Accurate determination of internalization for target binding antibody using papain digestion and flow cytometry

Internalization of monoclonal antibodies (MAbs) binding to the targeted cells has drawn great attention to both scientists and anti-cancer drug developmental professionals. Internalization of conjugated MAb is thought to be one of the major mechanisms for tumor cell destruction, and can be studied using several biochemical and microscopic approaches. Here we report a new method based on papain digestion followed by flow cytometry (FCM). This method can identify whether the binding MAb has internalized into the cell, with an additional advantage of accurately quantifying the internalized MAb without altering cell morphology after papain digestion. With this method, we studied the internalization degrees of 3A4 (a mouse anti-human CD45RA MAb) at different time points: 5.3% (15 min), 7.3% (30 min), 36.9% (60 min), 69.2% (120 min), and 72.6% (180 min). This methodology can facilitate our understanding of the efficiency of MAb internalization and allows us to evaluate the targeted killing capacity of the MAb. Our technique can serve as a reference model for future targeted drug development using MAbs. In summary, we established a simple and useful evaluation tool for MAb drug development and research.