A Simple Plate Based Assay Using pH Sensor Dye to Screen for Internalizing Antibody

Receptor mediated internalization is a key mechanism of action (MOA) for antibody drug conjugates (ADCs). However, current methods of studying antibody internalization have several limitations including: 1) A multistep process not suitable for screening; 2) Low signal-to-background ratios; 3) Not suitable for kinetic measurements. We have developed a method that mitigates problems associated with traditional internalization assays. The method includes: 1) A water soluble, bright and photo-stable pH sensor dye (pHAb) to study internalization; 2) An optimized method for conjugating pHAb dyes to antibodies directly from the cell media; 3) A 96-well, plate-based assay with high signal-tobackground ratios for real time measurement of internalization and screening internalization.