Anti-BDCA2 antibodies and uses thereof

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to BDCA2 and a composition comprising the same. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding fragments of the invention, vectors and host cells for expressing the antibodies or antigen-binding fragments of the invention, as well as therapeutic and diagnostic methods and uses of the antibodies or antigen-binding fragments of the invention.

Description

Anti-BDCA2 antibodies and uses thereof Technical Field

[0001] The present invention relates to the field of monoclonal antibodies and / or engineered antibodies. Specifically, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the present invention, vectors and host cells for expressing the antibodies or antigen-binding fragments thereof of the present invention, and therapeutic and diagnostic / detection methods and uses of the antibodies or antigen-binding fragments thereof of the present invention. Background Art

[0002] With the deepening of medical research, it has been found that many diseases are related to dysfunction of the immune system. Research on the immune system will help us further understand its pathogenesis and seek more effective treatment options.

[0003] The immune system is a highly complex system composed of many cell types, including but not limited to T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess a complex and delicate system to control their interactions and responses. Cells utilize activation and inhibition mechanisms and feedback loops to maintain control of their responses and minimize the negative consequences of uncontrolled immune responses (e.g., autoimmune diseases). A large number of signaling molecules and their interactions are involved in the multiple signaling pathways associated with immunity.

[0004] Blood DC Antigen 2 (BDCA2) is a C-type lectin expressed on human plasmacytoid dendritic cells (pDCs) (Dzionek et al., J. Immunol., 165:6037-6046 (2000)). BDCA2 consists of a single extracellular carbohydrate recognition domain (CRD) at its C-terminus, which belongs to the class II C-type lectin group; a transmembrane region extending from asparagine residue 45 to isoleucine residue 213; and a short cytoplasmic tail at its N-terminus, which does not contain a signaling motif. BDCA2 transmits intracellular signals through the cognate transmembrane adaptor FcεRIγ and induces a B cell receptor (BCR)-like signaling cascade.

[0005] Summary of the Invention

[0006] The present invention provides anti-BDCA2 antibodies or antigen-binding fragments thereof, which possess advantages such as high affinity and specificity for human and cynomolgus macaque BDCA2. These antibodies or antigen-binding fragments can be used as stand-alone therapies or in combination with other therapies or other agents for the treatment of, for example, inflammatory conditions or diseases.

[0007] The anti-BDCA2 antibodies described herein inhibit the production and / or secretion of inflammatory cytokines and chemokines by human plasmacytoid dendritic cells (pDCs). Furthermore, the anti-BDCA2 antibodies described herein can downregulate the levels of CD32a and / or CD62L on the surface of pDCs. Furthermore, the anti-BDCA2 antibodies disclosed herein can mediate the endocytosis of BDCA2 from the surface of pDCs. Furthermore, the anti-BDCA2 antibodies described herein can be used to deplete pDCs via ADCC or CDC and can be used to treat or prevent immune disorders, such as inflammatory disorders and autoimmune disorders.

[0008] In one aspect, the present invention provides an anti-BDCA2 antibody or an antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region:

[0009] The heavy chain variable region comprises:

[0010] (I) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or

[0011] (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or

[0012] (III) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or

[0013] (IV) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or

[0014] (V) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; or

[0015] (VI) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:46, and SEQ ID NO:47, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:46, and SEQ ID NO:47, respectively; or

[0016] (VII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:50, and SEQ ID NO:47, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:50, and SEQ ID NO:47, respectively; or

[0017] (VIII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; or

[0018] (IX) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; and / or

[0019] The light chain variable region comprises:

[0020] (I) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, by 1, 2, or 3 amino acids; or

[0021] (II) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, by 1, 2, or 3 amino acids; or

[0022] (III) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, by 1, 2, or 3 amino acids; or

[0023] (IV) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, by 1, 2, or 3 amino acids; or

[0024] (V) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or

[0025] (VI) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:49, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:49, respectively; or

[0026] (VII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:51, and SEQ ID NO:49, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:51, and SEQ ID NO:49, respectively; or

[0027] (VIII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or

[0028] (IX) LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:86, SEQ ID NO:15, and SEQ ID NO:16, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:86, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0030] (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or

[0031] (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or

[0032] (III) a heavy chain variable region comprising HCDR1 and HCDR2 as shown in SEQ ID NO:25 and SEQ ID NO:26, respectively, and HCDR3 as shown in SEQ ID NO:27 or SEQ ID NO:37; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; or

[0033] (IV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or

[0034] (V) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or

[0035] (VI) a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO:31, a HCDR2 as set forth in SEQ ID NO:46 or 50, and a HCDR3 as set forth in SEQ ID NO:47; and a light chain variable region comprising a LCDR1 as set forth in SEQ ID NO:34, a LCDR2 as set forth in SEQ ID NO:48 or SEQ ID NO:51, and a LCDR3 as set forth in SEQ ID NO:49; or

[0036] (VII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; and a light chain variable region comprising LCDR1 with amino acid sequences as shown in SEQ ID NO:14 or SEQ ID NO:86, LCDR2 with amino acid sequences as shown in SEQ ID NO:15 and LCDR3 with amino acid sequences as shown in SEQ ID NO:16, respectively.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:

[0038] (I) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:61; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:62; or

[0039] (II) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:66; or

[0040] (III) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:67, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:67; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:68; or

[0041] (IV) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:69; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:70, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:70; or

[0042] (V) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:71; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:68; or

[0043] (VI) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:72; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:73; or

[0044] (VII) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:76; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:77; or

[0045] (VIII) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:80; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:79, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:79; or

[0046] (IX) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:82; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:83.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0048] (I) a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 87-110, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 87-110; and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 135-158, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 135-158; or

[0049] (II) a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111-134, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 111-134; and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 159-182, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 159-182; or

[0050] (III) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:89, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:89; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:137, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:137.

[0051] (IV) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 154, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 154.

[0052] (V) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 114, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 114; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 162, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 162.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

[0054] (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 87-110, and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 135-158; or

[0055] (II) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 111-134, and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 159-182; or

[0056] (III) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 137;

[0057] (IV) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 106, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 154;

[0058] (V) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 114, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 162.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is a humanized antibody.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv and sdAb.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1 subtype.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises or consists of the following sequence:

[0063] (I) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 183, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 186, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or

[0064] (II) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 184, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 187, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or

[0065] (III) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 185, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 188, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises or consists of the following sequence:

[0067] (I) a heavy chain with an amino acid sequence as shown in SEQ ID NO: 183, and a light chain with an amino acid sequence as shown in SEQ ID NO: 186; or

[0068] (II) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 184, and a light chain having an amino acid sequence as shown in SEQ ID NO: 187; or

[0069] (III) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 185, and a light chain having an amino acid sequence as shown in SEQ ID NO: 188.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises or consists of the following sequence:

[0071] (I) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 183, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 186; or

[0072] (II) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 184, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 187; or

[0073] (III) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 185, and a light chain having the amino acid sequence of SEQ ID NO: 188.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises or consists of the following sequence:

[0075] (I) a heavy chain with an amino acid sequence as shown in SEQ ID NO: 191, SEQ ID NO: 192 or SEQ ID NO: 193, and a light chain with an amino acid sequence as shown in SEQ ID NO: 186; or

[0076] (II) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196, and a light chain having an amino acid sequence as shown in SEQ ID NO: 187.

[0077] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or an antigen-binding fragment thereof.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv or sdAb.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG1 subtype or IgG4 subtype.

[0080] In another aspect, the present invention provides an isolated anti-BDCA2 antibody or antigen-binding fragment thereof having one or more of the following properties:

[0081] (1) binds to the same or completely or partially overlapping epitope of the human BDCA2 protein as any anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention;

[0082] (2) competing with any anti-BDCA2 antibody or antigen-binding fragment thereof of the present invention for binding to an epitope of the human BDCA2 protein.

[0083] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have one or more of the following characteristics:

[0084] (1) Binds to human / cynomolgus monkey BDCA2 protein with high affinity, for example, exhibiting a 1.0×10-6 M or lower K D value, 5.0×10 -7 M or lower K D value, 2.5×10 -7 M or lower K D value, 1.0×10 -7 M or lower K D value, 5.0×10 -8 M or lower K D value, 2.5×10 -8 M or lower K D value, 1.0×10 -8 M or lower K D value, 5.0×10 -9 M or lower K D value, 2.5×10 -9 M or lower K D value, 1.0×10 -9 M or lower K D value, 5.0×10 -10 M or lower K D value, 2.5×10 -10 M or lower K D value, 1.0×10 -10 M or lower K D value, 5.0×10 -11 M or lower K D value or 2.5×10 -11 M or lower K D value;

[0085] (2) inhibiting CpG-A-stimulated human PBMC cells from releasing cytokine IFNα, for example, reducing the production of IFNα by target cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%;

[0086] (3) promoting the internalization of BDCA2 from the cell surface after binding to BDCA2, for example, causing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the BDCA2 molecules on the cell surface to be internalized.

[0087] In some embodiments, the antibodies described herein are monoclonal antibodies.

[0088] The present invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.

[0089] The present invention also provides a single-chain antibody comprising the light chain variable region and the heavy chain variable region of the antibody or antigen-binding fragment thereof described herein.

[0090] The present invention also provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described herein conjugated to a therapeutic agent or a diagnostic agent. Preferably, the therapeutic agent or the diagnostic agent is an anti-inflammatory drug or an immunosuppressant.

[0091] In yet another aspect, the present invention provides polynucleotide molecules encoding the anti-BDCA2 antibodies or antigen-binding fragments thereof described herein.

[0092] In yet another aspect, the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the vector is a eukaryotic expression vector.

[0093] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule described herein or the expression vector described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0094] In yet another aspect, the present invention provides a method for preparing the anti-BDCA2 antibody or antigen-binding fragment thereof described herein, comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0095] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-BDCA2 antibody or antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier or excipient.

[0096] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described herein, and one or more additional therapeutic agents.

[0097] In another aspect, the present invention provides a method for inhibiting the release of cytokine IFNα by immune cells of a subject, the method comprising contacting the anti-BDCA2 antibody or antigen-binding fragment thereof or pharmaceutical composition described herein with immune cells of the subject, preferably, the immune cells are CpG-A stimulated PBMC cells.

[0098] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein in the preparation of a medicament for treating and / or preventing a BDCA2-mediated disease, preferably an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0099] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a pharmaceutical combination as described herein, for use in treating and / or preventing a BDCA2-mediated disease, preferably an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, epidermal lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0100] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by BDCA2, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the pharmaceutical combination described herein. Preferably, the disease is an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0101] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein, preferably further comprising a drug delivery device.

[0102] In yet another aspect, the present invention provides a method for detecting the presence of BDCA2 in a sample using the antibody or antigen-binding fragment thereof described herein, or a detection composition containing the antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 shows the results of a FACS assay for binding of hybridoma antibodies to human CHOK1-hBDCA2 stably transfected cells expressing human BDCA2. Panels a and e refer to antibodies derived from different hybridomas, as indicated in the figure legends.

[0104] Figure 2 shows the results of FACS analysis of hybridoma antibody binding to 293F-cynoBDCA2 stably transfected cells expressing cynomolgus macaque BDCA2. Panels a and b relate to antibodies derived from different hybridomas, as indicated in the figure legend.

[0105] Figure 3 shows the results of a FACS assay for binding of anti-BDCA2 chimeric antibodies to human CHOK1-hBDCA2 cells. Panels a to c are from different chimeric antibodies, as indicated in the figure legends.

[0106] Figure 4 shows the endocytosis capacity assay for humanized antibodies. Panel a shows the results of the hu005 group of humanized antibodies at a concentration of 100 nM. Panel b shows the results of the hu005 group of humanized antibodies at a concentration of 10 nM. Panel c shows the results of the hu033 group of humanized antibodies at a concentration of 100 nM. Panel d shows the results of the hu033 group of humanized antibodies at a concentration of 10 nM.

[0107] Figure 5 shows the FACS analysis of cell binding ability of Fc-modified antibodies. Panel a shows the results of binding assays with CHOK1-Human hBDCA2 cells. Panel b shows the results of binding assays with 293-Cyno BDCA2 cells.

[0108] Figure 6 shows the endocytic capacity of humanized antibodies after Fc modification detected by FACS. Panel a shows the MFI result of the humanized antibody measured at a concentration of 100 nM. Panel b shows the MFI result of the humanized antibody measured at a concentration of 10 nM. Panel c shows the MFI result of the humanized antibody measured at a concentration of 1 nM. Panel d shows the surface signal percentage of the humanized antibody measured at a concentration of 100 nM. Panel e shows the surface signal percentage of the humanized antibody measured at a concentration of 10 nM. Panel f shows the surface signal percentage of the humanized antibody measured at a concentration of 1 nM.

[0109] FIG7 shows the inhibitory effect of the humanized antibody after Fc modification on IFNa secretion by PBMC induced by SLE-IC. DETAILED DESCRIPTION

[0110] definition

[0111] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0112] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined below. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms or expressions used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel), at least in part. The abbreviations of amino acid residues follow the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly provided in the text.

[0113] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0114] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0115] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in a claim, "consisting of..." will refer to only one of the listed numbers or one of the elements of the list.

[0116] As used herein, the words "a" and "an" should be understood to mean "at least one" unless the context clearly indicates otherwise.

[0117] The term "BDCA2" herein refers to a type II C-type lectin BDCA2 specifically expressed on pDCs, consisting of a single extracellular carbohydrate recognition domain (CRD) at the C-terminus, a transmembrane region from asparagine residue 45 to isoleucine residue 213, and a short cytoplasmic tail at the N-terminus (without a signaling motif). BDCA2 sends intracellular signals through the associated transmembrane adaptor FcεRIγ. Antibody-mediated binding of BDCA2 leads to the recruitment of spleen tyrosine kinase (SYK) to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcεRIγ. Activation of Syk leads to the activation of B cell linkers (BLNK), Bruton's tyrosine kinase (BTK), and phospholipase Cγ2 (PLCγ2), resulting in Ca2+ flow.

[0118] The term "BDCA2" includes variants, isoforms, species homologs of human BDCA2, BDCA2 from other species, and analogs that share at least one common epitope with BDCA2, unless otherwise indicated. The term encompasses unprocessed, full-length BDCA2 as well as any form of BDCA2 produced as a result of processing in cells. The term encompasses "full-length," unprocessed BDCA2 as well as any form of BDCA2 or any fragment thereof, such as a splice variant or allelic variant, produced by intracellular processing. In one embodiment, BDCA2 refers to full-length BDCA2 from humans or cynomolgus monkeys, or fragments thereof (such as a mature fragment lacking a signal peptide).

[0119] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0120] The term "human plasmacytoid dendritic cells (pDCs)" is a specialized population of bone marrow-derived cells that secrete type I interferon (IFN) in response to toll-like receptor (TLR) ligands. pDCs are a key link in cellular immunity, involved in the induction and initiation of immune responses and antigen tolerance. pDCs differ from ordinary DCs in that, for example, they acquire antigens through receptor-mediated endocytosis, among other things. Type I interferon produced by pDCs activated by pattern recognition receptors can also present antigens, thereby linking innate and acquired immune responses. At the same time, overactivation of pDCs may also have a negative impact on the immune response process, for example, potentially leading to autoimmune diseases.

[0121] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0122] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize BDCA2 activity in vivo or in vitro, IC 50, the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the target peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0123] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0124] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0125] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0126] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0127] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. In some preferred embodiments of the present invention, the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0128] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0129] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0130] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0131] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0132] The terms "murine antibody" or "hybridoma antibody," as used herein, refer to monoclonal antibodies against BDCA2 prepared according to the knowledge and skill in the art. To prepare these antibodies, a test subject is injected with the BDCA2 antigen, followed by isolation of hybridomas expressing antibodies with the desired sequence or functional properties. Hybridoma technology involves fusing two cell types while maintaining the key characteristics of both: spleen cells from mice immunized with an antigen and mouse myeloma cells. The key characteristic of mouse spleen cells (B lymphocytes) immunized with a specific antigen is their antibody secretion, but they cannot be cultured continuously in vitro. Mouse myeloma cells, on the other hand, can divide and proliferate indefinitely under culture conditions, thus possessing so-called immortality. Under the influence of a selective culture medium, only hybrid cells fused with B cells and myeloma cells can be cultured continuously, forming cell clones that possess both antibody secretion and cell immortality. In some embodiments, the present invention involves immunizing mice with the BDCA2 protein, then fusing the mouse spleen cells with myeloma cells to obtain hybridoma cells that express positive antibodies.

[0133] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are derived from antibodies ("parent antibodies") of rodents, etc., while the constant domain sequences are derived from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects compared to parent rodent antibodies. In some embodiments of the invention, the rodent is a mouse or rat. In some preferred embodiments of the invention, the affinity of the chimeric antibody for the antigen is not less than or almost not less than that of the parent mouse antibody.

[0134] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0135] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0136] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0137] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0138] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0139] As used herein, the term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Thus, "cross-reactivity" should be understood as the interspecies reaction of an antigen-binding molecule (e.g., antibody) with a similar molecule (e.g., BDCA2) expressed in a different species. The cross-reactivity specificity of monoclonal antibodies recognizing human BDCA2, monkey, and / or murine BDCA2 (mouse or rat) can be determined by FACS analysis.

[0140] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on ). Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technology is used to measure affinity, for example, the affinity between the antibody of the present invention and the antigen. In some preferred embodiments of the present invention, a specific method for measuring affinity is the BIAcore method herein.

[0141] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the K of the protein or cell is less than 1%. D 1.0×10 -6M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0142] The term "high affinity" for IgG antibodies refers to the K D 1.0×10 -6 M or less, preferably 5.0×10 - 8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D is 10 - 6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0143] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells that have antibodies bound to surface antigens on their cell membranes.

[0144] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies that, when bound to surface antigens, trigger the classic complement pathway, including formation of the membrane attack complex and target cell lysis.

[0145] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and "polynucleotide molecule" are used interchangeably herein (unless the context indicates otherwise) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited, the terms include nucleic acids containing analogs of known natural nucleotides that have similar binding properties to the reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0146] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of integrating with a genome or autonomously replicating, and comprising one or more polynucleotide molecules that have been linked (i.e., operably linked) in a functionally operable manner. In some preferred embodiments of the present invention, the recombinant construct comprises a polynucleotide of the present invention operably linked to a transcription initiation regulatory sequence that drives and / or directs transcription of the polynucleotide of the present invention in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to drive and / or direct expression of the polynucleotide of the present invention.

[0147] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0148] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure vector maintenance and, if desired, to provide for amplification within the host. In preferred embodiments of the present invention, the expression vectors of the present invention comprise a construct of the present invention and / or a polynucleotide of the present invention.

[0149] "Activation," "stimulation," and "treatment" as applied to the manipulation of a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0150] As used herein, the term "BDCA2-mediated disease" refers to a disease in which BDCA2 is involved in the onset, progression, or persistence of the disease, for example, involving BDCA2 activation (e.g., abnormal activation or overactivation), and in which BDCA2 involvement directly or indirectly produces at least one of the following effects: disease initiation / occurrence; increased / progressive pathological changes of the disease; expansion of the site / area affected by the disease; increased damage to the body caused by the disease; increased pain caused by the disease; insensitivity / resistance to treatment measures; decreased propensity for spontaneous recovery of the disease and worsening of the prognosis of the disease. In some embodiments of the present invention, the BDCA2-mediated disease is an inflammatory disease; in some specific embodiments of the present invention, the BDCA2-mediated disease is systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and / or polymyositis.

[0151] As used herein, the terms "treatment" or "treating" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0152] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey or a cynomolgus monkey.

[0153] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0154] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of the BDCA2 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, e.g., to treat, cure, prevent, or ameliorate the relevant medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0155] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0156] Anti-BDCA2 antibody

[0157] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2. The terms "anti-BDCA2 antibody," "anti-BDCA2," "BDCA2 antibody," or "antibody that binds to BDCA2" refer to antibodies that bind to the BDCA2 protein or fragment thereof with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting BDCA2.

[0158] In some embodiments, antibodies of the invention bind to human or cynomolgus monkey BDCA2 protein. In some embodiments, antibodies of the invention bind to CHOK1-human BDCA2 cells or 293F-cyno BDCA2 cells. In some embodiments, antibodies of the invention inhibit CpG-A-stimulated IFNα release from human PBMCs. In some embodiments, antibodies of the invention exhibit enhanced endocytosis.

[0159] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of BDCA2 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any BDCA2 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-BDCA2 antibodies can be produced by methods known in the art.

[0160] Antibodies derived from rodents (such as mice) may cause unwanted antibody immunogenicity when used as therapeutic drugs in vivo. Repeated use leads to the human body producing an immune response against the therapeutic antibody. Such immune response at least leads to loss of therapeutic efficacy, and in severe cases, leads to potentially lethal allergic reactions. One method of reducing the immunogenicity of rodent antibodies includes the production of chimeric antibodies, in which the mouse variable region is fused to the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, the retention of the complete rodent variable region in the chimeric antibody may still cause harmful immunogenicity in patients. Transplanting the complementary determining region (CDR) loop of the rodent variable domain onto the human framework (i.e., humanization) has been used to further minimize the rodent sequence (Jones et al. (1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534). In some embodiments, the antibodies of the present invention are chimeric antibodies. In some preferred embodiments, the antibodies of the present invention are humanized antibodies. In some preferred embodiments of the present invention, the affinity of the murine antibody, chimeric antibody and / or human antibody of the present invention to the antigen human BDCA2 or cynomolgus monkey BDCA2 is 1.0×10 -6 M or lower K D value, 5.0×10 -7 M or lower K D value, 2.5×10 -7 M or lower K D value, 1.0×10 -7 M or lower K D value, 5.0×10 -8 M or lower K D value, 2.5×10 -8 M or lower K D value, 1.0×10 -8 M or lower K D value, 5.0×10 -9 M or lower K D value, 2.5×10 -9 M or lower K D value, 1.0×10 -9 M or lower K D value, 5.0×10 -10 M or lower K D value, 2.5×10 -10 M or lower K D value, 1.0×10 -10 M or lower K D value.

[0161] The amino acid sequences of the light chain and heavy chain variable regions and CDRs of the anti-BDCA2 murine antibody of the present invention are shown in Table 2.

[0162] In some embodiments, chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal hybridoma antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0163] In some embodiments, the chimeric BDCA2 antibodies of the present invention can be prepared by operatively linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the constant regions comprised by the chimeric antibodies of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, and IgG4, with IgG1 being preferred.

[0164] In some embodiments, chimeric BDCA2 antibodies of the present invention can be obtained by "mixing and matching" chimeric light chain and chimeric heavy chain expression plasmids into expressing cells. BDCA2 binding of such "mixed and matched" antibodies can be tested using the above-described binding assays and other conventional binding assays (e.g., ELISA).

[0165] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of antibodies and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0166] Unless otherwise indicated, the boundaries of the CDRs of the antibodies of the invention can be determined by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).

[0167] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0168] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0169] The present invention mixes and pairs various chimeric heavy chain and light chain expression plasmids and transfects them into expression cells to produce anti-BDCA2 chimeric antibodies.

[0170] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0171] The amino acid sequences of the light and heavy chains of the anti-BDCA2 humanized antibodies Hu033-03, Hu033-20, and Hu005-04 of the present invention are shown below. The sequences of the remaining humanized antibodies outside the variable regions of LC and HC are identical to those of Hu033-03, Hu033-20, and Hu005-04.

[0172] Hu033-03-HC-IgG1: SEQ ID NO: 183 (HCDR1-HCDR3 are SEQ ID NOs: 52, 39, and 53, respectively)

[0173] Hu033-03-LC: SEQ ID NO: 186 (LCDR1-LCDR3 are SEQ ID NOs: 54, 55, and 42, respectively)

[0174] Hu033-20-HC-IgG1: SEQ ID NO: 184 (HCDR1-HCDR3 are SEQ ID NOs: 52, 39, and 53, respectively)

[0175] Hu033-20-LC: SEQ ID NO: 187 (LCDR1-LCDR3 are SEQ ID NOs: 54, 55, and 42, respectively)

[0176] Hu005-04-HC-IgG1: SEQ ID NO: 185 (HCDR1-HCDR3 are SEQ ID NOs: 11, 12, and 13, respectively)

[0177] Hu005-04-LC: SEQ ID NO: 188 (LCDR1-LCDR3 are SEQ ID NOs: 86, 15, and 16, respectively)

[0178] In some embodiments, the amino acid changes comprise amino acid deletions, insertions, or substitutions. In some embodiments, the anti-BDCA2 antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still share at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions when compared to the CDR regions depicted in the specified sequences. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions when compared to the framework regions in the specified sequences.

[0179] In some embodiments, the Fc regions of the humanized antibodies Hu033-03 and Hu033-20 of the present invention are mutated, including S239D / I332E, G236A / S239D / I332E, and G236A / I332E, to produce the following antibodies: Hu033-03-T1, Hu033-03-T2, Hu033-03-T3, Hu033-20-T1, Hu033-20-T2, Hu033-20-T3, whose light chain and heavy chain amino acid sequences are shown below:

[0180] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0181] Thus, in some embodiments, an antibody of the invention comprises a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination of Table A below.

[0182] Table A

[0183] In some embodiments, an antibody of the invention comprises a VH and a VL as shown in any combination of Table B below.

[0184] Table B

[0185] In some embodiments, an antibody of the invention comprises a VH and a VL as shown in any combination of Table C below.

[0186] Table C

[0187] In some embodiments, the BDCA2 antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof, as described in the following sections.

[0188] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0189] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0190] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0191] Antibody expression

[0192] In yet another aspect, the present invention provides a polynucleotide molecule encoding an anti-BDCA2 antibody or antigen-binding fragment thereof as described herein. The polynucleotide molecule may comprise a polynucleotide molecule encoding an amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or a polynucleotide molecule comprising an amino acid sequence encoding an antibody light chain and / or heavy chain.

[0193] In some embodiments, the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, insertion or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions depicted in the sequences described above.

[0194] In another aspect, the present invention provides an expression vector comprising a polynucleotide molecule as described herein, preferably, the vector is a eukaryotic expression vector. In some embodiments, the polynucleotide molecule as described herein is contained in one or more expression vectors.

[0195] In another aspect, the present invention provides a host cell comprising the polynucleotide molecule as described herein or the expression vector as described herein. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0196] In yet another aspect, the present invention provides a method for preparing an anti-BDCA2 antibody or antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or antigen-binding fragment thereof in a host cell as described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

[0197] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0198] In one embodiment, the present invention provides a method for preparing an anti-BDCA2 antibody, wherein the method comprises, upon introducing an expression vector into a mammalian host cell, producing the antibody by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown.

[0199] Standard protein purification methods can be used to recover antibodies from the culture medium. Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0200] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0201] Pharmaceutical compositions and pharmaceutical preparations

[0202] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-BDCA2 antibody or antigen-binding fragment thereof as described herein, a polynucleotide molecule as described herein, an expression vector as described herein, or a host cell as described herein, and a pharmaceutically acceptable carrier or excipient. It should be understood that the anti-BDCA2 antibodies or pharmaceutical compositions provided herein can be incorporated into formulations with suitable carriers, excipients, and other agents for co-administration, thereby providing improved transfer, delivery, tolerance, and the like.

[0203] The term "pharmaceutical composition" refers to a preparation that permits the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0204] Pharmaceutical formulations comprising the anti-BDCA2 antibodies described herein can be prepared by mixing an anti-BDCA2 antibody of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0205] The pharmaceutical compositions or formulations of the present invention may also include one or more other active ingredients as needed for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are immune checkpoint inhibitors or growth inhibitors, suitably combined in amounts effective for the intended use. In some embodiments, the pharmaceutical compositions of the present invention further include a composition of polynucleotide molecules encoding anti-BDCA2 antibodies.

[0206] In another aspect, the present invention provides a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, or the pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0207] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof described herein, the polynucleotide molecule described herein, the expression vector described herein, the host cell described herein, or the pharmaceutical composition described herein.

[0208] Medical uses and treatments

[0209] Any of the anti-BDCA2 antibodies or corresponding immunoconjugates provided herein can be used in therapeutic methods. It should also be understood that when discussing "antibodies," compositions comprising the antibodies are also encompassed. The anti-BDCA2 antibodies of the present invention can be used in therapeutically or prophylactically effective amounts in the therapeutic or prophylactic methods described in any embodiment of the present invention.

[0210] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the pharmaceutical composition described herein, or the pharmaceutical combination described herein in the preparation of a medicament for treating and / or preventing a BDCA2-mediated disease, preferably an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0211] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a pharmaceutical combination as described herein, for use in treating and / or preventing a BDCA2-mediated disease, preferably an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0212] In another aspect, the present invention provides a method for treating and / or preventing a disease or condition mediated by BDCA2, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the pharmaceutical combination described herein. Preferably, the disease is an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0213] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or local administration to the lesion, etc.

[0214] The term "treatment" refers to a clinical intervention intended to change the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any adverse feelings or direct or indirect pathological consequences of the disease, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving the prognosis. For the antibodies of the present invention, the ability to reduce the severity of the disease in one or more aspects is to exert a therapeutic effect. In certain embodiments, this is manifested as one or more of the following: an increase in the average lifespan (survival) of the patient; a delay in disease progression; a decrease in the need for medical care.

[0215] The present invention also provides for co-administering a therapeutically effective amount of one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to a subject. The antibodies of the present invention can be used alone or in combination with other therapeutic agents in a therapy. In some embodiments, the antibodies of the present invention are co-administered with at least one additional therapeutic agent.

[0216] Methods for diagnosis and detection

[0217] In yet another aspect, the present invention provides a method for detecting the presence of BDCA2 in a sample using the antibodies or antigen-binding fragments thereof described herein. The term "detecting," as used herein, includes both quantitative and qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other fluid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissue. In certain embodiments, BDCA2 is human BDCA2 or cynomolgus macaque BDCA2. The method comprises the steps of contacting the sample with an antibody or antigen-binding fragment thereof described herein, or a detection composition containing the antibody or antigen-binding fragment thereof, and detecting the presence of a conjugate or binding signal generated by binding of the antibody or antigen-binding fragment to BDCA2. For detection purposes, the antibodies or antigen-binding fragments described herein may be labeled to indicate the formation of a conjugate. In certain embodiments, the method may be an in vitro or in vivo method.

[0218] In some embodiments, BDCA2 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval. In one embodiment, an anti-BDCA2 antibody or antigen-binding fragment thereof is provided for use in a diagnostic or detection method.

[0219] The positive progress effect of the present invention is:

[0220] The antibodies targeting BDCA2 of the present invention have one or more of the following advantages:

[0221] 1. Compared to the prior art anti-BDCA2 antibody BIIB059, the antibodies of the present invention have stronger binding activity to CHOK1-humanBDCA2 cells and / or 293F-cynoBDCA2 cells;

[0222] 2. Compared with the prior art anti-BDCA2 antibody BIIB059, the antibody of the present invention has a better inhibitory effect on the release of cytokine IFNα from human PBMC cells stimulated by CpG-A;

[0223] 3. The antibodies of the present invention have higher internalization efficiency in cells expressing BDCA2;

[0224] 4. Compared with the prior art anti-BDCA2 antibody BIIB059, the humanized antibody of the present invention has a higher affinity for the human BDCA2 antigen.

[0225] 5. The BDCA2 gene is not expressed in non-primates such as mice, rats, and rabbits, and no primate models have been successfully established for the efficacy of BDCA2 antibodies for the primary diseases they treat (e.g., epidermal lupus erythematosus and systemic lupus erythematosus). Based on in vitro and clinical studies of the antibody BIIB059, it has been shown to induce BDCA2 internalization and inhibit type I interferon secretion in PBMCs in vitro, and to alleviate symptoms in clinical trials of epidermal lupus erythematosus and systemic lupus erythematosus (Werth VP et al. N Engl J Med 2022;387:321-31; Furie RA et al. N Engl J Med 2022;387:894-904). This suggests that the anti-BDCA2 antibodies of the present invention may have better clinical therapeutic effects for these diseases or conditions.

[0226] The present invention includes all combinations of the specific embodiments. Further embodiments of the present invention and the complete scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those familiar with the art from this detailed description. For all purposes, all disclosures, patents and patent applications cited herein, including citations, are incorporated herein by reference in their entirety. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other methods, and equivalent replacement modes well known to those skilled in the art, and preferred embodiments include but are not limited to embodiments of the present invention.

[0227] The present invention employs at least the following abbreviations:

[0228] his-tag stands for histidine tag;

[0229] Fc tag stands for fragment crystallizable tag;

[0230] ECD stands for extracellular domain;

[0231] PEI stands for polyethyleneimine;

[0232] BSA stands for bovine serum albumin;

[0233] PBS stands for phosphate-buffered saline;

[0234] FBS stands for fetal bovine serum;

[0235] CFSE stands for carboxyfluorescein diacetate succinimidyl ester;

[0236] APC stands for ectopic phycocyanin;

[0237] NA-PE represents phycoerythrin-labeled neutravidin;

[0238] PE stands for phycoerythrin;

[0239] TMB stands for 3,3',5,5'-tetramethylbenzidine;

[0240] HEPES: hydroxyethylpiperazine ethanesulfonic acid buffer;

[0241] DTT: dithiothreitol.

[0242] Example

[0243] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0244] Example 1. Preparation of recombinant protein for anti-BDCA2 antibody activity detection

[0245] The cDNA sequence encoding the extracellular domain of human BDCA2 (hBDCA2 or human BDCA2) (Asn45-Ile213) was synthesized by Genscript according to the Genbank reference sequence. Using conventional cloning techniques, the amplified fragment was cloned into a self-constructed eukaryotic expression plasmid containing a human Fc (hIgG1) fragment (pCP-EF1a-CMV-FcεRIγ) to produce the recombinant fusion protein expression plasmid Human BDCA2 Fc (pCP-EF1a-BDCA2-CMV-FcεRIγ). The recombinant Human BDCA2 Fc protein (the Fc region is located at the N-terminus of the BDCA2 extracellular domain and fused via a linker) was expressed in 293E cells and purified using size-exclusion chromatography. The protein was then identified by polyacrylamide gel electrophoresis.

[0246] The cDNA sequence encoding the extracellular domain (Asn45-Ile212) of cynomolgus macaque BDCA2 (Cyno BDCA2) was synthesized by Genscript according to the Genbank reference sequence, and six His expression sequences were added to the N-terminus of the cDNA. Using conventional cloning techniques, the fragment was cloned into a self-constructed eukaryotic expression plasmid system (pCP-EF1a-CMV-FcεRIγ) to produce the recombinant fusion protein expression plasmid Cyno BDCA2 His (pCP-EF1a-Cyno BDCA2-CMV-FcεRIγ). The recombinant Cyno BDCA2 His protein was expressed in 293E expression cells and purified using size exclusion chromatography. The protein was then identified by polyacrylamide gel electrophoresis.

[0247] After identification, the obtained two protein concentrations, total amount and endotoxin content were suitable for subsequent applications.

[0248] The amino acid sequence of Human BDCA2 Fc is shown in SEQ ID NO: 189, and the NCBI accession number used for the Human BDCA2 protein sequence is Q8WTT0. The amino acid sequence of Cyno BDCA2 His is shown in SEQ ID NO: 190, and the NCBI accession number used for the Cyno BDCA2 protein sequence is A0A2K5UWP4-1, which are used for activity detection of the antibodies of the present invention.

[0249] The sequence of the reference antibody BIIB059 that specifically binds to BDCA2 is derived from patent US9902775B2, wherein the heavy chain sequence corresponds to patent sequence No. 4 and the light chain sequence corresponds to patent sequence No. 3. It was produced using a conventional recombinant antibody production process.

[0250] Example 2: Construction of a BDCA2-expressing cell line for anti-BDCA2 antibody activity detection

[0251] The cDNA sequence encoding human BDCA2 was synthesized by Genscript according to the Genbank reference sequence. Using conventional cloning techniques, the amplified fragment was cloned into a self-constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro) to generate the lentiviral packaging plasmid Human BDCA2 (PLvx-EF1a-hBDCA2-CMV-hFcεRIγ-IRES-puro).

[0252] The cDNA sequence encoding cynomolgus macaque Cyno BDCA2 was synthesized by Genscript according to the Genbank reference sequence. Using conventional cloning techniques, the amplified fragment was cloned into the self-constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro), thereby generating the lentiviral packaging plasmid Cyno BDCA2 (PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro).

[0253] Transiently transfect 293T cells with the PLvx-EF1a-hBDCA2-CMV-hFcεRIγ-IRES-puro plasmid or PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro plasmid. Collect cell culture supernatants containing human BDCA2 or cyno BDCA2-expressing virus 48 and 72 hours after transfection and concentrate to 1E8 EU / mL.

[0254] CHOK1 cells, pre-cultured to the logarithmic growth phase, were digested and plated at 1E5 cells / well in 6-well cell culture plates. The cells were cultured overnight to allow attachment. Subsequently, increasing inoculation doses (100 μl and 200 μl) of concentrated human BDCA2 virus were added to the pre-attached 6-well plates. The infected cells were selected with puromycin-containing medium and assayed for antigen expression by flow cytometry. The cells were then subcloned. Flow cytometry using the reference antibody BIIB059 demonstrated significant BDCA2 binding relative to the hIgG control. Single clones with high antigen expression were selected and expanded for use as cell lines in later experiments.

[0255] 293F cells cultured to the logarithmic growth phase were digested and plated at 1E5 cells / well in 6-well cell culture plates. The cells were cultured overnight to allow attachment. Subsequently, the concentrated Cyno BDCA2 virus was added to the pre-attached 6-well plates in increasing infective doses (100 μl, 200 μl). The infected cells were selected with puromycin-containing medium and assayed for antigen expression by flow cytometry. The cells were then subcloned. Flow cytometry using the reference antibody BIIB059 demonstrated significant binding to BDCA2 relative to the hIgG control. Single clones with high antigen expression were selected and expanded for use as cell lines in later experiments.

[0256] Example 3. Preparation of mouse hybridoma cells

[0257] 3.1. Mouse immunization and serum titer detection

[0258] 3.1.1. Mouse immunization protocol

[0259] Three groups of mice (5 mice per group) were immunized using two different immunization routes, with 14-day intervals between immunizations. Group 1 consisted of five Balb / c mice (8-week-old female, purchased from SLAC) and Group 2 consisted of five SJL mice (8-week-old female, purchased from SLAC). They were immunized five times using a gene gun-based regimen of pCP-EF1a-BDCA2-CMV-FcεRIγ plus protein (humanBDCA2 Fc): the first four gene gun immunizations were at a dose of 4 μg per immunization; the fifth protein immunization was at a dose of 25 μg per mouse. Group 3 consisted of five SJL mice (8-week-old female, purchased from SLAC) immunized three times with purified protein (humanBDCA2 Fc), with the first immunization at 50 μg per mouse and the subsequent two immunizations at 25 μg per mouse. See Table 1.

[0260] Table 1 Mouse immunization scheme

[0261] 3.1.2. Mouse immune titer detection

[0262] Serum samples were collected from immunized mice at various time points and analyzed by ELISA and FACS. Based on the results of these tests (the final mouse serum test (TB) was used as the primary reference, detailed results are not shown), four mice were selected for two fusions: Fusion #F0223 involved Balb / c #9735, which had the highest fluorescence intensity in the G1 group, and SJL #9740, which had the highest fluorescence intensity in the G2 group; and Fusion #F0511 involved SJL #9947 and SJL #9948, which had the highest fluorescence intensity in the G3 group.

[0263] Cell fusion

[0264] On the fourth day after the last booster immunization, the mice were sacrificed and spleen cells were collected. The lymphocyte-rich suspension was ground in physiological saline and mixed with mouse myeloma cells Sp2 / 0 by conventional electroporation method, and cell fusion was performed by high-efficiency electrofusion.

[0265] After fusion, the cells were diluted into DMEM medium containing HT (hypoxanthine and thymidine), plated into 96-well plates according to the appropriate ratio, and cultured overnight in a 5% CO2, 37°C incubator. After 24 hours, DMEM medium containing 2*HAT (hypoxanthine, methotrexate, and thymidine) was added to screen for successfully fused cells (hybridomas).

[0266] The DMEM complete culture medium ratio is: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL penicillin-streptomycin + 1:100 OPI (oxaloacetate, pyruvate and insulin), and the incubator conditions are 8% CO2, 37°C.

[0267] Example 4. Screening of Mouse Hybridoma Cells and Purification of Anti-BDCA2 Mouse Antibodies

[0268] After approximately 10 days of screening and culturing the fused cells, the supernatant was assayed for BDCA2 antibody expression using an Acumen laser cell analyzer. Positive cells were CHOK1-humanBDCA2 and negative cells were CHOK1-blank. The positive control antibody was BIIB059. Based on the Acumen results, positive clones were selected and transferred to 24-well plates for expansion. After 3 days of culture, the supernatant from the expanded culture was assayed using the following methods: 1) FACS analysis of binding activity to CHOK1-humanBDCA2 cells and 293F-cynoBDCA2 cells; 2) ELISA analysis of binding activity to humanBDCA2 Fc protein and cynoBDCA2His protein; and 3) inhibition of IFNα production in CpG-A-stimulated PBMCs.

[0269] Among the polyclonal hybridoma cells derived from the F0223 fusion, 189 hybridoma cell lines were screened for antibodies specifically binding to hBDCA2 through binding to Acumen. Of these 189 binding-positive hybridoma cells, 28 hybridoma cell lines expressed antibodies that bound to both hBDCA2 and cyno BDCA2 in ELISA and to cell surface-expressed hBDCA2 and cyno BDCA2 in FACS. In an IFNα blocking assay, these 28 polyclonal cell lines inhibited CpG-induced IFNα secretion in PBMCs. These 28 hybridoma cell lines were subcloned. Subcloning yielded 18 blocking monoclonal cell lines, designated mAb001-mAb018.

[0270] Among the polyclonal hybridomas derived from the F0511 fusion, 168 hybridoma cell lines were screened for antibodies specifically binding to hBDCA2 through binding to Acumen. Among these 168 binding-positive hybridomas, 35 hybridoma cell lines expressed antibodies that bound to both hBDCA2 and cyno BDCA2 in ELISA and to cell surface-expressed hBDCA2 and cyno BDCA2 in FACS. In an IFNα blocking assay, these 35 polyclonal cell lines inhibited CpG-induced IFNα secretion in PBMCs. These 35 hybridoma cell lines were subcloned. Subcloning yielded 16 blocking monoclonal cell lines, designated mAb019 to mAb034.

[0271] After two fusions and screening, a total of 34 positive monoclonal clones were obtained. The secreted antibodies were purified and analyzed, and their concentration, quality, purity, and endotoxin content all met the requirements of subsequent experiments.

[0272] Example 5: Performance Determination of Anti-BDCA2 Mouse Antibodies

[0273] The obtained mouse antibodies all had good binding activity with humanBDCA2 Fc protein, and the binding affinity was EC 50 <0.3nM. Mouse antibodies have good binding ability with cynoBDCA2 His protein. Most antibodies have an EC binding capacity of <0.3nM. 50 <0.5nM.

[0274] All mouse antibodies bound to CHOK1-humanBDCA2 cells (see Figures 1a-1e for results). 50Most of the mouse antibodies bound to 293F-cynoBDCA2 cells (see Figures 2a-2e for results), and their EC 50 Better than or comparable to the control antibody BIIB059.

[0275] Thirty-two antibodies that bind to both CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells were tested for their ability to inhibit IFNα release in a CpG-A stimulated PBMC assay. All antibodies demonstrated moderate inhibitory activity. Among these, mAb005, mAb019, mAb020, mAb021, mAb022, mAb024, mAb027, mAb030, and mAb033 exhibited IC values ​​for inhibition of cytokine release. 50 They are 0.08667nM, 0.003323nM, 0.006146nM, 0.004047nM, 0.006232nM, 0.002744nM, 0.07004nM, 0.004762nM and 0.004083nM respectively.

[0276] The experimental method is as follows: mAb001-mAb034 are murine antibodies of the present invention, BIIB059 is the reference antibody described above, hIgG1 is a negative control for human IgG1, and mIgG1 is a negative control for mouse IgG1.

[0277] 5.1. ELISA to detect the binding activity of mouse antibodies to proteins

[0278] Dilute human BDCA2 Fc protein or cyno BDCA2 His protein to 1 μg / mL in PBS and add 100 μL / well to an ELISA microplate. Incubate overnight at 4°C. Block with ELISA blocking buffer (PBS phosphate buffer containing 1% BSA, pH 7.4, percentages are by weight) at 37°C for two hours. Add 10-fold serial dilutions of antibody to a total of eight concentrations (0.0001 nM to 100 nM, including a zero control) at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate three times and add anti-mouse IgG (Fab specific)-HRP (Sigma, A3682; 1:5000 dilution) secondary antibody at 100 μL / well and incubate at 37°C for 1 hour. The plate was washed three times, and 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009) was added. After incubation at 37°C for 10 minutes, 50 μL 1N hydrochloric acid was added to terminate the colorimetric reaction. The OD450nm value was read using an ELISA plate reader, and the curve was fitted using GraphPad Prism6 to calculate the EC 50 value.

[0279] 5.2. FACS detection of the binding activity of mouse antibodies to cells

[0280] CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells grown to the logarithmic growth phase were digested with TrypLE trypsin. After harvesting, the cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of cells were added to each well of a 3799 cell plate (Corning), centrifuged at 300 g, and the supernatant discarded. Antibodies were serially diluted in FACS buffer (as above), added to the centrifuged cells, and the cells were resuspended. Incubate at 4°C for one hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Add 100 μL of Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Invitrogen, A21202; 1:1000) secondary antibody, incubate at 4°C for one hour, wash twice with FACS buffer, resuspend in PBS, and analyze using a FACS (BD FACS Canto™ II) instrument. GraphPad Prism 6 was used to fit the curve and calculate the EC 50 value.

[0281] 5.3. Mouse Antibodies Inhibit CpG-A-Stimulated IFNα Release in Human PBMCs

[0282] 1) Thaw frozen human PBMCs and culture overnight in complete medium (1640+10% FBS+1×NEAA+1×L-Glutamine) for 18 hours. The next day, harvest the PBMCs, centrifuge at 600g for 8 minutes, and discard the supernatant. Resuspend the cells in medium (1640+10% FBS) to 8x10^6 / mL, and add 100 μL per well of a 3799 cell culture plate. Dilute the antibody to be tested in 10-step serial dilutions (final concentration, 0.0001 nM–10 nM) in medium (1640+10% FBS), and add 50 μL / well of the diluted antibody to the PBMCs. Resuspend the cells and incubate at 37°C for 6 hours. Add 50 μL of 2 μM CpG-A (Invitrogen, tlrl-2216-5) diluted in medium (1640+10% FBS) to each well, mix thoroughly, and incubate at 37°C for an additional 20 hours. On the third day, the cell culture plate was centrifuged at 400 g for 5 minutes, the cell culture supernatant was collected and the cytokine IFNα in the supernatant was detected.

[0283] 2) ELISA method to detect cytokine IFNα

[0284] Dilute the antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) to 4 μg / mL with PBS, add 100 μL per well to the CORNING ELISA plate, and incubate the plate overnight at 4°C. Afterwards, discard the plate-coated antibody, add 300 μL per well of blocking solution (PBS + 1% BSA) to the plate, and incubate overnight at 4°C. Dilute the supernatant to be tested with blocking solution to the appropriate concentration (select 1:3-1:7 depending on the situation), and add 100 μL per well to the coated ELISA plate. Dilute the IFNα detection standard with blocking solution in a 2-fold gradient (the highest concentration is 1000 pg / mL, 8 concentration points, and the last point is 0), and add 100 μL per well to the coated ELISA plate. Place the ELISA plate in a 37°C incubator and incubate for 1 hour. Wash the plate three times, add 100 μL of detection antibody MT2 / 4 / 6 (1:1000) to each well, and incubate at 37°C for 1 hour. Wash the plate three times, add 100 μL of secondary antibody SA-HRP (1:1000) to each well, and incubate at 37°C for 0.5 hours. Wash the plate three times, add 100 μL of TMB color development solution to each well, and stop the reaction by adding 50 μL of 1M HCl to each well after color development for the appropriate time. Read the OD450nm reading using an ELISA plate reader, calculate the inhibition rate, and use GraphPad Prism6 to make a fitting curve to calculate the IC 50 value.

[0285] Example 6. Determination of the variable region sequence of the anti-BDCA2 mouse hybridoma (monoclonal antibody) (according to Kabat)

[0286] Based on the ability of mouse antibodies to bind to antigens at the protein and cellular levels and their ability to inhibit the release of cytokine IFNα induced by CpG-A stimulation in the PBMC system, 16 hybridoma monoclonal clones were selected for VH / VL sequence determination.

[0287] The DNA coding sequence for the variable region of the anti-BDCA2 murine antibody was determined using a degenerate primer-based PCR method. Candidate hybridoma cells were cultured, harvested by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. This was used as a template for first-strand cDNA synthesis. The first-strand cDNA was then used as a template for subsequent PCR amplification of the corresponding variable region DNA coding sequence. The primer sequences used in the amplification reaction were complementary to the first framework region and constant region of the antibody variable region (Larrick, JW et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). To a 50 μl reaction system, 1 μl of cDNA, 5 μl of 10× PCR buffer, 1 μl of each upstream and downstream primer (25 pmol), 1 μl of dNTPs, 1 μl of 25 mmol pL MgCl₂, and 39 μl of H₂O were added. The reaction was pre-denatured at 95°C for 10 min, and 1 μl of Taq enzyme was added. PCR amplification was performed using a temperature cycler. The reaction conditions were denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 15 s for 32 cycles, followed by a 10-min incubation at 72°C. The PCR products were recovered and purified. The amplified products were sequenced to obtain the amino acid sequences of the heavy and light chain variable regions of the anti-BDCA2 mouse antibody.

[0288] Consensus sequences were searched against the germline and rearranged Ig variable region sequence database using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ). Complementarity-determining region (CDR) amino acid sequences were identified based on the Kabat (Wu, TT and Kabat, EA 1970 J. Exp. Med., 132:211-250) and IMGT systems (Lefranc M.-P. et al. 1999 Nucleic Acids Research, 27, 209-212) and by internet-based sequence analysis (http: / / www.imgt.org / IMGT_vquest / vquest and http: / / www.ncbi.nlm.nih.gov / igblast / ).

[0289] The amino acid sequences of the light and heavy chain variable regions and CDRs of the selected anti-BDCA2 murine antibodies are shown in Table 2. Multiple sequence alignment and phylogenetic tree classification were performed. Based on the results, the heavy chain variable regions (VH) of the 16 monoclonal antibody sequences were mainly divided into five categories: 1) mAb020, 022, 026; 2) mAb021, 027, 028, 030, 032, 034; 3) mAb019, 024, 033; 4) mAb001, 002; and 5) mAb005, 016. mAb028 and mAb030 differ in their heavy chain variable regions by only two amino acids, and mAb028 and mAb032 differ by only one amino acid. The light chain variable regions (VL) can be divided into three categories: 1) mAb019; 2) mAb021, 027, 028, 030, 032, 034; and 3) other antibodies. The light chain variable region sequences of mAb028, 030, and 032 are identical.

[0290] Table 2 CDR and variable region amino acid sequences of anti-BDCA2 mouse antibodies (KABAT scheme)

[0291] Example 7: Construction of anti-BDCA2 chimeric antibodies

[0292] Based on sequence analysis results, a total of 12 monoclonal antibody sequences were selected from the two fusions for the construction of human-mouse chimeric antibodies. During plasmid construction, the hIgG1 Fc region sequence was selected as the human constant region sequence. The heavy and light chain variable region coding sequences of the aforementioned anti-BDCA2 murine antibody were synthesized by Genscript. Expi 293F cells were used as the host cell for antibody expression. The resulting chimeric antibodies are: mab001c, mab002c, mab005c, mab016c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c, and mab033c.

[0293] The expressed chimeric antibodies were characterized in various ways.

[0294] Example 8. Screening of chimeric antibodies

[0295] 8.1. ELISA Method for Detecting the Binding Activity of Anti-BDCA2 Chimeric Antibodies to Proteins

[0296] Human BDCA2 Fc protein or cyno BDCA2 His protein was diluted to 1 μg / mL in PBS and added to an ELISA microplate at 100 μL / well for overnight incubation at 4°C. After coating, the plates were blocked with ELISA blocking buffer (PBS phosphate buffer containing 1% BSA, pH 7.4; percentages are by weight) at 37°C for two hours. A 10-fold serial dilution of the antibody was added to a total of eight concentrations (0.0001 nM–100 nM, including a zero control) at 100 μL / well for 1 hour at 37°C. The plates were washed three times and a secondary antibody (anti-human IgG (Fab specific)-peroxidase antibody (Sigma, A0293; 1:5000 dilution) was added at 100 μL / well for 1 hour at 37°C. The plate was washed three times, and 100 μL / well TMB colorimetric solution (Huzhou Yingchuang Biotechnology, EL0009) was added. After incubation at 37°C for 10 minutes, 50 μL 1N hydrochloric acid was added to terminate the colorimetric reaction. The OD450nm value was read using an ELISA plate reader, and the curve was fitted using GraphPad Prism6 to calculate the EC 50 value.

[0297] The results are as follows: The chimeric antibody tested bound to both humanBDCA2 Fc protein and cynoBDCA2 His protein, and the EC binding to humanBDCA2-Fc protein was 50 0.1-0.2 nM; most chimeric antibodies have an EC less than 0.6 nM 50 The values ​​were bound to cynoBDCA2 His protein.

[0298] 8.2. FACS Detection of Chimeric Antibody Binding Activity to Cells

[0299] CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells grown to the logarithmic growth phase were digested with TrypLE trypsin. After harvesting, the cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of cells were added to each well of a 3799 cell plate and centrifuged at 300 g. The supernatant was discarded. Antibodies were serially diluted in FACS buffer and added to the centrifuged cells. The cells were resuspended. Incubated at 4°C for one hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody and Alexa Fluor 488 (Sigma, A11013; 1:1000) secondary antibody were added, and the cells were incubated for one hour, washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS (BD FACS Canto™ II) instrument. The curves were fitted using GraphPad Prism 6, and the EC was calculated. 50 value.

[0300] As shown in Figure 3 (3a-3c), all the chimeric antibodies tested bound to CHOK1-humanBDCA2 cells, and their EC 50 The results were better than or equivalent to the control antibody BIIB059. At the same time, all the chimeric antibodies tested bound to 293F-cynoBDCA2 cells, and their EC 50 The activity was approximately 0.3-2 nM, which was superior to or comparable to the control antibody BIIB059.

[0301] 8.3. Detection of the ability of chimeric antibodies to inhibit the release of cytokine IFNα from human PBMC cells stimulated by CpG-A

[0302] Cryopreserved human PBMCs were thawed and cultured overnight in complete medium (1640+10% FBS+1×NEAA+1×L-Glutamine) for 18 hours. The next day, PBMCs were harvested and centrifuged at 600g for 8 minutes, and the supernatant discarded. Cells were resuspended in medium (1640+10% FBS) to 8x10^6 / mL, and 100 μL was added to each well of a 3799 cell culture plate. The antibody to be tested was diluted 10-fold in medium (1640+10% FBS) (final concentration 0.0001 nM–10 nM), and 50 μL / well of the diluted antibody was added to the PBMCs. Cells were resuspended and incubated at 37°C for 6 hours. Then, 50 μL of 2 μM CpG-A (Invivogen, tlrl-2216-5) diluted in medium (1640+10% FBS) was added to each well, mixed, and incubated at 37°C for an additional 20 hours. On the third day, the cell culture plate was centrifuged at 400 g for 5 minutes, the cell culture supernatant was collected and the cytokine IFNα in the supernatant was detected.

[0303] Dilute the MT1 / 3 / 5 antibodies (Mabtech, 3425-1H-20) to 4 μg / mL in PBS, add 100 μL per well of a CORNING ELISA plate, and incubate overnight at 4°C. Discard the coated antibody and add 300 μL per well of blocking solution (PBS + 1% BSA) to the plate. Incubate overnight at 4°C. Dilute the supernatant to be tested in blocking solution to the appropriate concentration (generally 1:3-1:7), and add 100 μL per well of the coated ELISA plate. IFNα detection standard is serially diluted in blocking solution (maximum concentration 1000 pg / mL, 8 concentration points, with the last point being 0), and add 100 μL per well of the coated ELISA plate. Incubate the ELISA plate in a 37°C incubator for 1 hour. Wash the plate three times, and add 100 μL per well of the detection antibody MT2 / 4 / 6 (1:1000) and incubate at 37°C for 1 hour. Wash the plate three times and add 100 μL of secondary antibody SA-HRP (1:1000) to each well. Incubate in a 37°C incubator for 0.5 hours. Wash the plate three times and add 100 μL of TMB colorimetric solution to each well. After color development for the appropriate time, add 50 μL of 1 M HCl to each well. Read the OD450 nm reading using an ELISA plate reader, calculate the inhibition rate, and use GraphPad Prism 6 to create a fitting curve and calculate the IC. 50 .

[0304] In the CpG-A stimulated PBMC cytokine release experiment, all 12 chimeric antibodies tested could inhibit the secretion of cytokine IFNα. Among them, the IC values ​​of mab005c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c and mab033c were the highest. 50They are: 0.008359nM, 0.01493nM, 0.01853nM, 0.01268nM, 0.03239nM, 0.01677nM, 0.008976,nM, 0.01645nM and 0.008331nM respectively.

[0305] 8.4. Detection of Chimeric Antibody Endocytosis Ability

[0306] CHOK1-humanBDCA2 cells grown to the logarithmic growth phase were digested with TrypLE trypsin. After harvesting, the cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of cells were added to each well of a 3799 cell plate and centrifuged at 300 g. The supernatant was discarded and the cells were cooled on ice. Antibody was serially diluted in FACS buffer (three concentrations: 100 nM, 10 nM, and 1 nM; and three groups: control at 0 hour; 4°C for 1 hour; and 37°C for 1 hour). After dilution, the cells were cooled on ice for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells and the cells were resuspended. Incubated at 4°C for 40 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Cells in the 0h group were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. Cells in the other two groups were resuspended in 100 μL of FACS buffer and incubated at 4°C and 37°C for 1 hour, respectively. Cells in the 4°C for 1 hour and 37°C for 1 hour were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. Cells in all three conditions were simultaneously incubated with 100 μL of the secondary antibody Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) at 4°C for one hour. Cells were washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated.

[0307] Chimeric antibodies exhibited internalization at concentrations of 100nM, 10nM, and 1nM. The 12 chimeric antibodies showed comparable endocytosis-mediated abilities, slightly outperforming the positive control antibody BIIB059. At 100nM, after one hour of internalization at 37°C, cell surface signal values ​​ranged from 50% to 60%.

[0308] Example 9: Humanization of Antibody Variable Regions

[0309] To humanize the antibody variable region, the human immunoglobulin gene database at NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) is first searched for human germline IgG genes homologous to the mouse antibody cDNA sequence. The amino acid sequence and precise boundaries of the variable region CDRs are then defined using the Kabat or IMGT numbering systems. In principle, a human IGHV with high homology to the mouse antibody is selected as the humanization template, and humanization of the antibody variable region is performed via CDR grafting.

[0310] From the murine antibody sequences obtained above, mAb005 and mAb033 were selected for humanization. The humanization process involved the following steps: A. Align the murine antibody gene sequence with the human germline antibody gene sequence to identify sequences with high homology; B. Analyze and examine HLA-DR affinity to select human germline framework sequences with low affinity; C. Utilize computer simulation and molecular docking to analyze the variable region and surrounding framework amino acid sequences to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, key amino acids within the murine antibody gene sequence that interact with human BDCA2 and maintain its spatial structure were identified. These amino acids were then grafted back into the selected human germline gene framework. Based on this, essential framework amino acid positions were identified to synthesize humanized antibodies. Multiple humanized antibody variable region sequences were generated. Various combinations of the designed humanized anti-BDCA2 antibody variable regions were used to generate humanized anti-BDCA2 antibodies. The amino acid sequences of these variable regions are shown in Table 3.

[0311] The CDRs of the Hu005 series humanized antibody variable regions (KABAT scheme) are shown below:

[0312] HCDR1: NYGVH (SEQ ID NO: 11)

[0313] HCDR2:VIWSGESTDYDAAFIS(SEQ ID NO:12)

[0314] HCDR3:RRSHYYGYVMDY(SEQ ID NO:13)

[0315] LCDR1:

[0316] LCDR2: AASNLES (SEQ ID NO: 15)

[0317] LCDR3: QQSNEDPPT(SEQ ID NO:16)

[0318] The CDRs of the Hu033 series humanized antibody variable regions (KABAT scheme) are shown below:

[0319] HCDR1: SYGMS (SEQ ID NO: 52)

[0320] HCDR2: TISSGDSYTYYPDSVKG(SEQ ID NO:39)

[0321] HCDR3:QIYYDYAYYFDF(SEQ ID NO:53)

[0322] LCDR1:RASESVSFRTSHLMH(SEQ ID NO:54)

[0323] LCDR2:GASNLES (SEQ ID NO:55)

[0324] LCDR3:QQSIEDPPT(SEQ ID NO:42)

[0325] Table 3 Variable region amino acid sequences of anti-BDCA2 humanized antibodies

[0326] Example 10: Screening of humanized anti-BDCA2 antibodies

[0327] 10.1. Binding Activity of Humanized Antibodies to BDCA2

[0328] CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells grown to the logarithmic growth phase were digested with TrypLE trypsin. After harvesting, the cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of the resuspended cells were added to each well of a 3799 cell plate and centrifuged at 300 g. The supernatant was discarded. The antibody to be tested was serially diluted 5-fold in FACS buffer (final concentration 0.012 nM to 200 nM). The diluted antibody was added to the centrifuged cells and the cells were resuspended. Incubate at 4°C for one hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Sigma, A11013; 1:1000) was added and the cells were incubated at 4°C for one hour, followed by two washes with FACS buffer. After resuspending the cells in PBS, the fluorescence signal of the antibody bound to the cell surface was analyzed using a FACS (BD FACS CantoTM II) instrument, and the curve was fitted using GraphPad Prism6 to calculate the EC of the antibody binding to the BDCA2 antigen. 50 Among them, BIIB059 antibody was selected as the positive control and hIgG1 was selected as the negative control.

[0329] Candidate antibodies derived from humanized mAb033 showed superior binding activity to both CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells compared to the positive control antibody BIIB059 (Table 4). SPR was used to determine the affinity of the antibodies for human BDCA2, and the selected antibodies showed stronger affinity for human BDCA2 than the positive control antibody BIIB059.

[0330] Table 4 Binding activity of mAb033 series humanized antibodies to BDCA2 Note: “-” means not detected.

[0331] The candidate antibody obtained by humanizing the mAb005 sequence is effective for EC binding to CHOK1-humanBDCA2 cells. 50 The value was better than that of the positive control BIIB059 antibody, as shown in Table 5. At the same time, the candidate antibody obtained by humanizing the mAb005 sequence also had good binding activity to 293F-cynoBDCA2 cells.

[0332] Table 5 Binding activity of mAb005 series humanized antibodies to BDCA2

[0333] 10.2. Humanized Antibodies Inhibit the Release of IFNα from Human PBMCs Stimulated by CpG-A

[0334] A series of humanized candidate antibodies were selected for functional testing of their ability to inhibit the release of cytokine IFNα from human PBMC cells stimulated by CpG-A. Frozen human PBMC cells were revived and cultured overnight in complete culture medium (1640+10% FBS+1×NEAA+1×L-Glutamine) for 18 hours. The next day, PBMC cells were collected and centrifuged at 600g for 8 minutes, and the supernatant was discarded. The cells were resuspended in culture medium (1640+10% FBS) to 8x10^6 / mL, and 100μL was added to each well of a 3799 cell culture plate. The antibody to be tested was diluted 10-fold in culture medium (1640+10% FBS) (0.0001nM-10nM, and a 0-point control), and the diluted antibody was added to the PBMC at 50μL / well. After resuspending the cells, they were incubated at 37°C for 6 hours. 2 μM CpG-A (Invitrogen, tlrl-2216-5) diluted in 50 μL of culture medium (1640 + 10% FBS) was added to each well, mixed, and incubated at 37°C for another 20 hours. On the third day, the cell culture plate was centrifuged at 400 g for 5 minutes, and the cell culture supernatant was collected and assayed for the cytokine IFNα. BIIB059 antibody was used as a positive control, and hIgG1 was used as a negative control.

[0335] Dilute the antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) to 4 μg / mL with PBS, add 100 μL per well to the CORNING ELISA plate, and incubate the plate overnight at 4°C. Then discard the plate-coated antibody, add 300 μL per well of blocking solution (PBS + 1% BSA) to the plate, and incubate overnight at 4°C. After blocking, dilute the supernatant to be tested with blocking solution to the appropriate concentration (generally 1:3-1:7), and add 100 μL per well to the coated ELISA plate. The IFNα detection standard is diluted with blocking solution in a 2-fold gradient (the highest concentration is 1000 pg / mL, 8 concentration points, and the last point is 0), and add 100 μL per well to the coated ELISA plate. Place the ELISA plate in a 37°C incubator and incubate for 1 hour. Wash the plate three times, add 100 μL of detection antibody MT2 / 4 / 6 (1:1000) to each well, and incubate at 37°C for 1 hour. Wash the plate three times, add 100 μL of secondary antibody SA-HRP (1:1000) to each well. Incubate at 37°C for 0.5 hour. Wash the plate three times, add 100 μL of TMB colorimetric solution to each well, and add 50 μL of 1 M HCl to each well after development for the appropriate time. Read the OD450nm reading using an ELISA plate reader, and create a fitting curve using GraphPad Prism6. Select the OD value corresponding to a 10 nM antibody concentration, and calculate the maximum inhibition rate according to the inhibition rate = (OD(IgG1) - OD(Ab)) / OD(IgG1).

[0336] In the experiment of PBMC releasing cytokines stimulated by CpG-A, all humanized antibodies tested could inhibit the secretion of cytokine IFNα. 50 The value was less than that of the positive control antibody BIIB059. For further results, see Table 6.

[0337] Table 6 Humanized antibodies inhibit the release of cytokine IFNα by human PBMC cells stimulated by CpG-A Note: “-” means not detected.

[0338] 10.3. Humanized Antibody Endocytosis Capacity Assay

[0339] CHOK1-humanBDCA2 cells cultured to the logarithmic growth phase were digested with TrypLE trypsin. After harvesting, the cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of cells were added to each well of a 3799 cell plate and centrifuged at 300 g. The supernatant was discarded and the cells were cooled on ice. Antibodies were serially diluted in FACS buffer (two concentrations: 100 nM and 10 nM; and three groups: 0 hour, 4°C for 1 hour, and 37°C for 1 hour). After dilution, the cells were cooled on ice for 15 minutes. After 15 minutes, the antibodies were added to the centrifuged cells and the cells were resuspended. Incubated at 4°C for 40 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Cells in the 0 hour group were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. Cells in the other two groups were resuspended in 100 μL of FACS buffer and incubated at 4°C and 37°C for 1 hour, respectively. Cells were fixed with paraformaldehyde for 10 minutes at room temperature (1 hour at 4°C) and 1 hour at 37°C, then washed twice with FACS buffer. All three groups were incubated with 100 μL of Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) at 4°C for one hour. Cells were then washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated.

[0340] The results are shown in FIG4 . Both Hu005 and Hu033 humanized antibodies were internalized at concentrations of 100 nM and 10 nM, and the ability of the humanized antibodies to mediate endocytosis was comparable to that of the positive control antibody BIIB059.

[0341] 10.4. Humanized Antibody Affinity Testing

[0342] The affinity of the humanized antibodies of the present invention was determined using a BIAcore assay: the surface of a CM5 chip channel was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC were obtained from an amino coupling kit). Anti-human IgG (Fc) antibody (diluted in sodium acetate, pH 5.0, at a concentration of 25 μg / mL) was injected for 420 seconds. Excess reactive carboxyl groups on the chip were then blocked by injection of 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds. The humanized antibody was diluted to 1 μg / mL in running buffer and injected into the detection channel flow cell at a flow rate of 10 μL / min for 60 seconds for capture. Recombinant human BDCA2 was diluted to 50 or 100 nM in 1× HBS-EP+ (pH 7.4) and then serially diluted 1:2 to 0.39 nM. hBDCA2 was injected into the detection channel at various concentration gradients at a flow rate of 30 μL / min. Two zero-concentration samples were used to subtract background signal. The binding and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively. Data analysis was performed using Biacore Insight Evaluation Software (Version 2.0.15.12933). After subtracting the signal from the reference channel (flow cell 1) and the zero-concentration signal, a 1:1 binding model was used for curve fitting and kinetic parameter calculation. The results are shown in Table 7 below.

[0343] Table 7 Affinity of humanized antibodies

[0344] Example 11 Fc mutation and antibody screening of humanized anti-BDCA2 antibodies

[0345] For the humanized antibodies Hu033-03 and Hu033-20 obtained in Example 10, mutations were performed at the Fc site to increase their affinity for Fc and FcγRIIA. A total of humanized antibodies Hu033-03-T1, Hu033-03-T2, Hu033-03-T3, Hu033-20-T1, Hu033-20-T2, and Hu033-20-T3 were designed. The resulting heavy and light chain coding sequences were synthesized by Genscript. The amino acid sequences of the heavy and light chains are shown in Table 8. Antibody expression was performed using 293F host cells, and the expressed and purified antibodies were subjected to various characterization tests.

[0346] Table 8: Variable region and full-length amino acid sequences of mutated anti-BDCA2 humanized antibodies

[0347] 11.1. Fc mutations increase antibody affinity for FcγRIIA

[0348] Antibody kinetics or affinity parameters were determined using BIAcore 8K and a SA chip with freshly immobilized biotinylated human FcγRIIA(R167) / FcγRIIA(H167). The experiments were run at 25°C in 1×HBS-EP++ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20) running buffer.

[0349] The test antibody was diluted 1:2 in running buffer to a concentration of 5000 nM, 2500 nM, 1250 nM, 625 nM, 321.5 nM, 156.25 nM, 78.13 nM, 39.06 nM, 19.53 nM, or 9.77 nM. A gradient of each concentration was injected into flow cell 1 and flow cell 2 at a flow rate of 30 μL / min, with antibody association and dissociation times of 60 and 90 seconds, respectively. Two zero-concentration samples were used to subtract background signal. Samples were injected in a multi-cycle fashion. The analyte was fully dissociated from the chip surface at the end of each cycle, so no regeneration step was performed. Data analysis was performed using Biacore Insight Evaluation Software (Version 2.0.15.12933). After subtracting the signal from the reference channel (flow cell 1) and the two zero-concentration samples, affinity analysis was performed using either a 1:1 binding model or a steady-state model for curve fitting and calculation of kinetic parameters.

[0350] Compared to wild-type Fc, the mutated antibodies showed significantly improved affinity for human FcγRIIA (R167) or FcγRIIA (H167) isoforms. The results are shown in Tables 9-1 and 9-2.

[0351] Table 9-1 Affinity of anti-BDCA2 humanized antibodies for human FcγRIIA (H167) (1:1 binding)

[0352] Table 9-2 Affinity of anti-BDCA2 humanized antibodies for human FcγRIIA (R167) (1:1 binding)

[0353] 11.2. Antibodies with Fc mutations retain their binding affinity to BDCA2

[0354] The binding ability of the Fc-mutated antibodies to human BDCA2 and Cyno BDCA2-expressing cells was detected by FACA.

[0355] The experimental method is as follows.

[0356] Digest CHOK1-humanBDCA2 and 293F-cynoBDCA2 cells with TrypLE. After harvesting, resuspend the cells in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. Add 100 μL of cells to each well of a 3799 cell plate and centrifuge at 300 g. Discard the supernatant. Dilute the antibody in FACS buffer at a starting concentration of 200 nM, using a 1:5 dilution series. Add the diluted antibody to the centrifuged cells and resuspend the cells. Incubate at 4°C for one hour. Centrifuge the incubated cells at 300 g for 5 minutes and wash twice with FACS buffer. Add 100 μL of Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Invitrogen, A21202; 1:1000) secondary antibody, incubate at 4°C for one hour, wash twice with FACS buffer, resuspend in PBS, and analyze using a FACS (BD FACS Canto™ II) instrument. GraphPad Prism 6 was used to fit the curve and calculate the EC50 value.

[0357] The Fc-mutated antibodies showed slightly better binding to human BDCA2 than the wild-type Fc and significantly better binding to the reference antibody BIIB059. The Fc-mutated antibodies also showed better binding to Cyno BDCA2 than both the wild-type Fc and BIIB059. The results are shown in Figures 5a and 5b and Table 10.

[0358] Table 10 Binding ability of anti-BDCA2 humanized antibodies to human BDCA2

[0359] 11.3. Antibody Endocytosis Ability Remains Unchanged After Fc Mutation

[0360] The internalization ability of each antibody was detected by FACS.

[0361] CHOK1-humanBDCA2 cells were digested with TrypLE. After harvesting, cells were resuspended in FACS buffer (PBS + 2% FBS) to 2 x 10^6 / mL. 100 μL of cells were added to each well of a 3799 cell plate and centrifuged at 300 g. The supernatant was discarded and the cells were cooled on ice. Antibodies were serially diluted in FACS buffer to 1 nM, 10 nM, or 100 nM (five assay conditions were set for each antibody concentration: 0 hour; 4°C, 1 hour; 37°C, 1 hour; 4°C, 4 hours; 37°C, 4 hours). After dilution, the cells were cooled on ice for 15 minutes. After 15 minutes, the antibodies were added to the centrifuged cells and the cells were resuspended. Incubate at 4°C for 40 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Cells in the 0h group were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. Cells in the other four groups were resuspended in 100 μL of FACS buffer and incubated at 4°C or 37°C for 1 or 4 hours, respectively. At the designated time, cells in the corresponding 4°C and 37°C groups were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. Cells in all five groups were simultaneously incubated with 100 μL of the secondary antibody Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) at 4°C for one hour. Cells were then washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated.

[0362] The Fc-mutated antibodies were internalized at concentrations of 100 nM, 10 nM, and 1 nM. The three Fc modifications had no effect on antibody internalization. See Figures 6a-6f for details.

[0363] 11.4. Fc mutant antibodies more effectively inhibit the release of IFNα from human PBMCs stimulated by the antibody complex SLE-IC

[0364] The Fc-modified antibodies were functionally tested for their ability to inhibit the release of cytokine IFNα from human PBMC cells stimulated by antibody complexes. Frozen human PBMC cells were revived and cultured overnight in complete culture medium (1640+10% FBS+1×NEAA+1×L-Glutamine) for 18 hours. The next day, PBMC cells were collected, centrifuged at 600g for 8 minutes, and the supernatant was discarded. The cells were resuspended in culture medium (1640+10% FBS) to 8x10^6 / mL, and 100μL was added to each well of a 3799 cell culture plate. The antibody to be tested was diluted 5-fold in culture medium (1640+10% FBS) (0.0032nM-10nM, and a 0-point control), and the diluted antibody was added to the PBMC at 50μL / well. After resuspending the cells, they were incubated at 37°C for 6 hours. SLE-IC (2.5 μl of Sm / RNP-antigen (AROTEC, ART01-10) mixed with 10 μl of anti-RNP antibody (RayBiotech, MD-14-0513) diluted in 50 μL of culture medium (1640 + 10% FBS) was added to each well, mixed thoroughly, and incubated at 37°C for another 20 hours. On the third day, the cell culture plate was centrifuged at 400 g for 5 minutes, and the cell culture supernatant was collected and assayed for the cytokine IFNα. BIIB059 antibody was used as a positive control, and hIgG1 was used as a negative control.

[0365] To detect the cytokine IFNα in the supernatant, dilute the antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) to 4 μg / mL in PBS, add 100 μL per well of the CORNING ELISA plate, and incubate overnight at 4°C. Discard the plate-coated antibody and add 300 μL per well of blocking solution (PBS + 1% BSA) to the plate. Incubate overnight at 4°C. Dilute the supernatant to be tested with blocking solution to the appropriate concentration (generally 1:3-1:7), and add 100 μL per well of the coated ELISA plate. IFNα detection standard is diluted in blocking solution in a 2-fold gradient (maximum concentration 1000 pg / mL, 8 concentration points, and the last point is 0), and add 100 μL per well of the coated ELISA plate. Incubate the ELISA plate in a 37°C incubator for 1 hour. Wash the plate three times, add 100uL of detection antibody MT2 / 4 / 6 (1:1000) to each well, and incubate at 37°C for 1 hour. Wash the plate three times, add 100uL of secondary antibody SA-HRP (1:1000) to each well. Incubate at 37°C for 0.5 hours. Wash the plate three times, add 100uL of TMB color development solution to each well, and add 50uL of 1× HCl to each well when color development is appropriate. Read the OD value using an ELISA plate reader. 450 The concentration of IFNα in the supernatant was calculated using the fitted curve using GraphPad Prism6.

[0366] Compared to wild-type Fc antibodies and BIIB059, the Fc-modified antibody exhibited stronger IFNα inhibition under SLE-IC stimulation conditions, achieving complete inhibition of IFNα secretion at 0.016 nM (see Figure 7 and Table 11 for details).

[0367] Table 11 Mutated humanized antibodies inhibit the release of cytokine IFNα by human PBMC cells stimulated by SLE-IC Note: “-” indicates below the detection limit; “NA” indicates not detected.

[0368] Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the present invention as claimed should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for implementing the present invention will be apparent to those skilled in the art of molecular biology, immunology, or related fields and are intended to be within the scope of the appended claims.

[0369] Sequence Listing (all amino acid sequences, for the definition / explanation of the sequences, see the previous section of this article):

Claims

1. An anti-BDCA2 antibody or antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region: The heavy chain variable region comprises: (I) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or (III) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or (IV) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or (V) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; or (VI) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:46, and SEQ ID NO:47, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:46, and SEQ ID NO:47, respectively; or (VII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:50, and SEQ ID NO:47, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:31, SEQ ID NO:50, and SEQ ID NO:47, respectively; or (VIII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; or (IX) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; and / or The light chain variable region comprises: (I) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, by 1, 2, or 3 amino acids; or (II) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, by 1, 2, or 3 amino acids; or (III) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, by 1, 2, or 3 amino acids; or (IV) LCDR1, LCDR2, and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; or LCDR1, LCDR2, and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, by 1, 2, or 3 amino acids; or (V) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or (VI) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:49, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:48, and SEQ ID NO:49, respectively; or (VII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:51, and SEQ ID NO:49, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:34, SEQ ID NO:51, and SEQ ID NO:49, respectively; or (VIII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or (IX) LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:86, SEQ ID NO:15, and SEQ ID NO:16, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:86, SEQ ID NO:15, and SEQ ID NO:16, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NO:52, SEQ ID NO:39, and SEQ ID NO:53, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:42, respectively; or (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (III) a heavy chain variable region comprising HCDR1 and HCDR2 as shown in SEQ ID NO:25 and SEQ ID NO:26, respectively, and HCDR3 as shown in SEQ ID NO:27 or SEQ ID NO:37; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; or (IV) heavy chain variable region, comprising amino acid sequences as shown in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or (V) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:41, SEQ ID NO:15, and SEQ ID NO:42, respectively; or (VI) a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO:31, a HCDR2 as set forth in SEQ ID NO:46 or 50, and a HCDR3 as set forth in SEQ ID NO:47; and a light chain variable region comprising a LCDR1 as set forth in SEQ ID NO:34, a LCDR2 as set forth in SEQ ID NO:48 or SEQ ID NO:51, and a LCDR3 as set forth in SEQ ID NO:49; or (VII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; and a light chain variable region comprising LCDR1 with amino acid sequences as shown in SEQ ID NO:14 or SEQ ID NO:86, LCDR2 with amino acid sequences as shown in SEQ ID NO:15 and LCDR3 with amino acid sequences as shown in SEQ ID NO:16, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region: (I) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:61; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:62; or (II) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:65; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:66; or (III) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:67, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:67; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:68; or (IV) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:69; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:70, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:70; or (V) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:71; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:68; or (VI) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72, or comprises the amino acid sequence shown in SEQ ID NO: 72 and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 73, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 73; or (VII) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:76; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:77; or (VIII) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:80; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:79, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:79; or (IX) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:82; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:

83.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 87-110, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 87-110; and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 135-158, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 135-158; or (II) a heavy chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 111-134, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 111-134; and a light chain variable region comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 159-182, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 159-182; or (III) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:89, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:89; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:137, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

137. (IV) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 106; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 154, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

154. (V) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 114, or comprising a sequence similar to SEQ ID NO: 114 and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 162, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 162; or (VI) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequence shown in any combination of Table C.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv and sdAb.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably of IgG1 subtype.

8. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody comprises or consists of the following sequence: (I) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 183, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 186, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (II) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 184, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 187, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (III) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 185, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 188, or at least 95%, 96%, 97%, 98% or 99% sequence identity thereto.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody comprises or consists of the following sequence: (I) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 183, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 186; or (II) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 184, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 187; or (III) a heavy chain having 1, 2 or 3 amino acid differences from the amino acid sequence of SEQ ID NO: 185, and a light chain having the amino acid sequence of SEQ ID NO:

188.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein the antibody comprises or consists of the following sequence: (I) a heavy chain with an amino acid sequence as shown in SEQ ID NO: 191, SEQ ID NO: 192 or SEQ ID NO: 193, and a light chain with an amino acid sequence as shown in SEQ ID NO: 186; or (II) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196, and a light chain having an amino acid sequence as shown in SEQ ID NO:

187.

11. A polynucleotide molecule encoding the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. An expression vector comprising the polynucleotide molecule according to claim 11, preferably, the expression vector is a eukaryotic expression vector.

13. A host cell comprising the polynucleotide molecule according to claim 11 or the expression vector according to claim 12, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

14. A method for producing the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising expressing the antibody or antigen-binding fragment thereof in the host cell according to claim 13 under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

15. An immunoconjugate comprising the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, conjugated to at least one therapeutic or diagnostic agent, preferably an anti-inflammatory drug or an immunosuppressant.

16. A pharmaceutical composition comprising the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the polynucleotide molecule according to claim 11, the expression vector according to claim 13, the host cell according to claim 11, the immunoconjugate according to claim 13, and a pharmaceutically acceptable carrier or excipient.

17. A method for inhibiting excess cytokine release from immune cells of a subject, the method comprising contacting the anti-BDCA2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the immunoconjugate according to claim 15, or the pharmaceutical composition according to claim 16 with immune cells of the subject, preferably, the immune cells are CpG-A-stimulated PBMCs.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the immunoconjugate according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating and / or preventing a disease mediated by BDCA2, wherein the disease is preferably an inflammatory disease; more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

19. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the immunoconjugate according to claim 15, or the pharmaceutical composition according to claim 16.

20. A method for detecting the presence of BDCA2 in a sample using the antibody or antigen-binding fragment thereof, or a detection composition containing the antibody or antigen-binding fragment thereof, according to any one of claims 1 to 10, comprising the step of contacting the antibody or antigen-binding fragment thereof, or a detection composition containing the antibody or antigen-binding fragment thereof, with the sample.

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