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P12.05 Cellular biosensors to study protein interactions relevant to immunotherapy

April, 04, 2024 | Select Oncology Journal Articles

Background

Many cancer immunotherapies rely on cell surface protein engagements in trans. Checkpoint inhibitors block certain interactions between cancer cells and immune cells. Chimeric antigen receptor (CAR)-based therapies redirect immune cells towards tumors by binding of surface antigens on cancer cells. This highlights the importance of such cellular interactions and provides a rationale to develop an easy-to-handle, flexible and cost-effective tool to study them. Here we present a novel immunoassay that fulfills these requirements and we show its utility to address research questions arising in the context of immunotherapies.1

Materials and Methods

Our assay makes use of T cell lines equipped with reporter genes, that are translated upon activation of the TCR signaling pathway.2 To create cellular biosensors, the reporter cells are transduced to express chimeric receptors. These receptors consist of an extracellular domain of interest fused to a CD3 intracellular signaling domain. The cellular biosensors can then be probed with cells expressing the respective interacting receptor or ligand. This induces a fluorescent signal that is evaluable by flow cytometry.

Results

The assay provides quantitative information on the inhibitory potency of immune checkpoint inhibitors to the PD-L1/PD1 axis and can be used to characterize small molecule inhibitors blocking the PD1/PD-L1 interaction. While some are highly active, surprisingly others do not interfere with PD1 binding to PD-L1 even at high concentrations. We demonstrate that cellular biosensors can function to probe defined cellular populations for the expression of interaction partners to orphan ligands. In this setting, we do not identify a receptor to the putative immune checkpoint B7-H3 on T cells suggesting alternative mechanisms for B7-H3 mediated immunosuppression. Finally, we apply cellular biosensors to validate binding of CAR antigen recognition domain. Variable chains of three B7-H3 specific antibodies are arranged as single chain fragments (scFv) and evaluated in a cellular biosensor assay. While a mirzotamab derived scFv induces a strong signal, binding of an omburtabmab-scFv is considerably weaker and no signal can be detected with an enoblituzumab derived scFv.

Conclusions

In summary, we present a novel type of immunoassay to study interactions between membrane proteins in trans and highlight potential uses in the context of immunotherapy. It is suitable to study immune checkpoints and can be used to characterize respective inhibitory drugs. It also enables unbiased screening of cellular samples for the expression of interaction partners to orphan ligands. Lastly, it is applicable for the validation of antigen binding domains for CARs. This may accelerate the development of new, functional CAR constructs and could furthermore serve as a method to characterize the role of antigen mutations for immune escape.

References

  • Funk MA, Leitner J, Gerner MC, et al. Interrogating ligand-receptor interactions using highly sensitive cellular biosensors. Nat Commun. 2023;14:7804.

  • Jutz S, Hennig A, Paster W, et al. A cellular platform for the evaluation of immune checkpoint molecules. Oncotarget. 2017;8(39):64892-64906.

  • M.A. Funk: None. J. Leitner: None. C. Battin: None. S. Gumpelmair: None. S. Theurich: None. P. Steinberger: None.

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