Monoclonal antibody targeting human plasma cell-like dendritic cells and application thereof

Abstract

A fully humanized mouse is immunized by using a BDCA-2 extracellular domain (ECD) antigen, positive clones are screened out through a hybridoma technology, and the specific monoclonal antibody targeting human plasma cell-like dendritic cells is obtained through gene sequencing and recombinant expression purification. The antibody disclosed by the invention has high binding capacity to human BDCA-2, and can be used for developing drugs or diagnostic reagents for treating or preventing inflammatory diseases or autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and provides a monoclonal antibody targeting human plasmacytoid dendritic cells and application thereof. Background Art

[0002] BDCA-2, full name 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 (pDC), a subtype of dendritic cells that plays a key role in antiviral immunity.

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

[0004] In immune response, BDCA-2 regulates immune response by inhibiting the production of type I interferon. When BDCA-2 is activated, a series of signal transduction pathways are triggered, including the activation of tyrosine kinase (Syk), the aggregation of B cell junction protein (SLP65) and the activity of phosphatidylinositol-specific phospholipase (PLCγ2), which together inhibit the activation of NFKB (nuclear factor κB), thereby reducing the production of type I interferon.

[0005] Since type I interferon plays a central role in the pathophysiology of autoimmune diseases such as lupus erythematosus, BDCA-2 is considered a potential therapeutic target. Currently, some targeted drugs targeting BDCA-2 have entered the clinical trial stage, such as BIIB059 monoclonal antibody (Litifilimab) developed by Biogen, which is used to treat diseases such as lupus erythematosus.

[0006] Chinese patent document CN105829534A discloses an anti-human BDCA-2 antibody, which binds to human BDCA-2 and regulates the function of plasmacytoid dendritic cells through human BDCA-2. The chimeric antibodies BDC3-12A2, BDC3-12F5 and BDC13-32E3 in the document have binding activity EC50s of 59, 57 and 96 ng / mL for the extracellular region of human BDCA-2, respectively. The binding activity EC50s of fully human antibodies for the extracellular region of human BDCA-2 are 80, 88 and 203 ng / mL, respectively. Summary of the invention

[0007] The present invention aims at the deficiencies of the prior art and provides a monoclonal antibody with higher binding ability to human BDCA-2.

[0008] The specific technical solutions of the present invention are as follows:

[0009] A monoclonal antibody or an antigen-binding fragment thereof targeting human plasmacytoid dendritic cells, comprising a heavy chain variable region and a light chain variable region:

[0010] (1) The heavy chain variable region of the antibody comprises a complementary determining region HCDR1 as shown by amino acid residues 26-36, a complementary determining region HCDR2 as shown by amino acid residues 51-66, and a complementary determining region HCDR3 as shown by amino acid residues 99-109 of SEQ ID NO:1; the light chain variable region of the antibody comprises a complementary determining region LCDR1 as shown by amino acid residues 24-34, a complementary determining region LCDR2 as shown by amino acid residues 50-54, and a complementary determining region LCDR3 as shown by amino acid residues 88-95 of SEQ ID NO:2;

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

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

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

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

[0015] The monoclonal antibody of the present invention may be of any isotype, such as IgM or IgG antibody, such as IgG1 or IgG2.

[0016] The antibody of the present invention may be:

[0017] (1) Fab, a fragment comprising 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 one heavy chain;

[0018] (2) Fab', an antibody molecule fragment that can be obtained by treating an intact antibody with pepsin followed by reduction to produce an intact light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule;

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

[0020] (4) Fv, a gene fragment containing the light chain variable region and the heavy chain variable region expressed as two chains;

[0021] (5) Single-chain antibodies (e.g., scFv), genetically engineered molecules containing a light chain variable region and a heavy chain variable region connected by a suitable polypeptide linker into a genetically fused single-chain molecule;

[0022] (6) dimers of single-chain antibodies (scFv2), defined as dimers of scFv (also known as “minibodies”);

[0023] (7) VH single-domain antibody, an antibody fragment consisting of the heavy chain variable region.

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

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

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

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

[0028] Another object of the present invention is to provide the use of the antibody or polynucleotide of the present invention in the preparation of a therapeutic or preventive drug or diagnostic reagent for an inflammatory disease or autoimmune disease. The inflammatory disease or autoimmune disease is selected from systemic sclerosis, fibrosis, pemphigus vulgaris, systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis, polymyositis and dermatomyositis, psoriasis, rheumatoid arthritis, Graves' disease, morphea, inflammatory bowel disease, morphea, type I diabetes, Sjogren's disease or Hashimoto's disease.

[0029] The present invention has the following advantages:

[0030] (1) The present invention uses BDCA-2 extracellular domain (ECD) antigen to immunize fully humanized mice, screens positive clones through hybridoma technology, and obtains specific monoclonal antibodies targeting human plasmacytoid dendritic cells through gene sequencing and recombinant expression purification. The antibodies 3C12H11 and 10D2C4 can significantly bind to the extracellular region of human BDCA-2, with EC50 of 13.33 and 18.17 ng / mL, respectively, which are significantly better than the existing technology.

[0031] (2) The BDCA-2 antibodies 3C12H11 and 10D2C4 of the present 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 antibodies have no binding ability to cells expressing human and cynomolgus monkey BDCA-2. This indicates that the 3C12H11 and 10D2C4 of the present invention have high specificity and binding ability to cell membrane-expressed human BDCA-2 and human plasmacytoid dendritic cells. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0034] Figure 3 The binding ability of the antibody of the present invention to cynomolgus monkey BDCA-2 overexpressing cells (FACS). DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to specific drawings and specific embodiments, but the protection scope 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 BDCA-2 extracellular domain (ECD) antigen (Human CLEC4C / BDCA2 Protein, His Tag, Asn45-Ile 213, ACROBiosystems, CLC-H5245). After the standard immunization and administration process, the serum titer produced by immunization of fully humanized mice can reach 1E 4 ~1E 5 (3 times of the blank serum OD450 value was used as the reference value), and mice with higher titers were selected for final immunization. Cell fusion of spleen cells and SP2 / 0 mouse myeloma cells was performed 3 days after the final immunization, and the fusion cells were screened according to microscopic observation after electroporation to prepare hybridomas. Positive clones were screened using the BDCA-2 extracellular domain (ECD) and the eukaryotic cell 293T overexpressing the human BDCA-2 membrane receptor obtained by the lentiviral packaging method (see Example 3 for the method), and positive clones with binding ability were selected from the reference value of three times higher than the background value. 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 the recombinant human BDCA-2 extracellular domain protein

[0041] Recombinant human BDCA-2 ECD protein was coated on a 384-well ELISA plate and placed in a 4°C refrigerator overnight. The supernatant was discarded, and PBS containing 2% skim milk powder was added after washing with PBST, and the plate was blocked at 37°C for 1 hour. After washing with PBST, the antibody to be tested was added and transferred to a 384-well plate, and the plate was incubated at 37°C for 1 hour in a constant temperature incubator. The positive antibody Litifilimab (CAS: 2407378-48-5, AbMole, catalog number M55040) and an irrelevant isotype control antibody (Anti-SARS-CoV-2 Nucleocapsid Antibody, Human IgG1 (AS95) (MALS verified), ACROBiosystems, NUN-CH14) were added to the plate. After discarding the antibody to be tested and washing, the enzyme-labeled secondary antibody was added and incubated at 37°C in the dark for 1 hour. The supernatant was discarded, and after washing, the enzyme-labeled substrate was added for color development for 10 minutes. Within 30 minutes after adding the stop solution, use an ELISA reader to measure the absorbance at 450 nm, analyze the data and calculate the EC50. Figure 1 As shown. The results showed that the 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 better than the positive control antibody EC50 value of 41.76 ng / mL. The isotype control antibody (as a negative control) had no binding ability to human BDCA-2 ECD.

[0042] Example 3 Binding ability of the BDCA-2 monoclonal antibody of the present invention to BDCA-2 on the surface of human and cynomolgus monkey cells

[0043] The lentiviral packaging and stable transfection technology was used to first prepare plasmids, select double enzyme digestion and vector, and PCR technology, mix BDCA-2 fragments with vectors for PCR, and then plate and extract to obtain high-concentration plasmids carrying BDCA-2 genes, ready for cell transfection. 293T cells were cultured at 5-8×10 6 The cells were inoculated into a 10 cm culture dish so that the cell confluence reached 80-90% on the day of transfection and placed in a 37°C, 5% CO 2Culture in an incubator. On the day of transfection, use a pipette to take 4.05μg of pSPAX2-K, 2.7μg of pMD2.GK and 5.4μg of BDCA-2 gene plasmid and dissolve them in a 1.5mL EP tube containing 250uL Opti-MEM medium and mix them. The amount of PEI used is 1.5 times the total amount of plasmid. Then dissolve 18μg of PEI in an EP tube containing culture medium and mix them. Add the diluted PEI to the diluted plasmid DNA solution. Mix the solution thoroughly and incubate at room temperature. After the incubation, discard the old culture medium in the 293T cell culture dish, supplement with serum-free culture medium, and then evenly and slowly add PEI-plasmid complex to the culture dish. After 2h, supplement the culture medium with FBS to a concentration of 10% (1mL). After 6h, replace the culture medium in the culture dish with 10mL of fresh complete culture medium and place it at 37℃ and 5% CO 2 Continue to culture in the incubator. Collect the virus supernatant after 48 or 72 hours. Centrifuge the collected virus solution at 4°C and 2000g for 10 minutes, take the supernatant and filter it with a 0.45um filter membrane. This filtrate is the virus supernatant required for infection. Use this supernatant to infect 293T cells, construct a plasmid carrying the BDCA-2 gene and the pueomycin resistance gene, transfer the plasmid into 293T cells, use puromycin to screen 293T cells that stably express human BDCA-2 or 293T cells that express cynomolgus monkey BDCA-2, transfer the cells to a 96-well V-type plate at 300,000 / well, wash the cells, add appropriate gradient concentrations of BDCA-2 antibody and isotype control antibody, and incubate at 4°C for 30 minutes; after washing, add APC-conjugated F(ab')2 fragment goat anti-human IgG Fcγ fragment specific secondary antibody at a dilution of 1:200, and incubate the cells at 4°C for 30 minutes; after washing twice, detect by flow cytometry. The data were analyzed using flow cytometry software and the EC50 was calculated. Figure 2 and Figure 3 As shown. The results show that the BDCA-2 antibody of the present invention can significantly bind to the human BDCA-2 receptor expressed on the membrane, and the binding ability is better than that of the positive control antibody, and does 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 result shows that the recombinant antibodies 3C12H11 and 10D2C4 have high specificity and binding ability to human BDCA-2 expressed on the cell membrane and human plasmacytoid dendritic cells.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof targeting human plasmacytoid dendritic cells, comprising a heavy chain variable region and a light chain variable region, characterized in that: (1) The heavy chain variable region of the antibody comprises a complementary determining region HCDR1 as shown by amino acid residues 26-36, a complementary determining region HCDR2 as shown by amino acid residues 51-66, and a complementary determining region HCDR3 as shown by amino acid residues 99-109 of SEQ ID NO:1; the light chain variable region of the antibody comprises a complementary determining region LCDR1 as shown by amino acid residues 24-34, a complementary determining region LCDR2 as shown by amino acid residues 50-54, and a complementary determining region LCDR3 as shown by amino acid residues 88-95 of SEQ ID NO:2; Alternatively, (2) the heavy chain variable region of the antibody has a complementarity determining region HCDR1 shown by amino acid residues 26-37, a complementarity determining region HCDR2 shown by amino acid residues 52-69, and a complementarity determining region HCDR3 shown by amino acid residues 100-108 of SEQ ID NO:3; the light chain variable region of the antibody has a complementarity determining region LCDR1 shown by amino acid residues 24-40, a complementarity determining region LCDR2 shown by amino acid residues 56-62, and a complementarity determining region LCDR3 shown by amino acid residues 96-104 of SEQ ID NO:

4.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, 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 heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID NO:3, and the light chain variable region amino acid sequence is as shown in SEQ ID NO:

4.

3. The monoclonal antibody or antigen-binding fragment thereof 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 as shown in SEQ ID NO:7, and the light chain variable region amino acid sequence is as shown in SEQ ID NO:

8.

4. A polynucleotide characterized in that It encodes the antibody according to any one of claims 1-3.

5. A pharmaceutical composition, characterized in that The method comprises the antibody according to any one of claims 1 to 3 or the polynucleotide according to claim 4.

6. Use of the antibody according to any one of claims 1 to 3 or the polynucleotide according to claim 4 in the preparation of a therapeutic or preventive drug or a diagnostic reagent for an inflammatory disease or an autoimmune disease.

7. The use according to claim 6, characterized in that The inflammatory disease or autoimmune disease is selected from systemic sclerosis, fibrosis, pemphigus vulgaris, systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis, polymyositis and dermatomyositis, psoriasis, rheumatoid arthritis, Graves' disease, morphea, inflammatory bowel disease, morphea, type I diabetes, Sjögren's disease, or Hashimoto's disease.

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