Monitoring T-Cell Activation in the Tumor Microenvironment by PET Imaging of the Chemokine CXCL9

Noninvasive monitoring of immune responses is important for increasing the efficacy of cancer immunotherapy. Although several approaches exist, few methods directly report on T-cell activation. We aimed to develop a novel PET probe targeting C-X-C motif chemokine ligand 9 (CXCL9), a chemokine specifically induced by interferon gamma (IFN-γ), a cytokine that is produced by activated T cells and group 1 innate lymphoid cells. CXCL9 binds to glycosaminoglycans, which are expressed on cell surfaces and the extracellular matrix, to recruit or position cells in the tumor microenvironment. In cancers, expression of CXCL9 has been associated with improved survival and predicts and reflects responses to immunotherapy. The pronounced upregulation of CXCL9 by IFN-γ and CXCL9's extracellular accessibility and site-specific accumulation make it a compelling biomarker for detecting T-cell activation. Methods: We developed a PET tracer targeting CXCL9 based on a high-affinity, antihuman CXCL9 nanobody (h2A12), which was isolated from a llama-derived phage display library and labeled with ¹⁸F. The tracer was evaluated in cell culture, a subcutaneous xenograft model, and a humanized mouse model of T-cell engager therapy. Results: The h2A12 nanobody demonstrated high specificity for human CXCL9 (K _d, 4.07 ± 0.44 nM) with no cross-reactivity to related chemokines. In xenograft models, [¹⁸F]F-h2A12 showed significant uptake in CXCL9-expressing tumors (10.33 ± 1.23 %IA/g) compared with control tumors (0.25 ± 0.04 %IA/g) at 2 h after injection, with excellent tumor-to-background ratios. In the humanized model, T-cell engager therapy induced CXCL9 expression that peaked at day 7, corresponding with increased [¹⁸F]F-h2A12 uptake in treated tumors (2.61 ± 0.50 %IA/g) versus controls (0.67 ± 0.12 %IA/g). Conclusion: [¹⁸F]F-h2A12 PET imaging enables noninvasive visualization of CXCL9 expression as a biomarker of immune activation. This approach offers potential applications in monitoring immunotherapy responses and studying immune-mediated diseases.