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQTPLFP
(SEQ ID NO: 2467)


I085D10
365
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLHP
(SEQ ID NO: 2563)


I085D11
366
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLAP
(SEQ ID NO: 2510)


I085D12
367
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTTLRF
(SEQ ID NO: 2495)


I085E01
368
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAVLHF
(SEQ ID NO: 2620)


I085E02
369
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLRL
(SEQ ID NO: 2575)


I085E07
370
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDALSF
(SEQ ID NO: 2568)


I085E08
371
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLDP
(SEQ ID NO: 2603)


I085E09
372
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPPRF
(SEQ ID NO: 2628)


I085E10
373
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SROLLLFPSEPLWP
(SEQ ID NO: 2668)


I085E11
374
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLSN
(SEQ ID NO: 2716)


I085E12
375
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHLPLTP
(SEQ ID NO: 2431)


I085F01
376
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLLF
(SEQ ID NO: 2551)


I085F02
377
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLQL
(SEQ ID NO: 2376)


I085F03
378
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYTPLLF
(SEQ ID NO: 2682)


I085F04
379
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLAF
(SEQ ID NO: 2707)


I085F05
380
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLYF
(SEQ ID NO: 2706)


I085F06
381
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAHLLF
(SEQ ID NO: 2586)


I085F07
382
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAOPLRF
(SEQ ID NO: 2410)


I085F09
383
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDHAFFV
(SEQ ID NO: 2439)


I085F10
384
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDSGFA
(SEQ ID NO: 2662)


I08SF11
385
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSYLEF
(SEQ ID NO: 2339)


I085F12
386
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLII
(SEQ ID NO: 2558)


I085001
387
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLHP
(SEQ ID NO: 2605)


I085G02
388
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLLL
(SEQ ID NO: 2613)


I085G03
389
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAAPLLF
(SEQ ID NO: 2403)


I085G04
390
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLDP
(SEQ ID NO: 2601)


I085G07
391
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAVLDI
(SEQ ID NO: 2629)


I085G08
392
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSEPLFF
(SEQ ID NO: 2664)


I085G09
393
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSVLWP
(SEQ ID NO: 2338)


I085G10
394
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHAPLQ
(SEQ ID NO: 2554)


I085011
395
141-249
163-173
189-195
228-238
1-123
26-35
5O-66
99-112
SRDLLLFPDSPLAP
(SEQ ID NO: 2445)


I085012
396
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLHP
(SEQ ID NO: 2576)


I085H10
397
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-112
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I086A03
398
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSMPLTF
(SEQ ID NO: 2695)


I086A04
399
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSILHP
(SEQ ID NO: 2438)


I086A05
400
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLSH
(SEQ ID NO: 2569)


I086A07
401
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDAALRF
(SEQ ID NO: 2421)


I086A09
402
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSHLSF
(SEQ ID NO: 2704)


I086A10
403
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLSS
(SEQ ID NO: 2624)


I086A11
404
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLTP
(SEQ ID NO: 2577)


I086A12
405
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYDPLHS
(SEQ ID NO: 2635)


I086B02
406
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHFPLHP
(SEQ ID NO: 2348)


I086B03
407
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAHPLLF
(SEQ ID NO: 2412)


I086B05
408
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFEPLII
(SEQ ID NO: 2457)


I086B06
409
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I086B07
410
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLYF
(SEQ ID NO: 2720)


I086B09
411
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLSF
(SEQ ID NO: 2579)


I086B10
412
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDDGLSS
(SEQ ID NO: 2428)


I086B11
413
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPISPLCF
(SEQ ID NO: 2530)


I086C03
414
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLYG
(SEQ ID NO: 2535)


I086C05
415
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSLFF
(SEQ ID NO: 2427)


I086C07
416
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQGPLRF
(SEQ ID NO: 2440)


I086C08
417
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAAPLAF
(SEQ ID NO: 2401)


I086C09
418
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHLPLLF
(SEQ ID NO: 2350)


I086C10
419
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTFPLIF
(SEQ ID NO: 2541)


I086C11
420
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDDPLSF
(SEQ ID NO: 2432)


I086C12
421
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTDSLLF
(SEQ ID NO: 2622)


I086D01
422
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLTP
(SEQ ID NO: 2630)


I086D04
423
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPYAPLYD
(SEQ ID NO: 2697)


I086D05
424
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLSF
(SEQ ID NO: 2461)


I086D06
425
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLDL
(SEQ ID NO: 2379)


I086D07
426
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTHLTF
(SEQ ID NO: 2365)


I086D08
427
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSSLDF
(SEQ ID NO: 2473)


I086D09
428
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNHPMFP
(SEQ ID NO: 2665)


I086D10
429
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLSSLEF
(SEQ ID NO: 2587)


I086D11
430
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLHP
(SEQ ID NO: 2610)


I086D12
431
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAHLRF
(SEQ ID NO: 2469)


I086E02
432
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYDPLHF
(SEQ ID NO: 2621)


I086E03
433
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDALQS
(SEQ ID NO: 2598)


I086E05
434
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTPLTF
(SEQ ID NO: 2567)


I086E07
435
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAAHLSF
(SEQ ID NO: 2398)


I086E09
437
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFSPLAP
(SEQ ID NO: 2464)


I086E10
438
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLDF
(SEQ ID NO: 2367)


I086E12
439
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLRF
(SEQ ID NO: 2522)


I086F02
440
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLRI
(SEQ ID NO: 2714)


I086F05
441
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTEPLQF
(SEQ ID NO: 2540)


I086F08
442
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDPLSA
(SEQ ID NO: 2643)


I086F09
443
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYNPPIF
(SEQ ID NO: 2653)


I086F11
444
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTPLLF
(SEQ ID NO: 2489)


I086G03
445
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLDL
(SEQ ID NO: 2513)


I086G04
446
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFDPLLI
(SEQ ID NO: 2454)


I086G05
447
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTDALRI
(SEQ ID NO: 2537)


I086G06
448
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAAPLTP
(SEQ ID NO: 2407)


I086G07
449
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPEGPLLF
(SEQ ID NO: 2448)


I086G09
450
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLSF
(SEQ ID NO: 2385)


I086G10
451
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPADSLSF
(SEQ ID NO: 2391)


I086H05
452
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLTH
(SEQ ID NO: 2679)


I089A01
453
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHDPLI
(SEQ ID NO: 2612)


I089A03
454
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLTPLLI
(SEQ ID NO: 2590)


I089A06
455
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTPLHF
(SEQ ID NO: 2485)


I089A07
456
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTDALYF
(SEQ ID NO: 2539)


I089A08
457
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYTPLLF
(SEQ ID NO: 2682)


I089A10
458
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHQPLTF
(SEQ ID NO: 2436)


I089A11
459
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTYLDF
(SEQ ID NO: 2572)


I089B01
460
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLHS
(SEQ ID NO: 2450)


I089B02
461
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I089B03
462
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLQP
(SEQ ID NO: 2528)


I089504
463
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTHPLLF
(SEQ ID NO: 2556)


I089B05
464
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLIF
(SEQ ID NO: 2712)


I089B06
465
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPMAPLSP
(SEQ ID NO: 2596)


I089B07
466
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSGLDA
(SEQ ID NO: 2374)


I089B08
467
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAAPLSP
(SEQ ID NO: 2405)


I089B09
468
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKSPILF
(SEQ ID NO: 2384)


I089B10
469
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLFF
(SEQ ID NO: 2571)


I089B11
470
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNSPLFP
(SEQ ID NO: 2388)


I089C01
471
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I089C02
472
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLLF
(SEQ ID NO: 2551)


I089C03
473
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYHPLLF
(SEQ ID NO: 2532)


I089C05
474
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSALRF
(SEQ ID NO: 2722)


I089C06
475
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSPYLSF
(SEQ ID NO: 2701)


I089C07
476
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLFD
(SEQ ID NO: 2683)


I089C09
477
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPFTF
(SEQ ID NO: 2507)


I089D01
478
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLVL
(SEQ ID NO: 2581)


I089D02
479
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I089D03
480
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I089D04
481
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLSP
(SEQ ID NO: 2717)


I089D05
482
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLFT
(SEQ ID NO: 2546)


I089D07
483
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNDPLLI
(SEQ ID NO: 2634)


I089D08
484
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHAPLQ
(SEQ ID NO: 2554)


I089D09
485
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHAFHE
(SEQ ID NO: 2677)


I089D11
486
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNHPLYP
(SEQ ID NO: 2663)


I089E01
487
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLFP
(SEQ ID NO: 2657)


I089E02
488
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQDPLHP
(SEQ ID NO: 2346)


I089E03
489
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDAPLFP
(SEQ ID NO: 2423)


I089E04
490
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLLI
(SEQ ID NO: 2453)


I089E06
491
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGSPLLF
(SEQ ID NO: 2491)


I089E09
492
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLTF
(SEQ ID NO: 2718)


I089E10
493
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTQPLSF
(SEQ ID NO: 2566)


I089E11
494
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLSPLWP
(SEQ ID NO: 2578)


I089F01
495
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTFPLLF
(SEQ ID NO: 2380)


I089F03
496
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLLL
(SEQ ID NO: 2580)


I089F04
497
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLLF
(SEQ ID NO: 2670)


I089F05
498
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLRI
(SEQ ID NO: 2459)


I089F06
499
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLLF
(SEQ ID NO: 2490)


I089F08
500
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTPLTF
(SEQ ID NO: 2567)


I089F09
501
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLAPLSF
(SEQ ID NO: 2555)


I089F10
502
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNQPLSF
(SEQ ID NO: 2667)


I089F11
503
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLEPMHF
(SEQ ID NO: 2565)


I089G01
504
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLTF
(SEQ ID NO: 2626)


I089G02
505
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHPLLF
(SEQ ID NO: 2687)


I089G03
506
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTPLVF
(SEQ ID NO: 2721)


I089G05
507
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGSPLTF
(SEQ ID NO: 2389)


I089G06
508
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLLF
(SEQ ID NO: 2514)


I089G07
509
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLDF
(SEQ ID NO: 2597)


I089G08
510
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHPLSF
(SEQ ID NO: 2688)


I089G11
511
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSFPLLF
(SEQ ID NO: 2671)


I089H10
512
141-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I090A02
513
142-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAKPLLF
(SEQ ID NO: 2416)


I090A03
514
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNSTLSF
(SEQ ID NO: 2678)


I090A04
515
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDAPLTP
(SEQ ID NO: 2426)


I090A05
516
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLLI
(SEQ ID NO: 2648)


I090A06
517
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTYPLSF
(SEQ ID NO: 2600)


I090A07
518
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTEPLVL
(SEQ ID NO: 2479)


I090A08
519
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTYPLHF
(SEQ ID NO: 2584)


I090B01
520
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLTF
(SEQ ID NO: 2627)


I090B03
521
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLTN
(SEQ ID NO: 2705)


I090B04
522
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLEA
(SEQ ID NO: 2520)


I090B05
523
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDHPLLF
(SEQ ID NO: 2442)


I090B06
524
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLSF
(SEQ ID NO: 2496)


I090B08
525
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRGPLRF
(SEQ ID NO: 2542)


I090B11
526
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFTPLTF
(SEQ ID NO: 2474)


I090B12
527
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQHPLSP
(SEQ ID NO: 2452)


I090C01
528
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPIVF
(SEQ ID NO: 2591)


I090C02
529
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLTF
(SEQ ID NO: 2702)


I090C03
530
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLRF
(SEQ ID NO: 2493)


I090C05
531
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTPLTF
(SEQ ID NO: 2567)


I090C06
532
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLDF
(SEQ ID NO: 2538)


I090C07
533
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAGFDS
(SEQ ID NO: 2498)


I090C08
534
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLSF
(SEQ ID NO: 2676)


I090C10
535
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGRPLTF
(SEQ ID NO: 2358)


I090D02
536
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAEHLLF
(SEQ ID NO: 2408)


I090D03
537
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLHP
(SEQ ID NO: 2351)


I090D04
538
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLTA
(SEQ ID NO: 2654)


I090D05
539
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLFE
(SEQ ID NO: 2529)


I090D06
540
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLDF
(SEQ ID NO: 2367)


I090D07
541
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFGTLRP
(SEQ ID NO: 2462)


I090D08
542
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLVF
(SEQ ID NO: 2723)


I090D09
543
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLAF
(SEQ ID NO: 2505)


I090D12
544
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLSF
(SEQ ID NO: 2579)


I090E04
545
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLLL
(SEQ ID NO: 2552)


I090E05
546
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPISF
(SEQ ID NO: 2588)


I090E06
547
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQGPLSF
(SEQ ID NO: 2443)


I090E07
548
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGSPLHP
(SEQ ID NO: 2484)


I090E09
549
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDPLSF
(SEQ ID NO: 2647)


I090E11
550
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDGLAP
(SEQ ID NO: 2700)


I090E12
551
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTSPLTF
(SEQ ID NO: 2582)


I090F01
552
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNGPLHP
(SEQ ID NO: 2649)


I090F02
553
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLSF
(SEQ ID NO: 2696)


I090F03
554
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLSF
(SEQ ID NO: 2526)


I090F04
555
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFFPLQF
(SEQ ID NO: 2460)


I090F05
556
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLDPLHF
(SEQ ID NO: 2359)


I090F06
557
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSEPLQL
(SEQ ID NO: 2666)


I090F07
558
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPFAPLRF
(SEQ ID NO: 2451)


I090F08
559
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLHPLIF
(SEQ ID NO: 2570)


I090F09
560
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I090F10
561
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLRI
(SEQ ID NO: 2527)


I090F11
562
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSNPLTF
(SEQ ID NO: 2698)


I090G01
563
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLEI
(SEQ ID NO: 2347)


I090G02
564
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLQF
(SEQ ID NO: 2395)


I090G04
565
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLAF
(SEQ ID NO: 2633)


I090G05
566
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLAF
(SEQ ID NO: 2472)


I090G06
567
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLAF
(SEQ ID NO: 2656)


I090607
568
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTPLDS
(SEQ ID NO: 2480)


I090008
569
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTPLTF
(SEQ ID NO: 2492)


I090G09
570
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSEPLRI
(SEQ ID NO: 2356)


I090610
571
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLDF
(SEQ ID NO: 2343)


I090612
572
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNRGLDL
(SEQ ID NO: 2669)


I091A02
573
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYDPLFM
(SEQ ID NO: 2724)


I091A03
574
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLYP
(SEQ ID NO: 2592)


I091A06
575
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLAF
(SEQ ID NO: 2594)


I091A11
576
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPITF
(SEQ ID NO: 2441)


I091B01
577
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRYPLFF
(SEQ ID NO: 2585)


I091B02
578
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLDF
(SEQ ID NO: 2361)


I091B04
579
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLQF
(SEQ ID NO: 2395)


I091B05
580
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLEL
(SEQ ID NO: 2475)


I091B07
581
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLTF
(SEQ ID NO: 2626)


I091B10
582
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLAF
(SEQ ID NO: 2342)


I091B11
583
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLDF
(SEQ ID NO: 2444)


I091B12
584
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHPLTF
(SEQ ID NO: 2690)


I091C02
585
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPAHPLVI
(SEQ ID NO: 2414)


I091C03
586
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLYP
(SEQ ID NO: 2378)


I091C04
587
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLTF
(SEQ ID NO: 2531)


I091C05
588
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTTPLHF
(SEQ ID NO: 2583)


I091C06
589
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I091C09
590
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLSF
(SEQ ID NO: 2415)


I091C11
591
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYHSYDI
(SEQ ID NO: 2650)


I091C12
592
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYATLSF
(SEQ ID NO: 2618)


IO9lDOl
593
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNSPLAP
(SEQ ID NO: 2672)


I091D02
594
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLQP
(SEQ ID NO: 2673)


I091D04
595
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQOPLSF
(SEQ ID NO: 2443)


I091D05
596
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLAP
(SEQ ID NO: 2606)


I091D06
597
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHSPLL
(SEQ ID NO: 2456)


I091D07
598
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNGALRF
(SEQ ID NO: 2645)


I091D09
599
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLRF
(SEQ ID NO: 2719)


I091E01
600
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDAPLHP
(SEQ ID NO: 2425)


I091E02
601
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLFP
(SEQ ID NO: 2689)


I091E03
602
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLWP
(SEQ ID NO: 2352)


I091E04
603
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKSPLAF
(SEQ ID NO: 2547)


I091E06
604
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLHP
(SEQ ID NO: 2576)


I091E07
605
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-1l2
SRDLLLFPNHPLTF
(SEQ ID NO: 2661)


I091E08
606
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLDS
(SEQ ID NO: 2607)


I091E09
607
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLDF
(SEQ ID NO: 2361)


I091E10
608
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLEF
(SEQ ID NO: 2711)


I091F01
609
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLFF
(SEQ ID NO: 2486)


I091F03
610
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPMAPLVG
(SEQ ID NO: 2599)


I091F05
611
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLAPLHP
(SEQ ID NO: 2553)


I091F06
612
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLGF
(SEQ ID NO: 2353)


I091F07
613
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I091F08
614
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQSPLLF
(SEQ ID NO: 2458)


I091F09
615
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEHLSF
(SEQ ID NO: 2354)


I091F10
616
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLDF
(SEQ ID NO: 2444)


I091F11
617
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLSP
(SEQ ID NO: 2549)


I091F12
618
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I091G01
619
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAALYP
(SEQ ID NO: 2386)


I091G03
620
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNDPLFO
(SEQ ID NO: 2355)


I091G04
621
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGAPLSP
(SEQ ID NO: 2478)


I091G05
622
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
ARDLLLFPAAPLWP
(SEQ ID NO: 2397)


I091G06
623
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPNDPLR
(SEQ ID NO: 2637)


I091G07
624
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLDP
(SEQ ID NO: 2345)


I091G09
625
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLFP
(SEQ ID NO: 2349)


I091G10
626
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSDPLVF
(SEQ ID NO: 2660)


I091G11
627
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGSPLTF
(SEQ ID NO: 2389)


I091G12
628
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSHLEF
(SEQ ID NO: 2655)


I104A01
629
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQSPLHP
(SEQ ID NO: 2455)


I104A07
630
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLFP
(SEQ ID NO: 2689)


I104A08
631
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLTF
(SEQ ID NO: 2617)


I104A09
632
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQNPLHP
(SEQ ID NO: 2506)


I104A10
633
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLCF
(SEQ ID NO: 2636)


I104A11
634
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLSF
(SEQ ID NO: 2611)


I104A12
635
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPMAPLRF
(SEQ ID NO: 2593)


I104B02
636
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLSF
(SEQ ID NO: 2557)


I104B04
637
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLYP
(SEQ ID NO: 2387)


I104B09
638
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLQF
(SEQ ID NO: 2395)


I104B11
639
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLTF
(SEQ ID NO: 2531)


I104C01
640
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLYP
(SEQ ID NO: 2710)


I104C04
641
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLLF
(SEQ ID NO: 2417)


I104C05
642
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRHPLLF
(SEQ ID NO: 2543)


I104C06
643
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SRDLLLFPHAPLE
(SEQ ID NO: 2524)


I104C07
644
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLHP
(SEQ ID NO: 2370)


I104C09
645
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHFPLIF
(SEQ ID NO: 2399)


I104C11
646
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLIF
(SEQ ID NO: 2644)


I104001
647
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNHAFDL
(SEQ ID NO: 2652)


I104002
648
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHTILYP
(SEQ ID NO: 2497)


I104D03
649
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDWPLYP
(SEQ ID NO: 2483)


I104D04
650
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLFL
(SEQ ID NO: 2511)


I104D07
651
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLHP
(SEQ ID NO: 2691)


I104D08
652
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPMDP
(SEQ ID NO: 2595)


I104D09
653
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLTF
(SEQ ID NO: 2500)


I104E01
654
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRATLEF
(SEQ ID NO: 2502)


I104E02
655
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHSPLFP
(SEQ ID NO: 2447)


I104E03
656
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNDPLVL
(SEQ ID NO: 2641)


I104E05
657
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLYI
(SEQ ID NO: 2463)


I104E11
658
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLSF
(SEQ ID NO: 2385)


I104E12
659
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I104F02
660
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLSP
(SEQ ID NO: 2616)


I104F03
661
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLRF
(SEQ ID NO: 2360)


I104F04
662
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGDPLDF
(SEQ ID NO: 2481)


I104F05
663
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHGPLTF
(SEQ ID NO: 2402)


I104F06
664
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLSP
(SEQ ID NO: 2573)


I104F07
665
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLIL
(SEQ ID NO: 2465)


I104F10
666
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNSPLSP
(SEQ ID NO: 2362)


I104F11
667
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQDPLVF
(SEQ ID NO: 2708)


I104F12
668
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKAPLVF
(SEQ ID NO: 2544)


I104G04
669
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLRF
(SEQ ID NO: 2559)


I104G05
670
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLAP
(SEQ ID NO: 2476)


I104G09
671
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTAPLNF
(SEQ ID NO: 2518)


I104611
672
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRHLLLFPQOPLSF
(SEQ ID NO: 2482)


I105A02
673
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHLPLNP
(SEQ ID NO: 2494)


I105A03
674
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I105A04
675
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPGAPLAP
(SEQ ID NO: 2487)


I105A08
676
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLYP
(SEQ ID NO: 2378)


I105A09
677
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLSF
(SEQ ID NO: 2557)


I105A11
678
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHSFDI
(SEQ ID NO: 2692)


I105B04
679
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLHP
(SEQ ID NO: 2658)


I105B05
680
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYSPLSF
(SEQ ID NO: 2676)


I105B07
681
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I105B08
682
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I105B10
683
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I105B11
684
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLSP
(SEQ ID NO: 2651)


I105B12
685
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLDPLII
(SEQ ID NO: 2560)


I105C02
686
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLAF
(SEQ ID NO: 2472)


I105C03
687
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSSPLSF
(SEQ ID NO: 2715)


I105C05
688
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I105C06
689
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLDF
(SEQ ID NO: 2367)


I105C08
690
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLTF
(SEQ ID NO: 2562)


I105C12
691
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQHGFDA
(SEQ ID NO: 2446)


I105D04
692
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLRE
(SEQ ID NO: 2360)


I105D06
693
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLSF
(SEQ ID NO: 2368)


I105D08
694
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLAF
(SEQ ID NO: 2608)


I105D09
695
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAAFDV
(SEQ ID NO: 2619)


I105D10
696
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHEPLFP
(SEQ ID NO: 2640)


I105D11
697
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSALTF
(SEQ ID NO: 2519)


I105E01
698
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDS
(SEQ ID NO: 2422)


I105E06
699
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I105E11
700
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNSPLHP
(SEQ ID NO: 2675)


I105F03
701
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLDS
(SEQ ID NO: 2409)


I105F06
702
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLHP
(SEQ ID NO: 2691)


I105F07
703
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSWPLTF
(SEQ ID NO: 2340)


I105F09
704
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I105F12
705
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPTYPLVF
(SEQ ID NO: 2604)


I105603
706
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLHP
(SEQ ID NO: 2370)


I105G08
707
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKHPLVF
(SEQ ID NO: 2366)


I105G09
708
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I105G10
709
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDA
(SEQ ID NO: 2419)


I105G11
710
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLLF
(SEQ ID NO: 2614)


I107A01
711
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRHPLVF
(SEQ ID NO: 2545)


I107A03
712
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLYP
(SEQ ID NO: 2501)


I107A06
713
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLDP
(SEQ ID NO: 2369)


I107A07
714
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLSP
(SEQ ID NO: 2371)


I107A09
715
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLSP
(SEQ ID NO: 2699)


I107A12
716
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLSF
(SEQ ID NO: 2564)


I107B02
717
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLFP
(SEQ ID NO: 2533)


I107B04
718
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLTF
(SEQ ID NO: 2420)


I107805
719
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I107C01
720
139-247
161-171
187-193
226-236
1-121
24-33
48-64
97-110
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I107C02
721
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMYV
(SEQ ID NO: 2504)


I107C04
722
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLHP
(SEQ ID NO: 2357)


I107C06
723
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLAP
(SEQ ID NO: 2510)


I107C08
724
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLEP
(SEQ ID NO: 2681)


I107C10
725
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHAFDL
(SEQ ID NO: 2674)


I107D01
726
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYAPLDF
(SEQ ID NO: 2361)


I107D04
727
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLSF
(SEQ ID NO: 2625)


I107D07
728
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSHSFDV
(SEQ ID NO: 2693)


I107D12
729
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDT
(SEQ ID NO: 2424)


I107E01
730
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPMLGLDL
(SEQ ID NO: 2499)


I107E05
731
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLDF
(SEQ ID NO: 2367)


I107E07
732
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRSPLLF
(SEQ ID NO: 2551)


I107E09
733
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKAPLTF
(SEQ ID NO: 2382)


I107F01
734
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLSP
(SEQ ID NO: 2623)


I107F05
735
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLAP
(SEQ ID NO: 2510)


I107F09
736
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLAP
(SEQ ID NO: 2394)


I107F10
737
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRTPLLF
(SEQ ID NO: 2373)


I107G01
738
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLSP
(SEQ ID NO: 2371)


I107G05
739
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLYP
(SEQ ID NO: 2387)


I107H02
740
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I107H06
741
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLSF
(SEQ ID NO: 2496)


I107H09
742
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLEM
(SEQ ID NO: 2536)


I107H10
743
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLAP
(SEQ ID NO: 2510)


I108A12
744
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I108B03
745
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLLF
(SEQ ID NO: 2515)


I108B04
746
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPLSPLVP
(SEQ ID NO: 2396)


I108C09
747
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDPLGF
(SEQ ID NO: 2353)


I108C11
748
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSLLF
(SEQ ID NO: 2429)


I108D10
749
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I108D11
750
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I108D12
751
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLNP
(SEQ ID NO: 2709)


I108E01
752
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I108E03
753
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKHPLRF
(SEQ ID NO: 2393)


I108E05
754
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLFP
(SEQ ID NO: 2533)


I108E07
755
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHAPLDP
(SEQ ID NO: 2369)


I108E08
756
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I108E09
757
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLSP
(SEQ ID NO: 2623)


I108E10
758
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLDL
(SEQ ID NO: 2509)


I108E11
759
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLEF
(SEQ ID NO: 2516)


I108F10
760
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLSP
(SEQ ID NO: 2371)


I108F12
761
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPFDA
(SEQ ID NO: 2508)


I108G01
762
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLRF
(SEQ ID NO: 2360)


I108002
763
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPDAPLAP
(SEQ ID NO: 2381)


I108007
764
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLAP
(SEQ ID NO: 2476)


I108G10
765
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSLLF
(SEQ ID NO: 2429)


I108011
766
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLTF
(SEQ ID NO: 2377)


I108G12
767
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLTF
(SEQ ID NO: 2377)


I108H01
768
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLHF
(SEQ ID NO: 2512)


I108H02
769
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPNAPLNP
(SEQ ID NO: 2615)


I108H06
770
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I108H08
771
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPASPLNP
(SEQ ID NO: 2364)


I111A06
772
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLHP
(SEQ ID NO: 2691)


I111B12
773
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I111C01
774
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQHGLDL
(SEQ ID NO: 2449)


I111D06
775
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRDPLLF
(SEQ ID NO: 2515)


I111E04
776
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I111E10
777
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLHP
(SEQ ID NO: 2691)


I111F07
780
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLYP
(SEQ ID NO: 2501)


I111G02
781
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKAPLDF
(SEQ ID NO: 2534)


I111H10
782
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPQHGFDA
(SEQ ID NO: 2703)


I113A04
783
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPSAPLWP
(SEQ ID NO: 2352)


I113A12
784
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQEPLAP
(SEQ ID NO: 2434)


I113B06
785
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLEP
(SEQ ID NO: 2411)


I113C06
786
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHGFDA
(SEQ ID NO: 2406)


I113004
787
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I113G05
788
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYSLLL
(SEQ ID NO: 2517)


I113G10
789
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHPLQF
(SEQ ID NO: 2413)


I113G11
790
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I113H06
791
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I113H07
792
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I113H09
793
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYTLLF
(SEQ ID NO: 2525)


I114C04
794
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHGFDA
(SEQ ID NO: 2406)


I114C12
795
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLHP
(SEQ ID NO: 2691)


I114D04
796
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I114D06
797
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I114D10
798
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYSLVL
(SEQ ID NO: 2521)


I114E01
799
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQEPLSP
(SEQ ID NO: 2435)


I114E02
800
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQESFSL
(SEQ ID NO: 2437)


I114E03
801
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPKAPLTF
(SEQ ID NO: 2382)


I114E11
802
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDSFFL
(SEQ ID NO: 2383)


I114H01
803
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I114H06
804
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHALDV
(SEQ ID NO: 2404)


I114H09
805
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I115A02
806
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPDHSFDL
(SEQ ID NO: 2684)


I115A07
807
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I115B10
808
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I115C05
809
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPRAPLYP
(SEQ ID NO: 2501)


I115C06
810
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHHSFDL
(SEQ ID NO: 2150)


I115C08
811
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I115C12
812
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDT
(SEQ ID NO: 2424)


I115D07
813
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I115E09
814
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHRFDL
(SEQ ID NO: 2418)


I115F06
815
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHYGMDV
(SEQ ID NO: 2685)


I115F07
816
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHYPLLF
(SEQ ID NO: 2686)


I115F12
817
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHHSFDL
(SEQ ID NO: 2150)


I115604
818
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHRFDL
(SEQ ID NO: 2418)


I115G05
819
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I115608
820
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHDSFDL
(SEQ ID NO: 2631)


I115H04
821
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHANLSP
(SEQ ID NO: 2503)


I115H07
822
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I115H09
823
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHRFDL
(SEQ ID NO: 2418)


I116A07
824
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPYEPLRF
(SEQ ID NO: 2642)


I116B01
825
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I116B12
826
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I116C06
827
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I116D07
828
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I116E02
829
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHRFDL
(SEQ ID NO: 2418)


I116E04
830
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHHSFDL
(SEQ ID NO: 2147)


I116F02
831
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRYLLLFPHHSFDL
(SEQ ID NO: 2150)


I116F11
832
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPHYPLLF
(SEQ ID NO: 2344)


I116G05
833
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
SRDLLLFPQAPLSP
(SEQ ID NO: 2699)


I001C09
834
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I006D07
835
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I007B03
836
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I007F11
837
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
DGIDILLVPAALMDV
(SEQ ID NO: 2160)


I007H08
838
144-254
166-179
195-201
234-243
1-128
26-37
52-69
102-117
DRYDILTGYYYYGMDV
(SEQ ID NO: 2129)


I008A09
839
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
DREAYYDILTGYYLYYYYMDV
(SEQ ID NO: 2172)


I008B01
840
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008C02
841
145-255
167-180
196-202
235-244
1-129
26-37
52-67
100-118
HVRDYDILTGYYRGHYFDY
(SEQ ID NO: 2167)


I008C03
842
143-250
164-174
190-196
229-239
1-127
26-35
50-65
98-116
EGSYDILTGYYVGVGRIVIDV
(SEQ ID NO: 2171)


I008C12
843
146-256
168-181
197-203
236-245
1-130
26-35
50-68
101-119
FNPTYDILTGYYIGGYFQH
(SEQ ID NO: 2155)


I012A06
844
147-254
169-179
195-201
234-243
1-129
26-37
52-67
100-118
GRWDYDLLTGEHLGYYFDY
(SEQ ID NO: 2162)


I016E05
845
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016F02
846
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I016F04
847
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016H07
848
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
GYHDPLTSYNYNWFDP
(SEQ ID NO: 2163)


I018C02
849
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018C10
850
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I018D07
851
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I018H08
852
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018H09
853
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I021B05
854
143-253
165-178
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I022E02
855
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGLDI
(SEQ ID NO: 2157)


I026E03
856
143-251
165-175
191-197
230-240
1-125
26-35
50-66
99-114
TDYDILTGYPMGYFDP
(SEQ ID NO: 2173)


I027A07
857
145-255
167-179
195-201
234-244
1-128
26-35
50-66
99-117
GGEYDILTGYYFGLGVYDY
(SEQ ID NO: 2170)


I028A06
858
142-253
164-176
192-198
231-242
1-126
26-35
50-66
99-115
GGDYDILTGLYYYGMDV
(SEQ ID NO: 2156)


I029D07
859
141-250
163-176
192-198
231-239
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I029F11
860
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I031C03
861
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031C07
862
148-258
170-183
199-205
238-247
1-131
26-35
50-66
99-120
SSPPRWYDALTGDSSYHSAMDV
(SEQ ID NO: 2169)


I031F09
863
145-255
167-179
195-201
234-244
1-127
26-35
50-66
99-116
DEGRDLLTGYYWPNFFDS
(SEQ ID NO: 2168)


I031G08
864
148-259
170-182
198-204
237-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031G10
865
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I031G11
866
145-255
167-179
195-201
234-244
1-127
26-35
50-66
99-116
DEGRDLLTGYYWPNFFDS
(SEQ ID NO: 2168)


I037E07
867
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
DGIDILLVPAALMDV
(SEQ ID NO: 2160)


I037E12
868
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
DGIDILLVPAALMDV
(SEQ ID NO: 2160)


I050A07
869
146-257
168-181
197-203
236-246
1-129
26-40
55-71
104-118
QDNDPLTGYKLGFDY
(SEQ ID NO: 2164)


I061D02
870
144-254
166-179
195-201
234-243
1-128
26-37
52-69
102-117
DRYDILTGYYYYGMDV
(SEQ ID NO: 2129)


I061E07
871
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I061H01
872
146-256
168-181
197-203
236-245
1-130
26-35
50-68
101-119
FNPTYDILTGYYIGGYFQH
(SEQ ID NO: 2155)


I001A03
873
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
ERHYYDILTGYQTGYGMDV
(SEQ ID NO: 2784)


I001A07
874
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001A08
875
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001A10
876
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001A12
877
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001B02
878
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
DRETKVGYGMDV
(SEQ ID NO: 2945)


I001B07
879
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001C06
880
143-253
165-178
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I001C08
881
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I001C12
882
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001D08
883
140-250
162-175
191-197
230-239
1-124
26-35
50-65
98-113
DSYDILTGYRGYYFDY
(SEQ ID NO: 2745)


I001D12
884
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001E05
885
143-253
165-178
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I001E07
886
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001G09
887
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I001H05
888
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
ERHYYDILTGYQTGYGMDV
(SEQ ID NO: 2784)


I001H08
889
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I003A01
890
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I003A06
891
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I003A07
892
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I003A10
893
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I003B03
894
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I003B04
895
140-248
162-172
188-194
227-237
1-122
25-34
49-65
98-111
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I003B09
896
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTOYSYYGMDV
(SEQ ID NO: 2135)


I003C01
897
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I003C02
898
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
GDYDILTGYPAECFQI
(SEQ ID NO: 2854)


I003C03
899
142-250
164-174
190-196
229-239
1-125
26-35
50-66
99-114
GDYDILTGYPAECFQI
(SEQ ID NO: 2854)


I003C12
900
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I003D04
901
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I003E05
902
142-253
164-176
192-198
231-242
1-125
26-35
50-66
99-114
GDYDILTOYPAECFQI
(SEQ ID NO: 2854)


I003F01
903
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I003F02
904
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I003001
905
145-254
168-179
195-201
234-243
1-127
26-35
50-66
99-116
GTGYDILTGYYMOSAFDQ
(SEQ ID NO: 2800)


I003005
906
144-255
166-179
195-201
234-244
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I003G06
907
146-256
168-181
197-203
236-245
1-129
26-35
50-66
99-118
DRGGNYDILTGYYFHHGVDV
(SEQ ID NO: 2914)


I003G11
908
143-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
DAQSYYDILTGYQSYAFDI
(SEQ ID NO: 2183)


I003H02
909
142-253
164-176
192-198
233-242
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I003H05
910
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I003H08
911
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTOYSYYGMDV
(SEQ ID NO: 2135)


I005A01
912
141-249
162-172
188-194
227-238
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I005A02
913
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
EGRDILTGVYYYGLDV
(SEQ ID NO: 2893)


I005B01
914
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I005B09
915
137-247
159-172
188-194
227-236
1-121
26-35
50-65
98-110
TYYDILTGRFFDI
(SEQ ID NO: 2866)


I005C01
916
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I005D02
917
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
DLRYDILTGYHDAFDI
(SEQ ID NO: 2890)


I005003
918
142-249
165-175
191-197
230-238
1-126
26-35
50-66
99-115
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I005E01
919
142-249
165-175
191-197
230-238
1-126
26-35
50-66
99-115
GTYYDILTGYFHYGMDV
(SEQ ID NO: 2774)


I005E08
920
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I005F01
921
142-248
164-174
190-196
229-238
1-124
26-35
50-66
99-113
DQHDILTGVYYGMDV
(SEQ ID NO: 2921)


I005F02
922
144-251
167-177
193-199
232-240
1-128
26-35
50-66
99-117
VSPSYDILTGYYLPHAFDV
(SEQ ID NO: 2849)


I005F04
923
137-247
159-172
188-194
227-236
1-121
26-35
50-65
98-110
TYYDILTGRFFDI
(SEQ ID NO: 2866)


I005F0S
924
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
PSYDILTGYLYYFDY
(SEQ ID NO: 2850)


I005G01
925
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
DLRYDILTGYHDAFDI
(SEQ ID NO: 2890)


I005G08
926
142-249
165-175
191-197
230-238
1-126
26-35
50-66
99-115
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I005H02
927
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
GQYYDILTGYNWFDP
(SEQ ID NO: 2857)


I006B01
928
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I006C09
929
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
GGYSSGWLRGGPYNWFDP
(SEQ ID NO: 2967)


I006D09
930
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
GDYDILTGYYIPLRDY
(SEQ ID NO: 2792)


I006E01
931
143-253
165-178
194-200
233-242
1-127
26-35
50-68
101-116
NLFDVWTLPYYYYMDV
(SEQ ID NO: 2965)


I006E07
932
143-250
166-176
192-198
231-239
1-127
26-35
50-66
99-116
ADYDILTGYSPLTYGMDV
(SEQ ID NO: 2762)


I006F01
933
140-250
162-175
191-197
230-239
1-124
26-35
50-68
101-113
MYYDILTGHNFDY
(SEQ ID NO: 2879)


I006F02
934
142-253
164-176
192-198
231-242
1-126
26-35
50-66
99-115
VSRDILTGNYYYYGMDV
(SEQ ID NO: 2817)


I006F07
935
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
GGYSSGWLRGGPYNWFDP
(SEQ ID NO: 2967)


I006G01
936
146-253
169-179
195-201
234-242
1-130
26-35
50-68
101-119
AGGYYDILTGRDYYYGMDV
(SEQ ID NO: 2877)


I006G04
937
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
RRYALDY
(SEQ ID NO: 2920)


I006H01
938
146-253
167-177
193-199
232-242
1-130
26-35
50-65
98-119
DRGSYDILTOYYTPPHYYGMDV
(SEQ ID NO: 2761)


I006H02
939
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
GGYSSGWLRGGPYNWFDP
(SEQ ID NO: 2967)


I007A01
940
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I007A08
941
139-249
161-174
190-196
229-238
1-123
26-35
50-66
99-114
SHYDILTGLNYWYFDY
(SEQ ID NO: 2746)


I007A11
942
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ENYDFLTGYYOAFDI
(SEQ ID NO: 2772)


I007A12
943
144-251
165-175
191-197
230-240
1-128
26-35
50-68
101-117
GIYDILTGYHWDGAFDI
(SEQ ID NO: 2892)


I007B04
944
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I007C04
945
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I007C08
946
142-249
163-173
189-195
228-238
1-126
26-35
50-65
98-115
IRLYCYSLTGYYPYGMDD
(SEQ ID NO: 2810)


I007C12
947
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
TNYDILTGYYQGVDY
(SEQ ID NO: 2782)


I007D07
948
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
GQYYDILTGYNWFDP
(SEQ ID NO: 2857)


I007D08
949
144-251
165-175
191-197
230-240
1-128
26-35
50-68
101-117
GIYDILTGYHWDDAFDI
(SEQ ID NO: 2872)


I007E03
950
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I007E10
951
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
DFYDILTGYPLGGMDV
(SEQ ID NO: 2741)


I007E11
952
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
DLPYYDILTGYSLTSGMDV
(SEQ ID NO: 2923)


I007F06
953
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I007F08
954
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
GRRYDILTGYYYYHHGMDV
(SEQ ID NO: 2811)


I007G07
955
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I007G09
956
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
DSGODILTGYYMPYFDY
(SEQ ID NO: 2847)


I007G10
957
142-249
163-173
189-195
228-238
1-126
26-35
50-65
98-115
VGLYYDILTGYYPSGMDV
(SEQ ID NO: 2805)


I007H07
958
147-257
169-182
198-204
237-246
1-131
26-35
50-68
101-120
SQAHYDILTGYYLWSYGMDV
(SEQ ID NO: 2875)


I007H11
959
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ESYDILTGYRHIYGMDL
(SEQ ID NO: 2891)


I008A02
960
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008A05
961
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008A06
962
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008A07
963
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DREYDLLTGYYLHAFDM
(SEQ ID NO: 2960)


I008A12
964
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ENYDFLTGYYGAFDI
(SEQ ID NO: 2772)


I008B02
965
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008B04
966
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I008B05
967
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
DHYDILTGLYYYGMDV
(SEQ ID NO: 2760)


I008B06
968
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008B07
969
140-247
163-173
189-195
228-236
1-124
24-33
48-64
97-113
GRRYDILTGYYKGPLDY
(SEQ ID NO: 2902)


I008B10
970
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
AYYDNLTGFLPYGMGV
(SEQ ID NO: 2947)


I008B11
971
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EGYDILTGYFLDYYHGMDV
(SEQ ID NO: 2753)


I008C06
972
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008C08
973
149-259
171-183
199-205
238-248
1-133
26-35
50-66
99-122
GPRGGPYYDILTGYYLSLSDAFDI
(SEQ ID NO: 2729)


I008C09
974
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
EYYDILTGYRDPYGMDV
(SEQ ID NO: 2973)


I008D01
975
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008D02
976
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008D03
977
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I008D04
978
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008D05
979
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008D06
980
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008D07
981
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DRGYYDILTOYYRGHGMDV
(SEQ ID NO: 2837)


I008D08
982
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
DLPYYDILTGYSLTSGMDV
(SEQ ID NO: 2923)


I008D12
983
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EEGFYDILTGYYGPGYFDY
(SEQ ID NO: 2974)


I008E01
984
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008E02
985
137-247
159-172
188-194
227-236
1-121
20-31
46-63
96-110
EGYDILTGYSKFLDY
(SEQ ID NO: 2906)


I008E03
986
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008E04
987
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTOYSFDGFDI
(SEQ ID NO: 2153)


I008E08
988
141-252
163-175
191-197
230-241
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I008E09
989
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
ERADYDILTGYYFYDMDV
(SEQ ID NO: 2833)


I008E12
990
141-251
163-176
192-198
231-240
1-125
26-37
52-67
100-114
FRYDILTSYYYGMDV
(SEQ ID NO: 2734)


I008F03
991
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008F06
992
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDL
(SEQ ID NO: 2153)


I008F07
993
143-250
164-174
190-196
229-239
1-127
26-35
50-65
98-116
GRRYDILTGYYYYHHGMDV
(SEQ ID NO: 2811)


I008F08
994
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
GHYDILTGYDDYYYOMDV
(SEQ ID NO: 2844)


I008F09
995
133-243
155-168
184-190
223-232
1-117
26-35
50-65
98-106
HDILTGFDY
(SEQ ID NO: 2904)


I008F10
996
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
SGYDILTGYLYGMDV
(SEQ ID NO: 2934)


I008F11
997
144-251
165-175
191-197
230-240
1-128
26-35
50-68
101-117
APYDILTGYSDYYOMDV
(SEQ ID NO: 2968)


I008G02
998
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008G03
999
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
GDYDPLTGYSFGVDV
(SEQ ID NO: 2941)


I008G04
1000
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I008G05
1001
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DOYYDILTGGFYYYYGMDV
(SEQ ID NO: 2899)


I008G11
1002
136-246
158-171
187-193
226-235
1-120
26-35
50-66
99-109
AYYDILTGLDY
(SEQ ID NO: 2966)


I008G12
1003
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DQQYDILTGYYIHYGMDV
(SEQ ID NO: 2964)


I008H02
1004
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
DQVDLLLMDHNYYMDV
(SEQ ID NO: 2918)


I008H03
1005
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I008H06
1006
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I008H09
1007
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DQQYDILTGYYIHYGMDV
(SEQ ID NO: 2964)


I008H11
1008
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
TKYDILTGYYYYYMDV
(SEQ ID NO: 2856)


I012B03
1009
141-249
163-175
191-197
230-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012B06
1010
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012B10
1011
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012C03
1012
143-255
165-178
194-200
233-244
1-126
26-35
50-66
99-115
TDRFGAKDVTSRWGMDV
(SEQ ID NO: 2814)


I012C06
1013
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGLSIVGATTOALDM
(SEQ ID NO: 2174)


I012C09
1014
142-250
164-174
190-196
229-239
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012D12
1015
146-256
168-180
196-202
235-245
1-129
26-35
50-66
99-118
DRGGNYDILTGYYFHHGVDV
(SEQ ID NO: 2914)


I012E07
1016
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012E08
1017
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I012E09
1018
141-247
163-173
189-195
228-236
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012F05
1019
141-249
163-173
189-195
228-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012F12
1020
142-251
164-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012G03
1021
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I012G05
1022
141-250
163-173
189-195
228-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I012G10
1023
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I012H09
1024
141-249
163-173
189-195
228-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I013A10
1025
148-259
170-182
198-204
237-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013A12
1026
149-256
171-181
197-203
236-245
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013B04
1027
149-256
172-182
198-204
237-245
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I013B09
1028
149-257
171-181
197-203
236-246
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013C02
1029
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYRSAMDV
(SEQ ID NO: 2818)


I013C04
1030
139-249
161-173
189-195
228-238
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I013D02
1031
138-248
160-173
189-195
228-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I013D03
1032
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I013D10
1033
146-257
168-181
197-203
236-246
1-129
26-35
50-66
99-118
GLRHVTLFGTGTRGHFYMDV
(SEQ ID NO: 2789)


I013E02
1034
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I013E05
1035
139-249
162-173
189-195
228-238
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I013E09
1036
148-260
170-183
199-205
238-249
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I013F03
1037
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I013F04
1038
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013F07
1039
147-260
170-185
201-207
240-249
1-129
26-35
50-66
99-118
AATTSQKRNKYAYYFYGMDV
(SEQ ID NO: 2131)


I013F09
1040
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I013F10
1041
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013H04
1042
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I013H07
1043
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I014A12
1044
143-253
165-178
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I014C06
1045
142-254
164-177
193-200
233-243
1-125
26-35
50-66
99-114
ODYDILTGYPAECFQI
(SEQ ID NO: 2854)


I014C10
1046
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I014C12
1047
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I014E06
1048
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I014F02
1049
143-251
166-176
192-198
231-240
1-125
26-37
52-67
100-114
AGYDLLTGYPFYFDS
(SEQ ID NO: 2757)


I016A08
1050
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I016A09
1051
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016C02
1052
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016C03
1053
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016C05
1054
148-255
169-179
195-201
234-244
1-132
26-35
50-66
99-121
VQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 2132)


I016C09
1055
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016C11
1056
148-255
169-179
195-201
234-244
1-132
26-35
50-66
99-121
VQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 2132)


I016D10
1057
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016D11
1058
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016E03
1059
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016E04
1060
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016F03
1061
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016F11
1062
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016G01
1063
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016G06
1064
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I016G12
1065
148-255
169-179
195-201
234-244
1-132
26-35
50-66
99-121
VQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 2132)


I016H10
1066
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I017A06
1067
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDOFDI
(SEQ ID NO: 2153)


I017A07
1068
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I017A11
1069
140-253
162-175
191-197
233-242
1-124
25-34
49-65
98-113
ATYDPLTGYSFDGLDI
(SEQ ID NO: 2157)


I017E12
1070
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I017G03
1071
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I017G07
1072
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I017H01
1073
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018A02
1074
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018A04
1075
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I018A05
1076
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018A11
1077
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018B02
1078
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018B08
1079
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018C04
1080
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018D02
1081
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018E06
1082
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018E08
1083
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018F04
1084
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018G06
1085
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I018H07
1086
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTOYSFDGFDI
(SEQ ID NO: 2153)


I019E05
1087
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
ERHYYDILTGYQTGYGMDV
(SEQ ID NO: 2784)


I019F06
1088
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
ERHYYDILTGYQTGYGMDV
(SEQ ID NO: 2784)


I019G12
1089
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I020D01
1090
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
DRETKVGYOMDV
(SEQ ID NO: 2945)


I020D05
1091
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGONYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I020E10
1092
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I020G12
1093
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I020H06
1094
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYHILTGYYIGNGAFDI
(SEQ ID NO: 2896)


I020H10
1095
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGENYDILTGYYIONGAFDI
(SEQ ID NO: 2903)


I021A11
1096
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EOGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I021B01
1097
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I021C11
1098
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EOGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I021D12
1099
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
DRETKVGYGMDV
(SEQ ID NO: 2945)


I021E10
1100
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTOYYIGNGAFDI
(SEQ ID NO: 2158)


I021G02
1101
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I022A08
1102
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I022B01
1103
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I022B10
1104
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I022C02
1105
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I022C04
1106
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I022C08
1107
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I022D06
1108
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I022E08
1109
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
ASYYDILTGYYKGAFDI
(SEQ ID NO: 2855)


I022F01
1110
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I022F04
1111
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I022F12
1112
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
GDYDILTGTYYYIDV
(SEQ ID NO: 2859)


I022011
1113
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I023D01
1114
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I023D04
1115
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I024B04
1116
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I024D01
1117
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I024F06
1118
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I024H01
1119
141-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I024H07
1120
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I025A01
1121
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I025A04
1122
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025A07
1123
141-249
163-173
189-195
228-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025801
1124
134-244
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I025B10
1125
142-253
164-176
192-198
233-242
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I025B12
1126
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I025C07
1127
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I025D11
1128
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATT0ALDM
(SEQ ID NO: 2174)


I025E04
1129
142-252
164-176
192-198
231-241
1-126
26-35
50-66
99-115
PLGITAVRGAKTDAFGI
(SEQ ID NO: 2929)


I025E05
1130
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025E07
1131
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025E10
1132
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I025F01
1133
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYODPFYYYYYMNV
(SEQ ID NO: 2755)


I025F08
1134
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GGSSQNFYGMDV
(SEQ ID NO: 2884)


I025003
1135
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025G08
1136
141-254
163-176
192-198
231-243
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025H02
1137
145-255
167-179
195-201
234-241
1-128
26-35
50-65
98-117
AGSGFHDILTGYYKGGYFDY
(SEQ ID NO: 2961)


I026A01
1138
143-249
165-175
191-197
230-238
1-125
26-35
50-66
99-114
GDYDILTGYPAECFQI
(SEQ ID NO: 2854)


I026B01
1139
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
GSVYDILTGTYYKSGMGV
(SEQ ID NO: 2733)


I026B06
1140
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026C06
1141
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026C10
1142
139-249
161-174
190-196
229-238
1-122
26-34
49-65
98-111
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I026C11
1143
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I026D09
1144
140-252
162-175
191-197
230-241
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I026E04
1145
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I026E06
1146
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
GYDDILTGYIMALDY
(SEQ ID NO: 2821)


I026E09
1147
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELOSSIVGATTGALDM
(SEQ ID NO: 2852)


I026F01
1148
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026F09
1149
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026F12
1150
141-256
163-176
192-202
237-245
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026G08
1151
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026010
1152
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I026G11
1153
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I026H02
1154
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I026H06
1155
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I026H10
1156
145-255
167-179
195-201
234-241
1-128
26-35
50-66
99-117
GGEYDILTGYYFGLGVYDY
(SEQ ID NO: 2170)


I027A09
1157
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I027B02
1158
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I027B05
1159
141-250
163-176
192-198
230-239
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I027C08
1160
139-249
161-174
190-196
229-238
1-122
26-34
49-63
96-111
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I027D02
1161
142-250
164-174
190-196
229-239
1-125
26-35
50-66
99-114
DPFGAVPGYYYYAMDV
(SEQ ID NO: 2826)


I027E03
1162
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I027E05
1163
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIV0ATTGALDM
(SEQ ID NO: 2174)


I027F04
1164
145-252
167-176
192-198
231-241
1-128
26-35
50-66
99-117
GPWYDPLFPPSGRHYGLDV
(SEQ ID NO: 2793)


I027F05
1165
141-254
163-176
192-198
231-243
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I027F11
1166
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSLVGATTGALDM
(SEQ ID NO: 2852)


I027006
1167
142-253
164-176
192-198
233-242
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I027007
1168
142-250
164-174
190-196
229-239
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I027H03
1169
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
GDYDILT0YPAECFQI
(SEQ ID NO: 2854)


I028A04
1170
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DMYYDILTGYYTGLAFDM
(SEQ ID NO: 2880)


I028A07
1171
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
VLNYDILTGYYYGMDV
(SEQ ID NO: 2832)


I028B08
1172
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I028B10
1173
148-258
170-183
199-205
238-247
1-132
26-35
50-68
101-121
DFGYYDILTGYYIGAFYAFDI
(SEQ ID NO: 2861)


I028C01
1174
142-250
165-175
191-197
230-239
1-126
26-37
52-69
102-115
GGHTCIIPTCHMGO
(SEQ ID NO: 2796)


I028C04
1175
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DMYYDILTGYYTGLAFDM
(SEQ ID NO: 2880)


I028C08
1176
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I028D04
1177
140-247
163-173
189-195
228-236
1-124
26-35
50-65
98-113
ATQDILTGYLYSGMDV
(SEQ ID NO: 2977)


I028D05
1178
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
EHYDILTGYSLLGMDV
(SEQ ID NO: 2907)


I028D12
1179
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DGYYDILTGYSVYYGMDV
(SEQ ID NO: 2938)


I028E06
1180
143-253
165-179
194-200
233-242
1-127
26-35
50-65
98-116
EGSYDILTGYYVGVORMDV
(SEQ ID NO: 2171)


I028E07
1181
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I028E08
1182
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I028F06
1183
146-256
168-180
196-202
235-245
1-130
26-35
50-66
99-119
DDRRGYYDILTGYYRFGSFDI
(SEQ ID NO: 2901)


I028F08
1184
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
DIDIGGDDS
(SEQ ID NO: 2954)


I028G08
1185
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
VSGYNSGYFESYDMDV
(SEQ ID NO: 2732)


I028G09
1186
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I028G10
1187
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I028H02
1188
142-249
165-175
191-197
230-238
1-126
26-37
52-69
102-115
SGEPCITLACNLGG
(SEQ ID NO: 2797)


I028H03
1189
148-256
169-179
195-201
234-245
1-132
26-35
50-66
99-121
DASEYYDILTGYYLATGRNWFDP
(SEQ ID NO: 2888)


I028H06
1190
145-255
167-180
196-202
235-241
1-129
26-35
50-66
99-118
DPSPYYDILTGYFLPYYMDV
(SEQ ID NO: 2843)


I028H09
1191
140-250
162-175
191-197
230-239
1-124
26-35
50-68
101-113
EIDDILTGYYMDV
(SEQ ID NO: 2905)


I029A10
1192
139-246
160-170
186-192
225-235
1-123
26-35
50-65
98-112
MNYDILTGLVNWFDP
(SEQ ID NO: 2786)


I029A12
1193
137-247
159-171
187-193
226-236
1-121
26-35
50-68
101-110
RDILTGFYDS
(SEQ ID NO: 2933)


I029811
1194
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I029C08
1195
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I029E10
1196
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I029F08
1197
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I029008
1198
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
GYYDILTGYQSDAFDI
(SEQ ID NO: 2927)


I030A02
1199
143-253
165-177
193-199
232-242
1-126
26-35
50-66
99-115
TERFGAKDVTARWGMDV
(SEQ ID NO: 2874)


I030A03
1200
141-253
163-175
191-197
230-242
1-124
26-35
50-66
99-113
ENYDILTGYYNFFDY
(SEQ ID NO: 2737)


I030A04
1201
141-252
163-176
192-198
231-241
1-124
26-35
50-66
99-113
RQYDILTGYYGGFDY
(SEQ ID NO: 2958)


I030A05
1202
141-249
163-175
191-197
230-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030A09
1203
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030A12
1204
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030B06
1205
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030B08
1206
141-247
163-173
189-195
228-236
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030B10
1207
143-251
165-175
191-197
230-240
1-125
26-35
50-66
99-114
ELGHREGGYWYSPYNV
(SEQ ID NO: 2838)


I030C03
1208
140-252
162-175
191-197
230-241
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030C06
1209
147-256
169-182
198-204
237-245
1-130
26-35
50-68
101-119
DPGNYDILTGYYYYYGMDV
(SEQ ID NO: 2935)


I030C08
1210
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
SGPGWFDP
(SEQ ID NO: 2870)


I030C09
1211
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030C10
1212
141-250
163-175
191-197
230-239
1-124
26-34
49-65
98-113
ELGLSIVGATT0ALDM
(SEQ ID NO: 2174)


I030C11
1213
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030C12
1214
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
SGPOWFDP
(SEQ ID NO: 2870)


I030D07
1215
141-249
163-173
189-195
228-238
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030D12
1216
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030E02
1217
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030E05
1218
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030E07
1219
142-251
165-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030E08
1220
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030E09
1221
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030E10
1222
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030F02
1223
142-252
164-176
192-198
231-241
1-125
26-37
52-67
100-114
AGYDLLTGYPFYFDS
(SEQ ID NO: 2757)


I030F05
1224
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030F06
1225
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYODPFYYYYYMNV
(SEQ ID NO: 2755)


I030F08
1226
141-254
163-176
192-198
231-243
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030F09
1227
142-253
164-176
192-198
231-242
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030F11
1228
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030F12
1229
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I030G03
1230
141-256
163-176
192-202
237-245
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030G07
1231
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I030009
1232
142-251
164-174
190-196
229-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030H05
1233
146-255
168-181
197-203
236-241
1-129
26-35
50-66
99-118
DRGGNYDILTGYYFHHGVDV
(SEQ ID NO: 2914)


I030H06
1234
148-258
170-182
198-204
239-247
1-130
26-37
52-69
102-119
ATKSYDILTRMYYYHMDV
(SEQ ID NO: 2748)


I030H10
1235
141-253
163-176
192-198
231-242
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I030H11
1236
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I031A01
1237
138-248
160-173
189-195
228-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031A03
1238
143-251
166-176
192-198
231-240
1-125
26-35
50-66
99-114
PYYDPLTAYTFQYFGN
(SEQ ID NO: 2806)


I031A12
1240
147-257
169-181
197-203
236-246
1-130
26-35
50-66
99-119
GREDTDKVKPWDRYYHYYMDV
(SEQ ID NO: 2972)


I031B03
1241
137-246
159-172
188-194
227-235
1-120
26-35
50-68
101-109
GLGHTDSDS
(SEQ ID NO: 2959)


I031B06
1242
143-253
165-177
193-199
232-242
1-126
26-35
50-66
99-115
AKGYYYDSSGASDVFDV
(SEQ ID NO: 2871)


I031B07
1243
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031B08
1241
149-260
171-183
199-205
238-249
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031B09
1245
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SNPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2840)


I031B11
1246
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031B12
1247
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031C01
1248
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031C02
1249
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
PFYDTLTSYVFQYFDH
(SEQ ID NO: 2137)


I031C04
1250
149-260
171-183
199-205
238-249
1-131
26-35
50-66
99-120
GRKDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2813)


I031C08
1251
139-248
161-171
187-193
226-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031C11
1252
149-257
171-181
197-203
236-246
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031D01
1253
146-256
168-180
196-202
235-245
1-129
26-35
50-66
99-118
AATTSQKHNKYAYYFYGMDV
(SEQ ID NO: 2131)


I031D04
1254
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031D06
1255
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
GREDTDKVKLWDRYYHYYYMDV
(SEQ ID NO: 2807)


I031D08
1256
145-257
167-180
196-202
235-246
1-128
26-35
50-66
99-117
VRPKLRYFDWLSRHDAFDL
(SEQ ID NO: 2820)


I031D09
1257
139-247
161-171
187-193
226-236
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031D11
1258
149-256
171-181
197-203
236-245
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I031D12
1259
146-254
168-178
194-200
233-243
1-128
26-35
50-66
99-117
DKAHOEYGRDYYYYYGMDV
(SEQ ID NO: 2735)


I031E01
1260
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031E05
1261
149-257
171-181
197-203
236-246
1-131
26-35
50-66
99-120
SGPPKWYDALTOHSSYHSAMDV
(SEQ ID NO: 2848)


I031E07
1262
148-259
170-182
198-204
237-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031E08
1263
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031E09
1264
139-246
162-173
189-195
228-235
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031E10
1265
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I031E11
1266
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031F01
1267
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031F04
1268
139-246
162-172
188-194
227-235
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031F06
1269
137-247
159-171
187-193
226-236
1-119
26-35
50-66
99-108
DTVRSGGMDV
(SEQ ID NO: 2804)


I031F10
1270
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031F11
1271
145-255
167-179
195-201
234-241
1-128
26-35
50-66
99-117
DKAHGEYGRDYYYYYGMDV
(SEQ ID NO: 2735)


I031F12
1272
138-249
160-172
188-194
227-238
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031G01
1273
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031G03
l274
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I031G05
1275
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031G06
1276
148-258
170-182
198-204
237-247
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031G07
1277
149-259
171-183
199-205
238-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMGV
(SEQ ID NO: 28l6)


I031G09
1278
148-263
170-183
199-209
244-252
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031G12
1279
146-256
168-180
196-202
235-245
1-129
26-35
50-66
99-118
AATTSQKHNKYAYYFYGMDV
(SEQ ID NO: 2131)


I031H01
1280
138-250
160-173
189-195
228-239
1-121
26-35
50-66
99-110
GYDSSAFRAFDI
(SEQ ID NO: 2136)


I031H02
1281
143-255
165-178
194-200
233-241
1-126
26-35
50-66
99-115
AKGYYYDSSGASDVFDV
(SEQ ID NO: 2871)


I031H03
1282
148-260
170-183
199-205
238-249
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I031H06
1283
145-257
167-179
195-201
234-246
1-128
26-35
50-66
99-117
DKAHGEYGRDYYYYYGMDV
(SEQ ID NO: 2735)


I031H09
1284
145-255
167-179
195-201
234-241
1-128
26-35
50-66
99-117
DKAHGEYGRDYYYYYGMDV
(SEQ ID NO: 2735)


I031H10
1285
144-256
166-179
195-201
234-245
1-127
26-35
50-66
99-116
DRGYTGYDRLVGGYYFDF
(SEQ ID NO: 2931)


I031H11
1286
136-246
158-170
186-192
225-235
1-119
26-35
50-66
99-108
DTVRSGGMDV
(SEQ ID NO: 2804)


I033A08
1287
144-254
166-179
195-201
234-243
1-128
26-37
52-69
102-117
DRYDILTGYYYYGMDV
(SEQ ID NO: 2129)


I033B11
1288
144-254
166-179
195-201
234-243
1-128
26-37
52-69
102-117
DRYDILTGYYYYGMDV
(SEQ ID NO: 2129)


I033C01
1289
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I033C08
1290
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
EMGYDILTGYYLNYMDV
(SEQ ID NO: 2862)


I033D02
1291
138-245
161-171
187-193
226-234
1-122
26-35
50-66
99-111
GDYDILTGYYMDV
(SEQ ID NO: 2781)


I033D03
1292
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTOYSFDGFDI
(SEQ ID NO: 2153)


I033D05
1293
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I033D11
1294
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
VKRDILTGYVEGMDV
(SEQ ID NO: 2869)


I033D12
1295
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
GGPHYDILTGYYMAVGFDI
(SEQ ID NO: 2962)


I033E01
1296
139-249
161-173
189-195
228-238
1-123
26-35
50-66
99-112
DIDARLAALDAFDI
(SEQ ID NO: 2794)


I033E06
1297
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATHDPLTOYSFDGFDI
(SEQ ID NO: 2780)


I033E11
1298
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
HRSRSCSSTSCRNDAFDI
(SEQ ID NO: 2770)


I033E12
1299
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
EMGYDILTGYYLNYMDV
(SEQ ID NO: 2862)


I033F03
1300
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
EGAADYLNGQYFQD
(SEQ ID NO: 2768)


I033F08
1301
145-256
167-179
195-201
234-245
1-129
26-35
50-66
99-118
QKVYYDILTGYNYYYYGMDV
(SEQ ID NO: 2767)


I033F10
1302
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I033F12
1303
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
DIDIGUDDS
(SEQ ID NO: 2954)


I033G01
1304
143-253
165-179
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I033G03
1305
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
PQGVTLVRGAETDAFAI
(SEQ ID NO: 2925)


I033G08
1306
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I033H04
1307
140-247
161-171
187-193
226-236
1-124
25-34
49-65
98-113
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I037A05
1308
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I037B03
1309
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
SHYDILTRLNYWYFDL
(SEQ ID NO: 2950)


I037B04
1310
144-251
167-177
193-199
232-240
1-128
26-35
50-66
99-117
DPGYYDILTGYFHRYGMDV
(SEQ ID NO: 2922)


I037C04
1311
142-252
164-177
193-199
232-241
1-126
26-35
50-65
98-115
ENGDYDILTGQTFYGMDV
(SEQ ID NO: 2752)


I037C06
1312
141-249
163-173
189-195
228-238
1-125
26-35
50-66
99-114
LYYDILTGYHWDAFDI
(SEQ ID NO: 2882)


I037C08
1313
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
DGIDILLVPAALMDV
(SEQ ID NO: 2160)


I037D11
1314
136-246
158-171
187-193
226-235
1-120
26-35
50-66
99-109
SQWLEHDVFDI
(SEQ ID NO: 2864)


I037E06
1315
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
DRRDYDLLTRYYYYYGMDV
(SEQ ID NO: 2928)


I037F04
1316
144-251
165-175
191-197
230-240
1-128
26-35
50-65
98-117
KQRGDYDILTGYQLGYAFDI
(SEQ ID NO: 2808)


I037G01
1317
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
SHYDILTRLNYWYFDL
(SEQ ID NO: 2950)


I037G03
1318
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
DLGSFYDILTALRLENYGMDV
(SEQ ID NO: 2963)


I037G10
1319
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
DYYDILTKLPYGMDV
(SEQ ID NO: 2975)


I042A07
1320
144-251
167-177
193-199
232-240
1-128
26-35
50-66
99-117
VSPSYDILTGYYLPHAFDV
(SEQ ID NO: 2849)


I042A10
1321
142-249
165-175
191-197
230-238
1-126
26-35
50-65
98-115
GPRYYDILTGYRYNWFDP
(SEQ ID NO: 2801)


I042B03
1322
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
DIDDILTGYVLGMDV
(SEQ ID NO: 2924)


I042B12
1323
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I042D01
1324
136-246
158-171
187-193
226-235
1-120
26-35
50-66
99-109
QQWLPYDAFDI
(SEQ ID NO: 2839)


I042D03
1325
140-250
162-175
191-197
230-239
1-124
26-35
50-68
101-113
AYYDILTGYFFDI
(SEQ ID NO: 2873)


I042D10
1326
142-252
164-177
193-199
232-241
1-126
26-35
50-65
98-115
ERADYDILTGYYFYGMDV
(SEQ ID NO: 2802)


I042E10
1327
147-257
169-182
198-204
237-246
1-131
26-37
52-69
102-120
ERPYYDILTGYTVTYGMDV
(SEQ ID NO: 2798)


I042E11
1328
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
DEYDILTGLLQGMDV
(SEQ ID NO: 2883)


I042F08
1329
142-252
164-177
193-199
232-241
1-126
26-37
52-67
100-115
GDYDILTGYPLHAFDI
(SEQ ID NO: 2738)


I042F12
1330
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
DGYDILTGYYFGMDV
(SEQ ID NO: 2976)


I042G08
1331
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
EHYDILTGYSLLGMDV
(SEQ ID NO: 2907)


I042G10
1332
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
SHYDILTGLNYWYFDL
(SEQ ID NO: 2166)


I042H03
1333
143-253
165-179
194-200
233-242
1-127
26-35
50-65
98-116
GSLYYDILTGYYIGNAFDI
(SEQ ID NO: 2759)


I043A03
1334
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DGYYDILTGGFYYYYGMDV
(SEQ ID NO: 2899)


I043B02
1335
142-249
163-173
189-195
228-238
1-126
26-35
50-65
98-115
GGYYDILTGYLVYYGMDV
(SEQ ID NO: 2744)


I043B03
1336
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I043B06
1337
143-253
165-179
194-200
233-242
1-127
26-35
50-66
99-116
DQQYDILTGYHIDYYMDV
(SEQ ID NO: 2828)


I043B07
1338
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFD0FDI
(SEQ ID NO: 2153)


I043B09
1339
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
HVRDYDILTGYYRGHHFDY
(SEQ ID NO: 2727)


I043D11
1340
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I043E05
1341
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
TESNYDILTGYYWPSMDV
(SEQ ID NO: 2940)


I043F01
1342
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I043F04
1343
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I043F12
1344
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
TESNYDILTGYYWPSMDV
(SEQ ID NO: 2940)


I043H07
1345
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I044A11
1346
144-251
165-175
191-197
230-240
1-128
26-35
50-68
101-117
APYDILTGYSDYYGMDV
(SEQ ID NO: 2968)


I044B11
1347
139-249
161-173
189-195
228-238
1-123
26-35
50-66
99-112
DSDARLAALDAFDI
(SEQ ID NO: 2978)


I044C09
1348
140-250
162-174
190-196
229-239
1-124
26-35
50-66
99-113
GQFGVLPNYYYHMDV
(SEQ ID NO: 2943)


I044C10
1349
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
DIKRYNSNWPYYDYYMDV
(SEQ ID NO: 2726)


I044D03
1350
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DKQYYDILTGDPVEGGMDV
(SEQ ID NO: 2889)


I044D09
1351
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I044E07
1352
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLVTYGTDV
(SEQ ID NO: 2825)


I044E11
1353
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
SDDYDILTGNYVGSLLDY
(SEQ ID NO: 2758)


I044F07
1354
147-257
169-182
198-204
237-246
1-131
26-35
50-66
99-120
DGRLSYDILTGYYARDYYGMDV
(SEQ ID NO: 2912)


I044G02
1355
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I044G07
1356
149-259
171-184
200-206
239-248
1-133
26-35
50-66
99-122
DQNHPIYDILTGYYVPTGPLELKN
(SEQ ID NO: 2845)


I044H01
1357
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I050A01
1358
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
DMGYDILTGYYGAFDI
(SEQ ID NO: 2946)


I050B12
1359
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
DYYDVLTGFSLDGMDV
(SEQ ID NO: 2829)


I050C06
1360
142-248
165-175
191-197
230-237
1-124
26-35
50-65
98-113
DHYDVLTGSYLQAFDV
(SEQ ID NO: 2728)


I050C08
1361
142-253
164-177
193-199
232-242
1-125
26-37
52-67
100-114
GRYDFLTGYLRNFDY
(SEQ ID NO: 2731)


I050E01
1362
141-252
163-176
192-198
231-241
1-124
26-35
50-66
99-113
GHYDILTGYYFGFDY
(SEQ ID NO: 2886)


I050E10
1363
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
DMKVYYKYALDV
(SEQ ID NO: 2823)


I050H08
1364
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
DLRYDILTGYHDAFDI
(SEQ ID NO: 2890)


I051A04
1365
148-258
170-183
199-205
238-247
1-131
26-35
50-66
99-120
SSPPKWYDALTGHSSYHSAMDV
(SEQ ID NO: 2159)


I051A08
1366
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
HRRARVVVPVPGAMDV
(SEQ ID NO: 2930)


I051A12
1367
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I051B05
1368
143-253
165-177
193-199
232-242
1-126
26-36
51-67
100-115
RSMIVVTTAPYDAFDL
(SEQ ID NO: 2785)


I051C06
1369
136-246
158-170
186-192
225-235
1-119
26-35
50-66
99-108
DTVRSGGMDV
(SEQ ID NO: 2804)


I051G12
1370
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DOSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I055A05
1371
134-241
156-169
185-191
224-233
1-117
26-35
50-66
99-106
SGPGWFDP
(SEQ ID NO: 2870)


I055A11
1372
134-241
156-169
185-191
224-233
1-117
26-35
50-66
99-106
SGPGWFDP
(SEQ ID NO: 2870)


I061A03
1373
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGSSIVGATTGALDM
(SEQ ID NO: 2852)


I061A04
1374
143-251
165-175
191-197
230-240
1-125
26-35
50-66
99-114
GDYDILTGYPAECFQI
(SEQ ID NO: 2854)


I061A08
1375
142-253
164-176
192-198
233-242
1-124
26-35
50-66
99-113
DNYDILTGYSRRFDP
(SEQ ID NO: 2942)


I061A09
1376
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I061A10
1377
141-249
163-173
189-195
228-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I061B07
1378
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I061B09
1379
143-253
165-178
194-200
233-242
1-127
24-33
48-64
97-116
EGGNYDILTGYYIGNGAFDI
(SEQ ID NO: 2158)


I061B12
1380
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I061C12
1381
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
TYYDILTGYHFDY
(SEQ ID NO: 2788)


I061D01
1382
137-247
159-172
188-194
227-236
1-121
26-35
50-68
101-110
GPGVIGNYDY
(SEQ ID NO: 2749)


I061D03
1383
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I061D04
1384
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
AVLRYSAGLQGAFDI
(SEQ ID NO: 2970)


I061D07
1385
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
VSGYNSGYFESYDMDV
(SEQ ID NO: 2732)


I061D09
1386
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
LNLEKTVVRGFGYFDL
(SEQ ID NO: 2952)


I061010
1387
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
DHYDILTGLYYYGMDV
(SEQ ID NO: 2760)


I061E01
1388
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
LNLEKTVVRGFGYFDL
(SEQ ID NO: 2952)


I061E05
1389
142-251
163-175
191-197
230-240
1-126
26-35
50-66
99-115
GGELVWFGESDYYGMDV
(SEQ ID NO: 2787)


I061E09
1390
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I061E12
1391
133-240
154-164
180-186
219-229
1-117
26-35
50-66
99-106
SQRLFIDS
(SEQ ID NO: 2842)


I061F01
1392
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
DRYYDILTGYYIPGLDDAFDI
(SEQ ID NO: 2887)


I061F09
1393
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
DSDARLAALDAFDI
(SEQ ID NO: 2978)


I061F10
1394
145-252
166-176
192-198
231-241
1-129
26-35
50-66
99-118
EESYYDILTGYYVHYYGMDV
(SEQ ID NO: 2743)


I061F11
1395
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYYFDGFDI
(SEQ ID NO: 2949)


I061G01
1396
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I061G03
1397
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
AYYDILTGFLPYDMDL
(SEQ ID NO: 2771)


I061G09
1398
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I061G10
1399
143-253
165-179
194-200
233-242
1-127
26-35
50-65
98-116
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I061G11
1400
137-247
159-171
187-193
226-236
1-121
26-35
50-68
101-110
RDILTGFYDS
(SEQ ID NO: 2933)


I061H05
1401
142-252
164-177
193-199
232-241
1-126
26-37
52-67
100-115
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I064A05
1402
142-249
163-173
189-195
228-238
1-126
26-35
50-68
101-115
DFYDILTGYQHGMDV
(SEQ ID NO: 2919)


I064A11
1403
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
HSKEYNWNYALDY
(SEQ ID NO: 2754)


I064B01
1404
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
TRMDVLTRYYSDF
(SEQ ID NO: 2750)


I064B02
1405
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
AFEDYDILTGYYHHDAFDI
(SEQ ID NO: 2911)


I064B12
1406
133-243
155-168
184-190
223-232
1-117
26-35
50-66
99-106
PSYHYMDV
(SEQ ID NO: 2740)


I064C06
1407
145-255
167-180
196-202
235-241
1-129
26-35
50-66
99-118
VNADYDILTGYPRDYYGMDV
(SEQ ID NO: 2819)


I064D01
1408
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I064D02
1409
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
EDATYYDILTGYYMGSYGMDV
(SEQ ID NO: 2763)


I064E01
1410
143-250
166-176
192-198
231-239
1-127
26-35
50-66
99-116
ETRKYTSSPPYNYYYMDV
(SEQ ID NO: 2736)


I064E02
1411
140-251
162-174
190-196
229-240
1-124
26-35
50-66
99-113
RDYDILTGYSRGFDP
(SEQ ID NO: 2725)


I064E03
1412
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DGIYDILTTLVSYYNOMDV
(SEQ ID NO: 2775)


I064E07
1413
140-250
162-175
191-197
230-239
1-124
26-35
50-65
98-113
GERDILTGYYLDGMDV
(SEQ ID NO: 2948)


I064E08
1414
140-250
162-174
190-196
229-239
1-124
26-35
50-66
99-113
ERGSYSSGYSGAFDV
(SEQ ID NO: 2898)


I064F05
1415
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
ESGGYSYGSRDYYGMDV
(SEQ ID NO: 2836)


I064F08
1416
145-252
166-176
192-198
231-241
1-129
26-35
50-66
99-118
DR0VGYDILTGRTYYYGMDV
(SEQ ID NO: 2900)


I064G06
1417
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065A12
1418
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DVSGHDILTGYSYRYFDV
(SEQ ID NO: 2795)


I065C04
1419
139-249
161-173
189-195
228-238
1-123
26-35
50-66
99-112
GQKNYYESSGYLEH
(SEQ ID NO: 2916)


I065C09
1420
140-250
162-174
190-196
229-239
1-124
26-35
50-66
99-113
GDYDILTGYYSHFDY
(SEQ ID NO: 2908)


I065E02
1421
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
AYDYDILTGYSYYFDY
(SEQ ID NO: 2895)


I065E04
1422
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I065F03
1423
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I065G06
1424
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I065G07
1425
141-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
GGNYDILTGYYIGAFDI
(SEQ ID NO: 2824)


I065G08
1426
138-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I065H06
1427
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
GYEYYDILTGYNELOAFDI
(SEQ ID NO: 2851)


I066A03
1428
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DGTYYDILTGYYNQYGMDV
(SEQ ID NO: 2915)


I066A08
1429
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I066A09
1430
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I066A10
1431
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
DRGYDILTGYYYYGMDV
(SEQ ID NO: 2876)


I066A11
1432
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
EVRDYDILTGYYISYMDV
(SEQ ID NO: 2778)


I066B02
1433
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I066B08
1434
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I066B10
1435
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
GLYFEDTNYRHGDAFDI
(SEQ ID NO: 2790)


I066C02
1436
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I066C11
1437
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066C12
1438
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I066D06
1439
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ENYDFLTGYYGAFDI
(SEQ ID NO: 2772)


I066D08
1440
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
HSKEYNWNYALDY
(SEQ ID NO: 2754)


I066D11
1441
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
ERSQFDFLTGVDRYHPMDV
(SEQ ID NO: 2956)


I066D12
1442
139-249
161-174
190-196
229-238
1-123
26-35
50-66
99-112
EGAADYLNGQYFQH
(SEQ ID NO: 2815)


I066E06
1443
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I066E12
1444
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
OMGDHYGMDV
(SEQ ID NO: 2161)


I066G05
1445
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
GLYFEDTNYRHGDAFDI
(SEQ ID NO: 2790)


I066G08
1446
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
VYYDILTGHPTYGMDV
(SEQ ID NO: 2791)


I066010
1447
144-254
166-178
194-200
233-243
1-128
26-35
50-68
101-117
GIYDILTGYHWDDAFDI
(SEQ ID NO: 2872)


I066G12
1448
143-254
165-177
193-199
232-243
1-127
26-35
50-66
99-116
ESTYDILTGSYHDYGLDV
(SEQ ID NO: 2822)


I066H04
1449
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
DRLHYDILTGHQTDDAFDI
(SEQ ID NO: 2885)


I067A07
1450
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
VLTNYDILTGYYREDAFDM
(SEQ ID NO: 2939)


I067A11
1451
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I067B08
1452
149-259
171-184
200-206
239-248
1-133
26-35
50-66
99-122
DRGASNYDILTGYYAPAQGVAFDI
(SEQ ID NO: 2969)


I067C08
1453
148-258
170-183
199-205
238-247
1-132
26-37
52-69
102-121
EGAHYDILTGHNYYHYGMDV
(SEQ ID NO: 2747)


I067C09
1454
143-253
165-179
194-200
233-242
1-127
26-35
50-66
99-116
ETRKYTSSPPYNYYYMDV
(SEQ ID NO: 2736)


I067D07
1455
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067E01
1456
142-248
164-174
190-196
229-238
1-124
26-35
50-66
99-113
DQHDILTGVYYGMDV
(SEQ ID NO: 2921)


I067E06
1457
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I067E07
1458
150-260
172-184
200-206
239-249
1-134
26-35
50-67
100-123
DYPOSEYDILTGYLFGYYYYGMDV
(SEQ ID NO: 2926)


I067E11
1459
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I067003
1460
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ARRVGVLGGKNAFEI
(SEQ ID NO: 2765)


I067G05
1461
140-250
162-174
190-196
229-239
1-124
26-35
50-66
99-113
DQHDILTGGYYGMDV
(SEQ ID NO: 2894)


I067G12
1462
141-252
163-176
192-198
231-241
1-125
26-35
50-66
99-114
ATYDPLTGYSFDOFDI
(SEQ ID NO: 2153)


I067H05
1463
146-256
168-180
196-202
235-245
1-130
26-35
50-68
101-119
EGTYYDILTGYYPLGYFDY
(SEQ ID NO: 2936)


I067H06
1464
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I068C09
1465
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
GGSSQNFYGMDV
(SEQ ID NO: 2884)


I068G03
1466
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I068G04
1467
143-252
165-178
194-200
233-241
1-126
26-35
50-66
99-115
GVVWVAYGDVGIYOFDV
(SEQ ID NO: 2937)


I068G07
1468
142-251
164-174
190-196
229-240
1-124
26-35
50-66
99-113
HDYYIMTAAHYYYDS
(SEQ ID NO: 2909)


I068G08
1469
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
GIGYDLLTGYFTGSPLDY
(SEQ ID NO: 2846)


I070F07
1470
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
DFYDILTGYHDAFDI
(SEQ ID NO: 2910)


I070G05
1471
140-250
162-175
191-197
230-239
1-124
26-35
50-68
101-113
DVDDILTGYSWDY
(SEQ ID NO: 2867)


I070H02
1472
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I071A01
1473
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
AAYDPLTGYSFDGFDI
(SEQ ID NO: 2783)


I071A03
1474
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DMHYDILTGYYTGLAFDM
(SEQ ID NO: 2917)


I071B08
1475
144-252
166-176
192-198
231-241
1-126
27-36
51-67
100-115
OGYDILTQYPAEFFHP
(SEQ ID NO: 2764)


I071E01
1476
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
DFGVIGDYRPFDY
(SEQ ID NO: 2777)


I071F11
1477
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
SSNPVYGLDV
(SEQ ID NO: 2957)


I071G11
1478
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I071H08
1479
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I074A02
1480
142-250
164-174
190-196
229-239
1-125
26-35
50-66
99-114
DDRDILTNYYLEYFQH
(SEQ ID NO: 2868)


I074A08
1481
148-259
170-182
198-204
237-248
1-131
26-35
50-66
99-120
SSPPKWYDALTGDSSYHSAMDV
(SEQ ID NO: 2165)


I074D10
1482
146-253
168-178
194-200
233-242
1-128
26-35
50-66
99-117
DKTLGDQLVEAYYYDGMDV
(SEQ ID NO: 2776)


I074E01
1483
146-255
168-178
194-200
233-241
1-128
26-35
50-66
99-117
LGRTSRDLLTGYHFYNMDV
(SEQ ID NO: 2944)


I074E02
1484
142-250
164-174
190-196
229-239
1-124
26-35
50-66
99-113
DDYDILTGSLYYFDS
(SEQ ID NO: 2803)


I074E08
1485
144-259
166-179
195-205
240-248
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I074F12
1486
142-250
164-174
190-196
229-239
1-124
26-35
50-66
99-113
DRADILTGYNDAFDI
(SEQ ID NO: 2739)


I074H06
1487
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I074H07
1488
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
GTOYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I074H08
1489
143-254
165-179
194-200
233-243
1-126
26-35
50-66
99-115
VSNDILTGWGGYNWFDP
(SEQ ID NO: 2955)


I075A07
1490
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
OTGYDILTGYYMOSAFDQ
(SEQ ID NO: 2800)


I075B01
1491
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075B04
1492
134-247
156-169
185-191
224-236
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075B06
1493
141-252
163-175
191-197
230-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075B08
1494
144-257
166-179
195-201
234-246
1-127
26-35
50-66
99-116
GTGYDILTGYYMOSAFDQ
(SEQ ID NO: 2800)


I075B09
1495
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
TYYDILTGYYAEYFQH
(SEQ ID NO: 2932)


I075B12
1496
141-251
163-176
192-198
231-240
1-124
26-35
50-66
99-113
SDYDILTGYYWVPAV
(SEQ ID NO: 2812)


I075C01
1497
148-259
170-183
199-205
238-248
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYFHYYYMDV
(SEQ ID NO: 2835)


I075C05
1498
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQORYLDL
(SEQ ID NO: 2175)


I075D05
1499
145-253
168-179
195-201
234-242
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSVFDP
(SEQ ID NO: 2897)


I075D07
1500
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
SYYDILTGYYHTPLDY
(SEQ ID NO: 2853)


I075D08
1501
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075E01
1502
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I075E03
1503
150-261
172-184
200-206
239-250
1-132
28-37
52-68
101-121
GGGYDILTGYSYPYLYYGLDV
(SEQ ID NO: 2865)


I075E04
1504
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
GRGYDVLTGYFTGSPLDY
(SEQ ID NO: 2881)


I075E05
1505
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075E10
1506
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075E11
1507
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
SGPGWFDP
(SEQ ID NO: 2870)


I075E12
1508
143-254
165-179
194-200
233-243
1-126
26-35
50-66
99-115
TDRFGAKDVTARWGMDV
(SEQ ID NO: 2979)


I075F02
1509
146-253
168-178
194-200
233-242
1-128
26-35
50-66
99-117
EQGYDILTGYYPEGGWFDP
(SEQ ID NO: 2834)


I075F04
1510
142-251
164-176
192-198
231-240
1-125
26-37
52-67
100-114
AGYDLLTGYPFYFDS
(SEQ ID NO: 2757)


I075F06
1511
146-254
168-178
194-200
233-243
1-128
26-35
50-66
99-117
GRNYYDFLTGYNFNLGLDY
(SEQ ID NO: 2830)


I075F07
1512
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
ENYDSLTGYYNYFDY
(SEQ ID NO: 2971)


I075F08
1513
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQRKAQDI
(SEQ ID NO: 2779)


I075F09
1514
147-257
169-181
197-203
236-246
1-129
26-35
50-66
99-118
LKAPYYDLLTGYHLPKWFDT
(SEQ ID NO: 2953)


I075F10
1515
135-243
157-167
183-189
222-232
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075F11
1516
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075G05
1517
141-252
163-175
191-197
230-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075G07
1518
141-252
163-175
191-197
230-241
1-124
26-35
50-66
99-113
GRYYDMLTRGGYFDY
(SEQ ID NO: 2858)


I075G08
1519
141-252
163-176
192-198
231-241
1-124
26-35
50-66
99-113
RQYDILTGYYGGFDY
(SEQ ID NO: 2958)


I075G11
1520
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
TDYDILTGYPMGYFDP
(SEQ ID NO: 2173)


I075G12
1521
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075H02
1522
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I075H03
1523
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075H06
1524
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075H08
1525
144-254
166-179
195-201
234-243
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I076A01
1526
144-253
166-176
192-198
231-242
1-126
26-35
50-66
99-115
DRRRDDLTGYLYDAFDS
(SEQ ID NO: 2878)


I076A03
1527
137-247
159-171
187-193
226-236
1-119
26-35
50-68
101-108
GYDTAMQY
(SEQ ID NO: 2951)


I076A06
1528
134-245
156-168
184-190
223-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076A07
1529
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
DRRDILTGSNFGQD
(SEQ ID NO: 2913)


I076A08
1530
144-253
166-176
192-198
231-242
1-126
26-35
50-66
99-115
MGHYDILTGYRHYGMDV
(SEQ ID NO: 2831)


I076B01
1531
145-257
167-179
195-201
236-246
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I076B03
1532
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076B07
1533
135-243
157-167
183-189
222-232
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076B08
1534
143-252
166-177
193-199
232-241
1-125
26-35
50-66
99-114
PYYDPLTAYTFQYFGN
(SEQ ID NO: 2806)


I076C04
1535
142-250
164-174
190-196
229-239
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I076C10
1536
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
GRYYDMLTRGGYFDY
(SEQ ID NO: 2858)


I076D01
1537
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
LDYDILTGYYPSGFDY
(SEQ ID NO: 2799)


I076D08
1538
141-251
163-175
191-197
230-240
1-124
26-37
52-67
100-113
RFYDLLTGYSAFDS
(SEQ ID NO: 2756)


I076D11
1539
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076D12
1540
142-250
164-174
190-196
229-239
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I076E04
1541
145-252
167-177
193-199
232-241
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076E07
1542
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
EYYDVLTGLFYYMDV
(SEQ ID NO: 2841)


I076E09
1543
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
DDRDILTNYYLEYFQH
(SEQ ID NO: 2868)


I076E11
1544
144-254
166-179
195-201
234-243
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076F01
1545
144-253
166-178
194-199
232-242
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076F03
1546
141-251
163-175
191-197
230-240
1-124
26-36
51-66
99-113
ODYDVLTGYLRKLDY
(SEQ ID NO: 2742)


I076F04
1547
135-245
157-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076F08
1548
142-250
164-174
190-196
229-239
1-124
26-36
51-66
99-113
VHYDILTGYLWAFDI
(SEQ ID NO: 2730)


I076F10
1549
141-252
163-175
191-197
230-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I076G09
1550
134-245
156-168
184-190
223-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076G10
1551
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
GRYYDMLTRGGYFDY
(SEQ ID NO: 2858)


I076G11
1552
144-259
166-179
195-205
240-248
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076G12
1553
147-257
169-181
197-203
236-246
1-130
26-35
50-66
99-119
NGYYDILTGYYLWDYYYGMDV
(SEQ ID NO: 2769)


I076H02
1554
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
ENYDSLTGYYNYFDY
(SEQ ID NO: 2971)


I076H04
1555
143-251
165-175
191-197
230-240
1-125
26-35
50-66
99-114
THYDILTGYYSHPLDY
(SEQ ID NO: 2863)


I076H05
1556
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I076H06
1557
141-252
163-176
192-198
231-241
1-124
26-35
50-66
99-113
VPYDILTGYWGAFDV
(SEQ ID NO: 2827)


I076H09
1558
144-256
166-179
195-201
234-245
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I076H10
1559
144-256
166-179
195-201
234-245
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I077D06
1560
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I078B04
1561
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I078E10
1562
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I002A01-K
1563
142-250
164-174
190-196
229-239
1-125
26-35
50-66
99-114
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I002A01-R
1564
142-250
164-174
190-196
229-239
1-125
26-35
50-66
99-114
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I026C04-K
1565
142-250
164-176
192-198
231-239
1-125
26-35
50-66
99-114
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I026C04-R
1566
142-250
164-176
192-198
231-239
1-125
26-35
50-66
99-114
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I067B10
1567
149-259
171-183
199-205
238-248
1-133
26-35
50-66
99-122
DRGAPNYDILTGYYAPAQGVAFDI
(SEQ ID NO: 2176)


I068C06
1568
134-241
156-169
185-191
224-233
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075F12
1569
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I003C06
1570
141-249
163-173
189-195
228-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025B06
1571
141-249
163-175
191-197
230-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I025B09
1572
141-249
163-175
191-197
230-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I026C04
1573
141-249
163-175
191-197
230-238
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I027B12
1574
142-250
164-174
190-196
229-239
1-125
26-34
49-65
99-114
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I030A10
1575
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I064C04
1576
147-257
169-182
198-204
237-246
1-131
26-35
50-66
99-120
DGRLSYDILTGYYARDYYGMDD
(SEQ ID NO: 2188)


I064C07
1577
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
SEGTIFGVD
(SEQ ID NO: 2178)


I065D04
1578
144-254
166-179
195-201
234-243
1-128
26-36
51-66
99-117
GKGYYDILTGYYRDNWFDP
(SEQ ID NO: 2181)


I065D08
1579
147-257
169-182
198-204
237-246
1-131
26-35
50-66
99-120
TPSSVYDLLTGYYHYFYSYMDV
(SEQ ID NO: 2189)


I065F08
1580
135-242
158-168
184-190
223-231
1-119
26-35
50-66
99-108
EKSAAGYFDY
(SEQ ID NO: 2190)


I067F05
1581
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ENYDSLTGYYGAFDI
(SEQ ID NO: 2185)


I068B04
1582
134-241
156-168
184-190
223-242
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I068B08
1583
141-252
163-175
191-197
231-241
1-124
26-34
49-65
98-113
KLGLSIVGATTGALDM
(SEQ ID NO: 2186)


I068C08
1584
143-254
165-178
194-200
233-243
1-126
26-35
50-66
99-115
EGMNDFINSHHYYTMDA
(SEQ ID NO: 2182)


I068F03
1585
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
AGNEY0HTERPADY
(SEQ ID NO: 2180)


I069B07
1586
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I071B03
1587
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072B09
1588
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTOYSFDGFDI
(SEQ ID NO: 2153)


I073F04
1589
136-246
158-171
187-193
226-235
1-120
26-35
50-66
99-109
SLATRPLGMDV
(SEQ ID NO: 2184)


I074B12
1590
142-252
164-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075A02
1591
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I075G01
1592
142-251
164-174
190-196
229-240
1-124
26-35
50-66
99-113
DHFDTLTGYFRRLDS
(SEQ ID NO: 2187)


I078D02
1593
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I078D08
1594
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
DAQSYYDILTGYQSYAFDI
(SEQ ID NO: 2183)


I078H08
1595
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I064A03
1596
150-257
171-181
197-203
236-246
1-134
26-35
50-66
99-123
GPSTTYYDILTGYYTPYYYYYYMDV
(SEQ ID NO: 3014)


I064B03
1597
145-255
167-179
195-201
234-241
1-129
26-37
52-67
100-118
HVRDYDILTGYYRGHYFDY
(SEQ ID NO: 2167)


I064B05
1598
140-250
162-174
190-196
229-239
1-124
26-35
50-66
99-113
ERGVVTAYGGDSFDL
(SEQ ID NO: 2985)


I064B11
1599
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
DRGPGLLSSFFES
(SEQ ID NO: 3033)


I064C02
1600
146-256
168-180
196-202
235-245
1-130
26-35
50-66
99-119
DEYYDILTGYQAPYYYYGMDV
(SEQ ID NO: 3068)


I064C03
1601
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ERGVVTAYGGDSFDL
(SEQ ID NO: 2985)


I064C11
1602
143-253
165-179
194-200
233-242
1-127
26-35
50-65
98-116
DVTYHDILTGYAGHEAFDI
(SEQ ID NO: 3055)


I064C12
1603
148-255
171-181
197-203
236-241
1-132
26-37
52-69
102-121
ESGRYDILTGYYSGGGGMDV
(SEQ ID NO: 3012)


I064D03
1604
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
DGANYDILTGYYTTTVYGMDV
(SEQ ID NO: 3072)


I064D04
1605
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
RSYDILTGYYTYGMDV
(SEQ ID NO: 3090)


I065B12
1629
139-249
161-174
190-196
229-238
1-123
26-35
50-66
99-112
EGAADYLNGQYFQH
(SEQ ID NO: 2815)


I064D06
1606
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
EGSSGYLVG
(SEQ ID NO: 2981)


I064E05
1607
146-256
168-180
196-202
235-245
1-130
26-37
52-67
100-119
KQRGDYDILTGYQLGYAFDI
(SEQ ID NO: 2808)


I064E06
1608
145-255
167-180
196-202
235-241
1-129
26-35
50-66
99-118
ERPGYDILTGYPSSIYGMDV
(SEQ ID NO: 3053)


I064F07
1609
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I064F09
1610
147-257
169-181
197-203
236-246
1-131
26-35
50-66
99-120
DTLGYDILTGYPPPYYYYDMDV
(SEQ ID NO: 2988)


I064F10
1611
143-253
165-177
193-199
232-242
1-127
22-31
46-62
95-116
DTLGYDILTGYPPPYYYYDMDV
(SEQ ID NO: 2988)


I064F11
1612
142-252
164-177
193-199
232-241
1-126
26-35
50-65
98-115
GRHYYDILTGYYNEAFDI
(SEQ ID NO: 3031)


I064G01
1613
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
NYYDVLTQSYYGMDV
(SEQ ID NO: 3077)


I064G04
1614
133-243
155-167
183-189
222-232
1-117
26-35
50-66
99-106
DNS0TYGY
(SEQ ID NO: 3084)


I064G08
1615
138-245
159-169
185-191
224-234
1-122
26-35
50-66
99-111
GGVTAGRSVYFDS
(SEQ ID NO: 2990)


I064G10
1616
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
SPNGDYSGYAWGLEY
(SEQ ID NO: 3085)


I064011
1617
138-248
160-173
189-195
228-237
1-122
26-35
50-65
98-111
YFDGSGYYPVSFSY
(SEQ ID NO: 3064)


I064012
1618
139-249
161-173
189-195
228-238
1-123
26-35
50-65
98-112
VNYDILTGLGYYFDY
(SEQ ID NO: 3049)


I064H03
1619
143-253
165-178
194-200
233-242
1-127
26-37
52-67
100-116
SYYDILTGRPYTDAFDI
(SEQ ID NO: 2989)


I064H04
1620
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
PLGITAVRGAKTDAFGI
(SEQ ID NO: 2929)


I064H06
1621
149-256
170-180
196-202
235-245
1-133
26-35
50-66
99-122
DRGASNYDILTGYYAPAQGVAFDI
(SEQ ID NO: 2969)


I065A02
1622
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065A04
1623
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065A06
1624
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065A07
1625
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DGGGYDILTGYQYYYGMDV
(SEQ ID NO: 2987)


I065B01
1626
145-255
167-180
196-202
235-241
1-129
26-35
50-65
98-118
WATYYDTLTGYRLKDHAGFDI
(SEQ ID NO: 3017)


I065B05
1627
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
SPGDDILTGYYKYYFDY
(SEQ ID NO: 3032)


I065B09
1628
146-253
167-177
193-199
232-242
1-130
26-35
50-66
99-119
DAGESYDILTGYYVIEGYMDV
(SEQ ID NO: 2986)


I065C02
1630
136-246
158-170
186-192
225-235
1-120
26-35
50-66
99-109
EGSWSGLDLDY
(SEQ ID NO: 3007)


I065C06
1631
141-253
163-175
191-197
230-242
1-125
26-35
50-66
99-114
ATYDPLT0YSFDGFDI
(SEQ ID NO: 2153)


I065C08
1632
141-250
163-176
192-198
231-239
1-125
26-35
50-66
99-114
VSGYNSGYFESYDMDV
(SEQ ID NO: 2732)


I065C10
1633
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
QGGQYDSPPLDV
(SEQ ID NO: 3002)


I065D01
1634
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
DRDYDILTDYSNYGMDV
(SEQ ID NO: 3074)


I065D03
1635
142-249
165-175
191-197
230-238
1-126
26-35
50-66
99-115
APLYDILTGYYIGGNDY
(SEQ ID NO: 3028)


I065D05
1636
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DKDYDILTGYWRDELLDY
(SEQ ID NO: 3040)


I065D06
1637
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
DPNYDILTGYYYYAMDV
(SEQ ID NO: 3062)


I065E01
1638
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
EFDQLLARGHGMDV
(SEQ ID NO: 3027)


I065E05
1639
137-241
158-168
184-190
223-242
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I065E06
1640
146-256
168-181
197-203
236-245
1-130
26-35
50-66
99-119
ARGSYD1LTGYYRPGDGYFDY
(SEQ ID NO: 3043)


I065E08
1641
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
GLYFEDTNYRHGDAFDI
(SEQ ID NO: 2790)


I065E09
1642
145-255
167-179
195-201
234-241
1-129
26-35
50-65
98-118
ERSYYDILTGYSPRSKYGMDV
(SEQ ID NO: 3021)


I065E12
1643
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065F04
1644
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ERGVVTAYGGDSFDL
(SEQ ID NO: 2985)


I065F05
1645
140-250
162-175
191-197
230-239
1-124
26-35
50-65
98-113
RYSDALTGYSLGAFDV
(SEQ ID NO: 3018)


I065F07
1646
145-252
166-176
192-198
231-241
1-129
26-38
53-69
102-118
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I065F09
1647
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
DYPIDVLTGRRTKNWFDP
(SEQ ID NO: 3013)


I065F12
1648
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
DQVDRLLMQYNYYMDA
(SEQ ID NO: 3047)


I065G01
1649
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I065G09
1650
143-253
165-179
194-200
233-242
1-127
26-35
50-68
101-116
DAYYDILTGWVYGMDV
(SEQ ID NO: 3030)


I065G10
1651
140-247
161-171
187-193
226-236
1-124
26-36
51-66
99-113
FRYDILTGYYYDMDV
(SEQ ID NO: 2983)


I065H05
1652
139-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
EYYDILTGYSGAFDI
(SEQ ID NO: 2984)


I065H07
1653
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
TRMDVLTRYYSDF
(SEQ ID NO: 2750)


I066A05
1654
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I066A06
1655
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
EGAADYLNGQYFQH
(SEQ ID NO: 2815)


I066A12
1656
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
DTRVIOIQLWERGAFDM
(SEQ ID NO: 3080)


I066B05
1657
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066B11
1658
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
PLGITAVRGAKTDAFGI
(SEQ ID NO: 2929)


I066C06
1659
144-254
166-178
194-200
233-243
1-128
26-35
50-65
98-117
GRRYYDILTGYSLGRGEMDV
(SEQ ID NO: 3009)


I066C10
1660
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066D02
1661
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGTSLMNYGTDV
(SEQ ID NO: 3048)


I066D07
1662
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
GPYDVLTGYLSGNFDY
(SEQ ID NO: 2992)


I066E01
1663
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
QOGQYDSPPFDV
(SEQ ID NO: 3001)


I066E03
1664
149-259
171-184
200-206
239-248
1-133
26-35
50-66
99-122
GEKARYYDILTGYYSAWGGYYMDV
(SEQ ID NO: 3045)


I066E04
1665
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
LNLEKTVIRGFGYFDL
(SEQ ID NO: 3081)


I066E05
1666
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
VGGYDILTGYYLRGMDV
(SEQ ID NO: 2997)


I066E07
1667
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066E09
1668
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066F01
1669
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
SPYDTLTGYVYNGVDV
(SEQ ID NO: 3058)


I066F03
1670
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I066F04
1671
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
VAAAGARTLGYFGMDV
(SEQ ID NO: 3071)


I066F07
1672
143-253
165-179
194-200
233-242
1-127
26-35
50-66
99-116
DVSGHDILTGYSYRYFDV
(SEQ ID NO: 2795)


I066F08
1673
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
SPMYYDRLTGFYPSGYFDS
(SEQ ID NO: 3036)


I066F11
1674
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I066F12
1675
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
GPSSAGTTIGLGSFDP
(SEQ ID NO: 3005)


I066G06
1676
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
ETRKYTSSPPYNYYYMDV
(SEQ ID NO: 2736)


I066G07
1677
133-243
155-168
184-190
223-232
1-117
26-30
45-61
94-106
DQFSVGGRHAFDL
(SEQ ID NO: 3054)


I066H02
1678
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I067A02
1679
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I067A03
1680
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067A06
1681
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I067A08
1682
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067A10
1683
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ERGVVTAYGGDSFDL
(SEQ ID NO: 2985)


I067B03
1684
142-253
164-177
193-199
232-242
1-126
26-35
50-66
99-115
PLGITAVRGAKTDAFGI
(SEQ ID NO: 2929)


I067B04
1685
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067C03
1686
134-241
156-169
185-191
224-233
1-117
26-35
50-66
99-106
DWGHWFDP
(SEQ ID NO: 2982)


I067C05
1687
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
SGSSLMTYGTDV
(SEQ ID NO: 3015)


I067C07
1688
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
EPYDILTGYYGSYFDY
(SEQ ID NO: 3041)


I067C10
1689
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067C12
1690
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
TYYDILTGYSGGGAFDY
(SEQ ID NO: 3024)


I067D01
1691
136-246
158-171
187-193
226-235
1-120
26-35
50-66
99-109
GSRVRGVTPDL
(SEQ ID NO: 3020)


I067D03
1692
137-241
158-168
184-190
223-242
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067D05
1693
146-256
168-180
196-202
235-245
1-130
26-35
50-66
99-119
ECSGSSCPARQPPYYQYYMDV
(SEQ ID NO: 2993)


I067D06
1694
137-241
158-168
184-190
223-242
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067D09
1695
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I067D12
1696
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
QGGQYDSPPLDV
(SEQ ID NO: 3002)


I067E02
1697
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067E04
1698
142-252
164-176
192-198
231-241
1-126
26-35
50-66
99-115
GAYYDILTGYYPYGMDV
(SEQ ID NO: 2860)


I067E05
1699
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DYRNYDILTGHPYYYGMDV
(SEQ ID NO: 2996)


I067F01
1700
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
QHYDILTGYSQEPFDI
(SEQ ID NO: 3022)


I067F03
1701
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DQTYYDILTGHYYYYGMDV
(SEQ ID NO: 3087)


I067F04
1702
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
EGAADYLNGQYFQH
(SEQ ID NO: 2815)


I067F08
1703
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
LGYYDILTGYRSDDY
(SEQ ID NO: 3029)


I067F10
1704
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMAYGTDV
(SEQ ID NO: 3016)


I067F11
1705
140-248
161-171
187-193
226-237
1-124
26-35
50-66
99-113
ENYDFLTGYYGAFDI
(SEQ ID NO: 2772)


I067G01
1706
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I067G09
1707
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I067H07
1708
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
GGLYDILTGRPATDDAFDI
(SEQ ID NO: 3035)


I068A07
1709
143-254
165-179
194-200
233-243
1-126
26-35
50-66
99-115
TDRFGAKDVTARWGMDV
(SEQ ID NO: 2979)


I068E05
1710
148-257
170-183
199-205
238-246
1-131
26-35
50-66
99-120
GREDTDKVKPWDRYYHYYYMDV
(SEQ ID NO: 2809)


I068E08
1711
135-247
157-169
185-193
226-236
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I068E11
1712
141-251
163-176
192-198
231-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I068F04
1713
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
ELGHREGGYWYSPYNV
(SEQ ID NO: 2838)


I068G05
1714
137-245
159-169
185-191
224-234
1-119
26-35
50-66
98-108
KNMGASAAADF
(SEQ ID NO: 3042)


I068G06
1715
140-250
162-174
190-196
229-239
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I068G11
1716
147-258
169-182
198-204
237-247
1-130
26-35
50-66
99-119
ESGSHYDLLTGLLVAANGFDV
(SEQ ID NO: 3044)


I069A09
1717
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I069A10
1718
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I069B06
1719
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I069B09
1720
139-249
161-174
190-196
229-238
1-123
26-35
50-66
99-112
PYYDILTGYFAFDI
(SEQ ID NO: 3026)


I069B12
1721
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGOYFDY
(SEQ ID NO: 2179)


I069C06
1722
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
VLPHYDILTGYSQNWFDP
(SEQ ID NO: 3000)


I069C09
1723
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
VLPHYDILTGYSQNWFDP
(SEQ ID NO: 3000)


I069D03
1724
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I069E09
1725
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DGYYDILTGYSYYGMDV
(SEQ ID NO: 2135)


I069E11
1726
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
VYYDILTGYNLFFDY
(SEQ ID NO: 2177)


I069F05
1727
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGOYFDY
(SEQ ID NO: 2179)


I069F07
1728
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I069F12
1729
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
GYYDILTGYYDAFDI
(SEQ ID NO: 3051)


I069G06
1730
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
DOYYDILTGYSGYYMDV
(SEQ ID NO: 3059)


I069G08
1731
145-252
166-176
192-198
231-241
1-129
26-35
50-66
99-118
DRLEYYDILTGYYYYYOMDV
(SEQ ID NO: 3039)


I069G11
1732
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I070A03
1733
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I070A09
1734
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I070B01
1735
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
SQSDYDILTGYYYYYGMDV
(SEQ ID NO: 3038)


I070B05
1736
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I070D03
1737
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I070D04
1738
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTSYYGGYFDY
(SEQ ID NO: 3034)


I070E01
1739
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
SQSDYDILTGYYYYYGMDV
(SEQ ID NO: 3038)


I070F01
1740
144-251
165-175
191-197
230-240
1-128
26-35
50-66
99-117
SQSNYDILTGYYYYYGMDV
(SEQ ID NO: 3067)


I070G10
1741
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I071A06
1742
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMODHYGMDV
(SEQ ID NO: 2161)


I071B02
1743
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I071D02
1744
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGTSLMNYGTDV
(SEQ ID NO: 3048)


I071D08
1745
146-256
168-181
197-203
236-245
1-130
26-37
52-66
99-119
VPYYYDTSGGYLOEYYYGMDV
(SEQ ID NO: 3010)


I071F01
1746
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGTSLMNYGTDV
(SEQ ID NO: 3048)


I071G09
1747
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072A01
1748
139-249
161-174
190-196
229-238
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I072A09
1749
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072B02
1750
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I072B10
1751
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGSSLMTYGTDV
(SEQ ID NO: 2773)


I072B11
1752
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDOFDI
(SEQ ID NO: 2153)


I072B12
1753
140-249
162-173
189-195
228-238
1-124
26-35
50-66
99-113
ENYDYLTGYYGAFDI
(SEQ ID NO: 2995)


I072C05
1754
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I072C10
1755
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTOYSFDGFDI
(SEQ ID NO: 2153)


I072D01
1756
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072D05
1757
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I072E01
1758
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072E04
1759
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EGSYDILTGYYVGVGRMDV
(SEQ ID NO: 2171)


I072E05
1760
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072E06
1761
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I072F03
1762
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I072F07
1763
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072F11
1764
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
DEYDILTGLLQGMDV
(SEQ ID NO: 2883)


I072G03
1765
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072G04
1766
137-247
159-171
187-193
226-236
1-121
26-35
50-68
101-110
RD1LTGFYDS
(SEQ ID NO: 2933)


I072G05
1767
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
GYRLNDWYGAFEI
(SEQ ID NO: 3079)


I072G09
1768
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072H03
1769
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I072H07
1770
137-247
159-172
188-194
227-236
1-121
26-35
50-66
99-110
AGTSLMNYGMDV
(SEQ ID NO: 3070)


I073A02
1771
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
GPYDILTGYYRDAFDI
(SEQ ID NO: 2998)


I073A03
1772
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
THYDILTGYYTADAFDI
(SEQ ID NO: 3019)


I073A04
1773
148-258
170-183
199-205
238-247
1-132
26-35
50-66
99-121
VQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 2132)


I073A05
1774
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073A06
1775
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073A09
1776
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073A10
1777
146-253
167-177
193-199
232-242
1-130
26-35
50-66
99-119
GDFGDYDILTGYYPVYYGMDV
(SEQ ID NO: 3082)


I073A11
1778
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
SYYDILTGYYPFGMDV
(SEQ ID NO: 3004)


I073B02
1779
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DLWYYDILTGYYLDDAFDI
(SEQ ID NO: 2999)


I073B05
1780
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
DLWYYDILTGYYLDDAFDI
(SEQ ID NO: 2999)


I073B06
1781
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I073B07
1782
138-248
160-173
189-195
228-237
1-122
26-35
50-66
99-111
TRMDVLTRYYSDF
(SEQ ID NO: 2750)


I073B08
1783
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073B11
1784
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073C01
1785
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
GYHDTLTSYNYNWFDP
(SEQ ID NO: 3006)


I073C02
1786
148-255
169-179
195-201
234-241
1-132
26-35
50-66
99-121
AQMDSEYYDLLTGINVGPYYFDY
(SEQ ID NO: 3076)


I073C04
1787
142-252
164-177
193-199
232-241
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073C07
1788
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
GMGDHYMDV
(SEQ ID NO: 3008)


I073C08
1789
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
EMGYDILTGYYLNYMDV
(SEQ ID NO: 2862)


I073C09
1790
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
QHYDILTGYSQEPFDI
(SEQ ID NO: 3022)


I073C11
1791
146-256
168-181
197-203
236-245
1-130
26-35
50-68
101-119
FNPTYDILTGYYIGGYFQH
(SEQ ID NO: 2155)


I073C12
1792
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073D01
1793
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073D03
1794
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I073D06
1795
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073D08
1796
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I073D10
1797
140-250
162-175
191-197
230-239
1-124
26-35
50-68
101-113
QYYDILTGYELDI
(SEQ ID NO: 3073)


I073D11
1798
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073E01
1799
148-258
170-183
199-205
238-247
1-132
26-37
52-69
102-121
EGAHYDILTGHNYYHYGMDV
(SEQ ID NO: 2747)


I073E02
1800
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073E03
1801
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSLDOFDI
(SEQ ID NO: 3003)


I073E05
1802
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
QHYDILTGYSQEPFDI
(SEQ ID NO: 3022)


I073E06
1803
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073E08
1804
140-250
162-175
191-197
230-239
1-124
26-35
50-66
99-113
ENYDFLTGYYGAFDI
(SEQ ID NO: 2772)


I073F01
1805
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F02
1806
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F03
1807
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F05
1808
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F07
1809
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
GEYDILTGYPYWYFDL
(SEQ ID NO: 3023)


I073F09
1810
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F11
1811
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073F12
1812
141-251
163-175
191-197
230-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073G03
1813
143-253
165-178
194-200
233-242
1-127
26-35
50-66
99-116
DGSYDILTGYYIDNYMDV
(SEQ ID NO: 2154)


I073G04
1814
143-253
165-178
194-200
233-242
1-127
26-35
50-65
98-116
GEGGYDILTGYLRGYGMDV
(SEQ ID NO: 3037)


I073G05
1815
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
GMGDHYGMDV
(SEQ ID NO: 2161)


I073G06
1816
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073G07
1817
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
GSYYDILTGISSLGMDV
(SEQ ID NO: 3063)


I073G08
1818
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
SRDLLLFPHYGMDV
(SEQ ID NO: 2133)


I073G09
1819
145-255
167-180
196-202
235-241
1-129
26-35
50-66
99-118
DRGHYDILTGYYIEPSGFDY
(SEQ ID NO: 3061)


I073G10
1820
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GPGVIGNYDY
(SEQ ID NO: 2749)


I073G12
1821
142-252
164-177
193-199
232-241
1-126
26-35
50-68
101-115
GGMIRAREDYYYMDV
(SEQ ID NO: 3083)


I073H01
1822
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073H03
1823
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073H05
1824
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073H06
1825
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I073H07
1826
138-245
159-169
185-191
224-234
1-122
26-35
50-66
99-111
TYYDILTGYYFDY
(SEQ ID NO: 3056)


I073H08
1827
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
ATYDPLTGYSFDGFDI
(SEQ ID NO: 2153)


I074A05
1828
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
LPPYDMLTGYYVGGGMDV
(SEQ ID NO: 3050)


I074A06
1829
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
AKPYTDFSRGSDADAFDV
(SEQ ID NO: 3065)


I074B03
1830
134-242
156-166
182-188
221-231
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I074B11
1831
140-251
162-175
191-197
230-240
1-123
26-35
50-66
99-112
RYGDPFYYYYYMNV
(SEQ ID NO: 2755)


I074C07
1832
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I074D03
1833
143-251
165-175
191-197
230-240
1-125
26-35
50-66
99-114
GGYDILTQYPAEFFHP
(SEQ ID NO: 2764)


I074D04
1834
134-246
156-169
185-191
224-235
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I074D05
1835
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
DRYYDILTKGDYYYGMDV
(SEQ ID NO: 3060)


I074D07
1836
151-262
173-186
202-208
241-251
1-134
26-35
50-66
99-123
VQGETYYDILTGYWGPKRDLYGMDV
(SEQ ID NO: 3069)


I074D08
1837
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
EL0LSIVVATTGALDM
(SEQ ID NO: 2980)


I074D11
1838
139-249
161-174
190-196
229-238
1-122
26-35
50-66
99-111
ESEGGDYTNPFGY
(SEQ ID NO: 2991)


I074E05
1839
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I074E07
1840
141-251
163-175
191-197
230-240
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I074E09
1841
147-258
169-182
198-204
237-247
1-130
26-35
50-68
101-119
DPGNYDILTGYYYYYGMDV
(SEQ ID NO: 2935)


I074E11
1842
138-241
160-170
186-192
225-233
1-121
26-35
50-66
99-110
VRLPHHHYFMAV
(SEQ ID NO: 3075)


I074H05
1843
144-254
166-178
194-200
233-243
1-126
26-35
50-66
99-115
ESS1TVNPPYYFYGMDV
(SEQ ID NO: 3025)


I075A03
1844
135-242
158-168
184-190
223-231
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I075A10
1845
135-241
157-169
185-191
224-233
1-117
26-35
50-66
99-106
DQORYLDL
(SEQ ID NO: 2175)


I075B07
1846
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
SPEGDYQPLSSNYNWLDP
(SEQ ID NO: 3011)


I075D11
1847
134-246
156-169
185-191
224-235
1-117
26-36
51-66
99-106
GKEGYNDN
(SEQ ID NO: 3089)


I075D12
1848
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I075G02
1849
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
SPEGDYQPLSSNYNWLDP
(SEQ ID NO: 3011)


I075G09
1850
143-253
165-177
193-199
232-242
1-126
26-35
50-66
99-115
MGHYDILTGYRHYGMDV
(SEQ ID NO: 2831)


I075G10
1851
140-250
162-174
190-196
229-239
1-122
26-35
50-66
99-111
GNYDILTGYPHDL
(SEQ ID NO: 3086)


I075H05
1852
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
SYYDILTGYYHTPLDY
(SEQ ID NO: 2853)


I075H07
1853
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
GSGYDLLTGYFTGSPLDY
(SEQ ID NO: 2766)


I076A11
1854
142-254
164-177
193-199
232-243
1-125
26-35
50-66
99-114
DDRDILTNYYLEYFQH
(SEQ ID NO: 2868)


I076A12
1855
144-256
166-178
194-200
233-245
1-127
26-35
50-66
99-116
GSGYDVLTGYFTGSPLDY
(SEQ ID NO: 3057)


I076B06
1856
142-249
164-174
190-196
229-238
1-124
26-35
50-66
99-113
GRYDILTGYFTSFDY
(SEQ ID NO: 3066)


I076B10
1857
142-254
164-177
193-199
232-243
1-125
26-35
50-66
99-114
DDRDILTNYYLEYFQH
(SEQ ID NO: 2868)


I076B12
1858
145-253
167-177
193-199
232-242
1-127
26-35
50-66
99-116
GTGYDILTGYYMGSAFDQ
(SEQ ID NO: 2800)


I076C06
1859
143-253
165-177
193-199
232-242
1-126
26-35
50-66
99-115
MGHYDILTGYRHYGMDV
(SEQ ID NO: 2831)


I076C11
1860
134-245
156-168
184-190
223-234
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076D06
1861
141-252
163-176
192-198
231-241
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I076E05
1862
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
GTGYDILTGYYMOSAFDQ
(SEQ ID NO: 2800)


I076E08
1863
135-243
157-167
183-189
222-232
1-117
26-35
50-66
99-106
DQGRYLDL
(SEQ ID NO: 2175)


I076F06
1864
134-245
156-169
185-191
224-234
1-117
26-36
51-66
99-106
RDVQGAPY
(SEQ ID NO: 3088)


I076G01
1865
144-254
166-178
194-200
233-243
1-127
26-35
50-66
99-116
VEGVYDILTGYSFDAFDI
(SEQ ID NO: 3078)


I076H01
1866
146-254
168-178
194-200
233-243
1-128
26-35
50-66
99-117
EQGYDILTGYYPEGGWFDP
(SEQ ID NO: 2834)


I076H03
1867
142-250
164-174
190-196
229-239
1-124
26-34
49-65
98-113
ELGLSIVGATTGALDM
(SEQ ID NO: 2174)


I077B05
1868
147-257
169-182
198-204
237-246
1-131
26-37
52-69
102-120
DKSYYDILTGYYYYYGMDV
(SEQ ID NO: 3052)


I077C10
1869
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYOGYFDY
(SEQ ID NO: 2179)


I077D01
1870
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I077D04
1871
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I077D11
1872
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I077D12
1873
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
EKYDILTGYYDAFDI
(SEQ ID NO: 3046)


I077E01
1874
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
EMGYDILTGYYLNYMDV
(SEQ ID NO: 2862)


I077E03
1875
142-252
164-177
193-199
232-241
1-126
26-35
50-66
99-115
EMGYDILTGYYLNYMDV
(SEQ ID NO: 2862)


I077E08
1876
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I077F05
1877
141-248
162-172
188-194
227-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I077G06
1878
141-251
163-176
192-198
231-240
1-125
26-35
50-66
99-114
MEYDILTGYYGOYFDY
(SEQ ID NO: 2179)


I077H02
1879
141-248
164-174
190-196
229-237
1-125
26-35
50-66
99-114
MEYDILTGYYGGYFDY
(SEQ ID NO: 2179)


I078B05
1880
143-253
165-179
194-200
233-242
1-127
26-35
50-66
99-116
ESHYDILTGYYSNPSFDI
(SEQ ID NO: 2994)


I079E02
1881
137-241
160-170
186-192
225-233
1-121
26-35
50-66
99-110
DSGSYYYDAFDI
(SEQ ID NO: 2194)


I079F11
1882
132-239
155-165
181-187
220-228
1-116
26-35
50-66
99-105
TGSGFDY
(SEQ ID NO: 2192)


I082G02
1883
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
DGYRTNDALDI
(SEQ ID NO: 2191)


I082H08
1884
132-242
154-167
183-189
222-231
1-115
26-35
50-66
99-104
DWDMDV
(SEQ ID NO: 2193)


I099D03
1885
137-247
159-172
188-194
227-236
1-120
26-35
50-66
99-109
DNGGGTIGFDY
(SEQ ID NO: 2195)


I079B05
1886
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
FVLDY
(SEQ ID NO: 2210)


I079B12
1887
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
WTSSGAFDI
(SEQ ID NO: 2205)


I079C01
1888
131-241
153-166
182-188
221-230
1-115
26-35
50-66
99-104
DWDMDV
(SEQ ID NO: 2193)


I079F06
1889
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
DNLHAAFDI
(SEQ ID NO: 2202)


I079F08
1890
138-248
160-172
188-194
227-237
1-122
26-35
50-66
99-111
YYYHSSGSDAFDI
(SEQ ID NO: 2206)


I080A03
1891
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I080A08
1892
136-247
158-171
187-193
226-236
1-119
26-35
50-66
99-108
VHSTGYAFEN
(SEQ ID NO: 2200)


I080B01
1893
144-254
166-178
194-200
233-243
1-126
26-35
50-66
99-115
EYSGYHYVEGGSYAMDV
(SEQ ID NO: 2201)


I080D03
1894
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I080E05
1895
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
EGGGDAYDVAPYYFDY
(SEQ ID NO: 2204)


I080G07
1896
138-245
161-172
188-194
227-234
1-120
26-35
50-66
99-109
EGPGYYYGMDV
(SEQ ID NO: 2209)


I080G09
1897
137-249
159-172
188-194
227-238
1-120
26-35
50-66
99-109
DNGGGTIGFDY
(SEQ ID NO: 2195)


I082A05
1898
131-240
153-165
181-187
220-229
1-115
26-35
50-66
99-104
DLDFDY
(SEQ ID NO: 2208)


I082B08
1899
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
DLGIAGTIYFDY
(SEQ ID NO: 2207)


I082C03
1900
138-245
161-171
187-193
226-234
1-122
26-35
50-66
99-111
DASRDIVVLPLAI
(SEQ ID NO: 2198)


I082D07
1901
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
WTSSGAFDI
(SEQ ID NO: 2205)


I082G01
1902
138-245
161-171
187-193
226-234
1-122
26-35
50-66
99-111
DRGSGWPNWYFDL
(SEQ ID NO: 2212)


I083B12
1903
139-247
161-171
187-193
226-236
1-121
26-35
50-66
99-110
ESGAGGYYYDDY
(SEQ ID NO: 2196)


I083G03
1904
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I084A01
1905
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084B02
1906
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084C04
1907
131-238
152-162
178-184
217-227
1-115
25-34
49-65
98-104
NLWGLDY
(SEQ ID NO: 2199)


I084C11
1908
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
GNAWGAFDI
(SEQ ID NO: 2211)


I079A01
1909
134-243
156-168
184-190
223-232
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I079A03
1910
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
GGMDWDFDY
(SEQ ID NO: 3183)


I079A04
1911
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
VDSSGYAYY
(SEQ ID NO: 3213)


I079A06
1912
133-240
154-164
180-186
219-229
1-117
26-35
50-66
99-106
DAAVTAEG
(SEQ ID NO: 3142)


I079A07
1913
136-246
158-170
186-192
225-235
1-120
26-35
50-66
99-109
GSNYSPDAFDI
(SEQ ID NO: 3112)


I079A10
1914
148-255
169-179
195-201
234-241
1-132
26-35
50-68
101-121
LPPDLRYCDGGICPGFDWLGP
(SEQ ID NO: 3163)


I079A11
1915
135-242
158-168
184-190
223-231
1-119
26-35
50-66
99-108
GPSYYYYMAV
(SEQ ID NO: 3114)


I079B02
1916
134-243
156-168
184-190
223-232
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I079B03
1917
136-246
158-170
186-192
225-235
1-120
26-35
50-66
99-109
GSNYSPDAFDI
(SEQ ID NO: 3112)


I079B04
1918
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
LLSDY
(SEQ ID NO: 3168)


I079B07
1919
138-245
159-169
185-191
224-234
1-122
26-35
50-66
99-111
DLSGSYFSRYFDY
(SEQ ID NO: 3193)


I079B09
1920
139-246
162-172
188-194
227-235
1-123
26-35
50-66
99-112
VEWEDIVVGSAFDI
(SEQ ID NO: 3128)


I079C02
1921
144-251
167-177
193-199
232-240
1-128
26-35
50-66
99-117
VTSLYSSSSGGYYYYGMDV
(SEQ ID NO: 3145)


I079C04
1922
132-239
155-165
181-187
220-228
1-116
26-35
50-66
99-105
GWRGVDY
(SEQ ID NO: 3195)


I079C05
1923
140-247
163-173
189-195
228-236
1-124
26-35
50-66
99-113
AGGNPRSGSLVYFDY
(SEQ ID NO: 3225)


I079C07
1924
137-241
158-168
184-190
223-242
1-121
26-35
50-66
99-110
GLDVYAIYGLDV
(SEQ ID NO: 3176)


I079D01
1925
144-254
166-179
195-201
234-243
1-128
26-35
50-66
99-117
EVRNYDLLTRSYLAGPLDN
(SEQ ID NO: 2751)


I079D02
1926
135-245
157-169
185-191
224-234
1-119
26-35
50-66
99-108
EIGWE0AFDI
(SEQ ID NO: 3178)


I079D04
1927
133-243
155-167
183-189
222-232
1-117
26-35
50-66
99-106
VRPGLMDV
(SEQ ID NO: 3132)


I079D06
1928
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
EAYTSSWAEFDF
(SEQ ID NO: 3190)


I079D07
1929
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
NITPLAMVGDF
(SEQ ID NO: 3146)


I079D08
1930
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
LIEDF
(SEQ ID NO: 3161)


I079D09
1931
131-238
152-162
178-184
217-227
1-115
26-35
50-66
99-104
DSGSPD
(SEQ ID NO: 3I08)


I079D11
1932
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I079E06
1933
136-241
158-168
184-190
223-242
1-120
26-35
50-66
99-109
EKRGSRRVFDI
(SEQ ID NO: 3093)


I079E08
1934
137-247
159-171
187-193
226-236
1-121
26-35
50-66
99-110
EAYASSWAEFDF
(SEQ ID NO: 3189)


I079E11
1935
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
PYGSGSYAFDI
(SEQ ID NO: 3185)


I079E12
1936
143-253
165-177
193-199
232-242
1-127
26-35
50-66
99-116
ARDYYDSSGYYVPDAFDI
(SEQ ID NO: 3I07)


I079F01
1937
133-241
154-164
180-186
219-230
1-117
26-35
50-66
99-106
GHFYGMDV
(SEQ ID NO: 3098)


I079F02
1938
148-253
169-179
195-201
234-242
1-132
26-35
50-68
101-121
LPPDLRYCDGGMCSGFDWLGP
(SEQ ID NO: 3219)


I079F03
1939
140-247
161-171
187-193
226-236
1-124
26-35
50-66
99-113
ESLLTEEYCGSDCYS
(SEQ ID NO: 3115)


I079F04
1940
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
NSAPPAPSMDV
(SEQ ID NO: 3099)


I079F09
1941
130-237
151-161
177-183
216-226
1-114
26-35
50-66
99-103
RYYDY
(SEQ ID NO: 3139)


I079F10
1942
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
NITPLAMVGDF
(SEQ ID NO: 3146)


I079F12
1943
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
ADYSNDYYMDV
(SEQ ID NO: 3166)


I079G02
1944
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
NITPLAMVGDF
(SEQ ID NO: 3146)


I079G05
1945
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
FPLESYYYMOV
(SEQ ID NO: 3124)


I079006
1946
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
GNSFGRTLDY
(SEQ ID NO: 3158)


I079G05
1947
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
DVPPPDGYLEV
(SEQ ID NO: 3192)


I079H06
1948
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
ASYPVPFDY
(SEQ ID NO: 3171)


I080A01
1949
132-242
154-166
182-188
221-231
1-115
26-35
50-66
99-104
GGWLDD
(SEQ ID NO: 3210)


I080A02
1950
134-245
156-169
185-191
224-234
1-117
26-35
50-66
99-106
EHSSSFDY
(SEQ ID NO: 3111)


I080A05
1951
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
EGEGDGYNVAPYYFDY
(SEQ ID NO: 3160)


I080A06
1952
143-250
166-176
192-198
231-239
1-125
26-35
50-66
99-114
EAGGSGSYHFSFPFDY
(SEQ ID NO: 3188)


I080A07
1953
136-247
158-171
187-193
226-236
1-119
26-35
50-66
99-108
TGIWGYYFDY
(SEQ ID NO: 3175)


I080A10
1954
142-252
164-176
192-198
231-241
1-125
26-35
50-66
99-114
DGNLNYDGSTDYGMDV
(SEQ ID NO: 3140)


I080B02
1955
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
LGRNYTSSWSLDY
(SEQ ID NO: 3181)


I080B03
1956
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VVGGYSSTLGTDV
(SEQ ID NO: 3096)


I080B05
1957
139-249
161-173
189-195
228-238
1-121
26-35
50-66
99-110
LGVARGREAFDL
(SEQ ID NO: 3206)


I080B06
1958
143-254
165-177
193-199
232-243
1-126
26-37
52-69
102-115
AVRSPGYYYYYMDV
(SEQ ID NO: 3125)


I080B07
1959
135-243
157-167
183-189
222-232
1-117
26-35
50-66
99-106
GRKPLFDY
(SEQ ID NO: 3141)


I080B08
1960
137-248
159-172
188-194
227-237
1-120
26-37
52-67
100-109
KQRREKYFDY
(SEQ ID NO: 3100)


I080B09
1961
143-254
165-179
194-200
233-243
1-126
26-35
50-66
99-115
EKAIIETTSGEADPFDI
(SEQ ID NO: 3151)


I080B10
1962
139-249
161-173
189-195
228-238
1-122
26-37
52-67
100-111
RPALRSLWYFDL
(SEQ ID NO: 3102)


I080B11
1963
138-248
160-172
188-194
227-237
1-121
26-35
50-68
101-110
LHCTGGSCGF
(SEQ ID NO: 3186)


I080B12
1964
141-253
164-179
195-201
234-242
1-123
26-35
50-66
99-112
NPYYYDSSEGFFDY
(SEQ ID NO: 3109)


I080C03
1965
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SGRQAYYYYGMDV
(SEQ ID NO: 3091)


I080C06
1966
146-254
168-178
194-200
233-243
1-128
26-36
51-66
99-117
DYYDGSSYSSGDYYYYMDV
(SEQ ID NO: 3227)


I080C07
1967
145-256
167-180
196-202
235-245
1-128
26-35
50-66
99-117
DSDLVVIPTAIQGRYYFDN
(SEQ ID NO: 3113)


I080C08
1968
138-249
160-173
189-195
228-238
1-121
26-35
50-66
99-110
GKRYSYGWYFDI
(SEQ ID NO: 3130)


I080C10
1969
132-243
154-167
183-189
222-232
1-115
26-35
50-66
99-104
DTPLDP
(SEQ ID NO: 3094)


I080C11
1970
138-249
160-173
189-195
228-238
1-121
26-35
50-66
99-110
EGDPTDNDAFDV
(SEQ ID NO: 3155)


I080C12
1971
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
DGPTYARPYYLDH
(SEQ ID NO: 3153)


I080D01
1972
138-245
161-171
187-193
226-234
1-120
26-35
50-66
99-109
DGTKYDWGFDY
(SEQ ID NO: 3220)


I080D02
1973
142-254
164-177
193-199
232-243
1-125
26-35
50-66
99-114
ETFSHCSGGSCYPFDY
(SEQ ID NO: 3212)


I080D04
1974
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
SGRQAYYYYGMDV
(SEQ ID NO: 3091)


I080D05
1975
138-246
160-170
186-192
225-235
1-120
26-35
50-66
99-109
EFFGYVYLTDY
(SEQ ID NO: 3165)


I080D08
1976
138-248
160-172
188-194
227-237
1-121
26-35
50-68
101-110
LHCTGGSCGF
(SEQ ID NO: 3186)


I080D09
1977
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
VDYTDYEMGAFEI
(SEQ ID NO: 3187)


I080D11
1978
136-247
158-171
187-193
226-236
1-119
26-35
50-66
99-108
VGNFGYYFEY
(SEQ ID NO: 3196)


I080D12
1979
137-245
159-169
185-191
224-234
1-119
26-35
50-68
101 -108
SSRNGGDY
(SEQ ID NO: 3214)


IOSCEOI
1980
138-246
160-170
186-192
225-235
1-120
26-35
50-66
99-109
DLSRVAGRFDY
(SEQ ID NO: 3164)


I080E04
1981
137-247
159-171
187-193
226-236
1-120
26-37
52-67
100-109
HDVYGDLFDY
(SEQ ID NO: 3211)


I080E06
1982
138-248
160-172
188-194
227-237
1-121
26-35
50-68
101-110
LHCSGGSCGF
(SEQ ID NO: 3221)


I080E07
1983
143-254
165-178
194-200
233-243
1-126
26-35
50-66
99-115
EGSIVGATLTINDAFDI
(SEQ ID NO: 3150)


I080E08
1984
138-249
160-173
189-195
228-238
1-121
26-35
50-66
99-110
GKRYSYGWYFDI
(SEQ ID NO: 3130)


I080E12
1985
132-242
154-166
182-188
221-231
1-114
26-35
50-66
99-103
DPFDY
(SEQ ID NO: 3134)


I080F04
1986
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
DGPTYARPYYLDH
(SEQ ID NO: 3153)


I080F05
1987
143-253
165-177
193-199
232-242
1-126
26-35
50-66
99-115
ESSGTLGEFSLELPFDY
(SEQ ID NO: 3203)


I080F06
1988
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
LGRNYTSSWSLDY
(SEQ ID NO: 3181)


I080F08
1989
132-240
154-164
180-186
219-229
1-114
26-35
50-66
99-103
NAFDY
(SEQ ID NO: 3121)


I080G03
1990
142-250
164-174
190-196
229-239
1-124
26-36
51-66
99-113
GRGYSSSSSVYOMDI
(SEQ ID NO: 3095)


I080G04
1991
133-241
156-171
187-193
226-233
1-115
26-35
50-66
99-104
VHSSGS
(SEQ ID NO: 3216)


I080G10
1992
145-252
167-177
193-199
232-241
1-127
26-35
50-66
99-116
KRGDFGVIRLHHYYGMDV
(SEQ ID NO: 3136)


l080G11
1993
137-247
159-171
187-193
226-236
1-120
26-37
52-67
100-109
HDVYGDLFDS
(SEQ ID NO: 3205)


I080H01
1994
142-252
164-176
192-198
231-241
1-124
26-37
52-67
100-113
LRPDADYGDYGFDY
(SEQ ID NO: 3218)


I080H02
1995
140-248
162-172
188-194
227-237
1-123
26-35
50-66
99-112
TSERGTYRQWDFDN
(SEQ ID NO: 3204)


I080H03
1996
136-246
158-170
186-192
225-235
1-119
26-35
50-66
99-108
EAGEVAAIDY
(SEQ ID NO: 3180)


I080H04
1997
138-249
160-173
189-195
228-238
1-121
26-35
50-66
99-110
GKRYSYGWYFDI
(SEQ ID NO: 3130)


I080H05
1998
137-247
159-171
187-193
226-236
1-120
26-37
52-67
100-109
HDVYGDLFDS
(SEQ ID NO: 3205)


I080H06
1999
138-249
160-173
189-195
228-238
1-121
26-35
50-66
99-110
OKRYSYGWYFDV
(SEQ ID NO: 3217)


I080H07
2000
138-248
160-172
188-194
227-237
1-121
26-35
50-68
101-110
LHCTGGSCGF
(SEQ ID NO: 3186)


I080H08
2001
140-251
162-175
191-197
230-240
1-122
26-35
50-66
99-111
ERGGRDGDYALDF
(SEQ ID NO: 3148)


I080H09
2002
141-249
163-173
189-195
228-238
1-123
26-36
51-66
99-112
RTPDHNGDSGPPDY
(SEQ ID NO: 3215)


I081A01
2003
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081A03
2004
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
ESLTGGAFDI
(SEQ ID NO: 3117)


I081A04
2005
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081A06
2006
130-237
151-161
177-183
216-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081A08
2007
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081A09
2008
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
GAGSRYFDL
(SEQ ID NO: 3118)


I081A10
2009
133-243
155-168
184-190
223-232
1-117
26-35
50-66
99-106
GGDRAFDI
(SEQ ID NO: 3119)


I081B01
2010
130-236
151-161
177-183
216-225
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081B04
2011
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
GNAWGAFDI
(SEQ ID NO: 2211)


I081B05
2012
133-243
155-168
184-190
223-232
1-117
26-35
50-66
99-106
GGDRAFDI
(SEQ ID NO: 3119)


I081B06
2013
133-240
154-164
180-186
219-229
1-117
26-35
50-66
99-106
VKRYYFDY
(SEQ ID NO: 3179)


I081B07
2014
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
ELTGANDAFDI
(SEQ ID NO: 3104)


I081B08
2015
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
RRYALDY
(SEQ ID NO: 2920)


I081B09
2016
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081B10
2017
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081B11
2018
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
GFALYKD
(SEQ ID NO: 3169)


I081C07
2019
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081C08
2020
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081D04
2021
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
EDLTGDAFDI
(SEQ ID NO: 3103)


I081D06
2022
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
GDAYFDY
(SEQ ID NO: 3147)


I081D08
2023
131-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
GDAYFDY
(SEQ ID NO: 3147)


I081D09
2024
130-238
152-162
178-184
217-227
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081D10
2025
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081D11
2026
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
EGLLDAFDI
(SEQ ID NO: 3200)


I081D12
2027
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081E02
2028
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081E03
2029
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081E05
2030
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081E06
2031
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
VGYGGKGDY
(SEQ ID NO: 3137)


I081E07
2032
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
GAGSRYFDL
(SEQ ID NO: 3118)


I081E10
2033
142-249
163-173
189-195
228-238
1-126
26-35
50-66
99-115
GLAPIVDGGMTNDAFDI
(SEQ ID NO: 3184)


I081F01
2034
130-239
152-164
180-186
219-228
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081F04
2035
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
RLIRKAR
(SEQ ID NO: 3170)


I081F05
2036
130-237
151-161
177-183
216-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081F06
2037
134-241
156-169
185-191
224-233
1-118
26-35
50-66
99-107
ERGNQAFDI
(SEQ ID NO: 3156)


I081F07
2038
132-239
153-163
179-185
218-228
1-116
26-35
50-66
99-105
RRYALDY
(SEQ ID NO: 2920)


I081F11
2039
130-237
151-161
177-183
216-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081G01
2040
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081G04
2041
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081G06
2042
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
SRSPYDAFDI
(SEQ ID NO: 3097)


I081G10
2043
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081H02
2044
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081H03
2045
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I081H04
2046
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
SNWGGDAFDI
(SEQ ID NO: 3202)


I081H06
2047
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
LAFDI
(SEQ ID NO: 3174)


I081H08
2048
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I082A02
2049
139-249
161-173
189-195
228-238
1-123
26-35
50-66
99-112
PAASSRGPKDAFDI
(SEQ ID NO: 3129)


I082A04
2050
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
LSGDS
(SEQ ID NO: 3122)


I082A08
2051
134-243
156-168
184-190
223-232
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I082A11
2052
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
FVLDY
(SEQ ID NO: 2210)


I082B06
2053
131-238
154-164
180-186
219-227
1-115
26-35
50-66
99-104
GNGKDV
(SEQ ID NO: 3135)


I082B09
2054
134-241
157-167
183-189
222-230
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I082B12
2055
131-241
153-166
182-188
221-230
1-115
26-35
50-66
99-104
DLDFDY
(SEQ ID NO: 2208)


I082C01
2056
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
VNDIVVVDMDV
(SEQ ID NO: 3143)


I082C05
2057
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
EKRGSRRVFDI
(SEQ ID NO: 3093)


I082C08
2058
136-241
158-168
184-190
223-242
1-121
26-35
50-66
99-110
LSNRNDNLRLDY
(SEQ ID NO: 3106)


I082D02
2059
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
FVLDY
(SEQ ID NO: 2210)


I082E05
2060
134-241
155-165
181-187
220-230
1-118
26-35
50-66
99-107
TWATNTFDM
(SEQ ID NO: 3152)


I082E06
2061
130-240
152-165
181-187
220-229
1-114
26-35
50-66
99-103
FDLDY
(SEQ ID NO: 3167)


I082E07
2062
139-246
162-172
188-194
227-235
1-123
26-35
50-66
99-112
VEWEDIVVGSAFDI
(SEQ ID NO: 3128)


I082F11
2063
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
GGDMTTVTTDY
(SEQ ID NO: 3177)


I082G07
2064
136-243
159-169
185-191
224-232
1-120
26-35
50-66
99-109
ADYSNDYYMDV
(SEQ ID NO: 3166)


I082G10
2065
138-249
160-173
189-195
228-238
1-118
26-35
50-66
99-107
EGVAAGEDY
(SEQ ID NO: 3123)


I082G11
2066
143-250
164-174
190-196
229-239
1-127
26-35
50-66
99-116
GPIYYFDGSAYEGYYFDY
(SEQ ID NO: 3222)


I082H04
2067
132-238
153-163
179-185
218-227
1-116
26-35
50-65
98-105
MNADAFEI
(SEQ ID NO: 3223)


I082H09
2068
139-246
160-170
186-192
225-235
1-123
26-35
50-66
99-112
PAASSRGPKDAFDI
(SEQ ID NO: 3129)


I083A06
2069
137-241
159-169
185-191
224-233
1-120
26-35
50-66
99-109
DSRPTNRAFHY
(SEQ ID NO: 3110)


I083A09
2070
138-248
160-172
188-194
227-237
1-121
26-35
50-68
101-110
LHCTGGSCGF
(SEQ ID NO: 3186)


I083A11
2071
136-248
158-171
187-193
226-237
1-119
26-35
50-66
99-108
VRDDSAGFDY
(SEQ ID NO: 3173)


I083B03
2072
139-247
161-171
187-193
226-236
1-121
26-35
50-66
99-110
VLVRGQYRGMDL
(SEQ ID NO: 3138)


I083B05
2073
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
VDYTDYEMGAFDL
(SEQ ID NO: 3172)


I083B06
2074
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
DRIAAAGGDAFDI
(SEQ ID NO: 3194)


I083B10
2075
139-246
162-172
188-194
227-235
1-121
26-35
50-66
99-110
DLYKNGYALFDS
(SEQ ID NO: 3197)


I083C01
2076
136-247
158-171
187-193
226-236
1-119
26-35
50-66
99-108
DEYSSLYMDV
(SEQ ID NO: 3201)


I083C02
2077
136-246
158-171
187-193
226-235
1-119
26-35
50-66
99-108
FGAGRLYDDY
(SEQ ID NO: 3224)


I083C07
2078
137-249
159-172
188-194
227-238
1-120
26-35
50-66
99-109
DNGGGTIGFDY
(SEQ ID NO: 2195)


I083C12
2079
136-246
158-171
187-193
226-235
1-119
26-35
50-66
99-108
DQGIETANDY
(SEQ ID NO: 3207)


I083D04
2080
146-256
168-181
197-203
236-245
1-129
26-35
50-66
99-118
DILPDYDFWNPNEDASSLDT
(SEQ ID NO: 3133)


I083D07
2081
150-262
173-188
204-210
243-251
1-132
26-35
50-66
99-121
DFQMVRGVFIANPPIYNYYGMDV
(SEQ ID NO: 3154)


I083D08
2082
143-254
165-178
194-200
233-243
1-126
26-35
50-66
99-115
DADEGLVEAETTNWFDS
(SEQ ID NO: 3126)


I083D10
2083
147-258
169-181
197-203
236-247
1-130
26-37
52-69
102-119
ATKSYDILTRMYYYHMDV
(SEQ ID NO: 2748)


I083D12
2084
134-242
156-166
182-188
221-231
1-116
26-35
50-66
99-105
DRTRMDV
(SEQ ID NO: 3182)


I083E02
2085
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I083E03
2086
136-248
158-171
187-193
226-237
1-119
26-35
50-66
99-108
DEIYNDAFDY
(SEQ ID NO: 3105)


I083E04
2087
144-255
166-179
195-201
234-241
1-127
26-35
50-66
99-116
DGDISDSPINNQNYAMDI
(SEQ ID NO: 3101)


I083E08
2088
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
RGGTSENYSGMDV
(SEQ ID NO: 3209)


I083E12
2089
135-245
157-170
186-192
225-234
1-118
26-35
50-66
99-107
DYPHNAFDI
(SEQ ID NO: 3127)


I083F02
2090
146-258
168-181
197-203
236-247
1-129
26-35
50-66
99-118
DVRSDRFWSGGYFHYSGMDV
(SEQ ID NO: 3131)


I083F04
2091
138-248
160-172
188-194
227-237
1-121
26-35
50-66
99-110
STLEVGATDFDY
(SEQ ID NO: 3199)


I083F06
2092
135-247
157-170
186-192
225-236
1-118
26-35
50-66
99-107
SDDWGAYHI
(SEQ ID NO: 3198)


I083F08
2093
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
ERGGRDGDYALDF
(SEQ ID NO: 3148)


I083F11
2094
137-248
159-172
188-194
227-237
1-120
26-35
50-66
99-109
ELVGAPGGFDP
(SEQ ID NO: 3191)


I083G04
2095
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
VDYTDYEMGAFDL
(SEQ ID NO: 3172)


I083G05
2096
139-249
161-173
189-195
228-238
1-121
26-35
50-68
101-110
SVAGRGNFDY
(SEQ ID NO: 3208)


I083G06
2097
139-250
161-174
190-196
229-239
1-122
26-35
50-66
99-111
ERGGRDGDYALDF
(SEQ ID NO: 3148)


I083G08
2098
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
EGGGDAYDVAPYYFDY
(SEQ ID NO: 2204)


I083G09
2099
132-242
154-166
182-188
221-231
1-114
26-35
50-66
99-103
DPFDY
(SEQ ID NO: 3134)


I083G11
2100
141-252
163-176
192-198
231-241
1-124
26-35
50-66
99-113
ALLGLPSDFSYYVDV
(SEQ ID NO: 3159)


I083H04
2101
142-253
164-177
193-199
232-242
1-125
26-35
50-66
99-114
EGEGDGYNVAPYYFDY
(SEQ ID NO: 3160)


I083H05
2102
135-243
157-167
183-189
222-232
1-117
26-35
50-66
99-106
TDYGGFDY
(SEQ ID NO: 3092)


I083H07
2103
139-247
161-171
187-193
226-236
1-121
26-35
50-66
99-110
GGVGDSRGVFDP
(SEQ ID NO: 3162)


I084A03
2104
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084A08
2105
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084B08
2106
135-242
156-166
182-188
221-231
1-119
26-35
50-66
99-108
ESLTGDAFDI
(SEQ ID NO: 3116)


I084C02
2107
136-243
157-167
183-189
222-232
1-120
26-35
50-66
99-109
SPLHFSDAFDI
(SEQ ID NO: 3120)


I084D03
2108
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084D05
2109
133-243
155-168
184-190
223-232
1-117
26-35
50-66
99-106
EVGGAFDI
(SEQ ID NO: 3157)


I084E01
2110
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084E06
2111
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084E10
2112
129-237
151-161
177-183
216-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084E12
2113
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084F04
2114
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084F07
2115
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084F12
2116
135-245
157-170
186-192
225-234
1-119
26-35
50-66
99-108
ESLTGDAFDI
(SEQ ID NO: 3116)


I084G12
2117
130-240
152-164
180-186
219-229
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I084H02
2118
130-237
153-163
179-185
218-226
1-114
26-35
50-66
99-103
DTTDY
(SEQ ID NO: 2203)


I099B05
2119
146-256
168-180
196-202
235-245
1-129
26-35
50-66
99-118
GAHYYDRSPSHLKSYWYFDL
(SEQ ID NO: 3149)


I099G09
2120
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I099H01
2121
140-248
162-172
188-194
227-237
1-122
26-35
50-66
99-111
LGRiNYTSSWSLDY
(SEQ ID NO: 3181)


I099H06
2122
139-249
161-173
189-195
228-238
1-122
26-35
50-66
99-111
VGIKAAAVDNFEY
(SEQ ID NO: 2197)


I099H08
2123
145-255
167-179
195-201
234-241
1-128
26-35
50-66
99-117
GGRYGYYYDGTOYVDAFDI
(SEQ ID NO: 3226)


I100A01
2124
137-247
159-172
188-194
227-236
1-120
26-35
50-66
99-109
DNGGGTIGFDY
(SEQ ID NO: 2195)


I100A10
2125
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
VRQQIADPPRSFFDP
(SEQ ID NO: 3144)


I100B03
2126
137-247
159-172
188-194
227-236
1-120
26-35
50-66
99-109
DNGGGTI0FDY
(SEQ ID NO: 2195)


I100B04
2127
137-247
159-172
188-194
227-236
1-120
26-35
50-66
99-109
DNGGGTIGFDY
(SEQ ID NO: 2195)


I100C03
2128
141-251
163-175
191-197
230-240
1-124
26-35
50-66
99-113
VRQQIADPPRSFFDP
(SEQ ID NO: 3144)


BAB2001
3240
140-247
163-173
189-195
228-236
1-124
31-35
50-65
99-113
EQYDTLTGSPYGMDV



BAB2080
3241
135-245
157-169
185-191
224-234
1-119
31-35
50-66
99-108
LNSLRGGHDY



BAB2015
3242
140-247
163-173
189-195
228-236
1-124
31-35
50-65
99-113
GASSGWYDYYYYMDV



BAB2019
3243
141-251
163-176
192-198
231-240
1-125
31-35
50-66
99-114
DSYDILTDYYNMIMDV



BAB2087
3244
144-253
167-177
193-199
232-242
1-128
31-35
50-66
99-117
GFTGYDILTDYYSVDYFDS



BAB2016
3245
141-251
163-176
192-198
231-246
1-125
31-35
20-66
99-114
DPRYDILTGYLYGMDV



BAB2034
3246
146-258
170-183
199-205
238-247
1-132
31-37
52-69
102-121
EGAHYDILTGHNYYHYGMDV



BAB2065
3247
141-250
162-175
191-197
230-239
1-125
31-35
50-66
99-114
ATYDPLTGYSFDGFD

## Claims

1. A method of antagonizing B Lymphocyte Stimulator activity in an animal having systemic lupus erythematosus or rheumatoid arthritis comprising administering to the animal an antibody comprising a first amino acid sequence that is at least 85% identical to amino acid residues 1-123 of SEQ ID NO:327 and a second amino acid sequence that is at least 85% identical to amino acid residues 141-249 of SEQ ID NO:327, and wherein the antibody binds B Lymphocyte Stimulator selected from the group consisting of:
(a) amino acid residues 1-285 of SEQ ID NO: 3228;
(b) amino acid residues 134-285 of SEQ ID NO: 3228; and
(c) a trimer comprising amino acid residues 134-285 of SEQ ID NO:3228.

2. A method of antagonizing B Lymphocyte Stimulator activity in a human patient having rheumatoid arthritis comprising administering to the human patient an antibody comprising a first amino acid sequence that is at least 85% identical to amino acid residues 1-123 of SEQ ID NO:327 and a second amino acid sequence that is at least 85% identical to amino acid residues 141-249 of SEQ ID NO:327, wherein the antibody binds B Lymphocyte Stimulator selected from the group consisting of:
(a) amino acid residues 1-285 of SEQ ID NO: 3228;
(b) amino acid residues 134-285 of SEQ ID NO: 3228; and
(c) a trimer comprising amino acid residues 134-285 of SEQ ID NO:3228.

3. The method of claim 2 further comprising administering one or more agents employed for treating rheumatoid arthritis selected from the group consisting of:
(a) a non-steroidal anti-inflammatory drug;
(b) an anti-malarial agent;
(c) a steroid;
(d) a cytotoxic agent;
(e) an immunosuppressive agent; and
(f) an anti-angiogenic factor.

4. The method of claim 2 further comprising administering one or more agents employed for treating rheumatoid arthritis selected from the group consisting of:
(a) a TNF antagonist;
(b) an anti-CD20 antibody;
(c) an interleukin-1 antagonist;
(d) CTLA4-Ig;
(e) methotrexate;
(f) leflunomide;
(g) sulfasalazine;
(h) auranofin;
(i) gold;
(j) azathioprine;
(k) penicillamine;
(l) cyclophosphamide; and
(m) cyclosporine.

5. A method of antagonizing B Lymphocyte Stimulator activity in a human patient having systemic lupus erythematosus comprising administering to the human patient an antibody comprising a first amino acid sequence that is at least 85% identical to amino acid residues 1-123 of SEQ ID NO:327 and a second amino acid sequence that is at least 85% identical to amino acid residues 141-249 of SEQ ID NO:327, wherein the antibody binds B Lymphocyte Stimulator selected from the group consisting of:
(a) amino acid residues 1-285 of SEQ ID NO: 3228;
(b) amino acid residues 134-285 of SEQ ID NO: 3228; and
(c) a trimer comprising amino acid residues 134-285 of SEQ ID NO:3228.

6. The method of claim 5 further comprising administering one or more agents employed for treating systemic lupus erythematosus selected from the group consisting of:
(a) a non-steroidal anti-inflammatory drug;
(b) an anti-malarial agent;
(c) a steroid;
(d) a cytotoxic agent;
(e) an immunosuppressive agent; and
(f) an anti-angiogenic factor.

7. The method of claim 5 further comprising administering one or more agents employed for treating systemic lupus erythematosus selected from the group consisting of:
(a) a TNF antagonist;
(b) an anti-CD20 antibody;
(c) an interleukin-1 antagonist;
(d) CTLA4-Ig;
(e) methotrexate;
(f) leflunomide;
(g) sulfasalazine;
(h) auranofin;
(i) gold;
(j) azathioprine;
(k) penicillamine;
(l) cyclophosphamide; and
(m) cyclosporine.

8. The method of claim 1, wherein the antibody is administered by a route selected from the group consisting of intravenous and subcutaneous.

9. The method of claim 2, wherein the antibody is administered by a route selected from the group consisting of intravenous and subcutaneous.

10. The method of claim 5, wherein the antibody is administered by a route selected from the group consisting of intravenous and subcutaneous.

11. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is a non-steroidal anti-inflammatory drug.

12. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is an anti-malarial agent.

13. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is a steroid.

14. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is a cytotoxic agent.

15. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is an immunosuppressive agent.

16. The method of claim 3, wherein the agent employed for treating rheumatoid arthritis is an anti-angiogenic factor.

17. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is a non-steroidal anti-inflammatory drug.

18. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is an anti-malarial agent.

19. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is a steroid.

20. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is a cytotoxic agent.

21. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is an immunosuppressive agent.

22. The method of claim 6, wherein the agent employed for treating systemic lupus erythematosus is an anti-angiogenic factor.

23. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is a TNF antagonist.

24. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is an anti-CD20 antibody.

25. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is an interleukin-1 antagonist.

26. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is CTLA4-Ig.

27. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is methotrexate.

28. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is leflunomide.

29. The method of claim 7, wherein the agent employed for treating systemic lupus erythematosus is cyclosporine.

30. The method of claim 1, wherein the first amino acid sequence is at least 95% identical to amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence is at least 95% identical to amino acid residues 141-249 of SEQ ID NO:327.

31. The method of claim 2, wherein the first amino acid sequence is at least 95% identical to amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence is at least 95% identical to amino acid residues 141-249 of SEQ ID NO:327.

32. The method of claim 5, wherein the first amino acid sequence is at least 95% identical to amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence is at least 95% identical to amino acid residues 141-249 of SEQ ID NO:327.

33. The method of claim 1, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

34. The method of claim 2, wherein the first amino acid sequence is comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

35. The method of claim 5, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

36. The method of claim 17, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

37. The method of claim 18, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

38. The method of claim 19, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

39. The method of claim 20, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

40. The method of claim 21, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

41. The method of claim 22, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

42. The method of claim 23, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

43. The method of claim 24, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

44. The method of claim 25, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

45. The method of claim 26, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

46. The method of claim 27, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

47. The method of claim 28, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

48. The method of claim 29, wherein the first amino acid sequence comprises amino acid residues 1-123 of SEQ ID NO:327 and the second amino acid sequence comprises amino acid residues 141-249 of SEQ ID NO:327.

49. A method of antagonizing B Lymphocyte Stimulator activity in a patient having systemic lupus erythematosus or rheumatoid arthritis comprising administering an antibody to the patient,
wherein the antibody comprises amino acid residues 26-35, 50-66, 99-112, 163-173, 189-195, and 228-238 of SEQ ID NO: 327, and
wherein the antibody binds B Lymphocyte Stimulator selected from the group consisting of:
(a) amino acid residues 1-285 of SEQ ID NO: 3228;
(b) amino acid residues 134-285 of SEQ ID NO: 3228; and
(c) a trimer comprising amino acid residues 134-285 of SEQ ID NO: 3228.

50. The method of claim 49, wherein the patient has systemic lupus erythematosus.

51. The method of claim 50, wherein the antibody is administered by a route selected from the group consisting of intravenous and subcutaneous.

52. The method of claim 50 further comprising administering one or more agents employed for treating systemic lupus erythematosus selected from the group consisting of:
a. a non-steroidal anti-inflammatory drug;
b. an anti-malarial agent;
c. a steroid;
d. a cytotoxic agent;
e. an immunosuppressive agent; and
f. an anti-angiogenic factor.

53. The method of claim 50 further comprising administering one or more agents employed for treating systemic lupus erythematosus selected from the group consisting of:
a. a TNF antagonist;
b. an anti-CD20 antibody;
c. an interleukin-1 antagonist;
d. CTLA4-Ig;
e. methotrexate;
f. leflunomide;
g. sulfasalazine;
h. auranofin;
i. gold;
j. azathioprine;
k. penicillamine;
l. cyclophosphamide; and
m. cyclosporine.

54. The method of claim 49, wherein the patient has rheumatoid arthritis.

55. The method of claim 54, wherein the antibody is administered by a route selected from the group consisting of intravenous and subcutaneous.

56. The method of claim 54 further comprising administering one or more agents employed for treating rheumatoid arthritis selected from the group consisting of:
a. a non-steroidal anti-inflammatory drug;
b. an anti-malarial agent;
c. a steroid;
d. a cytotoxic agent;
e. an immunosuppressive agent; and
f. an anti-angiogenic factor.

57. The method of claim 54 further comprising administering one or more agents employed for treating rheumatoid arthritis selected from the group consisting of:
a. a TNF antagonist;
b. an anti-CD20 antibody;
c. an interleukin-1 antagonist;
d. CTLA4-Ig;
e. methotrexate;
f. leflunomide;
g. sulfasalazine;
h. auranofin;
i. gold;
j. azathioprine;
k. penicillamine;
l. cyclophosphamide; and
m. cyclosporine.