Method for determining the biological activity of anti-cd20 monoclonal antibody drug adcp
A monoclonal antibody and drug technology, applied in the field of biomedicine, can solve the problems of lack of effective assay methods for ADCP biological activity of antibody drugs, and achieve the effects of stable and reliable test results, good application prospects, and simple operation
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Embodiment 1
[0099] Example 1 Construction of effector cells Jurkat / NFAT / CD32a-FcεRIγ stably expressing CD32a-FcεRIγ and NFAT-Luc reporter genes
[0100] 1. Experimental materials
[0101] Raji cells and Jurkat cells were purchased from ATCC, and all cells were cultured in a medium containing 90% (v / v) RPMI1640 and 10% (v / v) fetal bovine serum (FBS); Bright-Glo TM Luciferase detection system (E2650) was purchased from Promega; RPMI1640, FBS, puromycin and G418 were purchased from Gibco; bovine serum albumin (BSA) was purchased from Sigma; rituximab was from Roche company.
[0102] 2. Plasmid construction
[0103] Construction of plasmid pGL4[NFAT / puro]: after the whole gene synthesis of three repeated NFAT binding site nucleic acid sequences, KPNI+XhoI double enzyme digestion was used to synthesize the nucleic acid sequence and vector for connection, and the constructed plasmid pGL4[NFAT / puro] The nucleotide sequence is shown in SEQ ID NO: 3;
[0104] Construction of plasmid pcDNA3.1(+...
Embodiment 2
[0109] Example 2 Optimization of the method for measuring the biological activity of anti-CD20 monoclonal antibody drug ADCP
[0110] This example examines the working concentration range and different dilution ratios of rituximab in the assay method, the influence of different target cell (Raji) densities, different effect-to-target ratios (E:T), and different induction times on the assay results , the following optimization experiments were carried out.
[0111] 1. Optimization of working concentration range and dilution ratio
[0112] Rituximab was pre-diluted to 10 μg / mL (the initial working concentration was 5000 ng / mL), and then diluted 10 times according to the dilution ratio of 1:2, 1:3, 1:4, and 1:5, and the experiment was carried out. According to the dose-effect curve fitted by the chemiluminescence value, the optimal working concentration range and the optimal dilution ratio of rituximab were selected.
[0113] The results show that under the dilution ratio of 1:...
Embodiment 3
[0117] Example 3 Establishment of a method for measuring the biological activity of anti-CD20 monoclonal antibody drug ADCP
[0118] 1. Experimental method
[0119] (1) Sample preparation
[0120] Anti-CD20 monoclonal antibody (rituximab) was diluted to 10 μg / mL with diluent, and then serially diluted 1:4 times, and 10 concentration gradients were set up, with 2 to 3 duplicate wells for each concentration, and 50 μL / well was added to In 96-well white plate;
[0121] (2) Preparation of target cell Raji suspension
[0122] Collect well-growing Raji cells, centrifuge to discard the supernatant, resuspend the cells with diluent and count them, and adjust the cell density to 4×10 5 / mL spare.
[0123] (3) Preparation of effector cell Jurkat / NFAT / CD32a-FcεRIγ suspension
[0124] Collect well-growing Jurkat / NFAT / CD32a-FcεRIγ cells, centrifuge to discard the supernatant, resuspend the cells with diluent and count them, and adjust the cell density to 3.2×10 6 / mL, spare;
[0125...
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