Monoclonal antibodies targeting cynomolgus bdca-2 and uses thereof

Abstract

The present application relates to a kind of monoclonal antibody specifically targeting cynomolgus BDCA-2, the antibody has high binding ability to cynomolgus BDCA-2 cell, while for the weak binding of human BDCA-2 cell with high homology (>90%), high selective recognition of cynomolgus DC can be realized.This antibody can be applied to the development of specific detection method and kit of cynomolgus pDC, such as high sensitivity recognition, identification, staining or other scientific research analysis of cynomolgus DC.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and provides a monoclonal antibody targeting cynomolgus BDCA-2 and application thereof. BACKGROUND

[0002] BDCA-2, also known as CLEC4C or CD303, is a type II transmembrane glycoprotein belonging to the C-type lectin family. It is mainly expressed on plasmacytoid dendritic cells (pDC), a subset of dendritic cells that play 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 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 immune responses.

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

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

[0006] The genes of cynomolgus monkeys have high similarity to humans, and their physiological characteristics are also very similar to humans. In drug and vaccine trials, they are often used as stand-ins for humans to simulate human responses to drugs. SUMMARY

[0007] The present application provides a monoclonal antibody targeting cynomolgus BDCA-2 and its application to address the deficiencies of the prior art.

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

[0009] A monoclonal antibody or antigen-binding fragment thereof targeting cynomolgus BDCA-2, comprising a heavy chain variable region and a light chain variable region: the heavy chain variable region of the antibody has complementarity determining region HCDR1 shown by amino acid residues 26-35 of SEQ ID NO: 1, complementarity determining region HCDR2 shown by amino acid residues 50-66 of SEQ ID NO: 1, and complementarity determining region HCDR3 shown by amino acid residues 99-107 of SEQ ID NO: 1; the light chain variable region of the antibody has complementarity determining region LCDR1 shown by amino acid residues 24-34 of SEQ ID NO: 2, complementarity determining region LCDR2 shown by amino acid residues 50-56 of SEQ ID NO: 2, and complementarity determining region LCDR3 shown by amino acid residues 89-96 of SEQ ID NO: 2.

[0010] Further, the heavy chain variable region of the antibody has an amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 2.

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

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

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

[0014] The present application has the following advantages:

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

[0016] Figure 1 Binding capacity of the antibody of the present application to human BDCA-2 (ELISA).

[0017] Figure 2 Binding capacity of the antibody of the present application to human BDCA-2 overexpressing cells (FACS).

[0018] Figure 3 Binding capacity of the antibody of the present application to cynomolgus BDCA-2 overexpressing cells (FACS). DETAILED DESCRIPTION

[0019] The application will be described in detail below in conjunction with specific drawings and specific embodiments, but the scope of protection of the application is not limited to the following embodiments.

[0020] Example 1: Preparation of the BDCA-2 monoclonal antibody according to the application

[0021] A fully humanized mouse (Jiangsu Jiquan Yekang Biotechnology Co., Ltd., Catalog No: T058481) was immunized with a BDCA-2 extracellular domain (ECD) antigen (Human CLEC4C / BDCA2 Protein, His Tag, Asn45-Ile 213, ACROBiosystems, CLC-H5245). After a standard immunization process, the serum titer produced by the fully humanized mouse immunization reached 1E 4 ~1E 5 (3 times the OD450 value of the blank serum as the reference value), and a mouse with a higher titer was selected for final immunization. Three days after the final immunization, the spleen cells were fused with SP2 / 0 mouse myeloma cells to prepare hybridomas. The BDCA-2 extracellular domain (ECD) and the eukaryotic cells 293T overexpressing human BDCA-2 membrane receptors obtained by lentivirus packaging method were used for screening, and positive clones with binding ability were selected. After gene sequencing and recombinant expression and purification, the recombinant antibody 5B2A5 (the 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 the monoclonal antibody (Kabat)

[0023] .

[0024] Example 2: Binding ability of the BDCA-2 monoclonal antibody according to the application to the recombinant human BDCA-2 extracellular domain protein

[0025] Recombinant human BDCA-2 extracellular domain (ECD) was coated to 384-well enzyme-labeled plates and placed in a 4°C refrigerator overnight. After discarding the supernatant and washing with PBST, 2% skimmed milk powder-containing PBS was added and incubated at 37°C for 1 hour. After washing with PBST, the antibody to be tested was transferred to the 384-well plate, and the positive antibody Litifilimab (CAS: 2407378-48-5, AbMole, Catalog No. M55040) and irrelevant isotype control antibody (Anti-SARS-CoV-2 Nucleocapsid Antibody, Human IgG1 (AS95) (MALS verified), ACROBiosystems, NUN-CH14) were added to the experimental group, and incubated in a constant-temperature incubator at 37°C for 1 hour. After discarding the antibody to be tested and washing, enzyme-labeled secondary antibody was added and incubated at 37°C for 1 hour in the dark. After discarding the supernatant and washing, enzyme-labeled substrate was added for color development for 10 min. Within 30 min after adding the stop solution, the absorbance value at 450 nm was measured using an enzyme-labeled instrument, the data was analyzed, and the EC50 was calculated. The results are shown in Figure 1 The results show that the recombinant antibody 5B2A5 can significantly bind to human BDCA-2, with an EC50 value of 71.64 ng / mL, close to the positive control antibody. The isotype control antibody (as a negative control) has no binding ability to human BDCA-2 ECD.

[0026] Example 3 Binding ability of the BDCA-2 monoclonal antibody of the present application to human or cynomolgus monkey cell surface BDCA-2

[0027] The lentivirus packaging construction stable transfection strain technology was used. First, a plasmid was prepared, double enzyme digestion and vector were selected, and PCR technology was used to mix the BDCA-2 fragment with the vector, and plating and extraction were performed to obtain a high-concentration plasmid carrying the BDCA-2 gene, which was ready for cell transfection. 16 h before transfection, 293T cells were seeded at 5~8×10 6The cells were inoculated in 10 cm culture dishes at a cell amount so that the cell confluence reached 80-90% on the day of transfection, and were cultured in a 37°C, 5% CO2 incubator. On the day of transfection, 4.05 μg of pSPAX2-K, 2.7 μg of pMD2.G-K, and 5.4 μg of a plasmid containing the BDCA-2 gene were dissolved in a 1.5 mL EP tube containing 250 uL of Opti-MEM medium and mixed well. The amount of PEI used was 1.5 times the total amount of plasmid. Then 18 μg of PEI was dissolved in an EP tube containing the medium and mixed well. The diluted PEI was added to the diluted plasmid DNA solution. The solution was mixed well and incubated at room temperature. After incubation, the old medium in the 293T cell culture dish was discarded, and the medium was supplemented with serum-free medium, and then the PEI-plasmid complex was added to the culture dish slowly and evenly. After 2 h, FBS was added to the medium to a concentration of 10% (1 mL). After 6 h, the medium in the culture dish was replaced with 10 mL of fresh complete medium, and the culture was continued in a 37°C, 5% CO2 incubator. The virus supernatant was collected at 48 or 72 h. The collected virus liquid was centrifuged at 4°C, 2000 g for 10 min, and the supernatant was filtered with a 0.45 um filter membrane. The filtrate was the virus supernatant required for infection. The virus supernatant was used to infect 293T cells to construct a plasmid carrying the BDCA-2 gene and the puromycin resistance gene. The puromycin-resistant 293T cells stably expressing human BDCA-2 or expressing cynomolgus BDCA-2 were selected using puromycin. The cells were transferred to a 96-well V-shaped plate at a density of 300000 / well, and after washing the cells, appropriate gradient concentrations of BDCA-2 antibody and isotype control antibody were added, and the mixture was incubated at 4°C for 30 min. After washing, APC-conjugated F(ab') 2 fragment goat anti-human IgG Fcγ fragment specific secondary antibody was added at a dilution of 1:200, and the cells were incubated at 4°C for 30 min. After washing twice, the cells were detected by flow cytometry. The data were analyzed using flow cytometry software and the EC50 was calculated. The results are shown in Figure 2 and Figure 3 The results show that the BDCA-2 antibody 5B2A5 can significantly bind to membrane-expressed cynomolgus BDCA-2, and the binding ability is much higher than that of cells expressing human BDCA-2. The positive control antibody has little difference in binding ability to expressed cynomolgus BDCA-2 and human BDCA-2-expressing cells, and the isotype control antibody has no binding ability to human and cynomolgus BDCA-2-expressing cells. The results show that the recombinant antibody 5B2A5 has high selectivity and binding ability to cell membrane-expressed cynomolgus BDCA-2 and cynomolgus DC.

Claims

1. A monoclonal antibody targeting BDCA-2 or its antigen-binding fragment, 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 complementarity-determining region HCDR1 with the amino acid sequence GFTFSRNVMH, a complementarity-determining region HCDR2 with the amino acid sequence ATSSNGGRIYYADSVKG, and a complementarity-determining region HCDR3 with the amino acid sequence EGSGWHADK; the light chain variable region of the antibody has a complementarity-determining region LCDR1 with the amino acid sequence RASQSISSWLA, a complementarity-determining region LCDR2 with the amino acid sequence KASSLES, and a complementarity-determining region LCDR3 with the amino acid sequence QQYNSYWT.

2. The monoclonal antibody or its antigen-binding fragment 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 its antigen-binding fragment 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: Its code is the antibody according to any one of claims 1-3.

5. Use of the antibody according to any one of claims 1-3 or the polynucleotide according to claim 4 in the preparation of a detection reagent for cynomolgus monkey plasma cell-like dendritic cells.

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