BDCA2-binding protein and pharmaceutical use thereof

Abstract

The present application relates to a BDCA2-binding protein and a pharmaceutical use thereof. Specifically, the present application relates to a BDCA2-binding protein and a pharmaceutical use thereof in treating autoimmune diseases.

Description

BDCA2 binding protein and its medicinal uses

[0001] This disclosure claims priority to the following patent application: Chinese patent application filed on January 13, 2025, application number CN 202510047722.0, entitled "BDCA2 binding protein and its pharmaceutical use"; the entire contents of the aforementioned patent application are incorporated herein by reference. Technical Field

[0002] This application relates to a blood dendritic cell antigen 2 (BDCA2) binding protein, a pharmaceutical composition containing a BDCA2 binding protein, and its pharmaceutical use for treating or preventing autoimmune diseases. Background Technology

[0003] Plasma-like dendritic cells (pDCs) are a group of bone marrow-derived cells with surface-specific markers including blood dendritic cell antigen 2 (BDCA, or CD303), CD304 (BDCA4), CD123 (IL-3R), and CD45RA, accounting for 0.1%-0.6% of human peripheral blood. pDCs are a major source of IFN-I, primarily recognizing intracellular viruses or their own DNA and RNA via Toll-like receptors (TLRs), producing large amounts of type I and type III interferons. In vitro studies in mouse cells have shown that immune complexes containing RNA and DNA activate TLR7 and TLR9 through BCR-mediated B cell internalization or FcγR-mediated dendritic cell (DC) internalization, respectively, releasing IFN-I. The released IFN-I can activate more DCs and effector T cells, inducing... B cells transform into plasma cells that produce autoantibodies, exacerbating the inflammatory response.

[0004] BDCA2 is a C-type lectin specifically expressed on plasmacytoid dendritic cells (pDCs) in humans and primates. The protein structure of BDCA2 consists of a C-terminal extracellular carbohydrate recognition domain (CRD), a transmembrane region, and an N-terminal cytoplasmic amino acid fragment. The extracellular region of BDCA2 binds to a terminal galactose-modified double-stranded glycoprotein; IgG, IgA, and IgM are known ligands for BDCA2. The cytoplasmic region of BDCA2 lacks signal transduction domains; it primarily transmits signals through the associated transmembrane protein FcRγ, promoting phosphorylation of spleen tyrosine kinase (SyK), phosphatase Cr2 (PLCγ2), and Bruton's tyrosine kinase (BTK), ultimately leading to the release of intracellular Ca2+ ions and inhibiting the activation of NF-κB and NFAT. Studies have shown that treatment of TLR9-activated pDCs with anti-BDCA2 antibodies can inhibit the expression of IFN-I and its downstream transcripts. Currently, the most advanced anti-BDCA2 antibody is BIIB059 (Litifilimab) developed by Biogen, indicated for systemic lupus erythematosus (SLE) and cutaneous lupus erythematosus (CLE), and is undergoing phase III clinical trials. Clinical data indicate that this antibody has good therapeutic effects on SLE and CLE patients, effectively reducing IFNα secretion and demonstrating good safety and tolerability. Summary of the Invention

[0005] This disclosure provides a BDCA2 binding protein and its encoded nucleic acid, vector, host cell, pharmaceutical composition, and methods for treating and alleviating autoimmune diseases, as well as related pharmaceutical uses. The anti-human BDCA2 antibody of this disclosure has high affinity activity and is intended for the treatment of systemic lupus erythematosus, cutaneous lupus erythematosus, or autoimmune diseases associated with type I interferon overactivation.

[0006] BDCA2 binding protein

[0007] This disclosure provides a BDCA2-binding protein comprising a BDCA2-specific binding domain. In some embodiments, the domain comprises a heavy chain variable region (VH) and / or a light chain variable region (VL). In some embodiments, the BDCA2-specific binding domain in the BDCA2-binding protein is one or more.

[0008] In some embodiments, this disclosure provides a BDCA2 binding protein comprising sequences as shown in SEQ ID NO:87, SEQ ID NO:88 and SEQ ID NO:89.

[0009] Some implementations provide a BDCA2-binding protein comprising:

[0010] (1) Heavy chain variable region (VH) and light chain variable region (VL), wherein the VH includes HCDR1, HCDR2 and HCDR3, and the VL includes LCDR1, LCDR2 and LCDR3;

[0011] The LCDR1-3 is selected from LCDR1-3 as shown in SEQ ID NO:87, 88, 89, LCDR1-3 as shown in SEQ ID NO:96, 13, 14, or LCDR1-3 as shown in SEQ ID NO:97, 18, 98;

[0012] And (2) for binding the BDCA2 extracellular domain, which contains an amino acid sequence as shown in SEQ ID NO:99.

[0013] In some embodiments, the CDR of the VL of the BDCA2-binding protein of this disclosure has the following general formula:

[0014] In some embodiments, PR0031 and its modified molecules provided in this disclosure have a CDR region represented by the following general formula:

[0015] In some embodiments, PR0036 and its modified molecules provided in this disclosure have a CDR region represented by the following general formula:

[0016] In some embodiments, the BDCA2 binding protein VH comprises HCDR1, HCDR2, and HCDR3 from any of the VHs shown in SEQ ID NO: 58, 77, 5, 7, 37-42, 46-48, 51-56, and 78-79, and the VL comprises LCDR1, LCDR2, and LCDR3 from any of the VLs shown in SEQ ID NO: 73, 80, 6, 8, 43-45, 49-50, 57, 59, 60-72, 74, 76, and 81.

[0017] In some implementations, the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR is defined according to the Kabat numbering system.

[0018] In some specific implementations, the BDCA2-binding protein:

[0019] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:58, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:73; or,

[0020] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:77, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:80; or,

[0021] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:8; or,

[0022] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:58, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:59-72 and 74-76; or,

[0023] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:5, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6; or,

[0024] VH includes HCDR1, HCDR2, and HCDR3 as shown in any one of SEQ ID NO:37-42, and VL includes LCDR1, LCDR2, and LCDR3 as shown in VL:43; or,

[0025] VH includes HCDR1, HCDR2, and HCDR3 as shown in any of SEQ ID NO:37-42, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:44; or,

[0026] VH includes HCDR1, HCDR2, and HCDR3 as shown in any one of SEQ ID NO:51-56 and 77-79, and VL includes LCDR1, LCDR2, and LCDR3 as shown in VL:57; or,

[0027] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:77, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:80-81; or,

[0028] VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:78, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:80-81.

[0029] CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0030] In some implementations, the BDCA2-binding protein:

[0031] (a) VH contains HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, HCDR3 as shown in SEQ ID NO:17, and

[0032] VL includes LCDR1 as shown in any of SEQ ID NO:12, 36, 90-94, 27-28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in any of SEQ ID NO:14, 29-35;

[0033] or

[0034] (b) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in any of SEQ ID NO:11, 19-26, respectively, and VL includes LCDR1 as shown in any of SEQ ID NO:12, 27-28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively.

[0035] In some specific implementations, the BDCA2-binding protein:

[0036] (a-1) VH contains HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL contains LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0037] (b-1) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0038] (a-2) VH contains HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0039] (a-3) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or,

[0040] (a-4) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in any of SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:31, respectively; or,

[0041] (a-5) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in any one of SEQ ID NO:31, respectively; or,

[0042] (a-6) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in any of SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in any of SEQ ID NO:32, respectively; or,

[0043] (a-7) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:33, respectively; or,

[0044] (a-8) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:34, respectively; or,

[0045] (a-9) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:35, respectively; or,

[0046] (a-10) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:36, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0047] (a-11) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:90, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0048] (a-12) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:91, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0049] (a-13) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:92, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0050] (a-14) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:93, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0051] (a-15) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:94, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0052] (a-16) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or,

[0053] (a-17) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0054] (a-18) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or,

[0055] (b-2) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0056] (b-3) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:19, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0057] (b-4) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:20, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0058] (b-5) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:21, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0059] (b-6) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:22, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0060] (b-7) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:23, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0061] (b-8) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0062] (b-9) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0063] (b-10) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:26, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0064] (b-11) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0065] (b-12) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively; and VL includes LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or,

[0066] (b-13) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively.

[0067] In some embodiments, the BDCA2 binding protein comprises VH and VL, wherein: any HCDR contained in the VH has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or, any LCDR contained in the VL has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned LCDRs. In some specific embodiments, the amino acid mutations in the HCDRs or LCDRs are conserved substitutions.

[0068] In some embodiments, the BDCA2 binding protein provided in this disclosure comprises any one or any combination of any of the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., 2, 3, 4, 5, or 6).

[0069] In some embodiments, the BDCA2 binding protein comprises a VH as shown in any of SEQ ID NO: 58, 77, 5, 7, 37-42, 46-48, 51-56, 78-79 or having at least 80% or at least 90% identity with it, and / or comprises a VL as shown in any of SEQ ID NO: 73, 80, 6, 8, 43-45, 49-50, 57, 59, 60-72, 74, 76, 81 or having at least 80% or at least 90% identity with it.

[0070] In some specific implementations, the BDCA2-binding protein:

[0071] (A-1) VH contains a sequence as shown in SEQ ID NO:58 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:73 or having at least 80% or at least 90% identity with it; or,

[0072] (B-1) VH contains a sequence as shown in SEQ ID NO:77 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:80 or having at least 80% or at least 90% identity with it; or,

[0073] (A-2) VH contains a sequence as shown in SEQ ID NO:7 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:8 or having at least 80% or at least 90% identity with it; or,

[0074] (A-3) VH contains a sequence as shown in SEQ ID NO:46 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in any one of SEQ ID NO:49-50 or having at least 80% or at least 90% identity with it; or,

[0075] (A-4) VH contains a sequence as shown in SEQ ID NO:47 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in any one of SEQ ID NO:49-50 or having at least 80% or at least 90% identity with it; or,

[0076] (A-5) VH contains a sequence as shown in SEQ ID NO:48 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:50 or having at least 80% or at least 90% identity with it; or,

[0077] (A-6) VH contains a sequence as shown in SEQ ID NO:58 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in any one of SEQ ID NO:59-72, 74-76 or having at least 80% or at least 90% identity with it; or,

[0078] (B-2) VH contains a sequence as shown in SEQ ID NO:5 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:6 or having at least 80% or at least 90% identity with it; or,

[0079] (B-3) VH contains a sequence as shown in SEQ ID NO:37 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in any one of SEQ ID NO:43-44 or having at least 80% or at least 90% identity with it; or,

[0080] (B-4) VH contains a sequence as shown in SEQ ID NO:38 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in any one of SEQ ID NO:43-44 or having at least 80% or at least 90% identity with it; or,

[0081] (B-5) VH contains a sequence as shown in SEQ ID NO:39 or having at least 80% or at least 90% identity with it; VL contains a sequence as shown in SEQ ID NO:45 or having at least 80% or at least 90% identity with it; or,

[0082] (B-6) VH contains a sequence as shown in or having at least 80% or 90% identity with SEQ ID NO:40-42, and VL contains a sequence as shown in or having at least 80% or 90% identity with SEQ ID NO:44; or,

[0083] (B-7) VH contains a sequence as shown in or having at least 80% or 90% identity with any of SEQ ID NO:51-56, 77-79; VL contains a sequence as shown in or having at least 80% or 90% identity with any of SEQ ID NO:57; or,

[0084] (B-8) VH contains a sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:77-78; VL contains a sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:81; or,

[0085] (B-9)VH contains a sequence as shown in SEQ ID NO:78 or having at least 80% or at least 90% identity with it, and VL contains a sequence as shown in SEQ ID NO:80 or having at least 80% or at least 90% identity with it.

[0086] In some embodiments, the BDCA2-binding protein comprises VH and VL, wherein: the VH has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the aforementioned VHs; and / or, the VL has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the aforementioned VLs. In some specific embodiments, the amino acid mutations on VH and / or VL are conserved substitutions.

[0087] In some embodiments, the BDCA2 binding protein is an anti-BDCA2 antibody or an antigen-binding fragment.

[0088] In some implementations, the aforementioned antibody is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody, or a fragment thereof.

[0089] In some implementations, the aforementioned antibody is a recombinant antibody.

[0090] In some implementations, the aforementioned antibody is a monospecific antibody or a multispecific antibody (e.g., a bispecific antibody, a trispecific antibody, or a tetraspecific antibody).

[0091] In some embodiments, the aforementioned antibody or its antigen-binding fragment is Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibody (e.g., VH, VL, or VHH), scFv, linear antibody, single-chain antibody, peptide antibody, domain antibody, nanobody, peptide antibody, bivalent, trivalent, or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody, and any of the above epitope-binding fragments.

[0092] In some embodiments, the heavy chain variable region (VH) and / or light chain variable region (VL) of the BDCA2 binding protein are humanized, reversed by mutation, affinity maturation, removed by post-translational modification (PTM), removed by TCE, or modified to reduce viscosity.

[0093] In some embodiments, the BDCA2 binding protein undergoes viscosity-reducing modification to lower its viscosity. In some specific embodiments, viscosity can be determined as follows: antibody molecules are displaced in 520 buffer (10 mM HAc + 0% Sucrose, pH 5.2), and the sample is progressively concentrated to concentrations of 50 mg / mL, 100 mg / mL, and 150 mg / mL. Viscosity is then measured using a rheometer (Anton Paar, MCR 302e) and a RheoCompass. TM The software performs data analysis.

[0094] In some specific implementation schemes, the heavy-chain framework region of the genus template used in the humanization process is derived from IGHV3-11 and / or IGHV3-21, while the light-chain framework region of the genus template used in the humanization process is derived from IGKV3-11.

[0095] In some implementations, the BDCA2 binding protein comprises scFv, Fv, Fab, or Fab' fragments.

[0096] In some embodiments, the binding protein of this disclosure is capable of cross-linking immune effector cells with BDCA2-expressing cells. For example, the binding protein may be capable of cross-linking immune effector cells with BDCA2-expressing cells by binding to Fc receptors on immune effector cells. Various Fc receptors exist that are specific to different classes of antibodies, including IgG (γ receptor), IgE (η receptor), IgA (α receptor), and IgM (μ receptor). Therefore, in some embodiments, the binding protein is capable of binding to Fc receptors. In some embodiments, the binding protein is capable of binding to FcγR, such as receptors selected from FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, the binding protein is capable of binding to FcγRIIIa. In some embodiments, the binding protein is capable of binding to Fc receptors present on immune effector cells.

[0097] In some embodiments, the BDCA2-binding protein further comprises an immunoglobulin Fc region; in some embodiments, the BDCA2-binding protein can be bound to Fc receptors on immune effector cells via the Fc region. In some embodiments, the Fc region may be a segment that retains the ability to bind to one or more Fc receptors, such as one or more Fc receptors as defined above.

[0098] In some implementations, the Fc region is the Fc region of human IgG.

[0099] In some implementations, the Fc region is the Fc region of human IgG1, human IgG2, or human IgG4.

[0100] In some embodiments, the binding proteins of this disclosure provide altered effector functions that, in turn, affect the biological profile of the binding protein. For example, alterations to the glycosylation profile of the constant region domain of an immunoglobulin molecule can increase Fc receptor binding of the modified binding protein. In other cases, constant region modifications consistent with this disclosure can weaken complement binding. Other modifications to the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, thereby allowing for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region according to this disclosure can be readily performed using well-known biochemical or molecular engineering techniques within the knowledge of a person skilled in the art.

[0101] In some embodiments, the binding protein of this disclosure may have enhanced effector functions that increase the ability of the binding protein to interact with immune effector cells, such as increasing the ability of the binding protein to crosslink immune effector cells with BDCA2-expressing cells bound to the binding protein, or increasing the ability of the selective binding protein to induce ADCC. In some embodiments, the immunoglobulin Fc domain of the binding protein provided in this disclosure is modified compared to the corresponding wild-type Fc domain, wherein the modification results in increased affinity for one or more Fc receptors, preferably one or more Fcγ receptors. In some embodiments, the immunoglobulin Fc domain is modified compared to the corresponding wild-type Fc domain, wherein the modification results in enhanced antibody-dependent cell-mediated cytotoxic responses.

[0102] In some embodiments, the Fc region is an effector function-enhancing Fc region, such as an antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC)-enhancing Fc region. In some specific embodiments, the Fc region is an Fc region with fucose removed from Asn297, the amino acid positions of which are numbered according to the EU numbering system. In some specific embodiments, the Fc region contains mutations that enhance the effector function of the Fc region.

[0103] In some embodiments, the binding protein can be formulated with altered glycosylation profiles, such as fucosylated / low-fucosylated binding proteins with reduced fucosylate residue content or binding proteins with increased GlcNac structure. Such altered glycosylation profiles have been shown to increase the ADCC ability of the binding protein. These modifications can be achieved by any means known in the art, such as those described in the Examples section of this disclosure.

[0104] In some embodiments, the binding protein provided in this disclosure may be unfucosylated. The binding protein may not contain fucose. The binding protein may contain at least one N-linked glycan lacking a core fucose unit.

[0105] In some embodiments, the binding protein is unfucosylated at EU position 297 (also referred to herein as position N297) (Edelman et al., 1969, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 63(1):78-85). That is, the N-linked glycan at position N297 may be absent, may lack fucose, or may lack the core fucose unit.

[0106] In some embodiments, the BDCA2 binding protein has a heavy chain as shown in SEQ ID NO:84 or having at least 80% identity with it; a light chain as shown in SEQ ID NO:85 or having at least 80% identity with it; or a heavy chain as shown in SEQ ID NO:82 or having at least 80% identity with it; and a light chain as shown in SEQ ID NO:83 or having at least 80% identity with it.

[0107] In some embodiments, the BDCA2-binding protein comprises a heavy chain and a light chain, wherein: the heavy chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the aforementioned heavy chains; and / or, the light chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any of the aforementioned light chains. In some specific embodiments, the amino acid mutations on the heavy chain and / or light chain are conserved substitutions.

[0108] In the context of this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity; and "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity.

[0109] In some embodiments, a protein is provided that binds to or competes with the aforementioned BDCA2-binding protein of this disclosure for the same BDCA2 antigenic epitope.

[0110] In some embodiments, a protein is provided whose binding to BDCA2 is blocked by the aforementioned PSMA / TRGV9 binding protein of this disclosure.

[0111] In some embodiments, a protein is provided that binds to a different BDCA2 antigenic epitope than the aforementioned BDCA2-binding protein of this disclosure.

[0112] In some embodiments, a protein (or molecule) is provided that comprises any of the BDCA2-binding proteins disclosed herein. The protein (or molecule) may be a conjugate, coupling compound, or fusion protein formed with other compounds, peptides, nucleic acids, etc. For example, a conjugate may contain any detectable label, or a compound, peptide, nucleic acid, etc., with therapeutic activity.

[0113] In some embodiments, the BDCA2-binding protein of this disclosure binds to BDCA2, further promoting phosphorylation of spleen tyrosine kinase (SyK), phosphatase Cr2 (PLCγ2), and Bruton's tyrosine kinase (BTK). In some embodiments, the final BDCA2-binding protein of this disclosure can lead to the release of intracellular Ca ions, inhibiting the activation of NF-κB and NFAT.

[0114] In some implementations, the aforementioned BDCA2-binding protein has a function or property selected from at least one of the following:

[0115] (a) BDCA2 on cells is bound with EC50 at a concentration of ≤10 nM, such as ≤10 nM, ≤8 nM, ≤7 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.2 nM, or ≤0.1 nM. The EC50 is obtained by FACS detection, a commonly used affinity detection method in the art, such as that described in Example 5 of this disclosure.

[0116] (b) Inhibiting IFNα activity on cells with an IC50 of ≤0.1 nM, such as ≤0.1 nM, ≤0.09 nM, ≤0.08 nM, ≤0.07 nM, ≤0.06 nM, ≤0.05 nM, ≤0.04 nM, ≤0.03 nM, ≤0.02 nM, or ≤0.01 nM;

[0117] (c) Combined with BDCA2;

[0118] In some implementations, the aforementioned protein binds to the K of BDCA2. D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M;

[0119] (d) It binds with high affinity to FcγRIIIa (CD16a), the most important Fcγ receptor for mediating antibody-dependent cellular cytotoxicity (ADCC) of NK cells;

[0120] In some implementations, the aforementioned protein binds to the K+ receptor of the Fcγ receptor FcγRIIIa (CD16a). D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0121] Polynucleotides and carriers

[0122] This disclosure provides a polynucleotide encoding the BDCA2-binding protein; in some embodiments, the polynucleotide is DNA or RNA.

[0123] This disclosure provides a vector containing the aforementioned polynucleotide.

[0124] The nucleic acids disclosed herein may also be in vector form, may be present in a vector and / or may be part of a vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be, in particular, an expression vector, that is, a vector that provides for the in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system) expression of the BDCA2 binding protein. The expression vector typically contains at least one nucleic acid of this disclosure, operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the BDCA2 binding protein of this disclosure include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0125] The nucleic acids disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides disclosed herein, and / or can be isolated from suitable natural sources.

[0126] host cells

[0127] This disclosure provides a host cell that contains or expresses the said polynucleotide or the said vector.

[0128] In some implementations, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0129] For example, bacterial cells include cells containing Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0130] For example, fungal cells may include cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0131] For example, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0132] This disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0133] The cells disclosed herein cannot develop into complete plant or animal individuals.

[0134] Production or preparation method

[0135] This disclosure provides a method for preparing a BDCA2-binding protein, comprising expressing the polynucleotide or the vector in the host cell, and isolating the expressed BDCA2-binding protein from the host cell; in some embodiments, it further comprises a step of purifying the BDCA2-binding protein.

[0136] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving and ion exchange.

[0137] Pharmaceutical Composition

[0138] This disclosure also provides pharmaceutical compositions comprising the BDCA2 binding protein, or a polynucleotide or carrier encoding any of the aforementioned BDCA2 proteins.

[0139] In some embodiments, the pharmaceutical composition comprises an effective amount of the aforementioned BDCA2-binding protein, polynucleotide, or carrier for the prevention or treatment of disease.

[0140] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of BDCA2 binding protein, polynucleotide, and / or carrier per unit dose, or the amount of BDCA2 binding protein, carrier, and / or polynucleotide contained in a unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0141] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of BDCA2 binding protein per unit dose, or the amount of BDCA2 binding protein per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0142] In some embodiments, an article or product is provided comprising the aforementioned BDCA2-binding protein, polynucleotide, and / or carrier. In some embodiments, the article comprises a container and a label. Containers include, for example, bottles, syringes, and test tubes. The container contains a composition effective for treating a disease or condition. A label on or attached to the container indicates that the composition is intended to treat the selected disease or condition.

[0143] Methods of treating diseases and pharmaceutical uses

[0144] This disclosure provides methods for treating or preventing disease, including administering a therapeutically or preventively effective amount of the BDCA2-binding protein, the polynucleotide, the carrier, or the pharmaceutical composition to a subject in need.

[0145] In some embodiments, the BDCA2-binding protein, polynucleotide, carrier, or pharmaceutical composition is used to treat or delay the progression of an autoimmune disease. In some specific embodiments, the autoimmune disease is an autoimmune disease associated with type I interferon overactivation; in other specific embodiments, the autoimmune disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

[0146] This disclosure provides for any of the following uses of the BDCA2 binding protein, the polynucleotide, the carrier, or the pharmaceutical composition:

[0147] (1) Used in the preparation of medicines for the treatment or prevention of diseases;

[0148] (2) Used for the prevention or treatment of diseases;

[0149] (3) Use in the preparation of a medicament for the preparation of a drug that inhibits the activity and / or expression of type I interferon (e.g., IFNα);

[0150] (4) Used to inhibit the activity and / or expression of type I interferon (e.g., IFNα).

[0151] In some implementations, the disease is an autoimmune disease; in some specific implementations, the autoimmune disease is an autoimmune disease associated with type I interferon overactivation; and in some specific implementations, the autoimmune disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

[0152] In some embodiments, this disclosure provides a method for inhibiting the activity and / or expression of type I interferon (e.g., IFNα), comprising the step of contacting target cells with any of the aforementioned BDCA2-binding proteins, polynucleotides, carriers, or pharmaceutical compositions.

[0153] In some implementations, the target cells are isolated cells from outside the body.

[0154] In some implementations, the target cells are cells in the subject's body.

[0155] In some implementations, the subject suffers from an autoimmune disease. In some specific implementations, the autoimmune disease is an interferon overactivation-related autoimmune disease; in other specific implementations, the autoimmune disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

[0156] Terminology Definition

[0157] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0158] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0159] BDCA2, also known as CLEC4C, is a C-type lectin (sequence example: UniProtKB / Swiss-Prot number Q8WTT0), specifically expressed on pDCs in humans and primates. The protein structure of BDCA2 consists of a C-terminal extracellular carbohydrate recognition domain (CRD), a transmembrane region, and an N-terminal cytoplasmic amino acid fragment. The extracellular region of BDCA2 binds to a terminal galactose-modified double-stranded glycoprotein; IgG, IgA, and IgM are known ligands for BDCA2.

[0160] "BDCA2 binding protein" encompasses any protein capable of specifically binding to BDCA2 or any molecule containing said protein, including but not limited to anti-BDCA2 antibodies, such as those defined in this disclosure, their antigen-binding fragments, or conjugates thereof. In some embodiments, the BDCA2 binding protein may include a linker and / or a portion having effector function, such as a half-life extended portion (e.g., an immunoglobulin single variable domain binding to serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0161] In this disclosure, the terms "polypeptide," "peptide," or "protein" are used interchangeably to refer to a polymer of amino acid residues, or an aggregate of polymers of multiple amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are synthetic chemical analogs of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Polypeptide sequences are typically described as follows: the left-hand end of the polypeptide sequence is the amino terminus (N-terminus, N-terminus); the right-hand end of the polypeptide sequence is the carboxyl terminus (C-terminus, C-terminus).

[0162] "Antibody" encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding domains), as long as they exhibit the desired antigen-binding activity.

[0163] Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity varies. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ or λ chain. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains varies considerably, forming the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved frame regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0164] The antibodies disclosed herein can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable in many embodiments. Generally, the antibodies disclosed herein are recombinant antibodies. As used herein, “recombinant” refers to products such as cells or nucleic acids, proteins, or vectors, indicating that said cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that said cells are derived from such modified cells. For example, recombinant cells express genes not present in native (non-recombinant) cell forms or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

[0165] "Antigen-binding domains" encompass Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0166] The determination or definition of a contact ligand (CDR) can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0167] The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a computer program integration suite produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach” in PROTEINS, Structural, Function and Genetics Suppl., 3:194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods but still overlap at least partially with the Kabat CDR, although they may be shortened or lengthened depending on the prediction or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. Correspondences between various numbering systems are well known to those skilled in the art and are exemplified as shown in Table 1 below.

[0168] Table 1. Relationship between CDR numbering systems

[0169] The CDR amino acid residues in the VL and VH regions of the disclosed antibody conform to the known Kabat numbering system in terms of both number and position.

[0170] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0171] An "immunoglobulin domain" refers to a globular region of an antibody chain. The characteristic of an immunoglobulin domain is that it maintains the folding structure of the antibody molecule.

[0172] An immunoglobulin variable domain refers to an immunoglobulin domain that is essentially composed of four "frame regions" (frame region 1 or "FR1", "frame region 2 or "FR2", "frame region 3 or "FR3", and "frame region 4 or "FR4") and three "complementarity-determining regions" (complementarity-determining regions 1 or "CDR1", "complementarity-determining region 2 or "CDR2", and "complementarity-determining region 3 or "CDR3"), or "complementarity-determining regions". Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

[0173] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0174] "Humanized antibody," also known as CDR-grafted antibody, refers to an antibody generated by grafting a non-human CDR sequence into the variable region framework of a human antibody. This overcomes the strong immune response induced by chimeric antibodies due to their large non-human protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity. Examples of "humanization" include grafting mouse CDR sequences into the variable region framework of a human antibody, i.e., antibodies generated from different types of human germline antibody framework sequences. This overcomes the strong antibody-variable response induced by chimeric antibodies due to their large mouse protein components. Humanization methods include protein surface amino acid resurfacing and CDR grafting to a universal framework, which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the variable region framework sequence of the human antibody to maintain activity.

[0175] An epitope is a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining the binding of an epitope to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0176] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or a portion thereof with an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K). DQuantification can be achieved by using methods such as surface plasmon resonance (SPR) (Biacore) to measure the kinetics of complex formation and dissociation. D The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D =kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods, and even for complex mixtures, it can be calculated using methods such as those described by Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double-filtered nitrocellulose filter combined with determinations such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA, and FACS, as well as other assays exemplified elsewhere in this disclosure. Antibody binding kinetics and binding affinity can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, using Biacore. TM Evaluation can be performed using a system or KinExA. The Kelvin values ​​of individual antibody / antigen complexes can be compared. D The binding affinity is used to compare the binding affinity of different molecules associated with their interactions, for example, comparing the binding affinity of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by identifying and comparing the K-value of the target interaction (e.g., the specific interaction between an antibody and an antigen). D K values ​​and non-purposeful interactions D The value is evaluated.

[0177] "Conservative substitution" refers to the substitution with another amino acid residue that has properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

[0178] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two nucleic acid sequences or two polypeptides. When positions in two compared sequences are occupied by the same nucleotide or amino acid monomer—for example, if every position in two DNA molecules is occupied by the same nucleotide—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100%. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0179] The terms "nucleic acid" or "polynucleotide" are used interchangeably herein to refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0180] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors into which nucleic acid inserts are incorporated. Host cell includes progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cell includes cells transfected and / or transformed in vivo with nucleic acids of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

[0181] The term "pharmaceutical-acceptable excipient" or "pharmaceutical-acceptable excipient" includes any material that, when combined with an active ingredient, allows that ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0182] "Inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blockage. "Inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0183] "Inhibition of growth" or "growth inhibition" refers to suppressing the growth or proliferation of cells.

[0184] "Proliferative disease" refers to a condition associated with a certain degree of abnormal cell proliferation. In one implementation, a proliferative disease refers to cancer. "Tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disease," and "tumor" are not mutually exclusive when used in this disclosure.

[0185] "Cancer prevention" refers to delaying, suppressing, or preventing the onset of cancer in subjects whose cancer development or the initiation of tumor development has not been confirmed, but whose cancer susceptibility has been identified through methods such as genetic screening or other means. This also includes treating subjects with precancerous conditions to halt the progression of said precancerous conditions to malignancy or to cause their regression.

[0186] "Giving," "applying," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean treatment, such as of cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0187] "Treatment" means administering, either internally or externally, a therapeutic agent, such as a combination of any of the molecules or pharmaceutical compositions of this disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0188] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects in this disclosure may be animal or human subjects.

[0189] "Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And / or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to."

[0190] In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans. Attached Figure Description

[0191] Figure 1 shows the binding of the anti-BDCA2 chimeric antibody to a cell line that stably expresses recombinant human BDCA2.

[0192] Figure 2 shows the IFNα inhibitory activity of the anti-BDCA2 chimeric antibody on human peripheral blood single cells.

[0193] Figure 3 shows the binding of the humanized antibody against BDCA2 PR0031 to a cell line stably expressing recombinant human BDCA2.

[0194] Figure 4 shows the binding of the humanized antibody PR0036 against BDCA2 to a cell line stably expressing recombinant human BDCA2.

[0195] Figure 5 shows the binding of the PR0031-Hu08 TCE-depleted antibody to a cell line stably expressing recombinant human BDCA2.

[0196] Figure 6 shows the binding of the PR0036-Hu04 TCE-deactivated antibody to a cell line stably expressing recombinant human BDCA2.

[0197] Figure 7 shows the IFNα inhibitory activity of anti-BDCA2 antibody on human peripheral blood single cells.

[0198] Figure 8 shows the ADCC killing of stable cell lines expressing human BDCA2 by human peripheral blood single cells induced by anti-BDCA2 antibody. Detailed Implementation

[0199] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0200] Example 1. Preparation of extracellular domains and truncated proteins of human BDCA2 and cynomolgus monkey BDCA2

[0201] Recombinant human BDCA2-His tag fusion protein (purchased from ACRO, CLC-H5245) was obtained by fusing amino acids 45-213 of the human BDCA2 protein (GenBank accession number: NP_001358319.1) to the N-terminus of the protein. Recombinant monkey BDCA2-His tag fusion protein (purchased from ACRO, CLC-C52H4) was obtained by fusing amino acids 48-212 of the monkey BDCA2 protein (GenBank accession number: XP_005570080.1) to the N-terminus of the protein.

[0202] Human full-length BDCA2 protein sequence (Note: The underlined part is amino acids 45-213 of human BDCA2, i.e., the extracellular domain of BDCA2)

[0203] extracellular domain of human BDCA2 protein

[0204] Full-length BDCA2 protein sequence of cynomolgus monkey (Note: The underlined part represents amino acids 48-212 of the cynomolgus monkey's BDCA2, which belong to the extracellular domain of BDCA2.)

[0205] Example 2. Construction of a stable cell line expressing full-length BDCA2

[0206] HEK293T cells (purchased from ATCC) were transfected with lentiviral expression plasmids encoding human BDCA2 protein (GenBank, accession number: NP_001358319.1, SEQ ID NO:1) and human FcεRIγ(FCER1G) protein (GenBank, accession number: NP_004097.1, SEQ ID NO:3) or cynomolgus monkey BDCA2 protein (GenBank, accession number: XP_005570080.1, SEQ ID NO:2) and cynomolgus monkey FcεRIγ(FCER1G) protein (GenBank, accession number: XP_005541253.1, SEQ ID NO:4). Lentiviral cells were then packaged and infected with the lentivirus. Cell lines stably expressing recombinant full-length human BDCA2 protein or cynomolgus monkey BDCA2 protein were obtained through antibiotic stress selection. The overexpressed protein sequences in the above cell line construction are shown in Table 2.

[0207] Table 2. Protein sequences overexpressed during cell line construction

[0208] Specifically, the nucleotide sequence encoding the human or cynomolgus monkey BDCA2-FcεRIγ protein was cloned and ligated into a lentiviral expression vector, which was then transfected into HEK293T cells to package the lentivirus. HEK293T cells were transfected using a well-known lentiviral infection method. Anti-resistance selection was performed using DMEM medium (purchased from Gibco) containing puromycin. Subcloning was performed in 96-well plates using limiting dilution. The cells were then cultured in a cell culture incubator. After two weeks, the expression of BDCA2-FcεRIγ on the surface of lentivir-infected HEK293T cells was detected by FACS. Single-clone cell lines with high levels of human or monkey BDCA2-FcεRIγ expression were selected, expanded, and cryopreserved for future storage.

[0209] Example 3. Screening of murine anti-BDCA2 monoclonal antibodies

[0210] 3.1 Antibody Screening

[0211] The recombinant human BDCA2-His tag fusion protein from Example 1 was used as an immunogen. The recombinant human BDCA2-His tag fusion protein and the recombinant monkey BDCA2-His tag fusion protein from Example 1 were used for later screening.

[0212] Specifically, recombinant human BDCA2-His tag fusion protein was used as an immunogen to immunize five 6-8 week old female Balb / c mice. The initial immunization dose was 50 μg per mouse. Two weeks after the initial immunization, a booster immunization was performed at a dose of 25 μg per mouse, with subsequent booster immunizations spaced two weeks apart. One week after the second booster immunization, serum samples were collected, and antibody activity in mouse serum was detected using Protein ELISA and FACS. The Protein ELISA procedure was as follows: The plates were coated with 1 μg / mL recombinant human BDCA2-His tag fusion protein or recombinant monkey BDCA2-His tag fusion protein, incubated overnight at 4°C, blocked with 1% BSA / PBST buffer for 1 hour, and washed three times. Mouse serum was serially diluted 10-fold in blocking buffer, starting at a 1:100 ratio, incubated at 37°C for 1 hour, washed three times, and then incubated with HRP-labeled anti-mouse IgG Fc secondary antibody for 1 hour. Wash three times with PBST, add 100 μL of TMB substrate to each well, and terminate the reaction with 2M HCl after 15 minutes. Read the absorbance at 450 nm using a microplate reader. The FACS assay procedure is as follows: 1 × 10⁶ cells per well in the cell plate. 5 Centrifuge 100 μL of cells, discard the supernatant, and add serially diluted mouse serum. Incubate at 4°C for 1 hour. Wash twice with 1% FBS / PBS buffer, discard the supernatant, add Alexa Flour 647 goat anti-mouse secondary antibody (purchased from Jackson Immunological Research), and incubate at 4°C in the dark for 30 minutes. Wash twice with 1% FBS / PBS buffer, resuspend in 200 μL buffer, and perform FACS analysis.

[0213] Five days after the final immunization, mice were intraperitoneally injected with 25 μg of recombinant human BDCA2-His tag fusion protein. Spleens were then collected by euthanasia and milling. Red blood cells contaminated with 1% (w / w) NH4Cl were added to lyse the spleen cells, yielding a spleen cell suspension. The suspension was centrifuged at 1000 rpm and resuspended, and the cells were washed three times. Mouse spleen cells were mixed with mouse myeloma cells SP2 / 0 at a live cell ratio of 5:1, and cell fusion was performed using a high-efficiency electrofusion method. The fused cells were diluted with 20% fetal bovine serum and DMEM medium containing 1×HAT (w / w) into 96-well cell culture plates, 200 μL per well, for a total of 1×10⁶ cells. 5Cells were incubated at 37°C with 5% (v / v) CO2. After 14 days, hybridoma cell supernatants were screened using cell ELISA (HEK293T stable cell line expressing human BDCA2). Positive clones were amplified into 24-well cell plates and cultured further in DMEM medium containing 10% (w / v) HT fetal bovine serum at 37°C and 5% (v / v) CO2. After 3 days, the culture medium from the 24-well cell plates was centrifuged to collect the supernatant. The binding activity of the human BDCA2 expression cell lines HEK293T-human BDCA2-FcεRIγ and HEK293T-cyno BDCA2-FcεRIγ was determined by FACS. The binding to the recombinant human BDCA2-His tag fusion protein was detected by protein ELISA. Positive clones were selected for two rounds of monoclonalization, and monoclonal cell lines with excellent binding activity were screened using the above flow cytometry and protein ELISA methods. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted using Trizol, and reverse transcribed (PrimeScript) TM Reverse Transcriptase (Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified and sequenced by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503). Table 3 lists the variable region amino acid sequences of the anti-BDCA2 antibody obtained in this disclosure, and Table 4 shows the light and heavy chain CDR region amino acid sequences of the anti-BDCA2 antibody obtained in this disclosure.

[0214] Table 3. Variable region amino acid sequence of murine anti-BDCA2 antibody

[0215] Table 4. Variable region CDR sequence of murine anti-BDCA2 antibody (Kabat number)

[0216] 3.2 Preparation of anti-BDCA2 chimeric antibody

[0217] Against the backdrop of the human immunoglobulin constant region, the mouse variable region was expressed in mammalian host cells to provide chimeric antibodies. Specifically, the nucleotide sequence encoding the variable region of the mouse monoclonal antibody was cloned into a pcDNA3.4 vector containing the human heavy and light chain constant regions (human IgG1, kappa) protein sequence and transfected into CHOK1 cells. After 5 days, the cells were centrifuged to remove the cells, and the cell culture medium was collected and filtered. After adjusting the pH to 7.0, the harvested cell culture supernatant was loaded onto a protein A column, and the bound antibody was eluted with glycine, followed by neutralization of the elution buffer with 1M Tris. Detection revealed a chimeric anti-BDCA2 antibody with mouse VH / VL.

[0218] Example 4. Detection of cell surface antigen affinity of anti-BDCA2 chimeric antibody

[0219] The BDCA2 binding activity assay of anti-BDCA2 chimeric antibodies on cells assesses antibody binding strength by detecting the fluorescence signal of the bound antibody on the cell surface and evaluating the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules are mixed with 1×10⁻⁶ antibodies. 5 Cells were incubated at 4°C for 1 hour, excess antibody was washed away, and murine Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 1% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The cells used were the cell lines expressing human or cynomolgus monkey BDCA2-FcεRIγ protein prepared in Example 2. The control antibody was BIIB059 (see WO2014093396A). Table 5 summarizes the relevant characteristics of the antibodies (Figure 1).

[0220] Table 5. Detection of cell surface antigen affinity of anti-BDCA2 chimeric antibodies

[0221] Example 5. Inhibitory activity of anti-BDCA2 chimeric antibody against IFNα in human peripheral blood single cells

[0222] Inhibitory activity of anti-BDCA2 chimeric antibody against IFNα in human peripheral blood single cells. The experiment involved treating human peripheral blood single cells with different concentrations of chimeric antibody, followed by quantitative detection of IFNα production after a certain time period. Specifically, human peripheral blood single cells were resuspended in RPMI complete medium at 1×10⁻⁶ ppm. 6 / wells were seeded into 96-well plates, and serially diluted antibody and control molecules were added. The plates were incubated at 37°C for 2 hours. A fixed concentration of the stimulant CpG-A was then added, and the plates were incubated overnight at 37°C. The supernatant was collected, and the IFNα level was detected using a kit. The dose-response data were fitted to an S-shaped curve using nonlinear regression, and the IC50 value was calculated. Table 6 summarizes the relevant characteristics of the antibody (Figure 2).

[0223] Table 6. Detection of IFNα inhibitory activity of anti-BDCA2 chimeric antibody

[0224] Example 6. Humanization and Modification of Anti-BDCA2 Antibody

[0225] After predicting the structure of the murine monoclonal antibody through homology modeling, the CDR of the murine antibody was chimeric into a suitable human Germline framework (Bioinformation.2014;10(4):180-186;Methods Mol Biol.2019;1904:213-230). Then, a reversion mutation was introduced. Finally, the nucleotide sequence encoding the variable region of the humanized monoclonal antibody was cloned into a pcDNA3.4 vector containing the human heavy and light chain constant regions (human IgG1, kappa) protein sequence and transfected into Fut8 gene knockout CHOK1 cells to prepare fucose-free antibodies.

[0226] Based on the humanized antibody modification, since the heavy chain position 107 of PR0031 is an unpaired cysteine, during the humanization process, this cysteine ​​was replaced with alanine, valine, or serine, which have similar physicochemical properties to cysteine. The antibody sequences after humanization and unpaired cysteine ​​modification are shown in Tables 7 to 10 (Figures 3-6).

[0227] Table 7. Amino acid sequence of PR0031 after humanization modification

[0228] Table 8. Variable region sequence of PR0031 humanized antibody

[0229] Table 9. Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) after humanization of PR0036

[0230] Table 10. Variable region sequence of PR0036 humanized antibody

[0231] For humanized antibodies, in order to reduce the immunogenicity of the antibodies, possible T-cell epitope (TCE) sites are predicted using software. After predicting the structure of the humanized antibody through homology modeling, mutations are introduced at sites that may not affect antibody-antigen binding to reduce the number of predicted TCE sites. TCE-removed antibody molecules are then produced according to the aforementioned method. The antibody sequences after TCE removal modification are shown in Tables 11 and 12.

[0232] Table 11. Amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) after TCE removal in PR0031 and PR0036

[0233] Table 12. Variable region sequences of PR0031 and PR0036 antibodies after TCE removal.

[0234] Example 7. Detection of cell surface antigen affinity after humanization and TCE removal of anti-BDCA2 antibody

[0235] The BDCA2 binding activity assay of humanized and TCE-modified anti-BDCA2 antibodies on cells was performed by detecting the fluorescence signal of antibody binding on the cell surface, and the binding strength of the antibody was evaluated based on the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules were mixed with 1×10⁻⁶ antibodies. 5 Cells were incubated at 4°C for 1 hour to wash away excess antibody. Mouse Alexa Flour 647-labeled anti-human Fc antibody was added, and the cells were incubated at 4°C for 30 minutes. After washing away excess antibody, the cells were resuspended in 200 μL of 1% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The cells used were the cell line expressing human BDCA2-FcεRIγ protein prepared in Example 2. Tables 13 and 14 summarize the relevant characteristics of the antibody.

[0236] Table 13. Detection of cell surface antigen affinity after humanization and PTM modification of anti-BDCA2 antibody

[0237] Table 14. Detection of cell surface antigen affinity after TCE removal of anti-BDCA2 antibody

[0238] Example 8. Viscosity Modification of Anti-BDCA2 Antibody

[0239] Viscosity testing revealed that PR0036-Hu04-TCE01 and PR0031-Hu08-TCE04 had high viscosities, necessitating further modification of the variable region sequences to reduce their viscosity. Considering the charge and spatial structure of the heavy and light chain variable regions, mutations were introduced into the framework regions of both variable regions. The viscosity-modified antibody sequences of PR0036-Hu04-TCE01 and PR0031-Hu08-TCE04 are shown in Tables 15 to 20.

[0240] The stability-modifying mutants of PR0031-Hu08-TCE04 are: PR0031-Hu08-TCE04-Vis01 to PR0031-Hu08-TCE04-Vis03; the viscosity-modifying mutants are: PR0031-Hu08-TCE04-Vis05 to PR0031-Hu08-TCE04-Vis07.

[0241] The single-point mutant proteins of PR0036-Hu04-TCE01 are: PR0036-Hu04-TCE04-Vis01 to PR0036-Hu04-TCE04-Vis13; the multi-point mutant proteins obtained by recombination of the above mutation sites are PR0036-Hu04-TCE01-Vis14 to PR0036-Hu04-TCE01-Vis17.

[0242] Table 15. Variable region sequence after viscosity modification of PR0031-Hu08-TCE04 antibody

[0243] Table 16. Variable region sequence of PR0031-Hu08-TCE04 antibody after viscosity modification.

[0244] Table 17. Amino acid sequence of the light chain variable region (VL) after viscosity modification of PR0036-Hu04-TCE01 antibody.

[0245] Table 18. Variable region sequence of PR0036-Hu04-TCE01 antibody after viscosity modification.

[0246] Table 19. Amino acid sequences of the full-length heavy chain (HC) and full-length light chain (LC) of PR0036-Hu04-TCE01-Vis14 and PR0031-Hu08-TCE04-Vis04 antibodies after viscosity modification.

[0247] Table 20. Viscosity-modified sequences of full-length PR0036-Hu04-TCE01-Vis14-fl and PR0031-Hu08-TCE04-Vis04-fl antibodies.

[0248] Example 9. Detection of cell surface antigen affinity after viscosity modification with anti-BDCA2 antibody

[0249] The BDCA2 binding activity assay of the viscosity-modified anti-BDCA2 antibody on cells was performed by detecting the fluorescence signal of the antibody bound to the cell surface, and the binding strength of the antibody was evaluated based on the intensity of the fluorescence signal. Specifically, serially diluted antibody molecules and control molecules were mixed with 1×10⁻⁶ antibodies. 5Cells were incubated at 4°C for 1 hour to wash away excess antibody. Mouse Alexa Flour 647-labeled anti-human Fc antibody was added, and the cells were incubated at 4°C for 30 minutes. After washing away excess antibody, the cells were resuspended in 200 μL of 1% FBS / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer. The cells used were the cell line expressing human BDCA2-FcεRIγ protein prepared in Example 2. Tables 21 to 24 summarize the relevant characteristics of the antibody.

[0250] Table 21. Detection of cell surface antigen affinity after viscosity modification of PR0036-derived anti-BDCA2 antibody (1)

[0251] Table 22. Detection of cell surface antigen affinity after viscosity modification of PR0036-derived anti-BDCA2 antibody (2)

[0252] Table 23. Detection of cell surface antigen affinity after viscosity modification of PR0031-derived anti-BDCA2 antibody (1)

[0253] Table 24. Detection of cell surface antigen affinity after viscosity modification with anti-BDCA2 antibody (2)

[0254] Example 10. Surface plasmon resonance (SPR) assessment of the kinetics of the interaction between BDCA2 antibody and BDCA2 protein.

[0255] This study used the Biacore biomacromolecule interaction instrument and surface plasmon resonance (SPR) technology to determine the affinity kinetics of the interaction between BDCA2 antibody and human BDCA2 (Acrobiosystem, Cat#CLC-H5245, SEQ ID NO:1) and cynomolgus monkey BDCA2 (Acrobiosystem, Cat#CLC-C52H4, SEQ ID NO:2) proteins.

[0256] Affinity kinetics analysis of the interaction between BDCA2 antibody and human / cynomolgus monkey BDCA2 antigen: A Protein A biosensor chip (Cytiva, Cat#29-1275-56) was used. A specific concentration of the antibody to be tested was prepared as a ligand using HBS-EP+ buffer solution and captured into the Protein A chip's Fc2 (Flow cell 2, Fc2) channel at a flow rate of 10 μL / min for 30 s. A series of concentration gradients of human BDCA2 (150 nM–2.3475 nM) or cynomolgus monkey BDCA2 (150 nM–2.3475 nM) antigen protein were prepared using HBS-EP+ buffer solution as analytes, serially diluted 2-fold, resulting in 7 concentration points. These were injected into the chip's reference channel Fc1 (Flow cell 1, Fc1) and detection channel Fc2 at a flow rate of 30 μL / min. The binding time was 150 s, and the dissociation time was 400 s. Finally, regeneration was performed using 10 mM Glycine pH 1.5 (Cytiva, Cat#BR-1003-54). The regeneration conditions were: regeneration time 30 s, flow rate 30 μL / min. The buffer used in the experiment was HBS-EP + 10× buffer solution (Cytiva, Cat.#BR100669) diluted to 1× (pH 7.4) with DIWater.

[0257] Data statistics and analysis: The experimental data were fitted with a 1:1 Langmuir model using Biacore 8K insight Evaluation Software to obtain the binding parameter Ka, dissociation parameter Kd, or affinity KD values ​​(Table 25).

[0258] Table 25. Binding kinetic parameters of anti-BDCA2 antibodies with human / cynomolgus monkey BDCA2, respectively.

[0259] Example 11. Surface plasmon resonance (SPR) assessment of the kinetics of the interaction between BDCA2 antibody and human Fcγ receptor protein.

[0260] This experiment used the Biacore biomacromolecule interaction instrument and surface plasmon resonance (SPR) technology to determine the affinity kinetics of the interaction between BDCA2 antibody and human Fcγ receptor (human CD16a(F176), Acrobiosystem, Cat#CDA-H5220, SEQ ID NO:86) protein.

[0261] Affinity kinetics analysis of the interaction between BDCA2 antibody and human Fcγ receptor protein: A CM5 sensor chip (Cytiva, Cat#29149603) was used. Anti-histidine antibody was diluted to 20 μg / mL with sodium acetate (Cytiva, Cat#29234602) at pH 4.5 and injected into channels 1-8 of the CM5 chip over a period of 360 s. The signal response value after channel conjugation was greater than 7000 RU, sufficient for affinity kinetics assays. A specific concentration of human Fcγ receptor protein was prepared using HBS-EP+ buffer as a ligand and injected into channel Fc2 at a flow rate of 10 μL / min for a capture time of 30 s. BDCA2 antibody was used as the analyte, serially diluted 2-fold to seven concentration points, prepared with HBS-EP+ buffer to create a series of concentration gradients, which were then flowed through the amino-conjugated anti-histidine channels Fc1 and Fc2 of the CM5 chip. For human CD16a (F176) and human CD32b / c, the flow rate was 30 μL / min, with a binding time of 100 s and a dissociation time of 100 s. For human CD32a (H167) and human CD64, the flow rate was 30 μL / min, with a binding time of 80 s and a dissociation time of 200 s. All samples were regenerated using 10 mM Glycine at pH 1.5. The regeneration conditions were: regeneration time 30 s and flow rate 30 μL / min. The buffer used in the experiment was HBS-EP + 10× buffer solution diluted to 1× (pH 7.4) with DIWater.

[0262] Data statistics and analysis: The experimental data were fitted using Biacore 8K insight Evaluation Software with a 1:1 Langmuir model or a steady-state affinity model to obtain the binding parameter Ka, dissociation parameter Kd, or affinity KD values ​​(Table 26).

[0263] Recombinant human CD16a fusion protein (purchased from ACROBiosystems, CDA-H5220) was obtained by selecting amino acids 17-208 of human CD16a protein (GenBank accession number: NP_000560.6) and fusing the His tag to the C-terminus of the protein.

[0264] Human full-length CD16a protein sequence (Note: The underlined part refers to amino acids 17-208 of the human CD16a protein, i.e., the extracellular domain of the recombinant human CD16a protein.)

[0265] Table 26. Binding kinetic parameters of anti-BDCA2 antibody to human Fcγ receptor protein

[0266] SPR assay results showed that PR0036-Hu04-TCE01-Vis14 and PR0031-Hu08-TCE04-Vis04 had approximately 5 times higher affinity for FcγRIIIa (CD16a), the most important Fcγ receptor for mediating antibody-dependent cellular cytotoxicity (ADCC) of NK cells, than for BIIB059.

[0267] Example 12. Inhibitory activity of anti-BDCA2 antibody against IFNα in human peripheral blood single cells

[0268] Inhibitory activity of anti-BDCA2 antibody against IFNα in human peripheral blood single cells. The experiment involved treating human peripheral blood single cells with different concentrations of antibody, followed by quantitative detection of IFNα production after a certain time period. Specifically, human peripheral blood single cells were resuspended in RPMI complete medium at 1×10⁻⁶ ppm. 6 / wells were seeded into 96-well plates, and serially diluted antibody and control molecules were added. The plates were incubated at 37°C for 2 hours. A fixed concentration of the stimulant CpG-A was then added, and the plates were incubated overnight at 37°C. The supernatant was collected, and the IFNα level was detected using a kit. The dose-response data were fitted to an S-shaped curve using nonlinear regression, and the IC50 value was calculated. Table 27 summarizes the relevant characteristics of the antibody. Figure 7 shows that the anti-BDCA2 antibody has a superior ability to inhibit the release of cytokines from human peripheral blood single cells compared to BIIB059.

[0269] Table 27. Detection of IFNα inhibitory activity of anti-BDCA2 antibody

[0270] Example 13. ADCC killing activity of anti-BDCA2 antibody against stable cell lines expressing human BDCA2

[0271] The antibody-induced ADCC killing of stable human BDCA2-expressing cell lines by human peripheral blood single cells was detected by measuring lactate dehydrogenase (LDH) production, and antibody activity was evaluated based on absorbance. Specifically, serially diluted antibody molecules and control molecules were mixed with 1×10⁻⁶... 4 Stable cells of individual BDCA2 / CLEC4C and 2.5 × 10 5 Individual peripheral single cells were incubated at 37°C for 6 hours. After centrifugation, 50 μL of cell culture supernatant was collected, and 50 μL of LDH detection reagent was added. The cells were then incubated at room temperature with shaking for 30 minutes to terminate the reaction. The absorbance was read at 490 nM using a microplate reader.

[0272] Figure 8 shows that, compared with BIIB059, antibodies PR0036-Hu04-TCE01-Vis14-fl and PR0031-Hu08-TCE04-Vis04-fl significantly enhanced the ADCC killing function of human peripheral blood single cells against stable cell lines expressing human BDCA2.

Claims

1. A BDCA2-binding protein comprising: a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3; wherein, The LCDR1-3 is selected from: LCDR1-3 as shown in SEQ ID NO:87, 88, 89, LCDR1-3 as shown in SEQ ID NO:96, 13, 14, or LCDR1-3 as shown in SEQ ID NO:97, 18, 98; Furthermore, the BDCA2 binding protein is used to bind the BDCA2 extracellular domain, which contains the amino acid sequence shown in SEQ ID NO:

99.

2. BDCA2-binding protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) The VH includes HCDR1-3 as shown in SEQ ID NO:9, 10, 95, and the VL includes LCDR1-3 as shown in SEQ ID NO:96, 13, 14; or (2) The VH includes HCDR1-3 as shown in SEQ ID NO:15, 16, 17, and the VL includes LCDR1-3 as shown in SEQ ID NO:97, 18, 98.

3. BDCA2-binding protein, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:58, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:73; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:77, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:80; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:7, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:8; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:58, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:59-72 and 74-76; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:5, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in any one of SEQ ID NO:37-42, and VL includes LCDR1, LCDR2, and LCDR3 as shown in VL:43; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in any of SEQ ID NO:37-42, and VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:44; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in any one of SEQ ID NO:37-42, and VL includes LCDR1, LCDR2, and LCDR3 as shown in VL:45; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in any one of SEQ ID NO:51-56 and 77-79, and VL includes LCDR1, LCDR2, and LCDR3 as shown in VL:57; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:77, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:80-81; or, VH includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:78, and VL includes LCDR1, LCDR2, and LCDR3 as shown in any one of SEQ ID NO:80-81; CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

4. The BDCA2-binding protein according to any one of claims 1-3, wherein, (a) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, HCDR3 as shown in SEQ ID NO:17, and VL includes LCDR1 as shown in any of SEQ ID NO:27, 12, 36, 90-94 and 28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in any of SEQ ID NO:14 and 29-35; or (b) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in any of SEQ ID NO:24, 11 and 19-23, 25, 26, respectively, and VL includes LCDR1 as shown in any of SEQ ID NO:27, 12 and 28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; Preferably, (a-1) VH contains HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL contains LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-1) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-2) VH contains HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-3) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or, (a-4) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:30, respectively; or, (a-5) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:31, respectively; or, (a-6) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:32, respectively; or, (a-7) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:33, respectively; or, (a-8) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:34, respectively; or, (a-9) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:35, respectively; or, (a-10) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:36, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-11) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:90, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-12) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:91, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-13) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:92, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-14) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:93, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-15) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:94, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-16) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or, (a-17) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (a-18) VH includes HCDR1 as shown in SEQ ID NO:15, HCDR2 as shown in SEQ ID NO:16, and HCDR3 as shown in SEQ ID NO:17, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:18, and LCDR3 as shown in SEQ ID NO:29, respectively; or, (b-2) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-3) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:19, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-4) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:20, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-5) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:21, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-6) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:22, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-7) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:23, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-8) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-9) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively, and VL contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-10) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:26, respectively; and VL includes LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-11) VH contains HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:24, respectively, and VL contains LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-12) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively; and VL includes LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively; or, (b-13) VH includes HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:25, respectively; and VL includes LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14, respectively.

5. The BDCA2-binding protein according to any one of claims 1-4, wherein, (A) VH comprises a sequence as shown in any of SEQ ID NO:58, 7 and 46-48 or has at least 80%, preferably at least 90%, identity with it. VL contains a sequence as shown in or having at least 80% or at least 90% identity with any of SEQ ID NO:73, 8, 49-50 and 59; or (B) The VH comprises a sequence as shown in any of SEQ ID NO: 77, 5, 37-42, 51-56 and 78-79 or has at least 80%, preferably at least 90%, identity with it. VL includes sequences shown or having at least 80%, preferably at least 90%, of any of SEQ ID NO: 80, 6, 43-45, 57, 60-72, 74, 76 and 81; Preferably, (A-1) VH comprises a sequence as shown in SEQ ID NO:58 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:73 or having at least 80%, preferably at least 90%, identity with it; or, (B-1) VH comprises a sequence as shown in SEQ ID NO:77 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:80 or having at least 80%, preferably at least 90%, identity with it; or, (A-2) VH comprises a sequence as shown in SEQ ID NO:7 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:8 or having at least 80%, preferably at least 90%, identity with it; or, (A-3) VH comprises a sequence as shown in SEQ ID NO:46 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in any one of SEQ ID NO:49-50 or having at least 80%, preferably at least 90%, identity with it; or, (A-4) VH comprises a sequence as shown in SEQ ID NO:47 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in any one of SEQ ID NO:49-50 or having at least 80%, preferably at least 90%, identity with it; or, (A-5) VH comprises a sequence as shown in SEQ ID NO:48 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:50 or having at least 80%, preferably at least 90%, identity with it; or, (A-6) VH comprises a sequence as shown in SEQ ID NO:58 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in any one of SEQ ID NO:59-72, 74-76 or having at least 80%, preferably at least 90%, identity with it; or, (B-2) VH comprises a sequence as shown in SEQ ID NO:5 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:6 or having at least 80%, preferably at least 90%, identity with it; or, (B-3) VH comprises a sequence as shown in SEQ ID NO:37 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in any one of SEQ ID NO:43-44 or having at least 80%, preferably at least 90%, identity with it; or, (B-4) VH comprises a sequence as shown in SEQ ID NO:38 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in any one of SEQ ID NO:43-44 or having at least 80%, preferably at least 90%, identity with it; or, (B-5) VH comprises a sequence as shown in SEQ ID NO:39 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:45 or having at least 80%, preferably at least 90%, identity with it; or, (B-6) VH comprises a sequence as shown in SEQ ID NO:40-42 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:44 or having at least 80%, preferably at least 90%, identity with it; or, (B-7) VH comprises a sequence as shown in any one of SEQ ID NO:51-56, 77-79 or having at least 80%, preferably at least 90%, identity with it; VL comprises a sequence as shown in SEQ ID NO:57 or having at least 80%, at least 90%, identity with it; or, (B-8)VH comprises a sequence as shown in or having at least 80%, preferably at least 90%, of any of SEQ ID NO:77-78; VL comprises a sequence as shown in or having at least 80%, preferably at least 90%, of any of SEQ ID NO:81; or, (B-9)VH contains a sequence as shown in SEQ ID NO:78 or having at least 80%, preferably at least 90% identity with it, and VL contains a sequence as shown in SEQ ID NO:80 or having at least 80%, preferably at least 90% identity with it.

6. The BDCA2 binding protein according to any one of claims 1-5, wherein it is an anti-BDCA2 antibody or its antigen-binding fragment, preferably a murine antibody, a chimeric antibody, a humanized antibody or its antigen-binding fragment; the antigen-binding fragment is preferably scFv, Fv, Fab or Fab' fragment.

7. The BDCA2 binding protein as described in any one of claims 1-6, wherein it is humanized, reversed mutation, affinity maturation, removed post-translational modification (PTM), removed TCE, and / or modified to reduce viscosity; Preferably, the heavy-chain framework region of the genus template used in the humanization process is derived from IGHV3-11 and / or IGHV3-21, and the light-chain framework region of the genus template used in the humanization process is derived from IGKV3-11.

8. The BDCA2-binding protein according to any one of claims 1-7, further comprising an immunoglobulin Fc region; Preferably, the Fc region is selected from: (1) the Fc region of human IgG1, human IgG2 or human IgG4; (2) The Fc region is modified compared to the corresponding wild-type Fc region, wherein the modification results in increased affinity for one or more Fcγ receptors; or The Fc region is modified compared to the corresponding wild-type region, and said modification leads to enhanced antibody-dependent cytotoxicity (ADCC); or (3) The Fc region is free of fucosylation, preferably free of fucosylation at amino acid position 297 in the Fc region as determined by the EU numbering system.

9. The BDCA2-binding protein according to any one of claims 1-8, comprising a heavy chain and a light chain, wherein: The heavy chain comprises a sequence as shown in SEQ ID NO:82 or having at least 80%, preferably at least 90%, identity with it; the light chain comprises a sequence as shown in SEQ ID NO:83 or having at least 80% identity with it; or The heavy chain comprises a sequence as shown in SEQ ID NO:84 or having at least 80%, preferably at least 90%, identity with it; the light chain comprises a sequence as shown in SEQ ID NO:85 or having at least 80% identity with it.

10. A polynucleotide encoding the BDCA2-binding protein according to any one of claims 1-9; Preferably, the polynucleotide is DNA or RNA.

11. A vector comprising the polynucleotide of claim 10.

12. A host cell containing or expressing the polynucleotide of claim 10 or the vector of claim 11.

13. Methods for preparing BDCA2-binding proteins, including: The expression of the polynucleotide of claim 10 or the vector of claim 11 in a host cell, and the BDCA2 binding protein isolated and expressed from the host cell; Optionally, the process further includes a step of purifying the BDCA2-binding protein.

14. A pharmaceutical composition comprising the BDCA2 binding protein of any one of claims 1-9, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

15. A method of treating or preventing a disease, comprising administering to a subject in need a therapeutically or preventively effective amount of the BDCA2-binding protein of any one of claims 1-9, the polynucleotide of claim 10, the carrier of claim 11, or the pharmaceutical composition of claim 14, wherein: Preferably, the drug or drug composition is used to treat autoimmune diseases or to delay the progression of autoimmune diseases; More preferably, the autoimmune disease is an autoimmune disease associated with type I interferon overactivation; more preferably, the autoimmune disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

16. Use of the BDCA2 binding protein of any one of claims 1-9, the polynucleotide of claim 10, the carrier of claim 11, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating or preventing a disease; wherein: Preferably, the disease is an autoimmune disease; More preferably, the autoimmune disease is an autoimmune disease associated with type I interferon overactivation; more preferably, the autoimmune disease is systemic lupus erythematosus or cutaneous lupus erythematosus.

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