Abstract 5496: Target density matters: Binding kinetics of antibody-based therapeutics are highly influenced by target expression levels

Understanding antibody-based therapeutic behaviour under varying target expression levels iscrucial for effective tissue and cell-specific targeting. Traditional in vitro evaluations often relyon cancer cell line panels with differing receptor levels, which can be logistically challengingand yield inconsistent results. Standard techniques like SPR are poorly suited for analyzingmolecule binding to eukaryotic cells, as detection depth (∼200 nm) covers only ∼2% of a typical10-20 µm cell and unstable immobilization limits SPR to cell attachment studies. Similarlimitations apply to BLI and QCM. Other cell-based binding technologies have not gainedwidespread adoption due to challenges in automation, sensitivity, and throughput. Measuringhow binding is influenced by differential target expression is critical for tumor-targetingtherapeutics, as antigen density impacts safety through on-target, off-tumor toxicities andaffects efficacy by enabling tumor escape at low target expression levels.The purpose of this study was to investigate how target antigen density influences the bindingkinetics of therapeutic molecules under physiologically relevant conditions, addressinglimitations inherent to traditional kinetic assays. To achieve this, we utilized the novel IndEx-2system, which employs CHO-K1 cells engineered for tuneable expression of one or two targetantigens via chemically induced proximity-mediated induction. This was paired with single-cellInteraction Cytometry (scIC), a unique biosensor-based technology that measures real-timebinding directly on captured cells. By combining these two systems, we examined the bindingkinetics of trastuzumab to HER2 expressed at varying levels, serving as a model for tumourheterogeneity. HER2 expression was quantified using flow cytometry, while scIC was used toperform kinetic measurements on the same cells. The analysis revealed that higher HER2expression levels prolonged trastuzumab residence time due to avidity effects. Notably,differences in affinity (Kd) across HER2 densities were driven by variations in the dissociationrate (koff), rather than the association rate (kon), underscoring the critical influence of targetdensity on binding kinetics.This study demonstrates how target density can affect therapeutic binding kinetics, offeringcritical insights into drug efficacy and duration of action. By integrating scIC with the IndEx-2platform, we provide a robust framework for assessing kinetic behaviour of targetedtherapeutics in a cellular context, including bispecific binders and receptor internalisationstudies. These results pave the way for safer, more effective therapeutics, selected in morecomplex, and more relevant, biological systems. Citation Format: Eric Cruickshank, Nichole Cerutti, Hannah Findlay, Timothy London, Vivien Hafner, Andreas Kratzert. Target density matters: Binding kinetics of antibody-based therapeutics are highly influenced by target expression levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5496.