A reporter gene assay for determining antibody-dependent cell-mediated phagocytosis activity of HER2-targeted antibody drug conjugate.
- Author:
Ying CHEN
1
;
Can WANG
2
;
Qin ZHAO
2
;
Mingren WANG
2
;
Tiantian LI
2
;
Shanshan DONG
2
;
Hong SHAO
2
;
Weidong XU
2
Author Information
- Publication Type:Journal Article
- Keywords: anti-HER2 antibody drug conjugate; antibody-dependent cellular phagocytosis; biological activity; design of experiment; reporter gene assay
- MeSH: Humans; Receptor, ErbB-2/immunology*; Phagocytosis/drug effects*; Immunoconjugates/immunology*; Genes, Reporter; Antibody-Dependent Cell Cytotoxicity; Jurkat Cells
- From: Chinese Journal of Biotechnology 2025;41(8):3122-3130
- CountryChina
- Language:Chinese
- Abstract: To develop a method for determining the antibody-dependent cell-mediated phagocytosis (ADCP) activity of human epidermal growth factor receptor 2 (HER2)-targeted antibody drug conjugate (ADC) based on the reporter gene assay, we established an ADCP activity assay with Jurkat/NFAT/FcγRIIa cells as the effector cells and BT474 as the target cells. Then, the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were optimized by the method of design of experiment (DOE). The method showed a significant dose-response relationship, which was complied with the four-parameter equation: y=(A-D)/[1+(x/C)B]+D. The durability ranges of the target cell density, the ratio of effector to target cells, the target cell adhesion time, the incubation time for drug administration, and the induction time after adding effector cells were (2.5-4.0)×105 cells/mL, 3-5, 1.0-2.0 h, 0 h, and 5.0-6.0 h, respectively. The results of the methodological validation showed that the linear equation was y=1.106 8x-0.011 6, r=0.969 2. The established method showed the relative accuracy ranging from -6.59% to 2.98% and the geometric coefficient of variation less than 11% in the intermediate precision test. Furthermore, the method was target-specific. The method was then applied to the determination of ADCP activity of HER2-targeted ADC, demonstrating the result of (103.5±5.7)%. We developed a reporter gene assay for determining the ADCP activity of HER2-targeted ADC and the assay demonstrated high accuracy and good reproducibility, which proposes a highly efficient and approache for evaluating ADCP effect of this HER2-targeted ADC, and also provides a referable technique for characterizing the Fc effector functions of ADCs with diverse targets.
