DropArray™, a wall-less 96-well plate for uptake and immunofluorescence microscopy, confirms CD22 recycles

CD22 is a cell surface glycoprotein restricted to normal and malignant B-cells and is the target of several anti-CD22 antibody-based cancer therapies. For therapeutic antibody-payload conjugates, it is important to understand the subcellular trafficking of anti-CD22 antibodies to optimize antibody and/or linker-drug properties to maximize antitumor efficacy. It is agreed that anti-CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell-type dependent. No studies examined anti-CD22 trafficking to either pathway in B-cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray™, a novel wall-less 96-well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10-20 μL) volumes. We examined the time-course of trafficking of five different anti-CD22 antibodies in eight B-cell lines representing four B-cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti-CD22 antibodies conjugated to different therapeutic payloads.