Monoclonal antibody targeting cynomolgus monkey BDCA-2 and application thereof

Abstract

The invention relates to a monoclonal antibody specifically targeting cynomolgus monkey BDCA-2, which has high binding capacity to cynomolgus monkey BDCA-2 cells, and also has high binding capacity to cynomolgus monkey BDCA-2 cells with high homology (gt; 90%) human BDCA-2 cells are weak in binding, and high-selectivity recognition of cynomolgus monkey DC can be achieved. The antibody can be applied to the development of a specific detection method and a kit of cynomolgus monkey pDC, such as high-sensitivity identification, identification, dyeing or other scientific research analysis of cynomolgus monkey DC.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and provides a monoclonal antibody targeting cynomolgus monkey BDCA-2 and an 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] The genes of crab-eating macaques are highly similar to those of humans, and their physiological characteristics are also very similar to those of humans. They can often be used as substitutes for humans in drug and vaccine trials to simulate human responses to drugs. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a monoclonal antibody targeting cynomolgus monkey BDCA-2 and an application thereof.

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

[0009] A monoclonal antibody or an antigen-binding fragment thereof targeting cynomolgus monkey BDCA-2, comprising a heavy chain variable region and a light chain variable region: the heavy chain variable region of the antibody has a complementary determining region HCDR1 as shown by amino acid residues 26-35 of SEQ ID NO:1, a complementary determining region HCDR2 as shown by amino acid residues 50-66, and a complementary determining region HCDR3 as shown by amino acid residues 99-107; the light chain variable region of the antibody has a complementary determining region LCDR1 as shown by amino acid residues 24-34 of SEQ ID NO:2, a complementary determining region LCDR2 as shown by amino acid residues 50-56, and a complementary determining region LCDR3 as shown by amino acid residues 90-97.

[0010] Furthermore, 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.

[0011] In a specific example of the present invention, the heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO:3, and the light chain amino acid sequence is shown in SEQ ID NO:4.

[0012] Another object of the present invention is to provide a polynucleotide encoding the antibody of the present invention.

[0013] Another object of the present invention is to provide use of the antibody or polynucleotide of the present invention in preparing a detection reagent for cynomolgus monkey plasmacytoid dendritic cells.

[0014] The present invention has the following advantages:

[0015] The antibody of the present invention has a high binding ability to cynomolgus monkey BDCA-2 cells, and weakly binds to human BDCA-2 cells with high homology (>90%), and can achieve highly selective recognition of cynomolgus monkey DC. The antibody can be applied to the development of specific detection methods and kits for cynomolgus monkey pDC, such as high-sensitivity recognition, identification, staining or other scientific research analysis of cynomolgus monkey DC. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The binding ability of the antibody of the present invention to human BDCA-2 (ELISA).

[0017] Figure 2 The binding ability of the antibody of the present invention to human BDCA-2 overexpressing cells (FACS).

[0018] Figure 3 The binding ability of the antibody of the present invention to cynomolgus monkey BDCA-2 overexpressing cells (FACS). DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1: Preparation of the BDCA-2 monoclonal antibody of the present invention

[0021] 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 splenocytes and SP2 / 0 mouse myeloma cells was performed 3 days after the final immunization to prepare hybridomas. Screening was performed 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. After gene sequencing and recombinant expression purification, the recombinant antibody 5B2A5 (heavy chain amino acid sequence is shown in SEQ ID NO: 3, and the light chain amino acid sequence is shown in SEQ ID NO: 4) was obtained.

[0022] Table 1 CDR amino acid sequences of monoclonal antibodies (Kabat)

[0023]

[0024]

[0025] Example 2 Binding ability of the BDCA-2 monoclonal antibody of the present invention to the recombinant human BDCA-2 extracellular domain protein

[0026] The recombinant human BDCA-2 extracellular domain (ECD) 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 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. 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 experiment, and incubated at 37°C in a constant temperature incubator for 1 hour. 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 antibody 5B2A5 could significantly bind to human BDCA-2, with an EC50 value of 71.64 ng / mL, close to the positive control antibody. However, the isotype control antibody (as a negative control) had no binding ability to human BDCA-2 ECD.

[0027] Example 4 Binding ability of the BDCA-2 monoclonal antibody of the present invention to BDCA-2 on the surface of human or cynomolgus monkey cells

[0028] 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 FBS to the culture medium 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, 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 showed that the BDCA-2 antibody 5B2A5 could significantly bind to the membrane-expressed cynomolgus monkey BDCA-2, and the binding ability was much higher than that of cells expressing human BDCA-2. The positive control antibody had little difference in binding ability to cells expressing cynomolgus monkey BDCA-2 and human BDCA-2, and the isotype control antibody had no binding ability to cells expressing human and cynomolgus monkey BDCA-2. This result shows that the recombinant antibody 5B2A5 has a high selectivity and binding ability to cynomolgus monkey BDCA-2 expressed on the cell membrane and cynomolgus monkey DC.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof targeting BDCA-2, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region of the antibody has a complementary determining region HCDR1 shown by amino acid residues 26-35, a complementary determining region HCDR2 shown by amino acid residues 50-66, and a complementary determining region HCDR3 shown by amino acid residues 99-107 of SEQ ID NO:1; the light chain variable region of the antibody has a complementary determining region LCDR1 shown by amino acid residues 24-34, a complementary determining region LCDR2 shown by amino acid residues 50-56, and a complementary determining region LCDR3 shown by amino acid residues 90-97 of SEQ ID NO:

2.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: 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.

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

4.

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

5. Use of the antibody according to any one of claims 1 to 3 or the polynucleotide according to claim 4 in preparing a detection reagent for cynomolgus monkey plasmacytoid dendritic cells.

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