Recent advances in the field of immune-oncology led to the discovery of next-generation immune checkpoints (ICPs). Lymphocyte activation gene-3 (LAG-3), being the most widely studied among them, is being explored as a target for the treatment of cancer patients. Several antagonistic anti-LAG-3 antibodies are being developed and are prime candidates for clinical application. Furthermore, validated therapies targeting cytotoxic T-lymphocyte–associated protein-4, programmed cell-death protein-1, or programmed cell-death ligand-1 showed that only subsets of patients respond. This finding highlights the need for better tools for patient selection and monitoring. The potential of molecular imaging to detect ICPs noninvasively in cancer is supported by several preclinical and clinical studies. Here, we report on a single-domain antibody to evaluate whole-body LAG-3 expression in various syngeneic mouse cancer models using nuclear imaging. Methods: SPECT/CT scans of tumor-bearing mice were performed 1 h after injection with radiolabeled single-domain antibody. Organs and tumors of mice were isolated and evaluated for the presence of the radiolabeled tracer and LAG-3–expressing immune cells using a -counter and flow cytometry respectively. PD-1/LAG-3–blocking antibodies were injected in MC38-bearing mice. Results: The radiolabeled single-domain antibody detected LAG-3 expression on tumor-infiltrating lymphocytes (TILs) as soon as 1 h after injection in MC38, MO4, and TC-1 cancer models. The single-domain antibody tracer visualized a compensatory upregulation of LAG-3 on TILs in MC38 tumors of mice treated with PD-1–blocking antibodies. When PD-1 blockade was combined with LAG-3 blockade, a synergistic effect on tumor growth delay was observed. Conclusion: These findings consolidate LAG-3 as a next-generation ICP and support the use of single-domain antibodies as tools to noninvasively monitor the dynamic evolution of LAG-3 expression by TILs, which could be exploited to predict therapy outcome.

免疫肿瘤学领域的最新进展导致了下一代免疫检查点（ICP）的发现。其中研究最广泛的淋巴细胞激活基因 3 (LAG-3) 正在被探索作为癌症患者的治疗靶点。几种拮抗性抗 LAG-3 抗体正在开发中，是临床应用的主要候选者。此外，针对细胞毒性 T 淋巴细胞相关蛋白 4、程序性细胞死亡蛋白 1 或程序性细胞死亡配体 1 的经过验证的疗法表明，只有部分患者有反应。这一发现凸显了需要更好的患者选择和监测工具。多项临床前和临床研究支持分子成像在癌症中无创检测 ICP 的潜力。在这里，我们报告了一种单域抗体，用于使用核成像评估各种同基因小鼠癌症模型中的全身 LAG-3 表达。方法：注射放射性标记单域抗体1小时后，对荷瘤小鼠进行SPECT/CT扫描。分离小鼠的器官和肿瘤，并分别使用计数器和流式细胞术评估放射性标记示踪剂和表达 LAG-3 的免疫细胞的存在。 PD-1/LAG-3 阻断抗体被注射到携带 MC38 的小鼠中。结果：在 MC38、MO4 和 TC-1 癌症模型中注射后 1 小时，放射性标记的单域抗体即可检测到肿瘤浸润淋巴细胞 (TIL) 上 LAG-3 的表达。单域抗体示踪剂显示了用 PD-1 阻断抗体治疗的小鼠 MC38 肿瘤中 TIL 上 LAG-3 的补偿性上调。当PD-1阻断剂与LAG-3阻断剂联合使用时，观察到对肿瘤生长延迟的协同作用。 结论：这些发现巩固了 LAG-3 作为下一代 ICP 的地位，并支持使用单域抗体作为工具来无创监测 TIL 表达 LAG-3 的动态演变，从而可用于预测治疗结果。