Live Cell Labeling with Terpyridine Derivative Proligands to Measure Cytotoxicity Mediated by Immune Cells

Immunotherapy using immune checkpoint inhibitors and CAR‐T cells has revolutionized treatment for patients with malignant tumors. However, measuring tumor cell cytotoxicity mediated by immune effector cells in clinical laboratories has been difficult due to the requirement for radioactive substances. In this study, a series of novel terpyridine derivative proligands were synthesized, and a non‐radioactive cellular cytotoxicity assay using the newly synthesized compounds was developed for use in preclinical and clinical studies for cancer immunotherapy. Once internalized into target cells, the compounds are hydrolyzed by esterases, resulting in the intracellular accumulation of the negatively charged terpyridine derivatives. When the labeled target cells are recognized and killed by immune effector cells, the integrity of the cell membrane is disrupted, and the terpyridine derivatives are released. Upon combining the culture supernatant with europium (Eu3+), the cytotoxicity of immune effector cells for the target cells can be quantitatively determined by measuring the intensity of the Eu3+/ligand‐derived time‐resolved fluorescence. Thus, the assay developed in this study would facilitate the development of novel cancer immunotherapies. Gauging the effectors: Immune checkpoint inhibitors have revolutionized the treatment of cancer patients. Although many clinical trials have been conducted to develop novel immunotherapies, measuring cellular cytotoxicity has been difficult. In this study, a series of chelate‐forming proligands were synthesized to improve non‐radioactive cytotoxicity assays. This assay is ideal for measuring the effector functions of immune cells in clinical laboratories.