BDCA2 monoclonal antibody, fusion protein composed of same and TACI, and method and use therefor

Abstract

Provided are a monoclonal antibody specifically binding to BDCA2, and a bispecific fusion protein of BDCA2 and TACI constructed on the basis of the BDCA2 monoclonal antibody. Also provided are an immunoconjugate comprising the antibody, a bispecific molecule and a pharmaceutical composition, and diagnostic and therapeutic methods using the anti-BDCA2 antibody and BDCA2 / TACI bispecific fusion protein.

Description

BDCA2 monoclonal antibody and fusion protein composed of BDCA2 and TACI and methods and uses thereof Technical Field

[0001] The present invention belongs to the fields of molecular biology and biological products. Specifically, the present invention relates to monoclonal antibodies or fragments thereof, and bispecific fusion proteins comprising monoclonal antibodies or fragments thereof, as well as preparation methods and uses thereof. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease that affects multiple organs throughout the body, including the skin, joints, central nervous system, and kidneys, through immune complexes, autoantibodies, cellular immunity, and inflammatory responses (Kaul A, Gordon C, Crow MK, Touma Z, Urowitz MB, van Vollenhoven R, Ruiz-Irastorza G, Hughes G. Systemic lupus erythematosus. Nat Rev Dis Primers. 2016 Jun 16;2:16039.). Plasmacytoid dendritic cells (pDCs) are a special type of dendritic cells (DCs) that can sense nucleic acids through TLR7 or TLR9, producing large amounts of type I interferons (IFNs), triggering an innate immune response. (Saadeh D, Kurban M, Abbas O. Update on the role of plasmacytoid dendritic cells in inflammatory / autoimmune skin diseases. Exp Dermatol. 2016 Jun; 25(6): 415-21). In addition to pDCs, B cells also play an important role in the progression of SLE. B cells can not only secrete autoantibodies, but also act as antigen-presenting cells, affecting the function of T cells and then secreting a series of precursor inflammatory factors (Chan, VF., Tsang, HL., Tam, RY. et al. B-cell-targeted therapies in systemic lupus erythematosus. Cell Mol Immunol 10, 133–142 (2013)). Therefore, targeting pDCs and / or B cells is an effective approach to treat SLE.

[0003] BDCA2 (Blood DC antigen 2) is a C-type lectin receptor specifically expressed on the surface of human plasmacytoid dendritic cells (pDCs). It binds to the pDCs membrane surface receptor FcεRIγ, thereby activating the ITAM-regulated signaling pathway, causing downstream protein phosphorylation, and ultimately inhibiting CpG-induced pDCs from producing type I interferon (Gilliet M, Cao W, Liu YJ. Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nat Rev Immunol. 2008 Aug; 8(8): 594-606). Therefore, targeting BDCA2, further activating the BDCA2 downstream signaling pathway, and inhibiting the production of type I interferon is an effective approach for treating SLE.

[0004] TACI (Transmembrane activator and calcium-modulating cyclophilin ligand interactor) is a B cell surface receptor. Its extracellular region contains two cysteine-rich regions that can bind to BAFF (B cell activating factor of the tumor necrosis factor family) and APRIL (proliferation-inducing ligand). BAFF is a type II transmembrane protein that belongs to the TNF ligand superfamily and is primarily produced by myeloid cells such as monocytes, macrophages, neutrophils, and dendritic cells. BAFF exists in two main forms—membrane-bound and soluble—and its receptors include TACI, BCMA (B cell maturation antigen), and BAFF-R (BAFF receptor). APRIL is also a type II transmembrane protein that belongs to the TNF ligand superfamily and is primarily produced by myeloid cells. Unlike BAFF, APRIL is only secreted. APRIL and BAFF share two receptors: BCMA and TACI. TACI binds to BAFF and APRIL, inducing IgG-IgA class conversion in B cells and promoting the differentiation and survival of plasma cells. (Zhang Y, Li J, Zhang YM, Zhang XM, Tao J. Effect of TACI signaling on humoral immunity and autoimmune diseases. J Immunol Res. 2015; 2015: 247426.)

[0005] The present invention aims to construct a bifunctional molecule that simultaneously targets BDCA2 and BAFF / APRIL. On the one hand, it eliminates pDCs, activates the BDCA2 downstream signaling pathway, and inhibits the expression of type I interferon. On the other hand, it blocks the binding of BAFF / APRIL to its receptor, inhibiting the overactivation of B cells, potentially demonstrating a better therapeutic effect for SLE.

[0006] Summary of the Invention

[0007] The present invention provides novel anti-BDCA2 antibodies or antigen-binding fragments thereof, which have advantages such as high affinity and high specificity for human and cynomolgus monkey BDCA2. The present invention also provides a novel bispecific fusion protein that simultaneously targets BDCA2 and BAFF / APRIL. Polynucleotides encoding the antibodies, bispecific fusion proteins, or fragments thereof, vectors comprising the polynucleotides, host cells comprising the polynucleotides or vectors, methods for treating diseases associated with BDCA2 and / or BAFF / APRIL using the antibodies or bispecific fusion proteins, and uses of the antibodies or bispecific fusion proteins in treating, preventing, and / or diagnosing diseases associated with BDCA2 and / or BAFF / APRIL in individuals are also provided. The anti-BDCA2 antibodies, BDCA2×BAFF / APRIL bispecific fusion proteins, or antigen / target-binding fragments thereof provided herein can be used as stand-alone therapies or in combination with other therapies or other agents for the treatment of related diseases, such as inflammatory conditions / diseases and / or autoimmune conditions / diseases.

[0008] In one aspect, the present invention provides an anti-BDCA2 antibody or antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region VH comprises three CDRs, HCDR1, HCDR2, and HCDR3, and the light chain variable region VL comprises three CDRs, LCDR1, LCDR2, and LCDR3, wherein

[0009] (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 1; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 2;

[0010] (ii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 3; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 4; or

[0011] (iii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 13; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 15; or

[0012] (iv) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 14; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 15; or

[0013] (v) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) to (iv), for example, the amino acid changes are mutations that enhance antibody stability;

[0014] Preferably, the CDRs are identified by the Kabat numbering scheme.

[0015] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention comprises

[0016] (i) HCDR1 as set forth in SEQ ID NO:33, HCDR2 as set forth in SEQ ID NO:34, HCDR3 as set forth in SEQ ID NO:35; LCDR1 as set forth in SEQ ID NO:36, LCDR2 as set forth in SEQ ID NO:37, and LCDR3 as set forth in SEQ ID NO:38; or

[0017] (ii) HCDR1 as set forth in SEQ ID NO:39, HCDR2 as set forth in SEQ ID NO:40 or 49, HCDR3 as set forth in SEQ ID NO:41; LCDR1 as set forth in SEQ ID NO:42 or 48, LCDR2 as set forth in SEQ ID NO:43, and LCDR3 as set forth in SEQ ID NO:44; or

[0018] (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) or (ii), for example, the amino acid changes are mutations that enhance antibody stability.

[0019] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the heavy chain variable region VH

[0020] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 3, 13 or 14; or

[0021] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 3, 13 or 14.

[0022] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, the light chain variable region VL

[0023] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4 or 15; or

[0024] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 4 or 15.

[0025] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention,

[0026] (i) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0027] (ii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0028] (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0029] (iv) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0030] In some embodiments of the antibodies or antigen-binding fragments thereof of the present invention, VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0031] (i).SEQ ID NO:1 and SEQ ID NO:2;

[0032] (ii).SEQ ID NO:3 and SEQ ID NO:4;

[0033] (iii). SEQ ID NO: 13 and SEQ ID NO: 15;

[0034] (iv). SEQ ID NO:14 and SEQ ID NO:15.

[0035] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise an Fc or heavy chain constant region CH, for example, the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably an Fc region of IgG1; or the antibody heavy chain constant region CH is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably an heavy chain constant region of IgG1. In some more specific embodiments, the heavy chain constant region CH (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 5; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 5.

[0036] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain constant region with enhanced ADCC and / or extended half-life and / or increased affinity for FcRn, for example, having an altered glycosylation pattern, for example, hypofucosylated or afucosylated, or comprising an increased bisecting GlcNac structure, and / or, for example, carrying an amino acid substitution combination of M252Y / S254T / T256E according to EU numbering. In a specific embodiment, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54.

[0037] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention further comprise a light chain constant region, for example, a lambda or kappa light chain constant region. In some more specific embodiments, the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 6; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 6.

[0038] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, 11, 16 or 17, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0039] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 10, 12 or 18, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

[0040] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein

[0041] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0042] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0043] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0044] (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0045] In some more specific embodiments, the heavy chain and light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO:

[0046] (i).SEQ ID NO:9 and SEQ ID NO:10;

[0047] (ii). SEQ ID NO: 11 and SEQ ID NO: 12;

[0048] (iii). SEQ ID NO: 16 and SEQ ID NO: 18;

[0049] (iv). SEQ ID NO: 17 and SEQ ID NO: 18.

[0050] In some embodiments, the constant regions of the antibodies of the invention are afucosylated or have reduced fucosylation.

[0051] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are humanized antibodies or chimeric antibodies. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are monoclonal antibodies.

[0052] In some embodiments, the antigen-binding fragment of the present invention is selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (eg, scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

[0053] In another aspect of the present invention, a bispecific fusion protein that specifically binds to human BDCA2 and human BAFF and APRIL is provided, which comprises (i) a first target binding region that specifically binds to BDCA2, and (ii) a second target binding region that specifically binds to BAFF and / or APRIL.

[0054] In some embodiments of the bispecific fusion protein molecules of the present invention, the first target binding region and the second target binding region are connected by a linker, or are directly connected without a linker. In some more specific embodiments, the linker can be any universal flexible sequence known in the art or obtained in the future, typically a short flexible amino acid sequence. In some specific embodiments, the linker comprises 10-30 amino acids in length. In some embodiments, the linker is (GGGGS)n; (GSSSS)n; (GGGSG)n; (GGSGG)n; (GSGGG)n; (SGGGG)n; (GGTGS)n; (GTSPGG)n; (GNGGGS)n; (GGG)n; (DGGGS)n; (TGEKP)n; (GGRR)n; (EGKSSGSGSESKVD)n; (KESGSVSSEQLAQFRSLD)n; (GGRRGGGS)n; (LRQRDGERP)n; (LRQKDGGGSERP)n; (GSTSGSGKPGSGEGSTKG)n and / or G4S-GGSGG-G4S-SGGGG, etc., wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In one embodiment, the linker is (G4S)n or (GS4)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the linker is selected from GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4, and (G4S)4.

[0055] In some embodiments of the bispecific fusion protein molecules of the present invention, the first target binding region is an anti-BDCA2 antibody or antigen-binding fragment thereof or is derived from an anti-BDCA2 antibody or antigen-binding fragment thereof. Preferably, (i) the anti-BDCA2 antibody or antigen-binding fragment thereof is an anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention; or (ii) the anti-BDCA2 antibody or antigen-binding fragment thereof exhibits the same binding specificity as the anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention, e.g., binding affinity, avidity, binding epitope, effector function, etc.

[0056] In some embodiments of the bispecific fusion protein molecules of the present invention, the second target binding region comprises a BAFF / APRIL receptor or a functional fragment thereof that specifically binds to BAFF and APRIL. Preferably, the BAFF / APRIL receptor is a TACI molecule, for example, the TACI molecule (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 20; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 20; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 20.

[0057] In some more specific embodiments, the functional fragment of the BAFF / APRIL receptor comprises a CRD2 derived from TACI, such as a wild-type CRD2 of TACI, or a variant CRD2 comprising modifications relative to wild-type CRD2. In some more specific embodiments, the CRD2 of the TACI molecule (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 21; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 21; (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 21.

[0058] In some embodiments, the fusion protein molecule of the present invention comprises

[0059] (i) Chain A: anti-BDCA2 antibody light chain;

[0060] (ii) Chain B: an anti-BDCA2 antibody heavy chain linked to TACI or a functional fragment thereof at the N-terminus or C-terminus, with or without a linker.

[0061] In some more specific embodiments, the fusion protein molecule consists of 2 chains A and 2 chains B; preferably, the fusion protein molecule consists of 2 identical chains A and 2 identical chains B, that is, the fusion protein molecule is a homodimer.

[0062] In some more specific embodiments, chain B comprises the following elements in order from N-terminus to C-terminus:

[0063] (i) anti-BDCA2 heavy chain, linker and TACI; or

[0064] (ii) TACI, a linker, and an anti-BDCA2 heavy chain; or

[0065] (iii) anti-BDCA2 heavy chain, linker, TACI and polyserine residues, or

[0066] (iv) TACI, polyserine residue sequence, linker, and anti-BDCA2 heavy chain;

[0067] Herein, TACI is, for example, a full-length TACI molecule or a fragment thereof.

[0068] wherein the linker is preferably GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4 or (G4S)4; wherein the polyserine residue is SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or SSSSSSSSSS.

[0069] And optionally, the linker may be absent.

[0070] In some more specific embodiments, chain B comprises the following elements in order from N-terminus to C-terminus:

[0071] (i) anti-BDCA2 heavy chain, linker and CRD2 domain; or

[0072] (ii) a CRD2 domain, a linker, and an anti-BDCA2 heavy chain; or

[0073] (iii) anti-BDCA2 heavy chain, linker, CRD2 domain, and polyserine residues; or

[0074] (iv) CRD2 domain, polyserine residue sequence, linker, and anti-BDCA2 heavy chain;

[0075] wherein the linker is preferably GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4 or (G4S)4; wherein the polyserine residue is SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or SSSSSSSSSS.

[0076] And optionally, the linker may be absent.

[0077] In some more specific embodiments, chain A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some more specific embodiments, chain B comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 22-32 and 50-51 and 53, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0078] In some more specific embodiments, chain A comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and chain B comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NOs: 22-32 and 50-51 and 53, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0079] In some more specific embodiments, chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and chain B comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 22-32 and 50-51 and 53.

[0080] In some embodiments, the fusion protein molecule of the present invention comprises (e.g., chain B comprises) an antibody heavy chain constant region with enhanced ADCC and / or extended half-life and / or increased affinity for FcRn, for example, having an altered glycosylation type, for example, low fucosylation or afucosylation or comprising an increased bisecting GlcNac structure, and / or, for example, carrying an amino acid substitution combination of M252Y / S254T / T256E according to EU numbering. In a more specific embodiment, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54.

[0081] In some more specific embodiments, the fusion protein molecules of the present invention comprise an afucosylated or hypofucosylated antibody heavy chain constant region. In one embodiment, the anti-BDCA2 antibody or fragment thereof, or the BDCA2×BAFF / APRIL bispecific fusion protein or target binding fragment thereof of the present invention can inhibit excessive activation of plasmacytoid dendritic cells and / or eliminate plasmacytoid dendritic cells, inhibit IFNα release, inhibit B cell proliferation, treat or alleviate inflammatory conditions / diseases and / or autoimmune conditions / diseases, such as systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjögren's syndrome, ulcerative colitis, neuromyelitis optica, neuromyelitis spectrum disorder, primary Sjögren's syndrome, and myasthenia gravis.

[0082] In one embodiment, the anti-BDCA2 antibody or fragment thereof, or the BDCA2×BAFF / APRIL bispecific fusion protein or target binding fragment thereof of the present invention can treat tumors, for example, hematopoietic tumors.

[0083] In a third aspect, the present invention also provides a polynucleotide (nucleic acid) encoding the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein or antigen / target binding fragment thereof of the present invention, and a vector comprising the polynucleotide, preferably an expression vector.

[0084] In a fourth aspect, the present invention provides a host cell comprising the polynucleotide or vector of the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell commonly used in the art.

[0085] In a fifth aspect, the present invention provides a method for producing the anti-BDCA2 antibody or BDCA2×BAFF / APRIL bispecific fusion protein of the present invention, comprising the steps of (i) culturing the host cell of the present invention under conditions suitable for expressing the antibody or fusion protein of the present invention, and optionally, (ii) recovering the antibody or fusion protein of the present invention.

[0086] In a sixth aspect, the present invention provides a kit, pharmaceutical composition, combination product, or manufactured product comprising an anti-BDCA2 antibody or BDCA2 x BAFF / APRIL bispecific fusion protein of the present invention. In one embodiment, the pharmaceutical composition, combination product, or manufactured product provided herein further comprises an additional therapeutic agent, and optionally, a pharmaceutical excipient; preferably, the additional therapeutic agent is selected from anti-inflammatory agents, cytotoxic agents, immunosuppressants, other antibodies, small molecule drugs, and the like.

[0087] In a seventh aspect, the present invention provides uses of the anti-BDCA2 antibodies or BDCA2×BAFF / APRIL bispecific fusion proteins, kits, pharmaceutical compositions, combination products, or manufactured products of the present invention for treating, preventing, and / or diagnosing diseases associated with BDCA2 and / or diseases associated with BAFF / APRIL. In one embodiment, diseases associated with BDCA2 include systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, and ulcerative colitis. In one embodiment, diseases associated with BAFF / APRIL are, for example, systemic lupus erythematosus, neuromyelitis optica, neuromyelitis spectrum disorders, rheumatoid arthritis, IgA nephropathy, autoimmune glomerulonephritis, primary Sjögren's syndrome, multiple sclerosis (e.g., relapsing-remitting form), and myasthenia gravis (e.g., systemic form).

[0088] In one embodiment, the present invention provides an anti-BDCA2 antibody or BDCA2×BAFF / APRIL bispecific fusion protein, corresponding polynucleotide, vector, host cell, kit, pharmaceutical composition, combination product or article of manufacture for use in vivo or in vitro for one or more of the following:

[0089] - binds to human BDCA2 and human BAFF / APRIL; or

[0090] - activates the human BDCA2 signaling pathway; or

[0091] - Blockade of the BAFF / APRIL signalling pathway; or

[0092] - inhibiting the expression of type I interferon; or

[0093] -Exhibit effector functions against BDCA2, such as ADCC, ADCP, CDC, etc.;

[0094] - Shows neutralizing effect against BAFF / APRIL molecules;

[0095] - Kill cells expressing human BDCA2 protein; or

[0096] - Eliminate pDC cells in vivo

[0097] -Inhibit local or systemic inflammatory and / or immune responses.

[0098] In one embodiment, the present invention provides for use of an anti-BDCA2 antibody or BDCA2×BAFF / APRIL bispecific fusion protein, corresponding polynucleotides, vectors, host cells, kits, pharmaceutical compositions, combination products, or articles of manufacture in the preparation of a medicament for treating, preventing, and / or diagnosing systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, neuromyelitis spectrum disorder, primary Sjögren's syndrome, and / or myasthenia gravis.

[0099] In an eighth aspect, the present invention provides methods for inhibiting excessive activation of plasmacytoid dendritic cells and / or clearing plasmacytoid dendritic cells, methods for inhibiting IFNα release, methods for inhibiting B cell proliferation, and methods for treating or alleviating inflammatory disorders / diseases and / or autoimmune disorders / diseases, the methods comprising administering a therapeutically effective amount of an anti-BDCA2 antibody or BDCA2×BAFF / APRIL bispecific fusion protein of the present invention, or a pharmaceutical composition, combination product, or preparation of the present invention to a patient in need thereof. Description of the drawings:

[0100] FIG1 shows a diagram of the bispecific fusion protein structure.

[0101] FIG2 shows the binding of various anti-BDCA2 chimeric antibodies to cell lines expressing human BDCA2 protein.

[0102] FIG3 shows the killing ability of various anti-BDCA2 chimeric antibodies against cell lines expressing human BDCA2 protein via ADCC.

[0103] FIG4 shows the inhibitory effects of various anti-BDCA2 chimeric antibodies on TLR9 agonist-stimulated PBMCs, as shown by changes in the amount of IFN-α produced.

[0104] Figures 5a and 5b show the killing of cells expressing human BDCA2 protein by anti-BDCA2 humanized antibodies via ADCC.

[0105] FIG6 shows the inhibitory effect of anti-BDCA2 humanized antibodies on TLR9 agonist-stimulated PBMCs, as shown by changes in the amount of IFN-α produced.

[0106] Figure 7 shows the effect of anti-BDCA2 humanized antibody on pDC depletion in the huHSC-NCG mouse model. The number and percentage of pDC in the peripheral blood and the number and percentage of pDC in the spleen of treated mice are shown in panels a, b, c, and d, respectively.

[0107] FIG8 shows the results of a bispecific fusion protein sandwich ELSA assay.

[0108] FIG9 shows that bispecific fusion proteins and bispecific fusion proteins with different linker peptides inhibit the proliferation of B cells.

[0109] FIG10 shows the killing effect of bispecific fusion proteins and bispecific fusion proteins with different connecting peptides on cells expressing human BDCA2 protein.

[0110] FIG11 shows the inhibitory effects of bispecific fusion proteins and bispecific fusion proteins with different linker peptides on TLR9 agonist-stimulated PBMCs, as shown by changes in the amount of IFN-α produced.

[0111] Figures 12a-12d show the effect of the bispecific fusion protein on pDC depletion in the huHSC-NCG mouse model. The number and percentage of pDC in the peripheral blood and the number and percentage of pDC in the spleen of treated mice are shown in Figures 12a, 12b, 12c, and 12d, respectively.

[0112] Figures 13a-13e show the effects of the bispecific fusion protein in the KLH-induced delayed-type hypersensitivity (DTH) model in BALB / c mice.

[0113] FIG14 shows that a bispecific fusion protein having amino acid substitutions M252Y / S254T / T256E (YTE mutation) in the Fc region inhibits B cell proliferation.

[0114] FIG15 shows the killing effect of the bispecific fusion protein having a YTE mutation in the Fc region on cells expressing human BDCA2 protein.

[0115] FIG16 shows the inhibitory effect of the bispecific fusion protein having a YTE mutation in the Fc region on TLR9 agonist-stimulated PBMCs, as shown by changes in the amount of IFN-α produced.

[0116] Figures 17a-17d show the effect of bispecific fusion proteins with a YTE mutation in the Fc region on pDC depletion in the huHSC-NCG mouse model. The number and percentage of pDC in the peripheral blood and the number and percentage of pDC in the spleen of treated mice are shown in Figures 17a, 17b, 17c, and 17d, respectively.

[0117] Figures 18a-d show the effects of the bispecific fusion protein with a YTE mutation in the Fc region in the KLH-induced B-hFcRn mouse delayed-type hypersensitivity (DTH) model and the detection of antibody concentrations in mouse serum.

[0118] Figure 19a-h shows the bispecific fusion protein in MOG 35-55 Drug efficacy in the induced C57BL / 6J experimental allergic encephalomyelitis (EAE) model.

[0119] FIG20 shows the PK assay of bispecific fusion proteins with YTE mutation in the Fc region in B-hFcRn mice.

[0120] FIG21 shows the PK assay of the bispecific fusion protein with YTE mutation in the Fc region in cynomolgus monkeys. DETAILED DESCRIPTION

[0121] I. Definition

[0122] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0123] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0124] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0125] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0126] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0127] The term "BDCA2" herein refers to a type II C-type lectin BDCA2 specifically expressed on pDCs, consisting of a single extracellular carbohydrate recognition domain at the C-terminus, a transmembrane region from asparagine residue 45 to isoleucine residue 213, and a short cytoplasmic tail at the N-terminus (without a signaling motif). BDCA2 sends intracellular signals through the related transmembrane adaptor FcεRIγ. Antibody-mediated binding of BDCA2 leads to the recruitment of 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 linkers (BLNK), Bruton's tyrosine kinase (BTK), and phospholipase Cγ2 (PLCγ2), resulting in Ca 2+Activation of BDCA2 causes it to bind to the cell surface FcεRIγ receptor, which then internalizes and activates downstream signaling via the ITAM of the FcεRIγ receptor, inhibiting TLR7- and TLR9-induced type I interferon expression while simultaneously downregulating FcεRIγ receptor expression on the surface of pDCs. The BDCA2 antibody of the present invention binds to BDCA2 and causes its internalization, thereby producing therapeutic effects on a range of diseases, such as systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, and ulcerative colitis. In addition, the BDCA2 antibody of the present invention also has therapeutic effects on some hematopoietic tumors, especially BDCA2-positive hematological tumors, such as blastic plasmacytoid dendritic cell neoplasm. As used herein, the term "BDCA2-mediated disease" refers to a disease in which BDCA2 is involved in the onset, progression, or persistence of the disease, for example, involving BDCA2 activation (e.g., aberrant activation or overactivation), and in which BDCA2 involvement directly or indirectly results in at least one of the following: disease initiation / onset; increased / progressive pathological changes; expanded location / area of ​​disease impact; increased damage to the body caused by the disease; increased suffering caused by the disease; insensitivity / resistance to treatment measures; decreased propensity for spontaneous recovery of the disease and worsening of the disease prognosis. In some embodiments of the present invention, the BDCA2-mediated disease is an inflammatory disease, such as those described above; and in some embodiments of the present invention, the BDCA2-mediated disease is a tumor, such as those described above. The term "BDCA2" includes variants, isoforms, species homologs of human BDCA2, BDCA2 from other species, and analogs that share at least one common epitope with BDCA2, unless otherwise indicated. The term encompasses unprocessed, full-length BDCA2 as well as any form of BDCA2 produced by cell processing. The term encompasses "full-length," unprocessed BDCA2 as well as any form of BDCA2 resulting from intracellular processing, or any fragment thereof, such as a splice variant or allelic variant.In one embodiment, BDCA2 refers to full-length BDCA2 from humans or cynomolgus monkeys, or a fragment thereof (such as a mature fragment lacking a signal peptide).

[0128] "TACI," short for "Transmembrane activator and CAML-interactor," is one of the co-receptors for BAFF (B-cell activating factor, also known as BLyS, TNSF13B, THANK, TALL-1, zTNF4) and APRIL (proliferation-inducing ligand). Other receptors include BCMA and BAFF-R. Their ligand-receptor interactions can activate downstream pathways, promote the development and proliferation of B lymphocytes, and increase the expression of various immunoglobulins in serum. TACI is also expressed on the surface of mature T lymphocytes, and thus cellular immunity is also regulated by this mechanism. Overexpression of BAFF and APRIL is a cause of numerous autoimmune diseases, and inhibiting BAFF and APRIL is an effective therapeutic approach for autoimmune diseases such as systemic lupus erythematosus, neuromyelitis optica, neuromyelitis spectrum disorders, rheumatoid arthritis, IgA nephropathy, primary Sjögren's syndrome, relapsing-remitting multiple sclerosis, and generalized myasthenia gravis. At the same time, since BAFF and APRIL have a promoting effect on certain hematopoietic tumors, inhibition of BAFF and APRIL can also be used to treat such hematopoietic tumors. The term "disease associated with BAFF / APRIL" refers to any condition caused by, aggravated by, or otherwise associated with abnormal expression (e.g., increased expression) or abnormal activity of BAFF and / or APRIL. In one embodiment, the disease associated with BAFF / APRIL is an inflammatory disease, such as the inflammatory diseases described above; in some embodiments of the present invention, the disease associated with BAFF / APRIL is a tumor, such as the tumors described above.

[0129] Due to the high affinity of TACI for BAFF and APRIL, soluble TACI (e.g., the extracellular portion of TACI) can be used as a blocking molecule to bind to and block the binding between BAFF or APRIL and cell membrane receptors. The extracellular region of TACI contains two cysteine-rich domains (CRDs), but further studies have found that the N-terminal CRD is not involved in ligand binding. Therefore, it is believed that the second CRD, CRD2 (also referred to herein as D2), is the key part that determines the specific binding of TACI to BAFF / APRIL. In the context of the present invention, unless otherwise specified, the term "TACI" can cover any functional variant, truncation or variant thereof, and fragment or variant thereof of the natural TACI molecule, for example, the extracellular region of the natural TACI molecule or its functional variant, CRD1-2 domain, CRD2 domain, CRD2 domain N-terminal truncation, CRD2 domain C-terminal truncation, fragment consisting of amino acid residues 30-118, etc.

[0130] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0131] The term "human plasmacytoid dendritic cells (pDCs)" is a specialized population of bone marrow-derived cells that secrete type I interferon (IFN) in response to toll-like receptor (TLR) ligands. pDCs are a key link in cellular immunity, involved in the induction and initiation of immune responses and antigen tolerance. pDCs differ from ordinary DCs in that, for example, they acquire antigens through receptor-mediated endocytosis, among other things. Type I interferon produced by pDCs activated by pattern recognition receptors can also present antigens, thereby linking innate and acquired immune responses. At the same time, overactivation of pDCs may also have a negative impact on the immune response process, for example, potentially leading to autoimmune diseases.

[0132] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0133] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize BDCA2 activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the target peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0134] As used herein, the term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, with respect to its origin or source of derivation, is separated from naturally associated components with which it is found in nature, is substantially free of other proteins from the same species, is expressed by cells from a different species, or is not found in nature. Polypeptides that are chemically synthesized or synthesized in a cellular system different from the cells from which they naturally originate can be "isolated" from their naturally associated components. Proteins can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0135] The term "specific binding" or "specifically binds" when referring to the interaction of an antibody, binding protein, or peptide with a second chemical substance means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the second chemical substance; for example, an antibody recognizes and binds to a specific protein structure, but not proteins in general. If the antibody is specific for epitope "A", the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. Consistent with the present disclosure, a specific binding protein with a K of 10 nM or less D Binds to the corresponding antigen, e.g., 1 nM or less. The term "K D " refers to the equilibrium dissociation constant (the reciprocal of the equilibrium binding constant) and is used herein according to the definition provided in the art. The K of an antibody or binding protein binding to the corresponding antigen DValues ​​can be determined by known methods, including but not limited to fluorescence titration, competitive ELISA, calorimetry, such as isothermal titration calorimetry (ITC), flow cytometer titration analysis (FACS titration), bio-layer interferometry (BLI) and surface plasmon resonance (BIAcore). As used herein, the terms "antigen binding site" and "antigen binding specificity" are used interchangeably to refer to the region where an antibody actually binds to an antigen.

[0136] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0137] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0138] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0139] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0140] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. In some preferred embodiments of the present invention, the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0141] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0142] The term "single domain antibody" generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from a fish IgNAR) can confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include single domain antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks) (WO 2005 / 035572). Single domain antibodies derived from camelids are also referred to as VHH or "nanobodies" in this application. The term "VHH" or "VHH domain" is used herein to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, i.e., it consists of only one heavy chain variable region, comprising only one chain FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from C-terminus to N-terminus, also known as a single variable domain (sVD). Unlike conventional VHs of four-chain immunoglobulins, VHH domains do not need to be paired with light chain variable domains to form an antigen binding site. Single-domain antibodies are the smallest units currently known that can bind to target antigens. In some cases, for therapeutic applications of VHH, it is desirable to reduce its immunogenicity. Preferably, in some embodiments, the present invention provides antibodies or bispecific fusion proteins comprising humanized VHH domains.

[0143] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain, such as in the case of the Fc region of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies comprises a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies lacks a hinge region, but comprises a CH2 domain, a CH3 domain, and a CH4 domain. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Variant Fc regions with amino acid residue replacements in the Fc portion to alter antibody effector functions are known in the art (see, e.g., Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody can mediate one or more effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance rate of antibodies and antigen-antibody complexes. In some cases, these effector functions are ideal for therapeutic antibodies, but in other cases may be unnecessary or even harmful, depending on the therapeutic purpose. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to FcγR and complement C1q, respectively. In another embodiment, at least one amino acid residue is replaced in the constant region of an antibody, such as the Fc region of an antibody, thereby altering the effector functions of the antibody. Dimerization of two identical heavy chains of immunoglobulins is mediated by dimerization of the CH3 domains and stabilized by disulfide bonds in the hinge region connecting the CH1 constant domain to the Fc constant domains (e.g., CH2 and CH3). The anti-inflammatory activity of IgG depends on the sialylation of the N-linked glycans of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been determined so that appropriate IgG1 Fc fragments can be generated, thereby generating fully recombinant sialylated IgG1 Fc with greatly enhanced potency (see, Anthony et al., Science, 320: 373-376 (2008)). Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index.

[0144] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0145] "Complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy chain are typically referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The IMGT database (http: / / imgt.cines.fr / ) and the North CDR definition were based on affinity propagation clustering using a large number of crystal structures.

[0146] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0147] Unless otherwise indicated, the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations), and the terms "CDR" or "CDR sequence" encompass CDR sequences determined in any of the above ways.

[0148] CDRs can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplified CDRs of the present invention). In one embodiment, the CDRs of the antibodies of the present invention are positioned according to the Kabat numbering scheme.

[0149] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0150] Unless otherwise indicated, throughout the present invention, references to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues) refer to positions numbered according to the Kabat numbering system.

[0151] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0152] The term "multispecific" refers to the binding properties of an antibody / fusion protein / binding molecule having at least two antigen binding sites, each of which binds to a different epitope / binding site of the same target molecule / antigen or to a different epitope / binding site of a different target molecule / antigen. In one embodiment, the present invention provides multispecific antibodies / fusion proteins / binding molecules having binding specificity for a first target molecule / antigen and a second target molecule / antigen, also referred to as "bispecific antibodies / fusion proteins / binding molecules". In some embodiments, the bispecific antibodies / fusion proteins / binding molecules of the present invention comprise four binding sites and are typically tetravalent binding proteins. In some embodiments, the bispecific antibodies / fusion proteins / binding molecules of the present invention comprise six binding sites and are typically hexavalent binding proteins.

[0153] The term "agonism" refers to an increase in some parameter (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, this term includes substances that increase the activity of a given molecule by at least 5%, 10%, 20%, 30%, 40% or more. Thus, the agonism need not be 100%.

[0154] As used herein, "an antibody's activation / activation activity" or "internalization-promoting activity" against BDCA2 refers to the ability of the antibody to bind to BDCA2, promote its internalization following binding to the FcεRIγ receptor, and promote downstream signaling. Assays for measuring this activation activity can be conventional assays known in the art.

[0155] As used herein, "neutralizing activity" or "blocking activity" of an antibody against BAFF / APRIL refers to the ability of the antibody to block the signaling pathway induced by the binding of BAFF / APRIL to its native receptor. This activity can be determined using conventional assays known in the art, such as FACS assays, or reporter gene-based signaling pathway blocking assays.

[0156] Calculation of sequence identity between sequences is performed as follows.

[0157] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0158] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com), which has been integrated into the GAP program in the GCG software package. The Blossum 62 matrix or the PAM250 matrix is ​​used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6 is used. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0159] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0160] Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0161] As used herein, the term "k on” (also referred to as “Kon”, “kon”) refers to the association rate constant for the binding of a binding protein (e.g., an antibody) to an antigen to form a binding complex (e.g., an antibody / antigen complex) as known in the art. on ” is also referred to as the term “association rate constant” or “ka”, as used interchangeably herein. This value represents the rate of binding of an antibody to its target antigen or the rate of complex formation between the antibody and the antigen, as shown in the following formula:

[0162] Antibody ("Ab") + antigen ("Ag") → Ab-Ag.

[0163] As used herein, the term "k off ” (also known as “Koff”, “koff”) refers to the rate constant for the dissociation of a binding protein (e.g., an antibody) from a binding complex (e.g., an antibody / antigen complex), or the “dissociation rate constant,” as known in the art. This value represents the rate of dissociation of an antibody from its target antigen, or the rate at which the Ab-Ag complex separates into free antibody and antigen over time, as shown in the following formula:

[0164] Ab+Ag←Ab-Ag.

[0165] As used herein, the term "K D ” (also known as “K d ”) is intended to mean the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by the dissociation rate constant (k off ) divided by the binding rate constant (k on The binding rate constant (k on ), dissociation rate constant (k off ) and the equilibrium dissociation constant (K D ) is used to express the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Fluorescence-based techniques can be used, providing high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental methods and instruments can be used, such as (Biomolecular Interaction Analysis) assay (e.g., instruments available from BIAcore International AB, GE Healthcare, Uppsala, Sweden). Using e.g. Biomembrane interferometry (BLI) using the RED96 system (Pall Forte Bio LLC) is another affinity determination technique. Alternatively, the RED96 system available from Sapidyne Instruments (Boise, Idaho) can be used. (Kinetic Exclusion Assay) Test.

[0166] The term "isolated nucleic acid" refers to a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that has been separated by human intervention from all or part of the polynucleotides with which it is associated in nature; is operably linked to polynucleotides with which it is not naturally associated; or occurs as part of a larger sequence that is not found in nature.

[0167] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicate together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, the present disclosure is intended to include other forms of expression vectors, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play equivalent roles.

[0168] As used herein, "transformation" refers to any process by which exogenous DNA enters a host cell. Transformation can be performed under natural or artificial conditions using various methods well known in the art. Transformation can rely on any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell to be transformed and may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. Cells that transiently express the inserted DNA or RNA for a limited time are also included.

[0169] The term "recombinant host cell" (or simply "host cell") means a cell into which exogenous DNA has been introduced. In one embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding an antibody, for example, a host cell as described in U.S. Patent No. 7,262,028. These terms are intended to refer not only to a specific subject cell, but also to the offspring of such a cell. Because certain modifications may occur in offspring due to mutations or environmental influences, these offspring may actually be different from the parent cell, but are still included in the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotes and eukaryotic cells selected from any kingdom of life. In another embodiment, eukaryotic cells include protozoa, fungi, plants, and animal cells. In another embodiment, host cells include, but are not limited to, the prokaryotic cell line Escherichia coli; mammalian cell lines CHO, HEK 293, Jurkat, COS, NS0, SP2, and PER.C6; insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

[0170] The term "treat" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

[0171] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0172] The term "effective amount" refers to an amount or dosage of an antibody or composition of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; weight, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0173] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or composition can vary depending on a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0174] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0175] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0176] The terms "linker", "connecting peptide", "linker" or "peptide linker" are used interchangeably herein and refer to a peptide comprising one or more consecutive amino acids, such as small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). The connecting peptide typically comprises 5-50 amino acids in length, for example, 10, 15, 20, 25, or 30 amino acids in length. Those skilled in the art will appreciate that many commonly used linkers can be used in embodiments of the present invention.

[0177] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0178] II. Anti-BDCA2 Antibodies

[0179] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2. The terms "anti-BDCA2 antibody," "anti-BDCA2," "BDCA2 antibody," or "antibody that binds to BDCA2" refer to antibodies that bind to the BDCA2 protein or fragment thereof with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting BDCA2.

[0180] In some embodiments, antibodies of the invention bind to human or cynomolgus monkey BDCA2 protein. In some embodiments, antibodies of the invention bind to HEK293 / hBDCA2 / FcεRIγ cells and HEK293 / Cyno BDCA2 / FcεRIγ cells. In some embodiments, antibodies of the invention inhibit the release of the cytokine IFNα from human PBMCs stimulated by TLR9 agonists. In some embodiments, antibodies of the invention exhibit potent effector function.

[0181] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of BDCA2 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any BDCA2 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-BDCA2 antibodies can be produced by methods known in the art.

[0182] Antibodies derived from rodents (such as mice) may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use causes the human body to produce an immune response against the therapeutic antibody. Such immune responses at least lead to loss of therapeutic efficacy, and in severe cases, lead to potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, the retention of intact rodent variable regions in chimeric antibodies may still cause harmful immunogenicity in patients. Transplanting the complementary determining region (CDR) loops of rodent variable domains onto human frameworks (i.e., humanization) has been used to further minimize rodent sequences (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534). In some embodiments, the antibodies of the present invention are chimeric antibodies. In some preferred embodiments, the antibodies of the present invention are humanized antibodies. In some preferred embodiments of the present invention, the affinity of the murine antibody, chimeric antibody and / or human antibody of the present invention to the antigen human BDCA2 or cynomolgus monkey BDCA2 is 1.0×10 -6 M or lower K D value, 5.0×10-7 M or lower K D value, 2.5×10 -7 M or lower K D value, 1.0×10 -7 M or lower K D value, 5.0×10 -8 M or lower K D value, 2.5×10 -8 M or lower K D value, 1.0×10 -8 M or lower K D value, 5.0×10 -9 M or lower K D value, 2.5×10 -9 M or lower K D value, 1.0×10 -9 M or lower K D value, 5.0×10 -10 M or lower K D value, 2.5×10 -10 M or lower K D value, 1.0×10 -10 M or lower K D value, 5.0×10 - 11 M or lower K D value, 2.5×10 -11 M or lower K D value, 1.0×10 -11 M or lower K D value, 5.0×10 -12 M or lower K D value, 2.5×10 -12 M or lower K D value, 1.0×10 -12 M or lower K D value.

[0183] The amino acid sequences of the light chain and heavy chain variable regions and CDRs of the anti-BDCA2 murine antibody of the present invention are shown in Table 2.

[0184] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0185] In some embodiments, the chimeric BDCA2 antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant regions comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, and IgG4, with IgG1 being preferred.

[0186] In some embodiments, chimeric BDCA2 antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids into expressing cells. BDCA2 binding of such "mixed and matched" antibodies can be tested using the above-described binding assays and other conventional binding assays (e.g., ELISA).

[0187] The present invention mixes and pairs various chimeric heavy chain and light chain expression plasmids and transfects them into expression cells to obtain anti-BDCA2 chimeric antibodies.

[0188] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0189] In some embodiments, the amino acid changes comprise amino acid deletions, insertions, or substitutions. In some embodiments, the anti-BDCA2 antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still share at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions when compared to the CDR regions depicted in the specified sequences. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions when compared to the framework regions in the specified sequences.

[0190] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0191] Thus, in some embodiments, an antibody of the invention comprises a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination of Table A below.

[0192] Table A

[0193] In some embodiments, an antibody of the invention comprises a VH and a VL as shown in any combination of Table B below.

[0194] Table B

[0195] In some embodiments, the BDCA2 antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof, as described in the following sections.

[0196] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0197] Regarding modifications of the constant domain / Fc region, please refer to the detailed description of modifications below in this specification.

[0198] In some embodiments, the anti-BDCA2 antibodies or antigen-binding fragments thereof of the present invention have one or more of the following characteristics:

[0199] sex:

[0200] (i) exhibiting the same or similar binding affinity and / or specificity for BDCA2 as the antibodies of the present invention;

[0201] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the present invention to BDCA2;

[0202] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0203] (iv) competing with the antibodies of the present invention for binding to BDCA2;

[0204] (v) possess one or more biological properties of an antibody of the invention.

[0205] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0206] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0207] III. Bispecific Fusion Proteins of the Invention

[0208] The following describes in detail the components of the bispecific fusion proteins of the present invention and the bispecific fusion proteins composed thereof. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of the technical features of these components is contemplated by the present invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the bispecific fusion proteins of the present invention may include any combination of such features.

[0209] The terms "bispecific fusion protein that specifically binds to BDCA2 and BAFF / APRIL", "bispecific antibody against BDCA2 and BAFF / APRIL", "anti-BDCA2×BAFF / APRIL bispecific fusion protein", "BDCA2×BAFF / APRIL bispecific antibody" and the like mentioned herein refer to bispecific fusion proteins that can simultaneously bind to the targets BDCA2 and BAFF / APRIL with sufficient affinity.

[0210] In one aspect, the present invention provides a bispecific fusion protein that specifically binds BDCA2 and BAFF / APRIL. In some embodiments, the bispecific fusion protein comprises a first target-binding region and a second target-binding region, wherein the first target-binding region specifically binds BDCA2, and the second target-binding region specifically binds BAFF and APRIL. In some embodiments, the bispecific fusion protein comprises an antibody (anti-BDCA2) or a fragment thereof that specifically binds BDCA2, and a soluble TACI or a fragment thereof that specifically binds BAFF / APRIL.

[0211] As used herein, when referring to "TACI" constituting part of a bispecific fusion protein, unless otherwise specified, it may refer to any form of the wild-type natural TACI molecule that retains specific binding to BAFF and APRIL, such as full-length, soluble fragments, domains, variants, fusion proteins comprising the same, and the like.

[0212] In some embodiments, the first target binding region and the second target binding region are connected via a linker, or are directly connected without a linker.

[0213] In certain specific embodiments of the bispecific fusion proteins of the present invention, the anti-BDCA2 and TACI moieties can be contained in tandem on the same polypeptide chain (e.g., linked in tandem on a single polypeptide chain via a linker and / or Fc region). In certain specific embodiments, the bispecific fusion proteins of the present invention further comprise an Fc region. In the bispecific fusion proteins of the present invention, the anti-BDCA2 and TACI moieties can be linked to the Fc region in a tandem arrangement comprising both anti-BDCA2 and TACI, optionally with a linker fragment.

[0214] In some preferred embodiments of the bispecific fusion proteins of the present invention, the first target binding region is an anti-BDCA2 antibody or antigen-binding fragment thereof that: (i) is an anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention; or (ii) exhibits the same binding specificity as an anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention.

[0215] In some embodiments of the bispecific fusion protein of the present invention, the second target binding region comprises a BAFF / APRIL receptor or a functional fragment thereof that specifically binds to BAFF and APRIL. Preferably, the BAFF / APRIL receptor is TACI.

[0216] In some specific embodiments of the bispecific fusion protein of the present invention, the second target binding region is TACI, which (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 20; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 20; or (iii) comprises or consists of an amino acid sequence that has one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 20.

[0217] In certain specific embodiments of the bispecific fusion proteins of the present invention, the second target-binding region is a fragment of a full-length TACI molecule. Preferably, the fragment is the extracellular region of the full-length TACI molecule, or a functional variant thereof; more preferably, the fragment is CRD2 of the full-length TACI molecule, for example, CRD2 of a wild-type TACI molecule, or a variant CRD2 comprising modifications relative to wild-type CRD2.

[0218] In some specific embodiments of the bispecific fusion protein of the present invention, the second target binding region is CRD2 of the TACI molecule, which (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 21; (iii) comprises or consists of an amino acid sequence that has one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 21.

[0219] In some embodiments, the bispecific fusion protein of the present invention comprises (i) chain A: an anti-BDCA2 antibody light chain; and (ii) chain B: an anti-BDCA2 antibody heavy chain linked to TACI or a functional fragment thereof at the N-terminus or C-terminus with or without a linker.

[0220] In some more specific embodiments, the fusion protein molecule consists of 2 chains A and 2 chains B; preferably, the fusion protein molecule consists of 2 identical chains A and 2 identical chains B, that is, the fusion protein molecule is a homodimer.

[0221] Immunoglobulin constant domain

[0222] According to the bispecific fusion protein of the present invention, it is preferably included an immunoglobulin constant domain, such as an Fc region consisting of an immunoglobulin constant region CH2 and CH3. In some embodiments of the present invention, the present invention thus provides a bispecific fusion protein comprising an Fc region polypeptide chain consisting of an immunoglobulin heavy chain constant region CH2-CH3. The immunoglobulin constant domains included in the bispecific fusion protein according to the present invention, the CH2 and CH3 constant domains included in the Fc region can be independently of each other immunoglobulin constant domains from human IgG, for example, the constant domains of human IgG1, IgG2, IgG3 or IgG4, preferably the constant domains of human IgG1. In addition, the immunoglobulin constant domains included in the bispecific fusion protein according to the present invention, including constant regions CH2 and CH3, can be independently of each other native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof, preferably native sequence constant domains.

[0223] In a preferred embodiment, the Fc region contained in the bispecific fusion protein according to the present invention is an Fc region sequence from human IgG1, or an amino acid sequence having at least 90%, 95% or 99% identity thereto, or an amino acid sequence having no more than 1-10 (preferably 1-5) amino acid residues altered therefrom.

[0224] In some specific embodiments, the bispecific fusion protein according to the present invention may comprise a polypeptide chain having two Fc regions, for example, wherein the Fc regions of the two homologous polypeptide chains are capable of pairing and dimerizing. In such an embodiment, the first Fc region polypeptide and the second Fc region polypeptide constituting the dimerized Fc region of the antibody can both be from IgG, such as the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably the Fc domain of human IgG1; and can be independently of each other a native sequence Fc region and a variant Fc region as defined herein, provided that the first Fc region polypeptide and the second Fc region polypeptide can pair and dimerize. Preferably, the first Fc region and the second Fc region are native sequence Fc regions, in particular human IgG1 native sequence Fc regions.

[0225] In some embodiments, the constant domain / Fc region of the present invention has one or more amino acid substitutions compared to a native human constant domain / Fc region sequence, preferably, the one or more amino acid substitutions enhance the ADCC and / or CDC effect of the antibody and / or extend the half-life in vivo and / or increase the affinity for an Fc receptor (e.g., FcRn). In one embodiment, the constant domain / Fc region of the present invention has an amino acid substitution of M252Y / S254T / T256E compared to a native human constant domain / Fc region sequence (amino acid position numbering is according to the EU numbering scheme).

[0226] In some embodiments, the constant domain / Fc region of the invention is hypofucosylated or afucosylated, or is an antibody or fragment thereof or multispecific binding molecule with increased bisecting GlcNac structures.

[0227] In some embodiments, the constant domain / Fc region of the invention has altered glycosylation and / or altered amino acid sequence, e.g., is hypofucosylated or afucosylated and has the amino acid substitution combination of M252Y / S254T / T256E.

[0228] Regarding the modification of the constant domain / Fc region of the bispecific fusion protein, please refer to the detailed description of the modification below in this specification.

[0229] connector

[0230] In the bispecific fusion protein according to the present invention, different target binding regions can be connected using a linker. There is no specific limitation on the linker that can be used in the bispecific fusion protein of the present invention. Those skilled in the art can easily determine the applicable linker sequence based on the components to be connected and the connection position.

[0231] In embodiments of the present invention, those skilled in the art can select different linkers based on the connection position of different target binding regions, i.e., whether connected to the N-terminus or C-terminus of the Fc region. In one embodiment, the Fc region polypeptide chain is connected to a single target binding region or two different target binding regions in series at the N-terminus via a linker, wherein the linker preferably comprises (one or two) cysteine ​​residues, thereby enabling the formation of (one or two pairs of) disulfide bonds when the two polypeptide chains comprising the Fc region are paired to stabilize the structure of the bispecific fusion protein thus formed. Therefore, in some embodiments, the Fc region polypeptide chain contained in the bispecific fusion protein of the present invention carries a linker sequence comprising "CPPC" at the N-terminus. In other embodiments, the Fc region polypeptide chain contained in the bispecific fusion protein of the present invention carries a hinge region from an immunoglobulin at the N-terminus, such as a hinge region amino acid sequence comprising "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" or "EPKSSDKTHTCPPCP"; more preferably, the amino acid sequence "EPKSCDKTHTCPPCP".

[0232] In a preferred embodiment of the present invention, the linker is a flexible connecting peptide preferably 5-50 amino acids in length, more preferably comprising glycine (G) and / or serine (S) and / or threonine (T) residues. In some specific embodiments, the linker has a length of 5-50 amino acids, for example, 10, 15, 20, 25 or 30 amino acids in length, or an amino acid length falling between any two integers. In some embodiments, the linker is (GGGGS)n; (GSSSS)n; (GGGSG)n; (GGSGG)n; (GSGGG)n; (SGGGG)n; (GGTGS)n; (GTSPGG)n; (GNGGGS)n; (GGG)n; (DGGGS)n; (TGEKP)n; (GGRR)n; (EGKSSGSGSESKVD)n; (KESGSVSSEQLAQFRSLD)n; (GGRRGGGS)n; (LRQRDGERP)n; (LRQKDGGGSERP)n; (GSTSGSGKPGSGEGSTKG)n and / or G4S-GGSGG-G4S-SGGGG, etc., wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In a specific embodiment, the linker is (G4S)n or (GS4)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some most preferred embodiments, the linker is GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4, or (G4S)4.

[0233] Bispecific fusion protein embodiments of the present invention

[0234] In some embodiments, the bispecific fusion protein of the present invention comprises (i) chain A: an anti-BDCA2 antibody light chain; and (ii) chain B: an anti-BDCA2 antibody heavy chain connected to TACI or a functional fragment thereof at the N-terminus or C-terminus without a linker.

[0235] In some more specific embodiments, chain B comprises the following elements in order from N-terminus to C-terminus:

[0236] (i) anti-BDCA2 heavy chain, linker and TACI; or

[0237] (ii) TACI, a linker, and an anti-BDCA2 heavy chain; or

[0238] (iii) anti-BDCA2 heavy chain, linker, TACI and polyserine residues, or

[0239] (iv) TACI, polyserine residue sequence, linker, and anti-BDCA2 heavy chain;

[0240] wherein TACI is a full-length TACI molecule or a fragment thereof;

[0241] wherein the linker is preferably GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4 or (G4S)4; wherein the polyserine residue is SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or SSSSSSSSSS.

[0242] And optionally, the linker may be absent.

[0243] In some more specific embodiments, chain B comprises the following elements in order from N-terminus to C-terminus:

[0244] (i) anti-BDCA2 heavy chain, linker and CRD2 domain; or

[0245] (ii) a CRD2 domain, a linker, and an anti-BDCA2 heavy chain; or

[0246] (iii) anti-BDCA2 heavy chain, linker, CRD2 domain, and polyserine residues; or

[0247] (iv) CRD2 domain, polyserine residue sequence, linker, and anti-BDCA2 heavy chain;

[0248] wherein the linker is preferably GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4 or (G4S)4; wherein the polyserine residue is SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or SSSSSSSSSS.

[0249] And optionally, the linker may be absent.

[0250] In some more specific embodiments, chain A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some more specific embodiments, chain B comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 22-32 and 50-51 and 53, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0251] In some more specific embodiments, chain A comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and chain B comprises, or consists of, the amino acid sequence set forth in any one of SEQ ID NOs: 22-32 and 50-51 and 53, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0252] In some more specific embodiments, chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and chain B comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 22-32 and 50-51 and 53.

[0253] In some embodiments, the bispecific fusion protein of the present invention may comprise two bispecific antigen-binding polypeptides (e.g., chain B) comprising an Fc region arranged in tandem, wherein the two bispecific antigen-binding polypeptides are capable of forming a dimer by, for example, dimerization of the Fc region. Preferably, the two bispecific antigen-binding polypeptides are identical.

[0254] In some preferred embodiments, the bispecific fusion protein of the present invention may comprise two pairs of polypeptides (e.g., chain A and chain B), each of which comprises two bispecific antigen-binding polypeptides comprising an Fc region (e.g., chain B), wherein the two bispecific antigen-binding polypeptides are capable of forming a dimer by, for example, dimerization of the Fc region. Preferably, the two bispecific antigen-binding polypeptides are identical, and more preferably, the two pairs of bispecific antigen-binding polypeptides are identical.

[0255] In one embodiment, the bispecific fusion protein of the present invention comprises an amino acid modification, such as an amino acid substitution, addition or deletion, preferably an amino acid substitution, more preferably a conservative amino acid substitution.

[0256] In one embodiment, the two identical bispecific antigen-binding polypeptides of the bispecific fusion protein of the present invention are non-covalently linked or covalently linked. In one embodiment, the covalent link is a disulfide bond.

[0257] Regarding modification, please refer to the detailed description below in this specification.

[0258] In one embodiment, the BDCA2×BAFF / APRIL bispecific fusion protein of the present invention or its target binding fragment can inhibit excessive activation of plasmacytoid dendritic cells and / or eliminate plasmacytoid dendritic cells, inhibit IFNα release, inhibit B cell proliferation, treat or alleviate inflammatory conditions / diseases and / or autoimmune conditions / diseases, such as systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, neuromyelitis spectrum disorder, primary Sjögren's syndrome, myasthenia gravis, and various combinations thereof.

[0259] IV. Modification of Anti-BDCA2 Antibodies or Fragments thereof or Multispecific Binding Molecules

[0260] The molecules of the invention (eg, anti-BDCA2 antibody molecules or fragments thereof or multispecific binding molecules) can be mutated to obtain desired properties.

[0261] For example, the FR regions contained in the variable regions of the molecules of the present invention can be modified to reduce their immunogenicity or to remove T cell epitopes.

[0262] In one embodiment, the amino acid modifications described herein occur in regions outside of the CDRs (e.g., in the FRs). In one embodiment, the substitutions are conservative substitutions. Conservative substitutions refer to substitutions of one amino acid with another amino acid within the same class, e.g., substitutions of an acidic amino acid with another acidic amino acid, substitutions of a basic amino acid with another basic amino acid, or substitutions of a neutral amino acid with another neutral amino acid.

[0263] In certain embodiments, substitutions occur in the CDR regions of an antibody. Typically, the variants obtained are modified (e.g., improved) in certain biological properties (e.g., increased affinity) relative to the parent antibody and / or will have certain biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity matured antibody.

[0264] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. An Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0265] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thiomAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In one embodiment, cysteine ​​residues may be added or removed, e.g., to facilitate assembly of different chains of a molecule or to increase or decrease the stability of the molecule.

[0266] The molecules of the invention can also be engineered to introduce one or more modifications in the constant region, e.g., the heavy chain constant region, e.g., the Fc region, to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In addition, the molecules of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody), or modified to alter its glycosylation, thereby again altering one or more functional properties of the molecule.

[0267] In some embodiments, the heavy chain constant region or Fc region contained in the molecule of the present invention can be modified to adjust one or more properties of the antibody, such as changing its effector function, such as ADCC and / or CDC activity and / or changing its half-life in vivo, such as enhancing ADCC activity, enhancing CDC activity and / or enhancing ADCC and CDC activity, or extending the half-life of the antibody in vivo, and / or increasing affinity with an Fc receptor (e.g., FcRn). In some embodiments, the modification is one or more amino acid substitutions.

[0268] In some preferred embodiments, the one or more amino acid substitutions occur in the heavy chain constant region sequence at positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 3 01, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439. In some preferred embodiments, the one or more amino acid substitutions occur in one or more of the heavy chain constant region sequence positions L234, L235, G236, S239, F243, T256, D265, H268, D270, K290, R292, S298, Y300, V305, K326, A330, I332, E333, K334, A339, and P396 according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from one or more of N297A substitution, N297Q substitution, L235A substitution together with L237A substitution, L234A substitution together with L235A substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, D265A substitution, A327Q substitution, and P329A substitution according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions occur at one or more of positions 235, 239, 243, 292, 300, 330, 332, and 396 of the heavy chain constant region sequence according to the EU numbering system. In some preferred embodiments, the one or more amino acid substitutions are selected from at least one of S239D, L235V, F243L, R292P, Y300L, A330L, I332E, and P396L according to the EU numbering system.

[0269] In some preferred embodiments, the heavy chain constant region has one or more sets of mutations occurring simultaneously at a combination of positions selected from the group consisting of: (1) L235 / F243 / R292 / Y300 / P396, (2) F243 / R292 / Y300 / V305 / P396, (3) D270 / K326 / A330 / K334, (4) S 239 / A330 / I332, (5) S298 / E333 / K334, (6) L234 / L235 / G236 / S239 / H268 / D270 / S298, (7) M252 / S254 / T256, (8) L234 / L235 / D265, (9) G236 / S239 / I332 and (10) S239 / I332.

[0270] In some preferred embodiments, the heavy chain constant region has one or more mutations selected from the following combinations of mutations according to the EU numbering system: (1) L235V / F243L / R292P / Y300L / P396L, (2) F243L / R292P / Y300L / V305I / P396L, (3) D270E / K326D / A330M / K334E, (4) S239D / A330L / I332E, (5) S298A / E333A / K334A, (6)L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, (7)M252Y / S254T / T256 E, (8)L234A / L235A / D265A, (9)L234F / L235E / D265A, (10)G236A / S239D / I332E and (11)S239D / I332E.

[0271] In some embodiments, the molecules of the invention may be post-translationally modified to alter the effector function of the antibody, such as by changing the glycosylation pattern of the molecules of the invention.

[0272] In some embodiments, the glycosylation of the molecules of the present invention can be modified. For example, an aglycosylated antibody or binding molecule can be prepared (i.e., the antibody or binding molecule lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the molecules of the present invention for an antigen. Such carbohydrate modifications can be achieved by, for example, altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0273] Additionally or alternatively, antibodies or binding molecules with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies or fusion proteins with increased bisecting GlcNac structures. Such altered glycosylation has been shown to increase the ADCC ability of the antibody or binding molecule. Such carbohydrate modifications can be achieved, for example, by expressing the antibody or binding molecule in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing the molecules of the present invention, thereby producing antibodies or binding molecules with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6)-fucosyltransferase), and therefore antibodies or binding molecules expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. Ms704, Ms705, and Ms709 FUT8 - / -Cell lines were generated by targeted disruption of the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704 and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene (encoding a fucosyltransferase) such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating enzymes involved in α-1,6 linkages. EP 1,176,195 also describes cell lines with low or no activity for the enzyme that adds fucose to N-acetylglucosamine bound to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, thereby also resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields et al. (2002) J. Biol. Chem. 277:26733-26740). Antibodies or binding molecules with modified glycosylation profiles can also be produced in eggs, as described in PCT Publication WO 06 / 089231. Alternatively, antibodies or binding molecules with modified glycosylation profiles can be produced in plant cells, such as duckweed. Methods for producing antibodies in plant systems are disclosed in U.S. patent application filed August 11, 2006, corresponding to Alston & Bird LLP attorney docket number 040989 / 314911. PCT publication WO 99 / 54342 describes a cell line engineered to express glycosyltransferases that modify glycoproteins, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), whereby antibodies or binding molecules expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibody or binding molecule (see also Umana et al. (1999) Nat. Biotech. 17: 176-180). Alternatively, fucosidases can be used to cleave off fucose residues of antibodies; for example, fucosidase α-L-fucosidase removes fucosyl residues from antibodies or binding molecules (Tarentino et al. (1975) Biochem. 14: 5516-23).

[0274] Another modification of the molecules herein that the present invention contemplates is pegylation. Antibodies or binding molecules can be pegylated to, for example, extend the biological (e.g., serum) half-life of the antibodies or binding molecules. In order to pegylate antibodies or binding molecules, typically under conditions wherein one or more PEG groups are attached to the antibodies or binding molecules, the antibodies or binding molecules are reacted with polyethylene glycol (PEG), such as reactive esters or aldehyde derivatives of PEG. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any one of the forms of PEG that have been used to derivatize other proteins, such as single (C1-C10) alkoxypolyethylene glycol or aryloxypolyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or binding molecule to be pegylated is an antibody or binding molecule without glycosylation. Methods for pegylating proteins are known in the art and can be applied to molecules of the present invention. See, for example, EPO 154 316 and EP 0 401 384.

[0275] Therefore, the present invention relates to an antibody or fragment thereof or multispecific binding molecule with enhanced ADCC and / or extended half-life and / or increased affinity for Fc receptors (e.g., FcRn). In some embodiments, the antibody or fragment thereof or multispecific binding molecule with enhanced ADCC and / or extended half-life and / or increased affinity for Fc receptors (e.g., FcRn) is an antibody or fragment thereof or multispecific binding molecule with an altered glycosylation type, such as a low-fucosylated or defucosylated antibody or fragment thereof or multispecific binding molecule, or an antibody or fragment thereof or multispecific binding molecule with an increased bisecting GlcNac structure. In some embodiments, the antibody or fragment thereof with enhanced ADCC and / or extended half-life and / or increased affinity for Fc receptors (e.g., FcRn) carries an amino acid substitution combination of M252Y / S254T / T256E. In some embodiments, the antibody with enhanced ADCC and / or prolonged half-life and / or increased affinity for an Fc receptor (e.g., FcRn) has altered glycosylation and / or altered amino acid sequence, for example, is hypofucosylated or afucosylated and carries the amino acid substitution combination of M252Y / S254T / T256E in the Fc region.

[0276] As described herein, this can be achieved by modifying the antibody or fragment thereof or the multispecific binding molecule, or by expressing it in a host cell with altered glycosylation machinery.

[0277] V. Nucleic acids of the present invention, and vectors and host cells containing the same

[0278] In one aspect, the invention provides nucleic acids encoding any of the above antibodies, bispecific fusion proteins, or any fragments thereof (e.g., antigen / target binding fragments). The invention also encompasses nucleic acids that hybridize to the above nucleic acids under stringent conditions, nucleic acids having one or more substitutions (e.g., conservative substitutions), deletions, or insertions compared to the above nucleic acids, or nucleic acid sequences having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above nucleic acids.

[0279] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-6, 9-18, 20-32 and 50-53, or nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-6, 9-18, 20-32 and 50-53 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0280] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody / fusion protein or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. The nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0281] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies / fusion proteins or any peptide chains / fragments thereof. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human and / or monkey (e.g., cynomolgus monkey) BDCA2 and / or BAFF / APRIL binding ability.

[0282] In one embodiment, the nucleic acids encoding each chain of an antibody or bispecific binding molecule or fusion protein or fragment thereof or multispecific binding molecule of the present invention can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of an antibody or bispecific binding molecule or fusion protein or fragment thereof or multispecific binding molecule of the present invention can be introduced into the same or different host cells for expression. Therefore, in some embodiments, the production method of an antibody or bispecific binding molecule or fusion protein or fragment thereof or multispecific binding molecule of the present invention comprises the steps of culturing a host cell comprising a nucleic acid encoding each chain under conditions suitable for expressing each chain of the molecule to produce an antibody or bispecific binding molecule or fusion protein or fragment thereof or multispecific binding molecule of the present invention.

[0283] On the other hand, the present invention provides a vector comprising the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It is fully understood by those skilled in the art that the vectors commonly used in the technical field to which the present invention belongs can be applied to the present invention.

[0284] In one embodiment, the present invention provides a host cell comprising the nucleic acid or the vector.

[0285] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity that have been screened or selected in the initially transformed cells. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0286] The host cell may also be a cell with an altered glycosylation machinery as described herein, such as a cell in which the FUT8 gene is completely or partially knocked out or the function of the FUT8 gene or protein is completely or partially disrupted, such as a FUT8 knockout CHO cell.

[0287] VI. Compositions of the Invention

[0288] In some embodiments, the present invention provides compositions comprising any of the anti-BDCA2 antibodies, BDCA2×BAFF / APRIL bispecific fusion proteins, or antigen / target-binding fragments thereof described herein. Preferably, the compositions are pharmaceutical compositions. In one embodiment, the compositions further comprise a pharmaceutical excipient. The term "pharmaceutical excipient" refers to a diluent, adjuvant, carrier, excipient, or stabilizer administered with an active substance.

[0289] In one embodiment, a composition (e.g., a pharmaceutical composition) comprises an anti-BDCA2 antibody, a BDCA2×BAFF / APRIL bispecific fusion protein, or an antigen / target binding fragment thereof of the present invention, in combination with one or more other therapeutic agents (e.g., an anti-inflammatory agent, a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective agent, a small molecule drug, or an immunomodulatory agent).

[0290] The pharmaceutical composition of the present invention can also include one or more other therapeutic agents, which are required for the specific indications being treated, encompassing any substance effective in preventing or treating tumors (such as cancer) and inflammation (such as chronic inflammation, local inflammation or systemic inflammation), preferably having those therapeutic agents that do not adversely affect each other's activity. For example, glucocorticoids, etc. The therapeutic agent is suitably combined in an amount effective for the purpose of application.

[0291] VII. Preparation of Anti-BDCA2 Antibodies or BDCA2×BAFF / APRIL Bispecific Fusion Proteins of the Invention

[0292] In one embodiment, the present invention provides a method for producing an anti-BDCA2 antibody or a BDCA2×BAFF / APRIL bispecific fusion protein, wherein the method comprises culturing a host cell containing a nucleic acid encoding the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or a fragment thereof, or an expression vector containing the nucleic acid, under conditions suitable for expressing the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or a fragment thereof, and optionally isolating the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or a fragment thereof. In certain embodiments, the method further comprises recovering the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or a fragment thereof from the host cell (or host cell culture medium).

[0293] To recombinantly produce the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof of the present invention, nucleic acid encoding the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that specifically bind to nucleic acid encoding the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof.

[0294] The anti-BDCA2 antibodies, BDCA2×BAFF / APRIL bispecific fusion proteins, or fragments thereof of the present invention, prepared as described herein, can be purified by known techniques such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. The purity of the anti-BDCA2 antibodies, BDCA2×BAFF / APRIL bispecific fusion proteins, or fragments thereof of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.

[0295] VIII. Combination Products or Kits

[0296] In some embodiments, the present invention also provides a combination product comprising an anti-BDCA2 antibody, a BDCA2×BAFF / APRIL bispecific fusion protein, or a fragment thereof of the present invention, and one or more other therapeutic agents (e.g., a chemotherapeutic agent, other antibodies, a cytotoxic agent, an anti-infective agent, a small molecule drug, or an immunomodulatory agent).

[0297] In some embodiments, the combination product is used to prevent or treat diseases associated with BDCA2 and / or BAFF / APRIL and / or diseases mediated by BDCA2 and / or BAFF / APRIL. In some embodiments, the additional therapeutic agent is an existing standard of care therapeutic agent.

[0298] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.

[0299] In some embodiments, the present invention also provides a kit comprising an anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein or fragment thereof, pharmaceutical composition or combination product of the present invention, and optionally a package insert directing administration.

[0300] In some embodiments, the present invention also provides a pharmaceutical product comprising the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein or fragment thereof, pharmaceutical composition or combination product of the present invention, optionally further comprising a package insert for guiding administration.

[0301] IX. Uses and Treatment Methods of the Anti-BDCA2 Antibodies and BDCA2×BAFF / APRIL Bispecific Fusion Proteins of the Present Invention

[0302] Given their ability to bind to human BDCA2 and / or BAFF / APRIL, the antibodies, antigen-binding fragments thereof, and bispecific, multivalent binding proteins described herein can be used to detect BDCA2 and / or BAFF / APRIL, for example, in biological samples containing cells expressing one or both of those target antigens. The antibodies, antigen-binding fragments, and binding proteins disclosed herein can be used in conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or tissue immunohistochemistry. The present disclosure provides a method for detecting BDCA2 and / or BAFF / APRIL in a biological sample, comprising contacting the biological sample with an antibody, antigen-binding portion thereof, or binding protein disclosed herein, and detecting whether binding to the target antigen occurs, thereby detecting the presence of the target in the biological sample. The antibody, antigen-binding fragment, or binding protein can be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody / fragment / binding protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of suitable radioactive substances include 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153 Sm.

[0303] In one embodiment, a therapeutically effective amount of an anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof, or a pharmaceutical composition or combination product disclosed herein prevents or treats inflammatory disorders / diseases and / or autoimmune disorders / diseases and / or tumors such as hematopoietic tumors, inhibits excessive activation of plasmacytoid dendritic cells and / or eliminates plasmacytoid dendritic cells, inhibits IFNα release, and inhibits B cell proliferation.

[0304] In some specific embodiments, a therapeutically effective amount of an anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof, or a pharmaceutical composition or combination product disclosed herein prevents or treats systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes mellitus, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, neuromyelitis spectrum disorder, primary Sjögren's syndrome, myasthenia gravis, or any combination thereof.

[0305] In another aspect, the present invention relates to a method for preventing or treating inflammatory disorders / diseases and / or autoimmune disorders / diseases and / or tumors, such as hematopoietic tumors, inhibiting excessive activation of plasmacytoid dendritic cells and / or clearing plasmacytoid dendritic cells, inhibiting IFNα release, and inhibiting B cell proliferation, comprising administering to the subject an effective amount of an anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein or fragment thereof, or a pharmaceutical composition or combination product disclosed herein.

[0306] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, e.g., standard treatment regimens. Alternatively, or in combination, the subject is immunocompromised or at risk of being immunocompromised due to an infection.

[0307] In some embodiments, the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual an anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof, or pharmaceutical composition or combination product disclosed herein, in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents.

[0308] The anti-BDCA2 antibodies, BDCA2×BAFF / APRIL bispecific fusion proteins, or fragments thereof (and pharmaceutical compositions comprising the same) of the present invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether the administration is short-term or chronic, administration can be by any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0309] For the prevention or treatment of disease, the appropriate dosage of the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof, the severity and course of the disease, whether the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof, and the discretion of the attending physician. The anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof is suitably administered to the patient as a single treatment or over a series of treatments. The dosage and treatment regimen of the anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein, or fragment thereof can be determined by a skilled artisan.

[0310] It is understood that any of the above-mentioned prevention or treatment can be carried out using the composition or combination product of the present invention instead of anti-BDCA2 antibody, BDCA2×BAFF / APRIL bispecific fusion protein or fragment thereof.

[0311] X. Diagnosis and testing

[0312] In one aspect, the present invention also relates to methods for diagnosis and detection (eg, for diagnostic or non-diagnostic purposes) of anti-BDCA2 antibodies or fragments thereof or anti-BDCA2×BAFF / APRIL bispecific fusion proteins and compositions for diagnosis and detection comprising the same.

[0313] When the term "detection" is used herein, it includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as blood, serum, or plasma.

[0314] In certain embodiments, the anti-BDCA2 antibodies or fragments thereof or anti-BDCA2 x BAFF / APRIL bispecific fusion proteins provided herein can be used to detect the presence of BDCA2 in a biological sample.

[0315] In certain embodiments, the anti-BDCA2 x BAFF / APRIL bispecific fusion proteins provided herein can be used to detect the presence of BAFF / APRIL in a biological sample.

[0316] In certain embodiments, the method comprises contacting a biological sample with an anti-BDCA2 antibody or fragment thereof, or an anti-BDCA2 x BAFF / APRIL bispecific fusion protein as described herein, under conditions permissive for binding to BDCA2 and / or BAFF / APRIL, and detecting whether a complex forms between the anti-BDCA2 antibody or fragment thereof, or the anti-BDCA2 x BAFF / APRIL bispecific fusion protein and BDCA2 and / or BAFF / APRIL. Formation of a complex indicates the presence of BDCA2. The method can be in vitro or in vivo.

[0317] In certain embodiments, labeled anti-BDCA2 antibodies or fragments thereof, or anti-BDCA2 x BAFF / APRIL bispecific fusion proteins are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. In some embodiments, the label is, for example, a marker such as biotin or hFc.

[0318] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-BDCA2 antibody or fragment thereof or an anti-BDCA2 x BAFF / APRIL bispecific fusion protein of the present invention. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0319] In some embodiments, BDCA2 and / or BAFF / APRIL is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0320] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0321] Example

[0322] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art. Materials and reagents used in the following examples are commercially available unless otherwise specified.

[0323] Example 1: Hybridoma Screening

[0324] 1.1 Animal immunization

[0325] Construction of CHO-K1 / hBDCA2 / FcεRIγ cell line

[0326] The sequences encoding human BDCA2 (NP_569708) and FcεRIγ (UniProt Number: P30273) were cloned into a lentiviral vector, packaged into lentivirus, and transduced into CHO-K1 cells (ATCC, catalog number: CCL-61) using lentivirus. TM ), and then screened with 10 μg / mL puromycin (Gibco, catalog number: A1113802) and 600 μg / mL G418 (ThermoFisher catalog number: 10131027) to obtain a cell line (CHO-K1 / hBDCA2 / FcεRIγ) that highly expresses human BDCA2 and FcεRIγ.

[0327] Construction of HEK293 / Cyno BDCA2 / FcεRIγ cell line

[0328] The sequences encoding Cyno BDCA2 (UniProt Number: A0A2K5UWP4) and FcεRIγ (UniProt Number: P30273) were cloned into a lentiviral vector and packaged into lentivirus. Cyno BDCA2 and FcεRIγ were transduced into HEK293 cells (ATCC, Catalog Number: CRL-1573) using lentivirus. The cells were then screened with 1 μg / mL puromycin (Gibco, Catalog Number: A1113802) and 600 μg / mL G418 (ThermoFisher Catalog Number: 10131027) to obtain a cell line (HEK293 / Cyno BDCA2 / FcεRIγ) that highly expresses human BDCA2 and FcεRIγ.

[0329] Five BALB / c mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were immunized with human BDCA2-His protein (ACRO Biosystems, Catalog No. CLC-H5245). Mice were immunized every two weeks. Starting after the second immunization, venous blood was collected from the mice 7 days after each immunization. Mouse immune sera were analyzed by flow cytometry (FACS) using HEK293 / hBDCA2 / FcεRIγ cell lines and HEK293 / Cyno BDCA2 / FcεRIγ cell lines, and by ELISA using human BDCA2-His and cynomolgus monkey BDCA2-His (ACRO Biosystems, Catalog No. CLC-C52H4). Two mice were selected for animal fusion. Four days after the final booster immunization, spleens were harvested, and lymphocytes isolated from the spleens were used for fusion to generate hybridoma cells.

[0330] 1.2 Hybridoma Screening

[0331] Hybridoma cell culture supernatants were tested for primary screening by ELISA and FACS. In this assay, 20 clones that bound to human BDCA2-His protein and were able to bind to the HEK293 / hBDCA2 / FcεRIγ cell line and the HEK293 / Cyno BDCA2 / FcεRIγ cell line were selected for subcloning.

[0332] 1.3 Subcloning

[0333] Subclone the 20 clones selected in 1.2 to ensure monoclonality. Subclones were screened using a single-step cloning system and the parental clone was re-plated for subcloning. Twenty subclones were transferred to 96-well culture plates. Subclones were screened by ELISA and FACS. Subclones were selected for antibodies expressed in their cell culture supernatants that bind to human BDCA2 and cynomolgus BDCA2 proteins, as well as to the HEK293 / hBDCA2 / FcεRIγ and HEK293 / cyBDCA2 / FcεRIγ cell lines.

[0334] 1.4 Hybridoma Sequencing

[0335] The subcloned hybridoma cell line obtained in Example 1.3 was expanded, a portion of the cells were frozen, and a portion of the cells were used to extract RNA for reverse transcription and sequence the resulting cDNA. The obtained antibody variable region amino acid sequences are shown in Table 1 below.

[0336] Table 1: Antibody variable region amino acid sequences

[0337] Example 2: Construction, expression and purification of anti-BDCA2 chimeric antibodies

[0338] 2.1 Construction of anti-BDCA2 chimeric antibodies

[0339] The variable region amino acid sequences of the two murine antibodies listed in Table 1 were sent to Beijing Tsingke Biotech Co., Ltd. for codon optimization and coding gene synthesis. The genes encoding the VH and VL regions of the antibodies were sequentially inserted into expression vectors containing the human IgG1 heavy chain constant region (SEQ ID NO: 5) and the kappa light chain constant region (SEQ ID NO: 6) to generate plasmids for the murine chimeric antibodies.

[0340] 2.2 Expression and purification of anti-BDCA2 chimeric antibodies

[0341] Use ExpiCHO according to the manufacturer's product manual. TM ExpiCHO-S cells were co-transfected with plasmids encoding the heavy and light chains of the 2.1 murine chimeric anti-BDCA2 antibody using an expression system (ThermoFisher, Cat# A29133) to express the anti-BDCA2 chimeric antibody, along with a similarly expressed control antibody, litifilimab analogue (heavy chain: SEQ ID No: 7, light chain: SEQ ID No: 8). Cells were cultured for 10-12 days after transfection. When cell viability decreased to 60%-70% as measured by a cell counter, the supernatant was collected and the antibody expressed in the supernatant was purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibody was concentrated and sterile filtered. The purity of the antibody protein was assessed by SDS-PAGE and size exclusion, which demonstrated a purity of >90% and was suitable for use in further experiments.

[0342] Table 2: Chimeric antibody names and corresponding heavy and light chain combinations

[0343] Example 3: Binding affinity of anti-BDCA2 chimeric antibodies to human BDCA2 protein

[0344] The binding affinity of anti-BDCA2 chimeric antibodies to human BDCA2 protein (Cat#BCA-HM402, Kactus Biosystems) was measured using the ForteBio Octet RED96. According to the manufacturer's instructions, an AHC sensor (ForteBio, Cat#18-5060) was placed in Running Buffer (1X PBS Hyclone, Cat#SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate as follows: a) Baseline equilibration with Running Buffer for 100 seconds; b) Anti-BDCA2 chimeric antibodies were diluted to 5 μg / mL in Running Buffer and allowed to solidify for 200 seconds; c) Baseline equilibration with Running Buffer for 300 seconds; d) Human BDCA2 protein was diluted to 50 nM in Running Buffer, and binding was allowed for 200 seconds and dissociation for 600 seconds. The experimental data were fitted and calculated using the 1:1 binding model using Fortebio Data Analysis software.

[0345] The results are shown in Table 3 below.

[0346] Table 3. Binding affinity of anti-BDCA2 chimeric antibodies to human BDCA2 protein

[0347] Example 4: Binding of anti-BDCA2 chimeric antibodies to cell lines expressing human BDCA2 protein

[0348] 4.1. Construction of engineered cells expressing human BDCA2 protein

[0349] The sequences encoding human BDCA2 (NP_569708) and FcεRIγ (UniProt Number: P30273) were cloned into a lentiviral vector and packaged into lentivirus. Human BDCA2 and FcεRIγ were transfected into HEK293 cells (ATCC, Catalog No.: CRL-1573) using lentivirus. The cells were then screened with 1 μg / mL puromycin (Gibco, Catalog No.: A1113802) and 600 μg / mL G418 (ThermoFisher Catalog No.: 10131027) to obtain a cell line (HEK293 / hBDCA2 / FcεRIγ) that highly expresses human BDCA2 and FcεRIγ.

[0350] 4.2. BDCA2 Antibody Binding to HEK293 / hBDCA2 / FcεRIγ Engineered Cells

[0351] HEK293 / hBDCA2 / FcεRIγ cells were enzymatically digested to obtain a single-cell suspension, which was then centrifuged at 300g for 4 minutes at room temperature, after which the culture medium was discarded. The resulting cell pellet was washed once with PBS and resuspended in a serial dilution of anti-BDCA2 chimeric antibody in PBS (initial concentration: 100 nM, followed by 5-fold serial dilutions, for a total of 8 concentrations) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat. No. 109-116-098), was added and incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS and resuspending them, the fluorescence intensity of the PE was determined by flow cytometry.

[0352] As shown in the results in FIG2 , it can be seen that both anti-BDCA2 chimeric antibodies 37F2C6 and 26H10A11-1 can bind to cell lines expressing human BDCA2 protein.

[0353] Example 5: Killing of cell lines expressing human BDCA2 protein by anti-BDCA2 chimeric antibodies

[0354] Remove the frozen PBMCs from the liquid nitrogen tank and thaw them quickly in a 37-degree water bath. Transfer the reconstituted cells to a 15-ml centrifuge tube, add 9 ml of RPMI-1640 complete medium and mix thoroughly. Centrifuge at 350 g for 10 min. Then, separate NK cells from PBMCs using the NK cell separation kit (Stemcell, Cat. No. 17955). Resuspend the sorted NK cells in RPMI-1640 complete medium containing 200 IU / ml IL-2 and activate them overnight.

[0355] After enzymatic digestion of HEK293 / hBDCA2 / FcεRIγ cell line, single cell suspension was obtained, centrifuged at 300g for 4 minutes at room temperature, and the culture medium was discarded. The cell pellet was washed once with PBS. TMViolet Cell Proliferation Kit (Invitrogen, Catalog No. C34557) was used for staining. Anti-BDCA2 chimeric antibody was serially diluted (initial concentration: 100 nM, 5-fold serial dilutions, totaling 8 concentrations). The anti-BDCA2 chimeric antibody was mixed with IL-2-activated overnight NK cells. The stained HEK293 / hBDCA2 / FcεRIγ cells were then added to the wells containing the anti-BDCA2 chimeric antibody and NK cells at a 3:1 effector-target ratio. After incubation for 4 hours in a CO2 incubator, a 1:500 dilution of Propidium Iodide (Biyuntian, Catalog No. ST511) dye was added. The percentage of PI-positive target cells in the stained target cells was determined by flow cytometry.

[0356] As shown in Figure 3 , the anti-BDCA-2 chimeric antibodies exhibited ADCC effects and could induce primary NK cells to kill engineered cells expressing BDCA2. The chimeric antibodies 37F2C6 and 26H10A11-1 exhibited comparable cytotoxic activity against target cells HEK293 / hBDCA2 / FcεRIγ as the litifilimab analogue.

[0357] Example 6: Anti-BDCA2 chimeric antibody inhibits IFN-α production in PBMCs stimulated by TLR9 agonist

[0358] The IFN-α factor inhibition experiment was performed to determine whether the anti-BDCA2 chimeric antibody of the present invention could inhibit the production of IFN-α by human PBMC induced by TLR9 agonist.

[0359] Frozen PBMCs were removed from the liquid nitrogen tank and rapidly thawed in a 37°C waterbath. The reconstituted cells were transferred to a 15ml centrifuge tube, mixed with 9ml of 1640 complete medium, centrifuged at 350g for 10 minutes, and resuspended in fresh medium before counting. PBMCs were plated in a 96-well plate at 3×105 cells per well. Anti-BDCA2 chimeric antibody was serially diluted (initial concentration: 100 nM, followed by 5-fold serial dilutions for a total of 8 concentrations). The antibody and PBMCs were mixed and preincubated in a CO2 incubator for 30 minutes. TLR9 agonist (InvivoGen, Catalog No. tlrl-2216) was added at a final concentration of 1 μM, and the cells were incubated in a CO2 incubator for 24 hours. After 24 hours, the supernatant was collected and assayed for IFN-α using an HTRF assay kit (Cisbio, Catalog No. 62HIFNAPEG) for IFN-α.

[0360] As shown in the results in Figure 4 , anti-BDCA-2 chimeric antibodies can inhibit TLR9 agonist-induced IFN-α production in human PBMCs. The chimeric antibodies 37F2C6 and 26H10A11-1 clones exhibited comparable inhibitory effects on IFN-α as litifilimab analogues.

[0361] Example 7: Humanization and expression of murine anti-BDCA2 antibodies

[0362] 7.1 Humanization of mouse anti-BDCA2 antibody

[0363] The mouse antibody 37F2C6 was humanized using CDR grafting technology. The 37F2C6 sequence was searched and aligned in the IMGT database. The human germline gene sequence IGHV1-3*04, which showed high homology to the 37F2C6 heavy chain variable region, was selected as the humanized framework for the heavy chain variable region. The human germline gene sequence IGKV3-11*01, which showed high homology to the light chain variable region, was selected as the humanized framework for the light chain variable region. The CDRs of the 37F2C6 heavy and light chain variable regions were grafted into the corresponding humanized frameworks to form the humanized 37F2C6 antibody variable regions. To maintain the affinity of the antibody 37F2C6, the resulting humanized antibody variable regions were backmutated.

[0364] The humanized heavy chain variable region and light chain variable region sequences were obtained, respectively. Table 4 lists the variable region amino acid sequence of the 37F2C6 humanized antibody. The variable region amino acid sequence was sent to Beijing Tsingke Biotech Co., Ltd. for codon optimization and gene synthesis. The genes encoding the VH and VL regions of the antibodies were inserted into expression vectors containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 5) or the gene encoding the kappa light chain constant region (SEQ ID NO: 6), respectively, to obtain plasmids expressing the full-length heavy chain and full-length light chain of the anti-BDCA2 humanized antibody. Table 4 lists the names, variable region sequences, and heavy and light chain sequences of each antibody.

[0365] Table 4: Humanized antibody names and corresponding sequences

[0366] 7.2 Expression and purification of humanized anti-BDCA2 antibodies

[0367] The heavy and light chain encoding plasmids were co-transfected into ExpiCHO-S cells as described in Table 4 to express humanized anti-BDCA2 antibodies. Wild-type and ADCC-enhanced antibodies were obtained with or without the addition of a fucose inhibitor (Sigma, Cat. No. 344827-10 mg). The antibodies containing the fucose inhibitor were designated Hu37F2C6-8-A and Hu37F2C6-9-A, respectively, and purified as described in Example 2.2. The purified antibodies were concentrated, sterile filtered, and assayed for purity by SDS-PAGE and size exclusion chromatography (SEC).

[0368] 7.3 Physicochemical Analysis of Humanized Anti-BDCA2 Antibodies

[0369] The purity of the obtained humanized anti-BDCA2 antibody was confirmed by size exclusion chromatography. Specifically, 20 μg of the antibody sample was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na₂SO₄ (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. The results showed that the antibody was >90% pure and suitable for use in further experiments.

[0370] Example 8: Binding affinity of anti-BDCA2 humanized antibodies to human BDCA2 protein

[0371] In this experiment, the binding affinity of 37F2C6, Hu37F2C6-8, and Hu37F2C6-9 antibodies to human BDCA2 protein (KACTUS, Cat. No. BCA-HM402) was detected using ForteBio Octet RED96e according to the manufacturer's instructions.

[0372] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was pre-equilibrated in running buffer (1X PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) for 10 minutes at room temperature. BDCA2 kinetics assays were performed in a 96-well plate as follows: a) Baseline equilibration with running buffer for 180 seconds; b) Antibody diluted in running buffer was added to a final concentration of 5 μg / mL and allowed to immobilize for 200 seconds; c) Baseline equilibration with running buffer for 180 seconds; d) Human BDCA2 protein (100 nM) diluted in running buffer was added to each well at the following concentrations. Binding was allowed for 200 seconds, followed by dissociation for 180 seconds. e) Regeneration was performed in regeneration buffer (0.01 M Gly-HCl, pH 1.5) for 30 seconds. Experimental data were fitted to a 1:1 binding model using ForteBio Data Analysis software and the results are shown in the table below.

[0373] Table 5 Binding affinity of anti-BDCA2 humanized antibodies to human BDCA2 protein

[0374] Table 5 shows that the antibodies 37F2C6, Hu37F2C6-8, and Hu37F2C6-9 of the present invention bind to the human BDCA2 protein with high affinity.

[0375] Example 9: Killing of cells expressing human BDCA2 protein by anti-BDCA2 humanized antibodies

[0376] Two experiments were conducted to determine whether the anti-BDCA2 humanized antibody of the present invention could kill HEK293 / hBDCA2 / FcεRIγ cells and pDCs, using primary NK cells to kill HEK293 / hBDCA2 / FcεRIγ cells and autologous NK cells in a PBMC system to kill pDCs.

[0377] 9.1 Experimental system for primary NK cells killing HEK293 / hBDCA2 / FcεRIγ cells

[0378] The experimental method was the same as that in Example 5. As shown in the results of FIG5 a , the chimeric antibody 37F2C6 and its humanized antibodies Hu37F2C6-8 / Hu37F2C6-9 showed comparable cytotoxicity to the target cells HEK293 / hBDCA2 / FcεRIγ as did the litifilimab analogue.

[0379] 9.2 PBMC ADCC Experimental System

[0380] Frozen PBMCs were removed from the liquid nitrogen tank and thawed quickly in a 37-degree water bath. The reconstituted cells were transferred to a 15-ml centrifuge tube, 9 ml of 1640 complete medium was added and mixed, and the cells were centrifuged at 350 g for 10 min. The cells were resuspended in 1640 complete medium containing 200 IU / ml IL-2 and activated overnight. The PBMCs activated overnight with IL-2 were collected, resuspended, counted, and added to a 96-well plate, with 3×10^5 cells per well. BDCA2 antibody was serially diluted (initial concentration was 100 nM, 5-fold serial dilution, a total of 8 concentrations). The serially diluted antibodies were mixed with PBMCs and incubated in a carbon dioxide incubator for 48 hours. After 48 hours, the cells were collected and the number of pDCs in PBMCs was detected by flow cytometry. The pDCs flow cytometry detection method was as follows: Zombie Violet TM Live PBMCs were labeled with a Fixable Viability Kit (Biolegend, Catalog No. 423114) using a live / dead dye. T cells, NK cells, monocytes, and B cells (Biolegend, Catalog No. 348703) were then excluded using an APC anti-human lineage cocktail (CD3, CD14, CD19, CD20, CD56). CD123+ (Biolegend, Catalog No. 306014) / ILT7+ (Biolegend, Catalog No. 326408) cells were identified within the HLA-DR+ (Biolegend, Catalog No. 307644) / CD11c- (Biolegend, Catalog No. 301608) cell population, identifying pDCs.

[0381] From the results shown in FIG5 b , it can be seen that the humanized antibodies Hu37F2C6-8 / Hu37F2C6-9 and Litifilimab analogue have comparable cytotoxicity against pDCs, and the cytotoxicity of Hu37F2C6-8-A / Hu37F2C6-9-A against pDCs is superior to that of the control antibody Litifilimab analogue.

[0382] Example 10: Anti-BDCA2 humanized antibody inhibits IFN-α production in PBMCs stimulated by TLR9 agonist

[0383] The experimental method was the same as that in Example 6. As shown in the results in Figure 6, the chimeric antibody 37F2C6 clone and its humanized antibodies Hu37F2C6-8 / Hu37F2C6-9 showed comparable inhibitory effects on IFN-α as the litifilimab analogue.

[0384] Example 11: Effect of anti-BDCA2 humanized antibody on pDC elimination in the huHSC-NCG mouse model

[0385] The in vivo efficacy of a humanized BDCA2 antibody in depleting pDCs was investigated in a huHSC-NCG mouse model. huHSC-NCG mice were purchased from Jiangsu Gempharmatech Biotechnology Co., Ltd. After a one-week acclimation period, 15 huHSC-NCG mice were randomly assigned to three experimental groups, with five mice in each group. On the day of grouping (day 0), mice were intraperitoneally administered with a blank control (PBS) (G1), a litifilimab analogue (30 mg / kg) (G2), or Hu37F2C6-9-A (30 mg / kg) (G3). On day 3, peripheral blood and spleen samples were collected from each group for pDC analysis. As shown in Figure 7, the anti-BDCA2 humanized antibody group significantly reduced the proportion of pDCs in peripheral blood and the proportion and number of pDCs in the spleen compared to the PBS group.

[0386] Example 12: Anti-BDCA2 / D2, BDCA2 / TACI bispecific fusion protein

[0387] 12.1 Construction of Bispecific Fusion Proteins

[0388] The present invention constructed three bispecific fusion proteins with different symmetrical structures, as shown in Figure 1 . The anti-BDCA2 portion of each bispecific fusion protein is derived from the humanized 37F2C6-9 antibody described above. The TACI portion (abbreviated as TACI) is a fragment of full-length TACI, which corresponds to a truncated portion of the Telitacicept antibody, i.e., the underlined portion of the sequence. The CRD2 portion (abbreviated as D2) is a further truncated portion of the TACI amino acid sequence, as shown in the bolded and italicized sequence below. The amino acid sequence of the control antibody Telitacicept is as follows:

[0389] The CRD2 and TACI amino acid sequences were sent to Nanjing Tsingke Biotech Co., Ltd. for codon optimization and gene synthesis to obtain nucleic acids containing CRD2 and TACI coding sequences for subsequent bispecific antibody construction templates.

[0390] This application used standard construction methods to obtain three anti-BDCA2 / CRD2 and anti-BDCA2 / TACI bispecific fusion proteins with the structures shown in Figure 1. Their amino acid sequence numbers are listed in Table 6 below. The heavy chains of the bispecific fusion proteins were fused to the N-terminus and C-terminus of the humanized 37F2C6-9 antibody using PCR overlap technology, respectively, with the CRD2 sequence and the TACI sequence fused to the N-terminus of the humanized 37F2C6-9 antibody using PCR overlap technology.

[0391] The heavy chains of each bispecific fusion protein molecule were constructed as follows:

[0392] 37F2C6-HuVH3-D2 (SEQ ID NO: 22): Using synthetic CRD2 as a template, the nucleotide sequence encoding CRD2 was amplified, and the sequence was spliced ​​with the C-terminus of the nucleotide sequence encoding the complete Hu37F2C6-9 using overlapping PCR technology (the connecting peptide between the two sequences is: (G4S)3). The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-D2 molecule.

[0393] 37F2C6-HuVH3-3N-D2 (SEQ ID NO: 23): Using synthetic CRD2 as a template, the nucleotide sequence encoding CRD2 was amplified, and the sequence was spliced ​​with the N-terminus of the nucleotide sequence encoding the complete Hu37F2C6-9 using overlapping PCR technology (the connecting peptide between the two sequences is: (G4S)3). The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-3N-D2 molecule.

[0394] 37F2C6-HuVH3-3N-TACI (SEQ ID NO: 24): Using synthetic TACI as a template, the nucleotide sequence encoding TACI was amplified, and the sequence was spliced ​​with the N-terminus of the nucleotide sequence encoding the complete Hu37F2C6-9 using overlapping PCR technology (the connecting peptide between the two sequences is: (G4S)3). The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-3N-TACI molecule.

[0395] 37F2C6-HuVH3-CD2-DE (SEQ ID NO: 50): Using synthetic CRD2 as a template, the nucleotide sequence encoding CRD2 was amplified (the N-terminus of the CRD2 amino acid sequence was truncated and SLS was removed), and the sequence was spliced ​​with the C-terminus of the nucleotide sequence encoding Hu37F2C6-9 using overlapping PCR technology (the connecting peptide between the two sequences was: (G4S)3). The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-CD2-DE molecule.

[0396] 37F2C6-HuVH3-ND2-DE (SEQ ID NO: 51): Using synthetic CRD2 as a template, the nucleotide sequence encoding CRD2 was amplified (the C-terminus of the CRD2 amino acid sequence was truncated and EN was removed), and the sequence was spliced ​​with the N-terminus of the nucleotide sequence encoding the complete Hu37F2C6-9 using overlapping PCR technology (the connecting peptide between the two sequences was: (G4S)3). The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-ND2-DE molecule.

[0397] 12.2 Construction of Different Connecting Peptides (Linkers) for Bispecific Fusion Proteins

[0398] Taking the bispecific fusion protein heavy chain molecule 37F2C6-HuVH3-3N-D2 as an example, its connecting peptide was replaced from the original (G4S)3 to (GS4)2, (GS4)3, and (GS4)4, respectively, and co-transfected with the light chain 37F2C6-HuVL3 to obtain the bispecific fusion proteins BI-9ND2-2GS4, BI-9ND2-3GS4, and BI-9ND2-4GS4, respectively.

[0399] Similarly, the connecting peptide in the middle of 37F2C6-HuVH3-D2 was replaced from the original (G4S)3 to GS4, (GS4)2, and (GS4)3, respectively, and co-transfected with the light chain 37F2C6-HuVL3 to obtain the bispecific fusion proteins BI-9D2-1GS4, BI-9D2-2GS4, and BI-9D2-3GS4, respectively.

[0400] The bispecific fusion proteins BI-9D2-3G4S4S and BI-9D2-3G4S-8S keep the connecting peptide in the middle of 37F2C6-HuVH3-D2 unchanged (connecting peptide: (G4S)3), but add SSSS and SSSSSSSS at the C-terminus of CRD2, respectively, and are also co-transfected with the light chain 37F2C6-HuVL3.

[0401] 12.3 Construction of Bispecific Fusion Proteins with YTE Mutations in the Fc Region

[0402] 37F2C6-HuVH3-ND2-YTE (SEQ ID NO: 53): Using the constructed 37F2C6-HuVH3-3N-D2 as a template, point mutagenesis was performed. Mutations were made at M252Y, S254T, and T256E (according to EU numbering) using PCR overlap technology. The spliced ​​nucleotide sequence was cloned into an expression vector to express the 37F2C6-HuVH3-ND2-YTE molecule.

[0403] According to the specific composition shown in Table 6, the above expression vectors were combined accordingly and expressed under appropriate conditions to obtain the bispecific fusion protein in Figure 1.

[0404] The construction, expression, purification, and preliminary analysis steps of the bispecific fusion protein were the same as those in Example 7. The antibodies with the added fucose inhibitor were named: BI-9D2-A, BI-9ND2-A, BI-9NTACI-A, BI-9CD2-DE-A, BI-9ND2-DE-A, and BI-9ND2-A-YTE.

[0405] Table 6 Humanized bispecific fusion protein expression

[0406] Example 13: Binding Affinity of Bispecific Fusion Proteins, Bispecific Fusion Proteins with Different Linkers, and Bispecific Fusion Proteins with YTE Mutations in the Fc Region to Human BDCA2 Protein

[0407] 13.1 In this experiment, the binding affinity of the bispecific fusion protein to human BDCA-2 protein (KACTUS, Cat. No.: BCA-HM402) was detected using ForteBio Octet RED96e according to the manufacturer's instructions.

[0408] Briefly, an AHC sensor (ForteBio, Cat. No. 18-5060) was pre-equilibrated in running buffer (1X PBS, XiGene, Cat. No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) for 10 minutes at room temperature. BDCA2 kinetic assays were performed in a 96-well plate as follows: a) Baseline equilibration with running buffer for 180 seconds; b) Add each bispecific fusion protein diluted in running buffer to a final concentration of 5 μg / mL and allow to solidify for 200 seconds; c) Baseline equilibration with running buffer for 180 seconds; d) Add the following concentrations of human BDCA2 protein diluted in running buffer to each well: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. Binding was allowed for 200 seconds, and dissociation for 180 seconds. e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) was used for 30 seconds. The experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software; the results are shown in the table below.

[0409] Table 7 Binding affinity of bispecific fusion proteins and controls to human BDCA2 protein

[0410] Table 7 shows that the bispecific fusion protein of the present invention binds to the human BDCA2 protein with high affinity.

[0411] 13.2 The binding affinity of bispecific fusion proteins with different linker peptides to human BDCA2 protein was tested using the ForteBio Octet RED96e. The method was the same as in Example 8. The results are shown in the table below.

[0412] Table 8 Binding affinity of bispecific fusion proteins with different connecting peptides to human BDCA2 protein

[0413] Table 8 shows that the bispecific fusion proteins of the present invention with different connecting peptides bind to human BDCA2 protein with high affinity.

[0414] 13.3 The binding affinity of the bispecific fusion protein with the YTE mutation in the Fc region to the human BDCA-2 protein was detected using the ForteBio Octet RED96e, using the same method as Example 13.1.

[0415] Table 9 Binding affinity of bispecific fusion proteins with YTE mutation in the Fc region to human BDCA2 protein

[0416] Table 9 shows that the bispecific fusion protein of the present invention having a YTE mutation in the Fc region binds to the human BDCA2 protein with high affinity.

[0417] Example 14: Binding Affinity of Bispecific Fusion Proteins, Bispecific Fusion Proteins with Different Linkers, and Bispecific Fusion Proteins with YTE Mutations in the Fc Region to Human BAFF / APRIL Protein

[0418] 14.1 In this experiment, the binding affinity of the bispecific fusion protein to human BAFF protein (ACRO Biosystems, Catalog No. BAF-H52D4) and human APRIL protein (ACRO Biosystems, Catalog No. APL-H52D1) was determined using the ForteBio Octet RED96e according to the manufacturer's instructions. The method was the same as in Example 13.1. The results are shown in the table below.

[0419] Table 10 Binding affinity of bispecific fusion proteins and controls to human BAFF protein and human APRIL protein

[0420] Table 10 shows that the bispecific fusion protein of the present invention can specifically bind to human BAFF protein and human APRIL protein.

[0421] 14.2 The binding affinities of bispecific fusion proteins with different linker peptides to human BAFF and APRIL proteins were determined using the ForteBio Octet RED96e. The method was the same as in Example 8. The results are shown in the table below.

[0422] Table 11 Binding affinity of bispecific fusion proteins with different connecting peptides to human BAFF protein

[0423] Table 12 Binding affinity of bispecific fusion proteins with different connecting peptides to human APRIL protein

[0424] Tables 11 and 12 summarize the binding affinities of bispecific fusion proteins with different connecting peptides to human BAFF protein and human APRIL protein.

[0425] 14.3 The binding affinity of the bispecific fusion protein with the YTE mutation in the Fc region to human BAFF protein (BAF-H52D4) and human APRIL protein was tested using the ForteBio Octet RED96e, using the same method as in Example 13.1.

[0426] Table 13 Binding affinity of bispecific fusion proteins and controls to human BAFF protein and human APRIL protein

[0427] Table 13 shows that the bispecific fusion protein with a YTE mutation in the Fc region of the present invention can specifically bind to human BAFF protein and human APRIL protein.

[0428] Example 15: Bispecific fusion protein sandwich ELISA

[0429] 96-well plates were coated with 1 μg / ml human BDCA2 (ACRO Biosystems, Cat. No. CLC-H5245) at 50 μL / well and incubated overnight at 4°C. The plates were then incubated with blocking buffer (1% BSA in PBS) at 37°C for 1 hour. After blocking, the plates were washed once with PBST (0.05% Tween 20 in PBS). The bispecific fusion protein was serially diluted with diluent, added to the plates, and incubated at 37°C for 1 hour. After the incubation period, the plates were washed with PBST. Biotinylated human BAFF protein (KACTUS, Catalog No.: BAF-HM412B) was diluted with diluent to 1 μg / ml and added to the plate for incubation at 37°C for 1 hour. After incubation, the plate was washed again. Secondary antibody (horseradish peroxidase HRP-conjugated streptavidin, Jackson Immuno Research, Cat#016-030-084) was diluted with diluent and added to the plate for incubation at 37°C for 1 hour. After incubation, the plate was washed again. The plate was developed with TMB and then terminated with 1 M H2SO4. The absorbance values ​​at OD450nm-OD620nm were read on a microplate reader. The results are shown in Figure 8.

[0430] The results showed that the bispecific fusion protein can simultaneously bind to human BDCA2 protein and human BAFF protein using a double antigen sandwich binding method.

[0431] Example 16: Bispecific fusion proteins, bispecific fusion proteins with different connecting peptides, and bispecific fusion proteins with a YTE mutation in the Fc region inhibit B cell proliferation

[0432] Human B cells were isolated from PBMCs (Shanghai Rubai Biotechnology, Donor: 2202181749) using a B cell isolation kit (Miltenyi Biotec, Catalog No. 130-050-301). Cells were resuspended in RPMI-1640 complete medium supplemented with 20 μg / mL Anti-IgM (Jackson ImmunoResearch, Catalog No. 109-007-043), 20 ng / mL IL-4 (PeproTech, Catalog No. 200-04-100UG), 2 nM BAFF (ACRO Biosystems, Catalog No. BAF-H52D4), and 10 nM APRIL (ACRO Biosystems, Catalog No. APL-H52D1) to a cell density of 1E5 cells / mL. 100 μL of the cell suspension was seeded into a 96-well plate. 10X antibody was prepared with RPMI-1640 complete medium to a final concentration of 100nM, with a 4-fold gradient dilution and 8 points. 20μL of antibody was added to a 96-well plate, and each well was filled to 200μL with RPMI-1640 complete medium. After culturing the 96-well plate in a 37°C, 5% CO2 incubator for 4 days, the plate was centrifuged at 400g for 3min, 50μL of supernatant was removed, 50μL of Cell Counting-Lite 2.0 Luminescent Cell Viability Assay (Nanjing Novozyme Biotechnology Co., Ltd., Cat. No. DD1101-02) was added, and luminescence was measured using a luminescence microplate reader (Tecan F200 Pro). The results are shown in Figures 9 and 14.

[0433] The results showed that the anti-BDCA2 / TACI bispecific fusion proteins could inhibit B cell proliferation induced by BAFF and APRIL, and the inhibitory activities of BI-9D2-A, BI-9ND2-A and BI-9NTACI-A were superior to those of the TACI fusion protein Telitacicept. The linker region of the bispecific fusion protein was modified, and bispecific fusion proteins with different linker peptides could also inhibit B cell proliferation induced by BAFF and ARPIL, and the overall inhibitory activity was superior to that of the TACI fusion protein Telitacicept. The modification of the linker region did not affect the inhibitory effect of the bispecific protein on B cell proliferation. The Fc region of the bispecific fusion protein was mutated by YTE. The modified bispecific fusion protein BI-9ND2-A-YTE could also inhibit B cell proliferation, and its inhibitory activity was superior to that of the TACI fusion protein Telitacicept. The YTE mutation did not affect the inhibitory effect of the bispecific fusion protein on B cell proliferation.

[0434] Example 17: Cytotoxicity of cells expressing human BDCA2 protein by bispecific fusion proteins, bispecific fusion proteins with different connecting peptides, and bispecific fusion proteins with a YTE mutation in the Fc region

[0435] The experimental method is the same as that of Example 9, and the results are shown in Figures 10 and 15.

[0436] Results showed that the bispecific fusion proteins BI-9D2-A, BI-9ND2-A, and BI-9NTACI-A were all able to eliminate pDCs and exhibited superior ADCC activity to the litifilimab analogue. Modification of the linker region of the bispecific fusion proteins, resulting in bispecific fusion proteins with different linker peptides, also demonstrated pDC elimination and superior ADCC activity to the litifilimab analogue. Furthermore, the modified bispecific fusion protein BI-9ND2-A-YTE, with a YTE mutation in the Fc region, also demonstrated pDC elimination and superior ADCC activity to the litifilimab analogue. The YTE mutation did not affect the bispecific fusion proteins' ability to kill cells expressing human BDCA2 protein.

[0437] Example 18: Bispecific fusion proteins, bispecific fusion proteins with different linker peptides, and bispecific fusion proteins with a YTE mutation in the Fc region inhibit IFN-α production in PBMCs stimulated by TLR9 agonists

[0438] The experimental method is the same as that of Example 6, and the results are shown in Figures 11 and 16.

[0439] The results showed that TLR9 agonists can promote the release of IFN-α from PBMCs, while the bispecific fusion proteins BI-9D2-A and BI-9NTACI-A can both inhibit the release of IFN-α, with inhibitory effects comparable to those of the litifilimab analogue. The linker regions of the bispecific fusion proteins BI-9D2-A and BI-9ND2-A were modified, and bispecific fusion proteins with different linker peptides could also inhibit the release of IFN-α. The modification of the linker did not affect the inhibitory effect of the bispecific protein on IFN-α. The Fc region of the bispecific fusion protein BI-9ND2-A was mutated with YTE, and the modified bispecific fusion protein BI-9ND2-A-YTE could also inhibit the release of IFN-α.

[0440] Example 19: Effects of Bispecific Fusion Proteins and Bispecific Fusion Proteins with YTE Mutations in the Fc Region on Eliminating pDCs in the huHSC-NCG Mouse Model

[0441] 19.1 The in vivo efficacy of BDCA2-TACI bispecific fusion protein in depleting pDCs was studied in the huHSC-NCG mouse model.

[0442] For the experiments in this article, huHSC-NCG mice were purchased from Jiangsu Gempharmatech Biotechnology Co., Ltd. (Gempharmatech Co., Ltd). After the one-week adaptation period, 25 huHSC-NCG mice were randomly divided into 5 experimental groups, with 5 mice in each group. On day 0, the blank control group (PBS) (G1), Litifilimab analogue (10 mg / kg) (G2), BI-9D2-A (10 mg / kg) (G3), BI-9ND2-A (10 mg / kg) (G4), and BI-9NTACI-A (10 mg / kg) (G5) were intraperitoneally administered to the mice. On day 3, peripheral blood and spleen of each group of mice were collected for pDCs detection. As shown in Figures 12a-12d, each dual antibody group significantly reduced the proportion of pDCs in peripheral blood and spleen compared with the PBS group. BI-9D2-A and BI-9ND2-A significantly reduced the number and percentage of pDCs in peripheral blood and spleen.

[0443] 19.2 The in vivo efficacy of the BDCA2-TACI bispecific fusion protein with a YTE mutation in the Fc region in depleting pDCs was studied in the huHSC-NCG mouse model.

[0444] For the experiments in this article, huHSC-NCG mice were purchased from Jiangsu Gempharmatech Co., Ltd. After one week of adaptation, 15 huHSC-NCG mice were randomly divided into 5 experimental groups, with 5 mice in each group. On day 0, the blank control group (PBS) (G1), Litifilimab analogue (10 mg / kg) (G2), and BI-9ND2-A-YTE (10 mg / kg) were intraperitoneally administered to the mice. On day 3, peripheral blood and spleen of each group of mice were taken for pDCs detection. As shown in Figures 17a-17d, the dual antibody group can significantly reduce the number and proportion of pDCs in the peripheral blood and spleen compared with the PBS group, and the YTE mutation does not affect the clearance effect of the bispecific fusion protein on pDCs.

[0445] Example 20: Effects of bispecific fusion proteins and bispecific fusion proteins with YTE mutations in the Fc region in the KLH-induced delayed-type hypersensitivity (DTH) model in mice

[0446] Effects of 20.1 Bispecific Fusion Protein in KLH-Induced Delayed-Type Hypersensitivity (DTH) Model in BALB / c Mice

[0447] The effects of the bispecific fusion protein were investigated in a KLH (Sigma, Catalog No. SRP6195)-induced delayed-type hypersensitivity (DTH) model in BALB / c mice. BALB / c mice were purchased from Gempharmatech Co., Ltd. After a one-week acclimation period, 6-8-week-old BALB / c mice were randomly divided according to body weight into a healthy control group (6 mice), an induced model group (6 mice), and two antibody-administered model groups (6 x 2 = 12 mice). The day of grouping was defined as D0. The modeling group and the efficacy group received KLH (10 μg / g × mouse body weight (g)) induction (ip) on day 0 (D0) and day 12 (D12), respectively. The healthy control group received saline injection (ip) on days 4 and 11. The experimental group received the bispecific fusion protein on days 4 and 11, administered ip in a volume of 10 μL / g × mouse body weight (g). The doses were BI-9ND2-A 20 mpk and Telitacicept 10 mpk. The order of administration remained consistent for each administration. D20 was the endpoint, and serum was collected for anti-KLH antibody levels (IgM) by ELISA (Life Diagnostics, Catalog No.: KLHM-1). After euthanization, the spleens of the mice were harvested and weighed. Flow cytometry analysis of spleen immune cells was performed. The spleen flow cytometry analysis included the following proteins: CD45 (Biolegend, catalog number: 103140), CD3 (BD, catalog number: 562600), CD4 (BD, catalog number: 560181), PD1 (Biolegend, catalog number: 135231), CD185 (BD, catalog number: 551959), B220 (Biolegend, catalog number: 103206), CD19 (Biolegend, catalog number: 115546), and CD138 (Biolegend, catalog number: 142506).

[0448] As shown in Figures 13a-13e, KLH IgM levels were significantly increased in the KLH Vehicle group compared to the saline Vehicle group, and BI-9ND2-A significantly inhibited KLH IgM expression. Flow cytometry results showed that the bispecific fusion protein BI-9ND2-A significantly eliminated KLH IgM expression in mouse plasma cells, and the elimination effect was better than that of the control antibody Telitacicept. In addition, BI-9ND2-A also significantly inhibited KLH IgM expression in mouse B cells, and the inhibitory effect was also better than that of the control antibody Telitacicept.

[0449] Effects of Bispecific Fusion Proteins with a YTE Mutation in the Fc Region in the KLH-Induced B-hFcRn Mouse Delayed-Type Hypersensitivity (DTH) Model

[0450] The effects of bispecific fusion proteins harboring a YTE mutation in the Fc region were investigated in a KLH (Sigma, Catalog No. SRP6195)-induced B-hFcRn mouse delayed-type hypersensitivity (DTH) model. B-hFcRn mice were purchased from Biocytogen. After a one-week acclimation period, 6-8-week-old B-hFcRn mice were randomly divided according to body weight into a healthy control group (8 mice), an induced model group (8 mice), and two antibody-administered model groups (8 x 2 = 16 mice). The day of grouping was defined as D0. The modeling group and the efficacy group were induced (ip) with KLH (10 μg / g × mouse body weight (g)) on D0 and D12, respectively. The healthy control group was injected with normal saline (ip) on D4, D7, D10, D13 and D16. The bispecific fusion protein was administered ip on D4, D7, D10, D13 and D16. The administration route was ip, the administration volume was 10 μL / g × mouse body weight (g), and the administration dose was BI-9ND2-A-YTE 20 mpk and Telitacicept 10 mpk.

[0451] The order of administration remained consistent for each administration. D20 was the endpoint. Serum was collected and the antigen concentration in the mouse serum was measured.

[0452] After euthanization, the mice were harvested and weighed. Flow cytometry analysis of splenic immune cells was performed for CD45 (Biolegend, Catalog No. 103140), CD3 (Biolegend, Catalog No. 100326), CD4 (Biolegend, Catalog No. 100526), ​​B220 (Biolegend, Catalog No. 103240), and CD138 (Biolegend, Catalog No. 142519). As shown in Figure 18a, flow cytometry was used to assess the percentage of B cells in the spleen. The bispecific fusion protein BI-9ND2-A-YTE, harboring a YTE mutation in the Fc region, significantly inhibited mouse B cell expression.

[0453] At the end of the experiment, mouse serum was collected and anti-KLH antibody levels (IgM and IgG) were measured using ELISA kits (Life Diagnostics, Catalog Nos. KLHM-1 and KLHG-1). As shown in Figures 18b and 18c, the KLH Vehicle group showed significantly increased anti-KLH IgM and IgG levels compared to the Vehicle group, and BI-9ND2-A-YTE significantly inhibited the expression of KLH IgM and IgG.

[0454] 72 hours after the first administration and 72 hours after the fifth administration, the mouse serum was collected to detect the antibody concentration in the mouse serum.

[0455] BI-9ND2-A-YTE was detected using a sandwich assay: 1 μg / mL human BDCA2 protein (ACRObiosystems, Cat# CLC-H5245) was coated onto a 96-well plate at 50 μL / well and incubated overnight at 4°C. The plate was blocked with blocking buffer (1% BSA in PBS) at 37°C for 1 hour. After blocking, the plate was washed once with PBST (0.05% Tween 20 in PBS). The bispecific antibody (BI-9ND2-A-YTE) was serially diluted with 2% mouse serum diluent (0.05% Tween 20 and 0.5% BSA in PBS) as a standard. Serum samples were diluted with mouse serum and the diluent at various dilutions, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed twice with PBST. Biotinylated human BAFF protein (KACTUS, Cat#BAF-HM412B) was diluted in diluent to 1 μg / ml, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBST. Secondary antibody (horseradish peroxidase-HRP-conjugated streptavidin, Jackson Immuno Research, Cat#016-030-084) was diluted in diluent and added to the plate for incubation at 37°C for 1 hour. After incubation, the plate was washed again and developed with TMB (Thermo Scientific, Cat#34029). The color was then stopped with 1 M H2SO4. The absorbance was then read in a microplate reader at OD450nm-OD620nm.

[0456] Telitacicept detection method: 0.5 μg / ml human BAFF protein (ACRObiosystems, Cat#BAF-H52D4) was coated on a 96-well plate at 50 μL / well and incubated overnight at 4°C. The plate was incubated at 37°C with blocking buffer (PBS solution containing 1% BSA) for 1 hour. After blocking, the plate was washed once with PBST solution (PBS solution containing 0.05% Tween 20). Telitacicept was gradiently diluted with a diluent containing 2% mouse blank serum (PBS solution containing 0.05% Tween 20 and 0.5% BSA) as a standard. The serum samples were diluted with mouse blank serum and diluent to different multiples, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBST solution. The secondary antibody (horseradish peroxidase HRP-conjugated goat anti-human IgG, Fcγ fragment specific, Jackson Immuno Research, Cat#109-035-098) was diluted in diluent and added to the plate for incubation at 37°C for 1 h. After incubation, the plate was washed again and developed with TMB (Thermo Scientific, Cat#34029). The color was then stopped with 1 M H2SO4, and the absorbance at OD450nm-OD620nm was read in a microplate reader.

[0457] As shown in Figure 18d, in the KLH-induced B-hFcRn mouse delayed-type hypersensitivity (DTH) model, at the same time point, the concentration of the bispecific fusion protein BI-9ND2-A-YTE with a YTE mutation in the Fc region was higher than that of Telitacicept, suggesting that compared with Telitacicept, BI-9ND2-A-YTE was cleared more slowly in mice and had better PK.

[0458] Example 21 Efficacy of the bispecific fusion protein in the MOG-induced C57BL / 6J experimental allergic encephalomyelitis (EAE) model

[0459] In MOG 35-55 The effects of the bispecific fusion protein were investigated in a C57BL / 6J experimental allergic encephalomyelitis (EAE) model induced by Sangon Biotech. For the experiments herein, C57BL / 6J mice were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. After a one-week acclimation period, 8-10 week old C57BL / 6J female mice were injected with 200 μl of 100 μg MOG. 35-55Complete Freund's adjuvant (sc) was administered, and PTX (Pertussis Toxin, Listlabs, catalog number: #180, 200 ng x mouse, i.p.) was injected 2 hours and 48 hours after injection. The day of induction was defined as D0. Mice were randomly divided into a model control group (6 mice) and an antibody-administered model group (6 x 2 mice = 12 mice) based on their disease progression and body weight on D15, D18, D21, and D24. Administration was via i.p. with the bispecific fusion protein described herein. The administration volume was 10 μL / g x mouse body weight (g). The dosage was BI-9ND2-A 20 mpk and Telitacicept 10 mpk. The order of administration remained consistent for each administration. The body weight and clinical symptoms of the mice were assessed and scored twice weekly. Grade 0: No clinical symptoms; Grade 1: Tail weakness; Grade 2: Tail weakness + hindlimb weakness; Grade 3: Tail weakness + hindlimb paralysis; Grade 4: Complete limb paralysis; Grade 5: Imminent death or death. At the endpoint of day 27, mice were euthanized and spleens and brains were harvested. Single-cell suspensions were prepared from the spleen and portions of the brain for flow cytometry analysis of immune cells. Spleen flow cytometry analysis revealed the following: DL, CD45, CD19, and CD138. Brain flow cytometry analysis revealed the following: DL, CD45, CD3, CD19, CD23, and CD21 / 35. Results are shown in Figures 19a-19h.

[0460] The results showed that compared with the G1 group (Vehicle), the body weight of mice in the G2 group (Telitacicept) and the G3 group (BI-9ND2-A) increased after administration, the clinical symptoms of the mice were significantly improved, and the EAE scores were significantly reduced, which was statistically significant.

[0461] After killing the mice, the spleen and brain were taken out, and after preparing into single cell suspension, the proportion of each cell was detected by flow cytometry. As shown in Figures 19c-19h, compared with the Vehicle group, the number and percentage of B cells in the spleen of mice decreased significantly after BI-9ND2-A administration, which shows that BI-9ND2-A can reduce B cells in the spleen of mice, and its efficacy is better than Telitacicept. In addition, after BI-9ND2-A administration, the number and percentage of plasma cells in the spleen also decreased significantly, indicating that BI-9ND2-A can partially clear plasma cells in the spleen, and the clearance effect is comparable to Telitacicept. CD21 / 35 and CD23 are surface markers of activated B cells. After BI-9ND2-A treatment, CD21 / 35 in the brain + and CD23 + The percentage of B cells decreased significantly, indicating that BI-9ND2-A can inhibit the activation of B cells in the brain.

[0462] Example 22: Related Detection of Bispecific Fusion Protein Fc Region after YTE Mutation

[0463] 22.1 Affinity testing of bispecific fusion proteins with YTE mutations in the Fc region to human FcRn

[0464] The binding affinity of BI-9ND2-A and BI-9ND2-A-YTE antibodies to human FcRn protein (ACRO Biosystems, Cat. No.: FCM-H82W4) was detected using ForteBio Octet RED96e.

[0465] The SA sensor (ForteBio, Catalog No. 18-5019) was placed in loading buffer (1X PBS, Cytiva, Catalog No. SH30256.01, containing 0.05% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. The FcRn kinetics assay was performed in a 96-well plate according to the following steps:

[0466] a) Equilibrate the baseline with loading buffer for 180 s;

[0467] b) Add human FcRn protein diluted in loading buffer to a final concentration of 1 μg / mL, and stop when the protein solidifies to 0.3 nm;

[0468] c) The baseline was equilibrated with blocking solution (1X PBS, Cytiva, Catalog No.: SH30256.01, containing 0.05% Tween 20, 0.1% BSA, pH 7.0) for 180 seconds.

[0469] d) Add BI-9ND2-A or BI-9ND2-A-YTE antibodies diluted in running buffer (1X PBS, Cytiva, Cat. No. SH30256.01, containing 0.05% Tween 20, 0.1% BSA, pH 6.0 / 7.4) to each well at the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM. Bind for 60 s (pH 6.0) and dissociate for 60 s (pH 6.0 / 7.4).

[0470] e) Regeneration buffer (1X PBS, Cytiva, Catalog No. SH30256.01, pH 7.7) was used for 30 seconds. Baseline equilibration with blocking buffer was performed for 60 seconds. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0471] Table 14 Binding affinity of BI-9ND2-A and BI-9ND2-A-YTE antibodies to human FcRn protein

[0472] Table 14 shows that the antibody of the present invention has an increased affinity for FcRn compared to BI-9ND2-A under the conditions of binding for 60 s (pH 6.0) and dissociation for 60 s (pH 6.0) in the running buffer, indicating that the YTE mutation of the bispecific fusion protein was successful.

[0473] PK evaluation of bispecific fusion proteins with a YTE mutation in the Fc region in B-hFcRn mice

[0474] B-hFcRn mice (purchased from Biocytogen) were intravenously injected with 10 mg / kg of the bispecific fusion protein BI-9ND2-A or BI-9ND2-A-YTE, a bispecific fusion protein with a YTE mutation in the Fc region. Serum was collected before, 0.167 hours, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, and 240 hours after administration. Antibody concentrations in mouse serum were determined using a sandwich assay. The sandwich assay was performed as follows: 1 μg / ml human BDCA2 protein (ACRObiosystems, Cat# CLC-H5245) was coated onto a 96-well plate at 50 μL / well and incubated overnight at 4°C. The plate was blocked with blocking buffer (1% BSA in PBS) at 37°C for 1 hour. After blocking, the plate was washed once with PBST (0.05% Tween 20 in PBS). Bispecific antibodies (BI-9ND2-A, BI-9ND2-A-YTE) were serially diluted in a diluent containing 2% mouse blank serum (PBS solution containing 0.05% Tween 20 and 0.5% BSA) as standards. Serum samples were diluted with mouse blank serum and the diluent to varying multiples, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed twice with PBST. Biotinylated human BAFF protein (KACTUS, Cat#BAF-HM412B) was diluted in the diluent to 1 μg / ml, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBST. The secondary antibody (horseradish peroxidase HRP-conjugated streptavidin, Jackson Immuno Research, Cat#016-030-084) was diluted in diluent and added to the plate for incubation at 37°C for 1 h. After incubation, the plate was washed again and developed with TMB (Thermo Scientific, Cat#34029). The color was then terminated with 1 M H2SO4, and the absorbance values ​​at OD450nm-OD620nm were read in a microplate reader. Finally, the pharmacokinetic parameters were calculated using WinNonlin software.

[0475] Table 15 PK parameters of BI-9ND2-A and BI-9ND2-A-YTE bispecific fusion proteins in B-hFcRn mice

[0476] The results are shown in Table 15 and Figure 20. Compared with the bispecific fusion protein BI-9ND2-A without Fc mutation, BI-9ND2-A-YTE T 1 / 2 The time was longer (48.1h), the AUC (0-∞) was higher, and the CL value was lower, indicating that after the Fc region was modified with YTE, BI-9ND2-A-YTE had better PK in B-hFcRn mice.

[0477] Example 23 PK testing of bispecific fusion proteins with YTE mutations in the Fc region in cynomolgus monkeys

[0478] Cynomolgus macaques (n=2, Nanjing Yunqiao Pure Biotechnology Co., Ltd.) were injected intravenously with a single dose of 10 mg / kg. Serum was collected before, 0.017 h, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, and 672 h after dosing. Serum antibody concentrations were determined using a sandwich assay. The sandwich assay was performed as follows: 1 μg / ml human BDCA2 protein (ACRObiosystems, Cat# CLC-H5245) was coated onto a 96-well plate at 50 μL / well and incubated overnight at 4°C. The plate was blocked with blocking buffer (1% BSA in PBS) at 37°C for 1 h. After blocking, the plate was washed once with PBST (0.05% Tween 20 in PBS). The bispecific antibody (BI-9ND2-A-YTE) was serially diluted with a diluent containing 1% cynomolgus macaque blank serum (PBS solution containing 0.05% Tween 20 and 0.5% BSA) as a standard. Serum samples were diluted with cynomolgus macaque blank serum and the diluent at various dilutions, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed twice with PBST solution. Biotinylated human BAFF protein (KACTUS, Cat#BAF-HM412B) was diluted with the diluent to 1 μg / ml, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBST solution. The secondary antibody (horseradish peroxidase HRP-conjugated streptavidin, Jackson Immuno Research, Cat#016-030-084) was diluted in diluent and added to the plate for incubation at 37°C for 1 h. After incubation, the plate was washed again and developed with TMB (Thermo Scientific, Cat#34029). The color was then terminated with 1M H2SO4. The absorbance values ​​at OD450nm-OD620nm were read in a microplate reader, and the pharmacokinetic parameters were calculated using WinNonlin software.

[0479] Table 16 PK parameters of bispecific fusion proteins with YTE mutation in the Fc region in cynomolgus monkeys

[0480] The results are shown in Table 16 and Figure 21. The bispecific fusion protein with YTE mutation in the Fc region has a good PK in cynomolgus monkeys. After a single dose of 10 mg / kg, T 1 / 2 It is 281.8h.

[0481] This application relates to the following biological sequences:

Claims

1. An anti-BDCA2 antibody or an antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein the heavy chain variable region VH comprises three CDRs, HCDR1, HCDR2 and HCDR3, and the light chain variable region VL comprises three CDRs, LCDR1, LCDR2 and LCDR3, wherein (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 1; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 2; (ii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:4; or (iii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 13; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 15; or (iv) the HCDR1, HCDR2 and HCDR3 are selected from the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15; or (v) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) to (iv), for example, the amino acid change is a mutation that enhances antibody stability; Preferably, the CDRs are identified by the Kabat numbering scheme.

2. The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 1, comprising (i) HCDR1 as shown in SEQ ID NO:33, HCDR2 as shown in SEQ ID NO:34, HCDR3 as shown in SEQ ID NO:35; LCDR1 as shown in SEQ ID NO:36, LCDR2 as shown in SEQ ID NO:37, and LCDR3 as shown in SEQ ID NO:38; or (ii) HCDR1 as shown in SEQ ID NO:39, HCDR2 as shown in SEQ ID NO:40 or 49, HCDR3 as shown in SEQ ID NO:41; LCDR1 as shown in SEQ ID NO:42 or 48, LCDR2 as shown in SEQ ID NO:43 and LCDR3 as shown in SEQ ID NO:44; or (iii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) or (ii), for example, the amino acid change is a mutation that enhances the stability of the antibody.

3. The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region VH (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 3, 13 or 14; or (ii) comprises the amino acid sequence of SEQ ID NO: 1, 3, 13 or 14 or consists of the amino acid sequence.

4. The anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the light chain variable region VL (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4 or 15; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 2, 4 or 15.

5. The anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (ii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iv) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

6. The anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: (i).SEQ ID NO:1 and SEQ ID NO:2; (ii).SEQ ID NO:3 and SEQ ID NO:4; (iii). SEQ ID NO: 13 and SEQ ID NO: 15; (iv). SEQ ID NO:14 and SEQ ID NO:

15.

7. The anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising an Fc or heavy chain constant region CH, for example, The Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4, preferably the Fc region of IgG1; or The antibody heavy chain constant region CH is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1, Optionally, the sequence of the heavy chain constant region has one or more amino acid substitutions compared to the sequence of a natural human heavy chain constant region, preferably, the one or more amino acid substitutions enhance the ADCC and / or CDC effect of the antibody and / or prolong the in vivo half-life and / or increase the affinity for FcRn, More preferably, the one or more amino acid substitutions are the amino acid substitution combination of M252Y / S254T / T256E according to EU numbering, for example, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

54.

8. The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 7, wherein the heavy chain constant region CH (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5; or (ii) comprises the amino acid sequence of SEQ ID NO: 5 or consists of the amino acid sequence. 9 . The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 1 , further comprising a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

10. The anti-BDCA2 antibody or antigen-binding fragment thereof of claim 9, wherein the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6; or (ii) comprises the amino acid sequence of SEQ ID NO: 6 or consists of the amino acid sequence.

11. The anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-10, comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, 11, 16 or 17, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

12. The anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-11, comprising a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 10, 12 or 18, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

13. The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 11 or 12, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises the amino acid sequence of SEQ ID NO:9, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; and the light chain comprises the amino acid sequence of SEQ ID NO:10, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; (ii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 11, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; and the light chain comprises the amino acid sequence of SEQ ID NO: 12, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; (iii) the heavy chain comprises, or consists of, an amino acid sequence of SEQ ID NO:16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of an amino acid sequence of SEQ ID NO:18, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iv) the heavy chain comprises the amino acid sequence of SEQ ID NO:17, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; and the light chain comprises the amino acid sequence of SEQ ID NO:18, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

14. The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 13, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO: (i).SEQ ID NO:9 and SEQ ID NO:10; (ii). SEQ ID NO: 11 and SEQ ID NO: 12; (iii). SEQ ID NO: 16 and SEQ ID NO: 18; (iv). SEQ ID NO:17 and SEQ ID NO:

18.

15. The anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof has ADCC enhancement and / or half-life extension, for example, it has an altered glycosylation type, for example, it is hypofucosylated or defucosylated or contains an increased bisecting GlcNac structure, and / or for example, carries an amino acid substitution combination of M252Y / S254T / T256E. 16 . The anti-BDCA2 antibody or antigen-binding fragment thereof according to claim 1 , which is a humanized antibody or a chimeric antibody. 17 . The anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 , which is a monoclonal antibody.

18. The anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody) or linear antibody.

19. A fusion protein molecule that specifically binds to human BDCA2 and human BAFF and / or APRIL.

20. The fusion protein molecule of claim 19, comprising a first target binding region and a second target binding region, wherein the first target binding region specifically binds to BDCA2, and the second target binding region specifically binds to BAFF and / or APRIL.

21. The fusion protein molecule of claim 20, wherein the first target binding region and the second target binding region are connected via a linker, or are directly connected without a linker.

22. The fusion protein molecule of claim 20 or 21, wherein the first target binding region is an anti-BDCA2 antibody or an antigen-binding fragment thereof or is derived from an anti-BDCA2 antibody or an antigen-binding fragment thereof, preferably, (i) the anti-BDCA2 antibody or an antigen-binding fragment thereof is the anti-BDCA2 antibody or an antigen-binding fragment thereof of claims 1-18; or (ii) the anti-BDCA2 antibody or an antigen-binding fragment thereof exhibits the same binding specificity as the anti-BDCA2 antibody or an antigen-binding fragment thereof of claims 1-18.

23. The fusion protein molecule of any one of claims 19-22, wherein the second target binding region comprises a BAFF / APRIL receptor or a functional variant or fragment thereof that specifically binds to BAFF and APRIL.

24. The fusion protein molecule of claim 23, wherein the BAFF / APRIL receptor is TACI, for example, a natural TACI molecule or a functional variant thereof, or a functional fragment / truncation of a natural TACI molecule or a functional variant thereof, for example, the functional fragment is the extracellular region, CRD1-2, CDR2, CRD2 truncation, or a fragment consisting of amino acids 30-118 of a TACI molecule, Preferably, the TACI (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 20; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 20; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:

20.

25. The fusion protein molecule of claim 23 or 24, wherein the functional fragment comprises CRD2 or is CRD2.

26. The fusion protein molecule of claim 25, wherein the CRD2 is CRD2 of wild-type TACI, such as CRD2 of a wild-type TACI molecule, or is a variant CRD2 comprising modifications relative to wild-type CRD2.

27. The fusion protein molecule of claim 25 or 26, wherein the CRD2 is the CRD2 of the TACI molecule, such as the CRD2 of the TACI molecule (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 21; (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:

21.

28. The fusion protein molecule according to any one of claims 19 to 27, comprising (i) Chain A: anti-BDCA2 antibody light chain; (ii) Chain B: anti-BDCA2 antibody heavy chain, which is linked to TACI at the N-terminus or C-terminus with or without a linker.

29. The fusion protein molecule of claim 28, wherein chain B comprises the following elements in sequence from N-terminus to C-terminus: (iii) anti-BDCA2 heavy chain, linker and TACI; or (iv) TACI, a linker and an anti-BDCA2 heavy chain; or (vi) anti-BDCA2 heavy chain, linker, TACI and polyserine residue sequence, or (viii) TACI, a polyserine residue sequence, a linker, and an anti-BDCA2 heavy chain; in, TACI is a full-length TACI molecule or a fragment thereof, wherein the polyserine residue sequence is SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS or SSSSSSSSSS, And optionally, the linker may be absent.

30. The fusion protein molecule of claim 29, wherein TACI is a CRD2 domain.

31. The fusion protein molecule of any one of claims 28 to 30, wherein the linker can be any universal flexible sequence, such as a short flexible amino acid sequence; preferably, the linker comprises 10 to 30 amino acids in length; more preferably, the linker is (GGGGS)n; (GSSSS)n; (GGGSG)n; (GGSGG)n; (GSGGG)n; (SGGGG)n; (GGTGS)n; (GTSPGG)n; (GNGGGS)n; (GGG)n; (DGGGS)n; (TGEKP)n; (GGRR)n; (EGKSSGSGSESKVD)n; (KESGSVSSEQLAQFRSLD)n; (GG RRGGGS)n; (LRQRDGERP)n; (LRQKDGGGSERP)n; (GSTSGSGKPGSGEGSTKG)n and / or G4S-GGSGG-G4S-SGGGG, etc., wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9; More preferably, the linker is (G4S)n or (GS4)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9; Most preferably, the linker is selected from GS4, G4S, (GS4)2, (G4S)2, (GS4)3, (G4S)3, (GS4)4, (G4S)4.

32. The fusion protein molecule of any one of claims 28-31, wherein the fusion protein molecule consists of 2 chains A and 2 chains B, preferably, the fusion protein molecule consists of the same 2 chains A and the same 2 chains B.

33. The fusion protein molecule of any one of claims 28 to 32, wherein chain A comprises the amino acid sequence shown in SEQ ID NO: 18 or 52 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 1 An amino acid sequence with 98% or 99% identity or consisting of said amino acid sequence.

34. The fusion protein molecule of any one of claims 28-33, wherein chain B comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOs: 22-32 and 50-51 and 53, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

35. The fusion protein molecule of any one of claims 28 to 34, wherein Chain A comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 18 or 52, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and chain B comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 22-32 and 50-51 and 53, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, Preferably, chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 18 or 52, and chain B comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 22-32 and 50-51 and 53.

36. A fusion protein molecule according to any one of claims 19 to 35, comprising an antibody heavy chain constant region with enhanced ADCC and / or prolonged half-life and / or increased affinity for FcRn, for example, having an altered glycosylation type, for example, being hypofucosylated or defucosylated or comprising an increased bisecting GlcNac structure, and / or for example carrying an amino acid substitution combination of M252Y / S254T / T256E according to EU numbering, for example, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

54.

37. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding any one chain of the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1 to 18 or the fusion protein molecule of any one of claims 19 to 36.

38. An expression vector comprising the nucleic acid molecule of claim 37, preferably, the expression vector is pCDNA, such as pCDNA3.

1.

39. A host cell comprising the nucleic acid molecule of claim 37 or the expression vector of claim 38, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as HEK293 cells or 293T cells or CHO-S cells.

40. A method for preparing the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-18 or the fusion protein molecule of any one of claims 19-36, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 37 or the expression vector of claim 38 under conditions suitable for expression of the chains of the fusion protein, and optionally recovering the antibody from the host cell (or host cell culture medium).

41. A pharmaceutical composition or drug or preparation comprising the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or the fusion protein molecule according to any one of claims 19 to 36 and optionally a pharmaceutically acceptable excipient.

42. A pharmaceutical combination product comprising the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1 to 18 or the fusion protein molecule of any one of claims 19 to 36, and one or more other therapeutic agents (e.g., anti-inflammatory drugs, other antibodies or small molecule drugs).

43. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1 to 18 or the fusion protein molecule of any one of claims 19 to 36, or the pharmaceutical composition or formulation of claim 41; or the pharmaceutical combination product of claim 42.

44. The method of claim 43, wherein the disease is an inflammatory disorder / disease and / or an autoimmune disorder / disease, e.g., selected from systemic lupus erythematosus (SLE) (e.g., moderate or severe lupus), cutaneous lupus, discoid lupus, lupus nephritis, systemic sclerosis (scleroderma), morphea, psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD), dermatomyositis, polymyositis, psoriasis, and type I diabetes. Asthma, Behcet's disease, CREST syndrome, Crohn's disease, dermatomyositis, juvenile dermatomyositis, diabetes, discoid lupus erythematosus, pulmonary fibrosis, autoimmune glomerulonephritis, membranous glomerulopathy, juvenile rheumatoid arthritis (juvenile chronic arthritis), mixed connective tissue disease, multiple sclerosis, nephrotic syndrome, panniculitis, pemphigoid, pemphigus, pemphigus erythematosus, pemphigus foliaceus, pemphigus vulgaris, polymyalgia rheumatica, systemic sclerosis, progressive systemic sclerosis (scleroderma), morphea (localized scleroderma), multiple sclerosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, Raynaud's phenomenon / syndrome, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, neuromyelitis spectrum disorder, primary Sjögren's syndrome, myasthenia gravis, and any combination thereof.

45. The method of claim 43, wherein the disease is a tumor, e.g., a hematopoietic tumor.

46. ​​A method for inhibiting excessive activation of plasmacytoid dendritic cells and / or clearing plasmacytoid dendritic cells in a subject, comprising administering to the subject an effective amount of the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or the fusion protein molecule according to any one of claims 19 to 36, or the pharmaceutical composition or formulation according to claim 41; or the pharmaceutical combination product according to claim 42.

47. A method for inhibiting IFNα release in a subject, comprising administering to the subject an effective amount of the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-18 or the fusion protein molecule of any one of claims 19-36, or the pharmaceutical composition or formulation of claim 41; or the pharmaceutical combination product of claim 42.

48. A method for inhibiting B cell proliferation in a subject, comprising administering to the subject an effective amount of the anti-BDCA2 antibody or antigen-binding fragment thereof of any one of claims 1-18 or the fusion protein molecule of any one of claims 19-36, or the pharmaceutical composition or formulation of claim 41; or the pharmaceutical combination product of claim 42.

49. The method of any one of claims 43-44 and 46-48, wherein the subject suffers from inflammation and has BDCA2 expression or elevated BDCA2 expression in tissues or organs affected by the inflammation (e.g., compared to tissues / organs of healthy subjects, or compared to healthy tissues / organs of the same patient).

50. The method of claim 49, wherein the subject has BDCA2 nucleic acid present or elevated in the tissue or organ affected by inflammation (eg, compared to tissue / organ of a healthy subject, or compared to healthy tissue / organ of the same patient).

51. A method for detecting the presence of BDCA2 in a biological sample, comprising (i) contacting a biological sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 or the multi-specific fusion protein according to any one of claims 19 to 35 under conditions that allow the antibody or antigen-binding fragment thereof to bind to BDCA2, (ii) detecting whether a complex is formed between the antibody or multi-specific fusion protein and BDCA2, The formation of the complex indicates the presence of BDCA2.

52. A method for detecting the presence of BAFF and / or APRIL in a biological sample, comprising (i) contacting a biological sample with the multi-specific fusion protein of any one of claims 19 to 36 under conditions that allow it to bind to BAFF and / or APRIL, (ii) detecting whether a complex is formed between the multi-specific fusion protein and BAFF and / or APRIL, The formation of a complex therein indicates the presence of BAFF and / or APRIL.

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