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Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye
Nidhi Nath1, Becky Godat1, Chad Zimprich1
1Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States.
Abstract:
Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.
Insights
A novel pH-sensitive dye enables real-time measurement of antibody internalization into cancer cells, improving drug delivery and pharmacokinetic profiling for antibody therapeutics.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Receptor-mediated antibody internalization is crucial for anti-cancer antibody therapeutics like antibody-drug conjugates (ADCs).
- Current methods for studying antibody internalization are often multistep, low-throughput, and provide only endpoint data.
- Efficient internalization impacts drug delivery, receptor modulation, and pharmacokinetics, influencing therapeutic efficacy.
Purpose of the Study:
- To develop a new homogeneous, time- and concentration-dependent method for measuring antibody internalization.
- To utilize a novel hydrophilic and bright pH sensor dye (pHAb dye) for this purpose.
- To demonstrate the utility of the pHAb dye for studying receptor-mediated antibody internalization and for early-stage monoclonal antibody development.
Main Methods:
- Antibodies targeting cell surface receptors were conjugated with the pHAb dye, which is non-fluorescent at neutral pH and fluorescent at acidic pH.
- Cells expressing target receptors were incubated with the labeled antibodies.
- Internalization was measured by detecting the fluorescence signal generated as the dye enters acidic intracellular compartments (endosomes/lysosomes).
- Labeling strategies (amine and thiol chemistries) and dye-to-antibody ratios were investigated using Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR).
Main Results:
- The pHAb dye enables homogeneous, time- and concentration-dependent measurements of antibody internalization.
- The hydrophilic nature of the dye minimizes antibody aggregation.
- The method demonstrated successful labeling and internalization studies with therapeutic antibodies Trastuzumab and Cetuximab.
- Two novel approaches for high-throughput screening during early monoclonal antibody development were presented.
Conclusions:
- The pHAb dye provides a sensitive and efficient tool for studying receptor-mediated antibody internalization.
- This homogeneous assay format offers advantages over traditional methods, including higher throughput and real-time monitoring.
- The developed method and approaches are valuable for optimizing antibody-based cancer therapeutics and accelerating early-stage drug development.
