Background The field of therapeutic antibody developmenthas witnessed significant growth since the introduction of the firsttherapeutic monoclonal anti-CD3 antibody, Muronomab, in 1986 [1].Numerous investigations indicate that the dissociation rate of antibodies playsa predictive role in their clinical effectiveness [2, 3]. Therefore, it isimperative to conduct kinetic rate analyses to enhance the efficacy and safetyof these therapeutic agents. Transmembrane proteins such as PD-(L)1, CD3 orHER2 represent the most common targets. Their binding kinetics are influencedby neighbouring coreceptors as well as by their density and mobility within themembrane. Preserving these target molecules within their native cell membrane thusensures interaction kinetics that are physiologically relevant and offer high invivo predictability.

Materials and Methods Here, we investigated the real-time bindingkinetics of anti-CD3 and anti-PD-1antibodies with their targets for the first time directly on T cells usingReal-Time Interaction Cytometry (RT-IC). Single cells of an immortalized T cellline (Jurkat cells) were trapped on the surface of biosensor chips featuringflow-permeable biocompatible polymer cages. Afterwards, association anddissociation of fluorescently labelled antibodies were measured in real time.

Results First, we measured the real-time kinetic ratesof the immune checkpoint inhibitor nivolumab binding to its target PD-1. Thekinetic data rationalized the in vivo efficacy, as stable binding of theantibody enables T cell activation. Then, we demonstrated the reproducibilityof RT-IC measurements by measuring the interaction of anti-CD3 antibodies withCD3 in single-cell as well as multi-cell measurements. Next, we contrasted thebinding patterns of anti-CD3 antibodies between living and fixed Jurkat cells. Theinteraction data revealed distinct dissociation rates with a biphasicdissociation behaviour for fixed cells but a monophasic behaviour for livingcells.

Conclusions We presume that the observed variations indissociation behaviour stem from the preserved membrane fluidity in livingcells as opposed to fixed cells. In live cells, the sustained mobility ofreceptors within the membrane likely facilitates bivalent interactions(avidity) by enabling the clustering of receptor molecules. These findingsunderscore the significance of directly assessing binding kinetics on cellsurfaces, which will not only support the development of therapeutic antibodiesbut also of other immunotherapies like CAR-T cells or TCR-T cells.

References

1. Mullard A. FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov. 2021 May 5;20(7):491–5.

2. NauerthM, Weißbrich B, Knall R, Franz T, Dössinger G, Bet J, et al. TCR-ligandkoff rate correlates with the protective capacity of antigen-specific CD8+ Tcells for adoptive transfer. Sci Transl Med. 2013 Jul 3;5(192):192ra87.

3. AllardM, Couturaud B, Carretero-Iglesia L, Duong MN, Schmidt J, Monnot GC, et al. TCR-ligand dissociation rate is a robust and stable biomarker of CD8+ T cellpotency. JCI Insight. 2017 Jul 20;2(14):e92570, 92570.

References V. Hafner: A. Employment (full or part-time); Significant; Dynamic Biosensors GmbH. A. Marszal: A. Employment (full or part-time); Significant; Dynamic Biosensors GmbH. U. Rant: A. Employment (full or part-time); Significant; Dynamic Biosensors GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Dynamic Biosensors GmbH. N. Matscheko: A. Employment (full or part-time); Significant; Dynamic Biosensors GmbH. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Dynamic Biosensors GmbH.