Development of a Novel Optimization and Validation Method Based on DOE for the ADCC Potency of Anti-PD-1/PD-L1 Monoclonal Antibodies Using Reporter Genes / 中国药学杂志

ABSTRACT

OBJECTIVE: To develop a novel optimization and validation method based on design of experiment(DOE) for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of anti-programmed cell death 1 and anti-programmed cell death-ligand 1 (PD-1/PD-L1) monoclonal antibodies using reporter genes. METHODS: Jurkat-hFcγRIIIa-NFAT transgenic cell line was used as effector cells, 293FT-PD-1 cell line and CHO-PD-L1 cell line were used as target cells, respectively. The ADCC potency for anti-PD-1/PD-L1 monoclonal antibodies was detected with Luciferase detection system (BrightGloTM Luciferase Assay system), then the method was optimized and validated based on DOE. RESULTS: The anti-PD-1/PD-L1 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation: y=(A-D)/+D. The method was optimized and the testing parameters were determined as follows: the working concentration of anti-PD-1 monoclonal antibody was 10 000 ng•mL-1 to 4.833 ng•mL-1 and that of anti-PD-L1 was 2 000 ng•mL-1 to 0.488 ng•mL-1, the ratio of effector cells and target cells for anti-PD-1/PD-L1 monoclonal antibodies were 6:1 and 3:1, and the induction time for both of these antibodies was 20 h. The method possessed good specificity. The recovery rate test samples in the four different dilution groups were determined for 3 times, and the results showed that the relative potencies of anti-PD-1 monoclonal antibody were (51.74±2.22)%, (77.12±3.14)%, (118.71±2.83)% and (156.20±12.99)%, and the recoveries of which were (103.49±4.44)%, (102.83±4.19)%, (94.96±2.26)% and (104.14±8.66)%, respectively. While as for anti-PD-L1 monoclonal antibody, the relative potencies were (54.32±4.75)%, (75.24±4.25)%, (127.40±2.43)%, (156.82±3.27)% and the recoveries were (108.64±9.51)%, (100.33±5.67)%, (101.92±1.94)% and (104.55±2.18)%, respectively. The RSDs of the above results were all less than 10%. CONCLUSION: A novel optimization and validation method based on DOE for detecting ADCC potency of anti-PD-1/PD-L1 mAb is successfully developed. This detecting method based on reporter gene shows high specificity, good reproducibility and high accuracy, and might be used in the evaluation of ADCC potency of anti-PD-1/PD-L1 mAb.