Cross-Platform Analysis of the Binding and Function of Antibody-Drug Conjugates (ADCs)

Antibody-drug conjugates (ADCs) are one of several adaptations made to monoclonal antibodies (mAbs) to improve their anti-tumor activity.

ADCs unite both immunotherapeutic and chemotherapeutic interventions, to create an efficacious and targeted cancer treatment.   They leverage the highly specific nature of therapeutic mAbs, as a backbone for delivery of potent cytotoxic drugs. These mAbs have been adapted to include a linker in their constant region, onto which a toxic payload is attached. 

ADCs rely on internalization of the ADC into tumor cells to trigger the cytotoxic action of the payload drug, and therefore work to prevent release of the drug in the blood stream or healthy tissues. The payloads typically used are chemotherapeutic drugs, with mechanisms of action (MoAs) that interfere with processes such as microtubule polymerization, or that induce DNA damage.

Aside from the cytotoxic payload delivery to target cells, ADCs can maintain the Fc-mediated immune functions of the mAb on which they were based, for example antibody dependent cellular cytotoxicity (ADCC) activity. 

Due to the combination of MoAs that could be exerted by an ADC, it is important that we have a range of suitable in vitro assays to characterize their activity towards tumor cells. 

Instrumental analyses can be combined into workflows to build a comprehensive in vitro functional profile of ADCs.  This can be used to identify differences in the MoAs adopted by ADCs, which can be linked back to structural variations that may improve the efficacy of novel drugs.


This application note presents a combined iQue® Advanced Flow Cytometry and Incucyte® Live-Cell Imaging and Analysis approach, for quantifying the live-cell binding and function of two Trastuzumab (anti-HER2) based ADCs: Trastuzumab emtansine (Kadcyla®) and Trastuzumab deruxtecan (Enhertu®).  Trastuzumab emtansine, a treatment for HER2 over-expressing breast cancers, is one of the current FDA-approved anti-cancer ADC therapeutics.

The iQue® Platform measured binding, immune cell marker expression and cytokine release, to provide insight into how ADCs interact with both target and immune cells, and the associated changes in phenotype. An Incucyte® Live-Cell Analysis System captured temporal information on mAb target cell killing function, by quantifying target and immune cells throughout ADC cytotoxicity and ADCC assays. Live-cell data were complemented with Fcγ Receptor (FcγR) and antigen kinetic protein binding data generated using an Octet® Bio-Layer Interferometry (BLI) System.

  • Document type: Application Note
  • Page count: 14
  • Read time: 15 minutes
  • Authors: Kirsty McBain, David Apiyo and Nicola Bevan

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Key Takeaways

Key topics detailed in this application note include:

  • Protein Binding Kinetics
  • Antibody Internalization
  • ADC Cytotoxicity
  • Antigen Binding Characterization
  • Antigen Characterization
  • Enhertu® Bystander Killing
  • ADCC Activity
  • NK Activation and Cytokine Release

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