Antibody specifically binding to BDCA2 and BDCA2-TACI antibody fusion protein

Abstract

An antibody specifically binding to BDCA2 and a BDCA2-TACI antibody fusion protein. The antibody fusion protein contains the following elements which are fused together: (a) an antibody or antigen-binding fragment thereof that specifically binds to BDCA2; and (b) TACI or an active fragment thereof. The antibody and antibody fusion protein can inhibit pDC activity, reduce IFNα secretion, inhibit cell inflammatory responses, neutralize factors such as BAFF and APRIL released by monocytes, etc., and reduce B cell activation.

Description

An antibody specifically binding to bdca2 and bdca2-taci antibody fusion protein TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology. Specifically, the present application relates to an antibody specifically binding to BDCA2 and BDCA2-TACI antibody fusion protein. BACKGROUND

[0002] [According to Rule 26 Correction 12.11.2025] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, which is characterized by multiple system and multiple organ involvement, repeated relapse and remission, and the presence of a large number of autoantibodies in the body. If not treated in time, it will cause irreversible damage to the involved organs and eventually lead to the death of the patient. The main treatment for systemic lupus erythematosus in clinical treatment is glucocorticoid therapy to reduce disease damage. However, for patients with refractory or recurrent SLE, the use of biological agents can significantly increase the complete and partial remission rate of patients, reduce disease activity, disease recurrence rate and reduce hormone dosage. Because of the significant effect of biological agents, their treatment status is gradually improving, and a variety of biological agents have been tried for the treatment of SLE and have achieved certain clinical efficacy, but only three biological agents have been approved by the US Food and Drug Administration (FDA) and the National Medical Products Administration (NMPA) for the treatment of SLE: one is belimumab, which mainly neutralizes B cell activating factor BAFF and inhibits B cell activity, and has been approved for the treatment of non-renal SLE and renal SLE; another is itacil, which is a fusion protein of TACI extracellular domain Fc, mainly neutralizing B cell activating factor BAFF and APRIL, inhibiting B cell activity, and has been approved for the treatment of non-renal SLE; the third is anirulimab, which is a type I interferon (IFN) receptor antagonist, and has been approved for the treatment of moderate to severe systemic lupus erythematosus (SLE).

[0003] Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells that are mainly responsible for the secretion of type I interferons (IFNα), which play a crucial role in antiviral immunity. Although pDCs only account for 0.2%-0.8% of the peripheral blood, they are responsible for 95% of the IFNα in the blood. Studies have reported that pDCs are activated in SLE patients, migrate to peripheral tissues, and release a large amount of IFNα, which aggravates the damage to the target organs of SLE patients. At the same time, the abnormal expression of molecules on the surface of pDCs and the release of a large amount of serum inflammatory cytokine IFNα will further activate various immune cells and exacerbate the condition of SLE.

[0004] The combination of autoantibodies and autoantigens in SLE patients to form immune complexes can bind to the surface of pDCs FcγR (IgG), thereby activating the LTR7 / 9 signaling pathway of the cells to produce a large amount of IFNα. BDCA2 and ILT7 expressed on the surface of pDCs are negative feedback regulatory proteins of IFNα. BDCA2 / ILT7 expressed on the surface of pDCs cells binds to FcεRIγ (IgE) expressed on the surface of pDCs cells to form a complex, activates multiple protein kinases, and thereby inhibits the LTR7 / 9 signaling pathway and inhibits the production of IFNα and cytokines. Therefore, BDCA2 / ILT7 is a feedback regulatory protein of cytokines and IFNα, and BDCA2 plays a more important role and has been confirmed by 2-phase clinical trial data.

[0005] TACI is a membrane-bound receptor with an extracellular region containing two cysteine-rich pseudo-repeats, a transmembrane region and a cytoplasmic region that interacts with CAML (calcium modulator and cyclophilin ligand). TACI is associated with a subset of B cells and T cells. The TACI receptor binds to BAFF of the tumor necrosis factor ligand family. BAFF is a B cell-activating factor belonging to the TNF family. BAFF is mainly expressed on the surface of bone marrow cell membranes in the form of a trimer. BAFF on the surface of the cell membrane is hydrolyzed by a protease to form soluble BAFF into the blood circulation system. Soluble BAFF has the characteristics of polymerization and can form a 60-mer at most. In addition, BAFF can also interact with another protein APRIL of the same family to form a heterotrimer. At present, it is known that there are three BAFF receptors on the surface of B cells, which are BAFF-R, BCMA and TACI. BAFF acts on these three receptors to participate in the differentiation, maturation, survival and regulation of B cells. APRIL has two common receptors with BAFF, which are BCMA and TACI, and APRIL acts on these two receptors to participate in the survival and regulation of B cells. BAFF is very important for maintaining the balance of B cells in vivo, and overactivation of the BAFF signaling pathway can lead to the survival of self-reactive B cells and the production of autoantibodies to promote autoimmune reactions.

[0006] Based on this, we developed a monoclonal antibody targeting BDCA2 to further activate the immunoregulatory negative feedback of BDCA2 in SLE patients, inhibit pDC cell activation, and effectively reduce the concentration of IFNα. Further, in order to increase the effectiveness of this target and reduce the production of autoantibodies from the source, we developed a monoclonal antibody targeting BDCA2 and an antibody fusion protein of a B cell activity blocker TACI or an active fragment thereof, to further reduce the production of autoantibodies and INFα.

[0007] Therefore, the present application provides an antibody and an antibody fusion protein for treating immune diseases and / or B cell proliferation-related diseases, which have the advantages of specific recognition, synergistic effect, controllable toxicity, etc. SUMMARY

[0008] One object of the present application is to provide an antibody for treating immune diseases and / or B cell proliferation-related diseases.

[0009] Another object of the present application is to provide an antibody fusion protein for treating immune diseases and / or B cell proliferation-related diseases.

[0010] In a first aspect of the application, there is provided an antibody or antigen-binding fragment thereof that specifically binds to BDCA2, comprising a HCDR1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2, a HCDR3 as set forth in SEQ ID NO: 3; and, a LCDR1 as set forth in SEQ ID NO: 4, a LCDR2 as set forth in SEQ ID NO: 5, a LCDR3 as set forth in SEQ ID NO: 6.

[0011] In an alternative embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH as set forth in SEQ ID NO: 7, and a light chain variable region VL as set forth in SEQ ID NO: 8.

[0012] In a second aspect of the application, there is provided an antibody fusion protein comprising the following elements fused together:

[0013] (a) an antibody or antigen-binding fragment thereof that specifically binds to BDCA2; and

[0014] (b) a TACI or an active fragment thereof;

[0015] wherein the TACI or the active fragment thereof is linked to the C-terminus or N- terminus of the heavy chain of the antibody or antigen-binding fragment thereof that specifically binds to BDCA2, preferably the C-terminus.

[0016] In an alternative embodiment, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a heavy chain constant region CH, and the light chain comprises a light chain variable region VL and a light chain constant region CL.

[0017] In an alternative embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody.

[0018] In an alternative embodiment, the antibody or antigen-binding fragment thereof is a murine, chimeric, humanized or fully human antibody, or antigen-binding fragment thereof.

[0019] In an alternative embodiment, the antibody or antigen-binding fragment thereof has an Fc region, and the Fc region is derived from a human or a non-human mammal, preferably derived from a rodent (e.g. mouse, rat), a primate or a human.

[0020] In an alternative embodiment, the Fc region is selected from the group consisting of IgG, IgA, IgD, IgE and / or IgM.

[0021] In an alternative embodiment, the Fc region is selected from the group consisting of IgG1, IgG2, IgG3 and / or IgG4, preferably the Fc region is selected from IgG1.

[0022] In alternative embodiments, the Fc region comprises a native Fc fragment and an Fc mutant.

[0023] In alternative embodiments, the Fc mutant comprises substitution and / or truncation of amino acid residues based on the native Fc fragment.

[0024] In alternative embodiments, the Fc mutant comprises substitution of methionine (M) with leucine (L) at position 428 resulting in (M428L), and / or substitution of asparagine (N) with serine (S) at position 434 resulting in (N434S) based on the native Fc fragment, and wherein numbering is performed according to the EU index of Kabat et al., see WO2006053301A2.

[0025] In alternative embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3; and LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, LCDR3 as set forth in SEQ ID NO: 6.

[0026] In alternative embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH as set forth in SEQ ID NO: 7, and a light chain variable region VL as set forth in SEQ ID NO: 8.

[0027] In alternative embodiments, the TACI or active fragment thereof comprises the amino acid sequence of positions 1 to 165 (SEQ ID NO: 9), positions 1 to 109 (SEQ ID NO: 10), or positions 33 to 109 (SEQ ID NO: 11), or positions 68 to 109 (SEQ ID NO: 12) of TACI, or a truncation or variant thereof.

[0028] In alternative embodiments, the TACI or active fragment thereof is the amino acid sequence of positions 68 to 109 of TACI, or a truncation or variant thereof.

[0029] In alternative embodiments, the peptide linker is a peptide linker of 1 to 20 amino acids, preferably a peptide linker of 1 to 15 amino acids, more preferably a peptide linker of 4 to 10 amino acids, most preferably a peptide linker of 4 amino acids.

[0030] In alternative embodiments, the peptide linker has the sequence (GGGS) x or (GGGGS) xThe amino acid sequences shown are provided, where x is selected from 1, 2, or 3. GGGS is shown as SEQ ID NO: 15, and GGGGS is shown as SEQ ID NO: 16.

[0031] In an optional embodiment, the peptide linker is GGGS or GGGSGGGS.

[0032] In an optional implementation, the antibody fusion protein is a homodimer.

[0033] In an optional embodiment, the heavy chain of the antibody fusion protein is shown in SEQ ID NO: 13.

[0034] In an optional embodiment, the light chain of the antibody fusion protein is shown in SEQ ID NO: 14.

[0035] In a third aspect of the invention, a nucleic acid molecule is provided, said nucleic acid molecule encoding the antibody described in the first aspect of the invention or the antibody fusion protein described in the second aspect of the invention.

[0036] In a fourth aspect of the invention, a recombinant vector is provided, the recombinant vector comprising the nucleic acid molecule described in the third aspect of the invention.

[0037] In a fifth aspect of the invention, a recombinant cell is provided, the recombinant cell comprising the nucleic acid molecule described in the third aspect of the invention and / or the recombinant vector described in the fourth aspect of the invention.

[0038] In a sixth aspect of the present invention, a method for generating the antibody described in the first aspect of the present invention or the antibody fusion protein described in the second aspect of the present invention is provided, the method comprising the following steps:

[0039] The recombinant cells described in the fifth aspect of the invention are cultured under conditions suitable for expressing the antibody or antibody fusion protein, thereby expressing the antibody or antibody fusion protein; and the antibody or antibody fusion protein is isolated or purified.

[0040] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising an antibody as described in the first aspect of the invention or an antibody fusion protein as described in the second aspect of the invention and a pharmaceutically acceptable carrier.

[0041] In an eighth aspect of the invention, there is provided the use of the antibody described in the first aspect of the invention, the antibody fusion protein described in the second aspect of the invention, the nucleic acid molecule described in the third aspect of the invention, the recombinant vector described in the fourth aspect of the invention, or the recombinant cell described in the fifth aspect of the invention in the preparation of a pharmaceutical composition for treating immune diseases and / or B-cell hyperplasia-related diseases. Attached Figure Description

[0042] Figure 1 is the binding activity of mouse antibody Ms-113 to human BDCA2;

[0043] Figure 2 is the endocytosis activity of anti-BDCA2 mouse antibody;

[0044] Figure 3 is the inhibition activity of anti-BDCA2 mouse antibody;

[0045] Figure 4 is the binding activity determination of humanized antibody H-113-Z14;

[0046] Figure 5 is the inhibition activity determination of humanized antibody H-113-Z14;

[0047] Figure 6a is a structural schematic diagram of an antibody fusion protein fused at the C-terminus;

[0048] Figure 6b is a structural schematic diagram of an antibody fusion protein fused at the N-terminus;

[0049] Figure 7 is the detection result of different antibody fusion proteins blocking APRIL to activate HEK 293TACI luc reporter gene cell activity;

[0050] Figure 8 is the BDCA2 binding activity of different antibody fusion proteins;

[0051] Figure 9 is the endocytosis activity of different antibody fusion proteins;

[0052] Figure 10 is the BAFF binding activity of different antibody fusion proteins with different mutation sites;

[0053] Figure 11 is the BDCA2 binding activity of different antibody fusion proteins with different mutation sites;

[0054] Figure 12 is the binding ability test result of different antibody fusion proteins with human BDCA2 and BAFF double targets;

[0055] Figure 13 is the inhibition activity of antibody fusion protein H-113Z14-D2-M428L-N434S;

[0056] Figure 14 is the endocytosis activity of antibody fusion protein H-113Z14-D2-M428L-N434S;

[0057] Figure 15 is the in vivo efficacy-body weight change of antibody fusion protein H-113Z14-D2-M428L-N434S;

[0058] Figure 16 is the in vivo efficacy-urine protein change of antibody fusion protein H-113Z14-D2-M428L-N434S;

[0059] Figure 17 is the in vivo efficacy-lymphadenopathy score of antibody fusion protein H-113Z14-D2-M428L-N434S;

[0060] Figure 18 is the result of the antibody fusion protein blocking BAFF activation of HEK 293TACI luc reporter cell activity detection;

[0061] Figure 19 is the result of the antibody fusion protein binding to human BDCA2, BAFF double target site;

[0062] Figure 20 is the binding activity of the antibody fusion protein to human BDCA2;

[0063] Figure 21 is the binding activity of the antibody fusion protein to human BAFF;

[0064] Figure 22 is the binding activity of the antibody fusion protein to human APRIL;

[0065] Figure 23 is the result of the antibody fusion protein complement-dependent cytotoxicity test;

[0066] Figure 24 is the result of the antibody fusion protein inhibiting BAFF-induced B cell proliferation test;

[0067] Figure 25 is the result of the antibody fusion protein binding to hBDCA2 humanized mouse pDCs surface BDCA2 test;

[0068] Figure 26 is the result of the antibody fusion protein mediating hBDCA2 humanized mouse pDCs surface BDCA2 endocytosis test;

[0069] Figure 27 is the result of mouse arthritis clinical score;

[0070] Figure 28 is the result of the in vivo efficacy experiment positive rate of the antibody fusion protein (immature B cells);

[0071] Figure 29 is the result of the in vivo efficacy experiment positive rate of the antibody fusion protein (mature B cells);

[0072] Figure 30 is the change in mouse body weight;

[0073] Figure 31 is the result of mouse arthritis clinical score;

[0074] Figure 32 is the result of B cell subpopulation detection in collagen-induced DBA / 1 mouse arthritis model. DETAILED DESCRIPTION

[0075] The antibody and antibody fusion protein of the present application can inhibit pDCs activity, reduce IFNα secretion, inhibit cell inflammatory response, and neutralize BAFF and APRIL factors released by monocytes and the like, thereby reducing B cell activation.

[0076] In addition, the inventors have made an in-depth study and for the first time unexpectedly found that the antibody fusion protein obtained by fusing (a) an antibody or antigen binding fragment thereof specifically binding to BDCA2 and (b) TACI or an active fragment thereof has excellent biological activity, can effectively inhibit B cell activity, and increase TACI on the basis of BDCA2, can effectively block upstream signals, and reduce antibody production. Through the verification of the inventors, the antibody fusion protein of the present application has the advantages of specific recognition, synergistic effect, controllable toxicity, etc. In particular, the TACI target has already been marketed, and the combination of the above two targets has significant advantages in drug development.

[0077] The term "TACI" is a membrane-bound receptor, and wild-type human TACI comprises two extracellular cysteine-rich pseudo-repeat regions, a transmembrane region, and a cytoplasmic region interacting with CAML (calcium modulator and cyclophilin ligand). According to the NCBI protein database search results, the GenBank accession number corresponding to the full-length sequence of human TACI is BAE16555.1. The full-length amino acid sequence of TACI (i.e. 1-293) is MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMSCKTICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRRQRSGEVENNSDNSGRYQGLEHRGSEASPALPGLKLSADQVALVYSTLGLCLCAVLCCFLVAVACFLKKRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA.

[0078] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example, the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art. Unless otherwise specified, the variable region and CDR sequences herein are all applicable to the "Kabat" numbering rule.

[0079] The positive control BMK used in the present patent includes Ms-BMK and H-BMK. The variable region thereof is the same, and is derived from patent CN105452295B, the light chain variable region is the sequence SEQ ID NO: 23 in the patent, and the heavy chain variable region is the sequence SEQ ID NO: 24 in the patent. The complete Ms-BMK and H-BMK also need to increase the corresponding human or mouse constant region on the basis of the variable region. The heavy chain constant region of the Ms-BMK of the present patent is shown as SEQ ID NO: 17, and the light chain constant region is shown as SEQ ID NO: 18; the heavy chain constant region of the H-BMK is shown as SEQ ID NO: 19, and the light chain constant region is shown as SEQ ID NO: 20.

[0080] Example 1: Animal immunization

[0081] The immunogen hBDCA2-FcεRIγ-CHOK1 cells 5×10 6 The immunogen hBDCA2-FcεRIγ-CHOK1 cells 5×10 6 The immunogen hBDCA2-FcεRIγ-CHOK1 cells 5×106 hBDCA2-FcεRIγ-CHOK1 cells were immunized twice and on day 42, 5 x 106 6 hBDCA2-FcεRIγ-CHOK1 cells and 25 μg of hBDCA-HM (brand KACTUS, item BCA-HM402) were used for a boost. After the second immunization, the antiserum titer was evaluated by flow cytometry binding assay on hBDCA2-FcεRIγ-CHOK1 cells with sera collected by tail bleeds at different dilutions from 1 : 100 to 1 : 10000000. When the titer results were satisfactory, with detection of anti-human BDCA2 antibodies at a dilution > 1 : 100000, the mouse spleen and lymph nodes were harvested and cells were fused.

[0082] Example 2: Cell fusion

[0083] Myeloma cells SP2 / 0 (ATCC) were passaged one day before fusion to keep the cells in the logarithmic growth phase at the time of the experiment. The cells were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded and 10 mL of DMEM complete medium was added to mix the cells, ready for use. The spleen and lymph node cells used in the experiment were taken from Balb / c mice immunized four times with hBDCA2-FcεRIγ-CHOK1 cells and hBDCA-HM protein. The mice were sacrificed before the fusion and the spleen and lymph nodes were removed. The cells were ground through a mesh in 10 mL of DMEM complete medium and the cell suspension was centrifuged at 2000 rpm for 8 min, the supernatant was discarded and the cells were mixed in DMEM complete medium, ready for use. The feeder cells used in the selection culture of the fusions were taken from the macrophages of the peritoneal cavity of animals that had not been immunized. The macrophage suspension collected before the fusion was centrifuged at 100 rpm for 8 min, the supernatant was discarded and 25 mL of HAT selection medium was added to mix it, aliquoted in two 24-well culture plates to assist the growth of new hybrid B lymphocyte hybridomas.

[0084] The B lymphocytes and myeloma cells were mixed at a ratio of 1:2, and the suspension was centrifuged at 1000 rpm for 8 min, the precipitate was washed twice with the electrofusion solution. The precipitate was added to 1.2 mL of the electrofusion solution, and 0.4 mL of the cell suspension was injected into each of three multi-electrode small pools. The following electric field conditions were applied to each electrode small pool in sequence: the dielectric electrophoresis alternating current field sine signal had a frequency of 1 MHz and an amplitude of 250 V / cm, and was applied for 30 seconds. Then, a perforation RC electric pulse with an amplitude of 5 kV / cm, a pulse width of 20 μs, and a pulse number of 3 was immediately applied, with a time interval of 1 second. After standing at room temperature for 10 min, the fusion was washed out with a total amount of 5 mL of the PFM solution, and incubated at 37°C for 30 min. Then, the precipitate was obtained by centrifugation at 100 rpm for 8 min, 500 mL of HAT was added, and the mixture was uniformly mixed and injected into a 96-well culture plate, with a cell number of 1 x 10 4 cells / well (1.2 x 10 4 cells / well of feeder cells), and placed in a CO2 incubator at 37°C for culture.

[0085] Example 3: Primary screening of positive clones by FACS method

[0086] After 7 days of culture, the fusion supernatant was subjected to primary screening.

[0087] a. Cell treatment: The hBDCA2-FcεRIγ-CHOK1 cell suspension was centrifuged at 3000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended and counted in the diluent, and then the cells were evenly divided into the 96-well plate at a cell number of 3 x 10 5 cells / well. The diluent (1 x PBS) was washed once (3000 rpm, 3 min).

[0088] b. Incubation: 100 μL of the sample was mixed with the cells, and a blank control group (BLANK) and a negative control group (NC) were set, i.e., 100 μL of the diluent was mixed with the cells, and 100 μL of irrelevant antibody and 100 μL of positive control (Ms-BMK) were mixed with the cells, and incubated at 4°C for 1 h. After the incubation, the cells were washed once with 100 μL / well of the diluent (3000 rpm, 3 min).

[0089] c. Secondary antibody incubation: the secondary antibody was diluted according to the proportion, 100 μL of the secondary antibody was added to each of the sample, the irrelevant antibody group, and the positive control group, and 100 μL of the diluent was added to the blank control group, and incubated at 4°C for 30 min. After the incubation, the cells were washed twice with 100 μL / well of the diluent (3000 rpm, 3 min).

[0090] d. Flow cytometry detection: 50 μL / well of the diluent was used to resuspend the cells, and the fluorescence signal was detected by flow cytometry.

[0091] e. The cell wells with positive detection results were supplemented with liquid, and the next step was subjected to secondary screening.

[0092] Example 4: FACS method for re-screening positive clones

[0093] The fusion supernatant was re-screened after 24h of primary screening.

[0094] a. Cell treatment: hBDCA2-FcεRIγ-CHOK1 cells and CHOK1 cells were centrifuged at 3000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended and counted in diluent. The cells were evenly distributed in a 96-well plate at 3E5 cells / well, and the diluent (1x PBS) was washed once (3000 rpm, 3 min).

[0095] b. Incubation: 100 μL of sample was mixed with the cells, and a blank control group (BLANK) and a negative control group (NC) were set up, both of which were 100 μL of diluent mixed with the cells. An unrelated antibody and a positive control (Ms-BMK) control group were also set up, each of which was 100 μL mixed with the cells, and incubated at 4°C for 1 h. After incubation, the cells were washed once with 100 μL / well of diluent (3000 rpm, 3 min).

[0096] c. Secondary antibody incubation: The secondary antibody was diluted according to the proportion, 100 μL was added to each sample, unrelated antibody group, and positive control group, and 100 μL of diluent was added to the blank control group, and incubated at 4°C for 30 min. After incubation, the cells were washed twice with 100 μL / well of diluent (3000 rpm, 3 min).

[0097] d. Flow cytometry detection: 50 μL / well of diluent was resuspended, and the fluorescence signal was detected by flow cytometry.

[0098] g. The wells that only bound to hBDCA2-FcεRIγ-CHOK1 cells were defined as positive wells, and two rounds of limited dilution were used to monoclonalize the hybridoma cells.

[0099] Example 5: Preparation of antibodies

[0100] After obtaining a stable hybridoma cell line, the monoclonal antibody was mainly obtained by in vitro culture. The cell strain was expanded to a T75 culture bottle, and the cell coverage rate was 80-90%. The cell supernatant was discarded, and 30 mL of hybridoma production medium (hybridoma-SFM, Gibco) was added. The culture was incubated at 37°C and 5% CO2. After 2-3 days of culture, the hybridoma production medium (hybridoma-SFM, Gibco) was added, and the cell survival rate was less than 30%. Then, fresh viable cells were added. After 6-7 days of culture, the cell survival rate was less than 20%, and the culture supernatant was collected after low-speed centrifugation and stored at 4°C for standby.

[0101] Purification of antibodies with MabSelect SuRe-LX (GE) affinity chromatography column: ① Column loading, the purchased Protein A filler was appropriately loaded into the gravity chromatography column and rinsed with the balance buffer (PBS, pH 7.4) until equilibrium; ② Sample loading, the hybridoma cell culture supernatant filtered by 0.22 μm filter membrane was added to the loaded chromatography column, and the flow rate was controlled at 1 drop per second; ③ Equilibrium, after the sample was added, the equilibrium buffer was used for rinsing until equilibrium; ④ Elution, the elution buffer (50 mM acetic acid / sodium acetate, pH 3.5) was added to rinse the column and collect the eluate; ⑤ Regeneration, after the elution was completed, 2 column volumes of 0.1 M NaOH were added to rinse the column, and 2 column volumes of 20% ethanol were added for rinsing before the column was stored at 4°C. Finally, the antibody purity was identified by SDS-PAGE and SEC-HPLC methods, and the antibody concentration was determined by ultraviolet microspectrophotometry.

[0102] Example 6: Binding activity of anti-BDCA2 mouse antibody to human BDCA2 protein.

[0103] The binding activity of the antibody to human BDCA2 protein was determined by the ELISA detection method. The plate coated with hBDCA-HM (brand KACTUS, item number BCA-HM402) protein was incubated at 4°C overnight. 2% PBS-BSA was used for blocking, and PBS containing 0.05% Tween was used for washing 3 times. Gradient-diluted antibody to be detected was added as a primary antibody, and incubated at 37°C for 1 h. Anti-mouse-IgG-FC-HRP was used as a secondary antibody, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development. The binding of the antibody to human BDCA2 protein was detected, and the results are shown in Figure 1. The mouse antibody Ms-113 has a binding activity comparable to that of the positive control Ms-BMK.

[0104] Example 7: Endocytosis activity of anti-BDCA2 mouse antibody

[0105] PBMC was resuspended with diluent (PBS+2% FBS) and inoculated with 5x10 5 cells in a 96-well U-shaped plate. It was divided into a negative control group (NC), a BDCA2 fluorescent antibody direct labeling group (goat anti-human BDCA2, R&D Systems, AF1376), a positive control group (Ms-BMK), and an experimental group; the positive control group (Ms-BMK) and the experimental group were added with the corresponding antibodies (20 μg / mL), the negative control group was not added with the antibodies and was not incubated with the fluorescent secondary antibody, the BDCA2 fluorescent antibody direct labeling group was not added with the antibodies, and then incubated at 37°C for 1 h, and then incubated with goat anti-human BDCA2 (R&D Systems, AF1376) antibody on ice at 4°C for 30 min, fixed with 4% paraformaldehyde at room temperature for 15 min, and then incubated with anti-goat, anti-CD45, anti-CD123, and anti-BDCA4 antibodies at room temperature for 30 min, and the pDC cells were circled, which were CD45- CD123 + BDCA4 + pDC cells, and analyze the fluorescence intensity value of the binding signal of goat anti-human BDCA2 antibody to calculate the antibody-mediated endocytosis of pDC cell surface BDCA2. The results are shown in Figure 2, and the murine antibody Ms-113 has comparable endocytosis activity to the positive control Ms-BMK.

[0106] Example 8: Inhibition activity of anti-BDCA2 murine antibody

[0107] PBMC was resuspended with diluent (1640+3% FBS), 1x10 6 cells were inoculated in a 96-well flat-bottom plate, and the antibody (concentration gradient) was added. The cells were stimulated with 10 μg / mL of TLR9 ligand CpG ODN 2216. After incubation in a 37°C incubator for 18 h, the supernatant was collected by centrifugation, and 293ISRE reporter gene cells were used to detect the secretion of IFNα. 4x10 4 cells were inoculated in a 96-well flat-bottom plate, and the antibody (concentration gradient) was added. The cells were stimulated with 10 μg / mL of TLR9 ligand CpG ODN 2216. After incubation in a 37°C incubator for 18 h, the supernatant was collected by centrifugation, and 293ISRE reporter gene cells were used to detect the secretion of IFNα. 4x10

[0108] Example 9: Humanization of the antibody

[0109] By comparing the IMGT (https: / / www.imgt.org / ) human antibody heavy and light chain variable region gene database, the heavy and light chain variable region genes with high homology to the murine antibody were selected as templates, and the CDRs of the murine antibody MS-113 were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to the needs, the key amino acids in the framework sequence were back-mutated to the corresponding amino acids of the murine antibody to ensure the original affinity, i.e. to obtain a humanized monoclonal antibody. The CDR amino acid residues of the antibody are determined and annotated by the kabat numbering system. The humanized light chain template of the murine antibody is IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain template is IGHV4-38-2*01 and IGHJ1*01. The CDRs of the murine antibody are transplanted into the human templates, respectively, to obtain the corresponding humanized version. The modified humanized antibody is named H-113-Z14, and the specific sequence is as follows:

[0110] The amino acid sequence of VH is shown below:

[0111] The amino acid sequence of VL is shown below:

[0112] Example 10: Binding activity assay of humanized antibody

[0113] a. Cell treatment: hBDCA2-FcεRIγ-CHOK1 cell suspension was centrifuged at 3000 rpm for 3 min, and the supernatant was discarded. The cells were resuspended and counted, and 3E5 cells were evenly distributed in a 96-well plate. The cells were washed once (3000 rpm, 3 min) with diluent (1xPBS).

[0114] b. Incubation: The sample was initially diluted to 20 μg / mL, and 8 gradients were prepared by 4-fold dilution. 100 μL of the sample was mixed with the cells, and 100 μL of irrelevant antibody (H-IgG1) and positive control (H-BMK) were also mixed with the cells as controls. The mixture was incubated at 4°C for 1 h. After incubation, the cells were washed once with 100 μL of diluent (3000 rpm, 3 min).

[0115] c. Secondary antibody incubation: The secondary antibody was diluted according to the proportion, and 100 μL of each sample, irrelevant antibody, and positive control was added. 100 μL of diluent was added to the blank control group. The mixture was incubated at 4°C for 30 min. After incubation, the cells were washed twice with 100 μL of diluent (3000 rpm, 3 min).

[0116] d. Flow cytometry detection: 50 μL of diluent was added to each well, and the fluorescence signal was detected by flow cytometry.

[0117] The results are shown in Figure 4. The humanized antibody H-113-Z14 has stronger binding activity than the positive control H-BMK.

[0118] Example 11: Inhibition activity assay of humanized antibody

[0119] PBMC was resuspended with diluent (1640+3% FBS), and 1x10 6 cells were inoculated in a 96-well flat-bottom plate, and antibody (concentration gradient) was added. The cells were stimulated with 10 μg / mL of TLR9 ligand CpG ODN 2216. After incubation in a 37°C incubator for 18 h, the supernatant was collected by centrifugation, and 293ISRE reporter gene cells were used to detect the secretion of IFNα. 4x10 4The 96-well full white plate was incubated overnight, and the diluted supernatant was added, and the 96-well white plate was placed in a 37°C incubator for further incubation for 24 hours. The luciferase detection kit was used to detect the luminescence value, and the dose-effect curve of the known concentration of IFNα2b was drawn as a standard curve. Then, according to the standard curve and the luminescence value of the supernatant of the cells affected by the antibody, the amount of IFNα in the antibody supernatant was calculated. The results of the amount of IFNα and the inhibition rate of IFNα are shown in Figure 5 and Table 1, respectively. The humanized antibody H-113-Z14 has an inhibition activity comparable to the positive controls H-BMK and Ms-BMK, and has a more optimal inhibition rate of IFNα at a concentration of 0.1 μg / mL.

[0120] Table 1

[0121] Example 12: Preparation of antibody fusion protein

[0122] The constructed antibody fusion protein was finally purified through the conventional operations of gene synthesis, construction of expression vector, plasmid extraction, plasmid transfection, transient expression and fusion protein purification. The expression amount, purity and SDS-PAGE of the purified antibody fusion protein were detected, and the antibody fusion protein was confirmed and named as H-113Z14-D2-M428L-N434S. The full-length sequence of the heavy chain is as follows:

[0123] The full-length sequence of the light chain is as follows:

[0124] In the full-length sequence of the heavy chain, the first to 113th positions are the heavy chain variable region VH of the antibody specifically binding to BDCA2, the fine underlined part is HCDR1, HCDR2 and HCDR3, the thick underlined part is the heavy chain constant region CH1, the double underlined part is the IgG Fc fragment, wherein the mutations M428L and N434S are numbered according to the EU index of Kabat et al., and refer to WO2006053301A2, and the wavy line is the peptide linker. The bold mark is the TACI active fragment. In the full-length sequence of the light chain, the fine underlined part is LCDR1, LCDR2 and LCDR3, and the thick underlined part is the light chain constant region CL. The structure of the antibody fusion protein is shown in Figure 6a.

[0125] According to the same method, the antibody fusion protein in which TACI is connected to the N terminus of the antibody specifically binding to BDCA2 through a peptide linker was prepared and confirmed, and was named as antibody fusion protein-N, and the structure thereof is shown in Figure 6b.

[0126] The full-length sequence of the heavy chain is as follows:

[0127] The full-length sequence of the light chain is as follows:

[0128] Using the same method, an antibody fusion protein in which TACI is linked to the C-terminus of an antibody that specifically binds to BDCA2 via a peptide linker was prepared and confirmed, and named antibody fusion protein-YTE, the structure of which is shown in Figure 6a.

[0129] The full-length sequence of the heavy chain is as follows:

[0130] The full-length sequence of the light chain is as follows:

[0131] Using the same method, an antibody fusion protein in which TACI is linked to the C-terminus of an antibody that specifically binds to BDCA2 via a peptide linker was prepared and confirmed, and named antibody fusion protein-WT, the structure of which is shown in Figure 6a. The full-length heavy chain sequence is as follows:

[0132] The full-length sequence of the light chain is as follows:

[0133] Example 13: Detection of the activity of different antibody fusion proteins in blocking APRIL-activated HEK 293TACI luc reporter gene in cells.

[0134] HEK 293TACI luc reporter gene cells are stable TACI-expressing reporter gene cells obtained by transfecting HEK 293 cells with lentiviruses containing TACI and NF-κB-Luc luciferase reporter genes, followed by selection using puromycin and G418 pressure.

[0135] HEK 293TACI luc reporter cell 2×10 4 50 μL / well was seeded into each well of a 96-well blank plate and incubated at 37°C with 5% CO2 for 18 h. APRIL protein was diluted to 0.5 μg / mL (final concentration 0.25 μg / mL), and 55 μL / well was seeded into each well of a 96-well plate. H-113Z14-D2-M428L-N434S, antibody fusion protein-N, and irrelevant antibody were added to an initial concentration of 1800 nM (final concentration 900 nM). After one 3-fold dilution, all subsequent wells were 4-fold diluted, for a total of 6 gradients. 55 μL of the diluted sample solution was added to each well of the 96-well plate seeded with APRIL protein, and incubated at room temperature for 30 min. The cell culture plate was removed, the supernatant was discarded, and 100 μL / well of the pre-incubated sample and protein mixture was added to the corresponding cell culture well. The cell culture plate was then incubated at 37°C with 5% CO2 for 24 h. Add 100 μL of Bright-Luc Firefly Luciferase Reporter Assay Kit to each well, react for 10 min, and then detect the luminescence value using a microplate reader.

[0136] The results are shown in Figure 7. The blocking activity of H-113Z14-D2-M428L-N434S on APRIL-activated HEK 293T ACI luc reporter gene cell was better than that of antibody fusion protein-N.

[0137] Example 14: BDCA2 binding activity of different antibody fusion proteins

[0138] The binding activity of the antibody to human BDCA2 protein was determined by ELISA detection method. The plate coated with Human BDCA2, His (manufacturer: KATCUS, cat: BCA-HM402) protein was incubated overnight at 4°C, blocked with 2% PBS-BSA, and washed 3 times with PBS containing 0.05% Tween. Gradient-diluted H-113Z14-D2-M428L-N434S, antibody fusion protein-N was added as the primary antibody in a 96-well enzyme-labeled plate, starting at 1 μg / mL, 3-fold gradient dilution, a total of 11 gradients, with a blank control group, 100 μL / well, 37°C incubation for 1 h. Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (manufacturer: Jackson immuno, catalog number: 109-036-098) was diluted 1:10000 as the secondary antibody, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development. The binding of the antibody to human BDCA2 protein was detected. The results are shown in Figure 8. The binding activity of H-113Z14-D2-M428L-N434S to Human BDCA2, His was better than that of antibody fusion protein-N.

[0139] Example 15: Endocytosis activity of different antibody fusion proteins

[0140] PBMC was resuspended with diluent (PBS + 2% FBS), 4 x 10 5 / well, 100 μL / well, 37°C incubation for 1 h. Discard the supernatant after centrifugation, add Human DLEC / CLEC4C / BDCA-2 Antibody (R&D Systems, AF1376), 10 μg / ml, 80 μL / well, incubate on ice for 30 min, centrifuge and wash once. Add 4% paraformaldehyde, 100 μL / well, fix at room temperature for 15 min, centrifuge and discard the supernatant, add the mixture of surface marker dyes and secondary antibodies (recombinant anti-CD14 antibody (PerCp), PE / cyanine7 anti-human CD304 (neuropilin-1), IL3RA antibody (APC), secondary antibody PE anti-goat IgG) to each well, 100 μL / well, incubate at 4°C for 45 min, wash twice, resuspend in diluent, and detect by flow cytometry. pDCs are defined as CD14 - CD123 (IL-3RA) + BDCA4 (CD304) + The expression of BDCA2 on the surface of pDCs after incubation with the sample is detected by analyzing the binding signal fluorescence intensity value of the commercial polyclonal antibody Human DLEC / CLEC4C / BDCA-2 Antibody, and the sample-mediated endocytosis of BDCA2 on the surface of pDCs is calculated.

[0141] The results are shown in Figure 9. H-113Z14-D2-M428L-N434S has stronger endocytosis activity than antibody fusion protein-N.

[0142] Example 16: Half-life test of different antibody fusion proteins

[0143] SD rats (1 male and 1 female) were selected for rat PK test of each candidate molecule. The administration method was tail vein bolus, and the administration dose was 1 mpk. Blood samples were collected from the jugular vein / orbital vein of the test animals, and the actual blood collection time was recorded. The collected blood samples were placed at room temperature for half an hour, then centrifuged (centrifuge pre-cooled at 4°C, centrifugation conditions: 4°C, 3000 rpm, centrifugation for 10 min), and the serum was separated and transferred to a labeled centrifuge tube. ELISA method was used for blood concentration determination.

[0144] Coat Human BDCA2, His (KATCUS, Cat: BCA-HM402) 0.6 pg / mL, 100 pL / well, 4°C incubate overnight. Discard coating solution, 1 x PBST (0.05%) 300 pL / well, wash plate machine 3 times, 2% BSA (300 pL / well) block, 37°C incubate 1 h; discard blocking solution, 1 x PBST (0.05%) 300 pL / well, wash plate machine 4 times; dilute samples with 2% BSA, 100 pL / well into well plate, prepare standard curve solution with 2% BSA and blank rat serum added corresponding dilution, 100 pL / well into well plate, 37°C incubate 1 h; discard solution, 1 x PBST (0.05%) 300 pL / well, wash plate machine 4 times, dilute goat anti-human Fc-HRP (109-036-098) at 1:5000 ratio, 100 pL / well into well plate, 37°C incubate 45 min. Discard solution, 1 x PBST (0.05%) 300 pL / well, wash plate machine 6 times, tap well bottom solution on a piece of dust-free paper. Add TMB color developing solution into well, 100 pL / well, and wrap with aluminum paper, 37°C develop color for 3 min in the dark. Add stop solution (1 M hydrochloric acid) to stop color developing reaction, 100 pL / well. Read at 450 nm on a microplate reader, and analyze data.

[0145] Pharmacokinetic software was used to process the plasma drug concentration data using a non-compartment model. The linear-log trapezoidal method was used to calculate the relevant pharmacokinetic parameters t1 / 2, and the results are shown in Table 2. The half-life of H-113Z14-D2-M428L-N434S was longer than that of antibody fusion protein-N.

[0146] Table 2

[0147] Example 17: BAFF binding activity of antibody fusion proteins with mutations at different sites

[0148] The binding activity of the antibody to human BAFF protein was determined by ELISA detection method. The antibody fusion protein-YTE, antibody fusion protein-WT, H-113Z14-D2-M428L-N434S plate coated with antibody concentration of 0.5 μg / mL, 100 μL / well was incubated at 4°C overnight. Blocked with 2% PBS-BSA, washed with PBS containing 0.05% Tween 3 times. Biotinylated Human BAFF, His, Avitag (brand ACRO, item number BAF-H82Q2) was added to the 96-well enzyme-labeled plate as the primary antibody, 1.0 μg / mL starting, 3-fold gradient dilution, a total of 11 gradients, and set 1 blank control, 100 μL / well, 37°C incubation for 1 h. HRP-Conjugated Streptavidin (manufacturer: Thermo Scientific, item number: N100) was used as the secondary antibody at 1:10000, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development. The binding of the antibody to human BAFF protein was detected. The results are shown in Figure 10. The activity of H-113Z14-D2-M428L-N434S binding to Biotinylated Human BAFF, His, Avitag is better than that of antibody fusion protein-YTE.

[0149] Example 18: BDCA2 binding activity of antibody fusion proteins with mutations at different sites

[0150] The binding activity of the antibody to human BDCA2 protein was determined by ELISA detection method. The plate coated with 0.6 μg / mL Human BDCA2, His (manufacturer: KATCUS, cat: BCA-HM402) protein was incubated at 4°C overnight, blocked with 2% PBS-BSA, and washed with PBS containing 0.05% Tween 3 times. Gradient-diluted H-113Z14-D2-M428L-N434S, antibody fusion protein-YTE, and antibody fusion protein-WT were added to the 96-well enzyme-labeled plate as the primary antibody, with a starting concentration of 10 nM, 4-fold gradient dilution, a total of 8 gradients, and a blank, 100 μL / well, 37°C incubation for 1 h. Peroxidase AffiniPure Goat Anti-Human IgG, Fcγfragment specific (manufacturer: Jackson immuno, catalog number: 109-035-098) was diluted at 1:10000 as the secondary antibody, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development. The binding of the antibody to human BDCA2 protein was detected. The results are shown in FIG. 11. The binding activity of H-113Z14-D2-M428L-N434S to Human BDCA2, His was stronger than that of antibody fusion protein-YTE and antibody fusion protein-WT.

[0151] Example 19: Binding ability test of antibody fusion protein with different site mutations to human BDCA2 and BAFF double targets

[0152] The in vitro binding activity of H-113Z14-D2-M428L-N434S, antibody fusion protein-YTE, antibody fusion protein-WT with human BDCA2 and BAFF was analyzed by enzyme-linked immunosorbent assay. Human BDCA2, His (KATCUS, BCA-HM402) was diluted to 0.6 μg / mL with coating solution (1xPBS, pH 7.4) to 100 μL / well in a 96-well plate, gently shaken to mix, and incubated at 4°C overnight. After washing and blocking, H-113Z14-D2-M428L-N434S, antibody fusion protein-YTE, and antibody fusion protein-WT were diluted to a protein concentration of 100 nM, and 3-fold dilutions were performed to obtain 11 gradients. The blank control was set to 100 μL / well, and the mixture was incubated at 37°C for 1 h. Biotinylated Human BAFF, His, Avitag (ACRO, BAF-H82Q2) was diluted to 0.2 μg / mL, and 100 μL / well was added to the 96-well plate and incubated at 37°C for 1 h. The liquid in the 96-well plate was discarded, and 300 μL / well of washing solution was added to wash the plate 4 times. The secondary antibody HRP-Conjugated Streptavidin (Thermo Scientific, N100) was diluted 1:10,000 with sample diluent, and 100 μL / well was added to the 96-well plate and incubated at 37°C for 45 min. The liquid in the 96-well plate was discarded, and 300 μL / well of washing solution was added to wash the plate 6 times. TMB color developing solution was added to the 96-well plate at 100 μL / well, gently shaken to mix, and developed at 37°C for 3-4 min in the dark. The stop solution was added to the 96-well plate at 100 μL / well, gently shaken to mix, and the reaction was terminated. The 96-well plate was placed in a multifunctional enzyme label instrument, and the absorbance value was recorded at a detection wavelength of 450 nm. As shown in FIG. 12, the binding activity of H-113Z14-D2-M428L-N434S was stronger than that of antibody fusion protein-YTE.

[0153] Example 20: Half-life test of antibody fusion proteins with mutations at different sites

[0154] SD rats (1 male and 1 female) were selected for rat PK tests of each candidate molecule. The administration method was tail vein bolus injection, and the administration dose was 1 mpk. Blood samples were collected from the jugular vein / orbital vein of the test animals, and the actual blood collection time was recorded. The collected blood samples were placed at room temperature for half an hour and then centrifuged (centrifuge pre-cooled at 4°C, centrifugation conditions: 4°C, 3000 rpm, 10 min). The separated serum was transferred to a labeled centrifuge tube. The blood drug concentration was determined by ELISA method.

[0155] Detection method: coated 1.0 μg / mL of Monoclonal Anti-Human-IgG-Fc Antibody (manufacturer: Acro, Cat: IGG-AY69) 100 μL / well, 4℃ incubated overnight. Discard the coating liquid, 1 × PBST (0.05%) 300 μL / well, plate washing machine wash 3 times, 2% BSA (300 μL / well) blocking, 37℃ incubated for 1h; Discard the blocking liquid, 1 × PBST (0.05%) 300 μL / well, plate washing machine wash 4 times; The sample was diluted with 2% BSA, 100 μL / well was added to the well plate, 2% BSA and the corresponding dilution blank rat serum was prepared, 100 μL / well was added to the well plate, 37℃ incubated for 1h; Discard the liquid, 1 × PBST (0.05%) 300 μL / well, plate washing machine wash 4 times, dilute goat anti-human Fc-HRP (109-036-098) according to 1:5000 times, 100 μL / well was added to the well plate, 37℃ incubated for 45min. Discard the liquid, 1 × PBST (0.05%) 300 μL / well, plate washing machine wash 6 times, and the bottom liquid was wiped off with a dust-free paper. Add TMB color developing solution to the plate wells, 100 μL / well, and wrap it with aluminum paper, 37℃ avoid light color developing for 3min. Add stop solution (1M hydrochloric acid) to terminate the color developing reaction, 100 μL / well. Read at 450nm in the enzyme label instrument, and analyze the data.

[0156] The plasma drug concentration data was processed using a non-compartment model using pharmacokinetic software. The linear logarithmic trapezoidal method was used to calculate the relevant pharmacokinetic parameters t1 / 2, and the results are shown in Table 3. The half-life of H-113Z14-D2-M428L-N434S was longer than that of antibody fusion protein-YTE and antibody fusion protein-WT.

[0157] Table 3

[0158] Example 21: Inhibitory activity of antibody fusion protein

[0159] PBMC was resuspended with diluent (1640+3% FBS), 1 × 10 6 cells were inoculated in a 96-well flat-bottom plate, and 10 μg / mL of TLR9 ligand CpG ODN 2216 was added to stimulate the cells. Incubate in a 37℃ incubator for 18h, centrifuge to collect the supernatant, and use 293ISRE reporter gene cells to detect IFNα secretion. 4 × 10 4The 96-well white plates were incubated overnight, and the diluted supernatant was added. The 96-well white plates were placed in a 37°C incubator and incubated for another 24 hours. The luciferase detection kit was used to detect the luminescence value, and the dose-effect curve of the known concentration of IFNα2b was drawn as a standard curve. Then, according to the standard curve and the luminescence value of the supernatant of the cells treated with the antibody, the amount of IFNα in the supernatant of the antibody was calculated. The results are shown in Figure 13. H-113Z14-D2-M428L-N434S has an inhibitory activity comparable to that of H-113-Z14 and H-BMK, and has a lower secretion of IFNα2b than H-BMK at a low concentration.

[0160] Example 22: Endocytosis activity of antibody fusion protein

[0161] PBMC was resuspended with diluent (PBS+2%FBS), and 5x10 5 Cells were inoculated in a 96-well U-shaped plate, and the antibody (concentration gradient) was added. The plate was incubated at 37°C for 1 hour, and then incubated with goat anti-human BDCA2 (R&D Systems, AF1376) antibody at 4°C for 30 minutes. The plate was fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then incubated with anti-goat, anti-CD45, anti-CD123, and anti-BDCA4 antibodies at room temperature for 30 minutes. The pDC cells were circled as CD45 - CD123 + BDCA4 + cells, and the binding signal fluorescence intensity value of the goat anti-human BDCA2 antibody was analyzed to calculate the antibody-mediated endocytosis of pDC cell surface BDCA2. The results are shown in Figure 14. H-113Z14-D2-M428L-N434S has a stronger endocytosis activity than H-113-Z14 and H-BMK.

[0162] Example 23: In vivo efficacy of antibody fusion protein

[0163] Twenty MRL / lpr mice were randomly divided into four experimental groups; five C57BL / 6 healthy mice were used as normal control group. The drug administration was performed according to Table 4.

[0164] Table 4

[0165] From 8 weeks of age, the G3 and G4 groups were given intraperitoneal injections every other day for 8 weeks. The mice were weighed every week, and urine samples were collected from the animals every week starting at 8 weeks of age to detect the total protein level in the urine. The mice were observed every week for signs of illness, including body weight, lymph node, and skin lesions. The results are shown in Figures 15, 16, and 17. H-113Z14-D2-M428L-N434S is safe and can significantly reduce proteinuria levels and improve lymphadenopathy scores, and has a stronger ability to improve lymphadenopathy scores than the G3 group of etanercept.

[0166] Example 24: Detection of antibody fusion protein blocking BAFF activation of HEK 293TACI luc reporter cell activity

[0167] HEK 293TACI luc reporter cells are reporter cells stably expressing TACI obtained by puromycin and G418 pressure screening after transfecting HEK 293 cells with TACI lentivirus and NF-κB-Luc luciferase reporter lentivirus.

[0168] HEK 293TACI luc reporter cells were plated at 2x10 4 cells / well, 50 μL / well in a 96-well full-white plate, and incubated at 37°C in a 5% CO2 incubator for 18 h. BAFF protein was diluted to 0.5 μg / mL (final concentration 0.125 μg / mL), 30 μL / well was plated in a 96-well plate, H-113Z14-D2-M428L-N434S, etanercept (Rongchang Biopharmaceutical (Yantai) Co., Ltd., batch number: RC182202401003-1), Human IgG (catalog number: abs20037, Aibisheng Biotechnology Co., Ltd.) were diluted to an initial concentration of 1200 nmol / L (final concentration 300 nmol / L), 3-fold dilution, a total of 8 gradients, and 30 μL of the diluted sample solution was added to the 96-well plate with BAFF protein, incubated at room temperature for 30 min, and 50 μL of the pre-incubated sample and protein mixture was added to the corresponding cell wells. The cell plate was placed in a 37°C, 5% CO2 incubator for incubation for 24 h. Bright-Luc Firefly Luciferase Reporter Assay Kit, 100 μL / well, after 10 min of reaction, the luminometer was used to detect the luminescence value.

[0169] The results are shown in Figure 18. H-113Z14-D2-M428L-N434S has better blocking activity than the etanercept group.

[0170] Example 25: Antibody fusion protein and human BDCA2, BAFF double target binding ability test

[0171] The in vitro binding activity of H-113Z14-D2-M428L-N434S to human BDCA2 and BAFF was analyzed by enzyme-linked immunosorbent assay. Human BDCA2, His (KATCUS, BCA-HM402) was diluted to 0.6 μg / mL with coating solution (1x PBS, pH 7.4) and 100 μL / well was added to a 96-well plate, which was gently shaken and mixed, and then incubated at 4°C overnight. After washing and blocking, H-113Z14-D2-M428L-N434S was diluted to a protein concentration of 100 nM, and 3-fold dilutions were made to a total of 11 gradients. Litifilimab (heavy chain sequence: SEQ ID NO: 21, light chain sequence: SEQ ID NO: 22) and Tavolimab samples were diluted to a protein concentration of 100 nM with 3% skim milk as negative controls. The diluted samples were added to the 96-well plate at 100 μL / well, and incubated at 37°C for 1 h. The liquid in the enzyme-labeled plate was discarded, 300 μL / well of washing solution was added, and the plate was washed 4 times. The detection protein Biotinylated Human BAFF, His, Avitag (ACRO, BAF-H82Q2) was diluted to 0.2 μg / mL, and 100 μL / well was added to the 96-well plate and incubated at 37°C for 1 h. The liquid in the 96-well plate was discarded, 300 μL / well of washing solution was added, and the plate was washed 4 times. The secondary antibody HRP-Conjugated Streptavidin (Thermo Scientific, N100) was diluted 1:10,000 with sample diluent, and 100 μL / well was added to the 96-well plate and incubated at 37°C for 45 min. The liquid in the 96-well plate was discarded, 300 μL / well of washing solution was added, and the plate was washed 6 times. TMB color developing solution was added to the 96-well plate at 100 μL / well, gently shaken and mixed, and developed at 37°C for 3-4 min in the dark. The stop solution was added to the 96-well plate at 100 μL / well, gently shaken and mixed, and the reaction was terminated. The 96-well plate was placed in a multifunctional enzyme-labeled instrument, and the absorbance value was detected at a detection wavelength of 450 nm. The results are shown in FIG. 19. Litifilimab and Tavolimab did not bind to the double-target protein, and H-113Z14-D2-M428L-N434S had strong binding activity to Human BDCA2, His and Biotinylated Human BAFF, His, Avitag in a concentration-dependent manner, with an EC 50 value of 1.0957 nM.

[0172] Example 26: Binding ability of antibody fusion protein to human BDCA2

[0173] The binding activity of the antibody to human BDCA2 protein was determined by ELISA detection method. The plate coated with Biotinylated Human BAFF, His, Avitag (brand ACRO, item number BAF-H82Q2) protein was incubated at 4°C overnight. Blocked with 2% PBS-BSA, washed 3 times with PBS containing 0.05% Tween. Gradient dilution of the antibody to be detected was added as a primary antibody in a 96-well enzyme-labeled plate, and incubated at 37°C for 1 h. Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific was used as a secondary antibody, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development to detect the binding of the antibody to human BDCA2 protein. The results are shown in Figure 20, and H-113Z14-D2-M428L-N434S has strong binding activity to Human BDCA2, His in a concentration-dependent manner, with an EC 50 value of 0.03892 μg / mL.

[0174] Example 27: Binding ability of antibody fusion protein to human BAFF

[0175] The binding activity of the antibody to human BAFF protein was determined by ELISA detection method. The plate coated with Biotinylated Human BAFF, His, Avitag (brand ACRO, item number BAF-H82Q2) protein was incubated at 4°C overnight. Blocked with 2% PBS-BSA, washed 3 times with PBS containing 0.05% Tween. Gradient dilution of the antibody to be detected was added as a primary antibody in a 96-well enzyme-labeled plate, and incubated at 37°C for 1 h. Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific was used as a secondary antibody, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development to detect the binding of the antibody to human BAFF protein. The results are shown in Figure 21, and H-113Z14-D2-M428L-N434S has strong binding activity to Biotinylated Human BAFF, His, Avitag in a concentration-dependent manner, with an EC 50 value of 0.03554 μg / mL.

[0176] Example 28: Binding ability of antibody fusion protein to human APRIL

[0177] The binding activity of the antibody to human APRIL protein was determined by ELISA detection method. The plate coated with antibody concentration of 10 nM, 100 μL / well of Etanercept, H-113Z14-D2-M428L-N434S was incubated at 4°C overnight. Blocked with 2% PBS-BSA, washed with PBS containing 0.05% Tween 3 times. In the 96-well enzyme-labeled plate, biotinylated human APRIL, His, Avitag (brand ACRO, item number: APL-H82Q8) was added as a primary antibody, 1.0 μg / mL starting, 3-fold gradient dilution, a total of 7 gradients, and 1 blank control was set, 100 μL / well, 37°C incubation for 1 h. HRP-Conjugated Streptavidin (manufacturer: Thermo Scientific, item number: N100) was used as a secondary antibody at 1:10000, and the absorbance value of each well was read at a wavelength of 450 nm after TMB color development. The binding of the antibody to human BAFF protein was detected. The results are shown in Figure 22. The activity of H-113Z14-D2-M428L-N434S in binding to Biotinylated Human APRIL, His, Avitag was stronger than that of Etanercept.

[0178] Example 29: Antibody fusion protein complement-dependent cytotoxicity (CDC) test

[0179] Target cells CHOK1-BDCA2 were cultured in F12 ham (N3520-1L, Sigma) complete medium in a 10 cm culture dish at 37°C, 5% CO2 incubator, and passaged every three days or so. The cell culture plate was set up with antibody group, negative control group, and blank control group. The specific steps are as follows: CHOK1-BDCA2 cells in logarithmic growth phase were taken, washed with 3 mL PBS, and each was added with 1 mL trypsin for 3 min, then 3 mL complete medium was added to terminate digestion. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 3 min. Then 3 mL diluent was added to resuspend the cells, and the cell counter was used to count. The CHOK1-BDCA2 cell suspension was taken, and the cell density was adjusted to 3×10 5The antibody wells (3 replicates) and negative control wells (6 replicates) of the 96-well plate were added with 100 μL / well. Then, the starting concentration of the antibodies Litifilimab, H-113-Z14 (BDCA2 humanized monoclonal antibody), H-113Z14-D2-M428L-N434S, and Human IgG (abs20037, Aibio Biotech) diluted with diluent in the test group was 400 nmol / L (the final concentration was 100 nmol / L), 5-fold dilution, 7 gradients, 50 μL / well, mixed, and the plate was placed in a 37°C, 5% CO2 incubator for 15 min. Rabbit complement was diluted to a concentration of 20% with diluent, the cell culture plate was taken out, and 50 μL / well of rabbit complement was added to the test group, negative control group, and blank control group. At the same time, 50 μL / well of diluent was added to the negative control group, and 150 μL / well of diluent was added to the blank control group. Mix well, continue to incubate in a 37°C, 5% CO2 incubator for 1 h. Take out the culture plate, add CCK-8 color developing solution, 20 μL / well, mix well. Place the plate in a 37°C, 5% CO2 incubator for 4 h. Take out the 96-well cell culture plate, then place it in a microplate reader, set the plate to shake for 30 s before reading, and measure the absorbance at a wavelength of 450 nm. Subtract the background value of the blank control well from each well, and calculate the target cell lysis rate (%).

[0180] The data obtained were analyzed by plotting the curve using GraphPad Prism software Log (agonist) v.s. Response--Variable Slope. The results are shown in FIG. 23. H-113Z14-D2-M428L-N434S had a strong CDC effect on CHOK1-BDCA2 cells overexpressing human BDCA2, and was slightly better than H-113-Z14 and Litifilimab.

[0181] Example 30: Antibody fusion protein inhibits BAFF-induced B cell proliferation test

[0182] Preparation of B cell suspension: B cells (XFB-CD19B-05B-, Saisig Biotechnology) were taken out from the -80°C refrigerator, thawed in a water bath, added to a 15 mL centrifuge tube containing 4 mL of 1640 complete culture medium, centrifuged at 400 g for 10 min, the supernatant was discarded, and the cells were resuspended with 5 mL of diluent and counted with a cell counter. The B cell density was adjusted to 2.5 x 10 6 5 μL / well was added to the 96-well plate, i.e., 1 x 10​

[0183] BAFF control group: dilute BAFF with diluent to an initial concentration of 500 ng / mL (final concentration of 100 ng / mL), 20 μL / well, and then add 40 μL / well of diluent to the 96-well full white plate with B cells.

[0184] IgM control group: dilute IgM with diluent to an initial concentration of 50 μg / mL (final concentration of 10 μg / mL), 20 μL / well, and then add 40 μL / well of diluent to the 96-well full white plate with B cells.

[0185] IgM+BAFF control group: dilute IgM with diluent to an initial concentration of 50 μg / mL (final concentration of 10 μg / mL), 20 μL / well, dilute BAFF with diluent to an initial concentration of 500 ng / mL (final concentration of 100 ng / mL), 20 μL / well, and then add 20 μL / well of diluent to the 96-well full white plate with B cells.

[0186] Antibody group: dilute IgM with diluent to an initial concentration of 50 μg / mL (final concentration of 10 μg / mL), 20 μL / well, dilute BAFF with diluent to an initial concentration of 500 ng / mL (final concentration of 100 ng / mL), 20 μL / well, and then dilute antibodies Tavoliximab, H-113Z14-D2-M428L-N434S with diluent to an initial concentration of 500 nmol / L (final concentration of 100 nmol / L), 4-fold dilution, 8 concentration gradients. Dilute Human IgG (Abs20037, Aibisheng Biotechnology Co., Ltd.) to an initial concentration of 500 nmol / L (final concentration of 100 nmol / L), 4-fold dilution, 2 gradients. Add 20 μL / well of the diluted antibodies to the 96-well full white plate with IgM, BAFF, and B cells; blank control group: add 60 μL of diluent, and then place the 96-well full white plate in a 37°C cell incubator for incubation for 72 h. After 72 h, remove the cell plate and CellTiter-Glo Luminescent Cell Viability Assay detection kit (Promega, G7572), and then equilibrate to room temperature. Add 100 μL / well of CellTiter-Glo Luminescent Cell Viability Assay detection reagent to the 96-well full white plate, and then continue to incubate for 10 min. Detect the luminescence value by Luminescence detection method and enzyme-labeled instrument.

[0187] The antibody concentration (nmol / L) was plotted on the x-axis and the inhibition rate (%) on the y-axis. The obtained data were analyzed by plotting a curve using the Log(inhibitor) vs. Response-Variable Slope method in GraphPad Prism software. The results, shown in Figure 24, indicate that H-113Z14-D2-M428L-N434S exhibits superior inhibitory activity against BAFF-induced B cell proliferation compared to teltascept.

[0188] Example 31: Assay of antibody fusion protein binding to BDCA2 on the surface of hBDCA2 humanized mouse pDCs

[0189] Spleens were harvested from hBDCA2 humanized mice (source: Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.), ground, and lysed with erythrocytes for cell counting. Cells were then diluted to 2 × 10⁻⁶. 7 / ml, 50μL / well were seeded into a 96-well plate. 50μL of FVD (Fixable Viability Dye eFluoride) was added. TM Incubate the cells in 506 solution at 4°C for 30 min, then centrifuge at 500×g at 4°C for 5 min and discard the supernatant. Add 50 μL of blocking buffer and incubate at 4°C for 15 min. Dilute H-113Z14-D2-M428L-N434S, H-113-Z14, and Litifilimab to a concentration of 20 μg / mL (final concentration 10 μg / mL), add 50 μL / well to co-incubate with cells, incubate at 4°C for 30 min, then centrifuge at 500×g at 4°C for 5 min and discard the supernatant. Add 50 μL of surface-labeled dye and secondary antibody mixture (APC / Cy7 anti-mouse CD45 Antibody, PerCP / Cyanine 5.5 anti-mouse Siglec H Antibody, Pacific Blue) to each well. TM Incubate with anti-mouse / human CD45R / B220 Antibody, APC anti-mouse CD317 (BST2, PDCA-1) Antibody, and R-PE-Goat Anti-Human IgG secondary antibody at 4°C for 30 min. Add 150 μL of FACS solution, centrifuge at 500×g for 5 min at 4°C, discard the supernatant, and wash once more. Resuspend in 250 μL of FACS solution and analyze by flow cytometry. pDCs are defined as mCD45. + mSiglec-H + mCD45R / B220 + mCD317 +The binding of the antibody to hBDCA2 transgenic mouse pDCs was verified by the binding signal of the R-PE-Goat Anti-Human IgG secondary antibody.

[0190] Table 5 shows the fluorescence values ​​of H-113Z14-D2-M428L-N434S and related antibodies binding to pDCs of hBDCA2 transgenic mice; Figure 25 shows the flow cytometry chromatograms of H-113Z14-D2-M428L-N434S and related antibodies binding to pDCs of hBDCA2 transgenic mice. The results indicate that hBDCA2 humanized mice express BDCA2 on their surface, which binds to H-113Z14-D2-M428L-N434S.

[0191] Table 5

[0192] Example 32: Antibody fusion protein-mediated BDCA2 endocytosis assay on the surface of hBDCA2 humanized mouse pDCs

[0193] hBDCA2 humanized mice (source: Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were injected with H-113Z14-D2-M428L-N434S. Six hours after administration, the spleens of hBDCA2 mice were harvested, ground, and lysed with erythrocytes for cell counting. The cells were then diluted to 2×10⁻⁶. 7 / ml, 50μL / well were seeded into a 96-well plate. 50μL of LFVD (Fixable Viability Dye eFluoride) was added. TM After incubating the 506 solution at 4°C for 30 min, centrifuge at 500×g at 4°C for 5 min and discard the supernatant. Add 50 μL of blocking buffer and incubate at 4°C for 15 min. Add 50 μL of surface-labeled dye and commercial BDCA2 antibody mixture (APC / Cy7 anti-mouse CD45 Antibody, PerCP / Cyanine 5.5 anti-mouse Siglec H Antibody, Pacific Blue) to each well. TM Anti-mouse / human CD45R / B220 Antibody, APC anti-mouse CD317 (BST2, PDCA-1) Antibody, and FITC anti-human CD303 (BDCA-2) Antibody were incubated at 4°C for 30 min. 150 μL of FACS solution was added, and the mixture was centrifuged at 500 × g for 5 min at 4°C. The supernatant was discarded, and the mixture was washed once more. The mixture was resuspended in 250 μL of FACS solution and analyzed by flow cytometry. pDCs were defined as mCD45. + mSiglec-H +mCD45R / B220 + mCD317 + The expression of pDCs surface BDCA2 was verified by commercial FITC anti-human CD303 (BDCA-2) antibody.

[0194] The fluorescence values of commercial anti-BDCA2 antibody binding to pDCs of hBDCA2 transgenic mice injected with H-113Z14-D2-M428L-N434S and related antibody 6h were shown in Table 6; the flow cytometry of pDCs surface BDCA2 expression of hBDCA2 transgenic mice injected with H-113Z14-D2-M428L-N434S were shown in Figure 26. The results showed that the expression of pDCs surface BDCA2 of hBDCA2 humanized mice was reduced 6h after injection of H-113Z14-D2-M428L-N434S, which indicated that H-113Z14-D2-M428L-N434S could mediate the endocytosis of pDCs surface BDCA2 of hBDCA2 humanized mice.

[0195] Table 6

[0196] Example 33: In vivo efficacy experiment of antibody fusion protein

[0197] The emulsion preparation steps are as follows: connect a three-way tube to a 10 mL syringe, then seal; add 2 mL of complete Freund's adjuvant (incomplete Freund's adjuvant for booster immunization) and 2 mL of Col II 2 mg / mL glacial acetic acid solution into the 10 mL syringe respectively; push the syringe plunger to mix the complete Freund's adjuvant (incomplete Freund's adjuvant for booster immunization) and Col II solution in the syringe; clamp the syringe on the ring stand and place it in an ice water bath, keep low temperature during emulsification; homogenate at 3000 rpm with a high-speed stirrer to fully emulsify Col II and CFA (IFA for booster injection); transfer the emulsion into a 1 mL syringe for animal immunization. The emulsion should be injected as soon as possible and placed on ice throughout the process.

[0198] 8-week-old male DBA / 1JGpt mice (provided by Jiangsu Jizu Pharmaceutical Biotechnology Co., Ltd.) were immunized. First, the mice were anesthetized with 3% isoflurane at a gas flow rate of 2 L / min, and the hair at the root of the mouse tail was removed; on day 0, emulsion injection was performed at 3 sites on the mouse, 40 μL at a distance of 2 cm from the tail root (tail), 30 μL at two locations near the tail root (back), and a total of 100 μg of Col II (20022, Chondrex) was injected per mouse; on day 20, the CIA (collagen-induced arthritis) model mouse was immunized according to the same procedure as day 0, and 100 μL of Col II / IFA emulsion was injected near the last immunization site, and a total of 100 μg of Col II was injected per mouse. On day 30, the CIA model group animals were randomly divided into 5 groups (groups 2-6) according to the clinical score, 8 mice per group, as shown in Table 7.

[0199] Table 7

[0200] The mouse arthritis clinical score was performed as follows: the inflammation of each toe and joint was observed and recorded, and each paw was scored up to 4 points (a maximum of 16 points per mouse). 0: normal paw; 1: one or two toes inflamed and swollen; 2: ≥3 toes inflamed, no foot swelling, or mild swelling of the entire foot; 3: entire paw red and swollen; 4: severe swelling of the sole and toes, or rigidity of the sole and toes. The results of the score are shown in FIG. 27. From day 30, the Isotype (IgG-Fc) group of mice had significantly higher arthritis clinical scores than the non-model group, and the H-113Z14-D2-M428L-N434S three-dose groups all improved the arthritis clinical score.

[0201] The mice were sacrificed on day 69, and spleen B lymphocyte collection and staining were performed as follows: 1. The centrifuge was pre-cooled to 4°C in advance, and ice was prepared for standby, 1xPBS was pre-cooled in the refrigerator; 2. A 70-μm cell sieve was placed in a 35-mm culture dish and placed on ice; 3. The spleen was transferred to the cell sieve, and 5-7 mL of 4°C pre-cooled 1xPBS was added to the cell sieve. A 5-mL syringe plunger was used to gently grind the spleen until there were no red lumps; 4. After grinding was complete, the cell sieve was rinsed with 2 mL of 4°C pre-cooled 1xPBS, the cell suspension was mixed, and the cell suspension was transferred to a 15-mL centrifuge tube, which was centrifuged at 500g for 5 min at 4°C; 5. The supernatant was poured off, and the cell pellet was centrifuged at 4°C, and the supernatant was discarded; 6. 2 mL of red blood cell lysis solution was added to resuspend the cells, and the cells were lysed at room temperature for 5-8 min, with mixing every 1-2 min. The cell suspension gradually changed from turbid to transparent; 7. 8 mL of 1xPBS was added to terminate lysis, and the mixture was mixed gently; 8. The mixture was centrifuged at 500g for 5 min at 4°C, the supernatant was poured off, and the mixture was centrifuged again at 4°C, and the supernatant was discarded; 9. If the surface of the pellet was red or white, the lysis result was ideal, and the next step was performed; if the pellet was red overall, the steps 6-8 were repeated once, and then the next step was performed; 10. 1 mL of cell suspension was added, followed by 9 mL of 1xPBS, and the mixture was mixed gently; 11. 10 μL of cell suspension and 10 μL of AO / PI dye were mixed, 15-20 μL of the mixture was taken, and cell counting was performed. The cell concentration was adjusted to 6x10 6

[0202] ​The positive rate of immature B cells (CD19 / IgM) in the spleen of mice in the Isotype (IgG-Fc) group was significantly higher than that in the non-model group. The positive rate of immature B cells (CD19 / IgM) in the spleen of mice in the H-113Z14-D2-M428L-N434S three-dose groups was significantly lower than that in the Isotype (IgG-Fc) group, and the results are shown in Figure 28.

[0203] The positive rate of mature B cells (CD19 / IgD) in the spleen of mice in the H-113Z14-D2-M428L-N434S three-dose groups was significantly lower than that in the Isotype (IgG-Fc) group, and the results are shown in Figure 29.

[0204] Example 34: Pharmacodynamic effects in collagen-induced arthritis model of DBA / 1 mice

[0205] 8-9-week-old male DBA / 1 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. Type II collagen from chicken was dissolved in 0.1 M acetic acid (glacial acetic acid) to a concentration of 4 mg / mL, emulsified with an equal volume of Freund's complete adjuvant at 4°C overnight, and 100 μL of the mixed emulsion was injected into the base of the mouse tail in two injections, taking care to avoid the tail vein. The mice were injected twice at 0 days and 21 days according to the model. According to the incidence of arthritis and body weight data of the model group mice, the mice were divided into 5 groups, 5 mice in each group, and the grouping details are shown in Table 8.

[0206] Table 8

[0207] The mice were observed for clinical symptoms twice a day, and the body weight of the mice was measured and recorded three times a week. At the same time, from the start of collagen injection modeling on day 0 to the end of the experiment, the state of the mice was closely observed for changes in physical signs, especially changes in inflammation of the paws of the four limbs, and recorded. The end of the experiment was 1) the survival body weight curve of the animals; 2) the change curve of the arthritis score; 3) the distribution of immune cells: flow cytometry detection of T lymphocyte and B lymphocyte subsets.

[0208] The body weight is shown in Figure 30. During the experiment, the body weight of the mice in each group was stable, and no abnormal conditions were observed, indicating good safety.

[0209] Arthritis score and B cell subset detection. The arthritis score is shown in Figure 31. Except for the G1 blank control group, the remaining 5 groups of mice showed a more serious trend in arthritis. Compared with the G2 model control group, the scores of the G4 methotrexate (positive drug) and G5 test product (H-113Z14-D2-M428L-N434S) groups decreased effectively, and the score results of the G3 parallel isotype control treatment group did not show a significant decrease. Among them, G5 showed the best control of the arthritis score.

[0210] The results of B cell subpopulation detection in collagen-induced arthritis model of DBA / 1 mice by H-113Z14-D2-M428L-N434S are shown in Figure 32. Among them, H-113Z14-D2-M428L-N434S can significantly reduce the percentage of total B cells and different disease-related B cell subpopulations in CIA mice.

[0211] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to BDCA2, characterized in that, The antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2, a HCDR3 as set forth in SEQ ID NO: 3; and a LCDR1 as set forth in SEQ ID NO: 4, a LCDR2 as set forth in SEQ ID NO: 5, a LCDR3 as set forth in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH as set forth in SEQ ID NO: 7, and a light chain variable region VL as set forth in SEQ ID NO:

8.

3. An antibody fusion protein, characterized in that, The antibody fusion protein comprises the following elements fused together: (a) an antibody or antigen-binding fragment thereof that specifically binds to BDCA2; and (b) TACI or an active fragment thereof; wherein the TACI or active fragment thereof is linked to the C-terminus or N-terminus of the heavy chain of the antibody or antigen-binding fragment thereof that specifically binds to BDCA2 via a peptide linker.

4. The antibody fusion protein of claim 3, wherein, The heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a heavy chain constant region CH, and the light chain comprises a light chain variable region VL and a light chain constant region CL.

5. The antibody fusion protein of claim 3, wherein, The antibody or antigen-binding fragment thereof is a full-length antibody.

6. The antibody fusion protein of claim 3, wherein, The antibody or antigen-binding fragment thereof is a murine, chimeric, humanized or fully human antibody, or an antigen-binding fragment thereof.

7. The antibody fusion protein of claim 3, wherein, The antibody or antigen-binding fragment thereof comprises an Fc region, and the Fc region is derived from a human or a non-human mammal, preferably from a rodent (e.g., mouse, rat), a primate or a human.

8. The antibody fusion protein of claim 7, wherein, The Fc region is selected from the group consisting of IgG, IgA, IgD, IgE and / or IgM.

9. The antibody fusion protein of claim 8, wherein, The Fc region is selected from the group consisting of IgG1, IgG2, IgG3 and / or IgG4, preferably the Fc region is selected from IgG1.

10. The antibody fusion protein according to claim 8 or 9, characterized in that, The Fc region comprises a native Fc fragment and an Fc mutant.

11. The antibody fusion protein of claim 10, wherein, The Fc mutant comprises substitution and / or truncation of amino acid residues based on the native Fc fragment.

12. The antibody fusion protein of claim 11, wherein, The Fc mutant comprises substitution of methionine (M) with leucine (L) at position 428 resulting in (M428L), and / or substitution of asparagine (N) with serine (S) at position 434 resulting in (N434S), based on the native Fc fragment, and wherein the numbering is according to the EU index of Kabat et al.

13. The antibody fusion protein of claim 3, wherein, The antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2, a HCDR3 as set forth in SEQ ID NO: 3; and a LCDR1 as set forth in SEQ ID NO: 4, a LCDR2 as set forth in SEQ ID NO: 5, a LCDR3 as set forth in SEQ ID NO:

6.

14. The antibody fusion protein of claim 3, wherein, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH as set forth in SEQ ID NO: 7, and a light chain variable region VL as set forth in SEQ ID NO:

8.

15. The antibody fusion protein of claim 3, wherein, The TACI or active fragment thereof comprises the amino acid sequence of positions 1 to 165, positions 1 to 109, or positions 33 to 109, or positions 68 to 109 of TACI, or a truncation or variant thereof.

16. The antibody fusion protein of claim 3, wherein, The TACI or active fragment thereof is the amino acid sequence of positions 68 to 109 of TACI, or a truncation or variant thereof.

17. The antibody fusion protein of claim 3, wherein, The peptide linker is a peptide linker of 1 to 20 amino acids, preferably a peptide linker of 1 to 15 amino acids, more preferably a peptide linker of 4 to 10 amino acids, most preferably a peptide linker of 4 amino acids.

18. The antibody fusion protein of claim 17, wherein, The peptide linker has an amino acid sequence as shown in (GGGS) x or (GGGGS) x wherein x is selected from 1, 2 or 3.

19. The antibody fusion protein according to claim 17 or 18, characterized in that, The peptide linker is GGGS or GGGSGGGS.

20. The antibody fusion protein of claim 3, wherein, The antibody fusion protein is a homodimer.

21. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an antibody according to claim 1 or 2 or an antibody fusion protein according to any one of claims 3 to 20.

22. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule according to claim 21.

23. A recombinant cell, wherein, The recombinant cell comprises the nucleic acid molecule according to claim 21 and / or the recombinant vector according to claim 22.

24. A method of producing an antibody according to claim 1 or 2 or an antibody fusion protein according to any one of claims 3 to 20, characterized in that, The method comprises the following steps: culturing the recombinant cell according to claim 23 under conditions suitable for expression of the antibody or antibody fusion protein, thereby expressing the antibody or antibody fusion protein; and isolating or purifying the antibody or antibody fusion protein.

25. A pharmaceutical composition comprising, The pharmaceutical composition comprises an antibody according to claim 1 or 2 or an antibody fusion protein according to any one of claims 3 to 20 and a pharmaceutically acceptable carrier.

26. Use of an antibody according to claim 1 or 2, an antibody fusion protein according to any one of claims 3 to 20, a nucleic acid molecule according to claim 21, a recombinant vector according to claim 22, or a recombinant cell according to claim 23 for the manufacture of a pharmaceutical composition for the treatment of an immune disease and / or a B-cell proliferation related disease.

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