Category: Assay Development and Screening
Monoclonal antibodies (mAb) and antibody-drug conjugates (ADCs) are widely used biological therapeutics. A key property of these biologics is the extent and rate of internalization into different cells, which governs their efficacy, safety and pharmaco-dynamic profile. As a result, quantifying and comparing the rate of Ab internalization of drug candidates and production batches is a critical step in the biopharmaceutical selection and optimization process. Here, we describe an automated, novel and enabling cell-based Ab internalization assay that is turnkey, medium throughput and geared toward industrial biologics discovery. Internalization measurements are made over time on 96-well microplates using live-cell analysis (IncuCyte S3). Specifically, an antibody-binding fragment coupled to a pH-sensitive dye (FabFluor) was used to label and test mAbs using a single-step, no-wash protocol. As expected, an increase in fluorescence signal was observed as the mAb complex was internalized into the acidic lysosome. This approach was further validated using trastuzumab (Herceptin, Her-2) or rituximab (Rituxan, CD20) on pre-plated BT-474 or Raji cells, respectively. Cell images (10-20x) were taken and automatically analyzed for fluorescence area every 30 min for up to 48h. Both trastuzumab (BT-474) and rituximab (Raji) caused time- and concentration-dependent internalization (EC50 values 2.1 and 2.6 nM, respectively). The fluorescence signal was punctate, outside of the nucleus and strongly co-localized with a lysosomal marker (LysoSensor). In line with known marker expression profiles, specific internalization of mAbs to CD45, CD71 and CD3, but not CD20, was observed in Jurkat T-lymphocytes and CD45, CD71 and CD20, but not CD3 in Raji B cells. As a proof of concept for screening and direct comparison of test mAbs, 6 commercially available CD71 (transferrin receptor) Abs were labeled with mFabFluor reagent, serially diluted and added to HT-1080 osteosarcoma cells. 3 of the Abs produced a large internalization signal with detection
Daniel Appledorn– Director of R&D, Essen BioScience, Ann Arbor, MI
Director of R&D
Ann Arbor, MI
Dan is the Director of US Biology R&D at Essen BioScience. Dan joined Essen in the fall of 2010 and is leading a team of scientists to develop new applications, biological reagents and instruments for drug discovery with a focus on building assays for quantitative live cell analysis using the IncuCyte system.
Prior to joining Essen, Dan was a postdoctoral research fellow at Michigan State University where his primary focus was investigating the interaction between adenovirus vectors and the innate and adaptive immune systems in mouse models with the goal of developing gene therapy and/or vaccine vectors for the treatment of human disease.
Over the course of his 4 year postdoc, Dan’s pioneering work in this field resulted in several patents and 20+ publications describing the utility of Ad vectors in both gene therapy and vaccine platforms.