Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies
Chethana Kulkarni, James E Finley, Andrew J Bessire
1Global Biotherapeutics Technologies, Pfizer Worldwide R&D , Cambridge, Massachusetts 02139, United States.
Abstract:
As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolism-processes that are critical to ADC efficacy and toxicity-imaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a "clickable" ADC bearing an azide-functionalized linker-payload (LP) poised for "click" reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems.
Insights
Researchers developed a "clickable" antibody-drug conjugate (ADC) for studying cancer drug delivery. This method avoids altering ADC properties, unlike traditional fluorescent labels, enabling more accurate analysis of ADC behavior.
Area of Science:
- Chemical Biology
- Molecular Imaging
- Cancer Therapeutics
Background:
- Antibody-drug conjugates (ADCs) are crucial in cancer therapy, but understanding their molecular-level function (uptake, trafficking, catabolism) is vital.
- Current imaging studies using fluorophore-labeled ADCs may alter ADC behavior and efficacy.
- A small structural perturbation is desirable for accurate ADC studies.
Purpose of the Study:
- To develop a "clickable" ADC that minimizes structural changes for accurate molecular-level studies.
- To compare the efficacy and behavior of clickable ADCs versus traditional fluorophore-labeled ADCs.
- To investigate ADC uptake, trafficking, and internalization using advanced imaging techniques.
Main Methods:
- Development of an azide-functionalized linker-payload (LP) for "click" chemistry.
- Synthesis of a "clickable" ADC and comparison with a fluorophore-labeled ADC.
- In vitro potency assays in target-expressing cells.
- Live-cell confocal microscopy for uptake and trafficking studies.
Main Results:
- The clickable ADC demonstrated excellent potency, unlike the fluorophore-labeled ADC which showed reduced activity.
- Live-cell microscopy confirmed robust uptake and selective in situ reaction of the clickable ADC.
- Time-course studies indicated greater and faster net internalization of ADCs compared to the parent antibody.
Conclusions:
- "Clickable" ADCs offer a more accurate approach to studying ADC behavior by minimizing label-induced alterations.
- This chemical biology strategy enhances understanding of ADC processing in biological systems.
- The findings underscore the importance of label-free or minimally perturbing methods in ADC research.
