Determination of binding characteristics and cytotoxic activity of bispecific T cell engagers

By
  • Fabian Vandermeers (1)
  • Thomas Henri (1)
  • Florian Visée (1)
  • Sara Hully (1)
  • Arnaud Delobel (1)
  • Mick Verhagen (2)
  • Remon Van Geel (2)
  • Floris Van Delft (2)
Date
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T cell-based cancer immunotherapies have transformed oncology by enabling the targeted activation of immune responses against tumour cells. Among these approaches, bispecific T cell engagers (TCEs) play a key role by simultaneously binding tumour-associated antigens and CD3 receptors on T cells, triggering immune cell activation and tumour cell lysis. Characterising the binding properties and functional activity of these complex molecules is essential to ensure their quality, efficacy and safety. This application note presents a comprehensive analytical strategy combining multiple orthogonal techniques to assess affinity, kinetics, specificity and potency.

KEY HIGHLIGHTS OF THE METHOD

  • Combined use of Surface Plasmon Resonance (SPR) and BioLayer Interferometry (BLI) to determine binding affinity (KD) and kinetic parameters (ka, kd) for TCE–CD3 and TCE–HER2 interactions
  • Confirmation of consistent affinity measurements across SPR and BLI, with high-affinity TCEs showing KD values in the low nanomolar range (~2–4 nM)
  • Identification of avidity effects in multivalent TCE formats (e.g. 2:2), influencing apparent binding affinity and rebinding behaviour
  • Demonstration of very high affinity towards HER2 targets, with KD values reaching the picomolar range due to slow dissociation rates
  • Proof-of-concept of simultaneous dual binding (CD3 and HER2) using adapted BLI assay design

HOW THIS SUPPORTS YOUR PROGRAMME?

A robust and orthogonal analytical strategy is essential for the development and quality control of bispecific antibodies. This approach provides comprehensive insight into both binding and functional activity.

  • Enables accurate characterisation of affinity and kinetics across different molecular formats
  • Supports detection of format-dependent effects such as avidity and multivalency
  • Confirms specificity through cell-based binding assays using relevant and negative control cell lines
  • Correlates binding properties with biological activity through functional potency assays
  • Provides reliable ranking of candidate molecules based on affinity and potency
  • Facilitates informed selection of optimal TCE formats for further development

Together, these capabilities contribute to a deeper understanding of structure–function relationships in TCE development.

APPLICATIONS / USE CASES

  • Characterisation of bispecific T cell engagers targeting CD3 and tumour-associated antigens (e.g. HER2, CD20)
  • Quality control of engineered antibody formats generated using GlycoConnect® or KIH technologies
  • Evaluation of binding specificity using flow cytometry on CD3-positive and CD3-negative cell lines
  • Potency assessment through T cell activation assays using engineered reporter cells
  • Measurement of T cell-mediated cytotoxicity and cytokine secretion in co-culture systems
  • Comparative ranking of TCE candidates based on affinity and biological activity

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1 Quality Assistance S.A., Technoparc de Thudinie 2, 6536 Thuin, Belgium
2 Synaffix - A Lonza Company, Kloosterstraat 9, 5349 AB Oss, The Netherlands

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