BDCA2-targeted antibodies and their applications

Abstract

The present invention discloses an anti-BDCA2 antibody and its antigen-binding fragment, a polynucleotide encoding the antibody and its antigen-binding fragment, and a pharmaceutical composition comprising the antibody and its antigen-binding fragment. Furthermore, the use of the anti-BDCA2 antibody and its antigen-binding fragment described in the present invention in the treatment of plasmacytoid dendritic cell (pDC) or type I interferon (type I IFN)-associated immune diseases is also disclosed.

Description

Detailed description of the invention

[0001] Cross-references to related applications This application claims priority to PCT / CN2023 / 093331, filed on 10 May 2023, and incorporates its entire contents by reference.

[0002] Reference to electronically submitted sequence listings This application incorporates by reference an array listing submitted as an XML file titled "938A004WO01P_SL" created on April 28, 2024, with a size of 116,400 bytes. [Technical field] This invention relates to molecular biology, cell biology, and immunology. Provided herein are anti-BDCA2 antibodies and their use in the treatment of plasmacytoid dendritic cells (pDCs) or type I interferon (type I IFN)-related immune disorders. [Background technology] Plasma cell-like dendritic cells (pDCs) are responsible for the production of type I interferons (IFNs) and inflammatory cytokines, and are drivers of innate and adaptive immune responses. Both pDCs and type I IFNs are involved in various immune diseases. BDCA2 is a member of the transmembrane glycoprotein type C lectin family and is expressed only in pDCs. Therefore, there is a need for BDCA2-targeting reagents that can suppress the release of pDC-related type I IFNs, for example, for use in the treatment and prevention of autoimmune diseases. However, research on BDCA2 as a therapeutic target is limited, and the success in developing such BDCA2-targeting reagents has been limited. The composition and method provided herein meet these needs and offer relative advantages. [Overview of the prefecture] The present invention relates to (1) a light chain variable region (VL) comprising VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 18, respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR; or (2) a VL comprising VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively, as defined by Chothia, or VL The present invention provides an antibody or antigen-binding fragment that specifically binds to human BDCA2, comprising (b) a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the CDR; and / or (b) VH comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 14, 16, and 18, respectively; or a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VH CDR.

[0003] In some embodiments, the antibodies or antigen-binding fragments provided herein include VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences shown in Sequence IDs 8, 9, 10, 15, 17, and 18 as defined by Kabat.

[0004] In some embodiments, the antibodies or antigen-binding fragments provided herein include VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 14, 16, and 18 as defined by Chothia.

[0005] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26; and / or (b) VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0006] In some embodiments, the antibody or its antigen-binding fragment provided herein comprises VL and VH having the amino acid sequences shown in SEQ ID NOs. 26 and 27.

[0007] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising (a) VL having the amino acid sequence shown in SEQ ID NO: 26, VL-derived VL CDR1, VL CDR2, and VL CDR3; and / or (b) VH having the amino acid sequence shown in SEQ ID NO: 27, VH having VH-derived VH CDR1, VH CDR2, and VH CDR3.

[0008] In some embodiments, the antibody or its antigen-binding fragment provided herein is a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment provided herein is a humanized antibody or its antigen-binding fragment.

[0009] In some embodiments, the antibody or antigen-binding fragment provided herein comprises (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-31; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-37.

[0010] In some embodiments, the antibodies or antigen-binding fragments provided herein include VL and VH having the following amino acid sequences, respectively: (1) SEQ ID NOs. 28 and 34; (2) SEQ ID NOs. 28 and 35; (3) SEQ ID NOs. 28 and 36; (4) SEQ ID NOs. 28 and 37; (5) SEQ ID NOs. 29 and 34; (6) SEQ ID NOs. 29 and 35; (7) SEQ ID NOs. 29 and 36; (8) SEQ ID NOs. 29 and 37; (9) SEQ ID NOs. 30 and 34; (10) SEQ ID NOs. 30 and 35; (11) SEQ ID NOs. 30 and 36; (12) SEQ ID NOs. 30 and 37; (13) SEQ ID NOs. 31 and 34; (14) SEQ ID NOs. 31 and 35; (15) SEQ ID NOs. 31 and 36; or (16) SEQ ID NOs. 31 and 37.

[0011] In some embodiments, the present application relates to (1) a light chain variable region (VL) comprising (a) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13 respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23 respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR; or (2) a VL comprising (a) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13 respectively, or VL The present invention provides an antibody or antigen-binding fragment that specifically binds to human BDCA2, comprising (b) a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the CDR; and / or (b) VH having the amino acid sequences shown in SEQ ID NOs: 19, 21, and 23; or a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VH CDR.

[0012] In some embodiments, the antibodies or antigen-binding fragments provided herein include VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences shown in Sequence IDs 11, 12, 13, 20, 22, and 23 as defined by Kabat.

[0013] In some embodiments, the antibodies or antigen-binding fragments provided herein include VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 19, 21, and 23 as defined by Chothia.

[0014] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 40; and / or (b) VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 41.

[0015] In some embodiments, the antibody or antigen-binding fragment provided herein comprises VL and VH having the amino acid sequences shown in SEQ ID NOs. 40 and 41.

[0016] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to human BDCA2, comprising (a) VL having the amino acid sequence shown in SEQ ID NO: 40, VL CDR1, VL CDR2, and VL CDR3 derived from VL; and / or (b) VH having the amino acid sequence shown in SEQ ID NO: 41, VH having H CDR1, VH CDR2, and VH CDR3 of VH;

[0017] In some embodiments, the antibody or its antigen-binding fragment provided herein is a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment provided herein is a humanized antibody or its antigen-binding fragment.

[0018] In some embodiments, the antibody or its antigen-binding fragment provided herein is a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment provided herein is a humanized antibody or its antigen-binding fragment.

[0019] In some embodiments, the humanized antibody or its antigen-binding fragment provided herein includes (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-46; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-50.

[0020] In some embodiments, the humanized antibody or its antigen-binding fragment provided herein comprises VL and VH having the following amino acid sequences, respectively: (1) SEQ ID NOs. 42 and 47; (2) SEQ ID NOs. 42 and 48; (3) SEQ ID NOs. 42 and 49; (4) SEQ ID NOs. 42 and 50; (5) SEQ ID NOs. 43 and 47; (6) SEQ ID NOs. 43 and 48; (7) SEQ ID NOs. 43 and 49; (8) SEQ ID NOs. 43 and 50; (9) SEQ ID NOs. 44 and 47; (10) SEQ ID NOs. 44 and 48; (11) SEQ ID NOs. 44 and 49; (12) SEQ ID NOs. 44 and 50; (13) SEQ ID NOs. 45 and 47; (14) SEQ ID NOs. 45 and 48; (15) SEQ ID NOs. 45 and 49; (16) SEQ ID NOs. 45 and 50; (17) SEQ ID NOs. 46 and 47; (18) SEQ ID NOs. 46 and 48; (19) SEQ ID NOs. 46 and 49; or (20) SEQ ID NOs. 46 and 50.

[0021] In some embodiments, the antibodies or antigen-binding fragments provided herein are selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-domain antibodies (sdAb), and heavy-chain antibodies (HCAb).

[0022] In some embodiments, the antibody or antigen-binding fragment provided herein is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody or antigen-binding fragment provided herein is an IgG1 antibody.

[0023] In some embodiments, the antibody or antigen-binding fragment provided herein includes a light chain constant region (CL) having at least 85% sequence identity with κ CL (Cκ; SEQ ID NO: 51). In some embodiments, the antibody or antigen-binding fragment provided herein includes a light chain constant region (CL) having at least 85% sequence identity with λ CL (Cλ; SEQ ID NO: 52).

[0024] In some embodiments, the IgG1 antibody or its antigen-binding fragment provided herein includes a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 56.

[0025] In some embodiments, the heavy chain constant region (CH) of the IgG1 antibody provided herein contains wild-type IgG1 CH or contains at least one amino acid mutation that enhances the antibody's ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis). In some embodiments, the CH region of the IgG1 antibody provided herein has amino acid substitutions in L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334, P396, or any combination thereof, based on EU index numbers. In some embodiments, the CH region of the IgG1 antibody provided herein has amino acid substitutions of L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, or P396L, or any combination thereof, based on EU index numbers.

[0026] In some embodiments, the CH region of the IgG1 antibody provided herein is based on the EU index number, (i) S298A, E333A, and K334A; (ii) S239D and I332E; (iii) S239D, A330L, and I332E; (iv) G236A; (v) G236A, S239D, and I332E; (vi) G236A, A330L, and I332E; (vii) G236A, S239D, A33 (viii)F243L, R292P, Y300L, V305I, and P396L; (ix)L235V, F243L, R292P, Y300L, and P396L; (x)L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A; and (xi)D270E, K326D, A330M, and K334E are modified by amino acid substitutions selected from the group. In some embodiments, the CH region has an amino acid sequence selected from SEQ ID NOs. 62-86.

[0027] In some embodiments of the antibodies provided herein, the Fc region is not fucosylated.

[0028] The present application also provides an antibody or its antigen-binding fragment that specifically binds to human BDCA2 disclosed herein, and an antibody or its antigen-binding fragment that competes for binding to human BDCA2.

[0029] Embodiment 34: An antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 33, which is a bispecific antibody or a multispecific antibody.

[0030] Embodiment 35: A monoclonal antibody or its antigen-binding fragment, as described in any one of Embodiments 1 to 34.

[0031] Embodiment 36: The antibody or its antigen-binding fragment is: (1) K 50 nM or less as measured by SPR D An antibody or antigen-binding fragment according to any one of Embodiments 1 to 35, wherein (1) binds to human BDCA2; (2) inhibits the release of interferon-alpha (IFNα) from peripheral blood mononuclear cells (PBMCs); (3) selectively binds to plasmacytoid dendritic cells (pDCs) in human PBMCs; (4) exhibits natural killer cell (NK)-dependent ADCC activity against BDCA2-expressing cells; (5) exhibits neutrophil-dependent ADCC activity against BDCA2-expressing cells; or (6) exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

[0032] Embodiment 37: The antibody or its antigen-binding fragment (1) has a K of 50 nM or less as measured by SPR. Dbind to human BDCA2; (2) inhibit the release of IFNα from PBMC; (3) selectively bind to pDCs in human PBMC; (4) exhibit natural killer cell (NK)-dependent ADCC activity against BDCA2-expressing cells; (5) exhibit neutrophil-dependent ADCC activity against BDCA2-expressing cells; or (6) exhibit macrophage-dependent ADCP activity against BDCA2-expressing cells; or an antibody or an antigen-binding fragment thereof that specifically binds to human BDCA2, which is any combination of (1) to (6).

[0033] Embodiment 38: The antibody or an antigen-binding fragment thereof inhibits the release of IFNα in CpG-stimulated PBMC with an IC 50 of 0.008 nM or less; (2) inhibits the release of IFNα in IC-stimulated PBMC with an IC 50 of 0.0006 nM or less; (3) exhibits NK-dependent ADCC activity against BDCA2-expressing cells with an EC 50 of 0.01 nM or less as measured by a reporter assay; (4) exhibits NK-dependent ADCC activity against BDCA2-expressing cells with an EC 50 of 0.001 nM or less as measured by a cytotoxicity activity assay; (5) exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells with an EC 50 of 0.2 nM or less as measured by a reporter assay; or (6) exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells with an EC 50 of 2 nM or less as measured by a phagocytosis assay; or an antibody or an antigen-binding fragment thereof according to Embodiment 36 or 37, which is any combination of (1) to (6).

[0034] The antibody or an antigen-binding fragment thereof inhibits the release of IFNα in CpG-stimulated PBMC with an IC 50 in the range of 0.0005 nM to 0.008 nM; (2) inhibits the release of IFNα in IC-stimulated PBMC with an IC 50(3) inhibits IFNα release in IC-stimulated PBMCs; (4) EC in the range of 0.001 nM to 0.01 nM as measured by a reporter assay. 50 (4) EC2 in the range of 0.0001 nM to 0.001 nM, as measured by a cytotoxicity assay. 50 (5) 50 (6) EC2 in the range of 0.1 nM to 2 nM as measured by a phagocytic assay. 50 The antibody or antigen-binding fragment according to Embodiment 38, which exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

[0035] In some embodiments, the antibody or antigen-binding fragment provided herein exhibits neutrophil-dependent ADCC activity.

[0036] In some embodiments, the Application provides polynucleotides encoding polypeptides of antibodies or antigen-binding fragments disclosed herein.

[0037] In some embodiments, the present application provides vectors comprising polynucleotides disclosed herein.

[0038] In some embodiments, the present application provides a host cell comprising a polynucleotide disclosed herein or a vector disclosed herein.

[0039] In some embodiments, the host cells disclosed herein are (1) overexpressing N-acetylglucosaminyltransferase III (GnTIII), (2) lacking α-1,6-fucosyltransferase (FUT8), or (3) having a low fucose content, or any combination of (1) to (3).

[0040] In some embodiments, the present application provides a method for producing an antibody or an antigen-binding fragment that specifically binds to human BDCA2, comprising culturing the cells disclosed herein under conditions that enable the expression of the antibody or antibody fragment. In some embodiments, the method provided herein comprises isolating the antibody from the culture.

[0041] In some embodiments, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-BDCA2 antibody disclosed herein or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0042] In some embodiments, the present application provides a method for reducing type I interferon (IFN) levels in a subject requiring such reduction, comprising administering a therapeutically effective dose to the subject of the anti-BDCA2 antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, the type I interferon is IFNα.

[0043] In some embodiments, the present application provides a method for suppressing or depleting pDCs in a subject in need, comprising administering a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein to the subject.

[0044] In some embodiments, the present application provides a method for reducing autoimmunity in a subject in need, comprising administering to the subject an effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein.

[0045] In some embodiments, the subject has an autoimmune disease.

[0046] In some embodiments, the present application provides a method for treating autoimmune diseases associated with type I IFN or pDC in subjects in need, comprising administering to a subject a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein.

[0047] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).

[0048] In some embodiments, the method provided herein further comprises administering other therapeutic agents to the subject.

[0049] In some embodiments of the method provided herein, the subject is a human being.

[0050] In some embodiments, the present application provides the use of an anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for reducing type I IFN.

[0051] In some embodiments, the present application provides the use of an anti-BDCA2 antibody or its antigen-binding fragment disclosed herein in the manufacture of a pharmaceutical product that reduces type I IFN.

[0052] In some embodiments, the type I IFN is IFNα.

[0053] In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for inhibiting or depleting pDCs. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein in the manufacture of a pharmaceutical product that inhibits or depletes pDCs.

[0054] In some embodiments, the present application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for reducing autoimmunity. In some embodiments, the present application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein in the manufacture of a pharmaceutical product that reduces autoimmunity.

[0055] In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for the treatment of autoimmune diseases associated with type I IFN or pDC. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein in the manufacture of a pharmaceutical product for the treatment of autoimmune diseases associated with type I IFN or pDC.

[0056] In some embodiments, the autoimmune disease is SLE. [Brief description of the drawing] Figure 1 provides representative ELISA results showing that BDCA2 chimeric antibodies (cmAb03 and cmAb05), a reference antibody (Tab), and a negative control antibody (hIgG1) bind to the human BDCA2 protein.

[0057] Figure 2 provides representative flow cytometry results showing that BDCA2 chimeric antibodies (cmAb03 and cmAb05), a reference antibody (Tab), and a negative control antibody (hIgG1) bind to CHOK1-human BDCA2 cells.

[0058] Figures 3A–3E provide representative flow cytometry results showing that BDCA2 chimeric antibodies (cmAb03 (Figure 3A) and cmAb05 (Figure 3B)), a reference antibody (Tab (Figure 3C)), and a negative control antibody (hIgG1 (Figure 3D)) bind to cell surface BDCA2 on pDCs and other immune cells in human PBMCs.

[0059] Figure 4 provides representative results of an IFNα release assay in CpG-stimulated PBMCs. The inhibitory activity of BDCA2 chimeric antibodies (cmAb03 and cmAb05) against IFNα release is measured; results from a reference antibody (Tab) and a negative control antibody (hIgG1) are also shown.

[0060] Figures 5A-5B provide ELISA results showing that the humanized forms of cmAb03 (hu03; Figure 5A) and cmAb05 (hu05; Figure 5B), the reference antibody (Tab), and the negative control antibody (isotype) bind to the human BDCA2 protein.

[0061] Figures 6A-6B provide flow cytometry results showing that the humanized anti-BDCA2 antibodies hu03 (Figure 6A) and hu05 (Figure 6B), the reference antibody (Tab), and the negative control antibody (isotype) bind to CHOK1-human BDCA2 cells.

[0062] Figure 7 shows representative results of an IFNα release assay in CpG-stimulated PBMCs. The inhibitory activity of hu03 / hu05 on IFNα release is measured as instructed; results for reference antibody (Tab) and negative control antibody (isotype) are also shown.

[0063] Figure 8 shows representative results of an IFNα release assay in PBMCs stimulated with SLE-IC. The inhibitory activity of hu03 / hu05 on IFNα release is measured as instructed; the results for the reference antibody (Tab) are also shown.

[0064] Figures 9A and 9B provide representative results from the ADCP reporter assay (Figure 9A) and phagocytosis assay (Figure 9B), which show the phagocytic activity of hu03 / hu05, the reference antibody (Tab), and the negative control antibody (isotype) as indicated.

[0065] Figures 10A–10B provide representative results from the ADCC reporter assay (Figure 10A) and the cytotoxic activity assay (Figure 10B), which show the NK cell-dependent ADCC activity of hu03 / hu05, the reference antibody (Tab), and the negative control antibody (isotype) as indicated. [Modes for carrying out the invention] The present invention provides a novel antibody (including an antigen-binding fragment) that specifically binds to BDCA2 (e.g., human BDCA2). The application also discloses a pharmaceutical composition comprising a therapeutically effective amount of the antibody or its antigen-binding fragment. The application also discloses the use of the pharmaceutical composition for treating autoimmune diseases associated with plasmacytoid dendritic cells (pDCs) and / or type I interferon (IFN).

[0066] pDCs are subpastures of dendritic cells (DCs) in peripheral blood and secondary lymphoid organs. Although they make up only about 0.1%–0.8% of peripheral blood mononuclear cells (PBMCs), these cells are drivers of both innate and adaptive immune responses. pDCs enhance the innate immune response by inducing chemokines, recruiting myeloid cells, promoting monocyte recruitment and differentiation into antigen-presenting cells (APCs), inducing dendritic cell maturation and activation, and supporting the recruitment, activation, and cytotoxicity of spontaneous killer (NK) cells. pDCs also promote the adaptive immune response by promoting antigen presentation, supporting the activation and proliferation of antigen-specific CD4+ Th cells, driving the differentiation of CD4+ T cells into Th2 and Treg cells, promoting CD8+ T cell survival and activity, and improving B cell survival, maturation, differentiation, and autoantibody production.

[0067] Importantly, pDCs are a major source of type I IFN(α / β) and promote the function of NK cells, B cells, T cells, and myeloid DCs. Both pDCs and type I IFN are known to be involved in the development of immunological conditions such as autoimmune diseases. See, for example, Annu. Rev. Pathol. Mech. Dis. 2019, 14:369-93; J Immunol 2020, 205:2941-2950; Clinic Rev Allerg Immunol 59, 248-272(2020); Front. Immunol. 12:713779; Int. J. Mol. Sci. 2021, 22, 4190; Rheumatology 2017; 56:16621675.

[0068] Blood dendritic cell antigen 2 (BDCA2), also known as member C of the C-type lectin domain family 4 (CLEC4C) or member 7 of the C-type lectin superfamily, belongs to the calcium-dependent (C-type) lectin family. As a single transmembrane protein, full-length human BDCA2 is a protein consisting of 213 amino acids (Uniprot accession number Q8WTT0, SEQ ID NO. 1), which includes a cytoplasmic domain (amino acids 1-23), a transmembrane domain (amino acids 22-44), and an extracellular domain (amino acids 45-213).

[0069] MVPEEEPQDREKGLWWFQLKVWSMAVVSILLLSVCFTVSSVVPHNFMYSKTVKRLSKLREYQQYHPSLTCVMEGKDIEDWSCCPTPWTSFQSSCYFISTGMQSWTKSQKNCSVMGADLVVINTREEQDFIIQNLKRNSSYFLGLSDPGGRRHWQWVDQTPYNENVTFWHSGEPNNLDERCAIINFRSSEEWGWNDIHCHVPQKSICKMKKIYI(Sequence ID 1) Detailed information on human BDCA2 can be found in public databases via the following IDs: HGNC:13258; NCBI Entrez Gene:13258; Ensembl:ENSG00000239961; OMIM(registered trademark):606677; UniProtKB / Swiss-Prot:Q8WTT0. For the human BDCA2 gene (Uniprot NO:Q8WTT0-1, Q8WTT0-2), two alternative splicing transcription variants encoding two different isoforms have been described.

[0070] The BDCA2 sequence of the cynomolgus monkey is shown below: MVPEEEPQDREKGVWWFQLKVWSVAVVSILLLCVCFTVSSVASHNFMYSKTVKRLSKLQEYQQYYPSLTCVMEGKDMEDWSCCPTPWTSFQSSCYFISTVMQSWTKSQNNCSVMGADLVVINTKEEQDFITQNLKINSAYFLGLSDPKGWRHWQWVDQTPYNKNVTFWHSGEPNSPDERCAIINFRSEEWGWNDVHCHVPQKSICKMKKIYI(Sequence ID 7) BDCA2 is expressed only on the surface of pDCs and not on myeloid DCs or other peripheral blood leukocytes. Not bound by theory, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is useful for reducing pDC activity via NK / neutrophil-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) and / or macrophage-mediated ADCP (antibody-dependent cell-mediated phagocytosis), and is therefore useful for the treatment and prevention of (e.g.) autoimmune diseases.

[0071] Before further describing the present invention, it should be understood that this disclosure is not limited to the specific embodiments described herein, and that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit them.

[0072] 1.1 Definition Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings that are ordinarily understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms. Generally, the terms and techniques of the cell and tissue cultures, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0073] The term "one" refers to one or more of these entities; for example, "one antibody" should be understood to mean one or more antibodies.

[0074] As used in this Application, the term “and / or” is deemed to specifically disclose both the case in which each of the two specified features or components has the other and the case in which it does not. Accordingly, as used in this Application, the term “and / or” as used in the phrase “A and / or B” is intended to include “A and B,” “A or B,” “(alone) “A,” and “(alone) “B.” Similarly, as used in the phrase “A, B and / or C” is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; (alone) A; (alone) B; and (alone) C.

[0075] As used in this application, the term “approximately” is used to indicate a value that includes error variation inherent in the measurement device, the method of determining the value, or the variability in the subject of study. The term “approximately” encompasses the exact numerical value stated. In some embodiments, “approximately” means within positive or negative 10% of a given value or range. In certain embodiments, “approximately” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by “approximately”. In some embodiments, “approximately” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by “approximately”.

[0076] As used in this application, the term “antibody” and its grammatical equivalent refer to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, the antigen-binding site of which is usually located within the variable region of the immunoglobulin molecule. As used in this application, the term includes, but is not limited to, complete polyclonal antibodies, complete monoclonal antibodies, single-domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAb), light-chain antibodies (LCAb), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and other modified immunoglobulin molecules containing an antigen-binding site (e.g., bivariable-domain immunoglobulin molecules), insofar as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies may be any one of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM or their subtypes (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), based on the characteristics of their heavy chain constant regions, each known as α, δ, ε, γ, and μ, respectively. Unless otherwise specified, the term “antibody” as used in this application includes the “antigen-binding fragment” of a complete antibody. The term “antigen-binding fragment” as used in this application refers to a portion or fragment of a complete antibody that functions as the antigen-determining variable region of the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy-chain antibodies (HCAb), light-chain antibodies (LCAb), disulfide-bonded scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, bivariate region antibodies (DVD), single variable region antibodies (sdAb; e.g., camel antibody, alpaca antibody), single variable region of heavy-chain antibodies (VHH), and bispecific or multispecific antibodies formed from antigen fragments. A "bispecific" antibody is an artificial hybrid antibody having two different antigen-binding sites that recognize and specifically bind to two different targets.Bispecific antibodies can be produced by various methods, including hybridoma fusion and Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0077] As used in this application, the term "humanized antibody" refers to a non-human (e.g., mouse) antibody form as a specific immunoglobulin chain containing minimal non-human sequences, a chimeric immunoglobulin, or a fragment thereof. Generally, a humanized antibody is a human immunoglobulin. In some cases, the Fv framework region residues of a human immunoglobulin are substituted with corresponding residues from an antibody of a non-human species. In some cases, the CDR residues are substituted with residues from a CDR of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and / or binding ability. Humanized antibodies can be further modified to improve and optimize the antibody's specificity, affinity, and / or binding ability by substituting other residues within the Fv framework region and / or within the substituted non-human residues. As used in this application, the term "human antibody" refers to an antibody produced by a human, or an antibody prepared using any technique known in the art that has an amino acid sequence corresponding to an antibody produced by a human.

[0078] In the context of antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, with a variable region of approximately 120-130 or more amino acids in its amino-terminus and a constant region in its carboxyl-terminus. Based on the amino acid sequence of the heavy chain's constant region, the constant region can be one of five types known as α, δ, ε, γ, and μ. Different heavy chains differ in size, with α, δ, and γ containing approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains form five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM, which include four IgG subclasses: IgG1, IgG2, IgG3, and IgG4. The heavy chain may also be a human heavy chain.

[0079] In the context of antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, with a variable region of approximately 100-110 amino acids or more in the amino-terminal region and a constant region in the carboxyl-terminal region. The total length of a light chain is approximately 211-217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types called κ or λ. The amino acid sequences of light chains are well known in this art. The light chain may also be a human light chain.

[0080] The term "variable domain" or "variable region" generally refers to the light or heavy chain portion of an antibody located at the amino-terminus of the light or heavy chain, approximately 120–130 amino acids long in the heavy chain and approximately 100–110 amino acids long in the light chain, which each specific antibody utilizes for binding to and specificity of its particular antigen. The sequences of variable domains vary widely among different antibodies. Sequence variations are concentrated in the complementarity-determining region (CDR), while less mutated portions within the variable domain are called framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. The amino acid position numbers used in this application follow the EU index described in Kabat et al. (1991) Sequences of proteins of immunological interest. (USD Department of Health and Human Services, Washington, DC), 5th edition. The variable region may also be a human variable region.

[0081] CDR refers to one of the three highly variable regions (H1, H2, H3) within the non-framework region of the VHβ-sheet framework of an immunoglobulin (Ig or antibody), or one of the three highly variable regions (L1, L2, L3) within the non-framework region of the VLβ-sheet framework of an antibody. Therefore, CDRs are variable region sequences scattered within the framework region sequence. CDR regions are well known to those skilled in the art and are defined by various methods and systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines the most mutable region within the antibody variable (V) domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). Chothia's definition is based on the location of the structural loop region and defines the CDR region sequence as a residue that is not part of the conserved β-sheet framework and therefore can take on different three-dimensional structures (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terms are widely recognized in the art. Furthermore, the IMGT system is based on the degree and location of sequence variation within the variable region structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. Software programs (e.g., abYsis) are available and well known to those skilled in the art for analyzing antibody sequences and determining CDRs. The location of CDRs within typical antibody variable domains has been determined through multiple structural comparisons (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because the number of residues in the highly variable region differs between different antibodies, additional residues at typical positions are usually numbered by a, b, c, etc., following the residue number in a typical variable domain numbering scheme (Al-Lazikani et al., op. cit. (1997)). These nomenclature schemes are also well known to those skilled in the art.

[0082] For example, CDRs defined based on the Kabat (highly variable) or Chothia (structure) nomenclature are shown in the table below.

[0083] [Table 1]

[0084] 1 Residue numbers follow the nomenclature of Kabat et al. (mentioned above). 2 Residue numbers follow the nomenclature of Chothia et al. (mentioned above). It is also possible to introduce one or more CDRs into a molecule covalently or noncovalently to form an immunoadhesin. The CDR can be introduced as part of a larger polypeptide chain, covalently to another polypeptide chain, or noncovalently. The CDR allows the immunoadhesin to bind to a specific antigen of interest. The CDR region can be analyzed, for example, through the abysis website (http: / / abysis.org / ).

[0085] The terms “epitope” and “antigenic determinant” are used interchangeably in this application and refer to a site on the surface of a target molecule to which an antibody or its antigen-binding fragment binds, i.e., a local region on the antigen surface. The target molecule may include proteins, peptides, nucleic acids, carbohydrates, or lipids. An immunogenic epitope is a part of a target molecule that elicits an immune response in animals. An antigenic epitope of a target molecule is a part of the target molecule to which an antibody binds, as determined by any method well known in the art, such as immunoassay. Antigenic epitopes are not necessarily immunogenic. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and have specific stereochemical and electrical characteristics. The term “epitope” includes both linear epitopes and stereochemical epitopes. The region of a target molecule (e.g., polypeptide) that contributes to an epitope may be a sequence of amino acids in the polypeptide, or the epitope may be formed collectively from two or more discontinuous target molecule regions. Epitopes may or may not be three-dimensional surface features of the target molecule. When a protein denatures, epitopes formed by a sequence of amino acids (also called linear epitopes) are usually retained, while epitopes formed by the folding of the three-dimensional structure (also called structural epitopes) are usually lost. Epitopes typically contain at least three, and more commonly at least five, six, seven, or eight to ten, amino acids, each with a unique spatial structure.

[0086] As used in this application, the term “specifically binding” means that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with higher affinity, or a combination thereof, compared to alternative substances including related or unrelated proteins. For example, a binding site (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by immunoassay, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific reaction is at least twice the background signal or noise, and may be more than 10 times the background. For a discussion of antibody specificity, see, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336. A binding site that specifically binds to a target molecule can bind to the target molecule with higher affinity than its affinity to different molecules. In some embodiments, a binding site that specifically binds to a target molecule can bind to the target molecule with an affinity at least 20, 30, 40, 50, 60, 70, 80, 90, or at least 100 times higher than its affinity to a different molecule. In some embodiments, a binding site that specifically binds to a particular target molecule binds to a different molecule with an affinity undetectable by the measurement methods described herein or more known in the art. In some embodiments, "specifically binds" means that the binding site has a K of about 0.1 mM or less. D This means binding to the target molecule. In some embodiments, "specifically binding" means that the polypeptide or molecule has a K content of about 10 μM or less, or about 1 μM or less. D This means binding to the target. In some embodiments, "specifically binding" means that the polypeptide or molecule has a K content of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. DThis refers to binding to a target. Due to sequence homology between homologous proteins in different species, specific binding may include polypeptides or molecules that recognize proteins or targets in multiple species. Similarly, due to homology in specific regions within the polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize multiple proteins or targets. It should be understood that in some embodiments, a binding site (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, the expression "specifically binds" does not necessarily require (but may include) exclusive binding (i.e., binding to a single target). For this reason, in some embodiments, a binding site (e.g., an antibody) may specifically bind to multiple targets. For example, an antibody may contain two identical antigen-binding sites, each specifically binding to the same epitope on two or more proteins. In some alternative embodiments, an antibody may be bispecific and contain at least two antigen-binding sites with different specificities.

[0087] As used in this application, the term "binding affinity" generally refers to the overall strength of the non-covalent interaction between the binding site and the target molecule (e.g., antigen). Binding between the binding site and the target molecule is a reversible process, and binding affinity is usually expressed using the equilibrium dissociation constant (K). D ) is reported as K. D The dissociation rate (k off or K d ) and bond rate (k on or k a This is the ratio of the bond pairs. D The lower the value, the higher the affinity. Various methods for measuring binding affinity are known in the art and can be used for the purposes of this disclosure. Specific exemplary embodiments are as follows: In some embodiments, "K D " or "K D The value can be measured by assays known in the field, such as binding assays. DThis can be measured by radiolabeled antigen binding assay (RIA) (Chen et al., (1999) J. Mol Biol 293:865-881). Also, K D or K D The values ​​can also be measured by biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). Furthermore, K D or K D The values ​​can also be measured by surface plasmon resonance assay (SPR) using Biacore, such as BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Binding affinity is EC 50 It can also be quantified by EC 50 This is the ligand concentration at which half of the target molecules are bound in the binding assay.

[0088] The terms “polypeptide,” “peptide,” “protein,” and their grammatical equivalents, as used interchangeably in this application, refer to linear or branched amino acid polymers of any length. These may include unnatural or modified amino acids and may be interrupted by non-amino acids. Furthermore, polypeptides, peptides, or proteins may be modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other operations or modifications.

[0089] The term “mutant” in this application, with respect to a protein or polypeptide having specific sequence features (“reference protein” or “reference polypeptide”), refers to a different protein or polypeptide having one or more (e.g., about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions compared to the reference protein or reference polypeptide. Amino acid sequence changes may be amino acid substitutions. Amino acid sequence changes may be conserved amino acid substitutions. Amino acid sequence changes may be amino acid deletions. The mutant may be a fragment of the control protein or polypeptide. Functional mutants of a protein or polypeptide retain the basic structural and functional properties of the reference protein or polypeptide.

[0090] The terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents, as used interchangeably in this application, refer to nucleotide polymers or oligomers of any length. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base (such as methylated, hydroxymethylated, or glycosylated), a non-natural nucleotide, a non-nucleotide building block exhibiting a structure and / or function similar to a natural nucleotide (i.e., a “nucleotide analog”), and / or any substrate that can be introduced into the polymer by a DNA or RNA polymerase. The nucleic acid or polynucleotide may be heterogeneous or homogeneous in composition and may be isolated from natural sources, or produced artificially or synthetically. Furthermore, the nucleic acid may be DNA or RNA or a mixture thereof and may exist permanently or transiently in single-stranded or double-stranded forms (including homo-double-stranded, hetero-double-stranded, and hybrid states). Nucleic acid structures include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 4(14): 4503-4510 (2002) and U.S. Patent Nos. 5,034,506), loc nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122: 8595-8602 (2000)), and / or ribozymes.

[0091] In this application, as used in the context of two or more polynucleotides or polypeptides, “identical,” “percentage of identity,” and their grammatical equivalents refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence (with gaps introduced as necessary), and where conserved amino acid substitutions are not considered part of sequence identity. Percentage identity can be measured by sequence comparison software or algorithms, or by visual inspection. A variety of algorithms and software available for obtaining alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin software packages and their variations. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning that when compared and aligned for maximum correspondence, as measured by a sequence comparison algorithm or visual inspection, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity. In some embodiments, the identity exists within an amino acid sequence region whose length is at least about 10 residues, at least about 20 residues, at least about 40–60 residues, at least about 60–80 residues, or any integer value in between. In some embodiments, the identity exists within a region exceeding 60–80 residues, e.g., at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (e.g., the coding region of a target protein or antibody). In some embodiments, identity exists within a nucleotide sequence region having a length of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value in between.In some embodiments, identity exists within a region exceeding 60 to 80 bases, for example, a region of at least about 8 to 1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (such as the nucleotide sequence encoding the target protein).

[0092] As used in this application, the term "vector" and its grammatical equivalent refer to a medium that carries genetic material (e.g., polynucleotide sequences) that can be introduced into a host cell and replicated and / or expressed within the host cell. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selective sequences or markers that function for stable integration into host cell chromosomes. Furthermore, the vector may contain one or more selective marker genes and appropriate expression regulatory sequences. Possible selective marker genes provide, for example, antibiotic or toxin resistance, nutritional complementarity, or supply of essential nutrients not present in the culture medium. Expression regulatory sequences may include constitutive and inductive promoters, transcription enhancers, transcription termination sequences, etc., which are well known in the art. When two or more polynucleotides are co-expressed, the two polynucleotides may be inserted, for example, into a single expression vector or into different expression vectors. In the case of single-vector expression, the encoding polynucleotide may be functionally linked to a common expression regulatory sequence or to different expression regulatory sequences such as an inductive promoter and a constitutive promoter. The introduction of polynucleotides into host cells can be confirmed using methods well known in the art. Those skilled in the art will understand that polynucleotides can be expressed in amounts sufficient to produce the desired product (e.g., the anti-BDCA2 antibody or its antigen-binding fragment described herein), and that methods well known in the art can be used to optimize the expression level to obtain sufficient expression.

[0093] As used in this application, the term “code” and its grammatical equivalent refer to the inherent property of a particular nucleotide sequence in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, to function as a template in a biological process for synthesizing a limited nucleotide sequence (e.g., rRNA, tRNA, mRNA) or a limited amino acid sequence and other polymers or macromolecules having the resulting biological properties. Therefore, if a protein is produced by the transcription and translation of the mRNA corresponding to that gene, that gene codes for that protein. Unless otherwise specified, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate in form and code for the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0094] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions refer to polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the point where they are no longer in a form found in nature. In some embodiments, isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0095] As used in this application, “treatment” and its grammatical equivalents, relating to a disease or condition, or a subject having a disease or condition, refer to an act of suppressing, eliminating, reducing, and / or improving the symptoms, severity of symptoms, and / or frequency of symptoms related to the disease or condition being treated.

[0096] As used in this Application, the term “administration” and its grammatical equivalents refer to the act of delivering a therapeutic agent or pharmaceutical composition to the body of a subject, or causing delivery to the body of a subject, by the method described herein or by other methods known in the art. The therapeutic agent may be a compound, polypeptide, antibody, cell, or cell population. Administration of a therapeutic agent or pharmaceutical composition includes the act of prescribing a therapeutic agent or pharmaceutical composition to be delivered to the body of a subject. Exemplary forms of administration include oral forms such as tablets, capsules, syrups, and suspensions; injectable forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP) injectable forms; transdermal forms including creams, gels, powders, or patches; oral forms; powder inhalation, sprays, suspensions, and rectal suppositories.

[0097] As used in this application, “effective dose,” “therapeutic effective dose,” and their grammatical equivalents refer to administering a reagent to a subject in an amount that, when administered to the subject, produces a detectable beneficial effect against any symptom, aspect, or characteristic of a disease, disorder, or pathological condition, either alone or as part of a pharmaceutical composition, and as a single dose or as part of a series of doses. The therapeutic effective dose is determined by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, based on the subject’s age, weight, general health, the severity of the condition being treated, the clinician’s judgment, etc. Those skilled in the art can determine the appropriate “effective dose” in individual cases using conventional experiments.

[0098] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a material that is suitable for drug administration to an individual together with an activator and does not cause any harmful or undesirable biological effects or interactions with any other components of the pharmaceutical composition.

[0099] As used in this application, the term "subject" refers to any animal (e.g., mammal) that is the recipient of a particular treatment, and this includes (but is not limited to) humans, non-human primates, dogs, cats, rodents, etc. A subject may be human. A subject may have a particular disease or condition.

[0100] Scope: Throughout the disclosure of the present invention, various aspects of the invention are presented in range form. It should be understood that the range form is for convenience and brevity and should not be interpreted as a rigid limitation on the scope of the invention. Accordingly, a range description should be considered to specifically disclose all possible subranges and individual numerical values ​​that fall within that range. For example, a range description of 1 to 6 is considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values ​​within those ranges (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6). This applies regardless of the width of the range.

[0101] In this application, exemplary genes and polypeptides are described with reference to GenBank accession numbers, GI numbers, and / or sequence numbers. Those skilled in the art should understand that homologous sequences can be readily identified by referring to sequence sources (not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / )).

[0102] 1.2 Anti-BDCA2 antibody and antigen-binding fragment This application provides an antibody or an antigen-binding fragment thereof that specifically binds to BDCA2 (e.g., human BDCA2). In some embodiments, this application provides an anti-BDCA2 antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided in this application may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0103] In some embodiments, the present application provides antigen-binding fragments of anti-BDCA2 antibodies. In some embodiments, the antigen-binding fragments provided herein may be single-domain antibodies (sdAb), heavy-chain antibodies (HCAb), Fab, Fab', F(ab')2, Fv, single-chain variable fragments (scFv), or (scFv)2. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is a single-domain antibody (sdAb). In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is a heavy-chain antibody (HCAb). In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is Fab. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is Fab'. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is F(ab')2. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is Fv. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is scFv. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is a disulfide-linked scFv[(scFv)2]. In some embodiments, the antigen-binding fragment of the anti-BDCA2 antibody is a dAb.

[0104] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a recombinant antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a monoclonal antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a polyclonal antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises an antibody or its antigen-binding fragment from a camelid animal (e.g., camel, dromedary, llama). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a chimeric antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a humanized antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a human antibody or its antigen-binding fragment. In some embodiments, the application provides anti-BDCA2 human scFv.

[0105] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is isolated. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is substantially pure.

[0106] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a multispecific antibody or its antigen-binding fragment. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a bispecific antibody or its antigen-binding fragment. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises the anti-BDCA2 antibody or its antigen-binding fragment provided herein. In some embodiments, the bispecific antibody or its antigen-binding fragment comprises the anti-BDCA2 scFv provided herein.

[0107] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein includes a monovalent antigen-binding site. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment includes a single-specific binding site. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment includes a bivalent binding site.

[0108] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those skilled in the art. One exemplary method is screening a protein expression library, e.g., a phage display or ribosome display library. Phage displays are described (e.g.) Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; and WO 92 / 18619. In some embodiments, recombinant monoclonal antibodies are isolated from a phage display library expressing a variable region or CDR of a desired species. Screening of phage libraries can be achieved by various techniques known in the art.

[0109] In some embodiments, monoclonal antibodies are modified using DNA recombination technology to generate surrogate antibodies. In some embodiments, chimeric antibodies are generated by substituting the constant regions of the light and heavy chains of a mouse monoclonal antibody with the constant regions of a human antibody. In some embodiments, the constant regions are shortened or removed to generate desired antibody fragments from monoclonal antibodies. In some embodiments, site-directed mutagenesis or high-density mutagenesis of the variable region is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0110] In some embodiments, the present application provides anti-BDCA2 antibody clones Ab03 and Ab05. The sequence characteristics are as follows: The specific CDR sequences as defined herein are typically based on Kabat or Chothia's definitions. However, any general reference to one or more heavy-chain CDRs and / or one or more light-chain CDRs of a particular antibody should be understood to include all CDR definitions known to those skilled in the art. In some embodiments, the present application provides anti-BDCA2 antibodies having VL CDRs and / or VH CDRs of antibody clones Ab03 or Ab05 disclosed herein, wherein the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDR is defined by Kabat (as detailed below). In some embodiments, the CDR is defined by Chothia (as detailed below). In some embodiments, the CDR is defined by IMGT. In some embodiments, the CDR is defined by AbM. In some embodiments, the CDR is defined by Contact.

[0111] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is an antibody designated as cmAb03 (chimeric Ab03). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VL derived from cmAb03 (SEQ ID NO: 26). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VH derived from cmAb03 (SEQ ID NO: 27). The anti-BDCA2 antibody or its antigen-binding fragment provided herein may have both a VL and a VH derived from cmAb03. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VL containing VL CDRs 1, 2, and 3 of the VL derived from cmAb03 (SEQ ID NO: 26). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VH containing VH CDRs 1, 2, and 3 of the VH derived from cmAb03 (SEQ ID NO: 27). The anti-BDCA2 antibody or its antigen-binding fragment provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, wherein the VL CDRs 1, 2, and 3 and VH CDRs 1, 2, and 3 are derived from the VL and VH of cmAb03, respectively. The CDRs can be defined by any system known in the art. In some embodiments, the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDRs are defined by Kabat or Chothia as described in detail herein. In some embodiments, the CDRs are defined by IMGT. In some embodiments, the CDRs are defined by AbM. In some embodiments, the CDRs are defined by Contact.

[0112] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a variant of cmAb03. The cmAb03 variant may have a VL variant of cmAb03, which has up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 26. The cmAb03 variant may have a VL variant of cmAb03, which has up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 26. The cmAb03 variant may have a VH variant of cmAb03, which has up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 27. The cmAb03 variant may have a VH variant of cmAb03, which has up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 27. The amino acid substitutions, additions, and / or deletions may be within the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions, and / or deletions are not within the CDR. In some embodiments, variants of cmAb03 have up to about five conserved amino acid substitutions. In some embodiments, variants of cmAb03 have up to three conserved amino acid substitutions. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a humanized antibody or its antigen-binding fragment derived from cmAb03. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a human antibody or its antigen-binding fragment derived from cmAb03.

[0113] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises 1, 2, 3, 4, 5, and / or 6 CDRs of any of the antibodies described herein. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a light chain variable region (VL), the light chain variable region comprising 1, 2, and / or 3 light chain CDRs (VL CDRs) shown in Table 1a. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a heavy chain variable region (VH), the heavy chain variable region comprising 1, 2, and / or 3 heavy chain CDRs (VH CDRs) shown in Table 2a. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises 1, 2, and / or 3 VL CDRs shown in Table 1a and 1, 2, and / or 3 VH CDRs shown in Table 2a.

[0114] [Table 2]

[0115] [Table 3]

[0116] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 8; (2) VL CDR2 having the amino acid sequence shown in SEQ ID NO: 9; and / or (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 10; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 14 or 15; (2) VH CDR2 having the amino acid sequence shown in SEQ ID NO: 16 or 17; and / or (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 18; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0117] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 8; (2) VL CDR2 having the amino acid sequence shown in SEQ ID NO: 9; or (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 10; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 8; (2) VL CDR2 having the amino acid sequence shown in SEQ ID NO: 9; and (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 10; or a variant thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0118] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VL that specifically binds to BDCA2, wherein the VL comprises VL CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively, as defined by Kabat; or comprises variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VL that specifically binds to BDCA2, wherein the VL comprises VL CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively, as defined by Chothia; or comprises variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0119] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, including: (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 14 or 15; (2) VH CDR2 having an amino acid sequence shown in SEQ ID NO: 16 or 17; or (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 18; or a variant thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 14 or 15; (2) VH CDR2 having an amino acid sequence shown in SEQ ID NO: 16 or 17; and (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 18; or a variant thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0120] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VH that specifically binds to BDCA2, wherein the VH comprises VH CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NOs. 15, 17, and 18, respectively, as defined by Kabat; or comprises variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VH that specifically binds to BDCA2, wherein the VH comprises VH CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs. 14, 16, and 18, respectively, as defined by Chothia; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0121] In some embodiments, the present application provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, comprising (a) VL, including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) VH, including VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 18, respectively; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0122] In some embodiments, the present application provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, including VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 15, 17, and 18, respectively, as defined by Kabat; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the CDR.

[0123] In some embodiments, the present application provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, comprising (a) VL, comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) VH, comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 14, 16, and 18, respectively; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0124] In some embodiments, the present application provides antibodies or antigen-binding fragments thereof that specifically bind to BDCA2, including VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 14, 16, and 18, respectively, as defined by Chothia; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the CDR.

[0125] [Table 4] JPEG2026520857000005.jpg142169

[0126] [Table 5]

[0127] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0128] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26; and (b) VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, the antibody or antigen-binding fragment comprising VL and VH, the VL and VH having the amino acid sequences shown in SEQ ID NOs. 26 and 27, respectively.

[0129] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 85% sequence identity with SEQ ID NO: 26. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 90% sequence identity with SEQ ID NO: 26. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 95% sequence identity with SEQ ID NO: 26. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 98% sequence identity with SEQ ID NO: 26. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 26.

[0130] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 85% sequence identity with SEQ ID NO: 27. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 90% sequence identity with SEQ ID NO: 27. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 95% sequence identity with SEQ ID NO: 27. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 98% sequence identity with SEQ ID NO: 27. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: VH having the amino acid sequence shown in SEQ ID NO: 27.

[0131] In some embodiments, the Application provides a humanized antibody of cmAb03 (i.e., humanized Ab03, hu-cmAb03, hu-Ab03, or hu03). In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VL having an amino acid sequence selected from SEQ ID NOs. 28-31. In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VH having an amino acid sequence selected from SEQ ID NOs. 34-37. In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VL having an amino acid sequence selected from SEQ ID NOs. 28-31, and a VH having an amino acid sequence selected from SEQ ID NOs. 34-37. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a variant of humanized Ab03 provided herein. The aforementioned mutant may have a VL, which is a variant of humanized Ab03 VL having a maximum of approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs. The aforementioned mutant may have a VL, which is a variant of humanized Ab03 VL having a maximum of approximately 5 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs. The aforementioned mutant may have a VH, which is a variant of humanized Ab03 VH having a maximum of approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs. The aforementioned mutant may have a VH, which is a variant of humanized Ab03 VH having up to approximately five amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs: 34-37. In some embodiments, the variant of humanized Ab03 has up to approximately five conserved amino acid substitutions.

[0132] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, including the following: (a) VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 28-31; and / or (b) A VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.

[0133] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, the antibody or antigen-binding fragment comprising VL and VH, wherein VL and VH have the amino acid sequences shown in SEQ ID NOs. 28 and 34, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 28 and 35, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 28 and 36, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 28 and 37, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 29 and 34, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 29 and 35, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 29 and 36, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 29 and 37, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 30 and 34, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 30 and 35, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 30 and 36, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 30 and 37, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 31 and 34, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 31 and 35, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 31 and 36, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 31 and 37, respectively.

[0134] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 28. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 28. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 28. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 28. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 28.

[0135] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 29. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 29. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 29. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 29. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 29.

[0136] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 30. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 30. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 30. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 30. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 30.

[0137] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 31. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 31. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 31. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 31. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 31. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 31.

[0138] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 34. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 85% sequence identity with SEQ ID NO: 34. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 90% sequence identity with SEQ ID NO: 34. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 95% sequence identity with SEQ ID NO: 34. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 34. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 34.

[0139] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 85% sequence identity with SEQ ID NO: 35. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 90% sequence identity with SEQ ID NO: 35. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 95% sequence identity with SEQ ID NO: 35. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 35. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 35.

[0140] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 85% sequence identity with SEQ ID NO: 36. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 90% sequence identity with SEQ ID NO: 36. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 95% sequence identity with SEQ ID NO: 36. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 36. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 36.

[0141] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 85% sequence identity with SEQ ID NO: 37. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 90% sequence identity with SEQ ID NO: 37. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VH having at least 95% sequence identity with SEQ ID NO: 37. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 37. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 37.

[0142] In some embodiments, the present application provides an anti-BDCA2 antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment comprising a VL CDR derived from a VL (SEQ ID NO: 26, 28, 29, 30, or 31) described in the present application and / or a VH CDR derived from a VH (SEQ ID NO: 27, 34, 35, 36, or 37) described in the present application. Methods for identifying CDRs are well known in the art. For example, a publicly available software program (abYsis) is known to those skilled in the art for antibody sequence analysis and CDR determination.

[0143] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) VL comprising VL CDRs 1, 2, and 3 derived from VL having the amino acid sequence shown in SEQ ID NO: 26; and / or (b) VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 27.

[0144] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequences of SEQ ID NOs. 28-31; and / or (b) a VH comprising VH CDRs 1, 2, and 3 derived from a VH having an amino acid sequence selected from SEQ ID NOs. 34-37.

[0145] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VL CDR 1, 2, and 3 derived from VL having the amino acid sequence shown in SEQ ID NO: 31.

[0146] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 34. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: VH CDR 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 37.

[0147] In some embodiments, the present application provides an anti-BDCA2 antibody or its antigen-binding fragment, comprising the antibody or its antigen-binding fragment, a VL CDR derived from the VL (SEQ ID NOs. 26, 28, 29, 30, or 31) described in the present application, and / or a VH CDR derived from the VH (SEQ ID NOs. 27, 34, 35, 36, or 37) described in the present application. Methods for identifying CDRs are well known in the art. For example, software programs (abYsis) available on publicly accessible websites are known to those skilled in the art for antibody sequence analysis and CDR determination.

[0148] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) VL comprising VL CDRs 1, 2, and 3 derived from VL having the amino acid sequence shown in SEQ ID NO: 26; and / or (b) VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 27.

[0149] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequences of SEQ ID NOs. 28-31; and / or (b) a VH comprising VH CDRs 1, 2, and 3 derived from a VH having an amino acid sequence selected from SEQ ID NOs. 34-37.

[0150] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL containing VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VL CDR 1, 2, and 3 derived from VL having the amino acid sequence shown in SEQ ID NO: 31.

[0151] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 34. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: VH CDR 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 37.

[0152] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is an antibody designated as cmAb05 (chimeric Ab05). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VL derived from cmAb05 (SEQ ID NO: 40). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VH derived from cmAb05 (SEQ ID NO: 41). The anti-BDCA2 antibody or its antigen-binding fragment provided herein may have both a VL and a VH derived from cmAb05. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VL containing VL CDRs 1, 2, and 3 of the VL (SEQ ID NO: 40) derived from cmAb05. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a VH containing VH CDRs 1, 2, and 3 of the VH (SEQ ID NO: 41) derived from cmAb05. The anti-BDCA2 antibody or its antigen-binding fragment provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, wherein the VL CDRs 1, 2, and 3 and VH CDRs 1, 2, and 3 are derived from the VL and VH of cmAb05, respectively. The CDRs can be determined by any system definition known in the art. In some embodiments, the CDRs are defined by Kabat, Chothia, IMGT, AbM, or Contact. In some embodiments, the CDRs are defined by Kabat or Chothia, as described in detail herein. In some embodiments, the CDRs are defined by IMGT. In some embodiments, the CDRs are defined by AbM. In some embodiments, the CDRs are defined by Contact.

[0153] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a variant of cmAb05. The cmAb05 variant may have VL, which is a variant of cmAb05 VL having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 40. The cmAb05 variant may have VH, which is a variant of cmAb05 VL having up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 40. The cmAb05 variant may have VH, which is a variant of cmAb05 VH having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 41. The cmAb05 variant may have VH, which is a variant of cmAb05 VH having up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 41. The amino acid substitutions, additions, and / or deletions may be within the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions, and / or deletions are not within the CDR. In some embodiments, variants of cmAb05 have up to about 5 conserved amino acid substitutions. In some embodiments, variants of cmAb05 have up to 3 conserved amino acid substitutions. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a humanized antibody or its antigen-binding fragment derived from cmAb05. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a human antibody or its antigen-binding fragment derived from cmAb05.

[0154] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises one, two, three, four, five, and / or six CDRs of any of the antibodies described herein. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a light chain variable region (VL) containing one, two, and / or three light chain CDRs (VL CDRs) derived from Table 1b. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a heavy chain variable region (VH) containing one, two, and / or three heavy chain CDRs (VH CDRs) derived from Table 2b. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises one, two, and / or three VL CDRs derived from Table 1b and one, two, and / or three VH CDRs derived from Table 2b.

[0155] [Table 6]

[0156] [Table 7]

[0157] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 11; (2) VL CDR2 having the amino acid sequence shown in SEQ ID NO: 12; and / or (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 13; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 19 or 20; (2) VH CDR2 having the amino acid sequence shown in SEQ ID NO: 21 or 22; and / or (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 23; or variants thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0158] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (1) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 11; (2) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 12; or (3) a VL comprising a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: (1) VL CDR1 having the amino acid sequence shown in SEQ ID NO: 11; (2) VL CDR2 having the amino acid sequence shown in SEQ ID NO: 12; and (3) VL CDR3 having the amino acid sequence shown in SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0159] In some embodiments, the present application provides an antibody or antigen-binding fragment having a VL that specifically binds to BDCA2, wherein the VL includes VL CDR1, CDR2, and CDR3 having amino acid sequences shown in SEQ ID NOs. 11, 12, and 13, respectively, as defined by Kabat; or includes variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VL that specifically binds to BDCA2, wherein the VL includes VL CDR1, CDR2, and CDR3 of the amino acid sequence shown in SEQ ID NOs. 11, 12, and 13, respectively, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0160] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, including: (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 19 or 20; (2) VH CDR2 having an amino acid sequence shown in SEQ ID NO: 21 or 22; or (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 23; or a variant thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (1) VH CDR1 having an amino acid sequence selected from SEQ ID NO: 19 or 20; (2) VH CDR2 having an amino acid sequence shown in SEQ ID NO: 21 or 22; and (3) VH CDR3 having an amino acid sequence selected from SEQ ID NO: 23; or a variant thereof having about 3, about 5, about 8, about 10, about 12, or up to about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0161] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VH that specifically binds to BDCA2, wherein the VH includes VH CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23, respectively, as defined by Kabat; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof having a VH that specifically binds to BDCA2, wherein the VH comprises VH CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23, respectively, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0162] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: (a) VL, comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in VL CDR; and / or (b) VH, comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in VH CDR.

[0163] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 20, 22, and 23, respectively, as defined by Kabat; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the CDR.

[0164] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: (a) VL, comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in VL CDR; and / or (b) VH, comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 19, 21, and 23, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in VH CDR.

[0165] In some embodiments, the present application provides antibodies or antigen-binding fragments that specifically bind to BDCA2, including: VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, having the amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 19, 21, and 23, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the CDR.

[0166] [Table 8] JPEG2026520857000010.jpg180169

[0167] [Table 9]

[0168] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 41.

[0169] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, the antibody or antigen-binding fragment thereof comprising (a) VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 40; and (b) VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, the antibody or antigen-binding fragment comprising VL and VH, the VL and VH having the amino acid sequences shown in SEQ ID NOs. 40 and 41, respectively.

[0170] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 85% sequence identity with SEQ ID NO: 40. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 90% sequence identity with SEQ ID NO: 40. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 95% sequence identity with SEQ ID NO: 40. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VL having at least 98% sequence identity with SEQ ID NO: 40. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, including: a VL having the amino acid sequence shown in SEQ ID NO: 40.

[0171] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 41. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 85% sequence identity with SEQ ID NO: 41. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 90% sequence identity with SEQ ID NO: 41. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 95% sequence identity with SEQ ID NO: 41. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment has a VH having at least 98% sequence identity with SEQ ID NO: 41. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: VH having the amino acid sequence shown in SEQ ID NO: 41.

[0172] In some embodiments, the Application provides a humanized antibody of Ab05 (i.e., humanized Ab05, hu-cmAb05, hu-Ab05, or hu-05). In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VL having an amino acid sequence selected from SEQ ID NOs. 42-46. In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VH having an amino acid sequence selected from SEQ ID NOs. 47-50. In some embodiments, the humanized anti-BDCA2 antibody or its antigen-binding fragment provided herein comprises a VL having an amino acid sequence selected from SEQ ID NOs. 42-46, and a VH having an amino acid sequence selected from SEQ ID NOs. 47-50. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein is a variant of humanized Ab05 provided herein. The aforementioned variant may have a VL, which is a variant of humanized Ab05 VL having up to approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs.42-46. The aforementioned variant may have a VL, which is a variant of humanized Ab05 VL having up to approximately 5 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs.47-50. The aforementioned variant may have a VH, which is a variant of humanized Ab05 VH having up to approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs.42-46. The aforementioned mutant may have a VH, which is a variant of humanized Ab05 VH having up to approximately five amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from SEQ ID NOs: 47-50. In some embodiments, the variant of humanized Ab05 has up to approximately five conserved amino acid substitutions.

[0173] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, including the following: (a) VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 42-46; and / or (b) VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.

[0174] In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, wherein the antibody or antigen-binding fragment comprises VL and VH, wherein VL and VH have the amino acid sequences shown in SEQ ID NOs. 42 and 47, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 42 and 48, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 42 and 49, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 42 and 50, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 43 and 47, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 43 and 48, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 43 and 49, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 43 and 50, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 44 and 47, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 44 and 48, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 44 and 49, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 44 and 50, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 45 and 47, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 45 and 48, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 45 and 49, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 45 and 50, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 46 and 47, respectively.In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 46 and 48, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 46 and 49, respectively. In some embodiments, VL and VH have the amino acid sequences shown in SEQ ID NOs. 46 and 50, respectively.

[0175] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 42. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 42. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 42. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 42. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 42. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 42.

[0176] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 43. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 43. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 43. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 43. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 43. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 43.

[0177] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 44. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 44. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 44. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 44. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 44.

[0178] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 45. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 45. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 45. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 45. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 45. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 45.

[0179] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 46. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 46. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 46. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 46. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 46.

[0180] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 47. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 47. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 47. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 47. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 47. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 47.

[0181] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 48. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 48. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 48. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 48. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 48.

[0182] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 49. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 49. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 49. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 49. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 49. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 49.

[0183] In some embodiments, the present application provides a humanized antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 50. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 50. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 50. The humanized anti-BDCA2 antibody or antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 50. The humanized anti-BDCA2 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 50. In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: a VH having the amino acid sequence shown in SEQ ID NO: 50.

[0184] In some embodiments, the present application provides an anti-BDCA2 antibody or its antigen-binding fragment comprising: a VL CDR derived from the VL (SEQ ID NOs. 40, 42, 43, 44, 45, or 46) described herein and / or a VH CDR derived from the VH (SEQ ID NOs. 41, 47, 48, 49, or 50) described herein. Methods for identifying CDRs are well known in the art. For example, software programs (abYsis) available on publicly accessible websites are known to those skilled in the art for antibody sequence analysis and CDR determination.

[0185] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having the amino acid sequence shown in SEQ ID NO: 40; and / or (b) a VH comprising VH CDRs 1, 2, and 3 derived from a VH having the amino acid sequence shown in SEQ ID NO: 41.

[0186] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: (a) a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequences of SEQ ID NOs. 42-46; and / or (b) a VH comprising VH CDRs 1, 2, and 3 derived from a VH having an amino acid sequence selected from SEQ ID NOs. 47-50.

[0187] In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 42. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 43. In some embodiments, the present application provides an antibody or antigen-binding fragment that specifically binds to BDCA2, comprising: a VL comprising VL CDRs 1, 2, and 3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 45, comprising VL CDRs 1, 2, and 3 derived from a VL. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: a VL having the amino acid sequence shown in SEQ ID NO: 46, comprising VL CDRs 1, 2, and 3 derived from a VL.

[0188] In some embodiments, the present application provides a humanized antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 41. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 47. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, the present application provides an antibody or its antigen-binding fragment that specifically binds to BDCA2, comprising: VH comprising VH CDRs 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising: VH CDR 1, 2, and 3 derived from VH having the amino acid sequence shown in SEQ ID NO: 50.

[0189] The anti-BDCA2 antibody or its antigen-binding fragment may include a combination of any VL and any VH disclosed herein. In some embodiments, the VL and VH are linked via a linker. The linker may be flexible or rigid. In some embodiments, the linker has an amino acid sequence (GGGGS)n, where n=1, 2, 3, 4, or 5 (SEQ ID NO: 57). In some embodiments, the linker has an amino acid sequence (EAAAK)n, where n=1, 2, 3, 4, or 5 (SEQ ID NO: 58). In some embodiments, the linker has an amino acid sequence (PA)nPAP, where n=0, 1, 2, 3, or 4 (SEQ ID NO: 59).

[0190] In some embodiments, the anti-BDCA2 antibody provided herein is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0191] In some embodiments, the anti-BDCA2 antibody provided herein comprises a light chain and a heavy chain. The light chain may comprise a light chain constant region (CL) and a light chain variable region (VL). The heavy chain may comprise a heavy chain variable region (VH) and a heavy chain constant region (CH). VL / VH may be any VL / VH disclosed herein. In some embodiments, the light chain constant region (CL) has κ CL (Cκ; SEQ ID NO: 51). In some embodiments, the light chain constant region (CL) has λ CL (Cλ; SEQ ID NO: 52). In some embodiments, the heavy chain may comprise a heavy chain constant region (CH) derived from human IgA. In some embodiments, the heavy chain may comprise a heavy chain constant region (CH) derived from human IgD. In some embodiments, the heavy chain may comprise a heavy chain constant region (CH) derived from human IgE. In some embodiments, the heavy chain may comprise a heavy chain constant region (CH) derived from human IgG. In some embodiments, the heavy chain may include a heavy chain constant region (CH) derived from human IgM. In some embodiments, the heavy chain may include a heavy chain constant region (CH) derived from human IgG1 (e.g., SEQ ID NO: 53). In some embodiments, the heavy chain may include a heavy chain constant region (CH) derived from human IgG2 (e.g., SEQ ID NO: 54). In some embodiments, the heavy chain may include a heavy chain constant region (CH) derived from human IgG3 (e.g., SEQ ID NO: 55). In some embodiments, the heavy chain may include a heavy chain constant region (CH) derived from human IgG4 (e.g., SEQ ID NO: 56). The CH may further include a C-terminal lysine (K). Any and all combinations of VL / VH pairs that specifically bind to BDCA2 disclosed herein (e.g., human BDCA2) and CL / CH pairs disclosed herein or known in the art are expressly considered herein.

[0192] [Table 10]

[0193] [Table 11] JPEG2026520857000014.jpg98169

[0194] In some embodiments, the antibody provided in this application has a light chain constant region (CL) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 51. In some embodiments, the antibody provided in this application has a CL having the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the antibody provided in this application has a light chain constant region (CL) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 52. In some embodiments, the antibody provided in this application has a CL having the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the antibody provided in this application has a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 53. In some embodiments, the antibody provided in this application has a CH having the amino acid sequence shown in SEQ ID NO: 53. In some embodiments, the antibody provided in this application has a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 54. In some embodiments, the antibody provided in this application has a CH having the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 55. In some embodiments, the antibody provided in this application has a CH having the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 56. In some embodiments, the antibody provided in this application has a CH having the amino acid sequence shown in SEQ ID NO: 56.

[0195] In some embodiments, the present application further provides an antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment provided above for binding to BDCA2 (e.g., human BDCA2). An antibody that "competes with another antibody for binding to a target" means an antibody that (partially or completely) inhibits the binding of that other antibody to its target. Known competition experiments, such as the BIACORE® surface plasmon resonance (SPR) assay, can be used to determine whether two antibodies compete with each other for binding to a target, i.e., to what extent one antibody inhibits the binding of the other antibody to its target. In some embodiments, an anti-BDCA2 antibody or antigen-binding fragment competes with another antibody or antigen-binding fragment for binding to BDCA2 and inhibits the binding of that other antibody or antigen-binding fragment to BDCA2 by at least 50%, 60%, 70%, 80%, 90%, or 100%. Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0.H0l / pdb.prot4277, or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0196] In some embodiments, the Application provides an antibody or its antigen-binding fragment that competes with the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for binding to BDCA2 (e.g., human BDCA2). In some embodiments, the Application provides a chimeric Ab03 and an antibody or its antigen-binding fragment that competes for binding to BDCA2 (e.g., human BDCA2). In some embodiments, the Application provides a humanized Ab03 disclosed herein and an antibody or its antigen-binding fragment that competes for binding to BDCA2 (e.g., human BDCA2). In some embodiments, the Application provides a chimeric Ab05 and an antibody or its antigen-binding fragment that competes for binding to BDCA2 (e.g., human BDCA2). In some embodiments, the Application provides a humanized Ab05 disclosed herein and an antibody or its antigen-binding fragment that competes for binding to BDCA2 (e.g., human BDCA2).

[0197] Epitope mapping is a method for identifying binding sites, regions, or epitopes on target proteins to which antibodies bind. Numerous methods are known in this field for mapping epitopes on target proteins. These methods include (but not limited to) mutagenesis, including shotgun mutation, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display, ribosome / mRNA display); methods including proteolysis and mass spectrometry; and structural determination (e.g., X-ray crystallography and NMR). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is identified by assays including (but not limited to) N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion-exchange chromatography, and papain digestion.

[0198] The physical, chemical, and / or biological properties of the anti-BDCA2 antibodies or their antigen-binding fragments described herein can be analyzed by various methods known in the art. In some embodiments, the ability of the anti-BDCA2 antibody to bind to BDCA2 (e.g., human BDCA2) is tested. In some embodiments, the ability of the anti-BDCA2 antibody to bind to FcγR is tested. In some embodiments, the ability of the anti-BDCA2 antibody to bind to FcγRIIA / CD32A is tested. In some embodiments, the ability of the anti-BDCA2 antibody to bind to FcγRIIIA / CD16A is tested. Binding assays include (but are not limited to) BLI, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency of the antibody can be evaluated.

[0199] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is, for example, 10 -7 M or less, 5×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, or 10 -10 K below M D It binds to human BDCA2 with high affinity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is 10 -9 K below M D It binds to human BDCA2. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has high affinity, for example, about 10 -7 M, about 5 x 10 -8 M, about 10 -8 M, about 5 x 10 -9 M, about 10 -9 M, about 5 x 10 -10 M, or about 10 -10 M's K DIt binds to human BDCA2. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has a K of about 10 -9 M D and binds to human BDCA2. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has a K of 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, 10 -8 M to 10 -7 M, 10 -10 M to 5×10 -8 M, 10 -9 M to 5×10 -8 M, 10 -8 M to 5×10 -8 M, 10 -10 M to 10 -8 M, 10 -9 M to 10 -8 M, 10 -10 M to 5×10 -9 M, 10 -9 M to 5×10 -9 M, or 10 -10 M to 10 -9 M and binds to human BDCA2. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has a high affinity, for example, 10 D -10 M to 10 -9 M D and binds to human BDCA2. In some embodiments, K D is determined by BLI. In some embodiments, K D is determined by SPR. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has a high affinity, for example, 10 -7 M or less, 5×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, or 10 -10 M or less; or about 10 -7 ​M, approximately 5×10 -8 M, approximately 10 -8 M, approximately 5×10 -9 M, approximately 10 -9 M, approximately 5×10 -10 M, or approximately 10 -10 M; or 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M, 10 -8 M to 10 -7 M, 10 -10 M to 5×10 -8 M, 10 -9 M to 5×10 -8 M, 10 -8 M to 5×10 -8 M, 10 -10 M to 10 -8 M, 10 -9 M to 10 -8 M, 10 -10 M to 5×10 -9 M, 10 -9 M to 5×10 -9 M, or 10 -10 M to 10 -9 K within the range of M D binds to human BDCA2, which is the value measured by SPR.

[0200] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application binds to both human BDCA2 and cynomolgus BDCA2.

[0201] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application inhibits the IFNα release of PBMC. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application inhibits the IFNα release of PBMC by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application inhibits the IFNα release of PBMC by immune complex (IC) stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application inhibits the IFNα release of PBMC in vivo. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application reduces the IFNα level in vivo.

[0202] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an IC of 0.05 nM or less, 0.01 nM or less, 0.008 nM or less, 0.005 nM or less, 0.002 nM or less, 0.001 nM or less, 0.0008 nM or less, or 0.0005 nM or less 50 and inhibits the IFNα release of PBMC by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an IC of 0.008 nM or less 50 and inhibits the IFNα release of PBMC by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an IC in the range of 0.0005 nM to 0.01 nM, 0.0005 nM to 0.008 nM, 0.0005 nM to 0.005 nM, 0.0005 nM to 0.001 nM, 0.001 nM to 0.01 nM, 0.001 nM to 0.008 nM, 0.001 nM to 0.005 nM, 0.005 nM to 0.008 nM, or 0.005 nM to 0.01 nM 50 and inhibits the IFNα release of PBMC by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an IC in the range of 0.001 nM to 0.05 nM 50This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an IC50 in the range of 0.0005 nM to 0.005 nM. 50 This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an IC50 in the range of 0.0005 nM to 0.008 nM. 50 This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is used to control the IC of the reference antibody litifilimab. 50 ICs that are 60% or less, 50% or less, 40% or less, or 30% or less 50 This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is used to control the IC of the reference antibody litifilimab. 50 Less than 50% of ICs 50 This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is used to obtain 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% IC of the reference antibody litifilimab. 50 This inhibits IFNα release from PBMCs induced by CpG stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein provides 10-50% IC of the reference antibody litifilimab. 50 This inhibits IFNα release from PBMCs induced by CpG stimulation.

[0203] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an IC of 0.01 nM or less, 0.008 nM or less, 0.006 nM or less, 0.005 nM or less, 0.002 nM or less, 0.001 nM or less, 0.0008 nM or less, 0.0006 nM or less, 0.0005 nM or less, or 0.0002 nM or less.50 Therefore, it inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an IC of 0.0006 nM or less. 50 This inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein inhibits IC in the range of 0.0001nM to 0.01nM, 0.0001nM to 0.005nM, 0.0001nM to 0.001nM, 0.0001nM to 0.0006nM, 0.0001nM to 0.0005nM, 0.0005nM to 0.01nM, 0.0005nM to 0.005nM, 0.0005nM to 0.001nM, 0.001nM to 0.01nM, or 0.005nM to 0.01nM. 50 Therefore, it inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein inhibits IC in the range of 0.0001 nM to 0.0006 nM. 50 Therefore, it inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is used to stimulate IC in the range of 0.0001 nM to 0.0005 nM. 50 This inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein inhibits IC of 60%, 50%, 40%, or 30% of the reference antibody litifilimab. 50 Therefore, it inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an IC of 50% or less of that of the reference antibody litifilimab. 50 This inhibits IFNα release from PBMCs induced by IC stimulation. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein inhibits 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% of the IC of the reference antibody litifilimab. 50and inhibits the IFNα release of PBMCs by IC stimulation. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application is 10-50% of the IC of the reference antibody litifilimab 50 and inhibits the IFNα release of PBMCs by IC stimulation.

[0204] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application exhibits ADCC activity and ADCP activity against BDCA2-expressing cells, such as pDCs. The ADCC activity may be NK-dependent ADCC. The ADCC activity may be neutrophil-dependent ADCC. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application can deplete pDCs in vivo.

[0205] In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an EC of 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, 0.005 nM or less, or 0.001 nM or less as measured by a reporter gene assay 50 and exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an EC of 0.05 nM or less as measured by a reporter gene assay 50 and exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or antigen-binding fragment thereof described in the present application has an EC of 0.01 nM or less as measured by a reporter gene assay 50Therefore, it exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits EC levels in the range of 0.001nM~0.1nM, 0.005nM~0.1nM, 0.01nM~0.1nM, 0.05nM~0.1nM, 0.001nM~0.05nM, 0.005nM~0.05nM, 0.01nM~0.05nM, 0.001nM~0.01nM, 0.005nM~0.01nM, or 0.001nM~0.005nM, as measured by a reporter gene assay. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC in the range of 0.001 nM to 0.005 nM, as measured by a reporter gene assay. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC in the range of 0.001 nM to 0.01 nM, as measured by a reporter gene assay. 50 It exhibits NK-dependent ADCC activity.

[0206] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of ≤0.01 nM, ≤0.008 nM, ≤0.005 nM, ≤0.002 nM, ≤0.001 nM, ≤0.0005 nM, or ≤0.0001 nM, as measured by a cytotoxicity assay. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of 0.001 nM or less, as measured by a cytotoxicity assay. 50Therefore, it exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits EC levels in the range of 0.0001nM~0.0005nM, 0.0001nM~0.001nM, 0.0001nM~0.005nM, 0.0001nM~0.01nM, 0.0005nM~0.001nM, 0.0005nM~0.005nM, 0.0005nM~0.01nM, 0.001nM~0.005nM, or 0.001nM~0.01nM, as measured by a cytotoxicity assay. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC2 concentration in the range of 0.0001 nM to 0.001 nM, as measured by a cytotoxicity assay. 50 It exhibits NK-dependent ADCC activity.

[0207] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is present in an EC ratio of 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% of the reference antibody litifilimab. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is 10-50% EC of the reference antibody litifilimab. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has approximately 40% EC of the reference antibody litifilimab. 50 This then exhibits NK-dependent ADCC activity. In some embodiments, NK-dependent ADCC activity is measured by a cytotoxicity assay. In some embodiments, NK-dependent ADCC activity is measured by a reporter gene assay.

[0208] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits EC levels of ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.02 nM, or ≤0.01 nM, as measured by a reporter gene assay. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of 0.2 nM or less, as measured by a reporter gene assay. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of 0.1 nM or less, as measured by a reporter gene assay. 50 The antibody exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is measured by a reporter gene assay in concentrations of 0.01-10 nM, 0.01-5 nM, 0.01-2 nM, 0.01-1 nM, 0.01-0.5 nM, 0.01-0.2 nM, 0.01-0.1 nM, and 0.01-0. EC in the range of 0.05nM, 0.01~0.02nM, 0.05~10nM, 0.05~5nM, 0.05~2nM, 0.05~1nM, 0.05~0.5nM, 0.05~0.2nM, 0.05~0.1nM, 0.1~10nM, 0.1~5nM, 0.1~2nM, 0.1~1nM, 0.1~0.5nM, or 0.1~0.2nM 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC in the range of 0.01 to 0.2 nM, as measured by a reporter gene assay. 50It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC in the range of 0.01 to 0.1 nM, as measured by a reporter gene assay. 50 It then exhibits macrophage-dependent ADCP activity.

[0209] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits EC levels of ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.5 nM, ≤0.2 nM, ≤0.1 nM, ≤0.05 nM, ≤0.02 nM, or ≤0.01 nM, as measured by a phagocytosis assay. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of 2 nM or less, as measured by a phagocytic assay. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC of 1 nM or less, as measured by a phagocytic assay. 50 The antibody exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits EC levels in the range of 0.01-10 nM, 0.01-5 nM, 0.01-2 nM, 0.01-1 nM, 0.01-0.5 nM, 0.05-10 nM, 0.05-5 nM, 0.05-2 nM, 0.05-1 nM, 0.05-0.5 nM, 0.1-10 nM, 0.1-5 nM, 0.1-2 nM, 0.1-1 nM, or 0.1-0.5 nM, as measured by a phagocytosis assay. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has an EC in the range of 0.1 to 2 nM, as measured by a phagocytosis assay. 50It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein exhibits an EC in the range of 0.1 to 1 nM, as measured by a phagocytosis assay. 50 It then exhibits macrophage-dependent ADCP activity.

[0210] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein further exhibits neutrophil-dependent ADCC activity.

[0211] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein selectively binds to pDCs in human PBMCs. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein does not bind to T cells, B cells, NK cells, NKT cells, or monocytes in PBMCs.

[0212] The anti-BDCA2 antibody or its antigen-binding fragment provided herein may have one or more of the functional properties described above. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has (1) a K of 50 nM or less as measured by SPR. D (1) It binds to human BDCA2; (2) It inhibits the release of interferon-α (IFNα) from peripheral blood mononuclear cells (PB#MCs); (3) It selectively binds to plasmacytoid dendritic cells (pDCs) in human PBMCs; (4) It exhibits natural killer cell (NK)-dependent ADCC activity against BDCA2-expressing cells; (5) It exhibits neutrophil-dependent ADCC activity against BDCA2-expressing cells; or (6) It exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

[0213] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has a K content in the range of 0.5 to 10 nM, as measured by SPR. D It then binds to human BDCA2.

[0214] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an IC of 0.008 nM or less. 50 Therefore, inhibit IFNα release in CpG-stimulated PBMCs; (2) IC of 0.0006 nM or less 50 (3) inhibit IFNα release in IC-stimulated PBMCs; (4) EC of less than 0.01 nM as measured by reporter assay. 50 (4) It exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (4) It has an EC of less than 0.001 nM, as measured by a cytotoxic activity assay. 50 (5) It exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (6) as measured by reporter assay, EC is less than 0.2 nM 50 (6) The cells exhibit macrophage-dependent ADCP activity against BDCA2-expressing cells; or (6) EC2 is less than 2 nM as measured by a phagocytic assay. 50 The cells exhibit macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

[0215] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an IC in the range of (1) 0.0005 nM to 0.008 nM 50 Therefore, inhibit IFNα release in CpG-stimulated PBMCs; (2) IC in the range of 0.0001 nM to 0.0006 nM 50 (3) inhibits IFNα release in IC-stimulated PBMCs; (4) EC in the range of 0.001 nM to 0.01 nM as measured by a reporter assay. 50 (4) EC2 in the range of 0.0001 nM to 0.001 nM, as measured by a cytotoxicity assay. 50 (5) 50(6) EC2 in the range of 0.1 nM to 2 nM as measured by a phagocytic assay. 50 The cells exhibit macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

[0216] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an IC50 in the range of 0.001 nM to 0.005 nM. 50 This inhibits IFNα release from CpG-stimulated PBMCs. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an IC in the range of 0.0001 nM to 0.0006 nM. 50 This inhibits IFNα release from IC-stimulated PBMCs. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an EC in the range of 0.001 nM to 0.005 nM, as measured by a reporter gene assay. 50 The antibody exhibits NK-dependent ADCC activity against BDCA2-expressing cells. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein exhibits an EC level in the range of 0.0001 nM to 0.001 nM, as measured by a cytotoxicity assay. 50 It exhibits NK-dependent ADCC activity against BDCA2-expressing cells. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an EC level in the range of 0.02 nM to 0.12 nM, as measured by a reporter gene assay. 50 The antibody exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment provided herein has an EC2 concentration in the range of 0.1 nM to 0.5 nM, as measured by (6) a phagocytosis assay. 50 It exhibits macrophage-dependent ADCP activity in BDCA2-expressing cells; or any combination of (1) to (6).

[0217] 1.3 Mutants and Conjugates This disclosure also considers other variants and equivalent forms that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies or antibody fragments described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0218] A change refers to a modification of the amino acid sequence compared to the native antibody or polypeptide sequence due to the substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide. In some embodiments, amino acid substitutions are the result of conserved amino acid substitutions, i.e., substitutions by another amino acid with similar structural and / or chemical properties, such as leucine being replaced by serine. Insertions or deletions can be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the parent molecule. In some embodiments, biologically useful and / or relevant amino acid sequence changes can be determined by systematically introducing insertions, deletions, or substitutions into the sequence and evaluating the activity of the resulting mutant protein in comparison to the parent protein.

[0219] In some embodiments, the Application provides variants of the anti-BDCA2 antibody or its antigen-binding fragment described herein. In some embodiments, the Application provides variants of the anti-BDCA2 antibody clone Ab03 (cmAb03 or hu03). In some embodiments, the Application provides variants of the anti-BDCA2 antibody or its antigen-binding fragment described herein. In some embodiments, the Application provides variants of the anti-BDCA2 antibody clone Ab05 (cmAb05 or hu05). In some embodiments, the variant comprises 1 to 30 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant comprises 1 to 25 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant comprises 1 to 20 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant comprises 1 to 15 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant contains 1 to 10 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant contains 1 to 5 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the variant contains 1 to 3 amino acid substitutions, additions, and / or deletions in the parent antibody or its antigen-binding fragment. In some embodiments, the amino acid substitutions are located within the CDR of the antibody or its antigen-binding fragment. In some embodiments, the amino acid substitutions are not located within the CDR of the antibody or its antigen-binding fragment. In some embodiments, the amino acid substitutions are located within the framework region of the antibody or its antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and / or deletions are conservative amino acid substitutions.

[0220] In this field, the constant region of an antibody is known to mediate several effector functions, and these effector functions can vary depending on the antibody isotype. For example, the complement system is activated when the C1 component of complement binds to the Fc region (antigen-bound) of an IgG or IgM antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation can also stimulate inflammatory responses and be involved in autoimmune hypersensitivity reactions. Furthermore, the Fc region of an antibody can bind to cells that express Fc receptors (FcRs). There are Fc receptors specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). The binding of antibodies to Fc receptors on the cell surface triggers a variety of important biological responses, including the uptake and destruction of antibody-coated particles, removal of immune complexes, lysis of killer cells against antibody-coated target cells (known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transport, and regulation of immunoglobulin production.

[0221] As is known in this field, allotypes are polymorphic markers of IG subclasses corresponding to amino acid changes and are detected serologically using antibody reagents. Of these, the allotype of the human γ heavy chain of IgG is denoted as Gm ("γ marker"). Allotypes G1m, G2m, and G3m are present in the constant regions of the γ1, γ2, and γ3 chains encoded by the IHG1, IHG2, and IHG3 genes, respectively. The γ1 chain can express the G1m allele (combination of G1m allotypes): G1m3; G1m3,1; G1m17,1; G1m17,1,2; G1m17,1,27; Gm17,1,28; and Gm17,1,27,28. The C regions of the G1m3,1; G1m17,1; and G1m17,1,2 chains differ by 2, 3, and 4 amino acids compared to the G1m3 chain, respectively. The correspondence between the G1m allele and the IHG1 allele is known in this field; see, for example, Lefranc, Chapter 26 - IMGT (registered trademark) Immunoglobulin Repertoire Analysis and Antibody Humanization, Molecular Biology of B Cells (2nd edition), Academic Press, 2015, pp. 481-514 (Table 7). In IHG1 CH1, lysine at position 120 (K120) of chain G corresponds to the G1m17 allotype. Isoleucine I103 (chain F) is specific to the γ1 chain isotype. When arginine is expressed at position 120 (R120), the simultaneous presence of R120 and I103 corresponds to the expression of the G1m3 allotype. In the case of γ3 and γ4 isotypes that have R120 but have T at position 103, R120 corresponds to the expression of the nG1m17 isoallotype (the isoallotype or nGm can be detected by antibody reagents that distinguish this marker as an allotype in one IgG subclass and as an isotype in another). In IHG1 CH3, aspartate D12 and leucine L14 (chain A) correspond to G1m1, while glutamate E12 and methionine M14 correspond to the nG1m1 isoallotype. Glycine at position 110 corresponds to G1m2, but alanine does not correspond to any allotype (G1m2-negative chain).

[0222] Refer to the following exemplary allotypes of the human IgG1 heavy chain constant region (IgG1 CH). In some embodiments, the present application provides an IgG1 antibody having a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 and 62-66. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 53. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 62. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 63. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 64. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 65. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 66. The heavy chain constant region may further contain a C-terminal lysine (K).

[0223] [Table 12] JPEG2026520857000016.jpg254169JPEG2026520857000017.jpg46169

[0224] The γ2 chain can express the G2m allele. Position 45.1 (the first position of the transverse CD chain) corresponds to the presence or absence (G2m...) of a uniquely identifiable G2m allotype (G2m23). Valine V45.1 corresponds to G2m..., while methionine corresponds to G2m23.

[0225] The γ3 chain can express G3m alleles (combinations of G3m allotypes). G3m16(W83), G3m21(L82), and nG3m21(P82) are located on CH2. Other G3m allotypes form two mosaics on CH3. G3m26(R115), G3m5(R115,F116), G3m28(R115,Y116), nG3m5(H115,Y116), G3m14(M84,R115,F116), and G3m15(M39,H115,Y116) form the first mosaic. G3m11(S44), nG3m11(N44), G3m10(S44, I101), G3m24(S44, V101), G3m27(I101), G3m6(S44, E98), and G3m13(S44, Q98) form a second mosaic.

[0226] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgA antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgD antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgE antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgG antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgM antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgG1 antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgG2 antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes a constant region of a human IgG3 antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein comprises a constant region of a human IgG4 antibody. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment comprises a constant region of a human IgG1 antibody, wherein the IgG1 antibody may have any allotype known in the art. In some embodiments, the IgG1 antibody has allotypes G1m3;G1m3,1;G1m17,1;G1m17,1,2;G1m17,1,27;Gm17,1,28; or Gm17,1,27,28. In some embodiments, the IgG1 antibody has allotype G1m3. In some embodiments, the IgG1 antibody has allotype G1m3,1. In some embodiments, the IgG1 antibody has allotype G1m17,1. In some embodiments, the IgG1 antibody has allotype G1m17,1.2. In some embodiments, the IgG1 antibody has allotype Gm17,1.28.In some embodiments, the IgG1 antibody has allotypes Gm17, 1, 27, 28. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein comprises a constant region of a human IgG2 antibody, wherein the IgG2 antibody may have any allotype known in the art. In some embodiments, the IgG2 antibody has allotype G2m23. In some embodiments, the IgG2 antibody has allotype G2m.. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein comprises a constant region of a human IgG3 antibody, wherein the IgG3 antibody may have any allotype known in the art. In some embodiments, the IgG3 antibody has allotypes G3m16, G3m21, G3m26, G3m5, G3m28, G3m14, G3m15, G3m11, G3m10, G3m24, G3m27, G3m6, or G3m13. In some embodiments, the IgG3 antibody has allotype G3m16. In some embodiments, the IgG3 antibody has allotype G3m21. In some embodiments, the IgG3 antibody has allotype G3m26. In some embodiments, the IgG3 antibody has allotype G3m5. In some embodiments, the IgG3 antibody has allotype G3m28. In some embodiments, the IgG3 antibody has allotype G3m14. In some embodiments, the IgG3 antibody has allotype G3m15. In some embodiments, the IgG3 antibody has allotype G3m11. In some embodiments, the IgG3 antibody has allotype G3m10. In some embodiments, the IgG3 antibody has allotype G3m24. In some embodiments, the IgG3 antibody has allotype G3m27. In some embodiments, the IgG3 antibody has allotype G3m6. In some embodiments, the IgG3 antibody has allotype G3m13.

[0227] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein has at least one or more modifications or deletions in the constant region. In some embodiments, the antibody includes modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or to the light chain constant region (CL).

[0228] In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes multiple human constant regions. In some embodiments, the modification of the constant region includes the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain is removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer arm, which provides some of the molecular flexibility that would normally be conferred by the constant region. In some embodiments, the modified antibody includes a CH3 domain directly fused to the antibody hinge region. In some embodiments, the modified antibody includes a peptide spacer arm inserted between the hinge region and the modified CH2 and / or CH3 domain.

[0229] In some embodiments, an anti-BDCA2 antibody or its antigen-binding fragment includes an Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge may be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are well known to those skilled in the art. In some cases, Fc regions with amino acid changes have been identified in native antibodies. In some embodiments, the modified antibody (e.g., the modified Fc region) provides altered effector function, thereby affecting the antibody's biological profile. For example, in some embodiments, deletion or inactivation (by point mutation or other means) of the constant region during circulation reduces the Fc receptor binding of the modified antibody. In some embodiments, the constant region modification reduces the immunogenicity of the antibody. In some embodiments, the constant region modification prolongs the serum half-life of the antibody. In some embodiments, the constant region modification shortens the serum half-life of the antibody. In some embodiments, the constant region modification enhances the antibody's ADCC and / or complement-dependent cell-mediated cytotoxicity (CDC). In some embodiments, the constant region modification enhances the antibody's antibody-dependent cell-mediated phagocytosis (ADCP). In some embodiments, the constant region modification reduces or eliminates the antibody's ADCC and / or CDC. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region having corresponding IgG2 or IgG4 residues reduce effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody lacks one or more effector functions (e.g., an "effectorless" antibody). In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody lacks effector function. In some embodiments, the constant region modification improves or enhances the antibody's ADCC and / or ADCP. In some embodiments, the constant region is modified to remove a disulfide bond or oligosaccharide moiety.In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide one or more cytotoxins, oligosaccharides, or carbohydrate binding sites. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment includes a mutant Fc region designed by substitution at specific amino acid positions compared to the native Fc region.

[0230] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein comprises an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, A330, I332, K326, E333, K334, and P396 according to EU numbering.

[0231] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes an IgG1 heavy chain constant region comprising at least one amino acid substitution. The IgG1 heavy chain constant region may include an L234 substitution, for example, L234Y. The IgG1 heavy chain constant region may also include an L235 substitution, for example, L235Q or L235V. The IgG1 heavy chain constant region may also include a G236 substitution, for example, G236A or G236W. The IgG1 heavy chain constant region may also include an S239 substitution, for example, S239D or S239M. The IgG1 heavy chain constant region may also include an F243 substitution, for example, F243L. The IgG1 heavy chain constant region may also include an H268 substitution. The H268 substitution may be, for example, H268D. The IgG1 heavy chain constant region may include a D270 substitution. The D270 substitution may be, for example, D270E. The IgG1 heavy chain constant region may include an R292 substitution. The R292 substitution may be, for example, R292P. The IgG1 heavy chain constant region may include an S298 substitution. The S298 substitution may be, for example, S298A. The IgG1 heavy chain constant region may include a Y300 substitution. The Y300 substitution may be, for example, Y300L. The IgG1 heavy chain constant region may include a V305 substitution. The V305 substitution may be, for example, V305I. The IgG1 heavy chain constant region may include a K326 substitution. The K326 substitution may be, for example, K326D. The IgG1 heavy chain constant region may include an A330 substitution. The A330 substitution may be, for example, A330M or A330L. The IgG1 heavy chain constant region may also contain an I332 substitution. The I332 substitution may be, for example, I332E. The IgG1 heavy chain constant region may also contain an E333 substitution. The E333 substitution may be, for example, E333A. The IgG1 heavy chain constant region may also contain a K334 substitution. The K334 substitution may be, for example, K334A or K334E. The IgG1 heavy chain constant region may also contain a P396 substitution.The P396 substitution may also be, for example, P396L.

[0232] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein includes an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, and P396L based on EU index numbers. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M based on EU index numbers. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S298A, E333A, and K334A. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S239D and I332E. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S239D, A330L, and I332E. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitution G236A. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions G236A, S239D, and I332E. In some embodiments, the anti-BDCA2 antibody described herein and its antigen-binding fragment include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions G236A, A330L, and I332E.In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions G236A, S239D, A330L, and I332E. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions F243L, R292P, Y300L, V305I, and P396L. In some embodiments, the anti-BDCA2 antibody and its antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the anti-BDCA2 antibodies and their antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, the anti-BDCA2 antibodies and their antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions D270E, K326D, A330M, and K334E. All are numbered based on the EU index. Below are exemplary heavy chain constant regions (CH) of IgG1 isotypes having different mutations that improve or enhance the ADCC and / or ADCP of the antibodies. It is clearly considered that the antibody disclosed herein may include in the antibody any combination of mutations disclosed herein or otherwise known in the art, which enhance or enhance the ADCC and / or ADCP of the antibody, the heavy chain constant region (CH) of any immunoglobulin disclosed herein or otherwise known in the art (e.g., human IgG1).

[0233] [Table 13] JPEG2026520857000019.jpg254169JPEG2026520857000020.jpg254169JPEG2026520857000021.jpg254169JPEG2026520857000022.jpg253169JPEG2026520857000023.jpg254169JPEG2026520857000024.jpg254169JPEG2026520857000025.jpg38169

[0234] In some embodiments, the present application provides an IgG1 antibody having a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 67 to 86. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NOs. 67. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NOs. 68. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NOs. 69. In some embodiments, the IgG1 antibody provided in the present application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NOs. 70. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 71. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 72. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 73. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 74. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 75.In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 76. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 77. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 78. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 79. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 80. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 81. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 82. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 83. In some embodiments, the IgG1 antibody provided in this application has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 84.In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 85. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 86. The CH may further contain a C-terminal lysine (K).

[0235] In some embodiments, the variant may involve the addition of amino acid residues at the amino-terminus and / or carboxyl-terminus of an antibody or polypeptide. The length of the other amino acid residues may range from 1 to 100 residues or more. In some embodiments, the variant includes an N-terminal methionyl residue. In some embodiments, the variant includes another polypeptide / protein (e.g., an Fc region) to generate a fusion protein. In some embodiments, the variant is designed to be detectable and may include a detectable marker and / or protein (e.g., a fluorescent tag or enzyme).

[0236] The mutant antibodies or antigen-binding fragments described herein can be produced using methods known in the art, including (but not limited to) site-directed mutagenesis, alanine scan mutagenesis, and PCR mutagenesis. Methods for mutagenesis and nucleotide sequence modification are well known in the art. For example, see Walker and Gaastra (eds.) (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Methods Enzymol. 54:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY); U.S. Patent No. 4,873,192; and the references cited herein; these are incorporated herein by reference. Guidelines for appropriate amino acid substitutions that do not affect the biological activity of the target polypeptide can be found in the model described by Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), pp. 345-352; the entire document is incorporated herein by reference. Dayhoff et al.'s model uses a point-receptor mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine appropriate conserved amino acid substitutions. Conservative substitutions can be beneficial, such as replacing one amino acid with another amino acid that has similar properties. Examples of conserved amino acid substitutions based on the PAM 250 matrix in Dayhoff et al.'s model include, but are not limited to, Gly→Ala, Val→Ile→Leu, Asp→Glu, Lys→Arg, Asn→Gln, and Phe→Trp→Tyr.

[0237] When constructing anti-BDCA2 binding molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives, modifications are made so that the variant retains desired properties, such as the ability to specifically bind to BDCA2 and, in some embodiments, the ability to inhibit IFN-α release in the body and / or deplete pDCs. Clearly, any mutations made in the DNA encoding the variant polypeptide should not be outside the reading frame. In some embodiments, mutations made in the DNA do not generate complementary regions that can give rise to secondary mRNA structures.

[0238] In some embodiments, variants of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein may retain the same, equivalent, or greater ability to bind to BDCA2 as the parent antibody or its antigen-binding fragment. In some embodiments, the variants may have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more homology in amino acid sequence with the parent antibody or its antigen-binding fragment. In some embodiments, the variant of the anti-BDCA2 antibody or its antigen-binding fragment comprises the amino acid sequence of the parent anti-BDCA2 antibody or its antigen-binding fragment having one or more conserved amino acid substitutions. Conserved amino acid substitutions are well known in the art and include amino acid substitutions in which an amino acid having specific physical and / or chemical properties is replaced by another amino acid having the same or similar chemical and physical properties.

[0239] In some embodiments, a variant of an anti-BDCA2 antibody or its antigen-binding fragment comprises the amino acid sequence of a parent antibody or its antigen-binding fragment having one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-BDCA2 antibody or its antigen-binding fragment comprises the amino acid sequence of a parent-binding antibody or its antigen-binding fragment having one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities (e.g., BDCA2 binding) of the variant. In some embodiments, the one or more conservative amino acid substitutions and / or the one or more non-conservative amino acid substitutions enhance the biological activity of the variant, thereby improving the biological activity of the functional variant compared to the parent antibody or its antigen-binding fragment.

[0240] In some embodiments, the mutant may have 1, 2, 3, 4, or 5 amino acid substitutions in the binding site CDR (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3).

[0241] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is modified naturally or through artificial chemical modification. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment is chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protective / blocking groups, proteolytic cleavage, and / or binding to cellular ligands or other proteins. Any of these chemical modifications can be carried out by known techniques. The anti-BDCA2 antibody or its antigen-binding fragment may include one or more analogues of amino acids (e.g., including non-natural amino acids) and other modifications known in the art.

[0242] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein can be linked to at least one reagent to form an antibody complex. The complex may be an antibody conjugated to (e.g.) other proteins, carbohydrates, lipids, steroids, immunosuppressants, or mixed partial molecules. These antibody complexes include modifications that link the antibody to one or more polymers (but are not limited to these). For example, the antibody or its antigen-binding fragment may be linked to one or more water-soluble polymers. Linking to a water-soluble polymer reduces the likelihood of the antibody or its antigen-binding fragment precipitating in an aqueous environment (e.g., a physiological environment). Those skilled in the art can select a suitable water-soluble polymer based on the following considerations, which include (but are not limited to) whether the polymer / antibody complex is used for patient treatment, and if so, the pharmacological profile of the antibody (e.g., half-life, dose, activity, antigenicity, and / or other factors).

[0243] To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, at least one desired molecule or site is typically linked, covalently linked, or complexed with them. Such molecules or sites may be (but are not limited to) at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules linked to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antivirals, chelating agents, cytokines, growth factors, and oligo or polynucleotides. In contrast, a reporter molecule is defined as any site detectable by the assay. Non-limiting examples of reporter molecules complexed with antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, enzymes (e.g., enzymes that catalyze colorimetric, fluorescent, or bioluminescent reactions), substrates, and solid substrates such as biotin. Antibodies may contain one, two, or more of these labels.

[0244] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is modified naturally or through artificial chemical modification. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment is chemically modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protective / blocking groups, proteolytic cleavage, and / or binding to cellular ligands or other proteins. Any of these chemical modifications can be carried out by known techniques. The anti-BDCA2 antibody or its antigen-binding fragment may include one or more analogues of amino acids (e.g., including non-natural amino acids) and other modifications known in the art.

[0245] Antibody conjugates can be used to deliver cytotoxic agents to target cells. This type of cytotoxic agent can enhance antibody-mediated cytotoxicity and includes sites such as cytokines (those that directly or indirectly induce cell death), radioisotopes, chemotherapeutic agents (including prodrugs), bacterial toxins (e.g., Pseudomonas exotoxins, diphtheria toxin, etc.), plant toxins (e.g., lysine, gerdoanamycin, etc.), chemical conjugates (e.g., martansine toxin, calicheamicin, etc.), radioconjugates, and enzyme conjugates (e.g., ribonuclease conjugates, granzyme antibody-targeted enzyme / prodrug therapy).

[0246] Antibody conjugates are also used as diagnostic reagents. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is conjugated to a detectable substance or molecule, thereby making the reagent available for diagnosis and / or detection. Detectable substances include, but are not limited to, enzymes; prosthetic groups (e.g., biotin and flavin); fluorescent substances; bioluminescent materials such as luciferase; radioactive substances; positron-emitting metals; and magnetic metal ions.

[0247] Antibody diagnostics are generally classified into two types: those used for in vitro diagnostics, such as those in various immunoassays, and those used in in vivo diagnostic procedures (commonly referred to as "antibody-directed imaging"). A variety of suitable imaging agents are known in the art, as are methods for linking them to antibodies (see, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509). Imaging sites used may include paramagnetic ions, radioisotopes, fluorescent dyes, NMR detectables, MR hyperpolarizing molecules, targeted ultrasound bubbles, and X-ray imaging agents.

[0248] Paramagnetic ions considered as complexes include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other applications such as X-ray imaging include (but are not limited to) lanthanum(III), gold(III), lead(II), and bismuth(III). Alternative useful isotopes are those used in hyperpolarized MRI, such as carbon-13 and silicon-29.

[0249] Radioisotopes considered for use as complex or covalent incorporation in imaging and radiotherapy include astatine-211, actinium-225, carbon-14, bismuth-212, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-64, copper-67, europium-152, fluorine-18, gallium-68, gallium-67, gold-198, hydrogen-3, iodine-123, iodine-125, iodine-131, and indium-11. This includes 1, iron-52, iron-59, lead-212, lutetium-177, phosphorus-32, rhenium-186, rhenium-188, rubidium-82, rhodium-99, selenium-75, sulfur-35, samarium-153, strontium-92, strontium-89, thallium-201, thorium-227, technetium-94m, technetium-99m, yttrium-86, yttrium-90, zirconium-86, and / or zirconium-89. F-18, Zr-89, and Cu-64 are generally preferred in PET imaging. Lu-177, At-211, and Yt-90 are generally preferred in radiotherapy. The radiolabeled monoclonal antibodies and antibody fragments of this disclosure can be manufactured according to methods well known in the art. For example, monoclonal antibodies can be iodized by contacting sodium iodide and / or potassium iodide with a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. The monoclonal antibodies disclosed in this invention can be labeled with technetium-99m using a ligand exchange method, for example, by reducing the pertechnetium salt with a tin solution, chelating the reduced technetium onto a Sephadex column, and loading the column with the antibody. Alternatively, direct labeling techniques can be used, for example, by incubating the pertechnetium salt, a reducing agent such as SNCl2, a buffer such as a sodium-potassium phthalate solution, and the antibody.Intermediate functional groups incorporating chelating agents, commonly used to bind radioactive isotopes in metal ion form to antibodies, include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), monomer or dendrimer-type 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), deferoxamine (DFO), or 1-hydroxy-2(1H)-pyridone derivatives (e.g., 3,4,3-LI(l,2-HOPO) or HOPO).

[0250] Fluorescent labels considered as a complex include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cyan 3 (Cy3), Cyan 5 (Cy5), 6-FAM, Dansilchloride, Dichlorotriazinylaminofluorescein, Fluorescein Isothiocyanate (FITC), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, and Pacific Blue. Blue), phycoerythrin, REG, rhodamine green, rhodamine red, renographin, ROX, TAMRA, TET, tetramethylrhodamine isothiocyanate (TRITC), Texas Red, and / or umbelliferone.

[0251] Other types of antibodies considered in this invention are primarily intended for in vitro use, in which the antibody is bound to a secondary ligand and / or enzyme (enzyme tag), and when this comes into contact with a chromogenic substrate, it produces a colored product. Examples of suitable enzymes include β-galactosidase, acetylcholinesterase, urease, alkaline phosphatase, (horseradish) peroxidase, or glucose oxidase. Preferred secondary ligands are biotin and avidin, as well as streptavidin compounds.

[0252] Numerous methods are known in the art for linking or complexing antibodies to their complex portions. Some linking methods involve the use of metal chelate complexes, such as diethylenetriaminepentaanhydride (DTPA); ethylenediaminetetraacetic acid; monomer or dendrimer type 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA); DFO; HOPO; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3a-6a-diphenylglycouryl-3 (U.S. Patents 4,472,509 and 4,938,948) bound to antibodies. Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodates. Fluorescently labeled complexes can be prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Patent No. 4,938,948, imaging of breast cancer tumors was achieved using monoclonal antibodies, with a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate used to bind detectable imaging sites to the antibody.

[0253] Another known method of site-specific linking between molecules and antibodies involves the reaction of an antibody with a hapten-based affinity tag. Essentially, the hapten-based affinity tag reacts with amino acids within the antigen-binding site, thereby disrupting that site and blocking the specific antigen reaction.

[0254] Molecules containing azide groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light. Specifically, 2- and 8-azide analogs of purine nucleotides are used as site-specific optical probes to identify nucleotide-binding proteins in crude cell extracts. 2- and 8-azide nucleotides are also used for mapping nucleotide-binding domains in purified proteins and are available as antibody conjugates.

[0255] Furthermore, the derivatization of immunoglobulins under reaction conditions that do not alter the antibody binding site is also considered by selectively introducing thiol groups into the Fc region of the immunoglobulin. Antibody conjugates produced by this method have been disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, which is incorporated herein by reference). The literature also discloses site-specific ligation of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a glycan residue in the Fc region. This method generates antibodies with diagnostic and therapeutic prospects that are currently undergoing clinical evaluation.

[0256] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein can be conjugated with a steroid or immunosuppressant. In some embodiments, the antibody or its antigen-binding fragment is conjugated with a steroid or immunosuppressant to form an ADC (antibody-drug conjugate). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein can be conjugated with a steroid, the steroid being a corticosteroid. Corticosteroids may be, for example, dexamethasone, hydrocortisone, methylprednisolone, and prednisone. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein can be conjugated with an immunosuppressant, the immunosuppressant being an antimalarial agent (e.g., hydroxychloroquine, chloroquine), an antimetabolite (e.g., methotrexate, azathioprine, mercaptopurine), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), mycophenolic acid, mycophenolate mofetil, thalidomide, or acitretin.

[0257] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein may be conjugated to a cytotoxic agent or site. In some embodiments, the antibody or its antigen-binding fragment is conjugated to a cytotoxic agent to form an antibody-drug conjugate (ADC). In some embodiments, antibody-drug conjugates (ADCs) are a type of highly efficient biopharmaceutical designed as targeted therapy. An ADC consists of an antibody (a complete monoclonal antibody or an antibody fragment such as an scFv) conjugated to a bioactive cytotoxic / antiviral active ingredient or drug via a stable chemical linker with an unstable binding. Antibody-drug conjugates are an example of bioconjugates and immune conjugates. By combining the specific targeting ability of a monoclonal antibody with a cytotoxic drug, ADCs enable sensitive differentiation between healthy and diseased tissue. This means that, compared to conventional systemic approaches, ADCs target and attack diseased cells while minimizing impact on healthy cells.

[0258] In the development of ADC-based antitumor therapies, warheads (e.g., cell toxins) or cytotoxins are conjugated to antibodies that specifically target certain cellular markers (ideally proteins present only on diseased cells). The antibodies target these proteins in the body, binding themselves to the surface of diseased cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal within the target cell, which then takes up or internalizes the antibody and cytotoxin. After the ADC is internalized, the cytotoxic drug is released, either killing the cell or inhibiting cell replication. In other cases, the linker can be cleaved on the surface of the target cell or early endosome, thus eliminating the need for complete internalization. This targeting ideally results in a drug with fewer side effects and a wider therapeutic window than other drugs.

[0259] In some embodiments, the cytotoxic site of an ADC having the anti-BDCA2 antibody or its antigen-binding fragment described herein is a chemotherapeutic agent, which includes (but is not limited to) methotrexate, adriamycin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepine (PBD), or other intercalators. In some embodiments, the cytotoxic site is a microtubule inhibitor, which includes (but is not limited to) auristatin, mytansins (e.g., DM1 and DM4), and tubulicin. In some embodiments, the cytotoxic site is an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants, or animals, and includes (but is not limited to) diphtheria A chain, unbound active diphtheria toxin fragment, exotoxin A chain, lysine A chain, abrin A chain, modesine A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis This includes officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.In some embodiments, an antibody or its antigen-binding fragment is conjugated to one or more low-molecular-weight toxins such as calicheamicin, matansin, trichothenes, or CC1065.

[0260] Stable binding of antibodies to cytotoxic agents is a crucial aspect of ADCs. Linkers are based on a chemical basis including disulfides, hydrazones, or peptides (cleavable) or thioethers (non-cleavable) and control the distribution and delivery of cytotoxic agents to target cells. Both cleavable and non-cleavable linkers have been demonstrated to be safe in preclinical and clinical trials. The availability of superior, more stable linkers is altering the function of the chemical bond. Cleavable or non-cleavable linker species impart specific properties to cytotoxic (e.g., anticancer) drugs. For example, non-cleavable linkers retain the drug within the cell. Thus, the entire antibody, linker, and cytotoxic agent enter the target cell, where the antibody is broken down to the amino acid level. The resulting complex—amino acids, linker, and cytotoxic agent—becomes the active agent. In contrast, cleavable linkers are catalyzed by enzymes inside or on the host cell to release the cytotoxic agent. Common mechanisms of linker cleavage include protease sensitivity, pH sensitivity, and glutathione sensitivity. Another type of cleavable linker adds an additional molecule between the cytotoxic drug and the cleavage site. This linker technology allows researchers to create more flexible ADCs without altering the cleavage dynamics. Novel peptide cleavage methods based on Edman degradation have also been developed. Future directions for the development of ADCs include the development of site-directed complexes (TDCs) to further improve stability and therapeutic index, as well as alpha-emitting immune complexes and antibody-conjugated nanoparticles.

[0261] The anti-BDCA2 antibody or its antigen-binding fragment described herein can be conjugated to a solid carrier. These solid carriers include (but are not limited to) glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-BDCA2 antibody or its antigen-binding fragment is used for immunoassays. In some embodiments, the immobilized anti-BDCA2 antibody or its antigen-binding fragment is used for the purification of a target antigen (e.g., human BDCA2).

[0262] 1.4 Polynucleotides and Vectors The present application also provides polynucleotides encoding polypeptides described herein (e.g., anti-BDCA2 antibodies or their antigen-binding fragments). The term “polynucleotide encoding a polypeptide” encompasses both polynucleotides comprising only the coding sequence of a polypeptide and polynucleotides comprising other coding sequences and / or non-coding sequences. The polynucleotides of the present disclosure may be in the form of RNA or DNA. The DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. The single-stranded DNA may be a coding strand or a non-coding (antisense) strand. The polynucleotides of the present invention may be mRNA.

[0263] In this application, any polynucleotide encoding any anti-BDCA2 antibody or its antigen-binding fragment disclosed herein is explicitly considered. For illustrative purposes, in some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or an antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or the antigen-binding fragment is (1) a light chain variable region (VL) comprising (a) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10 respectively, or a variant thereof having up to about five amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 18 respectively, or a variant thereof having up to about five amino acid substitutions, additions, and / or deletions in the VH CDR; or (2) a VL comprising (a) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10 respectively, or VL (b) VH including variants of the CDR having up to approximately 5 amino acid substitutions, additions, and / or deletions in the CDR; and / or (b) VH including VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs: 14, 16, and 18; or variants of the VH CDR having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0264] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or an antigen-binding fragment thereof, comprising: (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26; and / or (b) VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27. The polynucleotide may be in the form of DNA. The polynucleotide may be in the form of mRNA.

[0265] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or its antigen-binding fragment comprising VL and VH disclosed herein, wherein VL comprises VL CDR1, CDR2, and CDR3, and VH comprises VH CDR1, CDR2, and CDR3, and the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 each have (1) the amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 15, 17, and 18; or (2) the amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 14, 16, and 18; or variants thereof having up to about five amino acid substitutions, additions, and / or deletions in the CDR. The polynucleotides may be in the form of DNA. The polynucleotides may be in the form of mRNA.

[0266] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or its antigen-binding fragment comprising VL and VH disclosed herein, wherein VL and VH each have the following amino acid sequences: (1) SEQ ID NOs. 28 and 34; (2) SEQ ID NOs. 28 and 35; (3) SEQ ID NOs. 28 and 36; (4) SEQ ID NOs. 28 and 35; (5) SEQ ID NOs. 29 and 34; (6) SEQ ID NOs. 29 and 35; (7) SEQ ID NOs. 29 and 36; (8) SEQ ID NOs. 29 and 37; (9) SEQ ID NOs. 30 and 34; (10) SEQ ID NOs. 30 and 35; (11) SEQ ID NOs. 30 and 36; (12) SEQ ID NOs. 30 and 37; (13) SEQ ID NOs. 31 and 34; (14) SEQ ID NOs. 31 and 35; (15) SEQ ID NOs. 31 and 36; or (16) SEQ ID NOs. 31 and 37. The polynucleotides may be in the form of DNA. The polynucleotides may be in the form of mRNA.

[0267] In this application, any polynucleotide encoding any anti-BDCA2 antibody or its antigen-binding fragment disclosed herein is explicitly considered. For illustrative purposes, in some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or an antigen-binding fragment thereof, wherein the anti-BDCA2 antibody or its antigen-binding fragment is (1) a light chain variable region (VL) comprising (a) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13 respectively, as defined by Kabat, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23 respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR; or (2) VL CDR1, VL CDR2, VL CDR3 having the amino acid sequences shown in SEQ ID NOs. 11, 12, and 13 respectively, as defined by Chothia. VL containing CDR3, or variants thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in VL CDR; and / or (b) VH containing VH CDR1, VH CDR2, VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 19, 21, and 23; or variants thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in VH CDR;

[0268] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or an antigen-binding fragment thereof, comprising: (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 40; and / or (b) VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 41. The polynucleotide may be in the form of DNA. The polynucleotide may be in the form of mRNA.

[0269] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or its antigen-binding fragment comprising VL and VH disclosed herein, wherein VL comprises VL CDR1, CDR2, and CDR3, and VH comprises VH CDR1, CDR2, and CDR3, and the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 each have (1) the amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 20, 22, and 23, respectively; or (2) the amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 19, 21, and 23; or variants thereof having up to about five amino acid substitutions, additions, and / or deletions in the CDR. The polynucleotides may be in the form of DNA. The polynucleotides may be in the form of mRNA.

[0270] In some embodiments, the polynucleotides provided herein encode an anti-BDCA2 antibody or its antigen-binding fragment comprising VL and VH disclosed herein, wherein VL and VH each have the following amino acid sequences: (1) SEQ ID NOs. 42 and 47; (2) SEQ ID NOs. 42 and 48; (3) SEQ ID NOs. 42 and 49; (4) SEQ ID NOs. 42 and 50; (5) SEQ ID NOs. 43 and 47; (6) SEQ ID NOs. 43 and 48; (7) SEQ ID NOs. 43 and 49; (8) SEQ ID NOs. 43 and 50; (9) SEQ ID NOs. 44 and 47; (10) SEQ ID NOs. 44 and 48; (11) SEQ ID NOs. 44 and 49; (12) SEQ ID NOs. 44 and 50; (13) SEQ ID NOs. 45 and 47; (14) SEQ ID NOs. 45 and 48; (15) SEQ ID NOs. 45 and 49; (16) SEQ ID NOs. 45 and 50; (17) SEQ ID NOs. 46 and 47; (18) SEQ ID NOs. 46 and 48; (19) SEQ ID NOs. 46 and 49; (20) SEQ ID NOs. 46 and 50. The polynucleotide may be in the form of DNA. The polynucleotide may be in the form of mRNA.

[0271] In some embodiments, the VL and VH are connected via a linker. The linker may be flexible or rigid. In some embodiments, the linker has an amino acid sequence (GGGGS)n, including n=1, 2, 3, 4, or 5 (SEQ ID NO: 57). In some embodiments, the linker has an amino acid sequence (EAAAK)n, including n=1, 2, 3, 4, or 5 (SEQ ID NO: 58). In some embodiments, the linker has an amino acid sequence (PA)nPAP, including n=0, 1, 2, 3, or 4 (SEQ ID NO: 59).

[0272] The present invention also provides variants of the polynucleotide described herein, the variants encoding, for example, fragments, analogs, and / or derivatives of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein. In some embodiments, the present invention provides polynucleotides having a nucleotide sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity with the polynucleotide sequence encoding the anti-BDCA2 antibody or its antigen-binding fragment described herein. In some embodiments, the present invention provides a polynucleotide having a nucleotide sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the polynucleotide sequence encoding the anti-BDCA2 antibody or its antigen-binding fragment described in this application.

[0273] As used in this application, the phrase “a polynucleotide having a nucleotide sequence having at least about 95% identity with the polynucleotide sequence” means that the nucleotide sequence of the polynucleotide is identical to that of the reference sequence, but the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence having at least 95% identity with the reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides of the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence, or at any location between those end positions, and may be individually scattered among the nucleotides in the reference sequence, or present in one or more consecutive groups within the reference sequence.

[0274] The polynucleotide variant may include changes in the coding region, the non-coding region, or both. In some embodiments, the polynucleotide variant includes changes that result in silent substitutions, additions, or deletions that do not alter the properties or activity of the encoded polypeptide. In some embodiments, the polynucleotide variant includes silent substitutions (due to genetic coding degeneracy) that leave the amino acid sequence of the polypeptide unchanged. Polynucleotide variants may be created for a variety of reasons, such as optimizing codon expression in a particular host (e.g., changing the codons in human mRNA to codons preferred by a bacterial host such as Escherichia coli). In some embodiments, the polynucleotide variant includes at least one silent mutation in the non-coding region or the coding region of the sequence.

[0275] In some embodiments, polynucleotide variants are created to regulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are created to improve the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are created to reduce the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant exhibits improved expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, the polynucleotide variant exhibits reduced expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0276] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) fused with a polynucleotide (e.g., a leader sequence that functions as a secretory sequence controlling polypeptide transport) that assists expression and secretion from a host cell within the same reading frame. The polypeptide may have a leader sequence that is cleaved by the host cell to form a “mature” form of the polypeptide.

[0277] In some embodiments, the polynucleotide includes the coding sequence of a polypeptide (e.g., an antibody) fused with a marker or tag sequence within the same reading frame. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS tag), which allows for efficient purification of the polypeptide fused to the marker. In some embodiments, when using a mammalian host (e.g., COS-7 cells), the marker sequence is a hemagglutinin (HA) tag derived from influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG TM It is a tag. In some embodiments, the marker can be used in combination with other markers or tags.

[0278] In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

[0279] The present application provides vectors and cells comprising the polynucleotide described herein. In some embodiments, the present application provides vectors comprising the polynucleotide provided herein. The vector may be an expression vector. In some embodiments, the vector provided herein comprises a polynucleotide encoding the anti-BDCA2 antibody or its antigen-binding fragment described herein. In some embodiments, the vector provided herein comprises a polynucleotide encoding a polypeptide as part of the anti-BDCA2 antibody or its antigen-binding fragment described herein.

[0280] In some embodiments, the Application provides recombinant expression vectors usable for amplification and expression of polynucleotides encoding the anti-BDCA2 antibody or its antigen-binding fragment described herein. For example, the recombinant expression vector is a replicable DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding the polypeptide chain of the anti-BDCA2 antibody, which is functionally linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene. In some embodiments, a viral vector is used. DNA regions are said to be "functionally linked" if they are functionally related to each other. For example, if a promoter controls the transcription of a sequence, the promoter is functionally linked to the coding sequence; or, if the arrangement of a ribosome binding site enables translation, it is functionally linked to the coding sequence. In some embodiments, structural elements designed for use in a particular expression system include a leader sequence that enables the host cell to secrete the translated protein extracellularly. In some embodiments, if the recombinant protein is expressed without a leader sequence or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0281] A variety of expression host / vector combinations can be utilized. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, including pCR1, pBR322, pMB9 and their derivatives, derived from Escherichia coli (E. coli), as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA phages. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is expressed from one or more vectors.

[0282] This application provides suitable host cells containing the vector described herein. In some embodiments, the host cells may be used for recombinant expression of the anti-BDCA2 antibody described herein. The host cells may include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under appropriate promoter control. Cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein (including antibody) production, are well known in the art.

[0283] Examples of suitable mammalian host cells include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary cancer), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from hamster renal fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may include a replication origin, appropriate promoters and enhancers linked to the target gene, other 5' or 3' wing non-transcriptional sequences, and non-transcriptional elements including essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and 5' or 3' non-translating sequences such as transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded, biologically functional proteins. Baculovirus systems for heterologous protein production in insect cells are well known to those skilled in the art.

[0284] The Disclosure also provides host cells containing the polypeptide described in the Application, a polynucleotide encoding the polypeptide described in the Application, or a vector comprising these polynucleotides. In some embodiments, the Application provides host cells containing a vector comprising the polynucleotide disclosed herein. In some embodiments, the host cells provided in the Application contain a vector comprising a polynucleotide encoding the anti-BDCA2 antibody described in the Application or its antigen-binding fragment. In some embodiments, the host cells provided in the Application contain a vector comprising a polynucleotide encoding the polypeptide as part of the anti-BDCA2 antibody described in the Application or its antigen-binding fragment. In some embodiments, the host cells provided in the Application contain a polynucleotide encoding the anti-BDCA2 antibody described in the Application or its antigen-binding fragment. In some embodiments, the cells produce the anti-BDCA2 antibody described in the Application or its antigen-binding fragment.

[0285] 1.5 Manufacturing method This application provides anti-BDCA2 antibodies and their antigen-binding fragments, including (but not limited to) monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and antigen-binding fragments thereof.

[0286] Methods for antibody production are well known in the art. References include Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); and Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), the entirety of which is incorporated herein by reference. In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those skilled in the art. For example, as described above, mice, rats, rabbits, hamsters, or other suitable host animals are immunized using hybridoma methods. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immune antigen is a human protein or a fragment thereof.

[0287] After immunization, lymphocytes are isolated and fused with appropriate myeloma cells, for example, using polyethylene glycol. Hybridoma cells are then selected using specialized media known in the art, with unfused lymphocytes and myeloma cells failing to survive the selection process. Hybridomas producing monoclonal antibodies against the target antigen can be identified by a variety of methods, including (but not limited to) immunoprecipitation, Western blotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), radioimmunoassay). Once hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, these clones can be subcloned by limiting dilution or other techniques. Hybridomas can be cultured in vitro or grown in vivo as ascites tumors in animals using standard methods. Monoclonal antibodies can be purified from culture media or ascites fluid according to standard methods in the art, including (but not limited to) affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0288] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which, upon transfection into host cells such as Escherichia coli (E. coli), monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, produce monoclonal antibodies.

[0289] The polynucleotides of antibodies or their antigen-binding fragments provided herein can be prepared, manipulated, and / or expressed using any known or available, authorized techniques in the art. In some embodiments, the polynucleotides of antibodies or their antigen-binding fragments provided herein can be prepared recombinantly. A variety of vectors can be used. Examples of vectors include plasmids, self-replicating sequences, and transposition factors. Exemplary transposon systems such as Sleeping Beauty and PiggyBac, which can be stably incorporated into the genome (e.g., Ivics et al., Cell, 91 (4): 501-510 (1997); Cadinanos et al., (2007) Nucleic Acids Research. 35 (12): e87), can be used. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), phages such as λ phage and M13 phage, and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses (baculovirus), baculoviruses (papillomavirus), papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells. TM pLenti6 / V5-DEST TM Examples include pLenti6.2 / V5-GW / lacZ(Invitrogen).

[0290] In some embodiments, the vector is an episomal vector or a vector maintained outside the chromosome. As used in this application, the term “episomal” refers to a vector that is replicable but not incorporated into host chromosomal DNA and is not gradually lost from a dividing host cell, and also means that the vector replicates outside the chromosome or episomal. The vector is designed to have a sequence encoding a DNA origin of replication ("ori") of lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically a lymphotropic herpesvirus or gamma herpesvirus origin of replication corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), squirrel monkey herpesvirus (Herpes virus saimiri, HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Generally, the host cell contains viral replication transactivator proteins that activate replication.

[0291] The “expression regulatory sequences,” “regulatory elements,” or “regulatory sequences” present in an expression vector refer to sequences that interact with host cell proteins for transcription and translation, such as the untranslated region of the vector replication origin, selection markers, promoters, enhancers, translation initiation signals (ribosome-binding site sequences or Kozak sequences), introns, polyadenylated sequences, and 5' and 3' untranslated regions. The intensity and specificity of these elements may vary. Depending on the vector system and host used, any number of appropriate transcription and translation elements, including ubiquitin promoters and inducible promoters, may be used.

[0292] Representative universal expression regulatory sequences usable in this disclosure include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) promoter (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rouss sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the vaccinia virus-derived H5, P7.5, and P11 promoters, the elongation factor 1-α (EF1α) promoter, the early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), and glyceraldehyde 3-phosphate dehydrogenase. This includes, but is not limited to, dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa β, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0293] Representative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters (e.g., promoters of genes encoding glucocorticoids or estrogen receptors: induced by corresponding hormone treatment), metallothionein promoters (induced by various heavy metal treatments), MX-1 promoters (induced by interferon), the "GeneSwitch" mifepristone regulatory system (Sirin et al., 2003, Gene, 323:67), the kmet-inducible gene switch (WO 2002 / 088346), and tetracycline-dependent regulatory systems. The anti-BDCA2 antibody or its antigen-binding fragment described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise specified, the invention utilizes conventional techniques within the scope of the art in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields.

[0294] The polypeptides described herein can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, or combinations thereof. In some embodiments, recombinant expression vectors are used to express polynucleotides encoding the polypeptides described herein. For example, a recombinant expression vector may be a replicable DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding a polypeptide, operably linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene. In some embodiments, the coding sequences of the polypeptides disclosed herein may be linked to such an expression vector for their expression in mammalian cells. In some embodiments, viral vectors are used. DNA regions are said to be "operably linked" if they are functionally related to each other. For example, if a promoter controls the transcription of a sequence, the promoter is functionally linked to the coding sequence; or, if the arrangement of a ribosome binding site enables translation, it is functionally linked to the coding sequence. In some embodiments, structural elements designed for use in a yeast expression system include a leader sequence that enables the host cell to secrete the translated protein extracellularly. In some embodiments, when recombinant protein is expressed without a leader sequence or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0295] A diverse range of expression host / vector combinations are available. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under appropriate promoter control. Cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein (including antibody) production, are well known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as pCR1, pBR322, pMB9 and their derivatives, derived from Escherichia coli (E. coli), as well as plasmids with broader host ranges, such as M13 and other filamentous single-stranded DNA phages.

[0296] Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary cancer), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from hamster renal fibroblasts), HEK-293 (derived from human fetal kidney) cell lines and their variants. Mammalian expression vectors may also include non-transcription elements, such as origins of replication, appropriate promoters and enhancers linked to the target gene, other 5' or 3' wing non-transcription sequences, and 5' or 3' non-translating sequences, including essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. The expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded, biologically functional proteins. Baculovirus systems for heterologous protein production in insect cells are well known to those skilled in the art.

[0297] To prepare fucose-unmodified anti-BDCA2 antibodies and their antigen-binding fragments, the following host cells may be used: (1) cells overexpressing N-acetylglucosamine transferase III (GnTIII), (2) cells lacking α-1,6-fucosyltransferase (FUT8), or (3) cells with a low fucose content, or any combination of (1) to (3). In some embodiments, host cells overexpressing N-acetylglucosamine transferase III (GnTIII) are used. In some embodiments, host cells lacking α-1,6-fucosyltransferase (FUT8) are used. In some embodiments, host cells with a low fucose content are used. In some embodiments, CHO host cells are used.

[0298] Peptides can be synthesized completely or partially by chemical methods (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, AK, Therapeutic Peptides and Proteins, Formula, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). A variety of solid-phase techniques can be used for peptide synthesis (see, e.g., Roberge, Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)). Automated synthesis can also be achieved, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Furthermore, peptides can also be synthesized using combinatorial methods. Synthetic residues and polypeptides can be synthesized using a variety of procedures and methods known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (eds.), John Wiley & Sons, Inc., NY). Modified peptides can be generated by chemical modification methods (see, e.g., Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Furthermore, peptide sequences can be altered, derivatized, substituted, and modified using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)), and other techniques can be applied to cloned DNA to generate the peptide sequences, variants, fusions, chimeras, and their alterations, derivatives, substitutions, and modifications according to the present invention.

[0299] For the in vivo use of antibodies in humans, human antibodies are preferably used. Complete human antibodies are particularly desirable for the therapeutic treatment of human subjects. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods (including improvements on these techniques) using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887, 4,716,111, and PCT Patent Publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741; each of these patents is incorporated in whole into this application by reference. Human antibodies may also be antibodies whose heavy and light chains are encoded by nucleotide sequences from one or more sources derived from human DNA.

[0300] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared using a variety of techniques known in the art. In some embodiments, human antibodies are produced from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced from lymphocytes isolated from immunized individuals. In either case, cells that produce antibodies against the target antigen can be generated and isolated. In some embodiments, human antibodies are selected from a phage library expressing human antibodies. Alternatively, human antibodies and antibody fragments can be produced in vitro from the immunoglobulin variable region genomic repertoire of non-immune donors using phage display techniques. Techniques for the preparation and use of antibody phage libraries are well known in the art. Once antibodies are identified, higher affinity human antibodies can be produced using affinity maturation strategies known in the art, including (but not limited to) chain shuffling and site-directed mutagenesis. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. These mice, through immunization, can produce a complete repertoire of human antibodies without endogenous immunoglobulin production.

[0301] Furthermore, human antibodies can be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, the human heavy-chain and light-chain immunoglobulin gene complex can be introduced into mouse embryonic stem cells by random or homologous recombination. Alternatively, in addition to the human heavy-chain and light-chain genes, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells. By introducing the human immunoglobulin locus by homologous recombination, the mouse heavy-chain and light-chain immunoglobulin genes can be inactivated individually or simultaneously. For example, homozygous deletion of the antibody heavy-chain binding region (JH) gene in chimeric and germline mutant mice has been reported to completely suppress endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to create chimeric mice. Next, the chimeric mice are mated to produce pure offspring that express human antibodies. The transgenic mice are immunized by a selected antigen, for example, all or part of the polypeptide of the present invention, using conventional methods. For example, using conventional hybridoma technology, anti-BDCA2 antibodies against the anti-human BDCA2 antigen can be obtained from immunized transgenic mice. The human immunoglobulin genes possessed by transgenic mice are rearranged during the B-cell differentiation process, and then undergo class switching and somatic mutation. Therefore, this technology can be used to produce IgG, IgA, IgM, and IgE antibodies (including, but not limited to, IgG1(γ1) and IgG3) that can be used for therapeutic purposes. For a review of this technology for human antibody production, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)).For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies, and for protocols for producing such antibodies, see, for example, PCT Patent Publications WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598. Each of these patents is incorporated in whole by reference into this application. Furthermore, human antibodies against selected antigens can be provided by commissioning companies such as Abgenix, Inc. (Fremont, California) and Genpharm (San Jose, California) using the same technology as described above. For a specific discussion on how introducing human germline immunoglobulin gene arrays into germline mutant mice induces human antibody production upon antigen administration, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0302] Human antibodies can also be obtained from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) region genome libraries of non-immune donors. In this technology, antibody V region genes are cloned in-frame into major or secondary coat protein genes of filamentous phages such as M13 and fd, and presented as functional antibody fragments on the surface of phage particles. Since filamentous particles contain single-stranded DNA copies of the phage genome, functional selection of antibodies also leads to selection of the genes encoding antibodies that exhibit those characteristics. Therefore, phages mimic some properties of B cells. Phage display can be performed in various forms; for a review, see, for example, Johnson and Chiswell, Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments are available for phage display. Clackson et al., Nature, 352:624-628 (1991), isolated a diverse group of anti-oxazolone antibodies from a small, randomly combined library constructed from V genes derived from non-immune mouse spleens. Constructing a V gene repertoire from non-immune human donors and isolating antibodies against numerous different antigens (including autoantigens) is fundamentally possible according to the techniques described in Marks et al., J. Mol. Biol., 222:581-597 (1991) or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patents No. 5,565,332 and No. 5,573,905. These documents are incorporated herein by reference in their entirety.

[0303] Human antibodies can also be produced from activated B cells in vitro (see U.S. Patents 5,567,610 and 5,229,275, which are incorporated herein by reference in their entirety). Furthermore, human antibodies can also be produced in vitro using hybridoma technology, such as the method described by Roder et al. (Methods Enzymol., 121:140-167 (1986)).

[0304] Alternatively, in some embodiments, non-human antibodies are humanized. This involves modifying specific sequences or regions of an antibody to increase its similarity to antibodies that occur naturally in humans. In some embodiments, the antigen-binding region is partially humanized. Various methods for producing humanized antibodies are known in the art. Methods for achieving high-affinity binding with humanized antibodies are also known in the art. Non-limiting examples of this method include hypermutation of variable regions and selection of cells expressing these high-affinity antibodies (affinity maturation). In addition to the use of display libraries, non-human animals such as rodents can be immunized using specific antigens (e.g., recombinant BDCA2 or its epitope). In some embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be produced and isolated using methods known in the art and / or disclosed herein. In some embodiments, mice can be immunized with an antigen (e.g., recombinant BDCA2 or its epitope).

[0305] Methods used for the production of humanized antibodies include, but are not limited to, the following techniques: CDR implantation (see, for example, European Patent No. EP 239,400, International Patent Publication No. WO 91 / 09967, U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated in whole as reference), veneering, or resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596, Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498, Studnicka et al., 1994, Protein See Engineering, 7(6):805-814, Roguska et al., 1994, PNAS, 91:969-973 (each of these documents is incorporated in its entirety by reference), chain shuffling (see, for example, U.S. Patent No. 5,565,332, which is incorporated in its entirety by reference), and the technologies disclosed in the following documents: for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Patent Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). The entirety of each of these documents is incorporated into this application as reference.Generally, antigen binding can be altered, and preferably improved, by substituting framework residues in the framework region of a CDR donor antibody with corresponding residues. These framework substitutions are identified by methods well known in the art, such as identifying framework residues important for antigen binding by modeling the interaction between the CDR and framework residues, and identifying atypical framework residues at specific positions by sequence comparison. (See, for example, Queen et al., U.S. Patent No. 5,585,089, and Riechmann et al., 1988, Nature, 332:323, these documents in their entirety incorporated herein by reference).

[0306] Humanized antibodies incorporate one or more non-human amino acid residues. These non-human amino acid residues are generally called "import" residues and are usually incorporated from the "import" variable region. Therefore, humanized antibodies contain one or more CDRs derived from non-human immunoglobulin molecules and a human-derived framework region. Antibody humanization is well known in the art and can be basically carried out by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences, i.e., CDR transplantation (EP 239,400; PCT Patent Publication No. WO 91 / 09967, U.S. Patents No. 4,816,567, No. 6,331,415, No. 5,225,539, No. 5,530,101, No. 5,585,089, No. 6,548,640; the contents of these patents are incorporated into this application in their entirety by reference). In these humanized chimeric antibodies, the incomplete human variable region is replaced with the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced with residues at homologous positions in rodent antibodies. Antibody humanization can also be achieved by veneerization or surface reconstruction (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); Roguska et al., PNAS, 91:969-973 (1994)), or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated in their entirety by reference.

[0307] The selection of human variable regions (both light and heavy chains) used in the preparation of humanized antibodies reduces antigenicity. According to the so-called "best-fit" method, variable region sequences of rodent antibodies are screened against the entire library of known human variable region sequences. The human sequence that most closely resembles the rodent sequence is then adopted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987). The contents of these publications are incorporated in their entirety as references). Alternatively, a specific framework is used, derived from the consensus sequences of all human antibodies belonging to a particular subgroup of light or heavy chains. The same framework can be applied to multiple different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of these documents are incorporated in their entirety as references).

[0308] Antibodies can be humanized while retaining high affinity for target antigens and other advantageous biological properties. For example, humanized antibodies can be prepared using parental sequence analysis techniques and multiple conceptual humanized products based on three-dimensional models of parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and well known to those skilled in the art. Computer programs are available that describe and display possible three-dimensional structures of selected candidate immunoglobulin sequences. By examining these displays, it becomes possible to analyze residues that may play a function in the candidate immunoglobulin sequence, i.e., residues that affect the target antigen binding ability of the candidate immunoglobulin. This method allows for the selection and combination of FR residues from receptor and transduction sequences to achieve desired antibody properties, such as improved affinity for target antigens. Generally, CDR residues are directly and most prominently involved in antigen binding.

[0309] Humanized antibodies retain similar antigen specificity to the original antibody, such as the ability to bind to the human BDCA2 antigen. However, certain humanization methods can improve the affinity and / or specificity of the antibody to specific antigens using the “directed evolution” method described in Wu et al., J. Mol. Biol., 294:151 (1999) (the contents of this document are incorporated in their entirety as reference).

[0310] The binding of the anti-BDCA2 antibody or its antigen-binding fragment described in this application to human BDCA2 can be verified, for example, by standard ELISA. Briefly, purified BDCA2 is coated onto a microtiter plate and then blocked with bovine serum albumin. Antibody diluent (e.g., diluted BDCA2-immunized mouse plasma) is added to each well and incubated. After washing the plate, it is incubated with a secondary reagent labeled with horseradish peroxidase (HRP) (e.g., goat anti-human IgG Fc-specific polyclonal reagent for human antibodies). After washing, the plate is color-developed and analyzed with a spectrophotometer. Next, further screening can be performed using immunized mouse serum by flow cytometry to determine whether the antibody binds to cell lines expressing human BDCA2 but not to control cell lines that do not express BDCA2. Briefly, the binding of the anti-BDCA2 antibody can be evaluated by incubating BDCA2-expressing CHO cells with the anti-BDCA2 antibody. After washing the cells, binding can be detected with an anti-human IgG antibody. For flow cytometry analysis, FACS or a flow cytometer (Becton Dickinson, San Jose, CA) can be used. Mice exhibiting the highest titer can be used for cell fusion.

[0311] The aforementioned ELISA assay can be used for antibody screening and, consequently, for selecting hybridomas that produce antibodies showing positive reactivity to the BDCA2 immunogen. Subsequently, hybridomas that produce antibodies that bind to BDCA2 with high affinity can be subcloned and analyzed in more detail. Then, one clone retaining the reactivity of the parent cell (as determined by ELISA) can be selected from each hybridoma and used for cell banking and antibody purification.

[0312] To purify the anti-BDCA2 antibody, selected hybridomas are grown and used for the purification of the monoclonal antibody. The supernatant can be filtered and concentrated before affinity chromatography. To confirm the purity of the eluted IgG, gel electrophoresis and high-performance liquid chromatography can be performed. The buffer solution can be changed and the concentration determined. The monoclonal antibody is then aliquoted and stored.

[0313] To determine whether the selected anti-BDCA2 monoclonal antibody binds to a specific epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of the biotinylated MAb can be detected using a streptavidin-labeled probe. A competitive experiment using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using the aforementioned BDCA2-coated ELISA plate.

[0314] To determine the isotype of purified antibodies, isotype ELISA can be performed using reagents specific to the antibody of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, 1 pg / mL of anti-human immunoglobulin is coated into the wells of a microtiter plate overnight at 4°C. After blocking with 1% BSA, the plate is reacted with a test monoclonal antibody or purified isotype control at a concentration of 1 f ig / mL or less at room temperature for 1-2 hours. Then, each well is reacted with a human IgG1 or human IgM-specific alkaline phosphatase-labeled probe. The plate is then colorimetrically analyzed according to the method described above.

[0315] To test the binding of monoclonal antibodies to BDCA2-expressing living cells, flow cytometry can be applied as described in the examples. Briefly, cell lines expressing membrane-bound BDCA2 (grown under standard culture conditions) are mixed with monoclonal antibodies of various concentrations prepared in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with fluorescently labeled anti-IgG antibodies under the same conditions as primary antibody staining. Using a FACScan instrument, the sample can be analyzed by gating single cells based on forward and side scattering characteristics and determining the binding of the labeled antibody. In addition to or as an alternative to flow cytometry, an alternative measurement method using fluorescence microscopy can be employed. Cells can be stained using the same method as described above, and observation with a fluorescence microscope can be performed. This method allows for the visualization of individual cells, but the sensitivity may decrease depending on the antigen density.

[0316] The reactivity of anti-BDCA2 antibodies or their antigen-binding fragments with the BDCA2 antigen can be further investigated by Western blotting. Briefly, cell extracts are prepared from BDCA2-expressing cells and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked with 20% mouse serum, and then probed with the monoclonal antibody of the test. IgG binding is detected with anti-IgG alkaline phosphatase and colored using BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0317] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-BDCA2 antibodies include standard measurement methods well known in this field, such as biolayer interferometry (BLI) using the Gator system (Probe Life) or the Octet-96 system (Sartorius AG), or BIACORE TM BIACORE using 2000 SPR devices (Biacore AB, Uppsala, Sweden) TM Examples include surface plasmon resonance (SPR) analysis.

[0318] 1.6 Pharmaceutical Compositions This application further provides a pharmaceutical composition comprising the anti-BDCA2 antibody disclosed herein or an antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-BDCA2 antibody disclosed herein or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is useful for suppressing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the pharmaceutical composition is useful for treating diseases or conditions associated with type I IFN (e.g., IFNα) or pDC.

[0319] In some embodiments, the pharmaceutical composition provided herein comprises an anti-BDCA2 antibody or its antigen-binding fragment provided herein. The anti-BDCA2 antibody or its antigen-binding fragment may be present at various concentrations. In some embodiments, the pharmaceutical composition provided herein comprises 1 to 1000 mg / mL of the soluble anti-BDCA2 antibody or its antigen-binding fragment provided herein. Those skilled in the art can easily adjust the dosage, for example, increasing the dosage may be necessary if the purity decreases.

[0320] The present application further provides a kit for preparing a pharmaceutical composition comprising the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein. In some embodiments, the kit comprises the anti-BDCA2 antibody or its antigen-binding fragment contained in one or more containers and a pharmaceutically acceptable carrier. In another embodiment, the kit may comprise the anti-BDCA2 antibody or its antigen-binding fragment for administration to a subject. In a specific embodiment, the kit comprises instructions for the preparation and / or administration of the anti-BDCA2 antibody or its antigen-binding fragment.

[0321] In some embodiments, the present application provides pharmaceutical compositions suitable for topical administration, comprising an anti-BDCA2 antibody or its antigen-binding fragment or cells provided herein.

[0322] pharmaceutically acceptable carriers usable with the compositions provided herein include any physiologically compatible solvents, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic agents, absorption retarders, and the like. In some embodiments, the carriers are suitable for intravenous, intramuscular, subcutaneous, transdermal, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., an anti-BDCA2 antibody or its antigen-binding fragment) may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate the active ingredient.

[0323] The present application further provides a pharmaceutical composition or formulation that improves the stability of an anti-BDCA2 antibody or its antigen-binding fragment, enabling long-term storage. In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) an anti-BDCA2 antibody or its antigen-binding fragment disclosed herein; (b) a buffer; (c) a stabilizer; (d) salts; (e) a leavening agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years or longer. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.

[0324] A buffer useful in a pharmaceutical composition or formulation disclosed herein may be a weak acid or weak base used to maintain the acidity (pH) of the solution near a predetermined value after the addition of other acids or bases. A suitable buffer can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also be based on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) and an isotonic agent, which is potentially pH-adjusted with acids or bases well known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0325] Stabilizers are added to pharmaceuticals to stabilize the product. These reagents can stabilize proteins in various ways. Common stabilizers include (but are not limited to) amino acids such as glycine, alanine, lysine, arginine, or threonine; carbohydrates such as glucose, sucrose, trehalose, raffinose, or maltose; polyhydric alcohols such as glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight; or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the FIX polypeptide in the lyophilized formulation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0326] Leavening agents are added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating accurate weighing and handling. Common leavening agents include (but are not limited to) lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0327] A surfactant is an amphiphilic substance having hydrophilic and hydrophobic groups. Surfactants may be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include (but are not limited to) alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, higher alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbate, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0328] The pharmaceutical compositions disclosed herein may further include one or more buffer systems, preservatives, tonicity agents, chelating agents, stabilizers and / or surfactants, and various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0329] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

[0330] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized and a physician or patient adds a solvent and / or diluent before use.

[0331] The pharmaceutical compositions disclosed herein may further contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium pyrosulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0332] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, polyhydric alcohols such as ethanol, glycerol, propylene glycol, and polyethylene glycol and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, the use of a coating material such as lecithin can maintain the desired particle size in the dispersion system, and the use of a surfactant can maintain appropriate fluidity.

[0333] These compositions may further contain additives such as preservatives, humectants, emulsifiers, and dispersants. To prevent the presence of microorganisms, this can be ensured by incorporating both antimicrobial and antifungal agents, such as parahydroxybenzoic acid, chlorobutanol, phenol, and sorbic acid, along with the aforementioned sterilization treatment. It is also desirable to include isotonic agents such as sugars and sodium chloride in the compositions. Furthermore, the inclusion of absorption retarders such as aluminum monostearate and gelatin can ensure sustained absorption of the injectable drug form.

[0334] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for preparing sterile injectable solutions or dispersions. The use of such media and reagents with pharmacologically active substances is well known in the art. Unless conventional media or reagents are incompatible with the active compound, their use in the pharmaceutical compositions described herein should be considered. The pharmaceutical compositions or formulations may or may not contain preservatives. Auxiliary active compounds can be introduced into the compositions.

[0335] Pharmaceutical compositions or formulations are typically sterile and stable under manufacturing and storage conditions. The compositions can be prepared as solutions, microemulsions, liposomes, or other ordered structures suitable for high concentrations of the drug. The carrier may be a solvent or dispersion medium containing water, ethanol, glycerol, polyhydric alcohols such as propylene glycol and liquid polyethylene glycol, and suitable mixtures thereof. For example, coatings such as lecithin can be used to maintain a desired particle size in the dispersion system, and surfactants can be used to maintain appropriate fluidity. Often, the compositions may contain isotonic agents such as sugars, mannitol, sorbitol, or polyalcohols such as sodium chloride. The inclusion of absorption retarders such as monostearate or gelatin in the composition can enable sustained absorption of the injectable composition.

[0336] If necessary, a sterile injection solution can be prepared by mixing the components or combinations of components listed above with a predetermined amount of the active compound in a suitable solvent, and then sterilizing by filtration. Generally, the dispersion system is prepared by mixing the active compound with a sterile medium containing the necessary components from the basic dispersion medium and the components listed in this application. In the case of sterile powders used in the preparation of sterile injection solutions, there are preparation methods such as vacuum drying and freeze-drying (lyophilization), and in these drying methods, a powder containing the active component and any desired component is obtained from a solution that has been previously sterile filtered.

[0337] In the pharmaceutical compositions or formulations disclosed herein, the amount of active ingredient combined with the carrier material may vary. In some embodiments, the amount of active ingredient combined with the carrier material is the amount that exerts a therapeutic effect. Generally, expressed as a percentage, this amount ranges from about 0.01% to about 99% of the active ingredient, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0338] The pharmaceutical compositions disclosed herein can be prepared with a carrier that prevents the rapid release of the active ingredient, and these include controlled-release formulations such as implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Various methods for preparing these formulations are protected by patents or are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems (J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

[0339] In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein can be prepared to ensure appropriate in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the active ingredient described herein can cross the BBB, it can be prepared, for example, in a liposome. For methods of producing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548, and 5,399,331. The liposome may contain one or more portions that enable selective transport to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Examples of targeted moieties include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 granted to Low et al.), mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PGBloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), and p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090). See also K. Keinanen; ML. Laukkanen (1994) FEBS Lett. 346:123 and J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

[0340] 1.7 Methods and Applications The antibodies or their antigen-binding fragments, compositions, and methods described herein have diverse in vitro and in vivo applications, including reducing type I IFN release from BDCA2-expressing cells (e.g., pDCs). In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment described herein is a humanized antibody or its antigen-binding fragment. For example, the anti-BDCA2 antibody or its antigen-binding fragment described herein can be applied in vitro or to cells in ex vivo culture, or to a human subject (e.g., administered in vivo) with the aim of selectively suppressing type I IFN release or suppressing pDC activity in various diseases. Accordingly, this application provides a method for reducing autoimmunity in a subject, comprising administering the anti-BDCA2 antibody or antigen-binding moiety described herein to the subject, thereby reducing type I IFN or pDC-related autoimmunity in the subject.

[0341] The present invention also provides methods for using anti-BDCA2 antibodies or their antigen-binding fragments disclosed herein, polynucleotides encoding these anti-BDCA2 antibodies or their antigen-binding fragments, vectors comprising these polynucleotides, or pharmaceutical compositions having these antibodies or their antigen-binding fragments for reducing cytokine (e.g., type I IFN) release from BDCA2-expressing cells (e.g., pDCs) or for treating diseases or conditions associated with type I IFN-related or pDC-related diseases (e.g., autoimmune diseases).

[0342] In some embodiments, the Application provides a method for reducing type I IFN (e.g., IFNα) in a subject who needs it, comprising administering a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein to the subject. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for reducing type I IFN (e.g., IFNα). In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment provided herein for the preparation of a pharmaceutical that reduces type I IFN (e.g., IFNα). In some embodiments, the Application provides a method for reducing type I IFN (e.g., IFNα) in a subject who needs it, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Application provides the use of the pharmaceutical composition disclosed herein for reducing type I IFN (e.g., IFNα). In some embodiments, the Application provides the use of the pharmaceutical composition provided herein in the manufacture of a pharmaceutical that reduces type I IFN (e.g., IFNα).

[0343] In some embodiments, the Application provides a method for suppressing or depleting pDCs in a subject in need, comprising administering a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein to the subject. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for suppressing or depleting pDCs. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment provided herein for the preparation of a pharmaceutical that suppresses or depletes pDCs. In some embodiments, the Application provides a method for suppressing or depleting pDCs in a subject in need, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Application provides the use of the pharmaceutical composition disclosed herein for suppressing or depleting pDCs. In some embodiments, the Application provides the use of the pharmaceutical composition provided herein for the preparation of a pharmaceutical that suppresses or depletes pDCs. In some embodiments, the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein depletes pDCs in a subject in need.

[0344] In some embodiments, the Application provides a method for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in a subject who needs it, comprising administering a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein to the subject. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment provided herein for the preparation of a pharmaceutical for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the Application provides a method for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in a subject who needs it, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Application provides the use of the pharmaceutical composition disclosed herein for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the present application provides the use of the pharmaceutical compositions provided herein in the manufacture of pharmaceuticals that reduce autoimmunity associated with type I IFN (e.g., IFNα) or pDC.

[0345] In some embodiments, the Application provides a method for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC in a subject who requires it, comprising administering a therapeutically effective amount of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein to the subject. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment disclosed herein for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the Application provides the use of the anti-BDCA2 antibody or its antigen-binding fragment provided herein in the manufacture of a pharmaceutical product for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the Application provides a method for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC in a subject who requires it, comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Application provides the use of the pharmaceutical composition disclosed herein for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the present application provides the use of pharmaceutical compositions in the manufacture of pharmaceuticals for treating autoimmune diseases associated with type I IFNs (e.g., IFNα) or pDCs.

[0346] As is known in the art, dysregulation or hyperactivation of pDC or type I IFN (e.g., IFNα) release leads to undesirable activation of the immune system and is involved in the pathogenesis of various diseases, including autoimmune diseases. In some embodiments, diseases or conditions related to pDC or IFNα that can be treated by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein include lupus; lupus erythematosus such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), and discoid lupus erythematosus (DLE); and lupus nephritis (LN). In some embodiments, the disease or condition that can be treated by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein is lupus. In some embodiments, the disease or condition that can be treated by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein is SLE. In some embodiments, the disease or condition that can be treated by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein is CLE. In some embodiments, the disease or condition treatable by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein is DLE. In some embodiments, the disease or condition treatable by the anti-BDCA2 antibody or its antigen-binding fragment or pharmaceutical composition provided herein is LN.

[0347] In some embodiments, the methods provided herein can promote beneficial therapeutic responses to autoimmune reactions. In some embodiments, the methods provided herein result in improvement of disease-related symptoms, such as a decrease in IFNα levels, a decrease in pDC number or activity, or a reduction in one or more disease-related symptoms. Thus, improvement of the disease can be identified as a complete response, for example. In some embodiments, the clinical response can be evaluated using screening techniques such as magnetic resonance imaging (MRI) scans, radiography, computed tomography (CT) scans, flow cytometry or fluorescence-activated cell sorting (FACS) analysis, histology, macroscopic pathology, and blood chemistry analysis (including, but not limited to, detectable changes by ELISA, RIA, chromatography, etc.).

[0348] The actual dose level of the active ingredient (i.e., anti-BDCA2 antibody or its antigen-binding fragment) in the pharmaceutical composition described herein can be modified to obtain an effective amount of the active ingredient that achieves the desired therapeutic response and does not cause toxicity to the patient for a particular patient, composition, and dosage form. The selected dose level depends on various pharmacokinetic factors, including the activity of the particular composition described herein, the route of administratio...

Claims

1. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (1) Defined by Kabat, (a) Light chain variable regions (VLs) comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) Heavy chain variable regions (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 15, 17, and 18, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in VH CDR; or (2) Defined by Chothia, (a) VLs comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in VL CDR; and / or (b) an antibody or antigen-binding fragment thereof comprising VH, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs: 14, 16, and 18, respectively; or a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in VH CDR.

2. The antibody or antigen-binding fragment according to claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences shown in Sequence IDs 8, 9, 10, 15, 17, and 18, as defined by Kabat.

3. The antibody or antigen-binding fragment according to claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences shown in SEQ ID NOs. 8, 9, 10, 14, 16, and 18, as defined by Chothia.

4. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (a) A VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26; and / or (b) An antibody or an antigen-binding fragment thereof comprising a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

27.

5. The antibody or its antigen-binding fragment according to claim 4, comprising VL and VH having the amino acid sequences shown in SEQ ID NOs. 26 and 27, respectively.

6. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (a) VL comprising VL CDR1, VL CDR2, and VL CDR3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 26; and / or (b) An antibody or antigen-binding fragment thereof comprising VH having the amino acid sequence shown in Sequence ID No. 27, comprising VH CDR1, VH CDR2, and VH CDR3 derived from VH;

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, which is a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment.

8. The antibody or antigen-binding fragment according to claim 7, wherein the antibody or antigen-binding fragment is a humanized antibody or an antigen-binding fragment thereof.

9. (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 28 to 31; and / or (b) The antibody or antigen-binding fragment according to claim 8, comprising: (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 37;

10. The antibody or its antigen-binding fragment according to claim 9, comprising VL and VH, each having the following amino acid sequences. (1) SEQ ID NOs. 28 and 34; (2) SEQ ID NOs. 28 and 35; (3) SEQ ID NOs. 28 and 36; (4) SEQ ID NOs. 28 and 37; (5) SEQ ID NOs. 29 and 34; (6) SEQ ID NOs. 29 and 35; (7) SEQ ID NOs. 29 and 36; (8) SEQ ID NOs. 29 and 37; (9) SEQ ID NOs. 30 and 34; (10) SEQ ID NOs. 30 and 35; (11) SEQ ID NOs. 30 and 36; (12) SEQ ID NOs. 30 and 37; (13) SEQ ID NOs. 31 and 34; (14) SEQ ID NOs. 31 and 35; (15) SEQ ID NOs. 31 and 36; or (16) SEQ ID NOs. 31 and 37.

11. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (1) Defined by Kabat, (a) Light chain variable regions (VLs) comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs: 11, 12, and 13, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) Heavy chain variable regions (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs. 20, 22, and 23, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in VH CDR; or (2) Defined by Chothia, (a) VLs comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences shown in SEQ ID NOs: 11, 12, and 13, respectively; or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) an antibody or antigen-binding fragment thereof comprising VH, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences shown in SEQ ID NOs: 19, 21, and 23, respectively; or a variant thereof having up to approximately five amino acid substitutions, additions, and / or deletions in VH CDR.

12. The antibody or antigen-binding fragment according to claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences shown in Sequence IDs 11, 12, 13, 20, 22, and 23, as defined by Kabat.

13. The antibody or antigen-binding fragment according to claim 1, comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, each having the amino acid sequences shown in SEQ ID NOs. 11, 12, 13, 19, 21, and 23, as defined by Chothia.

14. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (a) A VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 40; and / or (b) An antibody or an antigen-binding fragment thereof comprising a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

41.

15. The antibody or its antigen-binding fragment according to claim 14, comprising VL and VH having the amino acid sequences shown in SEQ ID NOs. 40 and 41.

16. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, (a) VL comprising VL CDR1, VL CDR2, and VL CDR3 derived from a VL having the amino acid sequence shown in SEQ ID NO: 40; and / or (b) An antibody or antigen-binding fragment thereof comprising VH having the amino acid sequence shown in SEQ ID NO: 41, including VH CDR1, VH CDR2, and VH CDR3 derived from VH;

17. The antibody or antigen-binding fragment according to any one of claims 11 to 16, which is a chimeric antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, or a human antibody or its antigen-binding fragment.

18. The antibody or antigen-binding fragment according to claim 17, which is a humanized antibody or an antigen-binding fragment thereof.

19. The antibody or antigen-binding fragment thereof according to claim 18, (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-46; and / or (b) The antibody or antigen-binding fragment according to claim 18, comprising: (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 47 to 50;

20. The antibody or antigen-binding fragment thereof according to claim 19, comprising VL and VH having the following amino acid sequences, respectively. (1) Sequence IDs 42 and 47; (2) Sequence IDs 42 and 48; (3) Sequence IDs 42 and 49; (4) Sequence IDs 42 and 50; (5) Sequence IDs 43 and 47; (6) Sequence IDs 43 and 48; (7) Sequence IDs 43 and 49; (8) Sequence IDs 43 and 50; (9) Sequence IDs 44 and 47; (10) Sequence IDs 44 and 48; (11) Sequence IDs 44 and 49; (12) Sequence IDs 44 and 50; (13) Sequence IDs 45 and 47; (14) Sequence IDs 45 and 48; (15) Sequence IDs 45 and 49; (16) Sequence IDs 45 and 50; (17) Sequence IDs 46 and 47; (18) Sequence IDs 46 and 48; (19) Sequence IDs 46 and 49; or (20) Sequence IDs 46 and 50.

21. Fab, Fab', F(ab') ２ , Fv, scFv, (scFv) ２ An antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, selected from the group consisting of a single-domain antibody (sdAb) and a heavy-chain antibody (HCAb).

22. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, which is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

23. The antibody according to claim 22, which is an IgG1 antibody.

24. The antibody according to claim 23, comprising a light chain constant region (CL) having at least 85% sequence identity with κCL (Cκ; SEQ ID NO: 51).

25. The antibody according to claim 23, comprising a light chain constant region (CL) having at least 85% sequence identity with λCL (Cλ; SEQ ID NO: 52).

26. The antibody according to claim 23, comprising a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 56.

27. The antibody according to any one of claims 23 to 26, wherein the heavy chain constant region (CH) of the IgG1 antibody contains a wild-type IgG1 CH or contains at least one amino acid mutation that enhances the antibody's ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis).

28. The antibody according to claim 27, wherein the CH region of the IgG1 antibody has an amino acid substitution in L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334, P396, or any combination thereof, based on the EU index number.

29. The antibody according to claim 27, wherein the CH region of the IgG1 antibody has an amino acid substitution in L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, or P396L, or any combination thereof, based on the EU index number.

30. The CH region of the IgG1 antibody is, based on the EU index number, (i) S298A, E333A, and K334A; (ii) S239D and I332E; (iii) S239D, A330L, and I332E; (iv) G236A; (v) G236A, S239D, and I332E; (vi) G236A, A330L, and I332E; (vii) G236A, S239D, A330L, and I332E; (viiii The antibody according to claim 27, modified by amino acid substitutions selected from the group consisting of (i) F243L, R292P, Y300L, V305I, and P396L; (ix) L235V, F243L, R292P, Y300L, and P396L; (x) L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A; and (xi) D270E, K326D, A330M, and K334E.

31. The antibody according to claim 30, wherein the CH region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 86.

32. The antibody according to any one of claims 23 to 31, wherein the Fc region of the IgG1 antibody is not fucosylated.

33. An antibody or antigen-binding fragment according to any one of claims 1 to 32 that competes for binding to human BDCA2 with an antibody or antigen-binding fragment thereof.

34. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, which is a bispecific antibody or a multispecific antibody.

35. The antibody or antigen-binding fragment according to any one of claims 1 to 34, which is a monoclonal antibody or an antigen-binding fragment thereof.

36. The antibody or its antigen-binding fragment is (1) K below 50 nM, as measured by SPR measurement. Ｄ It binds to human BDCA2; (2) Inhibit the release of interferon-alpha (IFNα) in peripheral blood mononuclear cells (PBMCs); (3) Selectively binds to plasmacytoid dendritic cells (pDCs) in human PBMCs; (4) It exhibits natural killer cell (NK)-dependent ADCC activity against BDCA2-expressing cells; (5) Exhibits neutrophil-dependent ADCC activity against BDCA2-expressing cells; or (6) An antibody or antigen-binding fragment according to any one of claims 1 to 35, which exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

37. An antibody or its antigen-binding fragment that specifically binds to human BDCA2, The antibody or its antigen-binding fragment (1) K below 50 nM, as measured by SPR measurement. Ｄ It binds to human BDCA2; (2) Inhibit IFNα release in PBMCs; (3) Selectively binds to pDCs in human PBMCs; (4) Exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (5) Exhibits neutrophil-dependent ADCC activity against BDCA2-expressing cells; or (6) An antibody or antigen-binding fragment that exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

38. The antibody or its antigen-binding fragment is (1) ICs with a particle size of 0.008 nM or less ５０ Therefore, inhibit IFNα release in CpG-stimulated PBMCs; (2) ICs with a mass of 0.0006 nM or less ５０ Therefore, inhibit IFNα release in IC-stimulated PBMCs; (3) EC of 0.01 nM or less, as measured by a reporter assay. ５０ Furthermore, it exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (4) The EC is less than 0.001 nM, as can be measured by a cytotoxicity assay. ５０ Furthermore, it exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (5)As measured by a reporter assay, an EC of 0.2 nM or less ５０ shows macrophage-dependent ADCP activity against BDCA2-expressing cells; or (6) EC of 2 nM or less, as measured by a phagocytic assay. ５０ The antibody or antigen-binding fragment according to claim 36 or 37, which exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

39. The antibody or its antigen-binding fragment is (1) ICs in the range of 0.0005 nM to 0.008 nM ５０ Therefore, inhibit IFNα release in CpG-stimulated PBMCs; (2) ICs in the range of 0.0001 nM to 0.0006 nM ５０ Therefore, inhibit IFNα release in IC-stimulated PBMCs; (3) EC in the range of 0.001 nM to 0.01 nM as measured by reporter assay ５０ Furthermore, it exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (4) EC in the range of 0.0001 nM to 0.001 nM, as measured by a cytotoxicity assay. ５０ Furthermore, it exhibits NK-dependent ADCC activity against BDCA2-expressing cells; (5) EC in the range of 0.01 nM to 0.2 nM as measured by reporter assay ５０ And, it exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or (6) EC in the range of 0.1 nM to 2 nM as measured by a phagocytic assay. ５０ The antibody or antigen-binding fragment according to claim 38, which exhibits macrophage-dependent ADCP activity against BDCA2-expressing cells; or any combination of (1) to (6).

40. An antibody or its antigen-binding fragment according to any one of claims 1 to 39, exhibiting neutrophil-dependent ADCC activity.

41. A polynucleotide encoding a polypeptide of an antibody or its antigen-binding fragment according to any one of claims 1 to 40.

42. A vector comprising a polynucleotide as described in claim 41.

43. A host cell comprising the polynucleotide described in claim 41, or the vector described in claim 42.

44. The host cell according to claim 43, wherein the host cell (1) overexpresses N-acetylglucosaminyltransferase III (GnTIII), (2) lacks α-1,6-fucosyltransferase (FUT8), or (3) has a low fucose content, or any combination of (1) to (3).

45. A method for producing an antibody or an antigen-binding fragment that specifically binds to human BDCA2, comprising culturing the cells described in claim 43 or 44 under conditions that enable the expression of the antibody or antibody fragment.

46. The method according to claim 45, comprising isolating the antibody from the culture.

47. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 40, and a pharmaceutically acceptable carrier.

48. A method for reducing type I interferon (IFN) in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 40.

49. The method according to claim 48, wherein the type I interferon is IFNα.

50. A method for suppressing or depleting pDCs in a subject requiring such suppression or depletion, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 40.

51. A method for reducing autoimmunity in a subject in need, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 40.

52. The method according to any one of claims 48 to 51, wherein the subject has an autoimmune disease.

53. A method for treating an autoimmune disease associated with type I IFN or pDC in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 40.

54. The method according to claim 52 or 53, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

55. The method according to any one of claims 48 to 54, further comprising administering other therapies to the subject.

56. The method according to any one of claims 48 to 55, wherein the subject is a human being.

57. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 40 for reducing type I IFN.

58. Use of an antibody or its antigen-binding fragment according to any one of claims 1 to 40 in the manufacture of a pharmaceutical product for reducing type I IFN.

59. The use according to claim 57 or 58, wherein the type I IFN is IFNα.

60. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 40 for suppressing or depleting pDCs.

61. Use of an antibody or its antigen-binding fragment according to any one of claims 1 to 40 in the manufacture of a pharmaceutical product for inhibiting or depleting pDCs.

62. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 40 to reduce autoimmunity.

63. Use of an antibody or its antigen-binding fragment according to any one of claims 1 to 40 in the manufacture of a pharmaceutical product for reducing autoimmunity.

64. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 40 for treating an autoimmune disease associated with type I IFN or pDC.

65. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 40 in the manufacture of a pharmaceutical product for treating an autoimmune disease associated with type I IFN or pDC.

66. The use according to claim 64 or 65, wherein the autoimmune disease is SLE.

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