biotinylated CCR4 + cells reflects on the receptor-ligand interaction. A strongly reduced biotinylation of CCR4 + cells upon addition of an inhibitor indicates an effective inhibitory binding capacity. Results In vitro binding assays using APEX proximity labeling were established to create a testing platform for inhibitors of the CCR4-CCL22 axis. For analysis of the murine and human system, CCR4-overexpressing B3Z cells and CCRF-CEM cells that endogenously express CCR4 were used, respectively.

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... n both settings, a strong biotinylation was achieved by adding the CCL22-APEX fusion protein as compared to only APEX as a control for unspecific biotinylation. Addition of two different CCR4 antagonists, C-021 and AZD-2098, lead to significant and dose-dependent reduction of target cell biotinylation. The same assay was conducted using CCR4transduced primary murine T cells, showing equivalent results. Further, we could demonstrate a decreased biotinylation of cells when recombinant CCL22 had been added, indicating competitive binding inhibition. Conclusion This work reveals a novel application of the APEX proximity labeling system to identify new chemokine-receptor interactions and to screen inhibitors for their binding capacity, facilitating further evaluation of promising inhibitors in functional experiments in vitro and in vivo. The adaption of the assay protocol for suspension cells and not only adherent cell lines also allows the use of primary murine as well as human cells. Overall, our results present a valuable tool for the evaluation of novel inhibitors of chemokine-receptor axes for immunotherapy of malignant diseases.