In vitro Bioassays to Model Complex Cellular Environments for Better Lead Selection

Comparing Kadcyla® (T-DM1) and Enhertu® (T-DXd) across assays of increasing complexity

Early in vitro testing provides critical insights to guide ADC lead selection, maximizing potency, understanding the mechanism of action (MoA), and predicting clinical performance. This case study demonstrates how Abzena’s suite of bioassays can distinguish between two HER2-targeted ADCs, Kadcyla and Enhertu, across simple 2D assays through to complex 3D co-culture models.

Endpoint Cytotoxicity

• CellTiter-Glo® Luminescence (2D Monolayer)
• – Both ADCs show similar EC₅₀ potencies on HER2⁺ cells after 96 h, but Kadcyla achieves slightly higher maximum cell killing (Figure 2).
• Real-Time, Cell-by-Cell Analysis (Incucyte®)
• – Enhertu triggers earlier induction of apoptosis, while Kadcyla delivers greater overall cytotoxicity over time, revealing kinetic differences in MoA (Figure 3).

Modeling Solid Tumors with 3D Spheroids

• Spheroid Viability Assay (Incucyte® Live-Cell Imaging)
• – In ultra-low attachment plates, both Kadcyla and Enhertu shrink HER2⁺ spheroids over 48 h. Consistent with 2D data, Kadcyla yields more rapid and pronounced spheroid killing at later time points, highlighting its superior penetration in compact tumor-like structures (Figure 4).

Heterogeneous Tumors & the Bystander Effect

• Co-Culture Bystander Killing
• – Enhertu’s cleavable payload mediates bystander killing of adjacent HER2⁻ cells (green), recapitulating its clinical efficacy in heterogeneous tumors. Kadcyla, by contrast, spares HER2⁻ cells, indicating limited bystander activity in mixed-cell populations (Figure 5).

Integrating the Immune Component

• ADCC and Payload Synergy (3D Co-Culture + PBMCs)
• – When peripheral blood mononuclear cells (PBMCs) are added, we observe additive antibody-dependent cellular cytotoxicity (ADCC) alongside direct payload-mediated killing. Enhertu eliminates a higher proportion of total cells than Kadcyla, modeling the in vivo synergy of immune effectors and payload (Figures 6–7).

Take the Next Step

Leverage Abzena’s advanced in vitro bioassays to ensure your ADC candidates are poised for clinical success.

Contact us to discuss how our tailored assay platforms can optimize your lead selection process.