Mutations in CCR4 arising after mogamulizumab treatment result in decreased CCR4 expression and reduced antibody dependent cellular cytotoxicity. (A) Diagram of resistance-associated CCR4 mutations (red circles) in 3 mogamulizumab-treated patients in relation to the mogamulizumab-binding epitope, transmembrane domains, and previously described GoF mutations (blue circles). (B) Jurkat cells were transfected with either CCR4wt, CCR4M116R, or CCR4L21V. MFI of CCR4 by flow cytometry is shown using 2 CCR4 antibodies: 1G1 (left) and KM2160 (right). (C) Normalized copy ratio of reads binned across chromosome 3. Yellow line indicates segmented copy number alterations. (D) Antibody-dependent cellular cytotoxicity assay. Jurkat transfectants were cocultured with a Jurkat reporter cell line expressing FcγRIIIa and firefly luciferase under an NFAT response element. Cells were incubated overnight with or without the human anti-CCR4 antibody KM0761 prior to detection of luciferase activity. Erros bars indicate standard deviation. (E) IHC staining of CCR4 in skin biopsies of 3 patients before (top) and after (both) progression on mogamulizumab. Each row of images represents paired biopsies from the same patient. (F) Response to mogamulizumab in the skin (black) and blood (red) in a patient with Sézary syndrome and a C329X GoF mutation of CCR4.