ImmunoPET Imaging of TIM-3 in Murine Melanoma Models

T cell immunoglobulin and mucin domain-containing-3 (TIM-3) is an immune checkpoint expressed mainly on CD4⁺ and CD8⁺ T cells. In addition to negatively regulating inflammatory T cell function, TIM-3 is a promising immunotherapy target. Herein, the aim is to develop an immuno-positron emission tomography (immunoPET) probe for noninvasively characterizing TIM-3 expression. Flow cytometry is used to detect the expression levels of TIM-3 in B16F10 cells. RMT3-23, a rat antimouse TIM-3-specific monoclonal antibody, is radiolabeled with ⁶⁴Cu and the performance of ⁶⁴Cu-NOTA-RMT3-23 is interrogated by immunoPET in murine melanoma models before and after radiation therapies. Biodistribution and immunofluorescent staining studies are carried out after the immunoPET imaging studies. TIM-3 is negatively expressed in B16F10 cells, and its expression is not induced by radiation therapies. ImmunoPET imaging with ⁶⁴Cu-NOTA-RMT3-23 precisely tracks the unique distribution of TIM-3-positive lymphocytes in immunocompetent melanoma models, and tumor uptake of the radiotracer is not affected by either single-dose or fractionated radiation therapies. The ⁶⁴Cu-NOTA-RMT3-23 immunoPET imaging results are further mirrored by the immunofluorescent staining studies. These results demonstrate the feasibility of ⁶⁴Cu-NOTA-RMT3-23 immunoPET in tracking TIM-3 and highlight a new opportunity to optimize TIM-3-targeted immunotherapies with this novel imaging strategy.