背景:淋巴细胞活化基因3(LAG-3)在活化的免疫细胞上表达,并已成为免疫检查点阻断的有希望的靶标。然而,关于LAG-3在肿瘤中的表达与患者预后之间的相关性,表明需要进一步研究LAG-3表达水平在肿瘤治疗中的意义。在这项研究中,68Ga-NOTA-XH05是一种靶向LAG-3的新型基于肽的正电子发射断层扫描(PET)示踪剂,用于非侵入性检测CpG寡核苷酸(CpG)治疗后黑色素瘤中LAG-3的表达,并探讨LAG-3表达与治疗效果之间的关系。
方法:示踪剂68Ga-NOTA-XH05在制备和纯化后通过高效液相色谱法鉴定。进行细胞摄取和阻断实验以验证示踪剂在体外的特异性。流式细胞术检测B16-F10皮下肿瘤中LAG-3的表达,并分析其与示踪剂摄取的相关性以评估示踪剂的特异性。在对单侧或双侧B16-F10皮下肿瘤模型进行CpG治疗后进行PET成像和生物分布研究,以评估68Ga-NOTA-XH05在监测免疫疗法疗效和CpG的横观效应方面的能力。
结果:纯化后,68Ga-NOTA-XH05表现出高的放射化学纯度和特异性。流式细胞术分析显示肿瘤中的LAG-3表达与68Ga-NOTA-XH05的摄取之间呈正相关。在用CpG处理的带有B16-F10的小鼠中,与对照组相比,使用68Ga-NOTA-XH05的PET成像显示出更高的肿瘤与血液比率(TBR)。此外,从CpG处理的小鼠获得的TBR值允许应答者和非应答者之间的区分。在双侧皮下肿瘤模型中,仅右侧肿瘤用瘤内注射CpG治疗,左侧肿瘤的TBR值明显高于对照组,表明68Ga-NOTA-XH05能有效监测CpG局部注射的全身效应。
结论:我们的发现强调了68Ga-NOTA-XH05在评估肿瘤内LAG-3表达水平和评估免疫治疗反应方面的检测能力。从而表明有希望的临床转化前景。
BACKGROUND: Lymphocyte activation gene 3 (LAG-3) is expressed on activated immune cells and has emerged as a promising target for immune checkpoints blockade. However, conflicting findings have been reported regarding the association between LAG-3 expression in tumors and patient prognosis, indicating the need for further investigation into the significance of LAG-3 expression levels in tumor therapies. In this study, 68Ga-NOTA-XH05, a novel peptide-based positron emission tomography (PET) tracer targeting LAG-3, was constructed to non-invasively detect LAG-3 expression in melanoma after CpG oligonucleotide (CpG) treatment and explore the relationship between LAG-3 expression and therapeutic effect.
METHODS: The tracer 68Ga-NOTA-XH05 was identified by high-performance liquid chromatography after being prepared and purified. Cell uptake and blocking essays were performed to verify the specificity of the tracer in vitro. The expression of LAG-3 in B16-F10 subcutaneous tumors was monitored by flow cytometry, and its correlation with the tracer uptake was analyzed to evaluate the tracer specificity. PET imaging and biodistribution studies were conducted after CpG treatment of unilateral or bilateral B16-F10 subcutaneous tumor models to assess the ability of 68Ga-NOTA-XH05 in monitoring immunotherapy efficacy and the abscopal effect of CpG.
RESULTS: Following purification, 68Ga-NOTA-XH05 exhibited high radiochemical purity and specificity. Flow cytometry analysis revealed a positive correlation between LAG-3 expression in tumors and the uptake of 68Ga-NOTA-XH05. In B16-F10 bearing mice treated with CpG, PET imaging using 68Ga-NOTA-XH05 demonstrated a higher tumor to blood ratio (TBR) compared with the control group. Furthermore, TBR values obtained from CpG-treated mice allowed for differentiation between responders and non-responders. In a bilateral subcutaneous tumor model where only right-sided tumors were treated with intratumoral injection of CpG, TBR values of left-sided tumors were significantly higher than those in the control group, indicating that 68Ga-NOTA-XH05 could effectively monitor the systemic effect of local CpG injection.
CONCLUSIONS: Our findings highlight the detection capability of 68Ga-NOTA-XH05 in assessing LAG-3 expression levels within tumors and evaluating response to immunotherapy, thereby suggesting promising clinical translational prospects.