Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

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.