Site-specific conjugation of novel fluorophores and tags to accelerate immunotherapy discovery and development
C. Langsdorf, R. Aggeler, B. Agnew, K. Chambers, A. Chen, N. Dolman, Y.-Z. Hu, S. Leonard, K. Oakleaf, M. Wickett, Y. Wu, W. Zhou; Thermo Fisher Scientific, Eugene, OR, USA
The basic cellular mechanisms of internalization and trafficking are important to many areas of cell biology, and especially to the proper function of therapeutic antibodies. Those antibodies intended for use as antibody drug conjugates should specifically bind to target cells and rapidly internalize into acidic compartments. Conversely, antibodies intended to kill cells via direct cell death, complement cascade, or effector cell killing should remain bound to the external surface of target cells as long as possible. However, the ability to study these internalization processes has historically been limited by the lack of tools to directly monitor the internalization and subsequent acidification of extracellular material.
Here we demonstrate three approaches to directly visualize internalization of therapeutic antibodies and ADCs in live cells. Two of these techniques employ site-specific antibody labeling to screen for and further characterize internalizing clones. Additionally, we demonstrate synthesis of antibody-drug conjugates via defined conjugation. The internalization of these ADCs into HER2- positive breast cancer cells shows a strong correlation with target cell killing. Further applications are enabled by site-specific conjugation of biotin, including fluorescent signal enhancement and cell depletion or enrichment. Finally we functionally characterize bispecific antibodies created via bioorthogonal antibody heterodimerization. We believe the insights provided by these approaches have the potential to accelerate biotherapeutic lead generation and development.
Credits: None available.
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