Advancing In Vivo Detection of T-Cell Function: Development and Preclinical Evaluation of ⁸⁹Zr-Ivuxolimab, a Human OX40 PET Tracer

The variable response to cancer immunotherapies highlights a critical gap in our ability to predict and monitor treatment efficacy. To address this, there is an urgent clinical need for advanced molecular imaging technologies that can noninvasively and precisely assess whole-body immune responses. The OX40 receptor (CD134), a potent costimulatory molecule on T cells, serves as a highly specific marker of T-cell activation, an early and crucial event in immunotherapy efficacy. In this study, we report the development of a human OX40-specific radiotracer based on a clinically evaluated therapeutic-ivuxolimab-and assess its utility for PET imaging of activated T cells in vivo. <b>Methods:</b> Deferoxamine conjugation and ⁸⁹Zr radiolabeling were optimized for ivuxolimab. In vitro specificity of the resultant tracer, ⁸⁹Zr-ivuxolimab, was then assessed using primary human T cells and stably transfected human OX40⁺ (huOX40⁺) human embryonic kidney 293 (HEK293) cells. In vivo specificity and biodistribution of ⁸⁹Zr-ivuxolimab were confirmed in subcutaneously implanted huOX40⁺ HEK293 or parental HEK293 tumor-bearing mice. To evaluate ⁸⁹Zr-ivuxolimab's utility for detecting T-cell activation in vivo, we used a transgenic human OX40 murine model of acute graft-versus-host disease. Ex vivo gamma counting, autoradiography, and immunohistochemistry were performed to verify tracer-binding specificity. <b>Results:</b> ⁸⁹Zr-ivuxolimab was reproducibly synthesized and showed significantly increased in vitro binding to activated human T cells versus resting cells (<i>P</i> < 0.0001) and increased binding to huOX40⁺ HEK293 cells versus HEK293 cells (<i>P</i> < 0.0001). Longitudinal PET/CT imaging of tumor-bearing mice over 5 d revealed markedly higher tracer accumulation in huOX40⁺ HEK293 tumors compared with HEK293 tumors (<i>P</i> < 0.0001). ⁸⁹Zr-ivuxolimab successfully detected T-cell activation in the spleen, mesenteric lymph node, and gastrointestinal tract of mice with graft-versus-host disease induced by transgenic murine T cells expressing human OX40, compared with control groups (total body irradiation, <i>P</i> < 0.0001; bone marrow, <i>P</i> < 0.001). Ex vivo gamma counting of tissues, autoradiography, and immunohistochemistry corroborated PET findings and confirmed tracer specificity for OX40. <b>Conclusion:</b> ⁸⁹Zr-ivuxolimab is a promising radiotracer for clinical translation as an imaging agent for activated T cells. Further investigation of its ability to monitor and predict response to different cancer immunotherapy modalities is warranted.