High Capacity Magnetic Antibody Capture Beads and pH Sensitive Dyes: Application in Therapeutic Antibody Drug Conjugates (ADCs)

With recent approval of two therapeutic Antibody Drug Conjugates (ADCs), Adcetris and Parjeta, the field is attracting lot of activity with about 30 ADCs at various stages of development. Concept of ADCs is simple - an antibody targeted to tumor specific receptor is conjugated to a toxic small molecule and then used to deliver toxin only to the tumor hence improving the efficacy of the small molecule and reducing the side effect of the small molecule. A key requirement for the ADCs is the ability of the antibody to bind to the receptor on the tumor cells and internalization of antibody. However, not all antibodies that bind even with high affinity to the receptors are internalized rather it appears that epitope on the receptor plays an important role in antibody internalization. Hence, methods that will allow selection of antibodies based on internalization assay early in the hybridoma screening stage, where number of samples are large, antibody concentration is low and sample volume are limiting, will significantly improve the chances for selecting right lead antibodies.

To enable easy and rapid selection for internalizing antibodies early in the hybridoma screening phase we developed a new method by combining an ‘on-bead’ antibody conjugation method with a new pH sensitive fluorescent dye. ‘On-Bead’ antibody–small molecule conjugation method uses high capacity magnetic Protein G and Protein A beads and allows labeling of antibodies using both the amine chemistry as well as the thiol chemistry. The method can be used with 1-96 samples in-parallel and with small sample volumes (100µl-1ml). In addition we developed a pH sensitive dye that is fluorescent only in the acidic pH below pH 6.0 found in endosomes and lysosomes. We used the ‘on-bead’ antibody conjugation method to label pH sensitive dyes to the Trastuzumab that internalizes on binding to its receptors and present a case study for a plate based screening assay for internalizing antibody.