Monoclonal antibodies targeting human plasmacytoid dendritic cells and uses thereof

Abstract

The application uses a BDCA-2 extracellular domain (ECD) antigen to immunize a fully humanized mouse, and through a hybridoma technology, a positive clone is screened, and through gene sequencing and recombinant expression purification, a specific monoclonal antibody targeting human plasmacytoid dendritic cells is obtained. The antibody has high binding capacity for human BDCA-2, and can be used for the development of therapeutic or prophylactic drugs or diagnostic reagents for inflammatory diseases or autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides a monoclonal antibody targeting human plasma cell-like dendritic cells and its application. Background Technology

[0002] BDCA-2, short for blood dendritic cell antigen 2, also known as CLEC4C or CD303, is a type II transmembrane glycoprotein belonging to the calcium-dependent lectin family. It is mainly expressed on plasmacytoid dendritic cells (pDCs), a subclass of dendritic cells that plays a crucial role in antiviral immunity.

[0003] The structure of BDCA-2 includes an extracellular Ca2+-dependent carbohydrate recognition domain (CRD), a transmembrane region, and a relatively short intracellular region. The CRD can bind to various glycosyl groups, such as galactose-terminal residues, while the intracellular region binds to FcεRIγ. This structure enables BDCA-2 to play a role in the immune response.

[0004] In the immune response, BDCA-2 regulates the immune response by inhibiting the production of type I interferon. When BDCA-2 is activated, it triggers a series of signaling pathways, including activation of tyrosine kinase (Syk), aggregation of B cell connective protein (SLP65), and activity of phosphatidylinositol-specific phospholipase (PLCγ2). These processes collectively inhibit the activation of NFKB (nuclear factor κB), thereby reducing the production of type I interferon.

[0005] Because type I interferon plays a central role in the pathophysiology of autoimmune diseases such as lupus, BDCA-2 is considered a promising therapeutic target. Currently, several targeted drugs against BDCA-2 have entered clinical trials, such as Biogen's BIIB059 monoclonal antibody (Litifilimab), used to treat lupus and other diseases.

[0006] Chinese patent document CN105829534A discloses an anti-human BDCA-2 antibody that binds to human BDCA-2 and regulates the function of plasmacytoid dendritic cells via human BDCA-2. The chimeric antibodies BDC3-12A2, BDC3-12F5, and BDC13-32E3 in this document exhibit EC50 binding activities against the extracellular region of human BDCA-2 of 59, 57, and 96 ng / mL, respectively. The fully human antibodies exhibit EC50 binding activities against the extracellular region of human BDCA-2 of 80, 88, and 203 ng / mL, respectively. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing a monoclonal antibody with a higher binding capacity to human BDCA-2.

[0008] The specific technical solution of this invention is as follows:

[0009] Monoclonal antibodies or antigen-binding fragments thereof targeting human plasmacytoid dendritic cells, including heavy chain variable regions and light chain variable regions:

[0010] (1) The heavy chain variable region of the antibody has a complementarity-determining region HCDR1, a complementarity-determining region HCDR2, and a complementarity-determining region HCDR3, as shown by amino acid residues 26-36 of SEQ ID NO:1; the light chain variable region of the antibody has a complementarity-determining region LCDR1, a complementarity-determining region LCDR2, and a complementarity-determining region LCDR3, as shown by amino acid residues 24-34 of SEQ ID NO:2;

[0011] Alternatively, (2) the heavy chain variable region of the antibody has a complementarity-determining region HCDR1 (26-37 amino acid residues), a complementarity-determining region HCDR2 (52-69 amino acid residues), and a complementarity-determining region HCDR3 (100-108 amino acid residues) as shown in SEQ ID NO:3; the light chain variable region of the antibody has a complementarity-determining region LCDR1 (24-40 amino acid residues), a complementarity-determining region LCDR2 (56-62 amino acid residues), and a complementarity-determining region LCDR3 (96-104 amino acid residues) as shown in SEQ ID NO:4.

[0012] Further, (1) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; or, (2) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0013] Furthermore, (1) the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:6;

[0014] Alternatively, (2) the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:7, and the light chain variable region amino acid sequence is shown in SEQ ID NO:8.

[0015] The monoclonal antibody described in this invention can be any isotype. It can be, for example, an IgM or IgG antibody, such as IgG1 or IgG2.

[0016] The antibody described in this invention can be:

[0017] (1)Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting an intact antibody with papain to produce a complete light chain and a portion of a heavy chain;

[0018] (2) Fab' is an antibody molecule fragment obtained by treating an intact antibody with pepsin and then reducing it to produce a portion of the intact light chain and heavy chain; each antibody molecule yields two Fab' fragments;

[0019] (3)(Fab')2 is an antibody fragment obtained by treating an intact antibody with pepsin but without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds;

[0020] (4)Fv contains gene segments that are expressed as two-stranded light chain variable regions and heavy chain variable regions;

[0021] (5) Single-chain antibodies (e.g., scFv) are genetically engineered molecules containing light chain variable regions and heavy chain variable regions and linked together by suitable polypeptide linkers to form genetically fused single-chain molecules.

[0022] (6) The dimer of a single-chain antibody (scFv2) is defined as a dimer of scFv (also known as a “micro antibody”);

[0023] (7) VH single domain antibody, an antibody fragment composed of heavy chain variable regions.

[0024] In the specific technical solution of the present invention, the antibody is any one of the following antibodies: antibody 3C12H11 with an amino acid sequence as shown in SEQ ID NO: 5, and antibody 10D2C4 with an amino acid sequence as shown in SEQ ID NO: 6.

[0025] The antibodies or fragments or derivatives thereof according to the present invention are preferably used in medicine, particularly for the treatment, diagnosis, prognosis and monitoring of diseases.

[0026] Another object of the present invention is to provide a polynucleotide encoding the antibody described herein.

[0027] Another object of the present invention is to provide a pharmaceutical composition comprising the antibody or polynucleotide described herein.

[0028] Another object of the present invention is to provide the use of the antibodies or polynucleotides described herein in the preparation of therapeutic or preventive remedies or diagnostic reagents for inflammatory diseases or autoimmune diseases. The inflammatory diseases or autoimmune diseases are selected from systemic sclerosis, fibrosis, pemphigus vulgaris, systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis, polymyositis and dermatomyositis, psoriasis, rheumatoid arthritis, Graves' disease, morphine scleroderma, inflammatory bowel disease, morphine scleroderma, type I diabetes, Sjögren's disease, or Hashimoto's disease.

[0029] The present invention has the following advantages:

[0030] (1) In this invention, fully humanized mice were immunized with the extracellular domain (ECD) antigen of BDCA-2. Positive clones were screened by hybridoma technology, and after gene sequencing and recombinant expression purification, specific monoclonal antibodies targeting human plasma cell-like dendritic cells were obtained with higher binding capacity to human BDCA-2: antibodies 3C12H11 and 10D2C4 can significantly bind to the extracellular domain of human BDCA-2, with EC50 values ​​of 13.33 and 18.17 ng / mL, respectively, which are significantly better than existing technologies.

[0031] (2) The BDCA-2 antibodies 3C12H11 and 10D2C4 of this invention can significantly bind to membrane-expressed human BDCA-2 and do not bind to cells expressing cynomolgus monkey BDCA-2, while the isotype control antibody has no binding ability to cells expressing human and cynomolgus monkey BDCA-2. This indicates that the 3C12H11 and 10D2C4 of this invention have high specificity and binding ability to membrane-expressed human BDCA-2 and human plasmacytoid dendritic cells. Attached Figure Description

[0032] Figure 1 The binding ability of the antibody described in this invention to human BDCA-2 (ELISA).

[0033] Figure 2 The binding ability (FACS) of the antibody described in this invention to human BDCA-2 overexpressing cells.

[0034] Figure 3 The binding ability (FACS) of the antibody described in this invention to BDCA-2 overexpressing cells of cynomolgus monkeys. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Example 1: Preparation of the BDCA-2 monoclonal antibody of the present invention

[0037] Fully humanized mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., catalog number: T058481) were immunized with the BDCA-2 extracellular domain (ECD) antigen (Human CLEC4C / BDCA2 Protein, His Tag, Asn45-Ile 213, ACROBiosystems, CLC-H5245). Following a standard immunization protocol, serum titers generated from immunization of fully humanized mice reached 1E. 4 ~1E 5 (Using three times the OD450 value of blank serum as the baseline), mice with high titers were selected for final immunization. Three days after final immunization, spleen cells were fused with SP2 / 0 mouse myeloma cells. After electroporation, fused cells were screened by microscopic observation to prepare hybridomas. Positive clones were screened using the BDCA-2 extracellular domain (ECD) and eukaryotic cells 293T overexpressing the human BDCA-2 membrane receptor obtained by lentiviral packaging (see Example 3 for the method). Positive clones with binding ability were selected from those with a background value three times higher than the baseline. After gene sequencing and recombinant expression purification, recombinant antibodies 3C12H11 (heavy chain amino acid sequence as shown in SEQ ID NO:5, light chain amino acid sequence as shown in SEQ ID NO:6) and 10D2C4 (heavy chain amino acid sequence as shown in SEQ ID NO:7, light chain amino acid sequence as shown in SEQ ID NO:8) were obtained, and their amino acid sequence information is shown in Table 1.

[0038] Table 1. CDR amino acid sequences of monoclonal antibodies (Kabat)

[0039]

[0040] Example 2: Binding ability of the BDCA-2 monoclonal antibody of the present invention to recombinant human BDCA-2 extracellular domain protein

[0041] Recombinant human BDCA-2ECD protein was coated onto 384-well microplates and incubated overnight at 4°C. The supernatant was discarded, and after washing with PBST, PBS containing 2% skim milk powder was added, and the plates were blocked at 37°C for 1 hour. After washing with PBST, the test antibody was added and transferred to 384-well plates, and incubated at 37°C for 1 hour. Positive antibody Litifilimab (CAS: 2407378-48-5, AbMole, catalog number M55040) and irrelevant isotype control antibody (Anti-SARS-CoV-2 Nucleocapsid Antibody, Human IgG1 (AS95) (MALS verified), ACROBiosystems, NUN-CH14) were added. After discarding the test antibody and washing, enzyme-labeled secondary antibody was added and incubated at 37°C in the dark for 1 hour. The supernatant was discarded, and after washing, enzyme-labeled substrate was added for color development for 10 minutes. Within 30 minutes of adding the stop solution, the absorbance at 450 nm was measured using a microplate reader. The data were analyzed and the EC50 was calculated. Results are as follows: Figure 1 As shown in the figure. The results showed that recombinant antibodies 3C12H11 and 10D2C4 could significantly bind to human BDCA-2, with EC50 values ​​of 13.33 and 18.17 ng / mL, respectively, which were superior to the positive control antibody's EC50 value of 41.76 ng / mL. The isotype control antibody (as a negative control) showed no binding ability to human BDCA-2 ECD.

[0042] Example 3: Binding ability of the BDCA-2 monoclonal antibody described in this invention to BDCA-2 on the surface of human and cynomolgus monkey cells.

[0043] Using lentiviral packaging to construct stable transfected cells, plasmids were first prepared. Double enzyme digestion and vectors were selected, along with PCR technology. The BDCA-2 fragment was mixed with the vector and PCR was performed, followed by plating and extraction to obtain a high concentration of plasmids carrying the BDCA-2 gene, ready for cell transfection. 16 hours before transfection, 293T cells were cultured at 5–8 × 10⁻⁶ cells / year. 6Cells were seeded in 10cm culture dishes to achieve 80-90% confluence on the day of transfection and cultured at 37°C in a 5% CO2 incubator. On the day of transfection, 4.05μg of pSPAX2-K, 2.7μg of pMD2.GK, and 5.4μg of plasmid containing the BDCA-2 gene were pipetted into a 1.5mL EP tube containing 250µL Opti-MEM medium and mixed thoroughly. The amount of PEI used was 1.5 times the total amount of plasmid. Therefore, 18μg of PEI was dissolved in an EP tube containing medium and mixed thoroughly. The diluted PEI was added to the diluted plasmid DNA solution. The solution was thoroughly mixed and incubated at room temperature. After incubation, the old medium in the 293T cell culture dish was discarded, serum-free medium was added, and then the PEI-plasmid complex was slowly and evenly added to the culture dish. After 2 hours, FBS was added to the medium to a concentration of 10% (1mL). After 6 hours, replace the culture medium in the petri dish with 10 mL of fresh complete culture medium and continue culturing in a 37°C, 5% CO2 incubator. Collect the viral supernatant after 48 or 72 hours. The collected viral fluid was centrifuged at 2000g for 10 min at 4℃. The supernatant was filtered through a 0.45µm filter membrane. This filtrate was the viral supernatant required for infection. This supernatant was used to infect 293T cells. A plasmid carrying the BDCA-2 gene and the puemycin resistance gene was constructed and transfected into 293T cells. Puemycin was used to screen for 293T cells stably expressing human BDCA-2 or cynomolgus monkey BDCA-2. Cells were transferred at a rate of 300,000 per well to 96-well V-plates. After washing the cells, appropriate gradient concentrations of BDCA-2 antibody and isotype control antibody were added, and the cells were incubated at 4℃ for 30 min. After washing, a 1:200 dilution of APC-conjugated F(ab')2 fragment goat anti-human IgG Fcγ fragment specific secondary antibody was added, and the cells were incubated at 4℃ for 30 min. After two washes, flow cytometry was used for analysis. Data were analyzed using flow cytometry software, and EC50 was calculated. Results are as follows: Figure 2 and Figure 3 As shown in the figure. The results show that the BDCA-2 antibody described in this invention can significantly bind to the membrane-expressed human BDCA-2 receptor, with a binding ability superior to the positive control antibody, and it does not bind to cells expressing cynomolgus monkey BDCA-2, while the isotype control antibody has no binding ability to cells expressing either human or cynomolgus monkey BDCA-2. These results indicate that the recombinant antibodies 3C12H11 and 10D2C4 have high specificity and binding ability to membrane-expressed human BDCA-2 and human plasmacytoid dendritic cells.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof targeting human blood dendritic cell antigen 2, comprising a heavy chain variable region and a light chain variable region, characterized in that: (1) The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; Alternatively, (2) the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: (1) The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:6; Alternatively, (2) the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:7, and the light chain variable region amino acid sequence is shown in SEQ ID NO:

8.

3. A polynucleotide, characterized in that... Its encoding is the antibody according to claim 1 or 2.

4. A pharmaceutical composition, characterized in that... Includes the antibody according to claim 1 or 2.

Images