Anti-BDCA-2 antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-BDCA-2 antibody as well as a preparation method and application thereof, and belongs to the field of antibodies. The invention provides a preparation method of an anti-BDCA-2 antibody, which comprises the following steps: carrying out ELISA detection screening on a hybridoma cell culture supernatant and an antigen protein to obtain a positive clone, and carrying out subcloning on the positive clone to obtain the anti-BDCA-2 antibody. The anti-BDCA-2 antibody comprises a heavy chain and a light chain, and 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 NO: 1-22. The anti-BDCA-2 antibody and related drugs provided by the invention can be used for inhibiting generation of IFN-alpha by pDC and preventing excessive activation of pDC. The anti-BDCA-2 antibody produced by the invention can effectively inhibit the activation of pDC cells and the generation of IFN-alpha, and has great potential in resisting virus infection and preventing some autoimmune diseases.

Description

Technical Field

[0001] 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

[0002] Blood Dendritic Cell Antigen 2 (BDCA-2) is a type II C-type lectin that plays an important role in the human immune system. [1] BDCA-2 is highly specifically expressed in plasmacytoid dendritic cells (pDCs). pDCs are a special type of cell in the immune system. They are widely present in lymphoid and non-lymphoid tissues. They are powerful interferon-producing cells and make important contributions to innate and adaptive immunity, especially in the early stages of viral infection and autoimmune diseases.

[0003] The expression of BDCA-2 on the surface of pDCs can regulate the function of pDCs. As an endocytic receptor, one of the main functions of BDCA-2 is endocytosis. Through endocytosis, BDCA-2 is able to capture and internalize antigens, further promoting antigen presentation, which is essential for immune response. Therefore, BDCA-2 helps the human immune system fight pathogens by participating in the process of antigen extraction and processing. However, unlike other endocytic receptors, BDCA-2 signaling may also inhibit the production of interferon by pDCs. Whenever BDCA-2 is activated, it blocks the production of interferon by inhibiting the signaling of Toll-like receptors 7 and 9 (TLR7 and TLR9), which are key receptors that drive interferon production in viral infections. In general, the main functions of BDCA-2 include: participating in the endocytosis and processing of antigens, and making fine adjustments to the disease response by inhibiting the response of pDCs to interferon production.

[0004] During viral infection, BDCA-2 inhibits the production of IFN-α by pDCs by inhibiting the TLR9 signaling pathway [2], preventing it from responding to viral infections. In addition, BDCA-2 also plays a key role in autoimmune diseases. In autoimmune diseases such as systemic lupus erythematosus (SLE), elevated levels of IFN-α can sometimes be detected, which may be due to overactivation of pDCs. As a role in inhibiting IFN-α production, BDCA-2 may help prevent excessive autoreactions and thus avoid triggering autoimmune diseases. In general, BDCA-2 plays a key role in regulating pDC responses, resisting viral infections, and preventing certain autoimmune diseases. Current research continues to explore the functions and clinical application prospects of BDCA-2.

[0005] [1]Wilson NR, Bover L, Konopleva M, Han L, et al. CD303(BDCA-2)-apotentialnovel target for therapy in hematologic malignancies[J]. LeukLymphoma.2022Jan; 63(1):19-30.

[0006] [2]Li Summary of the invention

[0007] In view of the above shortcomings, the present invention provides an anti-BDCA-2 antibody and a preparation method and application thereof. The antibody and related drugs can be used to inhibit the production of IFN-alpha by pDC and prevent the over-activation of pDC. The anti-BDCA-2 antibody produced by the present invention can effectively inhibit the activation of pDC cells and the production of IFN-alpha, and has great potential in resisting viral infection 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 pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics, protein nucleic acid chemistry, and hybridization described herein are well known and frequently used in the art.

[0010] In the present invention, "BDCA-2" refers to a blood dendritic cell antigen. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human BDCA-2 may cross-react with a BDCA-2 protein of another species, such as a monkey, in some cases. In other embodiments, an antibody specific for a human BDCA-2 protein may be completely specific for a human BDCA-2 protein without cross-reacting with other species or other types of proteins, or may cross-react with BDCA-2 proteins of some other species but not all other species.

[0011] In the present invention, "amino acid" includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to natural 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 single-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 in 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 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 three different complementary determining regions are referred to by numbers, such as LCDR1, LCDR2, and LCDR3.

[0013] In the present invention, "antigen binding portion", "antigen binding domain", "antigen binding region" or "antigen binding site" is the part of an antibody that contains 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 part of or at least one of its CDR domains.

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

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

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

[0017] In the present invention, "monoclonal antibody" or "monoantibody" refers to an antibody molecule composed of a single molecule. Monoclonal antibodies exhibit a single binding specificity and affinity for a specific epitope. In the present invention, murinized antibodies refer to antibodies 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, the preparation method comprising the following steps:

[0021] (1) Mouse B cells and myeloma cells are fused to obtain hybridoma cells;

[0022] (2) The 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, the antibody comprising a light chain and a heavy chain, the light chain comprising a light chain complementary determining region, and the heavy chain comprising a heavy chain complementary determining region;

[0027] The antibody light chain complementary determining region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 25, GAT and SEQ ID NO: 26; the antibody heavy chain complementary determining region comprises 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 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;

[0042] 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;

[0043] 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;

[0044] 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;

[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 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;

[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 yet another aspect, the present invention provides an expression vector encoding the above-mentioned nucleic acid.

[0069] In yet 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 antibody in preparing a drug.

[0071] Specifically, the drug achieves its effect by combining the anti-BDCA-2 antibody with BDCA-2, promoting BDCA-2 endocytosis, inhibiting pDC overactivation, and inhibiting pDC from producing IFN-α.

[0072] In another aspect, the present invention provides a pharmaceutical composition, comprising the above anti-BDCA-2 antibody, the above nucleic acid, the above expression vector and / or the above 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 the over-activation of pDCs.

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

[0077] Figure 1 Binding of anti-BDCA-2 antibody to human BDCA-2.

[0078] Figure 2Binding of anti-BDCA-2 antibody to monkey BDCA-2.

[0079] Figure 3 Binding of anti-BDCA-2 antibody to RPMI8226-hBDCA2 cells.

[0080] Figure 4 For internalization detection of anti-BDCA-2 antibody.

[0081] Figure 5 The anti-BDCA-2 antibody inhibits the IFN-α secretion of PBMC (Donor1) stimulated by TLR-ligand.

[0082] Figure 6 The anti-BDCA-2 antibody inhibits the IFN-α secretion of PBMC (Donor2) stimulated by TLR-ligand.

[0083] Figure 7 The anti-BDCA-2 antibody inhibits the IFN-α secretion of PBMC (Donor2) stimulated by TLR-ligand. DETAILED DESCRIPTION

[0084] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all 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 Freund's complete adjuvant containing 50 μg of antigen (200 μL / mouse): Connect the syringes containing the antigen and adjuvant respectively with a three-way valve. Push the syringe back and forth so that its contents flow from one side of the syringe to the other side, and push for 10 minutes until a stable emulsion is obtained. Use a syringe to inject the emulsified emulsion subcutaneously at multiple points on the back of 6-8 week old Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and commissioned by Suzhou Jima Gene Co., Ltd. to purchase and raise). A total of 5 mice were immunized. Two weeks later, an equal volume of emulsion prepared with PBS and incomplete Freund's adjuvant containing 25 μg of antigen (200 μL) was injected subcutaneously at multiple points again. Two weeks later, the mice were boosted with immunization, and blood was collected 7 days after immunization to detect the titer. When the antibody titer is high enough (≥1 / 50000), 3 days before fusion, the emulsion was injected with a mixture containing

[0091] 25 μg of antigen in PBS solution was injected subcutaneously into the tail vein for booster immunization. Three days after booster immunization, two mice were dissected and spleens were collected. Splenic lymphocytes of mice were isolated and fused with SP2 / 0 cells for hybridoma.

[0092] Example 1 Screening of molecules binding to BDCA-2 protein

[0093] 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 with high-affinity ELISA plates 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 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 to develop color for 5-10 minutes, and then 50 μL of 2M sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was read using an ELISA 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.

[0094] Fresh hybridoma cell pellets were collected from positive binding clones, with a cell number of 5×10 6 After quick freezing with liquid nitrogen, the monoclonal antibody was transported to Suzhou Genewise Biotechnology Co., Ltd. on dry ice for monoclonal antibody sequencing. The molecules with complete variable region sequences as shown in Table 1 below were obtained.

[0095] Table 1 Different clone sequences

[0096]

[0097]

[0098] Example 2 Construction and expression of anti-BDCA-2 antibodies

[0099] The light chain variable region and the light chain constant region (SEQ ID NO: 80) of the monoclonal antibody obtained by sequencing are directly spliced ​​to form a light chain, and the heavy chain variable region and the heavy chain constant region (SEQ ID NO: 81) of the monoclonal antibody obtained by sequencing are directly spliced ​​to form a heavy chain. The light chain and heavy chain of the antibody are the amino acid sequences shown in SEQ ID NO: 82 and SEQ ID NO: 83;

[0100] or the amino acid sequences shown in SEQ ID NO: 84 and SEQ ID NO: 85;

[0101] or the amino acid sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87;

[0102] or the amino acid sequences shown in SEQ ID NO: 88 and SEQ ID NO: 89;

[0103] or the amino acid sequences shown in SEQ ID NO: 90 and SEQ ID NO: 91;

[0104] or the amino acid sequences shown in SEQ ID NO: 92 and SEQ ID NO: 93;

[0105] or the amino acid sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95;

[0106] or the amino acid sequences shown in SEQ ID NO: 96 and SEQ ID NO: 97;

[0107] or the amino acid sequences shown in SEQ ID NO: 98 and SEQ ID NO: 99;

[0108] or the amino acid sequences shown in SEQ ID NO: 100 and SEQ ID NO: 101;

[0109] or the amino acid sequences shown in SEQ ID NO: 102 and SEQ ID NO: 103;

[0110] For the amino acid sequence of the light chain and the heavy chain, codon optimization was performed according to human host cells, and the gene was conventionally synthesized (the amino acid sequences of the light chain constant region and the heavy chain constant region are shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively), and the gene was cloned into the pTT5 vector (ampicillin resistance) through 5'EcoRI and 3'HindIII. The clones were selected for sequencing, and the bacteria with correct sequencing were selected for seed preservation and expanded culture of the bacteria, and the expanded bacteria were used for plasmid extraction. According to the same gene synthesis and vector construction method as mentioned 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.

[0111] Table 2 Control antibody sequences

[0112]

[0113]

[0114] The extracted plasmid was transfected into cells and the protein was isolated and purified as follows:

[0115] 1. HEK-293F cells were cultured in Free StyleTM 293 expression medium (Gibco, Cat#: 12338-018), and the cell density was measured. If the viability was greater than 95%, the cells were collected by centrifugation and the cell density was adjusted to 5×10 using a cell counter. 6 cells / mL.

[0116] 2. Take a 50mL centrifuge tube, add 10% MEM of the transfection system, add each plasmid, mix well, filter, let stand for 5 minutes, add PEI to the DNA suspension (the ratio of DNA to PEI is 1:3, and the amount of DNA added to each milliliter of cell culture medium is 1.5μg), gently mix (gently invert 2 to 3 times to mix), and let stand for 15 to 20 minutes. Then gently add the complex to the subpackaged cells, and gently shake the shaker while adding; the transfected HEK-293F cells are placed in a 37°C, 5% CO 2 The cells were cultured in an incubator at 120 rpm. After 10-12 days of culture, the supernatant was collected.

[0117] 3. Anti-BDCA-2 antibody was enriched and purified by 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 antibody was then placed in PBS at pH 7.0 and the antibody concentration was detected by NanoDrop.

[0118] Example 3 Cross-binding of anti-BDCA-2 antibodies to human and monkey BDCA-2

[0119] To determine whether the anti-BDCA-2 antibody cross-binds with human and monkey BDCA-2, ELISA detection was performed using recombinantly expressed human and monkey BDCA-2 proteins. The specific steps are as follows:

[0120] Recombinant human BDCA-2 and recombinant monkey BDCA-2 were used as antigens and coated on high-affinity ELISA plates overnight at 4°C. The coating amount for each well was 50 ng and blocked with 2% BSA at 37°C for 2 hours. 100 μL of each antibody diluted in multiples 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 with an ELISA reader, and the binding curve was plotted, as shown in the figure. Figure 1 , Figure 2 Calculate EC 50 The EC values ​​of each antibody 50 The values ​​are shown in Table 2.

[0121] Table 2 Anti-BDCA-2 antibodies bind to human and monkey BDCA-2 proteins

[0122]

[0123]

[0124] Example 4 Anti-BDCA-2 Antibodies Bind to Human BDCA-2

[0125] 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, catalog number FH0092).

[0126] 100 μL of culture medium was added to 5 RPMI8226 cells were plated on a 96-well V-bottom plate, and 50 μL of each anti-BDCA-2 antibody of each concentration was added. After incubation at 4°C for 1 hour, the 96-well plate was washed 3 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 plate was washed 3 times with PBS, and the cell fluorescence was detected by flow cytometry to draw a binding curve, as shown in Figure 2. Figure 3 Calculate EC 50 The EC values ​​of each antibody 50 The values ​​are shown in Table 3.

[0127] Table 3. Anti-BDCA-2 antibodies bind to human BDCA-2

[0128]

[0129]

[0130] Example 5 Detection of endocytosis of anti-BDCA-2 antibodies

[0131] Endocytosis was detected by FACS and high-content imaging using RPMI8226 cells stably overexpressing human BDCA-2. TM pHrodo TM The antibody to be tested was labeled with iFL Green Human IgG Labeling Reagent, and then the RPMI8226-hBDCA2 cells and the labeled antibody were incubated at 37°C for 16 hours, and the cells were collected and washed. High-content imaging was used to take pictures, and the results were as follows Figure 4 The fluorescence intensity was detected by flow cytometry, and the results are shown in Table 4.

[0132] Table 4. Flow cytometry detection of anti-BDCA-2 antibodies

[0133] Antibody MFI 12D5B12 2619 1C11F3 2405 4H6B5 1863 9H6C7 699 20B2C1 721 4A2C12 2352 14E6A12 1785 4B3F5 2645 5H6F9 3414 6G5H6 397 11G9B3 2735 PC 2272 iRed 224 Blank 73.7 Isotype 256

[0134] 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 had obvious endocytosis.

[0135] Example 6 Anti-BDCA-2 Antibody Inhibits TLR-ligand Stimulated PBMC IFN-α Secretion

[0136] In this example, human peripheral blood lymphocytes (PBMC, purchased from Shanghai Saili Biotechnology Co., Ltd., product number XFB-HP050B) were used as the research object, 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 the following table. Figure 5-7 Calculate IC 50The IC50 values ​​of each antibody are shown in Table 5.

[0137] Table 5. Anti-BDCA-2 Antibody IC 50 value

[0138]

[0139] The above results show that the anti-BDCA-2 antibody of the present invention can effectively inhibit the activation of pDC cells and the production of IFN-α.

[0140] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for preparing an anti-BDCA-2 antibody, It is 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, It is 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, It is 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, It is 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, It is 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 amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 3, and the amino acid sequence of the heavy chain variable region 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 light chain variable region amino acid sequence of the antibody is SEQ ID NO: 11, and the heavy chain variable region amino acid sequence is SEQ ID NO: 12; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 13, and the heavy chain variable region amino acid sequence is SEQ ID NO: 14; or the light chain variable region amino acid sequence of the antibody is SEQ ID NO: 15, and the heavy chain variable region amino acid sequence is 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 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; 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, It is 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, It is 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, It is characterized in that The nucleic acid encodes the antibody according to any one of claims 1-7.

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

10. A host cell, It is 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, It is 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, It is 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, It is characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

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