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Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands
Devin L Wakefield1, Ottavia Golfetto1, Raphael Jorand1
1Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, Duarte, CA, United States.
Introduction:
The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling.
Methods:
By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells.
Results:
We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases.
Discussion:
Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.
Insights
Combining trastuzumab and pertuzumab antibodies enhances HER2 receptor clustering in breast cancer cells. This approach, using advanced protein engineering, shows promise for developing novel HER2-targeted therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- HER2-positive metastatic breast cancer treatment often involves targeting the HER2 receptor.
- Trastuzumab and pertuzumab are monoclonal antibodies that target HER2 but have distinct mechanisms.
- The combination of trastuzumab and pertuzumab improves progression-free survival in patients.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the efficacy of combined HER2-targeted antibodies.
- To assess and optimize the clustering of HER2 receptors using protein engineering and quantitative single molecule localization microscopy (qSMLM).
- To evaluate the impact of antibody-mediated HER2 clustering on downstream signaling pathways.
Main Methods:
- Utilized quantitative single molecule localization microscopy (qSMLM) to visualize and quantify HER2 clustering in breast cancer cells.
- Engineered multivalent ligands, including meditope technology, to enhance HER2 receptor aggregation.
- Assessed the effects of different antibody formats (monovalent Fab, individual mAbs, mAb combinations, multivalent ligands) on HER2 organization.
- Measured the inhibition of epidermal growth factor (EGF)-dependent downstream kinase activation.
Main Results:
- Individual antibodies (trastuzumab or pertuzumab) significantly increased HER2 clustering compared to a monovalent Fab fragment.
- The combination of trastuzumab and pertuzumab induced the highest level of HER2 clustering.
- Engineered multivalent ligands, in combination with trastuzumab, further enhanced HER2 clustering.
- Meditope-based combinations showed greater early inhibition of EGF-dependent kinase activation compared to trastuzumab plus pertuzumab.
Conclusions:
- Monoclonal antibodies (mAbs) and multivalent ligands can effectively modulate HER2 receptor organization and activation.
- Enhanced HER2 clustering by therapeutic agents may represent a key mechanism for improved treatment efficacy.
- The developed approach holds potential for the future design of novel HER2-targeted cancer therapeutics.
