Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo

Area of Science:

• Oncology
• Biophysics
• Medical Imaging

Background:

• Human Epidermal growth factor Receptor 2 (HER2) drives aggressive breast cancer, making it a target for immunotherapy like trastuzumab (TZM).
• Clinical efficacy of TZM is limited by drug resistance and insufficient drug exposure, necessitating direct measurement of target engagement.
• Quantifying TZM-HER2 binding in vivo is crucial for understanding treatment response and optimizing therapy in live tumor models.

Purpose of the Study:

• To develop and validate a method for directly measuring TZM-HER2 binding dynamics in live, undisturbed tumor microenvironments.
• To quantify cellular target engagement and intracellular drug delivery of TZM in HER2-positive breast cancer models.
• To establish fluorescence lifetime imaging as a tool for monitoring immunoconjugate pharmacodynamics in vivo.

Main Methods:

• Utilized fluorescence lifetime Förster Resonance Energy Transfer (FLI-FRET) with near-infrared (NIR) microscopy (FLIM-FRET) and macroscopy (MFLI-FRET).
• Employed donor-labeled TZM and acceptor-labeled TZM to sense binding via fluorescence lifetime reduction.
• Applied the technique to HER2-positive human breast cancer cells and tumor xenografts in live animals.

Main Results:

• Successfully quantified the fraction of HER2-bound and internalized TZM in both cell culture and live animal tumor xenografts.
• Demonstrated direct measurement of TZM-HER2 binding dynamics using FLI-FRET imaging.
• Ex vivo immunohistological analysis confirmed TZM-HER2 complex binding and internalization within breast cancer cells.

Conclusions:

• FLI-FRET imaging provides a powerful, direct method to monitor cellular target engagement of TZM.
• This technique enables quantification of TZM internalization and intracellular drug delivery in live HER2-positive tumors.
• FLI-FRET imaging offers a valuable tool for assessing immunoconjugate pharmacodynamics and guiding personalized cancer treatment strategies.