CXCR3 在激活的 T 细胞上表达，在基于细胞和免疫检查点抑制剂 (ICI) 的免疫治疗期间，在 T 细胞募集到肿瘤微环境 (TME) 中发挥着至关重要的作用。本研究利用 64Cu 标记的 NOTA-α-CXCR3 抗体来评估 TME 中的 CXCR3 表达，并验证其作为体内潜在 T 细胞激活生物标志物。CXCR3 细胞浸润 MC38 肿瘤（B57BL/6 小鼠，未经治疗和经 αPD-治疗） 1/αCTLA-4 ICI) 使用荧光显微镜和流式细胞术进行定量。商业抗小鼠 CXCR3 抗体 (α-CXCR3) 与 2,2,2-(1,4,7-三氮杂环壬烷-1,4,7-三基)三乙酸 (NOTA) 进行位点特异性缀合，并用 64Cu 进行放射性标记。使用稳定转染鼠CXCR3的CHO细胞研究[64Cu]Cu-NOTA-α-CXCR3的饱和结合。分别使用不同摩尔活性（10 GBq/μmol 至 300 GBq/μmol）的 [64Cu]Cu-NOTA-α-CXCR3 在基线和 1 至 3 个 ICI 周期后进行生物分布和 PET 成像研究。Flow基线细胞计数分析证实 MC38 肿瘤中存在 CXCR3  T 细胞，在 ICI 后第五天显着增加（治疗组为 33.8 ± 17.4，对照为 8.8 ± 6.2 CD3 CXCR3 细胞/mg）。这些结果通过肿瘤冷冻切片的免疫荧光定性和定量证实。使用 [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) 对 MC38 荷瘤小鼠在 ICI 之前、期间和之后进行体内 PET 成像，揭示了次级淋巴器官中示踪剂积累的 CXCR3 特异性对摩尔活性的强烈依赖性。在 300 GBq/μmol（1.5 µg 抗体/小鼠）下，在淋巴结（6.33 ± 1.25 对照 vs. 3.95 ± 1.23%IA/g 阻断）和脾脏（6.04 ± 1.02 对照 vs. 3.84 ±）中观察到特异性信号。 0.79%IA/g 阻断），注射后 48 小时脾脏与肝脏的比率表明时间依赖性全身免疫反应，从 1.08±0.19（未治疗对照）稳定增加到 1.54±0.14（三个 ICI 周期）。本研究证明了使用免疫疗法对 CXCR3 上调进行体内成像的可行性抗体。然而，高摩尔活性和低抗体剂量对于淋巴结和脾脏的灵敏检测至关重要。由于二抗相关效应，检测治疗引起的肿瘤中 CXCR3 T 细胞数量的变化具有挑战性。尽管如此，CXCR3 仍然是 T 细胞激活成像的一个有前途的靶标，预计使用具有高亲和力和良好药代动力学的替代示踪剂可提高灵敏度。© 2024。作者。

CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo.CXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3.Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles).This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.© 2024. The Author(s).