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On-Bead Antibody-Small Molecule Conjugation and pH Sensor Fluorescent Dye for Screening of Internalizing Antibodies for ADC Applications

Part # PS212


Nidhi Nath, Becky Godat, Hélène Benink, Cesear Corona, Mark McDougall, Poncho Meisenheimer, Marjeta Urh
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711. E-mail: nidhi.nath@promega.com

A key requirement for candidate antibodies suitable for Antibody Drug Conjugate (ADCs) application is their ability to get internalized inside the cells. However, for ADCs application the early screening of antibodies during monoclonal antibody development phase is based on ELISA that select clones based on the affinity which does not reflect internalization characteristic of the antibody. We have developed a method that allows rapid screening of large number of antibodies for internalization in the cell. The method is a combination of two novel approaches, first, is the ‘on-bead’ antibody-small molecule conjugation that uses high capacity magnetic Protein A and Protein G beads to capture antibody directly from the cell media and conjugate with reactive small molecule. Second, is the conjugation of antibody with a pH sensor dye that becomes fluorescent only when antibody is internalized and trafficked to endosomes and lysosomes. We demonstrate several benefits for on-bead conjugation that include compatibility with 96-well plates, ability to process small to medium sample volumes and capability to test several different chemistries and small molecules in parallel. We further show that when ‘on-bead’ conjugation is used to attach pH sensor dye to antibody it enables a simple plate based method to be used to screen a large library of antibodies for internalization properties.

Printed in USA.