Abstract 3108: Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies
Authors: Holly Ryan W. 
Published: March 2024

Introduction: There is a growing need in the immunotherapy research field for tools to rapidly screen novel therapeutic antibodies, whether looking for cells expressing IgG, target binding, or tracking internalization. Thermo Fisher Scientific has developed the next generation of Invitrogen™ Zenon™ reagents to enable screening of potential therapeutic antibodies, working across a variety of sample types including primary B cells and hybridomas, while being compatible with a wide range of platforms including flow cytometry, high content imaging, and in-incubator imaging systems.

Methods: The Invitrogen™ Zenon™ Alexa Fluor™ Plus IgG Labeling Reagents to screen for antibody binding provide a fast, versatile, and reliable method to evaluate antibody binding to cell surface antigens. Zenon screening reagents are labeled with Invitrogen Alexa Fluor Plus 488, Alexa Fluor Plus 594, or Alexa Fluor Plus 647 dyes (i.e., labeling reagent). The labeling reagent is directed against the Fc portion of an intact mouse or human IgG primary antibody. The formation of the zenon-antibody complex is rapid and requires no purification steps. The Invitrogen™ Zenon™ pHrodo™ Deep Red Labeling Reagents for screening antibody internalization provides a highly sensitive method for monitoring antibody internalization. Zenon pHrodo Deep Red utilizes the same screening reagent as Zenon Alexa Fluor Plus reagents, replacing Alexa fluor dyes with the pH-sensitive dye, pHrodo Deep Red. The pHrodo Deep Red sensors have minimal fluorescence at neutral pH and only upon entry into the late endosome and lysosome do they brightly fluoresce. Zenon pHrodo Deep Red Labeling Reagents are perfectly suited for screening the endocytosis and degradation of potentially therapeutic antibodies including antibody-drug conjugates.

Results: Live cells stained with Zenon-labeled antibodies show remarkable signal to background in both flow and imaging applications, while retaining their signal following fixation. Therapeutic antibodies such as anti-Her2 labeled with Zenon pHrodo Deep Red show as bright fluorescent puncta upon lysosomal degradation in the Alexa Fluor 647 channel and co-localize with lysosomal markers such as Invitrogen™ LysoTracker Red dye. Both Zenon Alexa Fluor Plus and Zenon phrodo Deep Red are ideal reagents for screening antibody panels of up to 96 different antibodies.

Conclusions: Thermo Fisher Scientific has developed sensitive and rapid Zenon reagents to allow for high throughput therapeutic antibody screening. In addition to Zenon reagents, Thermo Fisher Scientific has developed additional tools that allow for specific monitoring of antibody and protein uptake into the lysosome for degradation. These tools not only greatly improve the workflow from our existing portfolio, but allow for increased sensitivity allowing the field to make decisive decisions when selecting candidate immunotherapies.

Acknowledgments: I would like to thank the whole team that worked to develop these new labeling reagents including: Maria Olson and Chris Langsdorf.

Citation Format: Ryan W. Holly. Antibody labeling reagents to rapidly screen for the binding and internalization of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3108.