Anti-BDCA-2 antibody, preparation method therefor and use thereof

Abstract

An anti-BDCA-2 antibody, a preparation method therefor, and a use thereof, relating to the field of antibodies. The preparation method for the anti-BDCA-2 antibody comprises: carrying out ELISA detection and screening on a hybridoma cell culture supernatant and an antigen protein to obtain a positive clone, and subcloning the positive clone to obtain the anti-BDCA-2 antibody. The anti-BDCA-2 antibody comprises a heavy chain and a light chain, wherein a variable region of the heavy chain and a variable region of the light chain are selected from amino acid sequences as shown in SEQ ID NOs: 1-22. The anti-BDCA-2 antibody and related drugs can be used for inhibiting the production of IFN-α by pDCs and preventing the over-activation of the pDCs. The anti-BDCA-2 antibody can effectively inhibit the activation of pDC cells and the production of IFN-α, and has great potential in resisting virus infection and preventing certain autoimmune diseases.

Description

An anti-BDCA-2 antibody and its preparation method and application

[0001] This application claims priority to Chinese patent application No. 2023116521493, filed with the Patent Office of China on December 5, 2023, entitled “An anti-BDCA-2 antibody, preparation method and use thereof,” the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of antibodies, and specifically relates to an anti-BDCA-2 antibody and a preparation method and application thereof. Background Art

[0003] Blood dendritic cell antigen 2 (BDCA-2) is a type II C-type lectin that plays an important role in the human immune system. BDCA-2 is highly specifically expressed in plasmacytoid dendritic cells (pDCs). pDCs are a specialized cell type in the immune system, ubiquitously present in both lymphoid and non-lymphoid tissues. They are potent interferon producers and contribute significantly to both innate and adaptive immunity, playing a key role in the early stages of viral infections and autoimmune diseases.

[0004] BDCA-2 expression on the surface of pDCs can regulate pDC function. As an endocytic receptor, one of BDCA-2's primary functions is endocytosis. Through endocytosis, BDCA-2 captures and internalizes antigens, further facilitating antigen presentation, which is crucial for immune responses. Therefore, by participating in antigen retrieval and processing, BDCA-2 aids the immune system in combating pathogens. However, unlike other endocytic receptors, BDCA-2 signaling may also inhibit interferon production by pDCs. Upon activation, BDCA-2 blocks interferon production by inhibiting signaling through Toll-like receptors 7 and 9 (TLR7 and TLR9), key receptors that drive interferon production during viral infection. Overall, BDCA-2's primary functions include participating in antigen endocytosis and processing, and fine-tuning disease responses by suppressing pDC responses to interferon production.

[0005] During viral infection, BDCA-2 inhibits the TLR9 signaling pathway, thereby suppressing the production of IFN-α by pDCs and preventing their response to viral infection. Furthermore, BDCA-2 plays a key role in autoimmune diseases. In autoimmune diseases such as systemic lupus erythematosus (SLE), elevated levels of IFN-α are sometimes detected, likely due to overactivation of pDCs. By inhibiting IFN-α production, BDCA-2 may help prevent excessive autoreactions and thus the initiation of autoimmune diseases. Overall, BDCA-2 plays a key role in regulating pDC responses, resisting viral infection, and preventing certain autoimmune diseases. Current research continues to explore the functions and clinical application prospects of BDCA-2.

[0006] Summary of the Invention

[0007] To address these shortcomings, the present invention provides an anti-BDCA-2 antibody, its preparation method, and its use. This antibody and related pharmaceuticals can be used to inhibit the production of IFN-alpha by pDCs, preventing overactivation of pDCs. The anti-BDCA-2 antibody produced by the present invention can effectively inhibit pDC activation and IFN-alpha production, and has great potential in combating viral infections and preventing certain autoimmune diseases.

[0008] the term:

[0009] Unless otherwise specified herein, scientific and technical terms used herein shall have the meanings understood by those of ordinary skill in the art. Generally, the nomenclature and techniques associated with the pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are well known and frequently used in the art.

[0010] As used herein, "BDCA-2" refers to a blood dendritic cell antigen. This term encompasses variants, homologs, orthologs, and paralogs. For example, an antibody specific for human BDCA-2 may, in some cases, cross-react with a BDCA-2 protein from another species, such as monkey. In other embodiments, an antibody specific for human BDCA-2 may be entirely specific for human BDCA-2 protein and not cross-react with proteins from other species or other types, or may cross-react with BDCA-2 proteins from some other species but not all other species.

[0011] As used herein, "amino acid" includes naturally occurring amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Natural amino acids are amino acids encoded by the genetic code. Amino acid analogs refer to amino acids having the same basic chemical structure as naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0012] In the present invention, "complementarity determining region" is also referred to as CDR herein. The complementary determining region disclosed herein refers to the antigen binding site within the heavy chain or light chain variable region of an antibody, that is, the amino acid residues in the antibody that cause antigen binding. The complementary determining regions of the light chain and the heavy chain together constitute the antigen binding site of the antibody. Generally speaking, the light chain or heavy chain of an antibody usually contains three cluster-determining complementary regions. When the complementary determining region herein refers to the complementary determining region of the heavy chain, it is abbreviated as HCDR, and the three different complementary determining regions are referred to by numbers, such as HCDR1, HCDR2, and HCDR3. When the complementary determining region herein refers to the complementary determining region of the light chain, it is abbreviated as LCDR, and the three different complementary determining regions are referred to by numbers, such as LCDR1, LCDR2, and LCDR3.

[0013] As used herein, an "antigen-binding portion," "antigen-binding domain," "antigen-binding region," or "antigen-binding site" is the portion of an antibody comprising amino acid residues that interact with an antigen and contribute to the antibody's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of, or at least one of, its CDR domains.

[0014] In the present invention, the "variable region" refers to the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes greatly.

[0015] In the present invention, "heavy chain" refers to the two longer, relatively larger, identical heavy chains (H chains) in an antibody; "light chain" refers to the two shorter, relatively smaller, identical light chains (L chains) in an antibody.

[0016] In the present invention, "similarity" refers to the proportion of identical and substitutable amino acids in the amino acid sequences of homologous proteins.

[0017] As used herein, "monoclonal antibody" or "mAb" refers to an antibody product composed of a single molecule. A monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. As used herein, "murinized antibody" refers to an antibody secreted by murine hybrid fusion cells obtained by fusing B cells from immunized mice with myeloma cells.

[0018] The term "EC 50"Also called half-maximal effect concentration, it refers to the antibody concentration that causes 50% of the maximum effect.

[0019] The term "EC 50 Also called half inhibitory concentration, IC 50 The value can be used to measure the ability of a drug to induce apoptosis, that is, the stronger the induction ability, the lower the value. Of course, it can also reversely indicate the degree of tolerance of a certain cell to a drug.

[0020] In one aspect, the present invention provides a method for preparing an anti-BDCA-2 antibody, comprising the following steps:

[0021] (1) Fusion of mouse B cells and myeloma cells to generate hybridoma cells;

[0022] (2) Hybridoma cell culture supernatant and antigen protein were tested by ELISA to obtain positive clones;

[0023] (3) Positive clones are subcloned and screened to obtain monoclonal antibodies;

[0024] (4) Encoding antibody nucleotide sequence and constructing expression vector;

[0025] (5) Transfecting host cells and obtaining anti-BDCA-2 antibodies through expression in the host cells.

[0026] In another aspect, the present invention provides an anti-BDCA-2 antibody, comprising a light chain and a heavy chain, wherein the light chain comprises a light chain complementary determining region, and the heavy chain comprises a heavy chain complementary determining region;

[0027] The antibody light chain complementary determining regions include LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 25, GAT, and SEQ ID NO: 26; the antibody heavy chain complementary determining regions include HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29;

[0028] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, LAS and SEQ ID NO: 31; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34;

[0029] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 35, WAS and SEQ ID NO: 36; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39;

[0030] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 40, RAS and SEQ ID NO: 41; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44;

[0031] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 45, SAS and SEQ ID NO: 46; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49;

[0032] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 50, AAS and SEQ ID NO: 51; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54;

[0033] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 55, WAS and SEQ ID NO: 56; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59;

[0034] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, YSS and SEQ ID NO: 61; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64;

[0035] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SAS and SEQ ID NO: 66; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69;

[0036] or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, RAS and SEQ ID NO: 71; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74;

[0037] Or the antibody light chain complementary determining region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 75, GTS and SEQ ID NO: 76; the antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79.

[0038] Specifically, the light chain variable region of the antibody includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

[0039] Specifically, the heavy chain variable region of the antibody includes SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

[0040] Specifically, the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2;

[0041] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region amino acid sequence of SEQ ID NO: 4;

[0042] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 5, and a heavy chain variable region amino acid sequence of SEQ ID NO: 6;

[0043] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region amino acid sequence of SEQ ID NO: 8;

[0044] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region amino acid sequence of SEQ ID NO: 10;

[0045] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region amino acid sequence of SEQ ID NO: 12;

[0046] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 13, and a heavy chain variable region amino acid sequence of SEQ ID NO: 14;

[0047] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region amino acid sequence of SEQ ID NO: 16;

[0048] or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 17, and a heavy chain variable region amino acid sequence of SEQ ID NO: 18;

[0049] Or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 19, and the heavy chain variable region amino acid sequence is SEQ ID NO: 20.

[0050] Or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 21, and the heavy chain variable region amino acid sequence is SEQ ID NO: 22.

[0051] Specifically, the light chain of the antibody is formed by splicing a light chain variable region and a light chain constant region; the heavy chain is formed by splicing a heavy chain variable region and a heavy chain constant region.

[0052] More specifically, the light chain variable region includes a kappa constant region.

[0053] Preferably, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 80.

[0054] More specifically, the heavy chain variable region includes an IgG1, IgG2, IgG3, IgG4 or IgG1-YTE heavy chain constant region.

[0055] Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81.

[0056] Specifically, the light chain amino acid sequence of the antibody is SEQ ID NO: 82, and the heavy chain amino acid sequence is SEQ ID NO: 83;

[0057] or the antibody has a light chain amino acid sequence of SEQ ID NO: 84 and a heavy chain amino acid sequence of SEQ ID NO: 85;

[0058] or the antibody has a light chain amino acid sequence of SEQ ID NO: 86 and a heavy chain amino acid sequence of SEQ ID NO: 87;

[0059] or the antibody has a light chain amino acid sequence of SEQ ID NO: 88 and a heavy chain amino acid sequence of SEQ ID NO: 89;

[0060] or the antibody has a light chain amino acid sequence of SEQ ID NO: 90 and a heavy chain amino acid sequence of SEQ ID NO: 91;

[0061] or the antibody has a light chain amino acid sequence of SEQ ID NO: 92 and a heavy chain amino acid sequence of SEQ ID NO: 93;

[0062] or the antibody has a light chain amino acid sequence of SEQ ID NO: 94 and a heavy chain amino acid sequence of SEQ ID NO: 95;

[0063] or the antibody has a light chain amino acid sequence of SEQ ID NO: 96 and a heavy chain amino acid sequence of SEQ ID NO: 97;

[0064] or the antibody has a light chain amino acid sequence of SEQ ID NO: 98 and a heavy chain amino acid sequence of SEQ ID NO: 99;

[0065] Or the light chain amino acid sequence of the antibody is SEQ ID NO: 100, and the heavy chain amino acid sequence is SEQ ID NO: 101.

[0066] Or the light chain amino acid sequence of the antibody is SEQ ID NO: 102, and the heavy chain amino acid sequence is SEQ ID NO: 103.

[0067] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned antibody.

[0068] In another aspect, the present invention provides an expression vector encoding the above nucleic acid.

[0069] In another aspect, the present invention provides a host cell, comprising the above-mentioned expression vector.

[0070] In another aspect, the present invention provides use of the above-mentioned antibody in preparing a drug.

[0071] Specifically, the drug achieves its effects by binding to BDCA-2 through anti-BDCA-2 antibodies, promoting BDCA-2 endocytosis, inhibiting excessive activation of pDCs, and inhibiting the production of IFN-α by pDCs.

[0072] In yet another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned anti-BDCA-2 antibody, the aforementioned nucleic acid, the aforementioned expression vector and / or the aforementioned host cell.

[0073] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0074] The technical effects achieved by the present invention are:

[0075] (1) The anti-BDCA-2 antibodies and related drugs provided by the present invention can be used to inhibit the production of IFN-α by pDCs and prevent excessive activation of pDCs.

[0076] (2) The anti-BDCA-2 antibodies produced by the present invention can effectively inhibit the activation of pDC cells and the production of IFN-α, and have great potential in resisting viral infections and preventing certain autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG1 shows the binding of anti-BDCA-2 antibodies to human BDCA-2.

[0078] FIG2 shows the binding of anti-BDCA-2 antibodies to monkey BDCA-2.

[0079] FIG3 shows the binding of anti-BDCA-2 antibodies to RPMI8226-hBDCA2 cells.

[0080] FIG4 shows the endocytosis detection of anti-BDCA-2 antibody.

[0081] FIG5 shows that anti-BDCA-2 antibody inhibits IFN-α secretion from PBMC (Donor1) stimulated by TLR-ligand.

[0082] FIG6 shows that anti-BDCA-2 antibodies inhibit TLR-ligand-stimulated IFN-α secretion from PBMC (Donor2).

[0083] FIG7 shows that anti-BDCA-2 antibodies inhibit TLR-ligand-stimulated IFN-α secretion from PBMC (Donor2). DETAILED DESCRIPTION

[0084] The present invention is described below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention so that the technical solutions of the present invention are more easily understood and grasped. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0085] The instruments used in the present invention are as follows:

[0086] 1. Microplate reader: Instrument manufacturer: Thermo Fisher; Instrument model: Multiskan FC.

[0087] 2. Flow cytometer: Instrument manufacturer: Coulter Beckman; Instrument model: CytoFLEX S.

[0088] 3. High-content imaging: Manufacturer: PerkinElmer; Instrument model: Operetta CLS.

[0089] Basic Experimental Example 1: Mouse Immunization and Hybridoma Fusion

[0090] Prepare an emulsion of equal volumes of PBS and complete Freund's adjuvant containing 50 μg of antigen (200 μL / mouse): Connect syringes containing the antigen and adjuvant, respectively, using a three-way stopcock. Push the syringe back and forth, allowing the contents to flow from one side to the other, for 10 minutes until a stable emulsion is obtained. The emulsion is injected subcutaneously into the back of 6-8 week-old Balb / c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and purchased and raised by Suzhou Jima Gene Co., Ltd.) using a syringe, for immunization of a total of five mice. Two weeks later, an equal volume of PBS and incomplete Freund's adjuvant (200 μL) containing 25 μg of antigen is injected subcutaneously into the back of the mice. Two weeks later, mice are boosted, and blood is collected 7 days after the booster for titer measurement. When antibody titers are sufficiently high (≥1 / 50,000), a booster is administered subcutaneously via the tail vein three days before fusion with 25 μg of antigen in PBS. Three days after the booster immunization, two mice were dissected and their spleens were collected. The spleen lymphocytes were isolated and hybridoma fused with SP2 / 0 cells.

[0091] Example 1 Screening of molecules binding to BDCA-2 protein

[0092] Recombinant human BDCA-2 (purchased from Suzhou Jinan Protein Technology Co., Ltd., NP: PLKAA-1, LOT: 20220816) and recombinant monkey BDCA-2 (purchased from Suzhou Jinan Protein Technology Co., Ltd., NP: PLKBB, LOT: 20211220) were used as antigens and coated on a high-affinity ELISA plate overnight at 4°C. The coating amount per well was 50 ng and blocked with 2% BSA at 37°C for 2 hours. 100 μL of hybridoma supernatant was incubated with recombinant human BDCA-2 and recombinant monkey BDCA-2 for 1 hour, respectively, and then the plate was washed three times with washing buffer. Freshly diluted enzyme-labeled antibody was added, incubated at 37°C for 30 minutes, and washed. 100 μL of TMB substrate solution was added and color was developed for 5-10 minutes, after which 50 μL of 2M sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was read using a microplate reader. If the read value was greater than 2.1 times the OD value of the negative control, it was a clone that positively bound to recombinant human BDCA-2 or recombinant monkey BDCA-2.

[0093] Fresh hybridoma cell pellets were collected from positive binding clones, with a cell count of 5 × 10 6 After quick freezing in liquid nitrogen, the cells were transported on dry ice to Suzhou Genewise Biotechnology Co., Ltd. for monoclonal antibody sequencing. The molecules with complete variable region sequences were obtained as shown in Table 1 below.

[0094] Table 1 Different clone sequences

[0095] Example 2 Construction and expression of anti-BDCA-2 antibodies

[0096] The light chain was formed by directly splicing the light chain variable region and light chain constant region (SEQ ID NO:80) of the sequenced monoclonal antibody, and the heavy chain was formed by directly splicing the heavy chain variable region and heavy chain constant region (SEQ ID NO:81) of the sequenced monoclonal antibody. The light and heavy chains of the antibody have the amino acid sequences shown in SEQ ID NO:82 and SEQ ID NO:83.

[0097] or the amino acid sequences shown in SEQ ID NO: 84 and SEQ ID NO: 85;

[0098] or the amino acid sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87;

[0099] or the amino acid sequences shown in SEQ ID NO: 88 and SEQ ID NO: 89;

[0100] or the amino acid sequences shown in SEQ ID NO: 90 and SEQ ID NO: 91;

[0101] or the amino acid sequences shown in SEQ ID NO: 92 and SEQ ID NO: 93;

[0102] or the amino acid sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95;

[0103] or the amino acid sequences shown in SEQ ID NO: 96 and SEQ ID NO: 97;

[0104] or the amino acid sequences shown in SEQ ID NO: 98 and SEQ ID NO: 99;

[0105] or the amino acid sequences shown in SEQ ID NO: 100 and SEQ ID NO: 101;

[0106] or the amino acid sequences shown in SEQ ID NO: 102 and SEQ ID NO: 103;

[0107] The amino acid sequences of the light and heavy chains were codon-optimized for human host cells, and the genes were conventionally synthesized (the amino acid sequences of the light and heavy chain constant regions are shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively). The genes were cloned into the pTT5 vector (ampicillin-resistant) using 5' EcoRI and 3' HindIII. Clones were selected for sequencing, and the cells with the correct sequence were selected for seed preservation and expanded for plasmid extraction. Following the same gene synthesis and vector construction methods as described above, a plasmid capable of expressing a control antibody was obtained, wherein the heavy chain of the control antibody is shown in SEQ ID NO: 109; the light chain is shown in SEQ ID NO: 110.

[0108] Table 2 Control antibody sequences

[0109] The extracted plasmid was transfected into cells and the protein was isolated and purified as follows:

[0110] 1. HEK-293F cells were cultured in Free Style™ 293 Expression Medium (Gibco, Cat#: 12338-018). Cell density was measured. If the viability was greater than 95%, cells were collected by centrifugation and the cell density was adjusted to 5×10 6 cells / mL.

[0111] 2. Add 10% MEM (10% transfection system) to a 50 mL centrifuge tube, add each plasmid, mix thoroughly, filter, and let stand for 5 minutes. Add PEI to the DNA suspension (DNA:PEI ratio 1:3, 1.5 μg DNA per mL of cell culture medium), mix gently (gently invert 2 to 3 times), and let stand for 15-20 minutes. Then, gently add the complex to the aliquoted cells, gently shaking the shaker. Incubate the transfected HEK-293F cells in a 37°C, 5% CO2 incubator at 120 rpm. After 10-12 days of culture, collect the supernatant.

[0112] 3. Anti-BDCA-2 antibodies were enriched and purified using a pre-equilibrated protein-G affinity column (GE, Cat#: 17040501) and eluted with elution buffer (20 mM citric acid, pH 3.0-pH 3.5). Anti-BDCA-2 antibodies were then diluted in PBS at pH 7.0, and the antibody concentration was determined by NanoDrop.

[0113] Example 3: Cross-binding of anti-BDCA-2 antibodies to human and monkey BDCA-2

[0114] To determine whether anti-BDCA-2 antibodies cross-bind to human and monkey BDCA-2, ELISA was performed using recombinantly expressed human and monkey BDCA-2 proteins. The specific steps are as follows:

[0115] Recombinant human BDCA-2 and recombinant monkey BDCA-2 were used as antigens and coated on a high-affinity enzyme-labeled plate overnight at 4°C with a coating amount of 50 ng per well. The plate was blocked with 2% BSA at 37°C for 2 hours. 100 μL of each antibody diluted in series was incubated with recombinant human BDCA-2 and recombinant monkey BDCA-2 for 1 hour, and then the plate was washed 3 times with washing solution. Freshly diluted enzyme-labeled antibody was added, incubated at 37°C for 30 minutes, and washed. 100 μL of TMB substrate solution was added, and after color development for 5-10 minutes, 50 μL of 2M sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was read using an enzyme reader, and the binding curve was plotted as shown in Figures 1 and 2. Calculate the EC 50 EC values ​​for each antibody 50 The values ​​are shown in Table 3.

[0116] Table 3 Anti-BDCA-2 antibodies bind to human and monkey BDCA-2 proteins

[0117] Example 4 Anti-BDCA-2 Antibody Binds to Human BDCA-2

[0118] To determine whether the anti-BDCA-2 antibody binds to human BDCA-2 expressed on the cell surface, FACS cell binding assay was performed using RPMI8226 cells stably overexpressing human BDCA-2 (purchased from Fuheng Biotechnology, cat. no. FH0092).

[0119] 100 μL of culture medium was added to 5 RPMI8226 cells were plated in 96-well V-bottom plates and 50 μL of serially diluted anti-BDCA-2 antibodies of various concentrations were added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBST. 1000-fold diluted PE-goat anti-human IgG (purchased from BioLegend, Cat. No. 366904) was added. After incubation at 4°C for 1 hour, the 96-well plates were washed three times with PBS. Cell fluorescence was detected using a flow cytometer, and binding curves were plotted as shown in Figure 3. EC values ​​were calculated. 50 EC values ​​for each antibody 50 The values ​​are shown in Table 4.

[0120] Table 4 Anti-BDCA-2 antibodies bind to human BDCA-2

[0121] Example 5 Detection of Endocytosis of Anti-BDCA-2 Antibodies

[0122] Endocytosis was detected by FACS and high-content imaging using RPMI8226 cells stably overexpressing human BDCA-2. TM pHrodo TM The test antibody was labeled with iFL Green Human IgG Labeling Reagent. RPMI8226-hBDCA2 cells were then incubated with the labeled antibody at 37°C for 16 hours. The cells were harvested and washed. Images were taken using high-content imaging, as shown in Figure 4. Fluorescence intensity was measured using flow cytometry, as shown in Table 5.

[0123] Table 5 Endocytosis detection of anti-BDCA-2 antibodies

[0124] Antibodies 12D5B12, 1C11F3, 4H6B5, 9H6C7, 20B2C1, 4A2C12, 14E6A12, 4B3F5, 5H6F9, and 11G9B3 showed obvious red fluorescence. Compared with the control group, the above anti-BDCA-2 antibodies showed significant endocytosis.

[0125] Example 6 Anti-BDCA-2 Antibody Inhibits TLR-ligand-Stimulated PBMC IFN-α Secretion

[0126] In this example, human peripheral blood lymphocytes (PBMC, purchased from Shanghai Saili Biotechnology Co., Ltd., product number XFB-HP050B) were used as the research subjects, and ODN2216 was used to stimulate PBMC to secrete IFN-α. While stimulating with ODN2216, a 5-fold diluted anti-BDCA-2 antibody was added to the paved cells. The cells added with ODN2216 and different concentrations of the antibody to be tested were placed in a 37°C cell culture incubator and incubated overnight (18 hours). The cell supernatant was taken and the IFN-α content in the supernatant was detected using a commercial Human IFN-α ELISA Kit (purchased from absin, product number abs51025-96T). An inhibition curve was drawn, and the results are shown in Figures 5-7. Calculate IC 50 The IC50 values ​​of each antibody are shown in Table 6.

[0127] Table 6 Anti-BDCA-2 Antibody IC 50 value

[0128] The above results indicate that the anti-BDCA-2 antibody of the present invention can effectively inhibit the activation of pDC cells and the production of IFN-α.

[0129] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A method for preparing an anti-BDCA-2 antibody, characterized in that: The preparation method comprises the following steps: (1) Mouse B cells and myeloma cells are fused to obtain hybridoma cells; (2) The hybridoma cell culture supernatant and antigen protein were tested by ELISA to obtain positive clones; (3) Positive clones are subcloned and screened to obtain monoclonal antibodies; (4) Encoding antibody nucleotide sequence and constructing expression vector; (5) Transfecting host cells and obtaining anti-BDCA-2 antibodies through expression in the host cells.

2. An anti-BDCA-2 antibody, characterized in that The antibody comprises a light chain and a heavy chain, wherein the light chain comprises a light chain complementary determining region, and the heavy chain comprises a heavy chain complementary determining region; the light chain complementary determining region of the antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 25, GAT and SEQ ID NO: 26; the heavy chain complementary determining region of the antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, LAS and SEQ ID NO: 31; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 35, WAS and SEQ ID NO: 36; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 40, RAS and SEQ ID NO: 41; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 45, SAS and SEQ ID NO: 46; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 50, AAS and SEQ ID NO: 51; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 55, WAS and SEQ ID NO: 56; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 60, YSS and SEQ ID NO: 61; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 65, SAS and SEQ ID NO: 66; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69; or the antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, RAS and SEQ ID NO: 71; the antibody heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74; Or the antibody light chain complementary determining region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 75, GTS and SEQ ID NO: 76; the antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO:

79.

3. The antibody according to claim 1, characterized in that The light chain variable region of the antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO:

21.

4. The antibody according to claim 1, characterized in that The heavy chain variable region of the antibody comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:

22.

5. The antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 2; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 3, and the heavy chain variable region amino acid sequence is SEQ ID NO: 4; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 5, and the heavy chain variable region amino acid sequence is SEQ ID NO: 6; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 7, and the heavy chain variable region amino acid sequence is SEQ ID NO: 8; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 9, and the heavy chain variable region amino acid sequence is SEQ ID NO: 10; or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 11, and a heavy chain variable region amino acid sequence of SEQ ID NO: 12; or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 13, and a heavy chain variable region amino acid sequence of SEQ ID NO: 14; or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 15, and a heavy chain variable region amino acid sequence of SEQ ID NO: 16; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 17, and the heavy chain variable region amino acid sequence is SEQ ID NO: 18; or the antibody has a light chain variable region amino acid sequence of SEQ ID NO: 19, and a heavy chain variable region amino acid sequence of SEQ ID NO: 20; Or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 21, and the heavy chain variable region amino acid sequence is SEQ ID NO:

22.

6. The antibody according to claim 1, characterized in that The light chain of the antibody is formed by splicing a light chain variable region and a light chain constant region; the heavy chain is formed by splicing a heavy chain variable region and a heavy chain constant region.

7. The antibody according to claim 6, characterized in that The light chain variable region includes a kappa constant region; the heavy chain constant region includes an IgG1, IgG2, IgG3, IgG4 or IgG1-YTE heavy chain constant region.

8. A nucleic acid, characterized in that The nucleic acid encodes the antibody according to any one of claims 1-7.

9. An expression vector, characterized in that The expression vector comprises the nucleic acid according to claim 8.

10. A host cell, characterized in that The host cell comprises the expression vector according to claim 9.

11. Use of the antibody according to any one of claims 2 to 7 in the preparation of a medicament.

12. The use according to claim 11, characterized in that: The drug achieves its effect by combining the anti-BDCA-2 antibody with BDCA-2, promoting BDCA-2 internalization, inhibiting pDC overactivation or inhibiting pDC from generating IFN-α.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-BDCA-2 antibody according to any one of claims 2 to 7, the nucleic acid according to claim 8, the expression vector according to claim 9 and / or the host cell according to claim 10.

14. The pharmaceutical composition according to claim 13, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

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