Anti-blood dendritic cell antigen 2 antibody and its use

Abstract

To provide an anti-blood dendritic cell antigen 2 antibody and its use. [Solution] Antibodies and antibody fragments that bind to BDCA2 are disclosed. Also disclosed are methods for using the above antibodies and antibody fragments to induce plasmacytoid dendritic cell death, inhibit the production or secretion of inflammatory cytokines and chemokines, and treat or prevent immune disorders such as inflammatory and autoimmune conditions. (i) an isolated antibody or its antigen-binding fragment that selectively binds to the extracellular domain of human BDCA2, and (ii) competes with BIIB059 for binding to the extracellular domain of human BDCA2.

Description

Technical Field

[0001] Citation of Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 735,362, filed December 10, 2012, and U.S. Provisional Application No. 61 / 763,270, filed February 11, 2013.

Background Art

[0002] Background Blood dendritic cell antigen 2 (BDCA2) is a C-type lectin expressed on human plasmacytoid dendritic cells (pDC) (Dzionek et al., J. Immunol., Vol. 165: 6037-6046 (20 00)), and is a specialized population of bone marrow-derived cells that secrete type I interferon (IFN) in response to toll-like receptor (TLR) ligands. BDCA2 consists of a single extracellular carbohydrate recognition domain (CRD) belonging to the group of type II C-type lectins at its C-terminus, a transmembrane region, and a short cytoplasmic tail at its N-terminus that does not possess a signaling motif. BDCA2 transmits intracellular signals via the associated transmembrane adapter FcεRIγ and induces a B cell receptor (BCR)-like signaling cascade.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0004] Summary [[ID= forty-two]] This disclosure is based, at least in part, on the identification and characterization of antibodies that bind to BDCA2. Such antibodies may reduce or inhibit the secretion of inflammatory cytokines and chemokines. The anti-BDCA2 antibodies described herein may also deplete pDCs by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In addition, the anti-BDCA2 antibodies described herein may also downregulate the levels of CD32a and / or CD62L on the surface of pDCs. Furthermore, the anti-BDCA2 antibodies of this disclosure may mediate the internal translocation of BDCA2 from the cell surface of pDCs. For at least these reasons, the anti-BDCA2 antibodies described herein are useful in the treatment or prevention of autoimmune and inflammatory conditions. This disclosure also shows that the anti-BDCA2 antibodies described herein may be combined with antimalarial agents to improve efficacy.

[0005] In one embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and competes with BIIB059 for binding to the extracellular domain of human BDCA2.

[0006] If the subsequent binding of BIIB059 to BDCA2 is completely or partially inhibited by pre-binding an anti-BDCA2 antibody or its antigen-binding fragment to BDCA2, the anti-BDCA2 antibody or its antigen-binding fragment will compete with BIIB059 for binding to BDCA2. For example, if the subsequent binding of BIIB059 to BDCA2 is completely inhibited by pre-binding an anti-BDCA2 antibody or its antigen-binding fragment to BDCA2, the anti-BDCA2 antibody or its antigen-binding fragment will compete with BIIB059 for binding to BDCA2. In certain embodiments, pre-binding an anti-BDCA2 antibody or its antigen-binding fragment to BDCA2 results in at least 30%, 50%, 70%, 80%, 90%, 95%, 98%, or 99% inhibition of the subsequent binding of BIIB059 to BDCA2.

[0007] In another embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and (i) inhibits the secretion of type I and / or type III interferons, in addition to other cytokines and chemokines, from plasmacytoid dendritic cells, or (ii) induces or enhances the depletion of plasmacytoid dendritic cells in vitro. In certain embodiments, the anti-BDCA2 antibody downregulates CD32a and / or CD62L from the surface of pDCs. In some embodiments, the anti-BDCA2 antibody mediates the internal translocation of BDCA2 from the cell surface of pDCs. In some embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey BDCA2 (SEQ ID NO: 73) and rhesus monkey BDCA2 (SEQ ID NO: 72). In certain embodiments, the isolated antibody or its antigen-binding fragment inhibits the secretion or production of type I interferon, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), type III interferon, macrophage inflammatory protein 1 (MIP-1)-α / CCL3, MIP-1β / CCL4, chemokine (CC motif) ligand 5 (CCL5 / RANTES), or interferon-γ-inducing protein 10 (IP-10 / CXCL10).

[0008] In some embodiments of the two aspects described above, the isolated antibody or its antigen-binding fragment may, if necessary, have the following characteristics: 0.5-3 μg / mL or 4 nM-10 nM EC 50 (Human BDCA2); 0.5-3 μg / mL or 5 nM-10 nM EC 50(Cynomolgus monkey BDCA2); pI of 7-7.5; does not bind to rat Clec4b2 or binds to rat Clec4b2 with a lower binding affinity than human BDCA2, cynomolgus monkey BDCA2, or rhesus monkey BDCA2; inhibits the production or secretion of chemokines such as MIP-1-α / CCL3, MIP-1β / CCL4, CCL5 / RANTES, IP-10 / CXCL10; heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, wherein heavy chain CDR1 is shown in Sequence ID No. 9 The antibody or its antigen-binding fragment has an amino acid sequence consisting of an amino acid sequence, or an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 8; heavy chain CDR2 has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 10; heavy chain CDR3 has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 11; and the variable heavy chain further comprises or consists of one, two, three, four, five, or six of heavy chains CDR1, CDR2, and CDR3, which include or consist of the amino acid sequence shown in SEQ ID NO: 24. In certain embodiments, the antibody or its antigen-binding fragment has a heavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 89, a heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 91, and a heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, the antibody or its antigen-binding fragment has a heavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 9, a heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 92, and a heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, the antibody or its antigen-binding fragment has a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 90, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 93, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, the EC50 (human BDCA2) of the isolated antibody or antigen-binding fragment is 4.5 nM, 4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM, 5.0 nM, 5.1 nM, 5.2 nM, 5.3 nM, 5.4 nM, or 5.5 nM. In a specific embodiment, the EC50 (human BDCA2) of the isolated antibody or antigen-binding fragment is 4.9 nM.In some embodiments, the EC50 (cynomolgus monkey BDCA2) of the isolated antibody or antigen-binding fragment is 4.0 nM, 4.1 nM, 4.2 nM, 4.3 nM, 4.4 nM, 4.5 nM, 4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM, or 5.0 nM. In a specific embodiment, the EC50 (cynomolgus monkey BDCA2) of the isolated antibody or antigen-binding fragment is 4.4 nM. In certain embodiments of this model, the antibody has a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7 to 15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL.

[0009] In another embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 comprises or comprises the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), or the amino acid sequence shown in SEQ ID NO: 9, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Heavy chain CDR2 comprises or comprises the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), or the amino acid sequence shown in SEQ ID NO: 10, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Heavy chain CDR3 comprises or comprises the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or the amino acid sequence shown in SEQ ID NO: 11, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 89, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 91, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 11, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 9, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 92, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 11, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. In another embodiment, the antibody or its antigen-binding fragment comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 90, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 93, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions; and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 94, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.These antibodies (i) bind to human BDCA2 or cynomolgus monkey BDCA2 but do not significantly bind to BDCA2 derived from phylogenetic species lower than primates, and / or (ii) inhibit the production of TLR7 / TLR9-induced type I interferon and other cytokines or chemokines by human pDCs, and / or (iii) mediate the internal translocation of BDCA2 from the surface of pDCs, and / or (iv) downregulate CD32a and / or CD62L from the surface of pDCs, and / or (v) deplete pDCs in vitro by ADCC or CDC. In certain embodiments of this model, the antibody has a human heavy chain constant region and a human light chain constant region.

[0010] In certain embodiments, an isolated antibody or its antigen-binding fragment that specifically binds to human BDCA2 comprises: a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), or having substitutions at one or two amino acid positions; a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), or having substitutions at one or two amino acid positions; and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or having substitutions at one or two amino acid positions. In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR1 comprising or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), a heavy chain CDR2 comprising or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), and a heavy chain CDR3 comprising or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11). In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3. The light chain CDR1 comprises or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR2 comprises or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR3 contains or comprises the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.In certain embodiments, light chain CDR1 comprises or consists of the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at one or two amino acid positions; light chain CDR2 comprises or consists of the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at one or two amino acid positions; and light chain CDR3 comprises or consists of the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at one or two amino acid positions. In other embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), a light chain CDR1 containing or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), a light chain CDR2 containing or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), and a light chain CDR3 containing or derived from the amino acid sequence QQANEDPRT (SEQ ID NO: 7).

[0011] In certain embodiments, an isolated antibody or its antigen-binding fragment that selectively binds to human BDCA2 comprises: a heavy chain CDR1 containing or derived from the amino acid sequence TYTMS (SEQ ID NO: 8), or having substitutions at one or two amino acid positions; a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), or having substitutions at one or two amino acid positions; and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or having substitutions at one or two amino acid positions. In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR1 comprising or derived from the amino acid sequence TYTMS (SEQ ID NO: 8), a heavy chain CDR2 comprising or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), and a heavy chain CDR3 comprising or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11). In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3. The light chain CDR1 comprises or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR2 comprises or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR3 contains or comprises the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.In certain embodiments, light chain CDR1 comprises or consists of the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at one or two amino acid positions; light chain CDR2 comprises or consists of the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at one or two amino acid positions; and light chain CDR3 comprises or consists of the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at one or two amino acid positions. In other embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence TYTMS (SEQ ID NO: 8), a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), a light chain CDR1 containing or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), a light chain CDR2 containing or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), and a light chain CDR3 containing or derived from the amino acid sequence QQANEDPRT (SEQ ID NO: 7).

[0012] In certain embodiments, the isolated antibody or antigen-binding fragment selectively binding to human BDCA2 comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTY (SEQ ID NO: 89), or having substitutions at one or two amino acid positions; a heavy chain CDR2 containing or derived from the amino acid sequence SPGDSFG (SEQ ID NO: 91), or having substitutions at one or two amino acid positions; and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or having substitutions at one or two amino acid positions. In other embodiments of this aspect, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTY (SEQ ID NO: 89), a heavy chain CDR2 containing or derived from the amino acid sequence SPGDSFG (SEQ ID NO: 91), and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11). In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 comprises or comprises the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR2 comprises or comprises the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR3 comprises or comprises the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.In certain embodiments, light chain CDR1 comprises or consists of the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at one or two amino acid positions; light chain CDR2 comprises or consists of the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at one or two amino acid positions; and light chain CDR3 comprises or consists of the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at one or two amino acid positions. In other embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTY (SEQ ID NO: 89), a heavy chain CDR2 containing or derived from the amino acid sequence SPGDSFG (SEQ ID NO: 91), a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), a light chain CDR1 containing or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), a light chain CDR2 containing or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), and a light chain CDR3 containing or derived from the amino acid sequence QQANEDPRT (SEQ ID NO: 7).

[0013] In certain embodiments, the isolated antibody or antigen-binding fragment selectively binding to human BDCA2 comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), or having substitutions at one or two amino acid positions; a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYY (SEQ ID NO: 92), or having substitutions at one or two amino acid positions; and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or having substitutions at one or two amino acid positions. In other embodiments of this aspect, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYY (SEQ ID NO: 92), and a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11). In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 comprises or comprises the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR2 comprises or comprises the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR3 comprises or comprises the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.In certain embodiments, light chain CDR1 comprises or consists of the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at one or two amino acid positions; light chain CDR2 comprises or consists of the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at one or two amino acid positions; and light chain CDR3 comprises or consists of the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at one or two amino acid positions. In other embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), a heavy chain CDR2 containing or derived from the amino acid sequence TISPGDSFGYY (SEQ ID NO: 92), a heavy chain CDR3 containing or derived from the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), a light chain CDR1 containing or derived from the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), a light chain CDR2 containing or derived from the amino acid sequence AASTLES (SEQ ID NO: 6), and a light chain CDR3 containing or derived from the amino acid sequence QQANEDPRT (SEQ ID NO: 7).

[0014] In certain embodiments, the isolated antibody or antigen-binding fragment selectively binding to human BDCA2 comprises a heavy chain CDR1 containing or derived from the amino acid sequence STYTMS (SEQ ID NO: 90), or having substitutions at one or two amino acid positions; a heavy chain CDR2 containing or derived from the amino acid sequence WVATISPGDSFGYY (SEQ ID NO: 93), or having substitutions at one or two amino acid positions; and a heavy chain CDR3 containing or derived from the amino acid sequence TRDIYYNYGAWFA (SEQ ID NO: 94), or having substitutions at one or two amino acid positions. In other embodiments of this aspect, the isolated antibody or antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence STYTMS (SEQ ID NO: 90), a heavy chain CDR2 containing or derived from the amino acid sequence WVATISPGDSFGYY (SEQ ID NO: 93), and a heavy chain CDR3 containing or derived from the amino acid sequence TRDIYYNYGAWFA (SEQ ID NO: 94). In other embodiments of this model, the isolated antibody or antigen-binding fragment comprises light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 comprises or comprises the amino acid sequence DYDGDSYMNWY (SEQ ID NO: 95), or the amino acid sequence shown in SEQ ID NO: 95, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR2 comprises or comprises the amino acid sequence LLIYAASTLE (SEQ ID NO: 96), or the amino acid sequence shown in SEQ ID NO: 96, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Light chain CDR3 comprises or comprises the amino acid sequence QQANEDPR (SEQ ID NO: 97), or the amino acid sequence shown in SEQ ID NO: 97, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions.In certain embodiments, light chain CDR1 comprises or consists of the amino acid sequence DYDGDSYMNWY (SEQ ID NO: 95), or the amino acid sequence shown in SEQ ID NO: 95, having substitutions at one or two amino acid positions; light chain CDR2 comprises or consists of the amino acid sequence LLIYAASTLE (SEQ ID NO: 96), or the amino acid sequence shown in SEQ ID NO: 96, having substitutions at one or two amino acid positions; and light chain CDR3 comprises or consists of the amino acid sequence QQANEDPR (SEQ ID NO: 97), or the amino acid sequence shown in SEQ ID NO: 97, having substitutions at one or two amino acid positions. In other embodiments, the isolated antibody or its antigen-binding fragment comprises a heavy chain CDR1 containing or derived from the amino acid sequence STYTMS (SEQ ID NO: 90), a heavy chain CDR2 containing or derived from the amino acid sequence WVATISPGDSFGYY (SEQ ID NO: 93), a heavy chain CDR3 containing or derived from the amino acid sequence TRDIYYNYGAWFA (SEQ ID NO: 94), a light chain CDR1 containing or derived from the amino acid sequence DYDGDSYMNWY (SEQ ID NO: 95), a light chain CDR2 containing or derived from the amino acid sequence LLIYAASTLE (SEQ ID NO: 96), and a light chain CDR3 containing or derived from the amino acid sequence QQANEDPR (SEQ ID NO: 97).

[0015] In certain embodiments of the above-described model, the antibody has a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7–15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL.

[0016] In another embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and comprises a VH heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52. In some embodiments of this embodiment, the isolated antibody or its antigen-binding fragment comprises a VL light chain CDR1, light chain CDR2, and light chain CDR3 as shown in any one of SEQ ID NOs: 54, 56, or 58. The CDRs may be Kabat-mediated CDRs or any of alternative CDRs. In certain embodiments, the antibody has a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region comprises a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7–15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody comprises a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL. In another embodiment, the disclosure features an isolated antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) domain that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and is at least 80% identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24), or a VH domain represented by any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52.These antibodies (i) bind to human BDCA2 or cynomolgus monkey BDCA2 but do not significantly bind to BDCA2 derived from phylogenetic species lower than primates, and / or (ii) inhibit the production of TLR7 / TLR9-induced type I interferon and other cytokines or chemokines by human pDCs, and / or (iii) mediate the internal translocation of BDCA2 from the surface of pDCs, and / or (iv) downregulate CD32a and / or CD62L from the surface of pDCs, and / or (v) deplete pDCs in vitro by ADCC or CDC.

[0017] In certain embodiments of this model, the antibody or antibody fragment comprises or consists of a VH domain that is at least 90% identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24), or the VH domain shown in any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52. In some embodiments of this model, the antibody or antibody fragment comprises or consists of a VH domain that is at least 95% identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24), or the VH domain shown in any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52. In other embodiments of this model, the VH domain of the isolated antibody or antigen-binding fragment is identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24), or the VH domain shown in any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52. In certain embodiments, the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments of this model, the antibody or its antigen-binding fragment comprises or consists of a variable light chain (VL) domain that is at least 80% identical to the amino acid sequence of the VL domain of BIIB059 (SEQ ID NO: 23), or the VL domain shown in any one of SEQ ID NOs: 54, 56, or 58. In some embodiments of this model, the antibody or its antigen-binding fragment comprises or consists of a VL domain that is at least 90% identical to the amino acid sequence of the VL domain of BIIB059 (SEQ ID NO: 23), or the VL domain shown in any one of SEQ ID NOs: 54, 56, or 58. In some embodiments of this model, the antibody or its antigen-binding fragment comprises or consists of a VL domain that is at least 95% identical to the amino acid sequence of the VL domain of BIIB059 (SEQ ID NO: 23), or the VL domain shown in any one of SEQ ID NOs: 54, 56, or 58. In some embodiments of this model, the antibody or its antigen-binding fragment includes or comprises a VH domain identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24) and a VL domain identical to the amino acid sequence of the VL domain of BIIB059 (SEQ ID NO: 23).In some embodiments of this model, the antibody or its antigen-binding fragment comprises or consists of a VH domain identical to the amino acid sequence of the VH domain shown in any one of SEQ ID NOs: 40, 42, 44, 46, 49, or 52, and a VL domain shown in any one of SEQ ID NOs: 54, 56, or 58. In certain embodiments, the antibody or its antigen-binding fragment comprises or consists of a heavy chain comprising or derived from the amino acid sequence shown in SEQ ID NO: 4, and a light chain comprising or derived from the amino acid sequence shown in SEQ ID NO: 3. These embodiments relate to all of the above embodiments and their embodiments. In certain embodiments, the antibody or its antigen-binding fragment is a humanized antibody. In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody. In other embodiments, the antibody or antigen-binding fragment is a polyclonal antibody, a chimeric antibody, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an Fsc fragment, an Fv fragment, an scFv, an sc(Fv)2, or a diabody. In some embodiments, the antibody has a constant region of the IgG1 heavy chain.

[0018] In another embodiment, the disclosure provides an isolated antibody or an antigen-binding fragment thereof, comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of an antibody produced by a hybridoma that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and is deposited with ATCC as designation number PTA-13450. In certain embodiments of this embodiment, the antibody or the antigen-binding fragment further comprises light chain CDR1, light chain CDR2, and light chain CDR3 of an antibody produced by a hybridoma deposited with ATCC as designation number PTA-13450. In certain embodiments of this embodiment, the antibody has a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7–15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL.In another embodiment, the present disclosure provides an isolated antibody or an antigen-binding fragment thereof, comprising variant heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of an antibody produced by a hybridoma deposited with ATCC as designation number PTA-13450, which selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1), and which comprises one, two, or three amino acid substitutions compared to each of the variant heavy chain CDR1, CDR2, and CDR3 of the antibody produced by the hybridoma deposited with ATCC as designation number PTA-13450. In certain embodiments of this model, the antibody or its antigen-binding fragment further comprises variant light chain CDR1, CDR2, and CDR3 of the antibody produced by a hybridoma deposited with ATCC under designation number PTA-13450, each containing one, two, or three amino acid substitutions compared to the respective light chain CDR1, CDR2, and CDR3 of the antibody produced by the hybridoma deposited with ATCC under designation number PTA-13450. In certain embodiments of this model, the antibody has a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7–15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL.In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL. In another embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and cross-blocks the binding of an antibody produced by a hybridoma deposited with ATCC as designation number PTA-13450. In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7 to 15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL. In yet another embodiment, the disclosure features an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) in the same epitope as the antibody produced by a hybridoma deposited with ATCC under designation number PTA-13450. In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 7 to 15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 10 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL.In a further embodiment, the Disclosure features an isolated antibody or an antigen-binding fragment thereof, comprising a VH domain that is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to the VH domain of an antibody produced by a hybridoma that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and is deposited with ATCC under designation number PTA-13450. In certain embodiments of this model, the isolated antibody or its antigen-binding fragment contains a VL domain that is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% identical to the VL domain of the antibody produced by the hybridoma deposited with ATCC as designation number PTA-13450.

[0019] In all five embodiments described above, the antibody or its antigen-binding fragment further (i) inhibits the secretion of type I interferon and / or type III interferon from plasmacytoid dendritic cells, in addition to other cytokines and chemokines, or (ii) induces or enhances the depletion of plasmacytoid dendritic cells in vitro. In some embodiments of the five embodiments described above, the antibody downregulates CD32a and / or CD62L on pDCs (compared to pDCs not contacted with the anti-BDCA2 antibody). In certain embodiments, the antibody mediates the internal translocation of BDCA2 from the surface of pDCs. In some embodiments of the five embodiments described above, the antibody or its antigen-binding fragment binds to cynomolgus monkey BDCA2 (SEQ ID NO: 73) and rhesus monkey BDCA2 (SEQ ID NO: 72). In certain embodiments of the five embodiments described above, the isolated antibody or its antigen-binding fragment inhibits the secretion or production of type I interferon, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), type III interferon, macrophage inflammatory protein 1 (MIP-1)-α / CCL3, MIP-1β / CCL4, chemokine (CC motif) ligand 5 (CCL5 / RANTES), or interferon-γ-inducing protein 10 (IP-10 / CXCL10). In certain embodiments of the five embodiments described above, the antibody or its antigen-binding fragment is a humanized antibody. In some embodiments of the five embodiments described above, the antibody or its antigen-binding fragment is a monoclonal antibody. In some embodiments of the five embodiments described above, the antibody or its antigen-binding fragment is a single-chain antibody. In other embodiments of the five embodiments described above, the antibody or antigen-binding fragment is a polyclonal antibody, a chimeric antibody, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an Fsc fragment, an Fv fragment, scFv, sc(Fv)2, or a diabody. In some embodiments of the five embodiments described above, the antibody has an IgG1 heavy chain constant region. In some embodiments of the five embodiments described above, the antibody has an IgG2 heavy chain constant region. In some embodiments of the five embodiments described above, the antibody has an IgG4 heavy chain constant region. In some embodiments of the five embodiments described above, the antibody is a hybrid of an IgG1 heavy chain constant region and an IgG4 heavy chain constant region.

[0020] In certain embodiments, the disclosure provides isolated cells that produce any of the antibodies or antigen-binding fragments described above.

[0021] In other embodiments, the disclosure provides pharmaceutical compositions comprising one of the antibodies or antigen-binding fragments described above and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises one of the antibodies or antigen-binding fragments described above, formulated in a composition comprising 10–25 mM citrate, 100–200 mM sodium chloride, and a pH of 5.5–6.5. In certain embodiments, the pharmaceutical composition optionally comprises Tween-80 (0.01–0.3%, e.g., 0.03%). In yet another embodiment, the pharmaceutical composition comprises one of the antibodies or antigen-binding fragments described above, formulated in a composition comprising 20 mM sodium citrate, 150 mM sodium chloride, and a pH of 6.0.

[0022] In another embodiment, the present disclosure provides a method for producing an anti-BDCA2 antibody. The method comprises the steps of supplying cells containing the heavy and / or light chains of a BDCA2 antibody, incubating the cells under conditions that enable antibody expression, and isolating the antibody. The method optionally includes the step of purifying the antibody. In certain embodiments, the cells are CHO cells. In other embodiments, the cells are 293 cells. In a particular embodiment, the anti-BDCA2 antibody is BIIB059. In one embodiment, the anti-BDCA2 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, having a heavy chain containing or derived from the sequence shown in SEQ ID NO: 4, and a light chain containing or derived from the sequence shown in SEQ ID NO: 3. In another embodiment, the anti-BDCA2 antibody or its antigen-binding fragment comprises or consists of VH CDR1 containing or derived from the amino acid sequence of SEQ ID NO: 9, VH CDR2 containing or derived from the amino acid sequence of SEQ ID NO: 10, and VH CDR3 containing or derived from the amino acid sequence of SEQ ID NO: 11. In further embodiments, the anti-BDCA2 antibody or its antigen-binding fragment may include VH CDR1 containing or derived from the amino acid sequence of SEQ ID NO: 9, VH CDR2 containing or derived from the amino acid sequence of SEQ ID NO: 10, VH CDR3 containing or derived from the amino acid sequence of SEQ ID NO: 11, or VL containing or derived from the amino acid sequence of SEQ ID NO: 5 It comprises or consists of CDR1, VL CDR2 containing or derived from the amino acid sequence of SEQ ID NO: 6, and VL CDR3 containing or derived from the amino acid sequence of SEQ ID NO: 7.

[0023] In another embodiment, the present disclosure provides a method for detecting the presence of plasmacytoid dendritic cells in a tissue. The method comprises the step of contacting the tissue with an anti-BDCA2 antibody. In certain embodiments, the tissue is a skin biopsy from a subject having systemic lupus erythematosus. In certain embodiments, the tissue is a skin biopsy from a subject having scleroderma. In certain embodiments, the tissue is a skin biopsy from a subject having maculoplastic scleroderma. In certain embodiments, the tissue is a skin biopsy from a subject having rheumatoid arthritis. In certain embodiments, the tissue is a skin biopsy from a subject having psoriasis. In certain embodiments, the tissue is a skin biopsy from a subject having dermatomyositis. In certain embodiments, the tissue is a skin biopsy from a subject having polymyositis. In certain embodiments, the tissue is a skin biopsy from a subject having inflammatory bowel disease. In specific embodiments, systemic lupus erythematosus is cutaneous lupus, discoid lupus, or lupus nephritis. The anti-BDCA2 antibody or its antigen-binding fragment can be labeled, for example, with a fluorophore (e.g., Alexa Fluor 647). In certain embodiments, the anti-BDCA2 antibody is BIIB059. In other embodiments, the anti-BDCA2 antibody is clone 124B3.13 (Dendritics). In certain embodiments, the method further includes the step of contacting the tissue with an anti-CD123 antibody.

[0024] In another aspect, the Disclosure provides a method for inducing the death of plasmacytoid dendritic cells in a subject requiring such death. The method involves administering one of the antibodies or antigen-binding fragments described herein to a subject, or contacting plasmacytoid dendritic cells expressing BDCA2 with one of the antibodies or antigen-binding fragments described herein.

[0025] In another embodiment, the present disclosure features a method for reducing the production of inflammatory cytokines or inflammatory chemokines by plasmacytoid dendritic cells in subjects requiring such reduction. The method comprises the steps of administering an effective amount of any of the antibodies or antigen-binding fragments described herein to a subject, or contacting plasmacytoid dendritic cells expressing BDCA2 with an effective amount of any of the antibodies or antigen-binding fragments described herein. In certain embodiments, the inflammatory cytokine or inflammatory chemokine is selected from the group consisting of type I interferon, IL-6, or TNF-α, type III interferon, MIP-1α / CCL3, MIP-1β / CCL4, CCL5 / RANTES, and IP-10 / CXCL10.

[0026] In another embodiment, the disclosure features a method for downregulating the expression of CD32a on the surface of plasmacytoid dendritic cells. The method comprises the step of contacting plasmacytoid dendritic cells with an anti-BDCA2 antibody as described herein. In certain embodiments, the anti-BDCA2 antibody has an IgG1 heavy chain constant region. In some embodiments, the antibody has an IgG2 heavy chain constant region. In some embodiments, the antibody has an IgG4 heavy chain constant region. In some embodiments, the antibody is a hybrid of an IgG1 heavy chain constant region and an IgG4 heavy chain constant region. In certain embodiments, the antibody is deglycosylated. In specific embodiments, the antibody is a deglycosylated hybrid of an IgG1 heavy chain constant region and an IgG4 heavy chain constant region.

[0027] In another embodiment, the disclosure features a method for downregulating the expression of CD32a(FcγRIIa) on the surface of plasmacytoid dendritic cells in human subjects requiring such regulation. The method comprises the step of administering an effective amount of the anti-BDCA2 antibody described herein to a human subject. In certain embodiments, the anti-BDCA2 antibody has an IgG1 heavy chain constant region. In some embodiments, the antibody has an IgG2 heavy chain constant region. In some embodiments, the antibody has an IgG4 heavy chain constant region. In some embodiments, the antibody is a hybrid of the IgG1 heavy chain constant region and the IgG4 heavy chain constant region. In certain embodiments, the antibody is deglycosylated. In specific embodiments, the antibody is a deglycosylated hybrid of the IgG1 heavy chain constant region and the IgG4 heavy chain constant region.

[0028] In another embodiment, the disclosure features a method for inhibiting the stimulation of plasmacytoid dendritic cells by immune complexes in a human subject requiring such inhibition. The method comprises the step of administering an effective amount of the anti-BDCA2 antibody described herein to a human subject. In some embodiments, the administration reduces the level of CD32a on the surface of pDCs. In some embodiments, the subject has type III hypersensitivity. In one embodiment, the human subject has SLE. In another embodiment, the human subject has rheumatoid arthritis. In yet another embodiment, the subject has Sjögren's syndrome. In certain embodiments, the anti-BDCA2 antibody has an IgG1 heavy chain constant region. In some embodiments, the antibody has an IgG2 heavy chain constant region. In some embodiments, the antibody has an IgG4 heavy chain constant region. In some embodiments, the antibody is a hybrid of the IgG1 heavy chain constant region and the IgG4 heavy chain constant region.

[0029] In another embodiment, the present disclosure features a method for downregulating (or de-regulating) the expression of CD62L (L-selectin) on the surface of plasmacytoid dendritic cells in a human subject requiring such regulation. The method comprises the step of administering an effective amount of an anti-BDCA2 antibody or antigen-binding fragment described herein to a human subject. In specific embodiments, the administration of the anti-BDCA2 antibody or antigen-binding fragment increases the level of one or more metalloproteinases. In certain embodiments, the downregulation of CD62L occurs via cleavage by metalloproteinases. In certain embodiments, the anti-BDCA2 antibody has an IgG1 heavy chain constant region. In some embodiments of the above five embodiments, the antibody has an IgG2 heavy chain constant region. In some embodiments of the above five embodiments, the antibody has an IgG4 heavy chain constant region. In some embodiments of the above five embodiments, the antibody is a hybrid of an IgG1 heavy chain constant region and an IgG4 heavy chain constant region.

[0030] In further embodiments, the Disclosure features a method for treating an inflammatory disorder in a subject requiring such treatment. The method involves administering to a subject requiring such treatment an effective amount of one of the anti-BDCA2 antibodies or antigen-binding fragments described herein. In some embodiments, the inflammatory disorder is selected from the group consisting of systemic lupus erythematosus (SLE), cutaneous lupus, discoid lupus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis, plaque scleroderma, psoriasis, type 1 diabetes mellitus, dermatomyositis, polymyositis, and Sjögren's disease. In one particular embodiment, the inflammatory disorder is SLE. In another particular embodiment, the inflammatory disorder is discoid lupus. In yet another particular embodiment, the inflammatory disorder is lupus nephritis. In yet another particular embodiment, the inflammatory disorder is cutaneous lupus. In some embodiments, the subject has general SLE. In some embodiments, the subject has moderate SLE. In certain embodiments, the subject has moderate SLE without severely active CNS and / or severely active renal complications. In certain embodiments, the subject has moderate SLE with severely active CNS and / or severely active renal complications. In certain embodiments, the subject has skin manifestations of SLE (e.g., cheek rash or discoid rash). In certain embodiments, the subject has severe SLE. In certain embodiments, the subject has severe SLE without severely active CNS and / or severely active renal complications. In certain embodiments, the subject has severe SLE with severely active CNS and / or severely active renal complications. Moderate or severe lupus refers to lupus staging (e.g., Guidelines). For Referral and Management of Systemic Lupus Erythematosus in Adults, Arthritis & Rheumatism, Vol. 42 (No. 9): pp. 1785-1795 (1999); Gladman, Prognosis and treatment of systemic lupus erythematosus, Curr. Opin. Rheumatol., Vol. 8: pp. 430-437 (1996); Kaluanian et al., Definition, See Classification, Activity and Damage Indices, Dubois' lupus eyrthematosus, 5th edition, Baltimore, Williams and Wilkins, pp. 19-30 (1997).

[0031] In another aspect, the present disclosure features a method for treating an autoimmune disease in a subject requiring such treatment. The method involves administering to the subject requiring such treatment an effective amount of one of the anti-BDCA2 antibodies or antigen-binding fragments described herein.

[0032] In certain embodiments of any of the above embodiments of the method, the subject is human. In certain embodiments of any of the above embodiments of the method, an anti-BDCA2 antibody or antigen-binding fragment is administered in combination with at least one of the following: an antimalarial agent (e.g., hydroxychloroquine), a TLR7 signaling inhibitor, a TLR9 signaling inhibitor, or a corticosteroid. In specific embodiments, the anti-BDCA2 antibody comprises the heavy-chain and light-chain CDR of BIIB059. In one embodiment, the anti-BDCA2 antibody comprises the heavy-chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 8, 10, and 11, respectively, and the light-chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises the heavy-chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 89, 91, and 11, respectively, and the light-chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 9, 92, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In yet another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 90, 93, and 94, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 95, 96, and 97, respectively. In certain embodiments, the anti-BDCA2 antibody further comprises an Fc region that binds to CD32a at an EC50 of at least about 7–15 μg / mL (e.g., 10, 11, 12 μg / mL). In a specific embodiment, the anti-BDCA2 antibody is BIIB059.

[0033] In another embodiment, the disclosure features a combination comprising an antimalarial agent (e.g., hydroxychloroquine) and an anti-BDCA2 antibody or its antigen-binding fragment. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR (or alternative CDR) of SEQ ID NO: 24. In another embodiment, the anti-BDCA2 antibody comprises the light chain CDR (or alternative CDR) of SEQ ID NO: 23. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR and light chain CDR of BIIB059. In one embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 8, 10, and 11, respectively, and the light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 89, 91, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 9, 92, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In yet another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 90, 93, and 94, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 95, 96, and 97, respectively. In certain embodiments, the anti-BDCA2 antibody further comprises an Fc region that binds to CD32a at an EC50 of at least about 7–15 μg / mL (e.g., 9, 10, 11, 12, 13, 14 μg / mL). In specific embodiments, the anti-BDCA2 antibody is BIIB059.

[0034] In another embodiment, the disclosure features a combination comprising an inhibitor of TLR7 and / or TLR9 signaling and an anti-BDCA2 antibody or its antigen-binding fragment. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR (or alternative CDR) of SEQ ID NO: 24. In another embodiment, the anti-BDCA2 antibody comprises the light chain CDR (or alternative CDR) of SEQ ID NO: 23. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR and light chain CDR of BIIB059. In one embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 8, 10, and 11, respectively, and the light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 89, 91, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 9, 92, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In yet another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 90, 93, and 94, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 95, 96, and 97, respectively. In certain embodiments, the anti-BDCA2 antibody further comprises an Fc region that binds to CD32a at an EC50 of at least about 7–15 μg / mL (e.g., 10, 11, 12 μg / mL). In specific embodiments, the anti-BDCA2 antibody is BIIB059.

[0035] In further embodiments, the disclosure features a combination comprising a corticosteroid and an anti-BDCA2 antibody or its antigen-binding fragment. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR (or alternative CDR) of SEQ ID NO: 24. In another embodiment, the anti-BDCA2 antibody comprises the light chain CDR (or alternative CDR) of SEQ ID NO: 23. In a specific embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR and light chain CDR of BIIB059. In one embodiment, the anti-BDCA2 antibody comprises the heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 8, 10, and 11, respectively, and the light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 89, 91, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 9, 92, and 11, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 5, 6, and 7, respectively. In yet another embodiment, the anti-BDCA2 antibody comprises heavy chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 90, 93, and 94, respectively, and light chain CDR1, CDR2, and CDR3 shown in SEQ ID NOs. 95, 96, and 97, respectively. In certain embodiments, the anti-BDCA2 antibody further comprises an Fc region that binds to CD32a at an EC50 of at least about 7–15 μg / mL (e.g., 9, 10, 11, 12, 13, 14 μg / mL). In specific embodiments, the anti-BDCA2 antibody is BIIB059.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. While similar or equivalent methods and materials may be used in the implementation or testing of the present invention, only exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. Conflicts of interest, including their definitions, are governed by this application. Materials, methods, and examples are illustrative and not intended to be limiting.

[0037] Other features and advantages of the present invention will become apparent from the following detailed description and claims. In certain embodiments, for example, the following are provided: (Item 1) (i) an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and (ii) competes with BIIB059 for binding to the extracellular domain of human BDCA2. (Item 2) Selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1), (i) Inhibit TLR-induced production of type I interferon, IL-6, TNF-α, CCL3, CCL4, IP10, and RANTES from plasmacytoid dendritic cells, or (ii) In vitro, induce or enhance the depletion of plasmacytoid dendritic cells, Isolated antibody or its antigen-binding fragment. (Item 3) An isolated antibody or antigen-conjugated fragment thereof, as described in item 1 or 2, that binds to cynomolgus monkey BDCA2 (SEQ ID NO: 73) and rhesus monkey BDCA2 (SEQ ID NO: 72). (Item 4) (i) Inhibit TLR-induced production of type I interferon, IL-6, TNF-α, CCL3, CCL4, IP10, and RANTES from plasmacytoid dendritic cells. (ii) In vitro, induce or enhance the depletion of plasmacytoid dendritic cells, The isolated antibody or its antigen-binding fragment as described in item 2. (Item 5) (i) selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1), and (ii) comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, The heavy chain CDR1 comprises the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), or the amino acid sequence shown in SEQ ID NO: 9, having substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The heavy chain CDR2 includes the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), or the amino acid sequence shown in SEQ ID NO: 10, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The heavy chain CDR3 contains the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or the amino acid sequence shown in SEQ ID NO: 11, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. Isolated antibody or its antigen-binding fragment. (Item 6) The heavy chain CDR1 comprises the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), or the amino acid sequence shown in SEQ ID NO: 9, having substitutions at one or two amino acid positions. The heavy chain CDR2 comprises the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), or the amino acid sequence shown in SEQ ID NO: 10, having substitutions at one or two amino acid positions. The heavy chain CDR3 includes the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), or the amino acid sequence shown in SEQ ID NO: 11, having substitutions at one or two amino acid positions. The isolated antibody or its antigen-binding fragment as described in item 5. (Item 7) The heavy chain CDR1 comprises the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), The heavy chain CDR2 comprises the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), The heavy chain CDR3 contains the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), The isolated antibody or its antigen-binding fragment as described in item 5. (Item 8) Including light chain CDR1, light chain CDR2, and light chain CDR3, The light chain CDR1 contains the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR2 contains the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. The light chain CDR3 contains the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, which has substitutions at the 1st, 2nd, 3rd, or 4th amino acid positions. An isolated antibody or its antigen-binding fragment as described in any one of items 5 to 7. (Item 9) The light chain CDR1 comprises the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), or the amino acid sequence shown in SEQ ID NO: 5, having substitutions at one or two amino acid positions. The light chain CDR2 comprises the amino acid sequence AASTLES (SEQ ID NO: 6), or the amino acid sequence shown in SEQ ID NO: 6, having substitutions at one or two amino acid positions. The light chain CDR3 comprises the amino acid sequence QQANEDPRT (SEQ ID NO: 7), or the amino acid sequence shown in SEQ ID NO: 7, having substitutions at one or two amino acid positions. The isolated antibody or its antigen-binding fragment as described in item 8. (Item 10) The heavy chain CDR1 comprises the amino acid sequence GFTFSTYTMS (SEQ ID NO: 9), The heavy chain CDR2 comprises the amino acid sequence TISPGDSFGYYYPDSVQG (SEQ ID NO: 10), The heavy chain CDR3 comprises the amino acid sequence DIYYYNYGAWFAY (SEQ ID NO: 11), The light chain CDR1 comprises the amino acid sequence KASQSVDYDGDSYMN (SEQ ID NO: 5), The light chain CDR2 comprises the amino acid sequence AASTLES (SEQ ID NO: 6), The light chain CDR3 contains the amino acid sequence QQANEDPRT (SEQ ID NO: 7), The isolated antibody or its antigen-binding fragment as described in item 8. (Item 11) (i) an isolated antibody or its antigen-binding fragment that selectively binds to the external domain of human BDCA2 (SEQ ID NO: 1) and (ii) contains a variable heavy chain (VH) domain that is at least 80% identical to the amino acid sequence of the VH domain of BIIB059 (SEQ ID NO: 24). (Item 12) The isolated antibody or antigen-binding fragment according to item 11, wherein the VH domain is at least 90% identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059. (Item 13) The isolated antibody or antigen-binding fragment according to item 11, wherein the VH domain is at least 95% identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059. (Item 14) The isolated antibody or antigen-binding fragment described in item 11, wherein the VH domain is identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059. (Item 15) The antibody or antigen-binding fragment described in item 11, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 4. (Item 16) An isolated antibody or antigen-binding fragment according to any one of items 11 to 15, comprising a variable light chain (VL) domain that is at least 80% identical to the amino acid sequence of the VL domain of BIIB059 (SEQ ID NO: 23). (Item 17) The isolated antibody or antigen-binding fragment according to item 16, wherein the VH domain is at least 90% identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059, and the VL domain is at least 90% identical to the amino acid sequence (SEQ ID NO: 23) of the VL domain of BIIB059. (Item 18) The isolated antibody or antigen-binding fragment according to item 16, wherein the VH domain is at least 95% identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059, and the VL domain is at least 95% identical to the amino acid sequence (SEQ ID NO: 23) of the VL domain of BIIB059. (Item 19) The isolated antibody or antigen-binding fragment according to item 16, wherein the VH domain is identical to the amino acid sequence (SEQ ID NO: 24) of the VH domain of BIIB059, and the VL domain is identical to the amino acid sequence (SEQ ID NO: 23) of the VL domain of BIIB059. (Item 20) The antibody or antigen-binding fragment according to item 16, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 4, and the light chain comprises the amino acid sequence of SEQ ID NO: 3. (Item 21) A humanized antibody, the antibody or its antigen-binding fragment as described in any one of items 1 to 20. (Item 22) A monoclonal antibody, as described in any one of items 1 to 20, or an antigen-binding fragment thereof. (Item 23) A single-chain antibody, the antibody or its antigen-binding fragment described in any one of items 1 to 20. (Item 24) An antibody or antigen-binding fragment thereof as described in any one of items 1 to 20, which is a polyclonal antibody, a chimeric antibody, a Fab fragment, an F(ab')2 fragment, a Fab' fragment, an Fsc fragment, an Fv fragment, scFv, sc(Fv)2, or a diabody. (Item 25) An antibody or antigen-binding fragment thereof, having an IgG1 heavy chain constant region, as described in any one of items 1 to 20. (Item 26) Isolated cells that produce an antibody or antigen-binding fragment described in any one of the above items. (Item 27) A pharmaceutical composition comprising an antibody or antigen-binding fragment described in any one of items 1 to 25 and a pharmaceutically acceptable carrier. (Item 28) A pharmaceutical composition comprising an antibody or antigen-binding fragment described in any one of items 1 to 25, formulated in a composition comprising 10 to 25 mM citrate, 100 to 200 mM sodium chloride, and a pH of 5.5 to 6.5. (Item 29) The pharmaceutical composition according to item 28, wherein the antibody or its antigen-binding fragment is formulated in a composition comprising 20 mM sodium citrate, 150 mM sodium chloride, and a pH of 6.0. (Item 30) A method for inducing the death of plasmacytoid dendritic cells in a subject, comprising the step of contacting plasmacytoid dendritic cells expressing BDCA2 with an antibody or antigen-binding fragment described in any one of items 1 to 25. (Item 31) A method for reducing the production of type I interferon, IL-6, TNF-α, CCL3, CCL4, IP10, and RANTES by plasmacytoid dendritic cells in a subject, comprising the step of contacting plasmacytoid dendritic cells expressing BDCA2 with a certain amount of an antibody or antigen-binding fragment described in any one of items 1 to 25. (Item 32) A method for treating an inflammatory disorder in a subject requiring treatment for an inflammatory disorder, comprising the step of administering to the subject requiring treatment an effective amount of an antibody or antigen-binding fragment described in any one of items 1 to 25. (Item 33) The method according to item 32, wherein the inflammatory disorder is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes mellitus, dermatomyositis, and polymyositis. (Item 34) The method according to item 32, wherein the inflammatory disorder is systemic lupus erythematosus, discoid lupus, lupus nephritis, or cutaneous lupus. (Item 35) The method according to item 32, wherein the inflammatory disorder is moderate to severe lupus with active central nervous system (CNS) and / or renal complications. (Item 36) The method according to item 32, wherein the inflammatory disorder is a moderate to severe disorder without active central nervous system (CNS) and / or renal complications. (Item 37) A method for treating an autoimmune disease in a subject requiring treatment for an autoimmune disease, comprising the step of administering to the subject requiring treatment an effective amount of an antibody or antigen-binding fragment described in any one of items 1 to 25. (Item 38) The method according to any one of items 30 to 37, wherein the subject is a human. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 shows a schematic description of BDCA2 signaling in plasmacytoid dendritic cells (see Geijtenbeek et al., Nature Reviews Immunology, Vol. 9: pp. 465-479 (2009)). [Figure 2] Figure 2 is a graph showing the binding of the hu24F4 Hx / L1 mutant to human BDCA2. [Figure 3] Figure 3 is a graph showing the binding of the hu24F4 Hx / L1 mutant to cynomolgus monkey BDCA2. [Figure 4]Figure 4 is a schematic map of pJP009, a plasmid encoding the anti-BDCA2 light chain. The nucleic acid sequence of the anti-BDCA2 light chain is under transcriptional regulation by the hCMV IE promoter and the hGH polyadenylation sequence. The aminoglycoside phosphotransferase gene (neomycin resistance gene) is under transcriptional regulation by the mouse phosphoglycerin kinase (muPGK) promoter and the polyadenylation sequence. The remaining sequence, including the β-lactamase gene, is for proliferation and selection in E. coli. [Figure 5] Figure 5 is a schematic map of pJP010, a plasmid encoding anti-BDCA2 heavy chain. The nucleic acid sequence of the anti-BDCA2 heavy chain is under transcriptional regulation by the hCMV IE promoter and the human growth hGH polyadenylation sequence. The dihydrofolate reductase (dhfr) gene is under transcriptional regulation by the SV40E promoter and the polyadenylation sequence. The remaining sequence, including the β-lactamase gene, is for growth and selection within E. coli. [Figure 6] Figure 6 is a line graph showing the binding of BIIB059 to cynomolgus monkey (A) plasmacytoid dendritic cells and human (B) plasmacytoid dendritic cells. Whole blood from cynomolgus monkeys (A) or humans (B) was incubated on ice with various concentrations of Alexa647-labeled BIIB059 antibody (circles) or human IgG isotype (squares). Data were collected using an LSRII-4 color FACS machine and analyzed using FlowJo and GraphPad Prism software. [Figure 7] Figure 7 is a line graph showing the results of the AlphaScreen assay for auto-assembly. Legend: diamond = BIIB059; square = 5c8; and triangle = LT105. [Figure 8] Figure 8 is a line graph showing the results of differential scanning fluorescence quantification testing the stability of BIIB059 under different conditions. This graph shows the data for 150 mM sodium chloride and 250 mM sucrose as a function of pH. [Figure 9]Figure 9 is a line graph showing the effect of stirring on agglomeration over a certain period of time. Agglomeration was suppressed by the addition of Tween 80. [Figure 10] Figure 10 is a line graph showing the direct binding of AC144 to human surface BDCA2 and cynomolgus monkey surface BDCA2. [Figure 11] Figure 11 is a series of graphs showing the results of size exclusion chromatography analysis for Fc fusion proteins. [Figure 12] Figure 12 is a graph showing the effect of calcium on the binding of BIIB059 to BDCA2. The binding of BIIB059 to BDCA2 is enhanced by the addition of calcium compared to EDTA, resulting in a signal that is approximately twice as strong. [Figure 13] Figure 13 is a graph showing the results of Octet's binding of BIIB059 to the human and cynomolgus monkey BDCA2 external domains. [Figure 14] Figure 14 is a graph showing that BIIB059 strongly inhibits IFNα from PBMCs stimulated by a TLR9 agonist. Each symbol represents the IC50 derived from an independent experiment, and the vertical line represents SEM. [Figure 15A] Figures 15A–C provide a series of graphs demonstrating that BIIB059 potently inhibits cytokines and chemokines derived from whole blood stimulated with TLR9 ligand. Figure 15A shows the inhibition of IFNα using heparinized venous blood from a healthy donor. Figure 15B compares the inhibition of IFNα using whole blood from two SLE patients (upper panel) with the results using whole blood from two healthy donors (lower panel). Figure 15C provides a series of bar graphs showing that BIIB059 treatment resulted in inhibition of many cytokines and chemokines. [Figure 15B]Figures 15A–C provide a series of graphs demonstrating that BIIB059 potently inhibits cytokines and chemokines derived from whole blood stimulated with TLR9 ligand. Figure 15A shows the inhibition of IFNα using heparinized venous blood from a healthy donor. Figure 15B compares the inhibition of IFNα using whole blood from two SLE patients (upper panel) with the results using whole blood from two healthy donors (lower panel). Figure 15C provides a series of bar graphs showing that BIIB059 treatment resulted in inhibition of many cytokines and chemokines. [Figure 15C] Figures 15A–C provide a series of graphs demonstrating that BIIB059 potently inhibits cytokines and chemokines derived from whole blood stimulated with TLR9 ligand. Figure 15A shows the inhibition of IFNα using heparinized venous blood from a healthy donor. Figure 15B compares the inhibition of IFNα using whole blood from two SLE patients (upper panel) with the results using whole blood from two healthy donors (lower panel). Figure 15C provides a series of bar graphs showing that BIIB059 treatment resulted in inhibition of many cytokines and chemokines. [Figure 16] Figure 16 is a bar graph showing that BIIB059 inhibits the expression of type I interferon. [Figure 17] Figure 17 is a diagram containing two line graphs showing that ligation of BDCA2 with BIIB059 inhibits TLR9-induced cytokine production in purified pDCs. [Figure 18] Figure 18 is a bar graph showing that ligation with BDCA2 suppresses the induction of IFN-α production in pDCs stimulated with SLE serum. [Figure 19] Figure 19A is a line graph showing that BDCA2 is internally translocated after ligation with BIIB059. Figure 19B is a line graph showing that internal translocation does not affect the inhibition of IFN-α production mediated by BIIB059. [Figure 20]Figure 20 is a series of line graphs showing the binding of BIIB059 to the Fcγ receptor. [Figure 21] Figure 21 shows the results of a C1q ELISA demonstrating the binding of human C1q to antibodies coated with increasing concentrations (0–15 μg / mL). [Figure 22A-B] Figures 22A–D are a series of graphs showing that BIIB059 mediates cell death via ADCC. CHO cell lineage (EAG2456 T1F2 clone 34.16.7) was used as target cells. BDCA2 expression levels on the surface of CHO cells were determined by FACS using an APC-labeled anti-BDCA2 mAb (clone AC144; Miltenyi). NK cells were used as effector cells. ADCC was evaluated using the Vybrant Cytotoxicity Assay Kit (Invitrogen) according to the manufacturer's instructions. The assay detects G6PD derived from damaged cells based on the G6PD-dependent reduction of resazurin, which fluoresces at 590 nm after excitation at 530 nm. The ADCC assay shown in Figure 22A was performed using CHO cells with high BDCA2 expression (Figure 22C), while the ADCC assay in Figure 22B used CHO cells with low BDCA2 expression (Figure 22D). [Figure 22C-D]Figures 22A–D are a series of graphs showing that BIIB059 mediates cell death via ADCC. CHO cell lineage (EAG2456 T1F2 clone 34.16.7) was used as target cells. BDCA2 expression levels on the surface of CHO cells were determined by FACS using an APC-labeled anti-BDCA2 mAb (clone AC144; Miltenyi). NK cells were used as effector cells. ADCC was evaluated using the Vybrant Cytotoxicity Assay Kit (Invitrogen) according to the manufacturer's instructions. The assay detects G6PD derived from damaged cells based on the G6PD-dependent reduction of resazurin, which fluoresces at 590 nm after excitation at 530 nm. The ADCC assay shown in Figure 22A was performed using CHO cells with high BDCA2 expression (Figure 22C), while the ADCC assay in Figure 22B used CHO cells with low BDCA2 expression (Figure 22D). [Figure 23] Figure 23 is a line graph showing that BIIB059 mediates cell death via CDC. CHO cells (EAG2456 T1F2 clone 34.16.7) were seeded at 5 × 10⁴ cells in 96-well collagen blackwell plates and incubated at 37°C for 48 hours. The plates were then washed and incubated at 37°C for 1 hour with rabbit serum complement and propidium iodide (PI) in the presence of effector-functional anti-BDCA2 mAbs (24F4S and BIIB059), effector-deficient mAbs (24F4S-Agly and 24F4A-Agly), or IgG1 isotype controls. Negative controls consisted of wells containing CHO cells, rabbit serum complement, and PI, without antibodies. [Figure 24]Figure 24 shows a series of graphs used to determine the EC50 for BIIB059 binding ("direct") and competitive BIIB059-A647 binding ("indirect") on cynomolgus monkey pDCs. Blood was collected from 12 cynomolgus monkeys once a week for a total of 3 weeks prior to in vivo injection of BIIB059. Flow cytometry was used to determine the EC50 for BIIB059 binding to BDCA2 on the pDC cell surface ("direct" method) and the amount of BDCA2 receptor available in the presence of BIIB059 ("indirect" method). Blood was incubated with BIIB059 in 6 titration volumes ranging from 40 to 0.04 μg / mL. Flow cytometry identified pDCs as CD20-CD14-CD123+HLA-DR+ and treated them with 10 ug / mL of anti-human IgG PE-labeled secondary antibody or BIIB059-A647-labeled antibody. The MFI for PE (white symbols graphed on the left y-axis (open symbol)) or the MFI for A647 (black symbols graphed on the right y-axis (closed symbol)) was calculated using FlowJo software and graphed using GraphPad Prism software (fitting a 4-parameter nonlinear regression curve to logarithmically transformed data). This figure shows representative graphs from 4 out of 12 cynomolgus monkeys. [Figure 25]Figure 25 is a representative graph showing the plateau binding of the anti-BDCA2 antibody BIIB059 to cell surface BDCA2 on pDCs in whole blood of cynomolgus monkeys. Blood was incubated with BIIB059 in 6 titrations ranging from 40 to 0.04 μg / mL. pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry and treated with anti-human IgG PE-labeled secondary antibody. The MFI of PE was calculated using FlowJo software, and the maximum binding percentage was calculated using the 40 μg / mL point as 100%. Each line represents an individual cynomolgus monkey from a total of 12 monkeys, and was graphed using GraphPad Prism software (fitting a 4-parameter nonlinear regression curve to logarithmically transformed data). Staining was repeated once a week for a total of 3 weeks. The dashed line confirms that BDCA2 receptor binding is saturated in all cynomolgus monkeys with BIIB059 at a concentration of 10 μg / mL. [Figure 26A]Figures 26A-C illustrate the staining levels of bound BIIB059 and free BDCA2 in cynomolgus monkeys treated with the medium. Figure 26A is a series of FACS histograms showing background PE staining in cynomolgus monkeys treated with the medium. Cynomolgus monkeys 1, 4, and 12 received a single IV injection of the medium control (sodium citrate) at time 0. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE fluorescence minus one (FMO) control (black histogram). Figure 26B is a graph of PE staining on pDCs derived from blood collected from three cynomolgus monkeys treated with the medium at the indicated time points. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 26C shows graphs of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with the medium, at the indicated time points. At each indicated time point, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with the medium, and the A647 staining on pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 26B-C]Figures 26A-C illustrate the staining levels of bound BIIB059 and free BDCA2 in cynomolgus monkeys treated with the medium. Figure 26A is a series of FACS histograms showing background PE staining in cynomolgus monkeys treated with the medium. Cynomolgus monkeys 1, 4, and 12 received a single IV injection of the medium control (sodium citrate) at time 0. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE fluorescence minus one (FMO) control (black histogram). Figure 26B is a graph of PE staining on pDCs derived from blood collected from three cynomolgus monkeys treated with the medium at the indicated time points. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 26C shows graphs of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with the medium, at the indicated time points. At each indicated time point, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with the medium, and the A647 staining on pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 27A]Figures 27A-C show that bound BIIB059 and BDCA2 receptors are no longer obtained on the surface of pDC cells after a single dose of 10 mg / kg of BIIB059 in cynomolgus monkeys. Figure 27A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with 10 mg / kg of BIIB059. Cynomolgus monkeys 3, 8, and 10 received a single IV injection of BIIB059 at time 0, at 10 mg / kg. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 27B is a graph of PE staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 27C is a graph of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. At each of the time points shown, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on the pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 27B-C]Figures 27A-C show that bound BIIB059 and BDCA2 receptors are no longer obtained on the surface of pDC cells after a single dose of 10 mg / kg of BIIB059 in cynomolgus monkeys. Figure 27A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with 10 mg / kg of BIIB059. Cynomolgus monkeys 3, 8, and 10 received a single IV injection of BIIB059 at time 0, at 10 mg / kg. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 27B is a graph of PE staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 27C is a graph of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. At each of the time points shown, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on the pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 28A]Figures 28A-C show that bound BIIB059 and BDCA2 receptors are no longer present on the pDC cell surface after a single dose of 1 mg / kg BIIB059 in cynomolgus monkeys. Figure 28A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with 1 mg / kg BIIB059. Cynomolgus monkeys 3, 8, and 10 received a single IV injection of BIIB059 at time 0, at 1 mg / kg. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 28B is a graph of PE staining on pDCs derived from blood collected from three cynomolgus monkeys treated with BIIB059 at the indicated time. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 28C shows graphs of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059 at the indicated time points. At each indicated time point, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 28B-C]Figures 28A-C show that bound BIIB059 and BDCA2 receptors are no longer present on the pDC cell surface after a single dose of 1 mg / kg BIIB059 in cynomolgus monkeys. Figure 28A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with 1 mg / kg BIIB059. Cynomolgus monkeys 3, 8, and 10 received a single IV injection of BIIB059 at time 0, at 1 mg / kg. One hour later, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 28B is a graph of PE staining on pDCs derived from blood collected from three cynomolgus monkeys treated with BIIB059 at the indicated time. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 28C shows graphs of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059 at the indicated time points. At each indicated time point, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 29A]Figures 29A-C show that bound BIIB059 and BDCA2 receptors are no longer found on the surface of pDC cells after a single subcutaneous (SC) administration of 0.2 mg / kg of BIIB059 in cynomolgus monkeys. Figure 29A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with SC 0.2 mg / kg of BIIB059. Cynomolgus monkeys 4, 6, and 12 received a single SC injection of BIIB059 at time 0, at 0. Whole blood was collected 1 hour later, and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 29B is a graph of PE staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 29C is a graph of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. At each of the time points shown, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on the pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 29B-C]Figures 29A-C show that bound BIIB059 and BDCA2 receptors are no longer found on the surface of pDC cells after a single subcutaneous (SC) administration of 0.2 mg / kg of BIIB059 in cynomolgus monkeys. Figure 29A is a series of FACS histograms showing BIIB059 staining in cynomolgus monkeys treated with SC 0.2 mg / kg of BIIB059. Cynomolgus monkeys 4, 6, and 12 received a single SC injection of BIIB059 at time 0, at 0. Whole blood was collected 1 hour later, and pDCs were identified as CD20-CD14-CD123+HLA-DR+ and treated with anti-human IgG PE (white histogram) or FACS buffer as a PE FMO control (black histogram). Figure 29B is a graph of PE staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. The MFI of PE was calculated using FlowJo software and graphed using GraphPad Prism software. Figure 29C is a graph of A647 staining on pDCs derived from blood samples taken from three cynomolgus monkeys treated with BIIB059, at the time shown. At each of the time points shown, 10 μg / mL of BIIB059-A647 was added to blood samples taken from three cynomolgus monkeys treated with BIIB059, and the A647 staining on the pDCs was assayed. The MFI of A647 was calculated using FlowJo software and graphed using GraphPad Prism software. [Figure 30-1] Figure 30 shows a series of graphs illustrating the PK / PD correlation observed in cynomolgus monkeys administered 1 mg / kg of BIIB059 intravenously and 10 mg / kg of BIIB059 intravenously. In each graph in this figure, the serum concentration of BIIB059 is plotted on the left y-axis (white symbols), and the density of the BDCA2 receptor is plotted on the right y-axis (black symbols). The accelerated clearance observed in cynomolgus monkey 5 may be due to immunogenicity to BIIB059. [Figure 30-2]Figure 30 shows a series of graphs illustrating the PK / PD correlation observed in cynomolgus monkeys administered 1 mg / kg of BIIB059 intravenously and 10 mg / kg of BIIB059 intravenously. In each graph in this figure, the serum concentration of BIIB059 is plotted on the left y-axis (white symbols), and the density of the BDCA2 receptor is plotted on the right y-axis (black symbols). The accelerated clearance observed in cynomolgus monkey 5 may be due to immunogenicity to BIIB059. [Figure 30-3] Figure 30 shows a series of graphs illustrating the PK / PD correlation observed in cynomolgus monkeys administered 1 mg / kg of BIIB059 intravenously and 10 mg / kg of BIIB059 intravenously. In each graph in this figure, the serum concentration of BIIB059 is plotted on the left y-axis (white symbols), and the density of the BDCA2 receptor is plotted on the right y-axis (black symbols). The accelerated clearance observed in cynomolgus monkey 5 may be due to immunogenicity to BIIB059. [Figure 31-1] Figure 31 shows a series of graphs illustrating the PK / PD correlation observed in cynomolgus monkeys treated with 0.2 mg / kg of BIIB059 via SC. In each graph in this figure, the serum concentration of BIIB059 is plotted on the left y-axis (white symbols), and the density of BDCA2 receptors is plotted on the right y-axis (black symbols). [Figure 31-2] Figure 31 shows a series of graphs illustrating the PK / PD correlation observed in cynomolgus monkeys treated with 0.2 mg / kg of BIIB059 via SC. In each graph in this figure, the serum concentration of BIIB059 is plotted on the left y-axis (white symbols), and the density of BDCA2 receptors is plotted on the right y-axis (black symbols). [Figure 32]Figure 32 is a series of bar graphs showing the results of ELISA or multiplex assays measuring the concentrations of inflammatory cytokines and chemokines produced by pDCs treated with CpG-A, CpG-A in the presence of anti-BDCA2, and CpG-A in the presence of isotype controls. Each bar represents the mean and standard deviation (SD) for two wells derived from a representative healthy human donor out of five tested cases. The vertical line indicates the SD. [Figure 33] Figure 33 is a series of bar graphs showing the results of ELISA or multiplex assays measuring the concentrations of inflammatory cytokines and chemokines produced by pDCs treated with Sm / RNP immune complexes, Sm / RNP immune complexes in the presence of anti-BDCA2, and Sm / RNP immune complexes in the presence of isotype controls. Each bar represents the mean and standard deviation (SD) for two wells derived from a representative healthy human donor out of five tested cases. Vertical lines indicate the SD. [Figure 34] Figure 34 is a series of bar graphs showing the results of a qPCR assay determining the effect of BIIB059 on the transcription of type I IFN subtypes in pDCs derived from healthy human donors stimulated with Sm / RNP IC. Each bar represents the mean relative multiplier change for a 4-well sequence derived from a representative donor out of the three cases tested (n=3), and the vertical line shows the standard deviation (SD). [Figure 35]Figure 35A shows the dose-dependent inhibition of TLR9-induced IFNα by BIIB059 mediated by PBMCs derived from one representative healthy human donor out of 18 tested cases. Each symbol represents the mean and standard deviation (SD) for two wells. Figure 35B shows the dose-dependent inhibition of TLR9-induced IFNα by BIIB059 mediated by PBMCs derived from one representative SLE patient out of 11 tested cases. Each symbol represents the mean and standard deviation (SD) for two wells. Figure 35C shows the IC50 values ​​for BIIB059-mediated inhibition of TLR9-induced IFNα production by PBMCs in healthy human donors (HD) compared to SLE patients (SLE). Each symbol represents an individual donor, and the vertical line indicates the SD. [Figure 36] Figure 36A shows the dose-dependent inhibition of TLR9-induced IFNα by BIIB059 in a whole blood assay from one representative case out of 12 tested. Each symbol represents the mean and standard deviation (SD) for two wells. Figure 36B shows the IC50 values ​​for inhibition of TLR9-induced IFNα production by BIIB059 in a whole blood assay compared to a PBMC assay. Each symbol represents an individual donor, and the vertical line indicates the SD. [Figure 37] Figure 37 shows the results of PBMCs from healthy human donors being stimulated with 1 μM TLR3 ligand (polyI:C) and treated with BIIB059 at concentrations ranging from 10 μg / mL to 0.5 ng / mL in a total assay volume of 250 μL per well in a 96-well plate. The plates were incubated overnight (18 hours) at 37°C and 5% CO2. 200 μL of supernatant was collected to evaluate IFNα levels by ELISA. Each symbol represents the mean IFNα level produced under each treatment condition. Data from two independent donors are shown. Vertical lines indicate the standard deviation (SD). [Figure 38]Figure 38A shows dose-dependent BIIB059-mediated internal transfer of BDCA2 from representative healthy human donors. Circles represent MFI (maximum staining) of 2D6 staining at various doses of BIIB059. Triangles represent MFI (maximum staining) of 2D6 in the presence of isotype controls. Diamonds represent MFI (background staining) with FMO controls. Figure 38B shows the EC50 for BIIB059-induced internal transfer of BDCA2 in pDCs from whole blood assays derived from healthy human donors (black circles; n=10 donors). The mean EC50 was 0.017 ± 0.005 μg / mL. [Figure 39] Figure 39 is a graphical representation of the mean fluorescence intensity (MFI) values ​​for gated CD14-CD20-HLA-DR+CD123+pDC stained with 2D6-FITC. The isotype (iso) represents the maximum staining, while the FMO (fluorescence minus one control), consisting of a FACS staining cocktail with 2D6-FITC subtracted, represents the background staining. This figure shows representative results from four independent experiments performed. [Figure 40] Figure 40 shows confocal images of human pDCs purified from peripheral blood and then incubated for 15 minutes with 10 μg / mL BIIB059~AF647 (white) in 5% CO2 at 4°C (left) or 5% CO2 at 37°C (right). The cell distribution of BIIB059 was evaluated by confocal microscopy, and representative images are shown for each condition. [Figure 41] Figure 41 is a graphical representation of the effect of internal translocation of BDCA2 on inhibiting IFNα production. This figure is representative of three independent experiments. [Figure 42] Figure 42 is a graph illustrating the correlation between the EC50 value for BIIB059-mediated BDCA2 internal transfer and the IC50 value for BIIB059-mediated inhibition of TLR9-induced IFNα production in a whole blood assay (n=10). The R2 value was 0.57. [Figure 43]Figure 43A shows the results of TLR9 localization within the LAMP1+ compartment, expressed as the mean and standard deviation (SD) of the Manders colocalization coefficient. Figure 43B shows the results of BIIB059 / BDCA2 localization within the TLR9+ compartment, expressed as the mean and SD of the Manders colocalization coefficient. Figure 43C shows the results of BIIB059 / BDCA2 localization within the LAMP1+ compartment, expressed as the mean and SD of the Manders colocalization coefficient. Each symbol represents an individual cell, the horizontal line represents the mean value, and the vertical line represents the SD. [Figure 44] Figure 44A shows histograms from representative experiments involving whole blood treated with 10 μg / mL BIIB059 (lightly shaded histogram), whole blood treated with 10 μg / mL isotype control (dotted line), or whole blood stimulated with the TLR9 ligand CpG-A (solid line). Figure 44B is a graphical representation (black squares) of the effect of BIIB059 treatment on whole blood, which results in the loss of CD62L. White squares represent isotype treatment (10 μg / mL). This figure represents three independent experiments. [Figure 45] Figure 45 is a graphical representation of CD62L surface expression assayed by flow cytometry. CD62L expression was measured in the presence of BIIB059 alone and with increasing concentrations of GM6001 (circles). White squares represent isotype-treated controls (10 μg / mL). Inverted triangles represent DMSO controls treated with BIIB059. This figure represents two independent experiments. [Figure 46A-BC]Figure 46A is a graphical depiction of the dose-dependent internal transfer of BDCA2 on the surface of pDCs derived from one representative healthy human donor (n=5) via BIIB059 and 24F4A-Agly. pDCs from a healthy human donor were isolated using a two-step magnetic bead separation procedure (MACS kit; Miltenyi Biotec). The pDCs were treated with increasing concentrations of BIIB059 (circles) or the non-glycosylated form of the antibody (24F4-Agly) (squares). Cells were also treated with 10 μg / mL of isotype control (triangles) and incubated at 37°C for 16 hours. The pDCs were then stained for surface expression of BDCA2 and CD32. Figure 46B is a histogram showing the levels of CD32 on isolated pDCs (n=5) treated with 10 μg / mL of BIIB059 (shaded) or isotype control (dotted line). Figure 46C is a histogram showing CD32 levels on isolated pDCs treated with 10 μg / mL of the unglycosylated form 24F4-A (shadowed) or isotype control (dotted histogram). The solid line represents unstained cells (n=5). Figure 46D is a graphical depiction of BIIB059-mediated dose-dependent downmodulation of CD32 on the surface of pDCs derived from one representative healthy human donor (n=5). Figure 46E is a histogram showing CD32 levels on isolated pDCs treated at 4°C for 1 hour in the presence of 10 μg / mL of BIIB059 (shadowed), the unglycosylated form (dashed line), or isotype control (dotted line). After incubation, pDCs were evaluated for CD32 surface expression. The solid black line represents unstained cells (n=3). Figure 46F is a histogram showing the levels of CD32 on isolated pDCs treated at 37°C for 1 hour in the presence of 10 μg / mL BIIB059 (shaded), non-glycosylated form (dashed line), or isotype control (dotted line). After incubation, pDCs were evaluated for CD32 surface expression. The solid black line represents unstained cells (n=3). [Figure 46D-EF]Figure 46A is a graphical depiction of the dose-dependent internal transfer of BDCA2 on the surface of pDCs derived from one representative healthy human donor (n=5) via BIIB059 and 24F4A-Agly. pDCs from a healthy human donor were isolated using a two-step magnetic bead separation procedure (MACS kit; Miltenyi Biotec). The pDCs were treated with increasing concentrations of BIIB059 (circles) or the non-glycosylated form of the antibody (24F4-Agly) (squares). Cells were also treated with 10 μg / mL of isotype control (triangles) and incubated at 37°C for 16 hours. The pDCs were then stained for surface expression of BDCA2 and CD32. Figure 46B is a histogram showing the levels of CD32 on isolated pDCs (n=5) treated with 10 μg / mL of BIIB059 (shaded) or isotype control (dotted line). Figure 46C is a histogram showing CD32 levels on isolated pDCs treated with 10 μg / mL of the unglycosylated form 24F4-A (shadowed) or isotype control (dotted histogram). The solid line represents unstained cells (n=5). Figure 46D is a graphical depiction of BIIB059-mediated dose-dependent downmodulation of CD32 on the surface of pDCs derived from one representative healthy human donor (n=5). Figure 46E is a histogram showing CD32 levels on isolated pDCs treated at 4°C for 1 hour in the presence of 10 μg / mL of BIIB059 (shadowed), the unglycosylated form (dashed line), or isotype control (dotted line). After incubation, pDCs were evaluated for CD32 surface expression. The solid black line represents unstained cells (n=3). Figure 46F is a histogram showing the levels of CD32 on isolated pDCs treated at 37°C for 1 hour in the presence of 10 μg / mL BIIB059 (shaded), non-glycosylated form (dashed line), or isotype control (dotted line). After incubation, pDCs were evaluated for CD32 surface expression. The solid black line represents unstained cells (n=3). [Figure 47]Figure 47A is a graphical representation of IFNα levels derived from isolated pDCs treated with increased concentrations of BIIB059 (squares), the non-glycosylated form of the antibody 24F4-A (circles), or a 10 μg / mL isotype control (triangles). pDCs were stimulated in the presence of CpG-A (75 μg / mL) or left unstimulated (inverted triangles). The pDCs were incubated at 37°C for 16 hours, the supernatant was collected, and IFNα was assayed by ELISA. A representative experiment from two performed experiments is shown. Figure 47B is a graphical representation of IFNα levels derived from isolated pDCs treated with increased concentrations of BIIB059 (squares), the non-glycosylated form of the antibody 24F4-A (circles), a 10 μg / mL isotype control (triangles), or a 10 μg / mL anti-human CD32 mAb. Sm / RNP immune complexes (ICs) were pre-formed by mixing Sm-RNP derived from bovine thymus with anti-RNP antibodies purified from the serum of SLE patients in serum-free medium for 30 minutes. Isolated cells were stimulated with the immune complex or treated with the antigen alone (unstimulated). Cells were incubated at 37°C for 16 hours, the supernatant was collected, and assayed for IFNα by ELISA. Representative figures from three experiments performed are shown. Each symbol represents the mean and standard deviation (SD) for two wells. [Figure 48]Figure 48A is a bar graph showing CD32 expression on isolated pDCs treated with immunocomplexes in the presence of 10 μg / mL BIIB059, 24F4-A, anti-CD32 mAb (AT10 clone), humanized anti-CD40 antibody, or isotype control. Cells were incubated at 37°C for 16 hours. pDCs were stained for surface expression of CD32 and CD40. Figure 48B is a bar graph showing IFNα levels measured by ELISA in the supernatant recovered from A. A representative figure (n=3) is shown. Figure 48C is a histogram showing CD40 expression on the surface of pDCs. The dotted line represents CD40 expression on the cell surface. The light-colored histogram represents the level of CD40 on pDCs after treatment with anti-CD40 antibody. The solid line represents unstained cells. [Figure 49] Figure 49 shows the effect of HCQ on the potency of BIIB059. Each symbol represents the IFNα concentration measured from an individual healthy human donor, and the vertical line indicates the standard deviation (SD). PBMCs derived from healthy human donors were treated with various concentrations of BIIB059 alone, HCQ alone, or a combination (BIIB059 + HCQ) in a total assay volume of 250 μL per well. BIIB059 concentrations ranged from 10 μg / mL to 0.1 ng / mL. HCQ concentrations ranged from 10 μM to 156 nM. 1 × 10⁶ PBMC cells per well were stimulated with 5 μM TLR7 ligand (R848). Plates containing PBMCs were incubated overnight (18 hours) at 37°C and 5% CO₂. 200 μL of supernatant was collected for evaluation in IFNα ELISA (PBL InterferonSource). [Figure 50]Figure 50 shows the effect of HCQ on the potency of BIIB059. Each symbol represents the IFNα concentration measured from a representative donor among two tested healthy donors, and the vertical line indicates the standard deviation (SD). PBMCs derived from heparinized venous blood of healthy human donors or SLE patients were isolated by discontinuous gradient centrifugation on Ficoll, washed in PBS, and resuspended in complete culture medium (RPMI with 3% FBS). PBMCs were treated with various concentrations of BIIB059 alone, HCQ alone, or a combination (BIIB059 + HCQ) in a total assay volume of 250 μL per well. BIIB059 concentrations ranged from 10 μg / mL to 0.1 ng / mL. HCQ concentrations ranged from 10 μM to 156 nM. Each well contained 1 × 10⁶ PBMC cells, stimulated with 1 μM TLR9 ligand (CPG-A). Plates containing PBMCs were incubated overnight (18 hours) at 37°C and 5% CO₂. 200 μL of supernatant was collected for evaluation using IFNα ELISA (PBL InterferonSource). [Figure 51] Figure 51 shows the distribution of circulating pDC percentages in whole blood of healthy cynomolgus monkeys, on the original scale (left panel) and logarithmic scale (right panel). Whole blood was collected weekly from 12 cynomolgus monkeys for a total of 4 weeks. Flow cytometry was used to identify pDCs as CD20-CD14-CD123+HLA-DR+. pDCs as a percentage of CD20-CD14- cells were calculated using FlowJo software. The graph was obtained using the R language for statistical calculations. [Figure 52] Figure 52 is a graphical representation (on a logarithmic scale) of the circulating pDC percentage (CD20-CD14-CD123+HLA-DR+) in the whole blood of healthy cynomolgus monkeys at different time points before IV injection of BIIB059. At the indicated time points, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry. The pDC percentage was calculated using FlowJo software and graphed using R software. [Figure 53]Figure 53 depicts the final fitted model for circulating pDC% in whole blood (on a logarithmic scale) of healthy cynomolgus monkeys at different time points before IV injection of BIIB059. A linear mixed-effects model for logarithmic (pDC%) values ​​with different time points as fixed coefficients and cynomolgus monkeys as random intercepts shows no difference between the ratios of geometric mean pDC% measured over different weeks (the p-value based on an F-test for all time effects equal to zero is 0.67). Graphs and statistical analyses were performed using the R language for statistical calculations. The black line shows the final fitted model, including only the fixed intercept and the random intercept for cynomolgus monkeys. The linear mixed-effects model was fitted using the lme4 package in R. [Figure 54] Figure 54 shows the circulating pDC percentage on a logarithmic scale before and after IV administration of sodium citrate, 1 mg / kg BIIB059, or 10 mg / kg BIIB059 in cynomolgus monkeys. At time 0, three cynomolgus monkeys were administered each dose group. At the indicated time points, whole blood was collected and pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry. The pDC percentage was calculated using FlowJo software and graphed using R software. [Figure 55] Figure 55 shows the final fitted model for circulating pDC percent on a logarithmic scale before and after IV administration of sodium citrate medium, 1 mg / kg BIIB059, and 10 mg / kg BIIB059 in cynomolgus monkeys. It is a linear mixed-effects model for logarithmic (pDC%) values ​​with fixed coefficients for administration groups, 1 hour, 6 hours, and over 28 days, as well as a random intercept for cynomolgus monkeys. The solid line shows the fitted model. The linear mixed-effects model was fitted using the lme4 package in R. Graphs and statistical analyses were calculated using the R language for statistical calculations. [Figure 56]Figure 56 shows the circulating pDC percentage in cynomolgus monkeys after SC administration of 0.2 mg / kg BIIB059. Cynomolgus monkeys 4, 6, and 12 received a single SC injection of 0.2 mg / kg BIIB059 at time 0. Of the three monkeys, monkey 6 received the mg / kg BIIB059 dose from the previously described study. Cynomolgus monkeys 4 and 12 received the dose from the previously described study. Whole blood was collected at the indicated time points, and pDCs were identified as CD20-CD14-CD123+HLA-DR+ by flow cytometry. The pDC percentage was calculated using FlowJo software and graphed using R software. [Figure 57] Figure 57 shows the final fitted model for circulating pDC percent after SC administration of 0.2 mg / kg BIIB059 in cynomolgus monkeys. A linear mixed-effects model for logarithmic (pDC%) values ​​is fitted, with fixed effects at continuous time and 1 hour post-treatment and cynomolgus monkeys as random intercepts. The solid line shows the fitted model. The linear mixed-effects model was fitted using the lme4 package in R. Graphs and statistical analyses were calculated using the R language for statistical computation. [Figure 58]Figure 58 shows a schematic representation of the cynomolgus monkey PK / PD experimental design. Nine cynomolgus monkeys underwent an intravenous (IV) administration study. Blood was collected from the monkeys before and after IV administration of the medium, 1 mg / kg BIIB059, or 10 mg / kg BIIB059, according to the indicated blood collection schedule. After this study, three cynomolgus monkeys underwent a subcutaneous (SC) administration study, receiving a single SC injection of 0.2 mg / kg BIIB059. At each blood collection time, a whole blood assay was performed. Whole blood from the cynomolgus monkeys was diluted 1:4 with complete RPMI 1640, stimulated with a final concentration of 200 μg / ml CPG-A in a 96-well round-bottom tissue culture plate, and incubated at 37°C and 5% CO2 for 18–20 hours. After culturing, the stimulated whole blood was centrifuged to collect serum. In the MxA bioassay, A549 cells were stimulated with collected serum at 37°C and 5% CO2 for 19–20 hours to induce the MxA protein. After 20 hours, the A549 cells were lysed, and the concentration of the MxA protein was detected by sandwich ELISA. The IFNα level (units per mL) was calculated by working backward from a calibration curve created by treating A549 cells with increasing doses of rIFNα. [Figure 59]Figure 59 is a graph showing the trend of reduction in TLR9-induced IFNα production compared to the mean value before treatment in cynomolgus monkeys treated with a single intravenous administration of BIIB059. Whole blood from cynomolgus monkeys treated with a single intravenous administration of 1 mg / kg BIIB059 or 10 mg / kg BIIB059 was diluted 1:4 with complete RPMI 1640 and stimulated with a final concentration of 200 μg / ml CPG-A(2216) in a 96-well round-bottom tissue culture plate, and incubated at 37°C and 5% CO2 for 18-20 hours. After the culture was complete, the stimulated whole blood was centrifuged and serum was collected. A549 cells were stimulated with the collected serum at 37°C and 5% CO2 for 19-20 hours to induce MxA protein. After 20 hours, A549 cells were lysed, and the concentration of MxA protein was detected by sandwich ELISA. IFNα levels (in units of 1 mL) were calculated by working backward from a calibration curve created by treating A549 cells with increasing doses of rIFNα. The mean pre-collection IFNα concentration was calculated for each monkey by averaging all IFNα measurements from pre-collection time points (-21, -14, -7 days, and T0). Then, IFNα% was calculated for each blood collection time point up to 14 days after BIIB059 administration by dividing the IFNα concentration at that time point by the pre-collection mean for that animal and multiplying by 100. These values ​​were then averaged for each treatment group. The graph shows the mean ± standard error of the mean. Graphs and statistical analyses were calculated using Excel and GraphPad 6.0 software (GraphPad, San Diego, CA). [Figure 60-1]Figure 60 is a graphical depiction of the decrease in TLR9-induced IFNα production in an ex vivo whole blood assay derived from cynomolgus monkeys treated intravenously with BIIB059. Whole blood from cynomolgus monkeys treated with a single intravenous dose of medium (top panel), 1 mg / kg of BIIB059 (middle panel), or 10 mg / kg of BIIB059 (bottom panel) was diluted 1:4 with complete RPMI 1640 and stimulated with a final concentration of 200 μg / ml of CPG-A(2216) in 96-well round-bottom tissue culture plates, and incubated at 37°C and 5% CO2 for 18–20 hours. After culturing, the stimulated whole blood was centrifuged and serum was collected. A549 cells were stimulated with the collected serum at 37°C and 5% CO2 for 19–20 hours to induce MxA protein. After 20 hours, A549 cells were lysed, and sandwich ELISA was performed to detect the concentration of MxA protein. IFNα levels (in units of 1 mL) were calculated by working backward from a calibration curve created by treating A549 cells with increasing doses of rIFNα. Two-way mixed-effects analysis of variance (ANOVA) was fitted to the calculated log10 values ​​of IFNα concentrations. IFNα values ​​were plotted against the blood collection date for each animal within each treatment group (on a log10 scale). Vertical lines indicate the grouping by blood collection date into pre-administration, up to 31 days post-administration, and beyond 31 days post-administration. Blood collection dates after 31 days were not used in the analysis. Model-based estimates of IFNα values ​​by geometric mean are represented by thick black horizontal lines within the pre-administration and post-administration regions of each panel. Graphs and statistical analyses were performed using the R language for statistical calculations. [Figure 60-2]Figure 60 is a graphical depiction of the decrease in TLR9-induced IFNα production in an ex vivo whole blood assay derived from cynomolgus monkeys treated intravenously with BIIB059. Whole blood from cynomolgus monkeys treated with a single intravenous dose of medium (top panel), 1 mg / kg of BIIB059 (middle panel), or 10 mg / kg of BIIB059 (bottom panel) was diluted 1:4 with complete RPMI 1640 and stimulated with a final concentration of 200 μg / ml of CPG-A(2216) in 96-well round-bottom tissue culture plates, and incubated at 37°C and 5% CO2 for 18–20 hours. After culturing, the stimulated whole blood was centrifuged and serum was collected. A549 cells were stimulated with the collected serum at 37°C and 5% CO2 for 19–20 hours to induce MxA protein. After 20 hours, A549 cells were lysed, and sandwich ELISA was performed to detect the concentration of MxA protein. IFNα levels (in units of 1 mL) were calculated by working backward from a calibration curve created by treating A549 cells with increasing doses of rIFNα. Two-way mixed-effects analysis of variance (ANOVA) was fitted to the calculated log10 values ​​of IFNα concentrations. IFNα values ​​were plotted against the blood collection date for each animal within each treatment group (on a log10 scale). Vertical lines indicate the grouping by blood collection date into pre-administration, up to 31 days post-administration, and beyond 31 days post-administration. Blood collection dates after 31 days were not used in the analysis. Model-based estimates of IFNα values ​​by geometric mean are represented by thick black horizontal lines within the pre-administration and post-administration regions of each panel. Graphs and statistical analyses were performed using the R language for statistical calculations. [Figure 61]Figure 61 is a graphical representation of the decrease in TLR9-induced IFNα production in an ex vivo whole blood assay derived from cynomolgus monkeys subcutaneously treated with BIIB059. Whole blood from cynomolgus monkeys treated with a single subcutaneous dose of 0.2 mg / kg of BIIB059 was diluted 1:4 with complete RPMI 1640 and stimulated with a final concentration of 200 μg / ml of CPG-A(2216) in a 96-well round-bottom tissue culture plate. The cells were incubated at 37°C and 5% CO2 for 18–20 hours. After incubation, the stimulated whole blood was centrifuged and serum was collected. A549 cells were stimulated with the collected serum at 37°C and 5% CO2 for 19–20 hours to induce MxA protein. After 20 hours, A549 cells were lysed and the concentration of MxA protein was detected by sandwich ELISA. IFNα levels (in units of 1 mL) were calculated by working backward from a calibration curve created by treating A549 cells with increasing doses of rIFNα. A one-way analysis of variance (ANOVA) with random effects was fitted to the log10 values ​​of the calculated IFNα concentrations. IFNα values ​​were plotted for each animal against the blood collection date (on a log10 scale). Vertical lines indicate the grouping of blood collection dates into pre-administration, up to 33 days post-administration, and beyond 33 days post-administration. Blood collection dates after 33 days were not used in the analysis. Model-based estimates of IFNα values ​​by geometric mean are represented by thick black horizontal lines within the pre-administration and post-administration regions of each panel. Graphs and statistical analyses were performed using the R language for statistical calculations. [Modes for carrying out the invention]

[0039] Detailed explanation BIIB059 is an exemplary monoclonal antibody that specifically binds to human BDCA2. The anti-BDCA2 antibody described herein inhibits the production and / or secretion of inflammatory cytokines and chemokines by pDCs. Furthermore, the anti-BDCA2 antibody described herein can downregulate the levels of CD32a and / or CD62L on the surface of pDCs. The anti-BDCA2 antibody described herein can also mediate the internal translocation of BDCA2 from the surface of pDCs. In addition, the anti-BDCA2 antibody described herein can be used to deplete pDCs by ADCC or CDC, and can be used to treat or prevent immunodeficiencies such as inflammatory and autoimmune conditions. The disclosure also shows that combining antimalarial agents with the anti-BDCA2 antibody described herein may result in improved efficacy compared to treatment with either agent alone.

[0040] BDCA2 BDCA2 is a type II C-type lectin specifically expressed on pDCs. BDCA2 consists of a single extracellular carbohydrate recognition domain (CRD) at its C-terminus, a transmembrane domain, and a short cytoplasmic tail at its N-terminus that lacks a signaling motif. BDCA2 transmits intracellular signals via its associated transmembrane adapter, FcεRIγ (see Figure 1). Antibody-mediated ligation of BDCA2 recruits spleen tyrosine kinase (SYK) to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcεRIγ. Activation of Syk leads to the activation of B-cell linker (Blnk), Bruton's tyrosine kinase (BTK), and phospholipase Cγ2 (PLCγ2), thus enabling Ca2 + This will result in movement.

[0041] The amino acid sequence of the human BDCA2 protein (Genbank accession number NP_569708.1) is shown below (transmembrane domains are italicized, and external domains are underlined). [ka] The amino acid sequence of human FcεRIγ (Genbank accession number NP_004097.1) is shown below. [ka]

[0042] The rat Clec4b2 (Genbank accession number NM_001005896), the most similar rat BDCA2 homolog, shares only 51.0% identity with human BDCA2. In contrast, cynomolgus monkey BDCA2 and rhesus monkey BDCA2 share 90.6% identity with human BDCA2. Furthermore, the identical cynomolgus monkey FcεRIγ protein sequences and rhesus monkey FcεRIγ protein sequences share 98.9% identity with human FcεRIγ protein.

[0043] Anti-BDCA2 antibodies can be prepared using human BDCA2 protein, cynomolgus monkey BDCA2 protein, and rhesus monkey BDCA2 protein as immunogens. To prepare human anti-BDCA2 antibodies, human BDCA2 protein can be used as an immunogen. Anti-human BDCA2 antibodies can then be screened to identify antibodies having one or more of the characteristics described herein (e.g., reducing the production / secretion of one or more of type I or type III interferon, IL-6, TNF-α, MIP-1-α, MIP-1β, CCL5, and IP-10 / CXCL10; depleting pDCs; competing with BIIB059 for binding to the extracellular domain of BDCA2; selectively binding to the extracellular domains of human, cynomolgus monkey, and rhesus monkey BDCA2 but not to rat Clec4b2; inhibiting disease development in a human psoriasis xenograft model).

[0044] Anti-BDCA2 antibody This disclosure includes the sequence of BIIB059, a monoclonal antibody that binds to human, cynomolgus monkey, and rhesus monkey BDCA2 but not to rat Clec4b2. BIIB059 does not bind to, or shows no significant binding to, BDCA2 derived from phylogenetic species lower than primates.

[0045] BIIB059 BIIB059 is a humanized IgG1 antibody that specifically recognizes BDCA2 on the surface of plasmacytoid dendritic cells. BIIB059 was derived from a mouse antibody (24F4) that binds to BDCA2 as follows: A plasmid encoding full-length human BDCA2 was injected into mice using a gene gun. Splenocytes derived from these mice were fused with myeloma cells, and the resulting hybridomas produced 24F4 antibody. The 24F4 antibody was manipulated into a wild-type human IgG1 framework to maintain full effector function. The predicted amino acid sequences of the mature BIIB059 heavy chain and mature BIIB059 light chain are shown below. The complementarity-determining regions (CDRs) 1, 2, and 3 of the variable light chain (VL) and variable heavy chain (VH) are shown in order from the N-terminus to the C-terminus of the mature VL and mature VH sequences, underlined and in bold. The antibody consisting of the mature heavy chain (SEQ ID NO: 4) and mature light chain (SEQ ID NO: 3) listed below is referred to as BIIB059. Mature BIIB059 light chain (LC) [ka] Mature BIIB059 heavy chain (HC) [ka] The variable light chain (VL) of BIIB059 has the following amino acid sequence: [ka] It has. The variable heavy chain (VH) of BIIB059 has the following amino acid sequence: [ka] It has. The amino acid sequence of the VL CDR of BII059 is listed below: [ka] The amino acid sequence of the VH CDR of BII059 is listed below: [ka]

[0046] As shown above, the modified Chothia / AbM CDR definition for VH CDR1 is 5 amino acids longer than the Kabat definition for this CDR. The additional 5 amino acids of the modified Chothia / AbM VH CDR1 are GFTFS (SEQ ID NO: 12).

[0047] The anti-BDCA2 antibody of this disclosure may also include “alternative CDRs” of BIIB059. “Alternative” CDRs mean CDRs (CDR1, CDR2, and CDR3) defined according to any one of the following: the Abysis Chothia definition, the modified Chothia / AbM CDR definition, or the contact definition. These alternative CDRs can be obtained, for example, by using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi). The heavy chain of BIIB059 is also available. The amino acid sequences of the "alternative" CDR1, CDR2, and CDR3 of the variable region and light chain variable region are compared with the CDRs defined according to Kabat in the table below. [ka]

[0048] Anti-BDCA2 antibodies may include heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, as well as light chain CDR1, light chain CDR2, and light chain CDR3, according to the Kabat definition, the Chothia definition by Abysis, the modified Chothia / AbM CDR definition, or the contact definition. These antibodies may have, for example, one, two, or three substitutions in one or more of the CDRs (i.e., one, two, three, four, five, or six). These antibodies (i) bind to human BDCA2 or cynomolgus monkey BDCA2 but do not significantly bind to BDCA2 derived from phylogenetic species lower than primates, and / or (ii) inhibit the production of TLR7 / TLR9-induced type I interferon and other cytokines or chemokines by human pDCs, and / or (iii) mediate the internal translocation of BDCA2 from the surface of pDCs, and / or (iv) downregulate CD32a and / or CD62L from the surface of pDCs, and / or (v) deplete pDCs in vitro by ADCC or CDC.

[0049] Human IgG antibodies are tetrameric molecules containing two light chains and two heavy chains. Each light chain of BIIB059 is covalently linked to the heavy chain via an interchain disulfide bond (LC Cys 218-HC Cys 225), and the heavy chains are paired with each other by two interchain disulfides (HC Cys 231-Cys 231 and Cys 234-Cys 234). All other cysteines form intramolecular disulfides that stabilize the constant and variable domains.

[0050] In certain embodiments, the anti-BDCA2 antibody includes a human heavy chain constant region and a human light chain constant region. In certain embodiments, the heavy chain constant region includes a CH1 domain and a hinge region. In some embodiments, the heavy chain constant region includes a CH3 domain. If the heavy chain constant region includes substitutions, such substitutions modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody is an IgG antibody. In specific embodiments, the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) with affinity between 7 μg / mL and 15 μg / mL. In certain embodiments, the antibody includes a human Fc region that binds to FcγRIIa(CD32a) with an EC50 of 10 μg / mL. In certain embodiments, the antibody contains a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 11 μg / mL. In certain embodiments, the antibody contains a human Fc region that binds to FcγRIIa(CD32a) at an EC50 of 12 μg / mL. Table 1 provides a list of the properties of the BIIB059 antibody. [Table 1-1] [Table 1-2]

[0051] BIIB059 exhibits physicochemical properties suitable for antibody therapeutics. The aggregation exhibited by this antibody is at a low level. The wild-type IgG1 framework contains a single N-linked glycosylation site intramolecularly and within BIIB059, and binds to the Fc receptor with affinity typical for molecules of this class. The calculated pI of 7.26 is somewhat low for an antibody. The charge heterogeneity detected within BIIB059 suggests that a significant proportion of BIIB059 contains modifications. This charge heterogeneity is at least partially explained by the glycosylation level of up to approximately 10% detected in purified batches of BIIB059. The folding Tm of BIIB059 is at the lower end of the typical value observed for antibodies, while the folding Tm of the CH2 and CH3 domains is typical for fully glycosylated IgG1 mAbs. Based on differential scanning fluorescence quantification and viscosity measurements, BIIB059 can be formulated at 50 mg / mL in, for example, 20 mM sodium citrate, 150 mM NaCl, pH 6.0. This antibody can also be formulated at much higher concentrations, such as 150–300 mg / mL (e.g., 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL).

[0052] BIIB059 is a fully humanized Fc-functional IgG1 mAb that exhibits high affinity for BDCA2 and binds equally well to both native human BDCA2 and native cynomolgus monkey BDCA2. BIIB059 is a potent inhibitor of all TLR9-induced type I interferon, as well as other cytokines and chemokines, by pDCs. BIIB059 is equally potent in inhibiting TLR9-induced type I interferon by pDCs derived from healthy human donors and SLE patients. BIIB059 specifically inhibits TLR9-induced type I interferon by pDCs and does not affect IFN production by other cell types induced by different TLR ligands. BIIB059 results in rapid internal translocation of BDCA2 from the cell surface. Upon stimulation, BDCA2 co-localizes with TLR9 in the endosomal / lysosomal compartment, where it is thought to be necessary for inhibiting TLR9 signaling. BIIB059 was found to induce the detachment of CD62L from the surface of human pDCs, which may affect their homing to target organs. In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) and in vitro complement-dependent cytotoxicity (CDC) studies suggest that BIIB059 may possess cell depletion activity in cell lines overexpressing BDCA2. However, the fact that BIIB059 results in the rapid and complete internal translocation of BDCA2 from the surface of pDCs makes it less likely that BIIB059 will cause sustained pDC depletion in vivo. The combination of BIIB059 with hydroxychloroquine (HCQ) resulted in further inhibition of TLR7 and TLR9-induced IFNα production by PBMCs derived from healthy human donors. These data highlight the potential further therapeutic benefits of BIIB059 when administered with antimalarial compounds such as HCQ.

[0053] Antibodies such as BIIB059 can be produced, for example, by preparing and expressing synthetic genes encoding the listed amino acid sequences, or by mutating human germline genes to result in genes encoding the listed amino acid sequences. Furthermore, this antibody and other anti-BDCA2 antibodies can also be obtained, for example, using one or more of the following methods.

[0054] Method for obtaining anti-BDCA2 antibodies Numerous methods are available for obtaining antibodies, particularly human antibodies. One exemplary method involves a step of screening a protein expression library, such as a phage display library or a ribosome display library. Phage displays are described, for example, in US5,223,409;Smith, Science, Vol. 228: pp. 1315-1317 (1985);WO92 / 18619;WO91 / 17271;WO92 / 20791;WO92 / 15679;WO93 / 01288;WO92 / 01047;WO92 / 09690; and WO90 / 02809. Fab, phage displays are described, for example, in U.S. Patent Nos. 5,658,727; 5,667,988; and 5,885,793.

[0055] In addition to using display libraries, other methods can also be used to obtain BDCA2-binding antibodies. For example, the BDCA2 protein or its peptides can be used as antigens in non-human animals, such as rodents, mice, hamsters, or rats. Furthermore, cells transfected with cDNA encoding BDCA2 can be injected into non-human animals as a means of producing antibodies that effectively bind to the cell surface BDCA2 protein.

[0056] In one embodiment, the non-human animal contains at least a portion of a human immunoglobulin gene. For example, a mouse strain lacking mouse antibody production can be used to manipulate a mouse strain containing a large fragment of the human Ig locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from the above gene with desired specificity can be produced and selected. For example, XENOMOUSE®, Green et al., Nature Genetics See Volume 7: pp. 13-21 (1994), US2003-0070185, WO96 / 34096, and WO96 / 33735.

[0057] In another embodiment, a monoclonal antibody is obtained from a non-human animal and then modified, for example, humanized or deimmunized. Winter describes exemplary CDR transplantation methods that may be used to prepare the humanized antibodies described herein (US5,225,539). All or some of the CDRs of a particular human antibody can be replaced with at least some of the CDRs of a non-human antibody. To obtain a useful humanized antibody that binds to BDCA2, it may only be necessary to replace the CDRs required for binding or binding determinants of such CDRs.

[0058] Humanized antibodies can be produced by replacing the sequence of the Fv variable region, which is not directly involved in binding to the antigen, with an equivalent sequence derived from the human Fv variable region. General methods for producing humanized antibodies are described in Morrison, SL, Science, vol. 229: pp. 1202-1207 (1985), Oi et al., BioTechniques, vol. 4: p. 214 (1986), and These methods are provided by US5,585,089;US5,693,761;US5,693,762;US5,859,205; and US6,407,213. These methods include isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of the immunoglobulin Fv variable region, derived from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those skilled in the art and can be obtained, for example, from hybridomas producing antibodies against the aforementioned targets, from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into a suitable expression vector.

[0059] The sequence of human germline cells is, for example, Tomlinson, IA et al., J. Mol. Biol., 227. Volume: 776-798 (1992); Cook, GP et al., Immunol. Today, Vol. 16: 237-242 (1995); Chothia, D. et al., J. Mol. Bio., Vol. 227: 799-817 (1992); and Tomlinson et al., EMBO J., Vol. 14: 4628-4638 This is disclosed on page (1995). The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, such as framework regions and CDRs. Human consensus framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0060] Non-human BDCA2-binding antibodies can also be modified by specific deletion or "deimmunization" of human T cell epitopes by the methods disclosed in WO98 / 52976 and WO00 / 34317. Briefly, the heavy chain and light chain variable regions of the antibody can be analyzed for MHC class II-binding peptides, and these peptides represent potential T cell epitopes (as defined in WO98 / 52976 and WO00 / 34317). To detect potential T cell epitopes, a computer modeling method called "peptide threading" can be applied, and in addition, a database of human MHC class II-binding peptides can be used as described in WO98 / 52976 and WO00 / 34317. H Within the array and V L Motifs present within the sequence can also be searched. These motifs bind to one of the 18 major MHC class II DR allotypes and thus constitute potential T cell epitopes. Detected potential T cell epitopes can be eliminated by substituting a small number of amino acid residues within the variable region, or preferably by a single amino acid substitution. Conservative substitutions are performed whenever possible. Amino acids common to positions in human germline antibody sequences can often be used, but are not limited to them. After identifying the deimmune alteration, V H and V L The nucleic acid encoding this can be constructed by mutagenesis or other synthetic methods (e.g., de novo synthesis, cassette replacement, etc.). The mutagenesized variable sequence can be fused to a human constant region, such as the human IgG1 constant region or the human κ constant region, as needed.

[0061] In some cases, potential T cell epitopes include residues that are known or predicted to be important for antibody function. For example, potential T cell epitopes typically tend to gravitate towards CDRs. In addition, potential T cell epitopes may occur at framework residues that are important for antibody structure and binding. Changes that eliminate these potential epitopes may require further scrutiny, for example, by creating and testing chains with and without the change. Where possible, potential T cell epitopes overlapping with CDRs can be eliminated by substitution outside the CDR. In some cases, a change within the CDR is the only option, and therefore, mutants with and without this substitution can be tested. In other cases, the substitution required to remove a potential T cell epitope is a substitution at a residue position within the framework that may be critical for antibody binding. In these cases, mutants with and without this substitution are tested. Therefore, depending on the case, several deimmunized heavy chain and light chain variable regions of the variants may be designed and tested for various heavy / light chain combinations to identify the optimal deimmunized antibody. The final selection of the deimmunized antibody can then be made by considering the binding affinity of different variants, along with the degree of deimmunization, particularly the number of potential T cell epitopes remaining within the variable region. Deimmunization can be used to modify any antibody, e.g., non-human sequences, such as synthetic antibodies, mouse antibodies, other non-human monoclonal antibodies, or antibodies isolated from display libraries.

[0062] Other methods can also be used to humanize antibodies. For example, other methods can reveal the three-dimensional structure of the antibody, the three-dimensional proximity of the binding determinant and framework, and the immunogenic peptide sequence. For example, WO90 / 07861; U.S. Patent No. 5,693,762; U.S. Patent No. 5,693,761; U.S. Patent No. 5,585,089; U.S. Patent No. 5,530,101; and U.S. Patent No. 6,407,213; Tempest et al. (1991) See Biotechnology, Vol. 9: pp. 266-271. Another method is, This is referred to as "Newmania Ring," and is described, for example, in US2005-008625.

[0063] Antibodies may contain a human Fc region, e.g., a wild-type Fc region or an Fc region with one or more modifications. In one embodiment, the constant region is altered, for example, by mutation, to modify the properties of the antibody (e.g., increasing or decreasing one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). For example, the human IgG1 constant region may be mutated at one or more residues, e.g., one or more of residues 234 and 237 (based on Kabat numbering). Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation. For example, antibodies may have mutations such as those described in U.S. Patents 5,624,821 and 5,648,260. Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of immunoglobulins, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al. (1993), Mol. Immunol., vol. 30: pp. 105-108). See also, for example, US2005-0037000. I want to be illuminated.

[0064] Affinity maturation In one embodiment, an anti-BDCA2 antibody or its antigen-binding fragment is modified, for example, by mutagenesis, to yield a pool of modified antibodies. These modified antibodies are then evaluated to identify one or more antibodies with altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased in vivo stability). One implementation uses display library technology to select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from a second library, for example, by using more stringency conditions or more competitive binding and washing conditions. Other screening methods may also be used.

[0065] In some implementations, mutagenesis targets regions known or potentially located at the binding interface. For example, if the identified binding protein is an antibody, mutagenesis can be directed to the CDR region of the heavy or light chain as described herein. Furthermore, mutagenesis can also be directed to framework regions in the vicinity of or adjacent to the CDR, for example, in particular framework regions within 10, 5, or 3 amino acids from the CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, for example, to perform stepwise improvements.

[0066] In one embodiment, mutagenesis is used to make an antibody more similar to the sequence of one or more germline cells. One exemplary germlining method may include the step of identifying one or more germline sequences that are similar to the sequence of the isolated antibody (e.g., the most similar in a particular database). Mutations (at the amino acid level) may then be introduced into the isolated antibody additionally, in combination, or both. For example, a nucleic acid library containing sequences encoding some or all possible germline mutations is constructed. The mutant antibodies are then evaluated to identify antibodies that have one or more additional germline residues compared to the isolated antibody and are still useful (e.g., functionally active). In one embodiment, as many germline residues as possible are introduced into the isolated antibody.

[0067] In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into the CDR region. For example, the germline CDR residue may be derived from a germline sequence similar to (e.g., most similar to) the variable region being modified. After mutagenesis, antibody activity (e.g., binding activity or other functional activity) can be evaluated to determine whether one or more germline residues are acceptable. Similar mutagenesis can also be performed within the framework region.

[0068] The selection of germline sequences can be carried out in different ways. For example, a germline sequence can be selected if it satisfies a predetermined criterion for selectivity or similarity, e.g., at least a certain percentage of identity compared to a non-human donor antibody, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be carried out using at least 2, 3, 5, or 10 germline sequences. For CDR1 and CDR2, the identification of similar germline sequences may involve the selection of one such sequence. For CDR3, the identification of similar germline sequences may involve the selection of one such sequence, but may also involve the use of two germline sequences contributing separately to the amino-terminal and carboxy-terminal portions. In other implementations, one or more germline sequences are used to form, for example, a consensus sequence.

[0069] The calculation of "sequence identity" between two sequences is performed as follows: The sequences are aligned for optimal comparison (for example, gaps can be introduced in one or both of the first amino acid or nucleic acid sequence and the second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences can be omitted for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package, with a Blossum 62 scoring matrix that has a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by the sequences.

[0070] In other embodiments, antibodies can be modified to alter their glycosylation pattern (i.e., to change it from the original or native glycosylation pattern). As used in this context, “altered” means deleting one or more carbohydrate moieties and / or adding one or more glycosylation sites to the original antibody. The addition of glycosylation sites to antibodies disclosed herein can be achieved by altering the amino acid sequence to include a consensus sequence of glycosylation sites, and such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on an antibody is by chemical or enzymatic coupling of glycosides to amino acid residues of the antibody. These methods are described, for example, in WO87 / 05330, and in Aplin and Wriston (1981), CRC Crit. Rev. Biochem., Vol. 22: pp. 259–306. Removal of any carbohydrate portion present on an antibody can be achieved chemically or enzymatically, as described in the Art (Hakimuddin et al. (1987), Arch. Biochem. Biophys., vol. 259: p. 52; Edge et al. (1981), Anal. Biochem., vol. 118: p. 131; and Thotakura et al. (1987), Meth. Enzymol., vol. 138: p. 350). For example, for modifications that extend the in vivo half-life by applying salvage receptor-binding epitopes, see U.S. Patent No. 5,869,046.

[0071] In one embodiment, the antibody has a CDR sequence different from that of the BIIB059 monoclonal antibody (e.g., a CDR by Chothia or a CDR by Kabat). The CDR sequence different from that of the BIIB059 monoclonal antibody may include amino acid changes, such as the substitution of 1, 2, 3, or 4 amino acids, if the CDR is 5 to 7 amino acids long, or 1, 2, 3, 4, 5, 6, or 7 amino acids within the CDR sequence if the CDR is 10 or more amino acids long. The substituted amino acids may have similar charge, hydrophobic, or stereochemical characteristics. In some embodiments, the amino acid substitution(s) are conserved substitutions. In other embodiments, the amino acid substitution(s) are non-conservative substitutions. Such substitutions are within the scope of the ordinary art of those skilled in the art. Antibodies containing substituted CDRs or antibody fragments thereof can be screened to identify antibodies having one or more of the features described herein (e.g., reducing the production / secretion of type I or type III interferon, IL-6, TNF-α, MIP-1-α / CCL3, MIP-1β / CCL4, CCL5 / RANTES, IP-10 / CXCL10; depleting pDCs; competing with BIIB059 for binding to the extracellular domain of BDCA2; selectively binding to the extracellular domains of human, cynomolgus monkey, and rhesus monkey BDCA2 but not to rat Clec4b2, or binding to rat Clec4b2 with a lower binding affinity than for human BDCA2, cynomolgus monkey BDCA2, or rhesus monkey BDCA2; inhibiting disease development in a human psoriasis xenograft model).

[0072] Unlike in the case of CDRs, more substantial structural changes to the framework region (FR) can be made without adversely affecting the antibody binding properties. Changes to the FR include, but are not limited to, humanization of non-human frameworks, or manipulation of certain framework residues that are important for stabilizing antigen contact or binding sites, such as changes in the class or subclass of the constant region, changes in specific amino acid residues that can alter effector functions such as Fc receptor binding (Lund et al., J. Immun., Vol. 147: pp. 2657-2662 (1991); Morgan et al., Immunology, Vol. 86: pp. 319-324 (1995)), or changes in the species from which the constant region originates.

[0073] Anti-BDCA2 antibodies may be in the form of a full-length antibody or in the form of a low molecular weight antibody (e.g., a biologically active antibody fragment or minibody), such as Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, and sc(Fv)2. Other anti-BDCA2 antibodies encompassed by this disclosure include single-domain antibodies (sdAb) containing a single variable chain, such as VH or VL, or their biologically active fragments. For example, Moller et al., J. Biol. Chem., vol. 285 (no. 49): pp. 38348-38361 (2010); Harmsen et al., Appl. Microbiol. Biotechnol., vol. 77 ( See also: Issue 1): pp. 13-22 (2007); US2005 / 0079574; and Davies et al. (1996), Protein Eng., Vol. 9 (No. 6): pp. 531-537. Similar to antibodies, sdAbs can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, sdAbs are much smaller than typical antibodies and even smaller than Fab fragments and single-chain variable fragments.

[0074] This specification provides compositions comprising a mixture of an anti-BDCA2 antibody or its antigen-binding fragment and one or more acidic variants thereof, for example, in which the amount of acidic variants is about 80%, 70%, 60%, 60%, 50%, 40%, 30%, 30%, 20%, 10%, 5%, or less than 1%. Also provided are compositions comprising an anti-BDCA2 antibody or its antigen-binding fragment containing at least one deamidation site, in which, for example, the pH of the composition is about 5.0 to about 6.5 such that at least about 90% of the anti-BDCA2 antibody is not deamidated (i.e., less than about 10% of the antibody is deamidated). In certain embodiments, less than about 5%, 3%, 2%, or 1% of the antibody is deamidated. The pH can be 5.0 to 6.0, such as 5.5 or 6.0. In certain embodiments, the pH of the composition is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0075] An "acidic mutant" is a variant of the target polypeptide that is more acidic than the target polypeptide (for example, as determined by cation exchange chromatography). An example of an acidic mutant is a deamidated mutant.

[0076] A "deamidation" mutant of a polypeptide molecule is a polypeptide in which one or more asparagine residues of the original polypeptide are converted to aspartic acid, that is, a polypeptide in which the neutral amide side chain is converted to a residue that has an overall acidic character.

[0077] As used herein with reference to compositions comprising an anti-BDCA2 antibody or its antigen-binding fragment, the term “mixture” means the presence of both the desired anti-BDCA2 antibody or its antigen-binding fragment and one or more of its acidic variants. The acidic variants may primarily consist of deamidated anti-BDCA2 antibodies, but may also include small amounts of other acidic variants.

[0078] In certain embodiments, the binding affinity (K D ) of an antibody mutated to abolish deamidation, the on-rate (K D on), and / or the off-rate (K D off) is the same as that of the wild-type antibody, e.g., K D , K D on, and / or K D off, and the difference is, for example, less than about 5-fold, 2-fold, 1-fold (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2%, or 1%.

[0079] In certain embodiments, an anti-BDCA2 antibody or an antigen-binding fragment thereof or a low molecular weight antibody thereof binds to BDCA2 on pDCs and inhibits or reduces the production and / or secretion of type I and type III IFNs, IL-6, TNF-α, and other inflammatory cytokines and chemokines (e.g., MIP-1α / CCL3, MIP-1β / CCL4, CCL5, and IP-10 / CXCL10) by pDCs; and / or depletes pDCs by ADCC or CDC or apoptosis; and / or when administered to a human patient or an animal model thereof having one or more of systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis, scleroderma, morphea, rheumatoid arthritis, polymyositis-dermatomyositis, psoriasis, Sjögren's syndrome, vasculitis, and type I diabetes, reduces the severity of the symptoms. In one embodiment, an anti-BDCA2 antibody or an antigen-binding fragment thereof or a low molecular weight antibody thereof inhibits the development of the disease in a human psoriasis xenograft model (Nestle et al., J. Exp. Med., 202(1):135-143 (2005)). These properties of the anti-BDCA2 antibody or an antigen-binding fragment thereof or a low molecular weight antibody thereof can be measured according to the methods described in the Examples and also by other methods known in the art.

[0080] Antibody fragment Antibody fragments (e.g., Fab, Fab', F(ab')2, Fabcb, and Fv) can be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Digestion of the whole antibody with papain yields an F(ab)2 or Fab fragment, digestion of the whole antibody with pepsin yields an F(ab')2 or Fab' fragment, and digestion of the whole antibody with plasmin yields a Fabc fragment.

[0081] Alternatively, antibody fragments can also be produced by recombination. For example, the nucleic acid encoding the target antibody fragment can be constructed, introduced into an expression vector, and expressed in a suitable host cell. For example, Co, MS et al., J. Immunol., Vol. 152: pp. 2968-2976. 1994); Better, M. and Horwitz, AH, Methods in Enzymology, Vol. 178: pp. 476-496 (1989); Plueckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, Vol. 121: pp. 652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989), Vol. 121: pp. 663-669 (1989); See also Bird, RE et al., TIBTECH, Vol. 9: pp. 132-137 (1991). Antibody fragments can be expressed within E. coli and secreted from E. coli, thereby enabling the easy production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology, Vol. 10: pp. 163-167 (1992)). By another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab fragments and F(ab')2 fragments with extended half-lives in vivo, which include salvage receptor-binding epitope residues, are described in U.S. Patent No. 5,869,046.

[0082] Mini body The minibodies of the anti-BDCA2 antibody include a diabody, a single chain (scFv), and a single chain (Fv)2 (sc(Fv)2).

[0083] A "diabody" is a bivalent minibody constructed by gene fusion (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 6444-6448 (1993); EP404, 097; WO93 / 11161). A diabody is a dimer consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain in a diabody are linked by a linker. The number of amino acid residues constituting the linker can be between 2 and 12 (e.g., 3 to 10 residues, or 5 residues or approximately 5 residues). Typically, the linker of polypeptides within a diabody is too short to allow VL and VH to bind to each other. Therefore, VL and VH encoded within the same polypeptide chain cannot form a single-chain variable region fragment, but instead can form a dimer with different single-chain variable region fragments. As a result, the diabody has two antigen-binding sites.

[0084] scFv is a single-chain polypeptide antibody obtained by linking VH and VL with a linker (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85: See pp. 5879–5883 (1988); and Plickthun, "The Pharmacology of Monoclonal Antibodies," Vol. 113, edited by Resenburg and Moore, Springer Verlag, New York, pp. 269–315 (1994). Linking VH and VL. The order is not particularly limited and can be arranged in any order. Examples of arrangement include [VH]linker[VL] or [VL]linker[VH]. The H chain V region and L chain V region within scFv may originate from any anti-BDCA2 antibody or its antigen-binding fragment described herein.

[0085] sc(Fv)2 is a minibody that forms a single chain by linking two VHs and two VLs with a linker (Hudson et al., J. Immunol. Methods (1999), 231). Volume: pp. 177-189 (1999). sc(Fv)2 can be prepared, for example, by linking scFv with a linker. The sc(Fv)2 of the present invention preferably contains an antibody in which two VHs and two VLs are arranged in the order VH, VL, VH, and VL ([VH]linker[VL]linker[VH]linker[VL]) starting from the N-terminus of the single-chain polypeptide, but the order of the two VHs and two VLs is not limited to the above arrangement and can be arranged in any order. Examples of arrangements are listed below. [ka] Typically, when linking four antibody variable regions, three linkers are required, and the linkers used may be the same or different. There are no specific limitations on the linkers used to link the VH and VL regions of the mini-bodies. In some embodiments, the linkers are peptide linkers. Any single-chain peptide containing approximately 3 to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 residues) can be used as a linker. Examples of such peptide linkers include: Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (Sequence No. 13); Ser Gly Gly Gly (Sequence No. 14); Gly Gly Gly Gly Ser (Sequence No. 15); Ser Gly Gly Gly Gly (Sequence No. 16); Gly Gly Gly Gly Gly Ser (Sequence No. 17); Ser Gly Gly Gly Gly Gly (Sequence No. 18); Gly Gly Gly Gly Gly Gly Ser ( Sequence ID 19); Ser Gly Gly Gly Gly Gly Gly (Sequence ID 20); (Gly Gly Gly Gly Ser (Sequence ID 21)n [In the array, n is one or more integers]; and (Ser Gly Gly Gly Gly (Sequence No. 22)) n [In the array, n is one or more integers.] Includes.

[0086] In certain embodiments, the linker is a synthetic compound linker (chemical crosslinking agent). Examples of commercially available crosslinking agents include N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0087] The amino acid sequences of the VH or VL within the minibody may include modifications such as substitutions, deletions, additions, and / or insertions. For example, modifications may occur in one or more CDRs of the anti-BDCA2 antibody or its antigen-binding fragment (e.g., BIIB059). In certain embodiments, the modification involves one, two, or three amino acid substitutions within one or more CDRs of the VH domain and / or VL domain of the anti-BDCA2 minibody. Such substitutions are made to improve the binding activity and / or functional activity of the anti-BDCA2 minibody. In other embodiments, one, two, or three amino acids may be deleted or added to the CDR of the anti-BDCA2 antibody or its antigen-binding fragment (e.g., BIIB059), insofar as binding activity and / or functional activity to BDCA2 exists when VH and VL are associated.

[0088] bispecific antibody A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. An exemplary bispecific antibody can bind to two different epitopes of the BDCA2 protein. In other such antibodies, the BDCA2 binding site can be combined with a binding site for another protein. Bispecific antibodies can be prepared as full-length antibodies or in their lower molecular weight forms (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

[0089] Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, vol. 305: 537-55). (Page 39 (1983)). In a different method, an antibody variable domain with the desired binding specificity is fused to the immunoglobulin constant domain sequence. The DNA encoding the immunoglobulin heavy chain fusion, and optionally the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into appropriate host cells. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, if high yields are obtained as a result of expressing at least two polypeptide chains in equal ratios, it is also possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0090] Another method described in U.S. Patent No. 5,731,168 allows for the manipulation of the interface between pairs of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. A preferred interface is C H3 This method involves replacing one or more small amino acid side chains originating from the interface of the first antibody molecule with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created on the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers above other undesirable end products, such as homodimers.

[0091] Bispecific antibodies include crosslinked antibodies or "heteroconjugated" antibodies. For example, in a heteroconjugated antibody, one antibody can be coupled to avidin and the other to biotin. Heteroconjugated antibodies can be prepared using any simple crosslinking method.

[0092] The "Diabody" technology provides an alternative mechanism for creating bispecific antibody fragments. The fragment contains a VH domain linked to a VL domain by a linker that is too short to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0093] Multivalent antibodies Polyvalent antibodies may be translocated (and / or catabolized) more quickly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies described herein may be polyvalent antibodies (e.g., tetravalent antibodies) having three or more antigen-binding sites, and which can be readily produced by recombinant expression of the nucleic acid encoding the antibody polypeptide chain. A polyvalent antibody may include a dimerization domain and three or more antigen-binding sites. An exemplary dimerization domain includes (or consists of) an Fc region or a hinge region. A polyvalent antibody may include (or consist of) three to about eight (e.g., four) antigen-binding sites. A polyvalent antibody may optionally include at least one polypeptide chain (e.g., at least two polypeptide chains), and the polypeptide chain(s) may include two or more variable domains. For example, the polypeptide chain(s) may be VD1-(X1) n -VD2-(X2) n -Fc [In the sequence, VD1 is the first variable domain, VD2 is the second variable domain, Fc is the polypeptide chain of the Fc region, X1 and X2 represent amino acid spacers or peptide spacers, and n is 0 or 1] may be included.

[0094] Conjugate Antibody The antibodies disclosed herein include polymers (e.g., polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive materials (e.g., 90 Y, 131I) These may be conjugated antibodies attached to a variety of molecules, including fluorescent substances, luminescent substances, haptens, enzymes, metal chelators, drugs, and toxins (e.g., calcheamicin, Pseudomonas exotoxin A, lysine (e.g., deglycosylated lysine A chain)).

[0095] In one embodiment, to improve the cytotoxic effect of anti-BDCA2 antibodies and consequently their therapeutic efficacy, antibodies are conjugated with highly toxic substances, including radioisotopes and cytotoxic agents. These conjugates can selectively deliver the toxic load to target sites (i.e., cells expressing antigens recognized by the antibody) while not targeting cells not recognized by the antibody. To minimize toxicity, conjugates are generally engineered based on molecules with short serum half-lives (hence the use of mouse sequences and IgG3 or IgG4 isotypes).

[0096] In certain embodiments, an anti-BDCA2 antibody or its antigen-binding fragment is modified by a portion that improves its stabilization and / or retention in circulation, e.g., in blood, serum, or other tissues, by, for example, at least 1.5, 2, 5, 10, or 50 times. For example, an anti-BDCA2 antibody or its antigen-binding fragment can be associated (e.g., conjugated) with a substantially non-antigenic polymer, such as a polymer, e.g., polyalkylene oxide or polyethylene oxide. The suitable polymer varies substantially by weight. Polymers with number-average molecular weights in the range of about 200 to about 35,000 daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500 daltons) can be used. For example, an anti-BDCA2 antibody or its antigen-binding fragment can be conjugated with a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG), or polypropylene glycol, polyoxyethylated polyols, copolymers thereof, and these block copolymers, assuming that the water solubility of the block copolymers is maintained. Further useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.

[0097] The conjugate antibodies described above can be prepared by chemically modifying the antibodies or their low molecular weight forms described herein. Methods for modifying antibodies are well known in the art (e.g., US5057313 and US5156840).

[0098] Methods for producing antibodies Antibodies can be produced in bacterial cells or eukaryotic cells. Some antibodies, such as Fab', can be produced in bacterial cells, for example, in E. coli cells. Antibodies can also be produced in eukaryotic cells, such as in transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFv') can be expressed in yeast cells, such as those of the Pichia genus (see, for example, Powers et al., J Immunol Methods, Vol. 251: pp. 123-35 (2001)), Hanseula genus, or Saccharomyces genus. To produce the antibody of interest, the polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Using standard molecular biology methods, a recombinant expression vector is prepared, transfected into host cells, transformants are selected, the host cells are cultured, and the antibody is recovered.

[0099] When expressing antibodies in bacterial cells (e.g., E. coli), the expression vector should have characteristics that allow for amplification of the vector within the bacterial cell. In addition, when using E. coli such as JM109, DH5α, HB101, or XL1-Blue as the host, the vector should have a promoter, for example, the lacZ promoter (Ward et al., Vol. 341: pp. 544-546 (1989)), the araB promoter (Better et al., Science, The vector must have a T7 promoter capable of efficient expression in E. coli, such as the one described in Vol. 240: pp. 1041-1043 (1988). Examples of such vectors include, for example, the M13 series vectors, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when using this expression vector, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. To produce antibody in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., Vol. 169: p. 4379 (1987)) must be used. It can be used as a signal sequence for secreting the body. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation.

[0100] When expressing antibodies in animal cells such as CHO cells, COS cells, and NIH3T3 cells, the expression vector must contain promoters necessary for expression within these cells, such as the SV40 promoter (Mulligan et al., Nature, Vol. 277: p. 108 (1979)), the MMLV-LTR promoter, and the EF1α promoter (Mizushima et al., Nucleic Acids Res. (Vol. 18:p. 5322 (1990)), or includes the CMV promoter. In addition to nucleic acid sequences encoding immunoglobulins or their domains, recombinant expression vectors may also contain further sequences such as sequences that regulate vector replication within host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, selection marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Examples of vectors with selection markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0101] In one embodiment, the antibody is produced within mammalian cells. An exemplary mammalian host cell for antibody expression is Chinese hamster ovary (CHO) cells (see, for example, Kaufman and Sharp (1982), Mol. Biol., Vol. 159: pp. 601-621). As described above, the dhfr used in conjunction with DHFR selection markers is described in Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA, Vol. 77: pp. 4216-4220. - This includes cells derived from transgenic animals, such as transgenic mammals. For example, mammary epithelial cells.

[0102] In an exemplary system for expressing an antibody, a recombinant expression vector encoding both the antibody heavy chain and antibody light chain of an anti-BDCA2 antibody (e.g., BIIB059) is transfected via calcium phosphate-mediated transfection using dhfr -The recombinant expression vector is introduced into CHO cells. Within the recombinant expression vector, the antibody heavy chain gene and antibody light chain gene are operatively linked to enhancer / promoter regulatory elements (e.g., CMV enhancer / AdMLP promoter regulatory elements, or SV40 enhancer / AdMLP promoter regulatory elements, derived from SV40, CMV, adenovirus, etc.) to drive high levels of gene transcription. The recombinant expression vector also contains a DHFR gene that allows selection of CHO cells transfected with the vector using methotrexate selection / amplification. The host cells of the selected transformants are cultured to enable expression of the antibody heavy and light chains, and the antibodies are recovered from the culture medium.

[0103] Antibodies can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A milk-specific promoter, as well as a transgene containing nucleic acids encoding the antibody of interest and a signal sequence for secretion, are constructed. The milk produced by such a female transgenic mammal contains the antibody of interest secreted therein. The antibody can also be purified from the milk and, in some applications, can be used directly. Animals containing one or more of the nucleic acids described herein are also provided.

[0104] The antibodies of this disclosure can be isolated from within host cells or from outside host cells (e.g., culture medium) and can be purified as substantially pure and homogeneous antibodies. Methods commonly used for isolation and purification of antibodies can be used to isolate and purify the antibodies, and are not limited to any specific method. Antibodies can be isolated and purified by appropriately selecting and combining methods such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed using liquid-phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Examples of columns using Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). This disclosure also includes highly purified antibodies using these purification methods.

[0105] Antibody characterization The BDCA2 binding properties of the antibodies described herein can be measured by any standard method, for example, one or more of the following: OCTET®, surface plasmon resonance (SPR), BIACORE® analysis, enzyme immunoassay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET).

[0106] The binding interaction between the target protein (anti-BDCA2 antibody) and the target (e.g., BDCA2) can be analyzed using the OCTET® system. This method utilizes multiple variations of the ForteBio analyzer (e.g., OCTET® QK e Using one of the following (QK) proteins, protein-protein interactions, binding specificity, and epitope mapping are determined. The OCTET® system provides a simple way to monitor binding in real time by measuring changes in polarization that propagate along a custom chip and then return to the sensor.

[0107] The binding interaction between a target protein (anti-BDCA2 antibody) and its target (e.g., BDCA2) can be analyzed using surface plasmon resonance (SPR). SPR, or biomolecular interaction analysis (BIA), detects biospecific interactions in real time without labeling any of the interacting substances. A change in mass at the binding surface of the BIA chip (indicating a binding event) results in a change in the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). This change in refractive index generates a detectable signal that is measured as an indicator of real-time reactions between biomolecules. Methods for using SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988). Surface Plasmons, Springer Verlag; Sjolander and Urbaniczky (1991), Anal. Chem., Vol. 63: pp. 2338-2345; Szabo et al. (1995), Curr. Opin. Struct. Biol., Vol. 5: pp. 699-705; and BIAcore Inter This is described in online resources provided by the national AB (Uppsala, Sweden). Using information from SPR, the equilibrium dissociation constant (K) for the binding of biomolecules to their targets is used. d ) and K on and K offThis allows us to obtain accurate and quantitative measures for reaction rate parameters, including [specific parameters].

[0108] Epitope is also used in BIACORE chromatography (Pharmacia BIAtechnology). Handbook, "Epitope Mapping," Section 6.3.2 (May 1994); also, John et al. See also (1993), J. Immunol. Methods, Vol. 160: pp. 191-198. It is also possible to directly map different antibodies that compete with each other for binding to human BDCA2 by using ) to evaluate their ability to bind to human BDCA2.

[0109] When using an enzyme immunoassay, an antibody-containing sample, such as the culture supernatant of antibody-producing cells or purified antibody, is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, and after washing, an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to evaluate the binding activity to the antigen.

[0110] Further general guidelines for antibody evaluation, such as Western blotting and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor Press (1988).

[0111] Deposit The mouse hybridoma BDCA2-1P24F4.1.1.1, which produces an anti-BDCA2 monoclonal antibody, was deposited with the American Type Culture Collection (ATCC) on January 15, 2013, in accordance with the provisions of the Budapest Convention on the International Recognition of Deposit of Microorganisms in Patent Proceedings, and holds accession number PTA-13450. The applicants acknowledge their obligation to replace the deposited material if, prior to the expiration of the patent granted herein, the depositary is unable to provide a sample when requested due to the condition of the deposited material. The applicants also acknowledge their responsibility to notify the ATCC of the granting of such patent, at which point the deposited material will be made publicly available. Prior to that point, the deposited material will be made available to the Commissioner of Patents in accordance with the provisions of 37 CFR §1.14 and 35 USC §112.

[0112] Antibodies with altered effector functions The interaction of antibodies and antibody-antigen complexes with immune system cells induces various responses, which are referred to herein as effector functions. Immune-mediated effector functions include two main mechanisms: antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Both of these are mediated by the constant region of immunoglobulin proteins. Therefore, the Fc domain of an antibody is the portion that defines the interaction with the immune effector mechanism.

[0113] IgG antibodies activate the effector pathway of the immune system by binding to members of the cell surface Fcγ receptor family and C1q of the complement system. Clustered antibodies ligate effector proteins, inducing a variety of responses, including the release of inflammatory cytokines, regulation of antigen production, endocytosis, and cell death. In some clinical applications, these responses are crucial to the efficacy of monoclonal antibodies. In other clinical applications, these responses induce undesirable side effects, such as inflammation and the disappearance of antigen-carrying cells. Therefore, the present invention further relates to BDCA2-binding proteins containing antibodies with altered effector function, for example, those with increased or decreased effector function.

[0114] The effector function of the anti-BDCA2 antibody of the present invention can be determined using one of many known assays. The effector function of the anti-BDCA2 antibody can be increased or decreased compared to the second anti-BDCA2 antibody. In some embodiments, the second anti-BDCA2 antibody may be any antibody that specifically binds to BDCA2. In other embodiments, the second BDCA2-specific antibody may be any of the antibodies of the present invention, such as BIIB059. In other embodiments, where the anti-BDCA2 antibody of interest has been modified to increase or decrease its effector function, the second anti-BDCA2 antibody may be unmodified or the parent antibody.

[0115] Effector function includes antibody-dependent cell-mediated cytotoxicity (ADCC), in which cell death is induced when an antibody binds to Fc receptors on cytotoxic T cells, natural killer (NK) cells, or macrophages, and complement-dependent cytotoxicity (CDC), which is cell death induced via the activation of the complement cascade (Daeron, Annu. Rev.). This has been reviewed in Immunol., Vol. 15: pp. 203-234 (1997); Ward and Ghetie, Therapeutic Immunol., Vol. 2: pp. 77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol., Vol. 9: pp. 457-492 (1991). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using standard assays known in the art (see, e.g., WO05 / 018572, WO05 / 003175, and US6,242,195).

[0116] Effector function can be avoided by using antibody fragments lacking the Fc domain, such as Fab, Fab'2, or single-chain Fv. An alternative is to use IgG4 subtype antibodies that bind to FcγRI but not so much to C1q, as well as FcγRII and FcγRIII. IgG2 subtypes also exhibit reduced binding to Fc receptors, but retain significant binding to H131 and C1q, which are allotypes of FcγRIIa. Therefore, further alterations to the Fc sequence are required to eliminate binding to all Fc receptors and C1q.

[0117] Multiple antibody effector functions, including ADCC, are mediated by Fc receptors (FcRs) that bind to the Fc region of the antibody. The antibody's affinity for a particular FcR can be modulated by altering the amino acid sequence and / or post-translational modifications of the antibody's Fc and / or constant region, and thus the antibody-mediated effector activity can also be modulated.

[0118] FcRs are defined by their specificity for immunoglobulin isotypes. For example, the Fc receptor for IgG antibodies is called FcγR, the Fc receptor for IgE antibodies is called FcεR, and the Fc receptor for IgA antibodies is called FcαR. Three subclasses of FcγR have been identified: FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). Both FcγRII and FcγRIII have two subclasses: FcγRIIa(CD32a) and FcγRIIB(CD32b); and FcγRIIIA(CD16a) and FcγRIIIB(CD16b). Each FcγR subclass is encoded by two or three genes, and alternative RNA splicing results in multiple transcripts, leading to a wide diversity of FcγR isoforms. For example, FcγRII(CD32) includes isoforms IIa, IIb1, IIb2, IIb3, and IIc.

[0119] The binding site for FcγR on human and mouse antibodies is residues 233-239 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Woof et al., Molec. Immunol., Vol. 23: pp. 319-330 (1986); Duncan et al., Nature, Vol. 332: pp. 563 (1988); Canfield and Morrison, J. Exp. Med., 1 Volume 73: pp. 1483-1491 (1991); Chappel et al., Proc. Natl. Acad. Sci According to the EU index in USA, Vol. 88: pp. 9036-9040 (1991). It has already been mapped to the so-called "lower hinge region" consisting of (numbered) residues 233-239. Of residues 233-239, P238 and S239 are among the residues mentioned as likely involved in binding. Other already mentioned areas likely involved in binding to FcγR are G316-K338 (human IgG) to human FcγRI (Woof et al., Mol. Immunol., Vol. 23: pp. 319-330 (1986)); K274-R301 (human IgG1) to human FcγRIII (Sarmay et al., Molec. Immunol., Vol. 21: pp. 43-51 (1984)); and Y407-R416 (human IgG) to human FcγRIII (Gergely et al., Biochem. Soc. Trans., Vol. 12: pp. 739-743 ( See, for example, 1984, and Shields et al., J Biol Chem, vol. 276: pp. 6591-6604 (2001), and Lazar GA et al., Proc Natl Acad Sci, vol. 103: pp. 4005-4010 (2006). The sequences or regions of these and other amino acid residues involved in FcR binding may be obvious to those skilled in the art from the examination of the crystal structure of the Ig-FcR complex (see, for example, Sondermann et al., 2000, Nature, vol. 406 (no. 6793): pp. 267-73; and Sondermann et al., 2002, Biochem Soc Trans., vol. 30 (no. 4): pp. 481-486). Accordingly, the anti-BDCA2 antibody of the present invention comprises modifications of one or more of the aforementioned residues (modifications that increase or decrease effector function, if necessary).

[0120] Another method for altering the effector function of monoclonal antibodies involves mutating amino acids on the surface of the monoclonal antibody that are involved in effector binding interactions (Lund, J. et al. (1991), J. Immunol., vol. 147(no. 8):2657~6). p. 2; Shields, RL et al. (2001), J. Biol. Chem., vol. 276(9): 6591-604).

[0121] In this field, methods for enhancing the effector function of antibodies are well known (e.g., Kelley et al., Methods Mol. Biol., Vol. 901: pp. 277-2793 (2012); Natsume et al.). Drug Des Devel Ther., Vol. 3: pp. 7-16 (2009); US8, 188, 23 1. See US7,960,512). In one embodiment, the BDCA2 antibody is 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 29 It has 1, 2, 3, 4, 5, 6, or 7 or more amino acid substitutions at positions selected from the group consisting of 1, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337 [where the numbering of residues within the Fc region is the numbering of the EU index by Kabat]. In certain embodiments, the BDCA2 antibody is D221K, D221Y, K222E, K222Y, T223E, T223K, H224E, H224Y, T225E, T225K, T225W, P227E, P227G, P227K, P227Y, P228E, P228G, P228K, P228Y, P230A, P230E, P230G, P230Y, A231E, A231G, A231K, A231P, A231Y, P232E, P232G, P232K, P232Y, E233A, E233D, E233F, E233G, E233H, E233I, E233K, E2 33L, E233M, E233N, E233Q, E233R, E233S, E233T, E233V, E233W, E233Y, L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234K, L234M, L234N, L234P, L234 Q, L234R, L234S, L234T, L234V, L234W, L234Y, L235A, L235D, L235E, L235F, L2 35G, L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R, L235S, L235T,L235V、L235W、L235Y、G236A、G236D、G236E、G236F、G236H、G236I、G236K、G236L、G236M、G236N、G236P、G236Q、G236R、G236S、G236T、G236V、G236W、G236Y、G237D、G237E、G237F、G237H、G237I、G237K、G237L、G237M、G237N、G237P、G237Q、G237R、G237S、G237T、G237V、G237W、G237Y、P238D、P238E、P238F、P238G、P238H、P238I、P238K、P238L、P238M、P238N、P238Q、P238R、P238S、P238T、P238V、P238W、P238Y、S239D、S239E、S239F、S239G、S239H、S239I、S239K、S239L、S239M、S239N、S239P、S239Q、S239R、S239T、S239V、S239W、S239Y、V240A、V240I、V240M、V240T、F241D、F241E、F241L、F241R、F241S、F241W、F241Y、F243E、F243H、F243L、F243Q、F243R、F243W、F243Y、P244H、P245A、K246D、K246E、K246H、K246Y、P247G、P247V、D249H、D249Q、D249Y、R255E、R255Y、E258H、E258S、E258Y、T260D、T260E、T260H、T260Y、V262A、V262E、V262F、V262I、V262T、V263A、V263I、V263M、V263T、V264A、V264D、V264E、V264F、V264G、V264H、V264I、V264K、V264L、V264M、V264N、V264P、V264Q、V264R、V264S、V264T、V264W、V264Y、D265F、D265G、D265H、D265I、D265K、D265L、D265M、D265N、D265P、D265Q、D265R、D265S、D265T、D265V、D265W、D265Y、V266A、V266I、V266M、V266T、S267D、S267E、S267F、S267H、S267I、S267K、S267L、S267M、S267N、S267P、S267Q、S267R、S267T、S267V、S267W、S267Y、H268D、H268E、H268F、H268G、H268I、H268K、H268L、H268M、H268P、H268Q、H268R、H268T、H268V、H268W、E269F、E269G、E269H、E269I、E269K、E269L、E269M、E269N、E269P、E269R、E269S、E269T、E269V、E269W、E269Y、D270F、D270G、D270H、D270I、D270L、D270M、D270P、D270Q、D270R、D270S、D270T、D270W、D270Y、P271A、P271D、P271E、P271F、P271G、P271H、P271I、P271K、P271L、P271M、P271N、P271Q、P271R、P271S、P271T、P271V、P271W、P271Y、E272D、E272F、E272G、E272H、E272I、E272K、E272L、E272M、E272P、E272R、E272S、E272T、E272V、E272W、E272Y、V273I、K274D、K274E、K274F、K274G、K274H、K274I、K274L、K274M、K274N、K274P、K274R、K274T、K274V、K274W、K274Y、F275L、F275W、N276D、N276E、N276F、N276G、N276H、N276I、N276L、N276M、N276P、N276R、N276S、N276T、N276V、N276W、N276Y、Y278D、Y278E、Y278G、Y278H、Y278I、Y278K、Y278L、Y278M、Y278N、Y278P、Y278Q、Y278R、Y278S、Y278T、Y278V、Y278W、D280G、D280K、D280L、D280P、D280W、G281D、G281E、G281K、G281N、G281P、G281Q、G281Y、V282E、V282G、V282K、V282P、V282Y、E283G、E283H、E283K、E283L、E283P、E283R、E283Y、V284D、V284E、V284L、V284N、V284Q、V284T、V284Y、H285D、H285E、H285K、H285Q、H285W、H285Y、N286E、N286G、N286P、N286Y、K288D、K288E、K288Y、K290D、K2 90H、K290L、K290N、K290W、P291D、P291E、P291G、P291H、P291I、P291Q、P291 T、R292D、R292E、R292T、R292Y、E293F、E293G、E293H、E293I、E293L、E293M、 E293N、E293P、E293R、E293S、E293T、E293V、E293W、E293Y、E294F、E294G、E29 4H、E294I、E294K、E294L、E294M、E294P、E294R、E294S、E294T、E294V、E294W 、E294Y、Q295D、Q295E、Q295F、Q295G、Q295H、Q295I、Q295M、Q295N、Q295P、Q 295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D, Y296E, Y296G, Y296H, Y296I, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S, Y296T, Y296V N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M, N297P, N297Q, N297R, N297S, N297T, N297V, N297W, N297Y, S298D, S298E, S298F, S298 H, S298I, S298K, S298M, S298N, S298Q, S298R, S298T, S298W, S298Y, T299A, T299D, T299E, T299F, T299G, T299H, T299I, T299K, T299L, T299M, T299N, T29 9P, T299Q, T299R, T299S, T299V, T299W, T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K, Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, Y 300W、R301D、R301E、R301H、R301Y、V302I、V303D、V303E、V303Y、S304D、S30 4H、S304L、S304N、S304T、V305E、V305T、V305Y、W313F、K317E、K317Q、E318H、E318L, E318Q, E318R, E318Y, K320D, K320F, K320G, K320H, K320I, K320L, K320N, K320P, K320S, K320T, K320V, K320W, K320Y, K322D, K322F, K322G, K322 H、K322I、K322P、K322S、K322T、K322V、K322W、K322Y、V323I、S324D、S324F、 S324G、S324H、S324I、S324L、S324M、S324P、S324R、S324T、S324V、S324W、S32 4Y, N325A, N325D, N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I, K326L, K 326P, K326T, A327D, A327E, A327F, A327H, A327I, A327K, A327L, A327M, A327N, A327P, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D, L328E L328F, L328G, L328H, L328I, L328K, L328M, L328N, L328P, L328Q, L328R, L328S, L328T, L328V, L328W, L328Y, P329D, P329E, P329F, P329G, P329H, P329 I, P329K, P329L, P329M, P329N, P329Q, P329R, P329S, P329T, P329V, P329W, P329Y, A330E, A330F, A330G, A330H, A330I, A330L, A330M, A330N, A330P, A33 0R, A330S, A330T, A330V, A330W, A330Y, P331D, P331F, P331H, P331I, P331L, P331M, P331Q, P331R, P331T, P331V, P331W, P331Y, I332A, I332D, I332E, I 332F, I332H, I332K, I332L, I332M, I332N, I332P, I332Q, I332R, I332S, I332T, I332V, I332W, I332Y, E333F, E333H, E333I, E333L, E333M, E333P, E333TE333Y, K334F, K334I, K33, It has one, two, three, four, five, six, or seven or more amino acid substitutions selected from the group consisting of 4L, K334P, K334T, T335D, T335F, T335G, T335H, T335I, T335L, T335M, T335N, T335P, T335R, T335S, T335V, T335W, T335Y, I336E, I336K, I336Y, S337E, S337H, and S337N [where the numbering of residues within the Fc region is the numbering of the EU index by Kabat]. In certain embodiments, the BDCA2 antibody is associated with the following mutations: S239D, S239D / I332E, S239D / I332E / A330L, S239D / I332E / G236A, S298A, A330L Includes one, two, or three of I332E, E333A, and K334A.

[0122] The presence of oligosaccharides (particularly the N-linked oligosaccharide at asparagine 297 in the CH2 domain of IgG1) is important for binding to FcγR and C1q. Effector function is improved by reducing the fucose content of the antibody (see, e.g., US8,163,551). In certain embodiments, the BDCA2 antibody has reduced amino acid substitutions that enhance fucosylation and effector function (e.g., one, two, or three of the following mutations: S298A, E333A, and K334A). Effector function can also be achieved by preparing and expressing the anti-BDCA2 antibodies described herein in the presence of an α-mannosidase I inhibitor (e.g., kifunensin) at inhibitor concentrations of approximately 60–200 ng / mL (e.g., 60 ng / mL, 75 ng / mL, 100 ng / mL, 150 ng / mL). Antibodies expressed in the presence of α-mannosidase I inhibitors primarily contain oligomannose-type glycans and generally increase ADCC activity and affinity for FcγRIIIA, while reducing binding to C1q.

[0123] The anti-BDCA2 antibodies of this disclosure, which have enhanced effector function, include antibodies that have increased binding affinity to one or more Fc receptors (FcRs) compared to the parental anti-BDCA2 antibody or a non-mutant anti-BDCA2 antibody. Therefore, anti-BDCA2 antibodies with increased binding affinity to FcRs include anti-BDCA2 antibodies that exhibit a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or greater increase in binding affinity to one or more Fc receptors compared to the parental anti-BDCA2 antibody or a non-mutant anti-BDCA2 antibody. In some embodiments, the anti-BDCA2 antibody with enhanced effector function binds to FcRs with approximately 10-fold higher affinity compared to the parental antibody or a non-mutant antibody. In other embodiments, the anti-BDCA2 antibody with enhanced effector function binds to FcRs with approximately 15-fold higher affinity or approximately 20-fold higher affinity compared to the parental antibody or a non-mutant antibody. The FcR receptor may be FcγRI(CD64), FcγRII(CD32), and FcγRIII, as well as their isoforms, and one or more of FcεR, FcμR, FcδR, and / or FcαR. In certain embodiments, an anti-BDCA2 antibody with enhanced effector function exhibits a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or greater increase in binding affinity to FcγRIIa.

[0124] To reduce effector function, combinations of different subtype sequence segments (e.g., combinations of IgG2 and IgG4) can be used to result in a greater reduction in binding to the Fcγ receptor than either subtype alone (Armour et al., Eur. J. Immunol., Vol. 29: pp. 2613-1624 (1999); Mol. Immunol., Vol. 40: pp. 585-593 (2003)). In addition, N-linked glycosylation sites can be removed as a means of reducing effector function. In the art, numerous Fc variants are known that alter and / or reduce affinity (and therefore effector function) to some or all Fc receptor subtypes. For example, US 2007 / 0224188;US 2007 / 0148171;US 2007 / 0048300;US 2007 / 0041966;US 2007 / 0009523;US 2007 / 0036799;US 2006 / 0275283;US 2006 / 0235208;US 2006 / 0193856;US 2006 / 0160996;US 2006 / 0134105;US 2006 / 0024298;US 2005 / 0244403;US 2005 / 0233382;US 2005 / 0215768;US 2005 / 0118174;US See 2005 / 0054832;US 2004 / 0228856;US 2004 / 132101;US 2003 / 158389;US 7,183,387;US 6,737,056;US 6,538,124;US 6,528,624;US 6,194,551;US 5,624,821;US 5,648,260.

[0125] The anti-BDCA2 antibodies of the present invention with reduced effector function include antibodies that have reduced binding affinity to one or more Fc receptors (FcRs) compared to the parental anti-BDCA2 antibody or a non-mutant anti-BDCA2 antibody. Therefore, anti-BDCA2 antibodies with reduced binding affinity to FcRs include anti-BDCA2 antibodies that exhibit a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or greater reduction in binding affinity to one or more Fc receptors compared to the parental anti-BDCA2 antibody or a non-mutant anti-BDCA2 antibody. In some embodiments, the anti-BDCA2 antibody with reduced effector function binds to FcRs with about one-tenth the affinity compared to the parental antibody or a non-mutant antibody. In other embodiments, the anti-BDCA2 antibody with reduced effector function binds to FcRs with about one-fifteenth or about one-twentieth the affinity compared to the parental antibody or a non-mutant antibody. FcR receptors may be FcγRI(CD64), FcγRII(CD32), and FcγRIII, as well as their isoforms, and one or more of FcεR, FcμR, FcδR, and / or FcαR. In certain embodiments, anti-BDCA2 antibodies with reduced effector function exhibit a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or greater reduction in binding affinity to FcγRIIa.

[0126] In the complement-antigen complex (CDC), antibody-antigen complexes bind to complement, resulting in the activation of the complement cascade and the formation of membrane attack complexes. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind to their cognitive antigens; therefore, the activation of the complement cascade is partially regulated by the binding affinity of immunoglobulins to the C1q protein. For the complement cascade to be activated, C1q must bind to at least two molecules of IgG1, IgG2, or IgG3 bound to antigenic targets, although in the case of IgM, binding to one molecule is sufficient (Ward and Ghetie, Therapeutic Immunology, Vol. 2: pp. 77-94). (1995), p. 80). To evaluate complement activation, for example, the CDC assay described in Gazzano-Santoro et al., J. Immunol. Methods, vol. 202: p. 163 (1996) can be performed.

[0127] It has been suggested that various residues of the IgG molecule, including Glu318, Lys320, and Lys322 residues on the CH2 domain, amino acid residue 331 located on a turn adjacent to the same β-chain, Lys235 and Gly237 residues located within the lower hinge region, and residues 231-238 located within the N-terminal region of the CH2 domain, are involved in binding to C1q (e.g., Xu et al., J. Immunol., Vol. 150: p. 152A (abstract) (1993); WO94 / 29351; Tao et al., J. Exp. Med., Vol. 178: pp. 661-667). 1993); Brekke et al., Eur. J. Immunol., Vol. 24: pp. 2542-2547 (1994); Burton et al., Nature, Vol. 288: pp. 338-344 (1980); Duncan and Winter, Nature, Vol. 332: pp. 738-740 (1988); Idusogie et al., J Immunol, Vol. 164: pp. 4178-4184 (2000); see US5,648,260 and US5,624,821).

[0128] Anti-BDCA2 antibodies with improved binding to C1q may contain amino acid substitutions at one, two, three, or four of the amino acid positions 326, 327, 333, and 334 of the human IgG Fc region [where the numbering of residues within the IgG Fc region is the numbering of the EU index by Kabat]. In one embodiment, the anti-BDCA2 antibody contains the following amino acid substitution: K326W / E333S, which is known to increase the binding of IgG1 antibodies to C1q (Steurer W. et al., J Immunol., Vol. 155 (No. 3): pp. 1165-1174 (1995)).

[0129] Anti-BDCA2 antibodies with reduced binding to C1q may contain amino acid substitutions at one, two, three, or four of the amino acid positions 270, 322, 329, and 331 of the human IgG Fc region [where the numbering of residues within the IgG Fc region is the numbering of the EU index by Kabat]. As an example in IgG1, two mutations in the COOH terminal region of the CH2 domain of human IgG1 (K322A and P329A) have been shown to prevent activation of the CDC pathway and result in a more than 100-fold reduction in binding to C1q (US6,242,195).

[0130] Therefore, in certain embodiments, the anti-BDCA2 antibody of the present invention shows an increase or decrease in binding to complement proteins compared to a second anti-BDCA2 antibody. In certain embodiments, the anti-BDCA2 antibody of the present invention shows an increase or decrease in binding to C1q of about 1.5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, or about 15 times or more compared to a second anti-BDCA2 antibody.

[0131] Therefore, in certain embodiments of the present invention, one or more of these residues may be modified, substituted, or removed, or one or more amino acid residues may be inserted, to increase or decrease the CDC activity of the anti-BDCA2 antibody provided herein.

[0132] In other certain embodiments, the present invention provides an anti-BDCA2 antibody that exhibits reduced binding to one or more FcR receptors but maintains its ability to bind to complement (e.g., to the same extent as, or in some embodiments, to a smaller extent than, that of a naturally occurring, non-mutant anti-BDCA2 antibody or a parental anti-BDCA2 antibody). Thus, the anti-BDCA2 antibody of the present invention may exhibit reduced binding to FcRs such as FcγRIIa (e.g., FcγRIIa expressed on platelets) while binding to and activating complement. Such an antibody that reduces or does not bind to FcγRIIa (e.g., FcγRIIa expressed on platelets) but is capable of binding to C1q and activating the complement cascade to at least some extent may maintain the desired effector function while reducing the risk of thromboembolic events. In alternative embodiments, the anti-BDCA2 antibody of the present invention exhibits reduced binding to one or more FcRs but maintains its ability to bind to one or more other FcRs. For example, see US2007-0009523, 2006-0194290, 2005-0233382, 2004-0228856, and 2004-0191244, which describe a variety of amino acid modifications that produce antibodies with reduced binding to FcRI, FcRII, and / or FcRIII, as well as amino acid substitutions that result in increased binding to one FcR but decreased binding to another.

[0133] Therefore, the effector function of an anti-BDCA2 antibody with a constant region can be modulated by altering the properties of the constant region, and in particular, the properties of the Fc region. In certain embodiments, an anti-BDCA2 antibody with increased or decreased effector function is compared to a second antibody, which may be a non-mutant, native antibody or parent antibody, that has effector function and includes a native constant region or Fc region that mediates the effector function.

[0134] The Fc region or constant region of the natural sequence contains an amino acid sequence identical to the amino acid sequence of the Fc region or constant chain region found in nature. Preferably, the control molecule used to evaluate relative effector function contains an Fc region of the same type / subtype as the test antibody or mutant antibody. The mutant Fc region or constant region, or a modified Fc region or constant region, contains an amino acid sequence different from the amino acid sequence of the heavy chain region of the natural sequence due to at least one amino acid modification (e.g., post-translational modification, amino acid substitution, amino acid insertion, or amino acid deletion). Therefore, the constant region of the mutant may contain one or more amino acid substitutions, amino acid deletions, or amino acid insertions that result in changes in post-translational modifications, including, for example, changes in glycosylation patterns. The parent antibody or parent Fc region is a mutant with normal effector function, used, for example, to construct a constant region (i.e., Fc) with altered or increased effector function.

[0135] Antibodies with altered (e.g., enhanced) effector function(s) can be produced by manipulating or creating antibodies with the constant region, Fc region, or heavy chain region of the mutant. Recombinant DNA technology and / or cell culture and expression conditions can be used to create antibodies with altered function and / or activity. For example, recombinant DNA technology can be used to manipulate one or more amino acid substitutions, deletions, or insertions within regions that affect antibody function, including effector function (e.g., the Fc region or constant region). Alternatively, alterations in post-translational modifications, such as glycosylation patterns, can be achieved by manipulating host cells, cell culture conditions, and the expression conditions under which the antibody is produced.

[0136] Certain embodiments of the present invention include one or more heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 9, VH CDR2 of SEQ ID NO: 10, and VH CDR3 of SEQ ID NO: 11; or one or more alternative heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 8, VH CDR2 of SEQ ID NO: 10, and VH CDR3 of SEQ ID NO: 11; or one or more alternative heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 89, VH CDR2 of SEQ ID NO: 91, and VH CDR3 of SEQ ID NO: 11; or one or more alternative heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 9, VH CDR2 of SEQ ID NO: 92, and VH CDR3 of SEQ ID NO: 11; or VH CDR1 of SEQ ID NO: 90, VH CDR2 of SEQ ID NO: 93, and VH CDR3 of SEQ ID NO: 94 The present invention relates to an anti-BDCA2 antibody comprising one or more surrogate heavy chain CDR sequences selected from CDR3, further comprising a mutant Fc region that confers increased or decreased effector function compared to the native Fc region or the parental Fc region. In further embodiments, the anti-BDCA2 antibody comprises at least two (or surrogate CDRs) of the CDRs, and in other embodiments, the antibody comprises all three (or surrogate CDRs) of the heavy chain CDR sequences. These anti-BDCA2 antibodies i) inhibit the secretion of type I and / or type III interferons from plasmacytoid dendritic cells in addition to other cytokines and chemokines, and / or (ii) induce or enhance plasmacytoid dendritic cell depletion in vitro.

[0137] Other embodiments of the present invention relate to an anti-BDCA2 antibody comprising one or more light chain CDR sequences selected from VL CDR1 of SEQ ID NO: 5, VL CDR2 of SEQ ID NO: 6, and VL CDR3 of SEQ ID NO: 7; or one or more alternative light chain CDR sequences selected from VL CDR1 of SEQ ID NO: 95, VL CDR2 of SEQ ID NO: 96, and VL CDR3 of SEQ ID NO: 97, further comprising a variant Fc region that confers increased or decreased effector function compared to the native Fc region or the parental Fc region. In further embodiments, the anti-BDCA2 antibody comprises at least two (or alternative CDRs) of the light chain CDRs, and in other embodiments, the antibody comprises all three (or alternative CDRs) of the light chain CDR sequences. These anti-BDCA2 antibodies (i) inhibit the secretion of type I and / or type III interferon from plasmacytoid dendritic cells, in addition to other cytokines and chemokines, and / or (ii) induce or enhance plasmacytoid dendritic cell depletion in vitro.

[0138] In a further embodiment of the present invention, an anti-BDCA2 antibody with increased or decreased effector function comprises all three light chain CDR sequences, or a light chain CDR substituted for SEQ ID NO: 3, and all three heavy chain CDR sequences, or a heavy chain CDR substituted for SEQ ID NO: 4.

[0139] In another embodiment, the present invention relates to an anti-BDCA2 antibody comprising a VL sequence including SEQ ID NO: 23, further comprising a mutant Fc region that confers reduced effector function compared to the native Fc region or the parental Fc region. In yet another embodiment, the present invention relates to an anti-BDCA2 antibody comprising a VH sequence including SEQ ID NO: 24, further comprising a mutant Fc region that confers reduced effector function compared to the native Fc region or the parental Fc region.

[0140] In the art, methods for generating variants of the aforementioned anti-BDCA2 antibodies, which are any of the variants and contain amino acid substitutions, are well-known. These methods include, but are not limited to, site-specific (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis on prepared DNA molecules encoding the antibody or at least the constant region of the antibody. Site-specific mutagenesis is well-known in the art (see, for example, Carter et al., Nucleic Acids Res., Vol. 13: 4431-4443 (1985), and Kunkel et al., Proc. Natl. Acad. Sci. USA, Vol. 82: 488 (1987)). PCR mutagenesis is also suitable for generating variants of the amino acid sequence of the starting polypeptide. See Higuchi, PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res., Vol. 17: 72 3-733 (1989). Another method for preparing sequence variants, cassette mutagenesis, is based on the techniques described in Wells et al., Gene, Vol. 34: 315-323 (1985).

[0141] Anti-BDCA2 antibodies with altered glycosylation Different glycoforms can deeply affect the properties of therapeutic agents, including pharmacokinetics, pharmacodynamics, receptor interactions, and tissue-specific targeting (Graddis et al., 2002, Curr Pharm Biotechnol., Vol. 3: 285-297). In particular, in the case of antibodies, the oligosaccharide structure affects the ​In addition to effector functions (e.g., inducing CDC, binding to complement complex C1, and binding to FcγR receptors which contribute to modulating the ADCC pathway), characteristics related to protease resistance, the serum half-life of antibodies mediated by the FcRn receptor, phagocytosis, and antibody feedback can also be affected (Nose and Wigzell, 1983; Leatherbarrow and Dwek, 1983; Leatherbarrow et al., 1985; Walker et al., 1989 ; Carter et al., 1992, PNAS, Vol. 89: 4285 - 4289).

[0142] Therefore, another means of modulating the effector function of an antibody involves changing the glycosylation of the antibody constant region. Changes in glycosylation include, for example, a decrease or increase in the number of glycosylated residues, a change in the pattern or position of glycosylated residues, as well as a change in the sugar structure(s). The oligosaccharides found on human IgG affect the degree of their effector functions (Raju, T.S., BioProcess International, April 2003, pp. 44 - 53), and the microheterogeneity of human IgG oligosaccharides can affect biological functions such as CDC and ADCC, binding to various Fc receptors, and binding to C1q protein (Wright A. and Morrison SL., TIBTECH, 1997, Vol. 15, pp. 26 - 32; Shields et al., J Biol Chem., 2001, Vol. 276(9): 6591 ~604; Shields et al., J Biol Chem., 2002, Vol. 277(30): 2673 pp. 3-40; Shinkawa et al., J Biol Chem., 2003, Vol. 278 (No. 5): pp. 3466-3473; Umana et al., Nat Biotechnol., February 1999, Vol. 17 (No. 2): pp. 176-1780). For example, the ability of IgG to bind to C1q and activate the complement cascade may depend on the presence, absence, or modification of the carbohydrate moiety located between the two CH2 domains (usually anchored to Asn297) (Ward and Ghetie, Therapeutic Immunology, Vol. 2: pp. 77-94 (1995)).

[0143] Glycosylation sites within Fc-containing polypeptides, such as antibodies like IgG antibodies, can be identified using standard techniques. Identification of glycosylation sites may be experimental, or based on sequence analysis or data modeling. Consensus motifs, i.e., amino acid sequences recognized by various glycosyltransferases, are described. For example, the consensus motif for N-linked glycosylation motifs is often NXT or NXS [where X can be any amino acid except proline]. Several algorithms for locating potential glycosylation motifs are also described. Therefore, to identify potential glycosylation sites within antibodies or Fc-containing fragments, antibody sequences are tested using publicly available databases, such as the website provided by the Center for Biological Sequence Analysis (see NetNGlyc service for predicting N-linked glycosylation sites and NetOGlyc service for predicting O-linked glycosylation sites).

[0144] In vivo studies have confirmed the reduced effector function of nonglycosyl antibodies. For example, nonglycosyl anti-CD8 antibodies are unable to deplete CD8-containing cells in mice (Isaacs, 1992, J. Immunol., Vol. 148: p. 3062), and nonglycosyl anti-CD3 antibodies do not induce cytokine release syndrome in mice or humans (Boyd, 1995, ibid.; Friend, 1999, Transplantation, Vol. 68: 1 (Page 632). Even in the non-glycosylated form of the BDCA2 antibody, the effector function is reduced.

[0145] Importantly, while the removal of glycans within the CH2 domain appears to have a significant effect on effector function, other functional and physical properties of the antibody remain unchanged. In particular, glycan removal has been shown to have little to no effect on serum half-life and antigen binding (Nose, 1983, cited above; Tao, 1989, cited above; Dorai, 1991, cited above; Hand, 1992, cited above; Hobbs, 1992, Mol. Immunol., Vol. 29: p. 949).

[0146] The anti-BDCA2 antibody of the present invention is modified or altered to provide multiple effector functions. It can induce an increase or decrease (compared to a second BDCA2-specific antibody). Methods for altering the glycosylation site of an antibody are described, for example, in US6,350,861 and US5,714,350, WO05 / 18572 and WO05 / 03175, and these methods can be used to produce anti-BDCA2 antibodies of the present invention that have altered glycosylation, reduced glycosylation, or no glycosylation.

[0147] Adaptation The anti-BDCA2 antibodies described herein can be used to treat or prevent a variety of immune disorders, including inflammatory and autoimmune disorders. Anti-BDCA2 antibodies are useful in treating or preventing such disorders because they at least inactivate or deplete pDCs and / or inhibit inflammatory cytokines and chemokines produced by pDCs, and / or downmodulate CD32a and / or inhibit stimulation of pDCs by immune complexes, and / or downmodulate or cause loss of CD62L. The anti-BDCA2 antibodies of this disclosure can be combined with antimalarial agents (e.g., HCQ) to improve the therapeutic effect in treating inflammatory and autoimmune disorders. Anti-BDCA2 antibodies can also be used to reduce the levels of cytokines and chemokines, such as type I interferon, type III interferon, IL-6, TNF-α, MIP1-α and MIP1-β, CCL5, and IP-10. Type I IFNs constitute a multi-member cytokine family, including 13 IFN-α subtypes, IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-δ, and IFN-τ (Theofilopoulos, Annu. Rev. Immunol., Vol. 23: pp. 307-36 (2005)). Type III interferons consist of three IFN-λ molecules called IFN-λ1, IFN-λ2, and IFN-λ3 (also referred to as IL29, IL28A, and IL28B, respectively). By depleting and / or reducing pDC function, the anti-BDCA2 antibodies described herein provide a more robust treatment approach than treatments that attempt to reduce specific IFN subtypes with neutralizing antibodies. In addition, pDC-focused treatment with anti-BDCA2 antibodies is more selective and potentially safer than general blockade of the IFN response. For example, the anti-BDCA2 antibodies described herein effectively eliminate type I IFN derived from pDCs while maintaining other sources of IFN that may be necessary in the event of a viral infection.

[0148] The term "treating" means administering any of the compositions described herein in an amount, form, and / or manner effective in improving, or preventing the progression or exacerbation of, a condition, symptom, or parameter associated with a condition, symptom, or disorder, to a statistically significant extent or to an extent detectable by a person skilled in the art.

[0149] Diseases or conditions that can be treated with the anti-BDCA2 antibodies described herein include, for example, systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), maculoplasty, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, and type 1 diabetes mellitus.

[0150] SLE is a chronic autoimmune disease in which multiple organs are damaged by immune complexes and tissue-binding autoantibodies (see Guidelines for Referral and Management of Systemic Lupus Erythematosus in Adults, Arthritis & Rheumatism, Vol. 42 (No. 9): pp. 1785-1795 (1999)). In SLE, autoantibodies are present and can precede the onset of clinical disease (Arbuckle et al., N. Engl. J. Med., Vol. 349 (No. 16): (pp. 1526-1533 (2003)). Internal translocation of autoantibody-containing immune complexes via Fc receptors leads to the production of type I interferon, which in turn promotes loss of tolerance and perpetuates a vicious cycle of autoimmunity (Means et al., Ann NY Acad Sci., Vol. 1062: pp. 242-251 (2005)). SLE is heterogeneous in terms of its clinical symptoms, course, prognosis, and genetic characteristics. African Americans share an increased risk of SLE and are often more severe than Caucasian patients. Early on, complement deficiency was recognized as a risk factor for developing SLE. More recently, it has been described that genetic polymorphisms associated with the type I interferon pathway confer susceptibility. For example, anti-double-stranded DNA autoantibodies and anti-Ro autoantibodies were associated with certain haplotypes of the transcription factor interferon regulator 5 (IRF5). Haplotypes also predicted high serum IFN-α levels in SLE patients (Niewold et al., Ann. Rheum. Dis., Vol. 71 (No. 3):4). pp. 63-68 (2012). High IFN-α levels correlate with an increased degree of multi-organ disease in SLE patients (Bengtsson et al., Lupus, Vol. 9 (No. 9): pp. 664-671 (2000)). Furthermore, so-called "interferon signatures" appear to be prominent in SLE. Interferon signatures represent the mRNA expression pattern of interferon-inducible genes. Type I interferon signatures have been found in the majority of SLE patients and are associated with increased disease activity (Baechler et al., Proc. Natl. Acad. Sci USA, Vol. 100 (No. 5): pp. 2610-265 (2003)). Currently, I FN-α monoclonal antibodies are in clinical trials, and Phase 1 results for cifalimumab and lontalizumab have confirmed a dose-dependent reduction of type I IFN signature in whole blood of SLE patients (McBride et al., Arthritis Rheum., Vol. 64 (No. 11): pp. 3666-3676 (2012); Yao et al., Arthritis Rheum., (No. 6): pp. 1785-1796 (2009)). Validated indices have been developed to assess disease activity and disease severity (e.g., moderate, severe) (e.g., Gladman, Prognosis and treatment of systemic lupus erythematosus, Curr. Opin.). See Rheumatol., Vol. 8: pp. 430-437 (1996); Kalunan et al., Definition, classification, activity and damage indices, Dubois' lupus eyrthematosus, 5th edition, Baltimore, Williams and Wilkins, pp. 19-30 (1997). ).

[0151] Systemic sclerosis, or systemic scleroderma, is a systemic connective tissue disease that is a subtype of systemic autoimmune disease or scleroderma. Systemic sclerosis is characterized by collagen deposition in the skin, and less commonly, collagen deposition in the kidneys, heart, lungs, and stomach. The female-to-male ratio for this disease is 4:1. The peak age of onset is between 30 and 50 years of age.

[0152] Psoriasis is an autoimmune disease that affects the skin. Psoriasis occurs when the immune system mistakenly identifies skin cells as pathogens, sending false signals that accelerate the growth cycle of skin cells. Psoriasis is associated with an increased risk of stroke and can be improved by treating high blood lipid levels. There are five types of psoriasis: plaque psoriasis, guttate psoriasis, reverse psoriasis, pustular psoriasis, and erythrodermic psoriasis. The most common form, plaque psoriasis, is generally seen as scaly patches of red and white in color appearing on the first layer of the epidermis. However, some patients do not show any skin signs or symptoms.

[0153] Rheumatoid arthritis is a chronic inflammatory disorder that affects many tissues and organs, but primarily attacks the flexible joints. The process involves an inflammatory response of the periarticular capsule, excess synovial fluid, and the development of fibrous tissue (pannus) within the synovium, secondary to swelling of synovial cells. The pathogenesis of the disease often leads to destruction of articular cartilage and joint rigidity. Rheumatoid arthritis can also cause diffuse inflammation in the lungs, the membranes surrounding the heart (pericardium), the pleura, and the whites of the eyes (sclera), and can also cause nodular lesions, which are the most common type of lesion in the subcutaneous tissue. The cause of rheumatoid arthritis is unknown, but autoimmunity plays a central role in both its chronicity and progression, and RA is considered a systemic autoimmune disease. Overexpression of TNFα and other pro-inflammatory cytokines has been observed in patients with arthritis (Feldmann et al., Prog Growth Factor Res., Vol. 4: pp. 247-255 (1992)). Furthermore, transgenic animals that overexpress human TNFα develop erosive polyarthritis with many disease-related features (Keffer et al., EMBO J., Vol. 10 (No. 13): pp. 4025-4031 (1991)). Analgesics and anti-inflammatory drugs, including steroids, are used to suppress symptoms, but disease-modifying antirheumatic drugs (DMARDs) are needed to inhibit or halt the underlying immune processes and prevent long-term damage. More recently, anti-TNFα antibody therapy (rituximab) has been used to manage the disease (Edwards et al., N. Engl. J. Med., Vol. 350 (No. 25): pp. 2572-2581 (2004)).

[0154] Inflammatory bowel disease (IBD) is an inflammatory condition affecting a group of parts of the colon and small intestine. The main types of IBD are Crohn's disease and ulcerative colitis (UC). The main difference between Crohn's disease and UC lies in the location and nature of the inflammatory changes: Crohn's disease can affect any part of the digestive tract from the mouth to the anus (stepping stones), but most cases begin in the terminal ileum, whereas UC is limited to the colon and rectum. Depending on the level of severity, IBD may require immunosuppression, such as prednisone, TNF inhibitors, azathioprine (Imuran), methotrexate, or 6-mercaptopurine, to control symptoms. More generally, treatment for IBD requires a form of mesalazine.

[0155] Dermatomyositis (DM) is a type of autoimmune connective tissue disorder associated with polymyositis (PM), characterized by inflammation of the muscles and skin. While DM most commonly affects the skin and muscles, it is a systemic disorder that can also affect the joints, esophagus, and lungs, and, less commonly, the heart.

[0156] Polymyositis (PM) ("inflammation of many muscles") is a type of chronic muscle inflammation (inflammatory myopathy) associated with dermatomyositis and inclusion body myositis.

[0157] Type 1 diabetes is a form of diabetes resulting from the autoimmune destruction of insulin-producing pancreatic beta cells. The subsequent insulin deficiency leads to increased glucose levels in the blood and urine. Classic symptoms include polyuria, polythirst, polyphagia, and weight loss.

[0158] Other diseases suitable for treatment with anti-BDCA2 antibodies described herein include asthma, Behçet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulosis, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, bullous pemphigoid, pemphigus, erythematous pemphigus, foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), macular sclerosis (regional sclerosis), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjögren's syndrome, and ulcerative colitis.

[0159] Subjects who are at risk of one of these disorders, have been diagnosed with one of these disorders, or have one of these disorders may be administered an anti-BDCA2 antibody in a dose that produces an overall therapeutic effect for a duration that produces an overall therapeutic effect. The anti-BDCA2 antibody may be administered alone (monotherapy) or in combination with other drugs (combination therapy). In one embodiment, the drug for use in combination therapy with the anti-BDCA2 antibody described herein is an antimalarial agent. In one embodiment, the drug for use in combination therapy with the anti-BDCA2 antibody described herein is a TLR7 and / or TLR9 signaling inhibitor. In another embodiment, the drug for use in combination therapy with the anti-BDCA2 antibody described herein is a corticosteroid. In certain embodiments, the agents for use in combination therapy with anti-BDCA2 antibodies described herein are antimalarial drugs and / or kinase inhibitors (e.g., BTK inhibitors (e.g., ibrutinib (PCI-32765), AVI-292, ONO-WG-307), JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, Tyk2 inhibitors). In specific embodiments, the agent for use in combination therapy with anti-BDCA2 antibodies described herein is hydroxychloroquine. The dosage and number of administrations for combination therapy may be, for example, a dosage and number of administrations that produce an additive or synergistic therapeutic effect. Furthermore, the administration of anti-BDCA2 antibodies (with or without the second agent) may be used as primary treatment, e.g., first-line treatment, or as secondary treatment, e.g., for subjects who have not responded well to previously administered treatments (i.e., treatments other than treatment with anti-BDCA2 antibodies).In some embodiments, the combination therapy comprises the use of an anti-BDCA2 antibody and one or more of the following agents: glucocorticoids, NSAIDs, prednisone, hydroxychloroquine, chloroquine, amodiaquine, pyrimethamine, proguanil, mefloquine, dapsone, primaquine, methotrexate, mycophenolate mofetil, azathioprine, thalidomide, cyclophosphamide, cyclosporin A, rapamycin, prostacyclin, phosphodiesterase inhibitors, endothelin antagonists, statins, ACE inhibitors, and calcium channel blockers. In other embodiments, the combination therapy comprises the use of an anti-BDCA2 antibody and any one or more of sulfasalazine, doxycycline, minocycline, penicillamine, tofacitinib, and leflunomide.

[0160] Pharmaceutical composition The anti-BDCA2 antibodies or antigen-binding fragments thereof described herein can be formulated, for example, as a pharmaceutical composition for administration to a subject for treating a disorder described herein. The pharmaceutical composition typically comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include pharmaceutically acceptable salts, such as, for example, acid addition salts or base addition salts (see, e.g., Berge, S.M. et al. (1977), J. Pharm. Sci., 66:1-19).

[0161] Pharmaceutical formulation is a well-established art, for example, Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms Further information is found in *and Drug Delivery Systems*, 7th edition, Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and in *Handbook of Pharmaceutical Excipients*, American Pharmaceutical Association, 3rd edition (2000) (ISBN: 091733096X), edited by Kibbe.

[0162] Pharmaceutical compositions can take various forms. These include liquid dosage forms, semi-solid dosage forms, and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form may depend on the intended method of administration and therapeutic application. Compositions for pharmaceuticals described herein are typically in the form of injectable or infusion solutions.

[0163] In one embodiment, the anti-BDCA2 antibody described herein is formulated with excipient materials such as sodium chloride, sodium citrate, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, Tween-80, and stabilizers. The anti-BDCA2 antibody described herein can be provided, for example, in a buffer solution of appropriate concentration and stored at 2–8°C. In some other embodiments, the pH of the composition is between approximately 5.8 and 6.6 (e.g., 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6).

[0164] The pharmaceutical composition may also include agents that reduce the aggregation of the BDCA2 antibody or its antigen-binding fragment when formulated. Examples of aggregation reducers include one or more amino acids selected from the group consisting of methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. These amino acids can be added to the formulation to concentrations ranging from about 0.5 mM to about 145 mM (e.g., 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM). The pharmaceutical composition may also include sugars (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and / or isotonic modifiers (e.g., sodium chloride, mannitol, or sorbitol) and / or surfactants (e.g., polysorbate-20 or polysorbate-80).

[0165] Such compositions can be administered parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular). In one embodiment, the composition of an anti-BDCA2 antibody or its antigen-binding fragment is administered subcutaneously. In another embodiment, the composition of an anti-BDCA2 antibody or its antigen-binding fragment is administered intravenously. As used herein, the terms “parenteral administration” and “parenterally administered” typically refer to methods of administration other than intestinal and local administration, usually by injection, and without limitation include, but are not limited to, intravenous injection and infusion, intramuscular injection and infusion, intra-arterial injection and infusion, intrathecal injection and infusion, intracapsular injection and infusion, intraorbital injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intraperitoneal injection and infusion, transtracheal injection and infusion, subcutaneous injection and infusion, subepidermal injection and infusion, intra-articular injection and infusion, subcapsular injection and infusion, subarachnoid injection and infusion, intraspinal injection and infusion, epidural injection and infusion, and intrasternal injection and infusion.

[0166] The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injection solutions can be prepared by incorporating the drugs described herein in the required amounts and as required, along with one or a combination thereof of the components listed above, in a suitable solvent, and then subjecting to filtration sterilization. Generally, dispersions are prepared by incorporating the drugs described herein into a sterile medium containing a base dispersion medium and other required components derived from the components listed above. For sterile powders for preparing sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, where the powder, with any further desired components added to the drugs described herein, is obtained from a pre-sterilized filtered solution. Proper fluidity of solutions can be maintained by using surfactants, for example, by using a coating such as lecithin, or, in the case of dispersions, by maintaining the required particle size. Sustained absorption of injection compositions can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0167] In certain embodiments, an anti-BDCA2 antibody or its antigen-binding fragment can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation comprising an implant and a microencapsulated delivery system. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or publicly known. For example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel See Dekker, Inc., New York (1978).

[0168] In one embodiment, the pharmaceutical formulation contains an anti-BDCA2 antibody or its antigen-binding fragment (e.g., BIIB059) at a concentration of approximately 0.5 mg / mL to 300 mg / mL (e.g., 1 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 250 mg / mL) and is formulated together with sodium citrate, sodium chloride, and optionally Tween-80 (0.01 to 0.1%, e.g., 0.03%, 0.05%, or 0.7%). The pH of the formulation may be between 5.5 and 7.5 (for example, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.3).

[0169] Administration Anti-BDCA2 antibodies or their antigen-binding fragments can be administered to subjects, such as human subjects, in a variety of ways. In many applications, the route of administration is one of the following: intravenous (IV) injection or IV infusion, subcutaneous (SC) injection, intraperitoneal (IP) injection, or intramuscular injection. Intra-articular delivery is also possible. Other parenteral administration methods can also be used. Examples of such methods include intra-arterial injection, intrathecal injection, intracapsular injection, intraorbital injection, intracardiac injection, intradermal injection, transtracheal injection, subepidermal injection, intra-articular injection, subcapsular injection, subarachnoid injection, intraspinal injection, and epidural injection, and intrasternal injection. In some cases, administration may be oral.

[0170] The route and / or method of administration of the antibody or its antigen-binding fragment may also be adjusted on an individual basis, for example, by monitoring the subject, for example, by using tomography to visualize the tumor.

[0171] The antibody or its antigen-binding fragment may be administered in a fixed dose or in doses in mg / kg units. The dose may also be selected to reduce or avoid antibody production against the anti-BDCA2 antibody. The administration regimen is adjusted to produce the desired response, e.g., a therapeutic response or a combination therapeutic effect. Generally, doses of the anti-BDCA2 antibody (and, if necessary, a second agent) can be used to administer the agent to the subject in a bioavailable amount. For example, doses in the range of 0.1–100 mg / kg, 0.5–100 mg / kg, 1 mg / kg–100 mg / kg, 0.5–20 mg / kg, 0.1–10 mg / kg, or 1–10 mg / kg can be administered. Other doses may also be used. In a specific embodiment, the antibody is administered to a subject requiring treatment with an anti-BDCA2 antibody at doses of 2 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 35 mg / kg, or 40 mg / kg.

[0172] The composition may contain an anti-BDCA2 antibody or its antigen-binding fragment in an amount of approximately 1 mg / mL to 100 mg / mL, or approximately 10 mg / mL to 100 mg / mL, or approximately 50 to 250 mg / mL, or approximately 100 to 150 mg / mL, or approximately 100 to 250 mg / mL.

[0173] In certain embodiments, the anti-BDCA2 antibody or its antigen-binding fragment in the composition is primarily in monomeric form, for example, at least about 90%, 92%, 94%, 96%, 98%, 98.5%, or 99% in monomeric form. The aggregates contained in certain compositions of anti-BDCA2 antibody or its antigen-binding fragment, for example, detected by UV at 280 nm, may be less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%. The fragments contained in certain compositions of anti-BDCA2 antibody or its antigen-binding fragment, for example, detected by UV at 280 nm, may be less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or 0.1%.

[0174] As used herein, the term "dose unit form" or "fixed dose" refers to a physically distinct unit suitable as a unit dose for the subject being treated. Each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, associated with the required pharmaceutical carrier and, if necessary, with other drugs. A single dose or multiple doses may be administered. Alternatively, or in addition, antibodies may be administered via serial infusion.

[0175] A certain dose of anti-BDCA2 antibody or its antigen-binding fragment may be administered at cyclical intervals over a sufficient period (treatment course) to include, for example, at least two, three, five, or ten or more doses, for example, once or twice daily, or about one to four times per week, or preferably weekly, every other week (every two weeks), every three weeks, or monthly, for example, over a period of about one to twelve weeks, preferably two to eight weeks, more preferably three to seven weeks, and even more preferably over four, five, or six weeks. Factors that may influence the dose and timing required to effectively treat the subject include, for example, the severity of the disease or disorder, the formulation, the route of delivery, previous treatments, the subject's overall health and / or age, and other pre-existing diseases. Furthermore, treatment of the subject with a therapeutically effective amount of the compound may consist of a single treatment, or preferably a series of treatments.

[0176] If a subject is at risk of developing an immunodeficiency as described herein, antibodies may be administered, for example, as a prophylactic measure, before the full onset of the immunodeficiency. The duration of such prophylactic measures may be a single dose of antibodies or a sustained treatment (e.g., multiple doses). For example, a subject at risk of or predisposed to the disorder may be treated with antibodies for several days, weeks, months, or even years to prevent the disorder from developing or becoming fulminant.

[0177] A pharmaceutical composition may contain a "therapeutic dose" of any of the agents described herein. Such a dose may be determined based on the effect of the administered agent, or, if multiple agents are used, based on the combined effect of the agents. The therapeutic dose may also vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the compound's ability to induce a desired response in the individual, such as improvement in at least one disorder parameter or improvement in at least one symptom of the disorder. The therapeutic dose is also the amount in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects.

[0178] In certain embodiments, the anti-BDCA2 antibody or its antigen-binding fragment is administered subcutaneously at concentrations ranging from approximately 1 mg / mL to approximately 300 mg / mL (e.g., 1 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 250 mg / mL). In one embodiment, the anti-BDCA2 antibody or its antigen-binding fragment is administered subcutaneously at a concentration of 50 mg / mL. In another embodiment, the anti-BDCA2 antibody or its antigen-binding fragment is administered intravenously at concentrations ranging from approximately 1 mg / mL to approximately 300 mg / mL. In a specific embodiment, the anti-BDCA2 antibody or its antigen-binding fragment is administered intravenously at a concentration of 50 mg / mL.

[0179] Devices and kits for treatment A pharmaceutical composition containing an anti-BDCA2 antibody or its antigen-binding fragment can be administered using a medical device. The device may be designed to be portable, room temperature storable, and easy to use in emergency situations, for example, by an untrained subject or by emergency personnel in a location away from medical facilities and other medical equipment. The device may include, for example, one or more housings for storing a pharmaceutical preparation containing an anti-BDCA2 antibody or its antigen-binding fragment, and may be configured to deliver one or more unit doses of the antibody. The device may also be configured to administer a second agent, such as a chemotherapeutic agent, as a single pharmaceutical composition also containing an anti-BDCA2 antibody or its antigen-binding fragment, or as two separate pharmaceutical compositions.

[0180] The pharmaceutical composition may be administered by syringe. The pharmaceutical composition may also be administered by needle-free subcutaneous injection devices, such as those disclosed in US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include US4,487,603 disclosing an implantable microinfusion pump for administering pharmaceuticals at a controlled rate; US4,486,194 disclosing a therapeutic device for administering pharmaceuticals via the skin; US4,447,233 disclosing a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate; US4,447,224 disclosing a variable-flow implantable infusion device for continuous drug delivery; US4,439,196 disclosing an osmotic drug delivery system with multi-chamber compartments; and US4,475,196 disclosing an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.

[0181] Anti-BDCA2 antibodies or their antigen-binding fragments may be provided in a kit. In one embodiment, the kit includes (a) a container containing a composition comprising an anti-BDCA2 antibody, and optionally (b) informational material. The informational material may be descriptive, directive, promotional, or other material relating to the use of the agent for the methods and / or therapeutic benefits described herein.

[0182] In one embodiment, the kit also includes a second agent for treating the disorders described herein (e.g., BTK inhibitors, antimalarial agents, glucocorticoids, NSAIDs, prednisone, hydroxychloroquine, amodiaquine, pyrimethamine, proguanil, sulfonamides, mefloquine, atovaquone, primaquine, artemisinin and derivatives, halofantrine, doxycycline, clindamycin, methotrexate, mycophenolate mofetil, azathioprine, cyclophosphamide, sulfasalazine, or leflunomide). For example, the kit includes a first container containing a composition comprising an anti-BDCA2 antibody and a second container containing the second agent.

[0183] The informational materials of the kit are not limited in form. In one embodiment, the informational materials may include information about the production of the compound, the molecular weight, concentration, and expiration date of the compound, and information about the batch or place of manufacture. In one embodiment, the informational materials relate to a method of treating a subject having or at risk of having an immunodeficiency as described herein by administering, for example, an anti-BDCA2 antibody or its antigen-binding fragment in an appropriate dose, dosage form, or method of administration (e.g., the dose, dosage form, or method of administration described herein). The information may be provided in various formats, including printed text, computer-readable materials, video recordings, or audio recordings, or information providing links or addresses to substantial materials on the internet, for example.

[0184] In addition to the antibody, the kit composition may also include other components such as solvents or buffers, stabilizers, or preservatives. The antibody can be supplied in any form, for example, preferably substantially pure and / or sterile liquid, dry, or lyophilized form. When the drug is supplied in liquid solution, the liquid solution is preferably an aqueous solution. When the drug is supplied in dry form, reconstitution is generally by adding a suitable solvent. The solvent, for example, sterile water or sterile buffer, may also be provided in the kit as needed.

[0185] A kit may comprise one or more containers for one or more compositions containing a drug. In some embodiments, the kit comprises separate containers, dividers, or compartments for the compositions and informational materials. For example, the compositions may be contained in a bottle, vial, or syringe, and the informational materials may be contained in a plastic sleeve or packet. In other embodiments, the individual elements of the kit are contained in a single, undivided container. For example, the compositions may be contained in a bottle, vial, or syringe with informational materials attached in the form of labels. In some embodiments, the kit comprises a plurality of individual containers (e.g., packs) each containing one or more unit dosage forms of the drug (e.g., dosage forms described herein). The containers may contain combined unit doses, for example, units containing both an anti-BDCA2 antibody or its antigen-binding fragment and a second drug, for example, in a desired ratio. For example, the kit comprises a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, each containing a single combined unit dose. The kit container may be airtight, waterproof (e.g., impermeable to changes or evaporation of moisture), and / or light-shielding.

[0186] The kit may include, if necessary, a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device may be provided pre-filled with one or both of the drugs, or it may be empty but suitable for filling.

[0187] Diagnostic use Anti-BDCA2 antibodies or their antigen-binding fragments can be used in diagnostic methods to detect the presence of BDCA2 in vitro (e.g., in biological samples such as tissue or biopsy) or in vivo (e.g., in vivo imaging of a subject). For example, a human anti-BDCA2 antibody or an effective human anti-BDCA2 antibody can be administered to a subject to detect BDCA2 in the subject. For example, the antibody can be labeled with a label detectable by MRI or a radioactive label. The subject can be evaluated using means for detecting the detectable label. For example, the subject can be scanned to evaluate the localization of the antibody within the subject. For example, the subject can be imaged by NMR or other tomography means.

[0188] Examples of labels useful for diagnostic imaging include: 131 I, 111 In, 123 I, 99m Tc, 32 P, 33 P, 125 I, 3 H, 14 C, and 188These include radiolabeling such as Rh, fluorescent labeling such as fluorescein and rhodamine, nuclear magnetic resonance activity labeling, positron-emitting isotopes detectable by positron emission tomography ("PET") scanners, chemiluminescent substances such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiators, such as isotopes detectable by short-range detection probes, can also be used. Protein ligands can be labeled with such reagents using known techniques. For example, see Wensel and Meares (1983), Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York; and Colcher et al. (1986), Meth. Enzymol., Vol. 121: pp. 802-816, for techniques relating to the radiolabeling of antibodies.

[0189] The subject can be “imaged” in vivo using known techniques, such as radionuclide scanning using a gamma camera or radiation tomography. See, for example, AR Bradwell et al., “Developments in Antibody Imaging,” Monoclonal Antibodies for Cancer Detection and Therapy, RW Baldwin et al. (eds.), pp. 65-85 (Academic Press, 1985). Alternatively, radiolabeling (e.g., 11 C, 18 F, 15 O, and 13 If N) emits positrons, a transaxial tomography (POSIRON emission) technique, such as Pet VI, is used at Brookhaven National Laboratory. A transaxial tomography scanner can also be used.

[0190] Magnetic resonance imaging (MRI) uses NMR to visualize the internal features of living subjects and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds, which offers clear advantages. Several MRI methods are summarized in EP0502814A. Generally, images are generated using differences in the relaxation time constants T1 and T2 of water protons in different environments. However, these differences may be insufficient to produce sharp, high-resolution images.

[0191] These differences in relaxation time constants can be enhanced by contrast agents. Examples of such contrast agents include several magnetic agents, paramagnetic agents (which mainly alter T1), and ferromagnetic or superparamagnetic agents (which mainly alter T2 response). Chelating agents (e.g., EDTA chelators, DTPA chelators, and NTA chelators) can be used to enhance some paramagnetic materials (e.g., Fe 3+ Mn 2+ , Gd 3+ ) can be bonded (and its toxicity reduced). Other agents may be in the form of particles with a diameter of 10 μm to less than approximately 10 nm, for example. The particles may have ferromagnetic, antiferromagnetic, or superparamagnetic properties. The particles may include, for example, magnetite (Fe3O4), γ-Fe2O3, ferrite, and other magnetic mineral compounds of transition elements. Magnetic particles may include one or more magnetic crystals with non-magnetic materials and one or more magnetic crystals without non-magnetic materials. The non-magnetic materials may include synthetic polymers or natural polymers (e.g., Sepharose, dextran, dextrin, starch, etc.).

[0192] The anti-BDCA2 antibody or its antigen-binding fragment also contains (i) substantially all of the naturally abundant fluorine atoms, 19(ii) Fluorine is an isotope, and therefore virtually all fluorine-containing compounds are NMR active; (ii) many chemically active polyfluorinated compounds, such as trifluoroacetic anhydride, are commercially available at relatively low cost; and (iii) many fluorinated compounds, such as perfluorinated polyethers used to carry oxygen as hemoglobin substitutes, are found to be medically acceptable for use in humans, insofar as they are NMR active. 19 It can also be labeled with a fluorine atom or a labeling group containing multiple such atoms. After such incubation time, whole-body MRI is performed using an instrument such as one of the instruments described by Pykett (1982), Scientific American, Vol. 246: pp. 78-88 to localize and image the distribution of BDCA2.

[0193] In another aspect, the disclosure provides a method for detecting the presence of BDCA2 in vitro in a sample (e.g., a biological sample such as serum, plasma, tissue, or biopsy). The method can also be used to diagnose disorders, such as autoimmune disorders (e.g., SLE), or to detect pDC levels in a sample. The method comprises (i) contacting a sample or a control sample with an anti-BDCA2 antibody, and (ii) evaluating the sample for the presence of BDCA2, for example, by detecting the formation of a complex between the anti-BDCA2 antibody and BDCA2, or by detecting the presence of the antibody or BDCA2. For example, the antibody can be immobilized on a support, for example, to detect the retention of the antigen on the support and / or the reverse. The antibody used can be labeled, for example, with a fluorophore. A control sample may be included. A positive control may be a sample known to have the disease or disorder being evaluated, and a negative control may be a sample derived from a subject who does not have the disease or disorder being evaluated. A statistically significant change in complex formation in a sample compared to a control sample may indicate the presence of BDCA2 in the sample. Generally, anti-BDCA2 antibodies can be used in applications including fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and cell sorting (e.g., fluorescence-activated cell sorting). In certain embodiments, the anti-BDCA2 antibody is BIIB059 or Dendritics clone 124B3.13. In some embodiments, the method further includes the step of immunostaining a tissue sample with an anti-CD123 antibody. The tissue sample may be, for example, a skin biopsy from a human patient with an autoimmune condition, e.g., SLE.

[0194] The following are embodiments of the present invention. These should not be considered to limit the scope of the present invention in any way. [Examples]

[0195] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are merely illustrative and not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without exercising their inventive ability and without departing from the scope of the invention.

[0196] (Example 1) Cloning of heavy and light chains of mouse anti-BDCA2 antibody 24F4 mouse hybridomas (IgG1, κ) were derived from Balb / c mice immunized with a gene gun using plasmid pEAG2456, a mammalian expression vector co-expressing full-length human BDCA2 cDNA and FcεRIγ cDNA. (See Example 17).

[0197] Following the manufacturer's recommended protocol, total intracellular RNA was prepared using the Qiagen RNeasy mini kit, derived from 24F4 mouse hybridoma cells. Using a random hexamer for priming, cDNA encoding the variable regions of the heavy and light chains was cloned from the total intracellular RNA by RT-PCR using the GE Healthcare First Strand cDNA Synthesis kit, following the manufacturer's recommended protocol.

[0198] To PCR amplify mouse immunoglobulin variable domains with intact signal sequences, a cocktail of degenerate forward primers hybridizing with signal sequences from multiple mouse immunoglobulin gene families and a single back primer specific to the 5' end of the mouse constant domain, as described in Current Protocols in Immunology (Wiley and Sons, 1999), was used. The 24F4 heavy chain variable domain was amplified using the following primers: 5' ACT AGT CGA CAT GRA CTT TGG GYT CAG CTT GRT TT 3'(R= A / G, and Y=C / T) (Sequence ID 25) and 5' AGG TCT AGA AYC TCC ACA CAC AGG RRC CAG TGG ATA GAC 3' (R=A / G, and Y=C / T) (Sequence ID It was amplified using (number 26). The 24F4 light chain variable domain with its signal sequence was amplified using the following primers: 5' ACT AGT CGA CAT GGA GWC AGA CAC ACT CCT GYT ATG GGT 3'(W=A / T, and Y=C / T)(SEQ ID NO: 27) and 5' GCG TCT AGA The signal was amplified using ACT GGA TGG TGG GAG ATG GA 3' (sequence number 28).

[0199] PCR products were gel-purified using the Qiagen Qiaquick gel extraction kit, following the manufacturer's recommended protocol. The purified PCR products were subcloned into the Invitrogen pCR2.1 TOPO vector using their respective TOPO cloning kits, also following the manufacturer's recommended protocol. Inserts derived from multiple independent subclones (a heavy-chain clone named pYL647 and a light-chain clone named pYL651) were sequenced to establish a consensus sequence.

[0200] Sequence variations between clones coincided with the degenerate positions of the primers. BLAST analysis of the variable domain sequences confirmed the identity of these immunoglobulins. The N-terminal sequences of the putative mature light chain and mature heavy chain matched the N-terminal sequences of the genuine 24F4 chain derived from Edman degradation data. The intact mass estimated from the hypothetical sequence assembled by adding the constant domain sequences put forth from the heavy chain cDNA and κ light chain cDNA of cloned Balb / c IgG1 to the putative mature variable domain sequence coincided with the intact mass of 24F4 derived from purified hybridomas, as determined by mass spectrometry.

[0201] The mouse 24F4 heavy chain variable domain (VH) is a member of mouse subgroup III(D). The sequences of the mouse 24F4 mature heavy chain variable domain are shown below, with CDR H1, CDR H2, and CDR H3 underlined in that order: [ka] The mouse 24F4 light chain variable domain (VL) is a member of mouse κ subgroup III. The sequence of the mouse 24F4 mature light chain variable domain is shown below, with CDR L1, CDR L2, and CDR L3 underlined in that order: [ka] The unpaired cysteine ​​is located at residue 95 within CDRL3 of the mouse 24F4 VL sequence mentioned above (according to Kabat nomenclature, this Cys is at residue 91).

[0202] (Example 2) Chimerization of mouse 24F4 antibody Using cDNA encoding the mouse 24F4 variable domain, we constructed a vector for expressing a mouse-human chimeric organism (ch24F4) in which the mu24F4 variable region was ligated to human IgG1 and human κ constant regions. First, the variable domain was manipulated by PCR to add a 5' Kozak sequence, and the human sequence and new restriction site were introduced into the FR4 / constant domain junction to fuse with the human immunoglobulin constant domain. The resulting variable region cDNA sequence in the plasmid was confirmed by DNA sequencing. The heavy chain variable domain within the plasmid pYL647 was used as a template for PCR with primers 5' GAT CCG CGG CCG CAC CAT GGA CTT TGG GTT CAG CTT G 3' (Sequence ID 31) (Adds NotI site and Kozak sequence) and 5' GAT GGG CCC TTG GTG GAA GCT GCA GAG ACA GTG ACC AGA G 3' (SEQ ID NO: 32) (FR4 / at the steady-state domain junction) pYL668 was generated by using it together with the human IgG1 CH1 sequence and ApaI site, amplifying a 0.45kb fragment, purifying it, and subcloning it into the Invitrogen pCRBluntIITOPO cloning vector. To construct a heavy chain chimeric organism, a 0.45 kb NotI-ApaI fragment derived from pYL668, a 24F4 heavy chain variable domain construct, and a 0.98 kb ApaI-BamHI fragment derived from pEAG1325 (a plasmid containing a sequence-confirmed huIgG1 heavy chain constant domain cDNA (genetically removed from the C-terminal lysine residue of IgG1)) were subcloned into the vector skeleton of the expression vector pV90 (the expression of heterologous genes within it is regulated by the CMV-IE promoter and human growth hormone polyadenylation signaling, which possesses a dhfr selection marker; see U.S. Patent No. 7,494,805) to create the expression vector pYL672. The heavy chain cDNA sequence within the resulting plasmid pYL672 was confirmed by DNA sequencing. The putative mature ch24F4-huIgG1 heavy chain protein sequence encoded by pYL672 is shown below: [ka] Furthermore, the non-glycosyl form of ch24F4 with minimal effector function was also constructed by subcloning a 0.45kb NotI-ApaI fragment derived from pYL668, a 24F4 heavy chain variable domain construct, and a 0.98kb ApaI-BamHI fragment derived from pEAG2412 (a plasmid containing the heavy chain constant domain cDNA of a sequence-confirmed S228P / N299Q huIgG4 / IgG1 hybrid with the C-terminal lysine residue of IgG1 genetically removed) into the vector backbone of the expression vector pV90, thereby generating the plasmid pYL670. The heavy chain cDNA sequence within the resulting plasmid pYL670 was confirmed by DNA sequencing. The sequence of the putative mature agly ch24F4-huIgG4 / G1 hybrid heavy chain protein encoded by pYL670 is shown below: [ka] The κ light chain variable domain within the plasmid pYL651 was used as a template for PCR with primers 5' GAT CCG CGG CCG CCA CCA TGG AGA CAG ACA CAC TCC TG 3' (SEQ ID NO: 35) (adding the 5' NotI site and Kozak sequence) and 5' CCA CCG TAC GTT TGA TTT CCA GCT TGG TGC 3' (SEQ ID NO: 36) (adding the human κ constant domain sequence at the FR4 / constant domain junction and the 3' BsiWI site). A 0.4kb fragment was amplified, purified, and processed in Invitrogen. pYL669 was generated by subcloning into the pCRBluntIITOPO cloning vector. The variable region cDNA sequence within the plasmid pYL669 was confirmed by DNA sequencing. To construct a light chain chimera, a 0.4kb NotI-BsiWI light chain variable domain fragment derived from pYL669 and a 0.34kb BsiWI-BamHI fragment derived from the plasmid pEAG1572 (which contains a sequenced human κ light chain constant domain cDNA) were subcloned into the vector skeleton of pV100 (whose heterologous gene expression is regulated by the CMV-IE promoter and human growth hormone polyadenylation signaling, which possesses a neomycin selection marker) to create the expression vector pYL671. The light chain cDNA sequence within the resulting plasmid pYL671 was confirmed by DNA sequencing. The putative mature ch24F4-human κ light chain protein sequence encoded by pYL671 is shown below: [ka]

[0203] Expression vectors (pYL670 or pYL672, which are ch24F4 heavy chain vectors, and pYL671, which is a ch24F4 light chain vector) were co-transfected into 293-EBNA cells, and the transfected cells were tested for antibody secretion and specificity (cells transfected with empty vectors (and molecularly cloned non-involved mAb vectors) were used as controls). Western blot analysis of the conditioned medium (color-developed with anti-human heavy chain and anti-human light chain antibodies) showed that cells transfected with ch24F4 synthesized and efficiently secreted heavy and light chains. The specificity of ch24F4 was confirmed by direct FACS binding to surface human BDCA2. Dilution titration FACS assays showed that the EC50 for binding of both ch24F4 mutants was equivalent to the EC50 for direct binding of mouse 24F4 mAb to surface-expressed human BDCA2. Stable CHO cell lines secreting ch24F4-huIgG1 κmAb and agly ch24F4-huIgG4 / G1 hybrid κmAb were generated by co-transfection with pYL672 / pYL671 and pYL670 / pYL671, respectively.

[0204] (Example 3) Removal of unpaired cysteine ​​residues in CDRL3 of the chimeric 24F4 antibody. Since unpaired cysteine ​​within exposed CDRs can lead to heterogeneity or instability in the product, ch24F4 mutants C95S and C95T were constructed by site-directed mutagenesis using pYL671, a ch24F4 light chain expression vector plasmid, as a template.

[0205] Site-directed mutagenesis was performed using the Agilent QuikChange II mutagenesis kit, following the manufacturer's recommended protocol. The C95S mutant was created using the mutagenesis primer 5' GCA ACC TAT TAC TGT CAA CAA AGT AAT GAG GAT CCT CGG AC 3' (SEQ ID NO: 38) and its reverse complement with a novel HincII site introduced. The plasmid pEAG2678 was constructed using the following method. The C95T mutant was created using the mutagenesis primer 5' CAA CCT ATT ACT GTC AGC AAA CTA ATG AAG ATC CTC GGA CGT TCG 3' (SEQ ID NO: 39) and its reverse complement with the BamHI site removed. The plasmid pEAG2679 was constructed using [a specific method / tool]. Mutant plasmids were identified by screening for changes in introduced restriction sites. The full-length light chain cDNA sequences within the resulting plasmids were confirmed by DNA sequencing. Wild-type ch24F4, as well as C95S and C95T mutant mAbs, were transiently expressed in 293E cells by co-transfection with pYL672 and pYL671, pEAG2678, or pEAG2679. Acclimation medium was collected two days after transfection. The titers of both mutants (assayed on an anti-human Fc chip using Octet) were similar to those of wild-type ch24F4, and Western blots of non-reducing SDS-PAGE did not show coarse aggregation or obvious clipping compared to wild-type ch24F4 mAb. Direct binding to surface BDCA2 by FACS showed that the apparent EC50 for binding with the C95S mutant was equivalent to that of wild-type ch24F4, while the EC50 for binding with the C95T mutant was a fraction of that. Conditioned media containing ch24F4 and C95 mutant mAbs were assayed for binding to the human BDCA2 external domain using Octet. Antibodies derived from conditioned media from transiently transfected cells were conjugated to an anti-human Fc chip, and then flowed onto monomeric huBDCA2 on the Octet chip to test for binding and dissociation. The binding and dissociation reaction rates by Octet were equivalent for wild-type ch24F4 and the C95S mutant, but the dissociation rate of the C95T mutant was faster than that of wild-type ch24F4. Based on these results, C95S was incorporated into the humanized 24F4 light chain CDRL3.

[0206] (Example 4) Exemplary humanized 24F4 heavy chain and humanized 24F4 light chain 7 humanized (hu)24F4 heavy chain (huIGHV3-21 * Examples of the 01 framework (24F4 VH CDR) and their corresponding DNA sequences are shown below. CDR1, CDR2, and CDR3 within each heavy chain are underlined in that order. Revertant mutations in the framework are shown in lowercase bold. Changes to CDR residues derived from mouse 24F4 are indicated by shading within the CDR sequence. The variable heavy chain CDR1 (CDR H1) is defined according to the Chothia definition, which is 5 amino acids longer than the Kabat definition, and the italicized residues within CDR H1 confirm the formation of five additional amino acids (i.e., GFTFS (SEQ ID NO: 12)) that make up the Chothia CDR H1. The N-terminal amino acids of the variable heavy chain domain (i.e., glutamic acid in the variants H0, H1, H2, and H3, and aspartic acid in the variants H4, H5, and H6) can directly contact the antigen and affect binding affinity. Kabat's buried residue at position 49 may affect the conformation of CDR2 in the heavy chain (serine in variants H0, H1, H2, and H3; as well as alanine in variants H4, H5, and H6). Kabat's residue at position 93 may affect heavy-light chain pairing (alanine in variants H0, H1, H2, and H3; as well as threonine in variants H4, H5, and H6). Shading of amino acid residues in the CDR H1, CDR H2, and CDR H3 regions differs from those in mouse 24F4. [ka] [ka] The amino acid sequence alignment of the variant forms H0-H6 is shown below: [ka]

[0207] Three humanized 24F4 light chains (huIGKV1-13) * Examples of the 02 framework (24F4 VL CDR) and their corresponding DNA sequences are shown below. CDR1, CDR2, and CDR3 within each light chain are underlined in that order. Ser91 (following Kabat numbering) which replaces Cys91 in all light chains is highlighted. The N-terminal amino acid of the variable light chain domain (i.e., alanine in variant L0, and aspartic acid in variants L1 and L2) can directly contact the antigen and affect binding affinity. Revertant mutations in the framework are shown in lowercase bold. The first variant (L0) contains the fewest revertant mutations, and the third variant (L2) contains the most (i.e., minimal "humanization"). [ka] The amino acid sequence alignment of the variant forms L0-L2 is shown below: [ka]

[0208] The humanized VH and VL amino acid sequences described above do not contain potential N-linked glycosylation sites or Asn-Gly deamidation sites. In germline sequences, methionine is observed in both the VH and VL domains, and since it is not exposed on the surface, the risk of methionine oxidation is considered to be minimal.

[0209] The solubility of proteins can correlate with their pI. The pI of the designed constructs was calculated using the amino acid pK values ​​in Bjellqvist et al. (Electrophoresis, Vol. 14: pp. 1023-31 (1993); Electrophoresis, Vol. 15: pp. 529-39 (1994)). The values ​​shown below were calculated using human IgG single-chain. The pI of each humanized antibody is significantly above 7, and therefore, a significant positive charge is expected at neutral pH. The values ​​in the table are the calculated pI values ​​of the complete combination antibody, with the net charge indicated in parentheses. Calculated value of molecular pI (net charge) Chimera 24F4 6.94 (-2) Humanized H4L1 7.26 (0)

[0210] (Example 5) Hx / L1 binding to BDCA2 All 21 possible variants of the hu24F4 heavy chain and hu24F4 light chain (described in Example 4), as well as ch24F4, were transiently expressed in 293E cells by co-transfection of the heavy chain plasmid and the light chain plasmid. All variants of hu24F4 were assembled and secreted at titers exceeding those of ch24F4 (determined by quantification of mAbs in conditioned medium by binding of Octet to an anti-human Fc tip). Western blot analysis of non-reducing SDS-PAGE for chimeric 24F4 mAbs and humanized 24F4 mAbs showed no evidence of coarse aggregation or obvious clipping compared to ch24F4.

[0211] The conditioned medium was assayed by direct binding FACS on stably transfected DG44 CHO cells co-expressing full-length BDCA2 and FcεRIγ cDNA (human or cynomolgus monkey) (the relevant expression vectors were pEAG2456 for human BDCA2 / FcεRIγ and pEAG2668 for cynomolgus monkey BDCA2 / FcεRIγ). Direct binding to surface human BDCA2 or surface cynomolgus monkey BDCA2 showed complete loss of binding for the H0, H1, and H2 series of the hu24F4 mutant, significant loss of binding affinity for the H3 series of the hu24F4 mutant, good retention of affinity for both the H4 and H5 series of the hu24F4 mutant, and moderate loss of binding for the H6 series of the hu24F4 mutant (Figures 2 and 3). Based on titer and apparent EC50 values ​​in direct-binding FACS analysis, H4 / L1 and H5 / L1 were identified as the "best" mutants of hu24F4.

[0212] Conditioning media containing ch24F4 and all hu24F4 mutant mAbs were assayed using Octet for binding to the human BDCA2 extradomain. Monomeric huBDCA2 extradomains were prepared from purified muIgG2a Fc-huBDCA2 fusion protein (related plasmid: pEAG2423) by proteolytic cleavage. Antibodies derived from conditioning media from transiently transfected cells were conjugated to anti-human Fc chips, and monomeric huBDCA2 was then flowed on Octet chips to test for binding and dissociation. The H4 and H5 series of hu24F4 mutants showed the best affinity for huBDCA2.

number

[0213] (Example 6) Enhanced affinity for hu24F4

[0214] To investigate the potential enhancement of hu24F4 affinity through substitution at the unpaired cysteine ​​position of the L1 CDR L3 of the hu24F4 variant (C95S in the hu24F4 light chain expression vector pYL740), several L1 variants were constructed by site-directed mutagenesis. The reversion to the unpaired cysteine, i.e., S95C, was constructed by site-directed mutagenesis resulting in the plasmid pYL749. The variants S95T, S95A, and S95V were constructed by site-directed mutagenesis resulting in the plasmids pYL750, pYL751, and pYL752, respectively. The full-length light chain cDNA sequences within the resulting plasmids were confirmed by DNA sequencing. The C95 mutant hu24F4 mAb was transiently expressed in 293E cells by co-transfection of the hu24F4 H4 heavy chain pYL746 plasmid or the hu24F4 H5 heavy chain pYL747 plasmid with the C95S pYL740 plasmid, S95C pYL749 plasmid, S95T pYL750 plasmid, S95A pYL751 plasmid, or S95V pYL752 plasmid within the hu24F4 L1 mutant light chain. Conditioning medium was collected 2 days after transfection. All mutant titers (assayed on an anti-human Fc chip using Octet) were similar, and Western blots of non-reducing SDS-PAGE showed no coarse agglutination or obvious clipping. Conditioning medium containing the C95 mutant mAb was assayed using Octet for binding to the human BDCA2 external domain. Antibodies derived from conditioned media using transiently transfected cells were bound to anti-human Fc chips, and then monomeric huBDCA2 was flowed onto Octet chips to test binding and dissociation. The C95A mutant showed the slowest dissociation rate.

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[0215] Based on these results, stable CHO cell lines were constructed for the hu24F4 H4 / L1 C95T and H4 / L1 C95A mutants, as well as the H5 / L1 C95T and H5 / L1 C95A mutants, which exhibited the slowest apparent dissociation rates. Octet binding studies were repeated for purified hu24F4 mAb. The C95A mutant was selected as the most promising candidate mutant. The sequences of the plasmids pYL746 (hu24F4 H4 heavy chain) and pYL751 (hu24F4 L1 light chain) were used to recode and construct expression vectors for generating a CHO-producing cell line.

[0216] The amino acid sequence of the putative mature hu24F4 L1 C95A light chain encoded by pYL751 is shown below (CDR L1, CDR L2, and CDR L3 are underlined): [ka]

[0217] The amino acid sequence of the putative mature hu24F4 H4-huIgG1 heavy chain encoded by pYL746 is shown below (CDR H1, CDR H2, and CDR H3 are underlined): [ka]

[0218] The antibody consisting of the mature heavy chain (SEQ ID NO: 4) and mature light chain (SEQ ID NO: 3) listed above is referred to as BIIB059.

[0219] (Example 7) Recoding of heavy chain and light chain genes To potentially improve expression, the nucleotide sequences of the light chain and heavy chain genes were recoded without altering the amino acid sequence. The modified DNA sequence of the anti-BDCA2 light chain gene is shown below. Amino acids 1-240 contain the light chain sequence. Amino acids 1-22 (lowercase nucleotides) contain the native light chain signal peptide. The mature N-terminus begins at amino acid 23(D). [ka]

[0220] The modified DNA sequence of the anti-BDCA2 heavy chain gene is shown below. Amino acids 1-470 contain the heavy chain sequence. Amino acids 1-19 (lowercase nucleotides) contain the native heavy chain signal peptide. The mature N-terminus begins at amino acid 20 (D). [ka] [ka] [ka]

[0221] (Example 8) Expression cassettes and expression vectors Heavy and light chain genes were excised and ligated into individual expression vectors. Each gene is under the transcriptional regulation of the human cytomegalovirus pre-initial promoter and the human growth hormone gene polyadenylated sequence.

[0222] The light chain-expressing plasmid pJP009 also contains an expression cassette for the neomycin phosphotransferase gene (neo), which includes the mouse phosphoglycerate kinase (muPGK) initial promoter sequence and the muPGK polyadenylated sequence (Figure 4). The heavy chain-expressing plasmid pJP010 also contains an expression cassette for the dhfr gene, which was used as a selection marker and methotrexate amplification marker. The key features of plasmids pJP009 and pJP010 are summarized below.

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[0223] Abbreviations: Human cytomegalovirus pre-early stage (hCMV IE), monkey virus 40 early stage (SV40E), mouse phosphoglycerate kinase (muPGK), human growth hormone (hGH), neomycin phosphotransferase gene (G418 resistance), dihydrofolate reductase gene (dhfr), bacterial gene for resistance to ampicillin (β-lactamase).

[0224] The complete nucleotide sequence of plasmid pJP009 is shown below. The three open reading frames are the 24F4 light chain, neomycin phosphotransferase, and β-lactamase. [ka] [ka] [ka] [ka]

[0225] The complete nucleotide sequence of plasmid pJP010 (Figure 5) is shown below. The three open reading frames are the 24F4 heavy chain, mouse dihydrofolate reductase, and β-lactamase. [ka] [ka] [ka] [ka]

[0226] (Example 9) Construction of cell systems The host cell line used was CHO-DG44, a Chinese hamster ovary dihydrofolate reductase (dhfr)-deficient host cell line. The DG44 host cell bank was tested and found to be negative for the presence of exogenous substances before use. DG44 host (CER-00-05-01) was used to construct a cell line expressing anti-BDCA2.

[0227] Plasmids pJP009 and pJP010, expressing the re-encoded light and heavy chains of anti-BDCA2, respectively, were transfected into host cell lines by electroporation. Transfected cells expressing dhfr were selected using α-nucleoside-deficient medium. Following selection in the αMEM nucleoside-free medium described above, the transfected pool was enriched for highly expressive cell lines using a combination of fluorescence-activated cell sorting and a Genetix Clonepix FL analyzer (CER-00-09-03). Cell colonies isolated by ClonePix FL were harvested from semi-solid medium into 96-well plates. Individual wells were grown and productivity was assessed. The cell line showing the highest titer in a frozen-batch culture analysis using a shaking flask (#49) was transferred to Research Animal Fermentation for growth in a 10 L bioreactor to generate material for characterization.

[0228] After an initial cell line screening, the best-producing cell line was selected for amplification. This best-producing cell line was subjected to methotrexate (MTX) amplification. The amplified pool was subcloned into a 384-well plate using a limiting dilution with a theoretical cell density of 0.5 cells per well. Clonality was verified by imaging each well of the 384-well plate using a Cellavista analyzer (Innovatis) to confirm the presence of a single cell per well.

[0229] Based on scaled-down flow-boiled culture in shaking flasks and product quality analysis, four clonal cell lines with the best amplification were selected. A pre-master cell bank (Pre-MCB) was constructed from these four cell lines, which were evaluated in a bioreactor. Based on bioreactor performance and product quality analysis, one most promising subclone was selected. The Pre-MCB vial of the most promising cell line was transferred to the manufacturing process for master cell bank generation.

[0230] (Example 10) Post-translational modification of the anti-BDCA2 antibody BIIB059 a) Oxidation EndoLysC peptide mapping using the anti-BDCA2 antibody BIIB059 revealed that Met-257, Met-433, and Trp-163 in the heavy chain are susceptible to oxidation. Oxidation levels ranged from 4% to 7%. The experimental data suggest that most of the oxidation is related to sample preparation. b) Deamidation EndoLysC peptide mapping analysis of the BIIB059 antibody revealed that approximately 2.5% of each of Asn-389, Asn-394, and Asn-395 in the heavy chain were deamidated (a combination of deamidation and succinimide formation), and approximately 2.5% of Asn-320 in the heavy chain was deamidated (in succinimide form). The total amount of succinimide form for Asp-32 and Asp-34 in the light chain was approximately 3%. Isomerization by combinations of Asp-32 and Asp-34 in the light chain was approximately 5%. Similar to oxidation, some of these modifications may be related to sample preparation. c) Saccharification Glycation is a non-enzymatic modification of amino groups on proteins caused by the reaction of these groups with glucose, a component of the culture medium. Glycation is routinely detected in proteins, and its levels vary widely depending on cell culture conditions. With the BIIB059 antibody, the level of glycation, as measured by intact mass spectrometry of non-reduced proteins, was approximately 10%. Peptide mapping analysis revealed approximately 0.46% glycation in the light chain Lys-107, 0.28% glycation in the light chain Lys-103, and approximately 0.2% glycation in the O-linked heavy chain Lys-295. d) Glycosylation No detectable O-linked glycosylation was observed in BIIB059. e) Other modifications (e.g., hydroxylysine) Analysis revealed that <1% of the heavy chain of the BIIB059 antibody was in a non-glycosyl form. The analysis did not show any Asn-to-Ser substitution within the antibody, nor were any unknown modifications or mutations found at a level of ≥1% within the antibody.

[0231] (Example 11) Direct binding of BIIB059 to the cell surface of plasmacytoid dendritic cells. A flow cytometry-based whole blood assay was developed to evaluate the binding of BIIB059 to BDCA2 on human plasmacytoid dendritic cells (pDCs) or cynomolgus monkey pDCs. Cynomolgus monkey peripheral blood (Toxikon, Inc., Bedford, MA) or human peripheral blood (Biogen Idec) was collected in recovery tubes with heparin sodium and maintained at room temperature. A FACS staining antibody cocktail for pDC identification was added to each whole blood aliquot, incorporating CD20 antibody, CD14 antibody, CD123 antibody, and HLA-DR antibody. Alexa647-labeled BIIB059 (Biogen Idec, lot number 17073-057) or an Alexa647-labeled hIgG isotype control was added to the FACS staining cocktail at concentrations of 0–40 μg / mL. The blood was incubated on ice protected from light for 30 minutes. After 30 minutes, 500 μL each of whole blood aliquots (cynomolgus monkey) or 100 μL each of whole blood aliquots (human) were treated with 10 mL (cynomolgus monkey) or 2 mL (human) of 1x Easy Lyse Buffer (Leinco Technologies) and incubated at 37°C for at least 1 hour. After incubation at room temperature for 10–15 minutes, the samples were centrifuged at 1400 rpm for 5 minutes. The supernatant was decanted, leaving only the leukocyte (WBC) pellet. Each WBC pellet was washed with 5 mL of FACS buffer (PBS with 1% BSA + 0.002% sodium azide + 1 mM CaCl2 + 1 mM MgCl2) and centrifuged at 1400 rpm for 5 minutes. The supernatant was decanted, and each WBC pellet was resuspended in 200 μL of FACS buffer and transferred to a 96-well round-bottom plate (Fisher Scientific). The plate was centrifuged at 1400 rpm for 5 minutes. The supernatant was released from the plate, and each WBC pellet was washed with 200 μL of FACS buffer. The plate was centrifuged at 1400 rpm for 5 minutes, and the supernatant was released from the plate. After washing (as described above), the WBCs were resuspended in 200 μL of 1% paraformaldehyde (PFA) in PBS, protected from light, and fixed at 4°C overnight.Immediately before flow cytometry analysis, WBCs were filtered using a 60-micron nylon mesh filter plate (Millipore). Each pellet was then transferred to a new 96-well round-bottom plate and centrifuged at 1400 rpm for 5 minutes. Each WBC pellet was resuspended in 250 μL of FACS buffer, and fluorescence intensity was measured on an LSRII 4-color FACS machine. Monochromatic correction was collected using an anti-mouse Ig Compensation Particle bead set (BD Biosciences). Analysis was performed using FlowJo and GraphPad Prism software. BIIB059 was administered to cynomolgus monkey cells and human cells at an EC of 1–2 μg / mL (7–13 nM). 50 The values ​​were similarly joined (Figure 6).

[0232] (Example 12) Evaluation of BIIB059's self-assembly The AlphaScreen assay is a homogeneous proximity assay that utilizes glutathione donor and acceptor beads (Perkin Elmer) that bind to human FcRIIa (CD32a) GST. Various concentrations of test antibodies were added to this mixture. Since the antibody binds to FcRIIa in a monovalent form, the only way to generate a signal is for both the donor and acceptor beads to bind to the antibody and then associate, bringing the beads within 200 nm, enabling the generation of singlet oxygen and the resulting emission of light. The level of luminescence detected by the Envision (Perkin Elmer) analyzer is proportional to the degree of self-association.

[0233] Figure 7 shows the Alpha Screen results for BIIB059 compared to 5c8 (negative control) and LT105 (positive control with strong auto-association).

[0234] (Example 13) Evaluation of nonspecific binding of BIIB059 Cross-interaction chromatography (CIC) is a high-throughput method for preliminary evaluation of the viscosity of mAb candidates (Jacobs et al., Pharm Res., Vol. 27 (No. 1): pp. 65-71 (2010) This method involves chemically coupling bulk polyclonal human IgG to an NHS-activated chromatography resin. The retention times of BIIB059 on non-derivative and IgG-derivative columns were then compared to a control panel of well-behaving and poorly behaving mAbs. In this method, BIIB059 showed no evidence of nonspecific binding, as evidenced by its short retention time and small K' value.

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[0235] (Example 14) Stability evaluation of BIIB059 The stability of BIIB059 was tested for initial research formulations across a range of buffer conditions using differential scanning fluorescence (SCFL). Protein unfolding was monitored on a 96-well Mx3005p real-time PCR system (Agilent Technologies) using 10 μg of protein in 50 μL of PBS (pH 7.0) supplemented with SYPRO orange fluorophores at a final concentration of 10-fold (based on 1000-fold in-stockings of Invitrogen). Samples were heated from 25°C to 95°C at 1°C / min, and fluorescence intensity was measured three times at each 1°C increment. Fluorescence intensity was plotted as a function of temperature. Tm was derived from these curves by taking the negative derivative ("-R'(T)" in the Mx3005p software) and selecting the local minimum of the derivative plot. When a basal buffer solution of 20 mM sodium citrate was used, the pH varied from 5.0 to 7.5, and the NaCl and sucrose concentrations varied from 50 to 250 mM.

[0236] Stability was similar across these buffer ranges. Figure 8 shows the data for 150 mM NaCl and 250 mM sucrose as a function of pH. Because it is difficult to reach high concentrations with sucrose using a research centrifuge concentrator, 20 mM sodium citrate, 150 mM NaCl, pH 6.0 was selected as the research formulation instead of sucrose.

[0237] (Example 15) Evaluation of the stirring stability of BIIB059 A 0.2 mL volume of BIIB059 mAb solution was mixed at 1 mg / mL in 20 mM sodium citrate, pH 6.0, and 150 mM NaCl, in a 2 mL glass vial (Waters; WAT270946C) at room temperature using a Lab-Line Instruments Model 4626 Titer Plate Shaker set to 600 rpm, and subjected to inversion shaking. Agglutination was assessed by monitoring the increase in turbidity at 320 nm using a Beckman DU640 spectrophotometer. BIIB059 showed time-dependent agglutination. Normally, wild-type human IgG1 antibodies do not agglutinate under these agitation conditions. As shown in Figure 9, agglutination was completely suppressed by adding 0.03% Tween 80, a common pharmaceutical excipient. Agglutination induced by agitation can sometimes be highly pH-dependent. Non-glycosyl IgG4 / IgG1 showed more rapid and widespread agglutination than BIIB059. Furthermore, aggregation of non-glycosyl IgG4 / IgG1 was also suppressed by the addition of Tween 80.

[0238] (Example 16) Viscosity evaluation of BIIB059 The stability and viscosity of the BIIB059 sample were measured at high concentrations of 150 mg / mL or higher to support the potential development of a product for subcutaneous administration. The BIIB059 solution was centrifuged in an ultra-concentrator tube to reduce its volume, and the achieved concentration was determined by UV scanning. Stability was determined by size exclusion chromatography after storage at 2–8°C for 1 and 2 weeks. Protein concentrations exceeding 200 mg / mL were easily achieved for small amounts of protein in a 20 mM citrate, pH 6, 150 mM NaCl buffer, and aggregates remained low (0.68%) even after 2 weeks at 2–8°C. Viscosity was measured using a Viscopro 2000 (Cambridge Viscosity) analyzer. The viscosity at 150 mg / mL in citrate / salt buffer was only 8 cP. These results support the feasibility of high-concentration BIIB059 formulations.

[0239] (Example 17) Cloning of the human BDCA2 gene The full-length human BDCA2 (huBDCA2) cDNA was subcloned into Invitrogen's pCR4TOPO cloning vector from Open Biosystems, and this plasmid was named pEAG2367. DNA sequencing confirmed that the cDNA is identical to the full-length human BDCA2 cDNA in the reference Genbank accession number NM_130441. The full-length open reading frame of huBDCA2 encoded by pEAG2420 is shown below, with the transmembrane domain predicted by TM-HMM underlined: [ka]

[0240] The full-length open reading frame of huFcεRIγ encoded by pEAG2413, identical to the reference sequence in Genbank accession number NP_004097, is shown below: [ka]

[0241] A CHO expression vector co-expressing both human BDCA2 cDNA and human FcεRIγ cDNA in a tandem transcription unit was constructed by subcloning a 2.11kb SpeI fragment derived from pEAG2413 into a phosphatase-treated, linearized 6.71kb SpeI vector skeleton of pEAG2420, resulting in a "univector" named pEAG2456. The human BDCA2 cDNA and FcεRIγ cDNA within pEAG2420 were sequenced. A stable CHO cell line that stably co-expresses BDCA2 cDNA and FcεRIγ cDNA was created by transfection with pEAG2456.

[0242] (Example 18) Cloning of the BDCA2 gene in cynomolgus monkeys and rhesus monkeys. The open reading frame of putative macaque BDCA2, encoded by one of the SNP morphologies observed in pEAG2384 and pEAG2383, is shown below. This SNP morphology is referred to below as the E73 SNP morphology of cynomolgus macaque BDCA2. A single sequence identical to the E73 SNP morphology of cynomolgus macaque BDCA2 was also observed in rhesus macaques. [ka]

[0243] In the second SNP morphology of cynomolgus monkey BDCA2, residue 73 (GAA=Glu, E), which was highlighted above, is lysine (AAA=Lys, K). This second SNP morphology is referred to as the K73 SNP morphology of cynomolgus monkey BDCA2. In human BDCA2, residue 73 is glutamic acid. The gap-processed alignment of the human BDCA2 sequence (top) and the macaque monkey BDCA2 sequence (bottom), which share 90.6% identity, is shown below. Potential N-linked glycosylation sites are shaded. Macaque monkey BDCA2 lacks one potential N-linked glycosylation site present in humans (NSS at 137-139 in humans vs. NSA in macaques). [ka]

[0244] The consensus FcεRIγ open reading frame for cynomolgus monkeys is shown below: [ka]

[0245] The cynomolgus monkey FcεRIγ cDNA sequence is a perfect match between the predicted rhesus monkey cDNA sequence (based on a short genome read) described in Genbank accession number XM_001115585 and the cynomolgus monkey sequence deposited by Genentech scientists as Genbank accession number AF485816. The cynomolgus monkey FcεRIγ protein sequence shares 98.9% identity with the human FcεRIγ protein, differing only by a single conserved substitution. The alignment between human FcεRIγ (top) and cynomolgus monkey FcεRIγ (bottom) is shown below: [ka]

[0246] A CHO expression vector co-expressing both cynomolgus monkey E73 SNP morphologies of BDCA2 cDNA and FcεRIγ cDNA as tandem transcription units was constructed by subcloning a 2.11kb SpeI fragment derived from pCN652 into a phosphatase-treated, linearized 6.72kb SpeI vector skeleton of pCN654, resulting in a "univector" named pEAG2668. The cynomolgus monkey BDCA2 cDNA and FcεRIγ cDNA within pEAG2668 were sequenced. A stable CHO cell line that stably co-expresses BDCA2 cDNA and FcεRIγ cDNA was created by transfection with pEAG2668.

[0247] (Example 19) Cross-reactivity between human BDCA2 and cynomolgus monkey BDCA2 To determine whether the E73 / K73 BDCA2 SNP in cynomolgus monkeys affected the binding of anti-BDCA2 antibodies, 293E cells were subjected to an expression vector (pEAG1458) containing an EGFP reporter, as well as BDCA2 cDNA and FcεRIγ. Expression vectors containing cDNA (human BDCA2:pEAG2420 and human FcεRIγ:pEAG2413; cynomolgus monkey E73 BDCA2:pCN652 or cynomolgus monkey K73 BDCA2:pCN656 and cynomolgus monkey FcεRIγ:pCN652) were co-transfected in a 1:1:1 molar ratio. Three days after transfection, cells were harvested and stained with PE-conjugated Miltenyi anti-human BDCA2 AC144 mAb (Miltenyi Biotec; model number 130-090-511) in direct binding dilution titration FACS, and gated to green EGFP-positive cells. Figure 10 shows the direct binding of AC144 to human surface BDCA2 and cynomolgus monkey surface BDCA2.

[0248] The apparent EC50 is essentially equivalent for both the E73 SNP and K73 SNP morphologies of human BDCA2 and cynomolgus monkey BDCA2. Based on these results, stable CHO transfects for full-length surface BDCA2 were generated using the human BDCA2 / FcεRIγ expression vector pEAG2456 and the cynomolgus monkey E73 SNP BDCA2 / FcεRIγ expression vector pEAG2668. These cell lines were used for triage of human / cynomolgus monkey cross-reactive anti-BDCA2 antibodies.

[0249] (Example 20) Fc fusion constructs of human and cynomolgus monkey BDCA2 external domains Five Fc fusion constructs of human and cynomolgus monkey BDCA2 ECD were manipulated. In three of the constructs, BDCA2 was fused to the human IgG1 hinge and the C-terminus of human IgG1 Fc via a G4S linker sequence. In two of the constructs, the G4S linker was replaced with ENLYFQC, a TEV protease cleavage site.

[0250] Since BDCA2 is a type II membrane protein (with its C-terminus located outside the cell), the design of the soluble Fc fusion protein involved adding the C-terminal extraterrestrial domain of BDCA2 (residues 45-213 in human BDCA2) to the C-terminus of the engineered IgG Fc, and its secretion was driven by an in-frame mouse κ light chain signaling sequence. The full-length huBDCA2 construct, pEAG2367, was used as a template for PCR, with primer 5' CAG TGT CTG TTT CAC TCC CGG GGG TGG CGG TGG TAG CAA TTT TAT GTA TAG C 3' (SEQ ID NO: 74) (Adds the 5' XmaI (Pro-Gly) and Gly4Ser linker immediately before the 5' end of the huBDCA2 external domain) and 5' CCA GGG AGA ATA GGA TCC TTA TAT GTA GAT CTT 3' (SEQ ID NO: 75) (3' The BamHI region on the side was used (added immediately after the huBDCA2 terminator). The 0.56kb PCR product was purified and subcloned into Invitrogen's pCRBluntIITOPO cloning vector to create pEAG2417, and its insert cDNA sequence was confirmed. A 0.53kb XmaI-BamHI fragment derived from pEAG2417 and a 0.75kb NotI-XmaI fragment derived from pEAG1397 (which contains an engineered huIgG1 Fc whose secretion is driven by an in-frame engineered mouse κ light chain signal sequence) were ligated with a 1.89kb BamHI-XbaI vector skeleton fragment and a 4.17kb XbaI-NotI vector skeleton fragment derived from the expression vector pV90 to create pEAG2421, an expression vector for the huIgG1 Fc-huBDCA2 fusion protein, and its cDNA insert sequence was confirmed. The estimated open reading frame, coded by pEAG2421, is shown below: [ka] κ light chain signal sequence: residues 1-19 above (italicized) Human IgG1 Fc: Residues 20-250 as listed above G4S linker: Residues 251-255 (bold) huBDCA2 external domain: residues 256-424 (underlined) above.

[0251] To construct an expression vector for the muIgG2a Fc-huBDCA2 fusion protein, a 0.53kb XmaI-BamHI fragment from pEAG2417 and a 0.75kb NotI-XmaI fragment from pEAG1442 (containing engineered mouse IgG2a Fc whose secretion is driven by an in-frame engineered mouse κ light chain signal sequence) were ligated with a 1.89kb BamHI-XbaI vector skeleton fragment and a 4.17kb XbaI-NotI vector skeleton fragment from the expression vector pV90 to produce pEAG2423, and its cDNA insert sequence was confirmed. The putative open reading frame encoded by pEAG2423 is shown below: [ka] κ light chain signa...

Claims

[Claim 1] The method or isolated antibody according to the specification.

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