Anti-BDCA2 antibody and its use

Abstract

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, as well as compositions containing them. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding fragments of the present invention, vectors and host cells for expressing the antibodies or antigen-binding fragments of the present invention, therapeutic and diagnostic methods, and uses of the antibodies or antigen-binding fragments of the present invention.

Description

Technical Field

[0001] Field of the Invention The present invention relates to the field of monoclonal antibodies and / or engineered antibodies. Specifically, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2 and a composition comprising the antibody or antigen-binding fragment thereof. The present invention further provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector and a host cell for expressing the antibody or antigen-binding fragment thereof, therapeutic and diagnostic / detection methods, and uses for the antibody or antigen-binding fragment thereof of the present invention.

Background Art

[0002] Background of the Invention With the progress of medical research, it has been found that various diseases are associated with immune system dysfunction, and research on the immune system has helped to further understand the etiology and explore more optimal treatment options.

[0003] The immune system is a very 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. The interactions and responses of these cells are under the control of a complex and delicate system. Cells utilize activation and suppression mechanisms as well as feedback loops to maintain control of the reaction and prevent, as much as possible, the negative consequences caused by uncontrolled immune reactions (e.g., autoimmune diseases). Multiple signaling pathways related to immunity involve a large number of signaling molecules and their interactions.

[0004] Blood dendritic cell 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 the C-terminus (belonging to the type II C-type lectin group), a transmembrane region from the asparagine residue at position 45 to the isoleucine residue at position 213, and a short cytoplasmic tail at the N-terminus (without a signaling motif). BDCA2 transmits intracellular signals via the associated transmembrane adapter FcεRIγ and induces a B cell receptor (BCR)-like signaling cascade. SUMMARY OF THE INVENTION

[0005] SUMMARY OF THE INVENTION The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to BDCA2 and has advantages such as high affinity and high specificity for human and cynomolgus monkey BDCA2. The anti-BDCA2 antibody or its antigen-binding fragment provided by the present invention can be used, for example, in the treatment of inflammatory pathologies / diseases as a single independent therapy or in combination therapy with other therapies and / or other drugs.

[0006] 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 may downregulate the levels of CD32a and / or CD62L on the surface of pDCs. Furthermore, the anti-BDCA2 antibodies of the present disclosure may mediate the intracellular translocation of BDCA2 from the surface of pDCs. Furthermore, the anti-BDCA2 antibodies described herein can also be used for pDC depletion by ADCC or CDC and can also be used for treating or preventing immune disorders, such as inflammatory disorders and autoimmune disorders.

[0007] 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, and the heavy chain variable region is (I) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or (II) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or (III) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or (IV) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; or (V) HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or (VI) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or (VII) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 50, and SEQ ID NO: 47, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 50, and SEQ ID NO: 47, respectively; or (VIII) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 39, and SEQ ID NO: 53, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 39, and SEQ ID NO: 53, respectively; or (IX) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; or HCDR1, HCDR2, and HCDR3 having 1, 2, or 3 amino acids different from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively comprising: and / or wherein the light chain variable region is (I) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or (II) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (III) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or (IV) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or (V) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 41, SEQ ID NO: 15, and SEQ ID NO: 42, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 41, SEQ ID NO: 15, and SEQ ID NO: 42, respectively; or (VI) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or (VII) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 51, and SEQ ID NO: 49, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 51, and SEQ ID NO: 49, respectively; or (VIII) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 42, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 42, respectively; or (IX) LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO: 86, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 having 1, 2, or 3 amino acid sequences different from the amino acid sequences set forth in SEQ ID NO: 86, SEQ ID NO: 15, and SEQ ID NO: 16, respectively comprising.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth 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 the amino acid sequences set forth 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 having the amino acid sequences set forth 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 having the amino acid sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (III) A heavy chain variable region comprising an HCDR1 and an HCDR2 having the amino acid sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively, and an HCDR3 shown in SEQ ID NO: 27 or SEQ ID NO: 37; and a light chain variable region comprising an LCDR1, an LCDR2, and an LCDR3 having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or (IV) A heavy chain variable region comprising an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; and a light chain variable region comprising an LCDR1, an LCDR2, and an LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or (V) A heavy chain variable region comprising an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; and a light chain variable region comprising an LCDR1, an LCDR2, and an LCDR3 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 15, and SEQ ID NO: 42, respectively; or (VI) A heavy chain variable region comprising an HCDR1 having the amino acid sequence shown in SEQ ID NO: 31, an HCDR2 shown in SEQ ID NO: 46 or SEQ ID NO: 50, and an HCDR3 shown in SEQ ID NO: 47; and a light chain variable region comprising an LCDR1 having the amino acid sequence shown in SEQ ID NO: 34, an LCDR2 shown in SEQ ID NO: 48 or SEQ ID NO: 51, and an LCDR3 shown in SEQ ID NO: 49; or (VII) A heavy chain variable region comprising an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a light chain variable region comprising an LCDR1 having the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 86, an LCDR2 shown in SEQ ID NO: 15, and an LCDR3 shown in SEQ ID NO: 16 comprising.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, (I) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 61; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62; or (II) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 66; or (III) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 67; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68; or (IV) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 69; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 70; or (V) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 71, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 71; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 68, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68; or (VI) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 72, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 72; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 73, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 73; or (VII) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 76, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 76; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 77, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 77; or (VIII) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 80, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 79, or contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79; or (IX) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 82; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 83.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 87 to 110, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 87 to 110; and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 135 to 158, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 135 to 158; or (II) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 111 to 134, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 111 to 134; and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 159 to 182, or comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 159 to 182; or (III) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 89; A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 137, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 137; or (IV) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106; A light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 154, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 154; or (V) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 114, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 162, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 162 comprising.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention (I) A heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 87 to 110 and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 135 to 158; or (II) A heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 111 to 134 and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 159 to 182; or (III) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 89 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 137; or (IV) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 106 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 154; or (V) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 114 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 162 comprising.

[0012] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody is selected from the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody is a humanized antibody.

[0013] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv and sdAb.

[0014] 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 of the IgG1 subtype.

[0015] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody has the sequence: (I) A heavy chain having an amino acid sequence shown in SEQ ID NO: 183 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence shown in SEQ ID NO: 186 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (II) A heavy chain having an amino acid sequence shown in SEQ ID NO: 184 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence shown in SEQ ID NO: 187 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (III) A heavy chain having an amino acid sequence shown in SEQ ID NO: 185 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and a light chain having an amino acid sequence shown in SEQ ID NO: 188 or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto comprising or consisting of.

[0016] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody has the sequence: (I) A heavy chain having the amino acid sequence shown in SEQ ID NO: 183 and a light chain having the amino acid sequence shown in SEQ ID NO: 186; or (II) A heavy chain having the amino acid sequence shown in SEQ ID NO: 184 and a light chain having the amino acid sequence shown in SEQ ID NO: 187; or (III) A heavy chain having the amino acid sequence shown in SEQ ID NO: 185 and a light chain having the amino acid sequence shown in SEQ ID NO: 188 comprising or consisting of.

[0017] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody has the sequence: (I) A heavy chain having 1, 2 or 3 amino acids different from the amino acid sequence shown in SEQ ID NO: 183, and a light chain having the amino acid sequence shown in SEQ ID NO: 186; or (II) A heavy chain having 1, 2 or 3 amino acids different from the amino acid sequence shown in SEQ ID NO: 184, and a light chain having the amino acid sequence shown in SEQ ID NO: 187; or (III) A heavy chain having 1, 2, or 3 amino acids different from the amino acid sequence shown in SEQ ID NO: 185, and a light chain having the amino acid sequence shown in SEQ ID NO: 188 comprising or consisting of.

[0018] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antibody has the sequence: (I) A heavy chain having the amino acid sequence shown in SEQ ID NO: 191, SEQ ID NO: 192, or SEQ ID NO: 193, and a light chain having the amino acid sequence shown in SEQ ID NO: 186; or (II) A heavy chain having the amino acid sequence shown in SEQ ID NO: 194, SEQ ID NO: 195, or SEQ ID NO: 196, and a light chain having the amino acid sequence shown in SEQ ID NO: 187 comprising or consisting of.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or an antigen-binding fragment thereof.

[0020] In some embodiments, for the antibody or antigen-binding fragment thereof of the present invention, the antigen-binding fragment is Fab, Fab’, F(ab’)2, Fv, scFv, and sdAb.

[0021] 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 of the IgG1 subtype.

[0022] In another aspect, the present invention has the following characteristics: (1) Binding to the same epitope as any one of the anti-BDCA2 antibodies of the present invention or its antigen-binding fragment, or an epitope that completely or partially overlaps; (2) Competing with any one of the anti-BDCA2 antibodies of the present invention or its antigen-binding fragment with respect to binding to an epitope of the human BDCA2 protein Provided is an isolated anti-BDCA2 antibody or antigen-binding fragment thereof having one or more of the following.

[0023] In another aspect, the present invention has the following characteristics: (1) Binding to human / cynomolgus BDCA2 protein with high affinity, e.g., a KD value of 1.0×10 -6 M or less, a KD value of 5.0×10 -7 M or less, a KD value of 2.5×10 -7 M or less, a KD value of 1.0×10 -7 M or less, a KD value of 5.0×10 -8 M or less, a KD value of 2.5×10 -8 M or less, a KD value of 1.0×10 -8 M or less, a KD value of 5.0×10 -9 M or less, a KD value of 2.5×10 -9 M or less, a KD value of 1.0×10 -9 M or less, a KD value of 5.0×10 -10 M or less, a KD value of 2.5×10 -10 M or less, a KD value of 1.0×10 -10 M or less, a KD value of 1.0×10 -10 M or less, a KD value of 5.0×10 -10 showing a KD value of 1.0×10 (2) Inhibiting CpG-A stimulation that promotes cytokine IFNα release from human PBMC cells, e.g., as a result, the production of IFNα by target cells is reduced 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%; (3) Promoting the intracellular translocation of BDCA2 from the cell surface when binding to BDCA2, e.g., as a result, 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 are intracellularly translocated Provided is an isolated anti-BDCA2 antibody or antigen-binding fragment thereof having one or more of the following.

[0024] In some embodiments, the antibody of the present invention is a monoclonal antibody.

[0025] The present invention also provides a multispecific antibody comprising the light chain variable region and the heavy chain variable region of the antibody described herein or an antigen-binding fragment thereof.

[0026] The present invention also provides a single-chain antibody comprising the light chain variable region and the heavy chain variable region of the antibody described herein or an antigen-binding fragment thereof.

[0027] The present invention also provides an immune complex comprising an antibody or an antigen-binding fragment thereof as described herein conjugated to a therapeutic or diagnostic agent, preferably, the therapeutic or diagnostic agent is an anti-inflammatory agent or an immunosuppressive agent.

[0028] In a further aspect, the present invention provides a polynucleotide molecule encoding the anti-BDCA2 antibody or an antigen-binding fragment thereof described herein.

[0029] In a further aspect, the present invention provides an expression vector comprising the polynucleotide molecule described herein, preferably, the vector is a eukaryotic expression vector.

[0030] In a further 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.

[0031] In yet another aspect, the present invention provides a method for producing an anti-BDCA2 antibody or an antigen-binding fragment thereof described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof in a host cell described herein under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0032] In a further aspect, the present invention provides a pharmaceutical composition comprising an anti-BDCA2 antibody or an antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier or excipient.

[0033] In a further aspect, the present invention provides a pharmaceutical combination comprising an antibody or an antigen-binding fragment thereof as described herein, or a pharmaceutical composition, together with one or more additional therapeutic agents.

[0034] In yet another aspect, the present invention provides a method for inhibiting cytokine IFNα release from immune cells of a subject, the method comprising contacting the immune cells of the subject with an anti-BDCA2 antibody or an antigen-binding fragment thereof or a pharmaceutical composition described herein, preferably, the immune cells are PBMC cells stimulated by CpG-A.

[0035] In a further aspect, the present invention provides the use of an antibody or an antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein, in the manufacture of a medicament for the treatment and / or prevention of BDCA2-mediated diseases, preferably, the disease is an inflammatory disease, more preferably, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis.

[0036] In a further aspect, the present invention provides an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein for use in the treatment and / or prevention of BDCA2-mediated diseases, preferably, said disease is an inflammatory disease, more preferably, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, cutaneous lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0037] In yet another aspect, the present invention provides a method of treating and / or preventing a BDCA2-mediated disease or disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein, preferably, said disease is an inflammatory disease, more preferably, the inflammatory disease is selected from the group consisting of systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0038] In a further aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein, preferably, further comprising a dosing device.

[0039] In yet another aspect, the present invention provides a method of detecting the presence of BDCA2 in a sample using an antibody or antigen-binding fragment thereof as described herein or a detection composition containing an antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0040]

Figure 1-1

Figure 1-2

Figure 1-3

Figure 2-1

Figure 2-2

Figure 2-3

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 4-3

Figure 5-1

Figure 5-2

Figure 6-1

Figure 6-2

Figure 6-3

Figure 7

Mode for Carrying Out the Invention

[0041] Detailed Description of the Invention Definitions In the practice of the present invention, unless otherwise specified, conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are used, which are within the scope of the skills in the art.

[0042] To make the present invention more easily understandable, specific technical terms and scientific terms are specifically defined below. Technical terms and scientific terms or expressions used in this specification have the meanings generally understood by those skilled in the technical field to which the present invention pertains, unless clearly defined elsewhere in this specification. Those skilled in the art can refer specifically, at least in part, to Current Protocols in Molecular Biology (Ausubel) regarding the definitions and terms in the technical field. The abbreviations of amino acid residues follow the standard three-letter code and / or one-letter code used in the technical field to refer to one of the 20 commonly used L-amino acids. As used herein, including in the claims, the singular form includes the corresponding plural form unless the context clearly indicates otherwise.

[0043] When the term "about" is used with a numerical value, it means a numerical value within a range having a lower limit 5% less than the specified numerical value and an upper limit 5% greater than the specified numerical value, including, but not limited to, ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for carrying out the disclosed method.

[0044] 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.

[0045] As used herein, the term "or" should be understood to have the same meaning as "and / or" defined above. For example, "or" or "and / or" is inclusive when inserted into a list of items, i.e., it should be interpreted as including at least one of the plurality of elements or list of elements, or two or more, or optionally an additional item not in the list. Only terms that clearly indicate otherwise, such as "only one of", "exactly one of", or "consisting of" used in the claims, refer to only one number or only one element in the list.

[0046] As used herein, the terms "a" and "an" are to be understood to mean "at least one" unless the context clearly dictates otherwise.

[0047] As used herein, the term "BDCA2" refers to the type II C-type lectin BDCA2 that is specifically expressed on pDCs, and consists of a C-terminal signal extracellular sugar chain recognition domain (CRD), a transmembrane region from the asparagine residue at position 45 to the isoleucine residue at position 213, and an N-terminal short cytoplasmic tail (without a signaling motif). BDCA2 transmits intracellular signals via the associated transmembrane adapter FcεRIγ. When BDCA2 is bound via an antibody, spleen tyrosine kinase (SYK) is recruited to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcRI. Activation of Syk results in activation of B cell linker (BLNK), Bruton's tyrosine kinase (BTK), and phospholipase C2 (PLC2), thereby mobilizing Ca 2+ is mobilized.

[0048] The term "BDCA2" includes variants, isoforms, species homologs, or BDCA2s of other species of human BDCA2, and analogs having at least one common epitope of BDCA2. Unless otherwise specified, the term includes full-length BDCA2 that has not been processed as well as any form of BDCA2 resulting from intracellular processing. This term includes "full-length", unprocessed BDCA2, as well as any form of BDCA2 resulting from intracellular processing, or any fragment thereof, such as a splice variant or allelic variant. In one embodiment, BDCA2 refers to the full-length of human or cynomolgus monkey BDCA2, or a fragment thereof (such as its mature fragment lacking the signal peptide).

[0049] The term "immune response" refers to, for example, the actions of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules including antibodies, cytokines, and complements by the aforementioned cells or the liver. By this action, selective damage, destruction, or elimination from the human 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 is brought about.

[0050] The term "human plasmacytoid dendritic cells (pDC)" refers to a special population of bone marrow-derived cells that secrete type I interferons (IFN) in response to Toll-like receptor (TLR) ligands. pDC play an important role in cellular immunity and are involved in the induction and priming of immune responses, as well as antigen tolerance. Unlike normal DC, pDC acquire antigens, for example, through receptor-mediated endocytosis. Type I interferons produced by pDC activated by pattern recognition receptors may also present antigens, thereby linking innate and adaptive immune responses. At the same time, overactivation of pDC can have an adverse effect on the immune response process and may, for example, result in autoimmune diseases.

[0051] The term "signaling pathway" or "signaling activity" refers to a biochemical causal relationship typically initiated by protein-protein interactions, such as the binding of a growth factor to its receptor, which results in the signal being transmitted from one part of the cell to another part of the same cell. Generally, transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that result in signal transduction. The final process typically involves nuclear events, thereby bringing about changes in gene expression.

[0052] The terms "active" or "bioactive", or the terms "biological property" or "biological characteristic" are used interchangeably herein and are not limited, but include 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 immunogenicity of the antibody. Other distinguishable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., typically cross-reactivity with non-human homologs of the target peptide, or cross-reactivity with other proteins or tissues), and the ability to maintain high expression levels of proteins in mammalian cells. The aforementioned properties or characteristics are observed, determined or evaluated using techniques well known in the art, and such techniques include, but are not limited to, ELISA, FACS or BIACORE plasmon resonance assays, unrestricted in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections of different origins including human, primate, or any other origin.

[0053] The term "antibody" refers to any form of antibody having the desired biological activity. Thus, this term 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.

[0054] The term "isolated antibody" refers to the purified state of a binding compound, in which case the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cellular debris and growth media. The term "isolated" does not mean that such substances are completely absent or that water, buffer, or salt is absent, as long as they are not present in an amount that significantly interferes with the experimental or therapeutic use of the binding compounds described herein.

[0055] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for natural mutations that may be present in trace amounts. Monoclonal antibodies are highly specific, whereas conventional (polyclonal) antibody preparations generally contain large amounts of antibodies against (or specific for) different epitopes. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.

[0056] The term "full-length antibody", when present in nature, refers to an immunoglobulin molecule containing at least four peptide chains, with 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 as VH herein) and a heavy chain constant region (abbreviated as CH herein). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions with hypervariable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0057] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of the antibody that retain at least some of the binding specificities of the parent antibody. Examples of antibody binding fragments include, but are not limited to, 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. In some preferred embodiments of the present invention, the antigen-binding fragment of the present invention is Fab, Fab’, F(ab’) 2and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. The binding fragment or derivative generally retains at least 10% of its antigen-binding activity when expressed in molar concentration. 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 contain conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0058] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment containing the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. The Fv polypeptide generally further contains a polypeptide linker between the VH domain and the VL domain, whereby the scFv can form the structure desired for antigen binding.

[0059] The term "domain antibody" is an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region. In certain cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of the bivalent domain antibody may target the same antigen or different antigens.

[0060] The term "bivalent antibody" contains two antigen-binding sites. In certain cases, both binding sites have the same antigen specificity. However, the bivalent antibody may be bispecific.

[0061] The term "diabody" refers to small antibody fragments having two antigen-binding sites, and these fragments contain a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). These domains are forced to pair with complementary domains on another chain by using a linker that is not short in order to enable pairing between the two domains on the same chain, creating two antigen-binding sites.

[0062] In the present disclosure, the terms "mouse antibody" or "hybridoma antibody" are monoclonal antibodies against BDCA2 prepared according to the knowledge and techniques in the art. The BDCA2 antigen was injected into a test subject during preparation, and hybridomas expressing antibodies having the desired sequences or functional characteristics were isolated. Hybridoma technology is by fusing two types of cells while maintaining the main characteristics of both. These two types of cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively. The main characteristic of mouse spleen cells (B lymphocytes) immunized with a specific antigen is their antibody-secreting function, not in vitro continuous culture, and mouse myeloma cells divide under culture conditions and can proliferate infinitely, that is, have so-called immortality. Only the hybrid cells obtained by fusing B cells and myeloma cells have the ability to continue culturing under the action of a selective medium and can create cell clones characterized by both antibody secretion and maintenance of cell immortality. In some embodiments, the present invention immunizes a mouse with the BDCA2 protein and fuses the spleen cells and myeloma cells of the mouse to obtain hybridoma cells capable of expressing positive antibodies.

[0063] The term "chimeric antibody" refers to an antibody that has the variable domain of a first antibody and the constant domain of a second antibody, where the first and second antibodies are derived from different species. Generally, the variable domain is obtained from an antibody such as a rodent antibody ("parent antibody"), while the constant domain sequence is obtained from a human antibody. Thus, the resulting chimeric antibody is less likely to induce a harmful immune response in human subjects compared to the parent rodent antibody. In some embodiments of the present invention, the rodent is a mouse or a rat. In some preferred embodiments of the present invention, the affinity of the chimeric antibody for the antigen is equal to or nearly equal to that of the parent mouse antibody.

[0064] The term "humanized antibody" refers to a form of antibody that contains sequences derived from both human antibodies and non-human (e.g., mouse, rat) antibodies. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, where 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 of human immunoglobulin sequence. A humanized antibody may optionally contain at least a portion of the human immunoglobulin constant region (Fc).

[0065] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycans produced by mice, mouse cells, or hybridomas derived from mouse cells. Similarly, the term "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat glycans if produced by rats, rat cells, or hybridomas derived from rat cells. Similarly, the term "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0066] An "isotype" antibody refers to a class of antibodies produced by heavy chain constant region genes (e.g., IgM, IgE, IgG, such as IgG1, IgG2, or IgG4). An isotype also includes a modified form of one of these classes, where the modification was made to alter the Fc function, for example, to enhance or reduce effector function or binding to Fc receptors.

[0067] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This 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 follows the EU numbering system, also called the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0068] The term "epitope" refers to a determinant of a protein to which an antibody can specifically bind. Epitopes usually consist of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural properties as well as specific charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent while binding to the latter is not.

[0069] As used herein, the term "cross-reactivity" refers to the binding of an antigen to a fragment of the same target molecule of human, monkey, and / or mouse (mouse or rat) origin. "Cross-reactivity" should be understood as an interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a cognate molecule (e.g., BDCA2) expressed heterologously. The cross-reactivity specificity of a monoclonal antibody that recognizes human BDCA2, monkey, and / or mouse BDCA2 (mouse or rat) can be determined by FACS analysis.

[0070] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ), and the equilibrium dissociation constant is the ratio of the dissociation and association rate constants (k dis and k on ), respectively. Affinity can be measured by common methods known in the art. In some embodiments of the present invention, the affinity, for example, the affinity between an antibody of the present invention and an antigen, is measured using surface plasmon resonance (SPR) technology. In some preferred embodiments of the present invention, one specific method for measuring affinity is the BIAcore method described herein.

[0071] The term "does not bind" to a protein or cell means that it does not bind to the protein or cell at all, or does not bind to the protein or cell with high affinity, i.e., it binds to the protein or cell with a K -6 of 1.0×10 -5 M or more, more preferably 1.0×10 -4 M or more, 1.0×10 -3 M or more, more preferably 1.0×10 -2 M or more, 1.0×10 D M or more.

[0072] The term "high affinity" for an IgG antibody means 1.0×10 -6 M or less, preferably 5.0×10-8 Less than M, more preferably 1.0×10 -8 Less than M, 5.0×10 -9 Less than M, more preferably 1.0×10 -9 K less than M D is meant. The "high affinity" binding to other antibody isotypes may vary. For example, the "high affinity" binding to the IgM isotype is 10 -6 Less than M, preferably 10 -7 Less than M, more preferably 10 -8 K less than M D is meant.

[0073] The terms "antibody-dependent cell-mediated cytotoxicity", "antibody-dependent cellular cytotoxicity" or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells whose cell membrane surface antigens are bound by an antibody.

[0074] 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 classical complement pathway, including the formation of the membrane attack complex and the lysis of target cells.

[0075] 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) in single-stranded or double-stranded form and polymers thereof. Unless explicitly limited otherwise, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to natural 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 of synthesizing said mRNA molecules, and methods of delivering therapeutic proteins in vivo). A particular nucleic acid sequence also implicitly encompasses, unless otherwise specified, its conservatively modified variants (e.g., degenerate codon substitutions), alleles, homologous molecular species, SNPs, and complementary sequences, in the same manner as an explicitly recited sequence. 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 a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

[0076] "Construct" refers to any recombinant polynucleotide molecule (plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule) of any origin that contains one or more polynucleotides linked in a functionally operative manner (i.e., operably linked) and is capable of integration with or autonomous replication from a genome. In some preferred embodiments of the invention, the recombinant construct comprises a polynucleotide of the invention operably linked to a transcriptional initiation regulatory sequence that drives and / or directs transcription of the polynucleotide of the invention in a host cell. Both heterologous promoters and non-heterologous (i.e., endogenous) promoters can be used to drive and / or direct the expression of the polynucleotide of the invention.

[0077] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introduction of 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 ligated. Another type of vector is a viral vector, into which additional DNA can be ligated within the viral genome. Certain vectors are capable of autonomous replication within the introduced host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. Furthermore, certain vectors can direct the expression of an operably linked gene. Such vectors are referred to herein as "expression vectors".

[0078] 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. An expression vector contains one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and, if necessary, provide amplification in the host. In a preferred embodiment of the present invention, the expression vector of the present invention comprises the construct of the present invention and / or the polynucleotide of the present invention.

[0079] The manipulations of "activation", "stimulation" and "treatment" with respect to a cell or receptor have the same meaning and, unless the context indicates otherwise or is explicitly stated, for example, a cell or receptor is activated, stimulated or treated with a ligand. "Ligand" includes natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compounds. "Ligand" also includes small molecules such as peptidomimetics of cytokines and peptidomimetics of antibodies. "Activation" can refer to the activation of a cell that is regulated by internal mechanisms as well as external or environmental factors. A "response", e.g., of a cell, tissue, organ, or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion, or movement within a biological compartment, rate of gene expression, or state of differentiation), which change is related to activation, stimulation, or treatment or to internal mechanisms such as genetic programming.

[0080] As used herein, the term "BDCA2-mediated disease" refers to a disease in which BDCA2 is involved in the onset, progression, or course of transition, for example, activation of BDCA2 (e.g., abnormal activation or over-activation), and this involvement of BDCA2 directly or indirectly affects at least one of the following: onset / occurrence of the disease, increase / deepening of pathological changes of the disease, expansion of the affected site / scope of the disease, deterioration of physical damage caused by the disease, increase in pain caused by the disease, sensitivity / resistance of the disease to treatment means, decrease in the self-healing tendency of the disease, and deterioration of the prognosis of the disease. In some embodiments of the present invention, the BDCA2-mediated disease is an inflammatory disease, and in some specific embodiments of the present invention, the BDCA2-mediated disease is systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and / or polymyositis.

[0081] As used herein, the term "treating" or "treatment" of any disease or disorder, in one embodiment, refers to improving the disease or disorder (i.e., slowing, stopping, or reducing the progression of at least one of the disease or its clinical symptoms). In another embodiment, "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be recognizable to the patient. In another embodiment, "treatment" refers to modulating the disease or disorder either physically (e.g., stabilization of recognizable symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for evaluating the treatment and / or prevention of diseases are generally known in the art unless specifically described herein.

[0082] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as 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 cynomolgus monkeys or those derived from cynomolgus monkeys.

[0083] "Combined" administration of one or more additional therapeutic agents includes co-administration and sequential administration in any order.

[0084] "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 that is effective to prevent or ameliorate one or more symptoms of a disease or condition, or the onset of a disease or condition, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject. Also, a therapeutically effective dose refers to an amount of an antibody or antigen-binding fragment thereof sufficient to effect an improvement in symptoms, e.g., an amount that treats, cures, prevents, or ameliorates a related condition, or increases the rate of treatment, cure, prevention, or amelioration of such a condition. A therapeutically effective dose refers to the case where the active ingredient alone is administered to a subject when administered alone. A therapeutically effective dose refers to the total amount of the active ingredient that produces a therapeutic effect when administered in combination, sequentially, or simultaneously. The effective amount of a therapeutic agent results in an increase of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% of the diagnostic criteria or parameters.

[0085] "Pharmaceutically acceptable carrier" refers to a non-toxic component to a subject other than the active ingredient in a pharmaceutical preparation or composition. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0086] Anti-BDCA2 antibody In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2. The terms "anti-BDCA2 antibody", "anti-BDCA2", "BDCA2 antibody" or "antibody that binds to BDCA2" refer to an antibody that can bind to the BDCA2 protein or a fragment thereof with sufficient avidity such that the antibody can be used as a diagnostic and / or therapeutic agent when targeting BDCA2.

[0087] In some embodiments, the antibody of the present invention binds to human or cynomolgus monkey BDCA2 protein. In some embodiments, the antibody of the present invention binds to CHOK1-human BDCA2 cells or 293F-cynoBDCA2 cells. In some embodiments, the antibody of the present invention inhibits CpG-A from stimulating human PBMC cells to release the cytokine IFNα. In some embodiments, the antibody of the present invention has greater intracellular trafficking ability.

[0088] To produce the antibody of the present invention, any suitable method for producing an antibody can be used. Any suitable form of BDCA2 can be used as an immunogen (antigen) for producing an antibody. By way of example, but not limitation, any BDCA2 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells that produce a monoclonal anti-BDCA2 antibody of mouse origin can be produced by methods well known in the art.

[0089] Antibodies derived from rodents such as mice, when used in vivo as therapeutic agents, may cause undesirable immunogenicity of the antibody. With repeated use, an immune response to the therapeutic antibody occurs in the human body. Such an immune response can at least result in loss of therapeutic effect and, in severe cases, may cause fatal anaphylaxis. One way to reduce the immunogenicity of rodent antibodies is the production of chimeric antibodies, in which case the murine variable region is fused to the human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, since the intact rodent variable region is retained in chimeric antibodies, there is still a possibility of causing harmful immunogenicity in patients. To further minimize rodent sequences, grafting of the complementarity-determining region (CDR) loops of the rodent variable domain into the human framework (i.e., humanization) is used (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 antibodies, chimeric antibodies and / or human-derived antibodies of the present invention for the antigen human BDCA2 or cynomolgus BDCA2 is 1.0×10 -6 KD value of M or less, 5.0×10 -7 KD value of M or less, 2.5×10 -7 KD value of M or less, 1.0×10 -7 KD value of M or less, 5.0×10 -8 KD value of M or less, 2.5×10 -8 KD value of M or less, 1.0×10 -8 KD value of M or less, 5.0×10 -9 KD value of M or less, 2.5×10 -9 KD value of M or less, 1.0×10 -9 KD value of M or less, 5.0×10 -10 KD value of M or less, 2.5×10 -10 KD value of M or less, 1.0×10 -10It is embodied as a KD value of less than M.

[0090] The amino acid sequences of the light and heavy chain variable regions and CDRs of the anti-BDCA2 mouse antibody of the present invention are shown in Table 2.

[0091] In some embodiments, the chimeric or humanized antibody of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody. DNA encoding the heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma using standard molecular biology techniques and manipulated to include non-mouse (e.g., human) immunoglobulin sequences.

[0092] In some embodiments, the hybridoma-derived immunoglobulin heavy and light chain variable regions are operably linked to human IgG constant regions using methods known in the art (e.g., U.S. Patent No. 4 to Cabilly et al.) to obtain chimeric heavy and light chains, thereby producing the chimeric BDCA2 antibody of the present invention. In some embodiments, the chimeric antibody of the present invention includes a constant region selected from any human IgG subtype such as IgG1, IgG2, IgG3, IgG4, preferably IgG1.

[0093] In some embodiments, the chimeric BDCA2 antibody of the present invention can be obtained from an expression cell that is "mixed and compatible" transfected with a chimeric light chain expression plasmid and a chimeric heavy chain expression plasmid, and the BDCA2 binding of such "mixed and compatible" antibodies can be tested using the above binding assay and other conventional binding assays (e.g., ELISA).

[0094] The exact amino acid sequence boundaries of the CDRs of the variable regions of the antibodies of the present invention can be determined using any of several well-known protocols, including the 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 the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)) based on the variability of the antibody sequence, 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 number of crystal structures.

[0095] The boundaries of the CDRs of the antibodies of the present invention can be determined by those skilled in the art according to any protocol in the art (e.g., different assignment systems or combinations), unless otherwise specified.

[0096] It should be noted that the CDR boundaries 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 defining an antibody having a specific CDR sequence as defined herein, the scope of the antibody includes antibodies in which the variable region sequence contains the specific CDR sequence, but due to the application of different schemes (e.g., different assignment systems or combinations), the claimed CDR boundaries are different from the specific CDR boundaries defined herein.

[0097] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although the CDRs are different for each antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimal overlapping region can be determined using at least two of the Kabat method, the Chothia method, the AbM method, the Contact method, and the North method, thereby providing a "minimal binding unit" for antigen binding. The minimal binding unit may be part of a CDR. As will be apparent to those skilled in the art, the remaining residues of the CDR sequence can be determined by the structure of the antibody and protein folding. Accordingly, any variant of the CDRs shown herein is also contemplated by the present invention. For example, in a variant of one CDR, the amino acid residues of the minimal binding unit may remain unchanged, and the remaining CDR residues according to the Kabat or Chothia definition may be replaced with conserved amino acid residues.

[0098] Mixture pairs of various chimeric heavy and light chain expression plasmids are used for transfection of the expression cells, which are anti-BDCA2 chimeric antibodies.

[0099] The humanized antibodies described herein may be those in which mouse CDR regions are inserted into human germline framework regions using methods known in the art. See U.S. Patent No. 5,225,539 to Winter et al. and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0100] The light and heavy chain amino acid sequences of the anti-BDCA2 humanized antibodies Hu033-03, Hu033-20, and Hu005-04 of the present invention are shown below. The sequences outside the variable regions of the LC and HC of the remaining humanized antibodies are identical to the sequences of the corresponding Hu033-03, Hu033-20, and Hu005-04.

[0101] Hu033-03-HC-IgG1: SEQ ID NO: 183 (SEQ ID NOS: 52, 39, and 53 for HCDR1 to HCDR3, respectively) TIFF2025518061000001.tif44164TIFF2025518061000002.tif6164Hu033-03-LC: SEQ ID NO: 186 (SEQ ID NOS: 54, 55, and 42 for LCDR1 to LCDR3, respectively) TIFF2025518061000003.tif25164Hu033-20-HC-IgG1: SEQ ID NO: 184 (SEQ ID NOS: 52, 39, and 53 for HCDR1 to HCDR3, respectively) TIFF2025518061000004.tif48164Hu033-20-LC: SEQ ID NO: 187 (SEQ ID NOS: 54, 55, and 42 for LCDR1 to LCDR3, respectively) TIFF2025518061000005.tif23164Hu005-04-HC-IgG1: SEQ ID NO: 185 (SEQ ID NOS: 11, 12, and 13 for HCDR1 to HCDR3, respectively) TIFF2025518061000006.tif47164Hu005-04-LC: SEQ ID NO: 188 (SEQ ID NOS: 86, 15, and 16 for LCDR1 to LCDR3, respectively) TIFF2025518061000007.tif25164

[0102] In some embodiments, the amino acid mutations include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-BDCA2 antibodies of the present invention or their antigen-binding fragments are mutated by amino acid deletions, insertions, or substitutions, but still have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above antibodies, particularly in the CDR regions shown by the above sequences. In some embodiments, the antibodies of the present invention have 1, 2, 3, 4, or 5 or fewer amino acids mutated by amino acid deletions, insertions, or substitutions in the CDR regions when compared with the CDR regions shown by specific sequences. In some embodiments, the antibodies of the present invention have 1, 2, 3, 4, or 5 or fewer amino acids mutated from amino acid deletions, insertions, or substitutions in the framework regions when compared with the framework regions within specific sequences.

[0103] In some embodiments, the Fc regions of the inventors' humanized antibodies Hu033-03 and Hu033-20 have mutations such as S239D / I332E, G236A / S239D / I332E, and G236A / I332E, resulting in the following antibodies: Hu033-03-T1, Hu033-03-T2, Hu033-03-T3, Hu033-20-T1, Hu033-20-T2, Hu033-20-T3, and their light and heavy chain amino acid sequences are shown below. Hu033-03-T1-HC: SEQ ID NO: 191 TIFF2025518061000008.tif48164Hu033-03-T2-HC: SEQ ID NO: 192 TIFF2025518061000009.tif48164Hu033-03-T3-HC: SEQ ID NO: 193 TIFF2025518061000010.tif48164Hu033-03-T1 / T2 / T3-LC: SEQ ID NO: 186 TIFF2025518061000011.tif26164Hu033-20-T1-HC: SEQ ID NO: 194 TIFF2025518061000012.tif47164Hu033-20-T2-HC: SEQ ID NO: 195 TIFF2025518061000013.tif48164Hu033-20-T3-HC: SEQ ID NO: 196 TIFF2025518061000014.tif48164Hu033-20-T1 / T2 / T3-LC: SEQ ID NO: 187 TIFF2025518061000015.tif23164

[0104] 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 those in a reference amino acid sequence when the amino acid sequences are aligned (with gaps introduced if necessary) so as to achieve the maximum percent sequence identity and conservative substitutions are not considered as part of the sequence identity. Sequences can be aligned using various methods in the art to determine the percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0105] Thus, in some embodiments, the antibodies of the invention comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any combination in Table A below.

[0106] TIFF2025518061000016.tif201162

[0107] In some embodiments, the antibodies of the invention comprise VH and VL as shown in any combination in Table B below.

[0108] TIFF2025518061000017.tif116162

[0109] In some embodiments, the antibodies of the invention comprise VH and VL shown in any combination in Table C below.

[0110] TIFF2025518061000018.tif201162

[0111] In some embodiments, the BDCA2 antibody is an IgG antibody, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof as described in the following sections.

[0112] In some embodiments, the Fc region variant can be made by introducing one or more amino acid modifications into the Fc region of the antibodies provided herein. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0113] In some embodiments, it may be desirable to generate a cysteine-engineered antibody, e.g., a "chioMAb" in which one or more residues of the antibody are replaced with cysteine residues.

[0114] In some embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known in the art and readily available. Suitable moieties for derivatizing the antibody 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 (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0115] Expression of the antibody In yet another aspect, the invention provides a polynucleotide molecule encoding an anti-BDCA2 antibody or an antigen-binding fragment thereof described herein. The polynucleotide molecule can include a polynucleotide molecule encoding the amino acid sequence of the variable region of the light chain and / or the heavy chain of the antibody, or a polynucleotide molecule comprising the amino acid sequence encoding the light chain and / or the heavy chain of the antibody.

[0116] In some embodiments, the polynucleotide molecules encoding the antibodies of the invention include those 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 corresponding coding regions of the CDRs shown in the above sequences.

[0117] In a further 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 included in one or more expression vectors.

[0118] In a further aspect, the present invention provides a host cell comprising a polynucleotide molecule as described herein or an expression vector as described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0119] In yet another aspect, the present invention provides a method for producing an anti-BDCA2 antibody or an antigen-binding fragment thereof as described herein, the method comprising expressing the antibody or the antigen-binding fragment thereof under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof in the host cell as described herein, and recovering the expressed antibody or the antigen-binding fragment thereof from the host cell.

[0120] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including several immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

[0121] In one embodiment, the present invention provides a method for producing an anti-BDCA2 antibody, the method comprising culturing host cells for a period sufficient to allow expression of the antibody in the host cells when the expression vector is introduced into mammalian host cells, or more preferably, to secrete the antibody into the culture medium in which the host cells are grown, thereby enabling production of the antibody.

[0122] The antibody can be recovered from the culture medium using standard protein purification methods. Antibody molecules produced as described herein can be purified by known state-of-the-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, etc. These will be readily apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any one of several well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0123] Antibodies expressed by different cell lines or expressed in transgenic animals may have different glycosylation patterns. 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 antibody. Also, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have greater potency than their fucosylated counterparts in vitro and in vivo, and their sugar structures are normal components of native human serum IgG and are less likely to cause immunogenicity.

[0124] Pharmaceutical Compositions and Preparations In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-BDCA2 antibody or an 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 will be understood that the anti-BDCA2 antibodies or pharmaceutical compositions provided by the present invention can be formulated with suitable carriers, excipients, and co-administered formulations with other agents, thereby providing improvements in introduction, delivery, tolerability, and the like.

[0125] The term "pharmaceutical composition" refers to a preparation that allows the bioactivity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0126] A pharmaceutical formulation containing the anti-BDCA2 antibody described herein, preferably in the form of an aqueous solution or a lyophilized formulation, can be prepared by mixing the anti-BDCA2 antibody of the present invention having the desired purity with one or more optional pharmaceutically acceptable excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).

[0127] The pharmaceutical composition or formulation of the present invention may further comprise one or more additional active ingredients, which are those necessary for the particular indication being treated and preferably active ingredients having complementary activities that do not adversely affect each other. In some embodiments, the additional active ingredients are immune checkpoint inhibitors, growth inhibitors, and are present in combination in amounts effective for the intended use. In some embodiments, the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide molecule encoding an anti-BDCA2 antibody.

[0128] In a further aspect, the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0129] In a further aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a polynucleotide molecule described herein, an expression vector described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0130] Pharmaceutical uses and methods of treatment Any of the anti-BDCA2 antibodies or corresponding immune complexes provided herein can be used in a method of treatment. Also, when "antibody" is discussed, it is understood to include compositions containing the antibody. The anti-BDCA2 antibodies of the present invention can be used in a therapeutically effective amount or a prophylactically effective amount in any of the methods of treatment or prevention described in embodiments of the present invention.

[0131] In a further aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein in the manufacture of a medicament for the treatment and / or prevention of BDCA2-mediated diseases, preferably, the disease is an inflammatory disease, more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0132] In a further aspect, the present invention provides an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein for use in the treatment and / or prevention of BDCA2-mediated diseases, preferably, said disease is 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.

[0133] In yet another aspect, the present invention provides a method of treating and / or preventing a BDCA2-mediated disease or disorder, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof as described herein, a pharmaceutical composition as described herein, or a pharmaceutical combination as described herein, preferably, said disease is an inflammatory disease, more preferably, the inflammatory disease is selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis and polymyositis.

[0134] In some embodiments, the modes of administration of the present invention include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraarterial administration, intra-articular (e.g., to the joints of arthritis) administration, inhalation administration, aerosol delivery, or topical administration.

[0135] The term "treatment" refers to a clinical intervention that attempts to change the natural course of a disease in an individual being treated. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of undesirable or direct or indirect pathological consequences of a disease, reduction in the rate of progression of a disease, amelioration or palliation of a condition, and remission or improvement of the prognosis. The antibodies of the present invention can, in one or more aspects, reduce the severity of a condition, i.e., exhibit their therapeutic effect. In certain embodiments, one or more of the following are manifested: extension of the average lifespan (survival period) of a patient, delay in the progression of a disease, and reduction in the need for medical treatment.

[0136] The present invention also provides for the co - administration of 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 may be used alone in a therapy or in combination with other therapeutic agents. In some embodiments, the antibodies of the present invention are co - administered with at least one additional therapeutic agent.

[0137] Methods for diagnosis and detection 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 quantitative or qualitative detection. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample includes cells or tissues. In certain embodiments, BDCA2 is human BDCA2 or cynomolgus BDCA2. The method includes 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 or absence of a complex or binding signal resulting from the binding of the antibody or antigen-binding fragment to BDCA2. For use in detection, the antibodies or antigen-binding fragments thereof described herein can be labeled to indicate whether a complex has formed. In certain embodiments, the method can be an in vitro method or an in vivo method.

[0138] In some embodiments, BDCA2 is detected before treatment, for example, before the start of treatment or before a particular treatment after a treatment interval. In one embodiment, an anti-BDCA2 antibody or antigen-binding fragment thereof for use in a diagnostic or detection method is provided.

[0139] The positive advances of the present invention are as follows: The BDCA2-targeting antibodies of the present invention include one or more of the following advantages: 1. The antibodies of the present invention show stronger active binding to CHOK1-human BDCA2 cells and / or 293F-cynoBDCA2 cells than the prior art anti-BDCA2 antibody BIIB059; 2. The antibodies of the present invention mediate stronger inhibition of the release of the cytokine IFNα from human PBMC cells stimulated with CpG-A than the prior art anti-BDCA2 antibody BIIB059; 3. The antibody of the present invention brings about a high intracellular translocation efficiency in BDCA2-expressing cells; 4. The humanized antibody of the present invention has a higher affinity for the human BDCA2 antigen than the prior art anti-BDCA2 antibody BIIB059. 5. In non-primates such as mice, rats, and rabbits, there is no expression of the BDCA2 gene, and the main diseases (such as cutaneous lupus erythematosus and systemic lupus erythematosus) treated with BDCA2 antibodies have not been successfully established in pharmacodynamic test models in primates. According to in vitro and clinical trials, the antibody BIIB059 shows an effect of inducing intracellular translocation of BDCA2 and inhibiting the type I interferon secretion function of PBMCs in in vitro experiments, and shows a symptom-relieving effect on cutaneous lupus erythematosus and systemic lupus erythematosus in clinical trial studies (Werth VP et al. N Engl J Med 2022; 387: 321-31. Furie RA et al. N Engl J Med 2022; 387: 894-904). Therefore, it can be presumed that the anti-BDCA2 antibody of the present invention has a better therapeutic effect on the above-mentioned diseases or disorders clinically.

[0140] The present invention includes all combinations of the specific embodiments described above. The further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided below. The detailed description and specific examples illustrate the preferred embodiments of the present invention, but it should be understood that various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description, and these descriptions and examples are provided for illustrative purposes only. All publications, patents, and patent applications cited herein, including the citations, are hereby incorporated by reference in their entirety as part of this specification for any purpose. The compounds of the present invention can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, those formed by combining them with other methods, and their equivalents well known to those skilled in the art. The preferred embodiments include, but are not limited to, the examples of the present invention.

[0141] In the present invention, at least the following abbreviations are used: His-tag means histidine tag; Fc-tag means crystallizable fragment tag; ECD means extracellular domain; PEI means polyethyleneimine; BSA means bovine serum albumin; PBS means phosphate buffered saline; FBS means fetal bovine serum; CFSE means carboxyfluorescein diacetate succinimidyl ester; APC means allophycocyanin; NA-PE means phycoerythrin-labeled Neutravidin; PE means phycoerythrin; TMB means 3,3',5,5'-tetramethylbenzidine; HEPES means 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer; DTT means dithiothreitol.

Example

[0142] The present invention will be described by the following examples, but it is not intended to limit the present invention in any way. The present invention is described in detail herein, and its specific embodiments are also 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 present invention.

[0143] Example 1 Preparation of Recombinant Protein for Detecting Activity of Anti-BDCA2 Antibody The cDNA sequence encoding the extracellular domain (Asn45-Ile213) of human BDCA2 (hBDCA2 or human BDCA2) was synthesized by Genscript Corporation according to the Genbank reference sequence. The amplified fragment was cloned into a eukaryotic expression plasmid system (pCP-EF1a-CMV-FcεRIγ) containing a human-derived Fc (hIgG1) fragment that was independently constructed, using conventional cloning techniques, to generate a recombinant fusion protein expression plasmid, human BDCA2 Fc (pCP-EF1a-BDCA2-CMV-FcεRIγ). The human BDCA2 Fc recombinant protein (the Fc region is located within the BDCA2 extracellular domain, in the N-terminal direction, and is fused by a linker) was obtained using expression cell 293E for expression and a size exclusion chromatography column for purification, and was identified by polyacrylamide gel electrophoresis.

[0144] The cDNA sequence encoding the extracellular domain (Asn45-Ile212) of cynomolgus BDCA2 (Cyno BDCA2) was synthesized by Genscript Corporation according to the Genbank reference sequence, and a 6-His expression sequence was added to the N-terminus of the cDNA. This fragment was cloned into an independently constructed eukaryotic expression plasmid system (pCP-EF1a-CMV-FcεRIγ) using conventional cloning techniques to generate a recombinant fusion protein expression plasmid, Cyno BDCA2 His (pCP-EF1a-Cyno BDCA2-CMV-FcεRIγ). The Cyno BDCA2 His recombinant protein was obtained using expression cell 293E for expression and a size exclusion chromatography column for purification, and was then identified by polyacrylamide gel electrophoresis.

[0145] The concentrations, total amounts, and endotoxin contents of both proteins obtained were confirmed to be appropriate for subsequent applications.

[0146] The amino acid sequence of human BDCA2 Fc shown in SEQ ID NO: 189, and the NCBI accession number Q8WTT0 used as the protein sequence of human BDCA2, the amino acid sequence of Cyno BDCA2 His shown in SEQ ID NO: 190, and the NCBI accession number A0A2K5UWP4-1 used as the protein sequence of Cyno BDCA2 were used in the activity assay of the antibodies of the present invention.

[0147] The reference antibody BIIB059 sequence that specifically binds to BDCA2 was obtained from Patent US9902775B2. The heavy chain sequence corresponds to Patent SEQ ID NO: 4, and the light chain sequence corresponds to Patent SEQ ID NO: 3. It was produced using the conventional production method of recombinant antibodies.

[0148] Example 2 Construction of BDCA2-expressing cell line for detecting the activity of anti-BDCA2 antibodies The cDNA sequence encoding human BDCA2 was synthesized by Genscript Corporation according to the Genbank reference sequence. The amplified fragment was cloned into an independently constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro) using conventional cloning techniques to generate the lentiviral packaging plasmid Human BDCA2 (PLvx-EF1a-hBDCA2-CMV-hFcεRIγ-IRES-puro).

[0149] The cDNA sequence encoding cynomolgus BDCA2 was synthesized by Genscript Corporation according to the Genbank reference sequence. The amplified fragment was cloned into an independently constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro) using conventional cloning techniques to generate the lentiviral packaging plasmid Cyno BDCA2 (PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro).

[0150] Using 293T as the host cell, by the transient method, the plasmid PLvx-EF1 a-hBDCA2-CMV-hFcεRIγ-IRES-puro or PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro was introduced into 293T cells. At 48 hours and 72 hours after transfection, the cell culture supernatant containing the virus expressing human BDCA2 or Cyno BDCA2 was collected, concentrated to 1E8 EU / mL.

[0151] CHOK1 cells were pre-cultured until the logarithmic growth phase, then digested, seeded at 1E5 / well in a 6-well cell culture plate, and cultured overnight for adhesion. Next, the concentrated human BDCA2 virus was added to the cells in the pre-adhered 6-well plate at a gradually increasing infection dose (100 μl, 200 μl), the infected cells were screened with puromycin-containing medium, then the antigen expression level was measured by flow cytometry, and the cells were subcloned. When flow cytometry was performed using the reference antibody BIIB059, the results showed significant binding of BDCA2 compared to the control hlgG. Monoclonal clones showing high antigen expression were collected and expanded in culture to obtain cell lines.

[0152] 293F cells were pre-cultured until the logarithmic growth phase, then digested, seeded at 1E5 / well in a 6-well cell culture plate, and cultured overnight for adhesion. Next, the concentrated cyno BDCA2 virus was added to the cells in the pre-adhered 6-well plate at a gradually increasing infection dose (100 μl, 200 μl), the infected cells were screened with puromycin-containing medium, then the antigen expression level was measured by flow cytometry, and the cells were subcloned. When flow cytometry was performed using the reference antibody BIIB059, the results showed significant binding of BDCA2 compared to the control hlgG. Monoclonal clones showing high antigen expression were collected and expanded in culture to obtain cell lines.

[0153] Example 3 Preparation of Mouse Hybridoma Cells 3.1 Immunization of Mice and Detection of Serum Titers 3.1.1 Protocol for Mouse Immunization A total of three groups of mice (five mice in each group) were immunized via two immunization routes with a 14-day interval between immunizations. Group 1 of five Balb / c mice (purchased from SLAC, BALB / c, female, 8 weeks old), and Group 2 of five SJL mice (purchased from SLAC, BALB / c, female, 8 weeks old) were immunized five times using a biological (pCP-EF1a-BDCA2-CMV-FcεRIγ) + protein (human BDCA2 Fc) mixed protocol. The first four biological immunizations were at a dose of 4 μg each time, and the fifth protein immunization was at a dose of 25 μg / mouse. Group 3 of five SJL mice (purchased from SLAC, female, 8 weeks old) was immunized a total of three times with pure protein (human BDCA2 Fc), at 50 μg per time for the first immunization and 25 μg per time for the second immunization. See Table 1.

[0154]

Table 1

[0155] 3.1.2 Detection of Mouse Immunity Titer Serum samples of immunized mice were collected at multiple time points, and ELISA and FACS assays were performed respectively. Based on the results of multiple assays (with the last mouse serum assay (TB) as the main reference factor, detailed results not shown), a total of four mice were selected for two fusions: For Fusion #F0223, the two mice with the highest fluorescence intensity, Balb / c #9735 from Group G1 and SJL #9740 from Group G2, were selected; for Fusion #F0511, the two mice with the highest fluorescence intensity, SJL #9947 and SJL #9948 from Group G3, were selected.

[0156] 3.2 Cell Fusion Four days after the final boost administration, the mice were sacrificed, spleen cells were collected, crushed with physiological saline, and then lymphocyte concentrated suspensions were collected and mixed with mouse myeloma cells Sp2 / 0 by normal electrotransfer, and cell fusion using high-efficiency electrofusion was performed.

[0157] The fused cells were diluted in DMEM medium containing HT (hypoxanthine, thymidine), seeded at a certain ratio in a 96-well plate, and incubated overnight in an incubator at 37°C with 5% CO 2 24 hours later, 2 * DMEM medium containing HAT (hypoxanthine, methotrexate, and thymidine) was added to screen for cells that had successfully fused (hybridoma cells).

[0158] The composition of the DMEM complete medium was 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL penicillin and streptomycin + 1:100 OPI (oxaloacetic acid, pyruvic acid, and insulin), and the incubator conditions were 8% CO 2 at 37°C.

[0159] Example 4 Screening of mouse hybridoma cells and purification of anti-BDCA2 mouse antibody After culturing and screening the fused cells for about 10 days, the expression of the BDCA2 antibody in the supernatant was detected using an Acumen laser cell detector. The positive cells for detection were CHOK1-human BDCA2, the negative cells were CHOK1-blank, and the positive control antibody was reference antibody BIIB059. According to the Acumen results, positive clones were collected in a 24-well plate for expansion culture. After culturing for 3 days, the supernatant of the expansion culture in the 24-well plate was collected using the following methods: 1) FACS regarding the binding activity to CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells, 2) ELISA regarding the binding activity to human BDCA2 Fc protein and cynoBDCA2 His protein, 3) Detection of the inhibitory activity in the experiment where CpG-A stimulates the production of cytokine IFNα by PBMC (inhibiting the production of IFNα).

[0160] 189 hybridoma cells obtained from the fusion of F0223 were screened by the Acumen binding assay, and the secreted antibodies were able to specifically bind to hBDCA2. Furthermore, after scaling up, these 189 hybridoma cells that were positive for binding were re-screened in 24-well plates. As a result, the antibodies expressed by 28 hybridoma cells were able to bind to hBDCA2 and cyno BDCA2 in the ELISA assay, bind to hBDCA2 and cyno BDCA2 expressed on the cell surface by FACS, and it was shown that they could inhibit the CpG-induced IFNα secretion from PBMC cells in the IFNα blocking experiment. Subsequently, the 28 hybridoma cells were subcloned. 18 blocking monoclonal cell lines numbered mAb001 - mAb018 were screened in the subclone.

[0161] 168 hybridoma cells obtained from the fusion of F0511 were screened by the Acumen binding assay, and the secreted antibodies were able to specifically bind to hBDCA2. Furthermore, after scaling up, these 168 hybridoma cells that were positive for binding were re-screened in 24-well plates. As a result, the antibodies expressed by 35 hybridoma cells were able to bind to hBDCA2 and cyno BDCA2 in the ELISA assay, bind to hBDCA2 and cyno BDCA2 expressed on the cell surface by FACS, and it was shown that they could inhibit the CpG-induced IFNα secretion from PBMC cells in the IFNα blocking experiment. Subsequently, the 35 hybridoma cells were subcloned. 16 blocking monoclonal cell lines numbered mAb019 - mAb034 were screened in the subclone.

[0162] After two rounds of fusion and screening, a total of 34 positive monoclonals were finally obtained. The secreted antibodies were purified and analyzed to ensure that their concentration, quality, purity, and endotoxin content met the requirements of subsequent experiments.

[0163] Example 5 Determination of the performance of anti-BDCA2 mouse antibodies All of the obtained mouse antibodies had good binding activity to human BDCA2 Fc protein with an EC 50 <0.3 nM as measured by ELISA. The mouse antibodies showed good binding to cynoBDCA2 His protein, and most of the antibodies showed an EC 50 <0.5 nM as measured by ELISA.

[0164] All of the mouse antibodies bound to CHOK1-human BDCA2 cells with an EC 50 equivalent to or better than that of the control antibody BIIB059 (see the results in Figures 1a - 1e). Most of the mouse antibodies bound to 293F-cynoBDCA2 cells with an EC 50 equivalent to or better than that of the control antibody BIIB059 (see the results in Figures 2a - 2e).

[0165] Thirty-two antibodies that bind to CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells were tested for their ability to inhibit cytokine IFNα release in the CpG-A-stimulated PBMC experiment. All of these antibodies had a certain degree of inhibitory activity. Among them, mAb005, mAb019, mAb020, mAb021, mAb022, mAb024, mAb027, mAb030, mAb033 had IC 50 values of cytokine release inhibition of 0.08667 nM, 0.003323 nM, 0.006146 nM, 0.004047 nM, 0.006232 nM, 0.002744 nM, 0.07004 nM, 0.004762 nM, and 0.004083 nM, respectively.

[0166] The above test procedures are shown below. mAb001 - mAb034 are mouse antibodies of the present invention, BIIB059 is a reference antibody as described above, hIgG1 is a human IgG1 negative control, and mIgG1j is a mouse IgG1 negative control.

[0167] 5.1 Detection of the binding activity of mouse antibodies to proteins by ELISA Human BDCA2 Fc protein or cynoBDCA2 His protein was diluted to 1 μg / mL with PBS and added to ELISA microplates at 100 μL / well and incubated overnight at 4 °C. The plates were blocked with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage represents mass percentage) at 37 °C for 2 hours. Ten-fold serial dilutions of the antibody were added at 8 concentrations (0.0001 nM to 100 nM, including the zero control) at a total of 100 μL / well. The plates were incubated at 37 °C for 1 hour, then washed three times, and anti-mouse IgG (Fab specific)-HRP (Sigma, A3682; diluted 1:5000) secondary antibody was added at 100 μL / well and then incubated at 37 °C for 1 hour. The plates were washed three times, TMB chromogenic solution (EL0009, Huzhou Ying Chuang Biotechnology) was added at 100 μL / well, incubated at 37 °C for 10 minutes, and then 50 μL of 1 N hydrochloric acid was added to stop the chromogenic reaction. The OD450nm value was read with an ELISA plate reader, and a curve was fitted with GraphPad Prism6 to calculate the EC 50 value.

[0168] 5.2 Detection of the binding activity of mouse antibodies to cells by FACS CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured to the logarithmic growth phase were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) at 2 × 10 6Resuspended to a concentration of / mL. The cells were added to a 3799 cell plate (Corning) at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The antibody was serially diluted with FACS buffer (same as above), the diluted antibody was added to the centrifuged cells, and the cells were resuspended. The cells were incubated at 4 °C for 1 hour. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Invitrogen, A21202; 1:1000) secondary antibody was added to the cells, incubated at 4 °C for 1 hour, washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS (BD FACS CantoTM II) instrument. Curves were fitted using GraphPad Prism6, and the EC 50 value was calculated.

[0169] 5.3. Ability of mouse antibodies to inhibit the release of cytokine IFNα from human PBMC cells stimulated by CpG-A 1) Thawed cryopreserved human PBMC cells and incubated them in complete medium (1640 + 10% FBS + 1×NEAA + 1×L-glutamine) for 18 hours overnight. The next day, the PBMC cells were collected, centrifuged at 600 g for 8 minutes, and the supernatant was discarded. The cells were resuspended in medium (1640 + 10% FBS) to a concentration of 8×l0 6 / mL and added to a 3799 cell culture plate at 100 μL / well. The test antibody was serially diluted in medium (1640 + 10% FBS) at 10 levels (final concentrations of 0.0001 nM to 10 nM), and the diluted antibody was added to the PBMC at 50 μL / well. The cells were resuspended and incubated at 37 °C for 6 hours. 50 μL of 2 μM CpG-A (Invitrogen, tlrl-2216-5) diluted in medium (1640 + 10% FBS) was added to each well, mixed, and incubated at 37 °C for 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 assayed.

[0170] 2) Detection of cytokine IFNα by ELISA The antibody MT1 / 3 / 5 (Mabtech, 3425 - 1H - 20) was diluted to 4 μg / mL with PBS and added to a CORNING ELISA plate at 100 μL / well, and the plate was coated by incubating overnight at 4°C. Next, the antibody used for coating the plate was discarded, and the blocking solution (PBS + 1% BSA) was added to the plate at 300 μL / well and incubated overnight at 4°C. The test supernatant was diluted to an appropriate concentration (selected from 1:3 to 1:7 as appropriate) with the blocking solution and added to the coated ELISA plate at 100 μL / well. The IFNα standard for detection was serially diluted 2-fold with the blocking solution (with the highest concentration being 1000 pg / mL, 8 concentration points, and the final point being 0), and added to the coated ELISA plate at 100 μL / well. The ELISA plate was incubated in an incubator at 37°C for 1 hour. The plate was washed 3 times, the detection antibody MT2 / 4 / 6 (1:1000) was added at 100 μL / well, and incubated in the incubator at 37°C for 1 hour. The plate was washed 3 times, the secondary antibody SA - HRP (1:1000) was added at 100 μL / well, and then incubated in the incubator at 37°C for 0.5 hour. The plate was washed 3 times, the TMB color development solution was added at 100 μL / well, and the reaction was stopped by adding 50 μL / well of 1M HC1 at an appropriate time point. The OD450nm reading was measured with an ELISA plate reader, and then the inhibition rate was calculated, and a curve was fitted with GraphPad Prism6 to calculate the EC 50 value.

[0171] Example 6 Determination of the variable region sequences of anti - BDCA2 mouse hybridomas (monoclonal antibodies) (according to Kabat's representation) Sixteen hybridoma monoclonals were selected for VH / VL sequencing based on the results of the ability of mouse antibodies to bind antigens at the protein and cell levels and the ability of antibodies to inhibit cytokine IFNα release induced by CpG - A stimulation in the PBMC system.

[0172] The DNA coding sequence corresponding to the variable region of the anti-BDCA2 mouse antibody was determined by a method based on PCR with degenerate primers. Cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. First-strand cDNA was synthesized using the total RNA as a template, and the corresponding variable region DNA coding sequence was amplified by pCR using this as a template. The primer sequences used in the amplification reaction are complementary to the first framework region and the constant region of the variable region of the antibody (Larrick, J.W., et al., 1990, Scand. J. Immunol., 32, 121-128 and Coloma, J. J. et al., (1991) BioTechniques, 11, 152-156). 1 μl of cDNA, 5 μl of 10×PCR buffer, 1 μl each of the upstream and downstream primers (25 pmol), 1 μl of dNTP, 1 μl of 25 mmol PL MgCl 2 1 μl, H 2 1 μl, and 39 μl of H

[0173] O were each added to a 50 μl reaction system, pre-denatured at 95°C for 10 minutes, 1 μl of Taq enzyme was added, and the temperature cycle was started to perform PCR amplification. The reaction conditions were 1 denaturation at 94°C, 1 minute of annealing at 58°C, 15 seconds of extension at 72°C for a total of 32 cycles, followed by incubation at 72°C for 10 minutes. The PCR product was recovered and purified. Sequencing of the amplification product was performed to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-BDCA2 mouse antibody.Consensus sequences were searched in the germline and rearranged Ig variable region sequence databases using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast / ). The amino acid sequences of the complementarity-determining regions (CDRs) were identified by sequence annotation and Internet sequence analysis (http: / / www.imgt.org / IMGT_vquest / vquest and http: / / www.ncbi.nlm.nih.gov / igblast / ) based on the Kabat (Wu, T.T and Kabat, E.A. 1970 J. Exp. Med., 132: 211-250) and IMGT systems (Lefranc M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212).

[0174] The amino acid sequences of the light and heavy chain variable regions and CDRs of the selected anti-BDCA2 mouse antibodies are shown in Table 2. Multiple sequence alignments and phylogenetic tree classifications were performed, and according to the results, the heavy chain variable regions VH of the 16 monoclonal antibody sequences were mainly classified into the following five major classes: 1) mAb020, 022, 026; 2) mAb021, 027, 028, 030, 032, 034; 3) mAb019, 024, 033; 4) mAb001, 002; 5) mAb005, 016, where the heavy chain variable regions of mAb028 and mAb030 differ by only two amino acids, and mAb028 and mAb032 differ by only one amino acid. The light chain variable regions VL could be classified into three major classes: 1) mAb019; 2) mAb021, 027, 028, 030, 032, 034; 3) the other antibodies.

[0175]

Table 2

[0176] Example 7 Construction of Anti-BDCA2 Chimeric Antibodies For the construction of human-mouse chimeric antibodies, a total of 12 monoclonal antibody sequences were selected according to the results of sequence detection, and the sequence of the hlgG1 Fc fragment was selected as the human constant region sequence during plasmid construction. The coding sequences of the heavy and light chain variable regions of the anti-BDCA2 mouse antibody were synthesized by Genscript Corporation. Antibody expression was performed using expi 293F as the host cell. The resulting chimeric antibodies were mab001c, mab002c, mab005c, mab016c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c, and mab033c, respectively, and the expressed chimeric antibodies were individually characterized.

[0177] Example 8 Screening of Chimeric Antibodies 8.1 Detection of the binding activity of anti-BDCA2 chimeric antibodies to proteins by ELISA Human BDCA2 Fc protein or cynoBDCA2 His protein was diluted to 1 μg / mL with PBS and added to an ELISA microplate at 100 μL / well, and incubated overnight at 4°C. Blocking was performed with an ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4, the percentage represents mass percentage) at 37°C for 2 hours. Ten-fold serial dilutions of the antibody were added at 8 concentrations (0.0001 nM to 100 nM, including the zero control). The plate was incubated at 37°C for 1 hour at 100 μL / well. The plate was washed three times, and an anti-human IgG (Fab specific)-HRP (Sigma, A0293; diluted 1:5000) secondary antibody was added at 100 μL / well and incubated at 37°C. The plate was washed three times, and a TMB chromogenic solution (EL0009, Huzhou Ying Chuang Biotechnology) was added at 100 μL / well and incubated at 37°C for 10 minutes, then 50 μL of 1 N hydrochloric acid was added to stop the chromogenic reaction. The OD450nm value was read with an ELISA plate reader, and a curve was fitted with GraphPad Prism6 to calculate the EC 50 value.

[0178] The results were as follows: The tested antibodies bound to both human BDCA2-Fc protein and cynoBDCA2 His protein. The EC 50 for binding to human BDCA2-Fc protein was 0.1 - 0.2 nM. Most antibodies bound to cynoBDCA2 protein with an EC 50 value of less than 0.6 nM.

[0179] 8.2. Detection of the binding activity of chimeric antibodies to cells by FACS method CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured until the logarithmic growth phase were trypsinized with TrypLEd. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2×10 6 / mL. The cells were added to a 3799 cell plate (Corning) at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The antibodies were serially diluted in FACS buffer (same as above), the diluted antibodies were added to the centrifuged cells, and the cells were resuspended. The cells were incubated at 4°C for 1 hour. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of donkey anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) secondary antibody was added to the cells, incubated at 4°C for 1 hour, washed twice with FACS buffer, resuspended in PBS, analyzed with a FACS (BD FACS CantoTM II) instrument, the curve was fitted with GraphPad Prism6, and the EC 50 value was calculated.

[0180] As shown in Figure 3 (3a - 3c), all the tested chimeric antibodies bound with an EC 50 equivalent to or better than that of the control antibody BIIB059 to CHOK1-human BDCA2 cells. At the same time, all the tested chimeric antibodies bound to 293F-cynoBDCA2 cells with an EC 50 of approximately 0.3 - 2 nM equivalent to or better than that of the control antibody BIIB059.

[0181] 8.3. Detection of the ability of a chimeric antibody to inhibit the stimulation of cytokine IFNα release from human PBMC cells by CpG-A Cryopreserved human PBMC cells were recovered and incubated overnight for 18 hours in complete medium (1640 + 10% FBS + 1×NEAA + 1×L-glutamine). The next day, the PBMC cells were collected, centrifuged at 600 g for 8 minutes, and the supernatant was discarded. The cells were resuspended in medium (1640 + 10% FBS) to a concentration of 8×10 6 / mL and added to a 3799 cell culture plate at 100 μL / well. The test antibody was serially diluted in medium (1640 + 10% FBS) to 10 dilutions (final concentrations 0.0001 nM to 10 nM), and the diluted antibody was added to the PBMC at 50 μL / well. The cells were resuspended and incubated at 37°C for 6 hours. 2 μM CpG-A (Invitrogen, tlrl-2216-5) diluted in 50 μL of medium (1640 + 10% FBS) was added to each well, mixed well, and incubation was continued at 37°C for 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 assayed.

[0182] The antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL with PBS and added to a CORNING ELISA plate at 100 μL / well for coating. The coated plate was incubated overnight at 4°C. Next, a blocking solution (PBS + 1% BSA) was added to the antibody-coated plate at 300 μL / well and incubated overnight at 4°C. The test supernatant was diluted with the blocking solution to an appropriate concentration (generally in the range of 1:3 to 1:7), and 100 μL / well was added to the coated ELISA plate. The IFNα detection standard was serially diluted 2-fold with the blocking solution (with the highest concentration being 1000 pg / mL, 8 concentration points, and the final point being 0), and 100 μL / well was added to the coated ELISA plate. The ELISA plate was incubated in an incubator at 37°C for 1 hour. The plate was washed three times, and the detection antibody MT2 / 4 / 6 (1:1000) was added at 100 μL / well and incubated in the incubator at 37°C for 1 hour. The plate was washed three times, and the secondary antibody SA-HRP (1:1000) was added at 100 μL / well and incubated in the incubator at 37°C for 0.5 hour. The plate was washed three times, the TMB chromogenic solution was added at 100 μL / well, and 1M HC1 was added at 50 μL / well at an appropriate time point to stop the reaction. The OD450nm reading was measured with an ELISA plate reader, the inhibition rate was calculated, a curve was fitted with GraphPad Prism6, and the EC 50 value was calculated.

[0183] All 12 chimeric antibodies tested were able to inhibit the secretion of the cytokine IFNα in the experiment of CpG-A-stimulated cytokine release from PBMC. The IC 50 values of mab005c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c, and mab033c were 0.008359 nM, 0.01493 nM, 0.01853 nM, 0.01268 nM, 0.03239 nM, 0.01677 nM, 0.008976 nM, 0.01645 nM, and 0.008331 nM, respectively.

[0184] 8.4. Detection of the intracellular translocation ability of chimeric antibodies CHOK1-human BDCA2 cells cultured until the logarithmic growth phase were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2×10 6 / mL. The cells were added to a 96-well plate at 100 μL / well, centrifuged at 300 g, the supernatant was discarded, and the cells were placed in an ice box for cooling. The antibody was serially diluted with FACS buffer (3 concentrations were set: 100 nM, 10 nM, 1 nM; 3 groups each: 0-time point control; 4°C, 1 hour; 37°C, 1 hour), then placed in an ice box and cooled for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells, the cells were resuspended, and incubated at 4°C for 40 minutes. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. The cells in the 0-time point group were fixed with paraformaldehyde at room temperature for 10 minutes, washed twice with FACS buffer, and the other two groups of cells were resuspended in 100 μL of FACS buffer and incubated at 4°C and 37°C for 1 hour respectively. The cells in the groups incubated at 4°C for 1 hour and 37°C for 1 hour were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. 100 μL of secondary antibody goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) was added to the three groups of cells simultaneously and incubated at 4°C for 1 hour. The cells were washed twice with FAC buffer, then resuspended in PBS, analyzed with a FACS instrument, and the percentage of surface signals was calculated.

[0185] Intracellular translocation of the chimeric antibody occurred at concentrations of 100 nM, 10 nM, and 1 nM, and the intracellular translocation ability mediated by 12 chimeric antibodies was slightly better, with no significant difference from the positive control antibody BIIB059. When intracellular translocation occurred at 100 nM at 37°C for 1 hour, the cell surface signal value was 50% - 60%.

[0186] Example 9 Humanization of the variable region of the antibody For humanization of the variable regions of antibodies, first, human germline IgG genes homologous to the cDNA sequences of mouse antibodies were searched in the human immunoglobulin gene database on the NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / ) website, and then their exact boundaries were identified using the Kabat numbering system or the IMGT numbering system. In particular, human IGHV with high homology to the mouse antibody was selected as the humanization template, and humanization of the variable regions of the antibody was performed by CDR grafting.

[0187] mAb005 and mAb033 were selected for humanization from the sequences of the mouse antibodies obtained above. The steps of the humanization process were as follows: a. Align the gene sequences of the mouse antibodies with the gene sequences of human germline antibodies to find sequences with high homology; b. Conduct an analysis to examine HLA-DR affinity and select human germline framework sequences with low affinity; c. Apply molecular docking and analyze the framework amino acid sequences of the variable regions and their surroundings using in silico technology to examine the spatial steric binding approach. Through analysis of calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy values, the major amino acids of the mouse antibody gene sequences that act on human BDCA2 and can maintain the spatial framework were determined, and then these amino acids were grafted back onto the human germline gene frameworks selected, and after identifying the amino acid sites in the framework regions to be retained based on this, humanized antibodies were synthesized. Based on this, various humanized antibody variable region sequences were obtained, various combinations of the designed variable regions of the humanized anti-BDCA2 antibodies were prepared, and humanized anti-BDCA2 antibodies with amino acid sequences of the variable regions shown in Table 3 were obtained.

[0188] The CDRs of the variable regions of the Hu005 series humanized antibodies (KABAT scheme) are shown below: HCDR1: NYGVH (SEQ ID NO: 11) HCDR2: VIWSGESTDYDAAFIS (SEQ ID NO: 12) HCDR3: RRSHYYGYVMDY (SEQ ID NO: 13) LCDR1: KASQSIDYDAIGYLN (SEQ ID NO: 86) LCDR2: AASNLES (SEQ ID NO: 15) LCDR3: QQSNEDPPT (SEQ ID NO: 16)

[0189] The CDRs of the variable regions of the Hu003 series of humanized antibodies (KABAT scheme) are shown below: HCDR1: SYGMS (SEQ ID NO: 52) HCDR2: TISSGDSYTYYPDSVKG (SEQ ID NO: 39) HCDR3: QIYYDYAYYFDF (SEQ ID NO: 53) LCDR1: RASESVSFRTSHLMH (SEQ ID NO: 54) LCDR2: GASNLES (SEQ ID NO: 55) LCDR3: QQSIEDPPT (SEQ ID NO: 42)

[0190]

Table 3

[0191] Example 10 Screening of Humanized Anti-BDCA2 Antibodies 10.1. Binding Activity of Humanized Antibodies to BDCA2 CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured until the logarithmic growth phase were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) at a density of 2 × 10 6Resuspended to a concentration of / mL. The suspended cells were added to a 3799-cell plate at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The antibody to be detected was serially diluted 5-fold with FACS buffer (final concentration 0.012 nM - 200 nM), the diluted antibody was added to the centrifuged cells, and the cells were resuspended. The cells were incubated at 4 °C for 1 hour. The incubated 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, incubated at 4 °C for 1 hour, and then washed twice with FACS buffer. The cells were resuspended in PBS, and then the fluorescence signal of the antibody bound to the cell surface was analyzed using a FACS (BD FACS CantoTM II) instrument. A curve was fitted with GraphPad Prism6, and the EC 50 value of the antibody binding to the BDCA2 antigen was calculated. The BIIB059 antibody was selected as the positive control, and hlgG1 was selected as the negative control.

[0192] Table 4 shows candidate antibodies with binding activity to CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells higher than that of the positive control BIIB059 antibody, obtained after humanization of the mAb033 sequence. When the affinity of the antibodies for human BDCA2 was determined using SPR, the selected antibodies had a stronger affinity for human BDCA2 than the positive control BIIB059 antibody.

[0193]

Table 4

[0194] As shown in Table 5, the candidate antibodies obtained from humanization of the mAb005 sequence bound to CHOK1-human BDCA2 cells with a better EC 50 value than the positive control BIIB059 antibody. At the same time, the candidate antibodies obtained from humanization of the mAb005 sequence also had good binding activity to 293F-cynoBDCA2 cells.

[0195]

Table 5

[0196] 10.2. Functional assay of the ability of a humanized antibody to inhibit CpG-A from stimulating cytokine IFNα release from human PBMC cells A series of humanized candidate antibody molecules that inhibit CpG-A-stimulated cytokine IFNα release from human PBMC cells were selected for functional assay. Cryopreserved human PBMC cells were thawed and incubated overnight for 18 hours in complete medium (1640 + 10% FBS + 1×NEAA + 1×L-glutamine). The next day, the PBMC cells were collected, centrifuged at 600 g for 8 minutes, and the supernatant was discarded. The cells were resuspended in medium (1640 + 10% FBS) to a concentration of 8×10 6 / mL and added to a 3799 cell culture plate at 100 μL / well. The test antibody was serially diluted 10-fold in medium (1640 + 10% FBS) (final concentrations 0.0001 nM to 10 nM, 0-point control), and the diluted antibody was added to the PBMC at 50 μL / well. The cells were resuspended and incubated at 37°C for 6 hours. 2 μM CpG-A (Invitrogen, tlrl-2216-5) diluted in 50 μL of medium (1640 + 10% FBS) was added to each well, mixed well, and incubation was continued at 37°C for 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 assayed. The BIIB059 antibody was selected as the positive control, and hIgG1 was selected as the negative control.

[0197] The antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL with PBS and added to a CORNING ELISA plate at 100 μL / well. The coated plate was incubated overnight at 4°C. Next, the plate coated with the antibody was drained, and the blocking solution (PBS + 1% BSA) was added to the plate at 300 μL / well and incubated overnight at 4°C. The test supernatant was diluted with the blocking solution to an appropriate concentration (generally in the range of 1:3 to 1:7) and added to the coated ELISA plate at 100 μL / well. The IFNα detection standard was serially diluted 2-fold with the blocking solution (with the highest concentration being 1000 pg / mL, 8 concentration points, and the final point being 0) and added to the coated ELISA plate at 100 μL / well. The ELISA plate was incubated in an incubator at 37°C for 1 hour. The plate was washed three times, and the detection antibody MT2 / 4 / 6 (1:1000) was added at 100 μL / well and incubated in an incubator at 37°C for 1 hour. The plate was washed three times, and the secondary antibody SA-HRP (1:1000) was added at 100 μL / well and incubated in an incubator at 37°C for 0.5 hour. After the plate was washed three times and the secondary antibody SA-HRP (1:1000) was added at 100 μL / well, it was incubated at 37°C for 0.5 hour. The plate was washed three times, the TMB chromogenic solution was added at 100 μL / well, and 1M HC1 was added at 50 μL / well at an appropriate time point to stop the reaction. The OD450nm reading was measured with an ELISA plate reader, and a curve was fitted with GraphPad Prism6. The concentration of the 10 nM antibody corresponding to the OD value was selected, and the maximum inhibition rate was Inhibition rate = (OD(IgG1) - OD(Ab)) / OD(IgG1) calculated as follows.

[0198] All the humanized antibodies tested were able to inhibit the secretion of the cytokine IFNα at an EC 50 value lower than that of the positive control antibody BIIB059 in the cytokine release experiment from PBMC stimulated with CpG-A. The results are further shown in Table 6.

[0199]

Table 6

[0200] 10.3. Detection of the intracellular translocation ability of humanized antibodies CHOK1-human BDCA2 cells cultured until the logarithmic growth phase were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2×10 6 / mL. The cells were added to a 3799-cell plate at 100 μL / well, centrifuged at 300 g, the supernatant was discarded, and the cells were placed in an ice box to cool. The antibody was serially diluted with FACS buffer (two concentrations were set: 100 nM, 10 nM; three groups each: 0 time point; 4 °C, 1 hour; 37 °C, 1 hour), then placed in an ice box and cooled for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells, the cells were resuspended, and incubated at 4 °C for 40 minutes. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. The cells in the 0 time point group were fixed with paraformaldehyde at room temperature for 10 minutes, washed twice with FACS buffer, and the other two groups of cells were resuspended in 100 μL of FACS buffer and incubated at 4 °C and 37 °C for 1 hour, respectively. The cells in the groups incubated at 4 °C for 1 hour and 37 °C for 1 hour were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. 100 μL of secondary antibody goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) was added to the three groups of cells simultaneously and incubated at 4 °C for 1 hour. The cells were washed twice with FAC buffer, then resuspended in PBS, analyzed with a FACS apparatus, and the percentage of surface signal was calculated.

[0201] The results are shown in Figure 4. The humanized antibodies in both groups of Hu005 and Hu033 caused intracellular translocation at concentrations of 100 nM and 10 nM, and the ability to mediate the intracellular translocation of the humanized antibodies was equivalent to that of the positive control antibody BIIB059.

[0202] 10.4. Detection of the Affinity of the Humanized Antibody The affinity of the humanized antibody of the present invention was determined by a BIAcore experiment: The surface of the channel of a CM5 chip was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC of an amino coupling kit), and an anti-human IgG (Fc) antibody (diluted with a sodium acetate solution at pH 5.0, concentration 25 μg / mL) was injected for 420 seconds. Next, 1 M ethanolamine was injected at a flow rate of 10 μL / min for 420 seconds to block the excess activated carboxyl groups on the chip. The humanized antibody was diluted to 1 μg / mL using a running buffer and then injected into the flow cell of the detection ion channel at a flow rate of 10 μL / min for 60 seconds for capture. Recombinant human BDCA2 was diluted to 50 or 100 nM with 1×HBS-EP+ (pH 7.4) and serially diluted 1:2 to 0.39 nM. The test sample hBDCA2 at each concentration gradient was injected into the detection ion channel at a flow rate of 30 μL / min, and the background signal was removed using two samples at 0 concentration. The binding time and dissociation time of the antigen and the antibody were 180 seconds and 400 seconds, respectively. Data analysis was performed using Biacore Insight Evaluation software (version 2.0, 15.12933). A 1:1 binding model was selected for curve fitting, and after subtracting the signal of the reference channel (flow cell 1) and the 0 concentration, the kinetic parameters were calculated. The results of the assay are shown in Table 7 below.

[0203]

Table 7

[0204] Example 11 Fc Mutation of the Humanized Anti-BDCA2 Antibody and Screening of the Antibody For the humanized antibodies Hu033-03 and Hu033-20 obtained in Example 10, the Fc region was selected for mutations to enhance the affinity for FcγRIIA. All of the humanized antibodies Hu033-03-T1, Hu033-03-T2, Hu033-03-T3, Hu033-20-T1, Hu033-20-T2, and Hu033-20-T3 were designed, and the resulting heavy and light chain coding sequences were synthesized by Genscripts. The amino acid sequences of the heavy and light chains are shown in Table 8. 293F was selected as the host cell for antibody expression, and various property evaluation tests were performed on the purified antibody after expression.

[0205]

Table 8

[0206] 11.1. Fc mutations enhance antibody affinity for FcγRIIA Antibody kinetics or affinity parameters were measured using a BIAcore 8K and a newly immobilized biotinylated human FcγRIIA(R167) / FcγRIIA(H167) SA chip. The experiment was carried out at 25 °C using a running buffer of 1×HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The antibody was diluted in the running buffer at ratios of 1:2 to concentrations 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, and the test antibody was injected into flow cell 1 and flow cell 2 at a flow rate of 30 μL / min at each concentration gradient. The binding time and dissociation time of the antibody were 60 seconds and 90 seconds, respectively. The background signal was removed using two 0-concentration samples. The samples were injected in a multi-cycle mode, and the analyte could be completely dissociated from the chip surface at the end of each cycle, and no regeneration process was applied. Data analysis was performed using Biacore Insight Evaluation software (version 2.0.15.12933). Affinity analysis was performed using a 1:1 binding model or a steady-state model for curve fitting and calculation of kinetic parameters after subtracting the reference channel (flow cell 1) and the two 0-concentration signals.

[0207] The affinity of the mutant antibody for both subtypes of human FcγRIIA(R167) and FcγRIIA(H167) is significantly enhanced compared to wild-type Fc. The results are shown in Tables 9-1 and 9-2.

[0208]

Table 9-1

[0209]

Table 9-2

[0210] 11.2. Antibodies with Fc mutations maintain invariant binding affinity for BDCA2 The binding ability of Fc mutant antibodies to human BDCA2 and Cyno BDCA2-expressing cells was detected by FACS.

[0211] The experimental method is as follows.

[0212] CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2×10 6 / mL. The cells were added to a 3799 cell plate (Corning) at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The antibody was diluted in a 1:5 gradient using FACS buffer with an initial concentration of 200 nM, and the diluted antibody was added to the centrifuged cells and the cells were resuspended. The cells were incubated at 4°C for 1 hour. The incubated cells were centrifuged at 300 g for 5 minutes and washed with FACS buffer. 100 μL of donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibidy-Alexa Fluor 488 (Invitrogen, A21202; 1:1000) secondary antibody was added, incubated at 4°C for 1 hour, washed twice with FACS buffer, resuspended in PBS, analyzed using a FACS (BD FACS CantoTM II) instrument, the curve was fitted with GraphPad Prism6, and the EC 50 value was calculated.

[0213] The binding ability of the Fc mutant antibody to human BDCA2 was slightly better than that of wild-type Fc and significantly better than that of the reference antibody BIIB059. The binding ability of the Fc mutant antibody to Cyno BDCA2 was superior to that of wild-type Fc and BIIB059. The results are shown in Figure 5a, Figure 5b, and Table 10.

[0214]

Table 10

[0215] 11.3. The intracellular translocation ability of the antibody remains unchanged even after Fc mutation. The intracellular translocation ability of each antibody was detected using FACS.

[0216] CHOK1-human BDCA2 cells were trypsinized with TrypLE. The cells were recovered and resuspended in FACS buffer (PBS + 2% FBS) to a concentration of 2×10 6 / mL. The cells were added to a 96-well plate at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The cells were then placed in an ice box to cool. The antibody was serially diluted to 1 nM, 10 nM, or 100 nM with FACS buffer (five sets of detection conditions were set for each antibody concentration: time 0; 4°C, 1 hour; 37°C, 1 hour; 4°C, 4 hours; 37°C, 4 hours). Then, it was placed in an ice box and cooled for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells, the cells were resuspended, and incubated at 4°C for 40 minutes. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. The cells in the time 0 group were fixed with paraformaldehyde at room temperature for 10 minutes, washed twice with FACS buffer, and the other four groups of cells were resuspended in 100 μL of FACS buffer and incubated at 4°C and 37°C for 1 hour or 4 hours, respectively. At 4°C and 37°C for the above-mentioned times, the cells in each group were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. At the same time, 100 μL of secondary antibody goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000) was added to the five groups of cells and incubated at 4°C for 1 hour. The cells were washed twice with FAC buffer, then resuspended in PBS, analyzed with a FACS instrument, and the percentage of surface signal was calculated.

[0217] The Fc mutant antibodies were found to translocate intracellularly at concentrations of 100 nM, 10 nM, and 1 nM. These types of Fc modifications have no effect on the intracellular translocation of the antibody. For details, see Figures 6a - 6f.

[0218] 11.4. The Fc mutant antibody has a strong inhibitory effect on the release of cytokine IFNα from human PBMC cells stimulated by the antibody complex SLE-IC. The function of the Fc-modified antibody to inhibit the release of cytokine IFNα from human PBMC cells stimulated by the antibody complex was detected. Cryopreserved human PBMC cells were thawed and incubated overnight, 18 hours, in complete medium (1640 + 10% FBS + 1×NEAA + 1×L-glutamine). The next day, the PBMC cells were harvested, centrifuged at 600 g for 8 minutes, and the supernatant was discarded. The cells were resuspended in medium (1640 + 10% FBS) to a concentration of 8×10 6 / mL and added to a 3799 cell culture plate at 100 μL / well. The test antibody was diluted in medium (1640 + 10% FBS) in a 5-fold gradient (0.0032 nM to 10 nM, and 0 control), and the diluted antibody was added to the PBMC at 50 μL / well. The cells were resuspended and incubated at 37 °C for 6 hours. SLE-IC (2.5 μl Sm / RNP-antigen (AROTEC, ART01-10)) mixed with 10 μl anti-RNP antibody (RayBiotech, MD-14-0513)) diluted in 50 μL medium (1640 + 10% FBS) was added to each well, mixed well, and incubation was continued at 37 °C for 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 assayed. The BIIB059 antibody was selected as the positive control, and hIgG1 was selected as the negative control.

[0219] Cytokine IFNα in the supernatant was detected. Antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL with PBS, added to CORNING ELISA plates at 100 μL / well, and incubated overnight at 4°C. Next, the plates coated with the antibody were drained, and blocking solution (PBS + 1% BSA) was added to the plates at 300 μL / well and incubated overnight at 4°C. The test supernatant was diluted with the blocking solution to an appropriate concentration (selected from 1:3 to 1:7 as appropriate) and added to the coated ELISA plates at 100 μL / well. The IFNα detection standard was serially diluted 2-fold with the blocking solution (with the highest concentration being 1000 pg / mL, 8 concentration points, and the final point being 0), and added to the coated ELISA plates at 100 μL / well. The ELISA plates were incubated in an incubator at 37°C for 1 hour. The plates were washed three times, and detection antibody MT2 / 4 / 6 (1:1000) was added at 100 μL / well and incubated in an incubator at 37°C for 1 hour. The plates were washed three times, and secondary antibody SA-HRP (1:1000) was added at 100 μL / well, and then incubated in an incubator at 37°C for 0.5 hour. The plates were washed three times, TMB chromogenic solution was added at 100 μL / well, and 1M HC1 was added at 50 μL / well at an appropriate time point. OD 450nm The reading value was read with an ELISA plate reader, and a curve was fitted with GraphPad Prism6 to calculate EC 50 .

[0220] The Fc-modified antibody had a stronger IFNα inhibitory effect compared to the wild-type Fc antibody and BIIB059 antibody under SLE-IC stimulation conditions, and achieved complete inhibition of IFNα secretion at 0.016 nM. For details, see Figure 7 and Table 11.

[0221]

Table 11

[0222] Various modifications and variations of the methods and systems described in this invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described with respect to specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications in the manner described for carrying out the invention will be apparent to those skilled in the art of molecular biology, immunology, or related fields and are intended to be included within the scope of the appended claims.

[0223] Sequence Listing (all are amino acid sequences; see the previous section of this specification for sequence definitions / descriptions): TIFF2025518061000031.tif116164TIFF2025518061000032.tif235164TIFF2025518061000033.tif249165TIFF2025518061000034.tif244164TIFF2025518061000035.tif243164TIFF2025518061000036.tif244165TIFF2025518061000037.tif242165TIFF2025518061000038.tif243165TIFF2025518061000039.tif242165TIFF2025518061000040.tif246165TIFF2025518061000041.tif245166TIFF2025518061000042.tif115166

Claims

1. An anti-BDCA2 antibody or its antigen-binding fragment, wherein the anti-BDCA2 antibody or its antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region. The aforementioned heavy chain variable region is (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 39, and SEQ ID NO: 53, respectively; or HCDR1, HCDR2, and HCDR3 having one, two, or three amino acids different from the amino acid sequences shown in SEQ ID NO: 52, SEQ ID NO: 39, and SEQ ID NO: 53, respectively; or (II) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; or (III) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; or (IV) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively; or (V) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively; or (VI) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; or (VII) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; or (VIII) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 50, and SEQ ID NO: 47, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 50, and SEQ ID NO: 47, respectively; or (IX) HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively; or HCDR1, HCDR2, and HCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 37, respectively. Includes: and / or The aforementioned light chain variable region is (I) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 42, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 42, respectively; or (II) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 86, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 86, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. (III) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; or (IV) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; or (V) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; or (VI) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; or (VII) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 15, and SEQ ID NO: 42, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 15, and SEQ ID NO: 42, respectively; or (VIII) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 48, and SEQ ID NO: 49, respectively; or (IX) LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 51, and SEQ ID NO: 49, respectively; or LCDR1, LCDR2, and LCDR3 having amino acid sequences that differ by one, two, or three amino acids from the amino acid sequences shown in SEQ ID NO: 34, SEQ ID NO: 51, and SEQ ID NO: 49, respectively; An anti-BDCA2 antibody or its antigen-binding fragment, including the above.

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

3. Including heavy chain variable regions and light chain variable regions, (I) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 82, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 82; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 83, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

83. (II) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 61, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 61; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 62, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 62; or (III) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 65, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 65; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 66, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 66; or (IV) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 67, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 67; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 68, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68; or (V) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 69, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 69; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 70, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 70; or (VI) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 71, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 71; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 68, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 68; or (VII) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 72, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 72; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 73, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 73; or (VIII) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 76, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 76; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 77, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 77; or (IX) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 80, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 79, or includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 79; The antibody or its antigen-binding fragment according to claim 1 or 2.

4. (I) Heavy chain variable regions containing an amino acid sequence represented by any one of SEQ ID NOs: 89, 106, 87-88, 90-105 and 107-110, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence represented by any one of SEQ ID NOs: 89, 106, 87-88, 90-105 and 107-110; and light chain variable regions containing an amino acid sequence represented by any one of SEQ ID NOs: 137, 154, 135-136, 138-153 and 155-158, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence represented by any one of SEQ ID NOs: 137, 154, 135-136, 138-153 and 155-158; or (II) Heavy chain variable regions containing an amino acid sequence shown in any one of SEQ ID NOs: 111-134, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence shown in any one of SEQ ID NOs: 111-134; and light chain variable regions containing an amino acid sequence shown in any one of SEQ ID NOs: 159-182, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence shown in any one of SEQ ID NOs: 159-182; or (III) Heavy chain variable regions containing the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 89; Light chain variable regions containing the amino acid sequence shown in SEQ ID NO: 137, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 137; (IV) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 106; Light chain variable region containing the amino acid sequence shown in SEQ ID NO: 154, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 154; (V) Heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 114, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 114; and light chain variable region containing the amino acid sequence shown in SEQ ID NO: 162, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 162; or (VI) Heavy chain variable region and light chain variable region containing any combination of amino acid sequences as shown in Table C The antibody or antigen-binding fragment according to claim 1, comprising:

5. The antibody is selected from the group consisting of mouse-derived antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. Furthermore / or, The antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and sdAb. Furthermore / or, The antibody or antigen-binding fragment according to claim 1, which is any IgG subtype such as IgG1, IgG2, IgG3, or IgG4, preferably an IgG1 subtype.

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

7. The following array: (I) A heavy chain having one, two, or three amino acids different from the amino acid sequence shown in SEQ ID NO: 183, and a light chain having the amino acid sequence shown in SEQ ID NO: 186; or (II) A heavy chain having one, two, or three amino acid sequences different from the amino acid sequence shown in SEQ ID NO: 184, and a light chain having the amino acid sequence shown in SEQ ID NO: 187; or (III) A heavy chain having one, two, or three amino acids different from the amino acid sequence shown in SEQ ID NO: 185, and a light chain having the amino acid sequence shown in SEQ ID NO:

188. The antibody or antigen-binding fragment according to claim 8, comprising or comprising Or, The antibody has the following sequence: (I) A heavy chain having the amino acid sequence shown in SEQ ID NO: 192, SEQ ID NO: 191, or SEQ ID NO: 193, and a light chain having the amino acid sequence shown in SEQ ID NO: 186; or (II) Heavy chain having the amino acid sequence shown in SEQ ID NO: 194, SEQ ID NO: 195, or SEQ ID NO: 196, and light chain having the amino acid sequence shown in SEQ ID NO: 187 The antibody or antigen-binding fragment according to claim 6, comprising or comprising.

8. A polynucleotide molecule encoding the anti-BDCA2 antibody or its antigen-binding fragment according to claim 1.

9. An expression vector comprising a polynucleotide molecule according to claim 8, preferably a eukaryotic expression vector.

10. A host cell comprising the polynucleotide molecule described in claim 8 or the expression vector described in claim 9, preferably a eukaryotic cell, more preferably a mammalian cell.

11. A method for producing an anti-BDCA2 antibody or its antigen-binding fragment according to claim 1, comprising expressing the antibody or its antigen-binding fragment in a host cell according to claim 10 under conditions suitable for the expression of the antibody or its antigen-binding fragment, and recovering the expressed antibody or its antigen-binding fragment from the host cell.

12. An immune complex comprising an anti-BDCA2 antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment is conjugated with at least one therapeutic or diagnostic agent, preferably an anti-inflammatory agent or an immunosuppressant.

13. A pharmaceutical composition comprising the anti-BDCA2 antibody or its antigen-binding fragment according to claim 1, the polynucleotide molecule according to claim 8, the expression vector according to claim 9, the host cell according to claim 10, the immune complex according to claim 12, and a pharmaceutically acceptable carrier or excipient.

14. An anti-BDCA2 antibody or its antigen-binding fragment according to claim 1, an immune complex according to claim 12, or a pharmaceutical composition according to claim 13, for use in a method to inhibit the excessive release of cytokines from target immune cells, The method comprises contacting target immune cells with the antibody or its antigen-binding fragment, immune complex, or pharmaceutical composition, wherein the immune cells are PBMC cells stimulated with CpG-A, as described in claim 1, the anti-BDCA2 antibody or its antigen-binding fragment according to claim 12, or the pharmaceutical composition according to claim 13.

15. An antibody or antigen-binding fragment thereof according to claim 1, an immune complex according to claim 12, or a pharmaceutical composition according to claim 13, for use in treating and / or preventing an inflammatory disease selected from systemic lupus erythematosus, discoid lupus, lupus nephritis, cutaneous lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic scleroderma (scleroderma), psoriasis, type 1 diabetes mellitus, dermatomyositis, and polymyositis.

16. A method for detecting the presence of BDCA2 in a sample using an antibody or its antigen-binding fragment according to any one of claims 1 to 9, or a detection composition containing the antibody or its antigen-binding fragment, the method comprising the step of contacting the sample with the antibody or its antigen-binding fragment or the detection composition containing the antibody or its antigen-binding fragment.

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