Cell-Based Assay Development and Standardization for Antibody–Drug Conjugates

ADC-111 is an Antigen-1-targeting antibody–drug conjugate with approximately eight payloads conjugated to one monoclonal antibody. The primary mechanism of action of ADC-111 is payload-induced cytotoxicity after antigen–antibody binding-mediated internalization. A cell-based assay using T24 cells engineered with Antigen-1 expression was developed and phase-appropriately validated to measure the potency of ADC-111 drug substance and drug product. This specific and stability-indicating assay detected potency loss resulting from degradation in the complementarity-determining region of the antibody, and correlated closely with the results from the Antigen-1 binding enzyme-linked immunosorbent assay, demonstrating that the ADC-111 cell-based assay is antigen-binding dependent. The potency measured by the cell-based assay also showed good correlation with varying drug–antibody ratio values, but ELISA did not. Notably, the ready-to-use format of the cell-based assay had performance comparable to that of the continuous culture using the same procedure, allowing both formats to be used interchangeably. In addition, the same parental cell line T24 was engineered with other tumor antigens, and the corresponding cell-based assay also showed good specificity, demonstrating the proof-of-concept of using a single parental cell line for multiple ADCs to standardize and accelerate cell-based assay development.