新型双模式近红外二区/MRI 纳米探针靶向 PD-L1，准确评估免疫疗法治疗三阴性乳腺癌的疗效。

BACKGROUND: Durable responses to immune-checkpoint blocking therapy (ICT) targeting programmed cell death protein-1/ligand-1 (PD-1/PD-L1) have improved outcomes for patients with triple negative breast cancer (TNBC). Unfortunately, only 19-23% of patients benefit from ICT. Hence, non-invasive strategies evaluating responses to therapy and selecting patients who will benefit from ICT are critical issues for TNBC immunotherapy. METHODS: We developed a novel nanoparticle-Atezolizumab (NPs-Ate) consisting of indocyanine green (ICG), gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA), human serum albumin (HSA), and Atezolizumab. The efficiency of Gd-DTPA linking was verified using mass spectrometry, and the size of NPs-Ate was characterized using Nano-flow cytometry. The synthesized NPs-Ate were evaluated for fluorescence stability, penetration depth, and target specificity. TNBC cell lines and tumor-bearing mice models were used to identify the feasibility of this dual-modal second near-infrared/magnetic resonance imaging (NIR-II/MRI) system. Additionally, ICT combination with chemotherapy or radiotherapy in TNBC tumor-bearing mice models were used to assess dynamic changes of PD-L1 and predicted therapeutic responses with NPs-Ate. RESULTS: Atezolizumab, a monoclonal antibody, was successfully labeled with ICG and Gd-DTPA to generate NPs-Ate. This demonstrated strong fluorescence signals in our NIR-II imaging system, and relaxivity (γ1) of 9.77 mM s. In tumor-bearing mice, the NIR-II imaging signal background ratio (SBR) reached its peak of 11.51 at 36 hours, while the MRI imaging SBR reached its highest as 1.95 after 12 hours of tracer injection. NPs-Ate specifically targets cells and tumors expressing PD-L1, enabling monitoring of PD-L1 status during immunotherapy. Combining therapies led to inhibited tumor growth, prolonged survival, and increased PD-L1 expression, effectively monitored using the non-invasive NPs-Ate imaging system. CONCLUSION: The NIR-II/MRI NPs-Ate effectively reflected PD-L1 status during immunotherapy. Real-time and non-invasive immunotherapy and response/prognosis monitoring under NIR-II/MRI imaging guidance in TNBC is a promising and innovative technology with potential for extensive clinical applications in the future.

背景：针对程序性死亡蛋白-1/配体-1（PD-1/PD-L1）的免疫检查点阻断疗法（ICT）的持久反应改善了三阴性乳腺癌（TNBC）患者的预后。不幸的是，只有 19-23%的患者受益于 ICT。因此，评估治疗反应和选择将从 ICT 中获益的患者的非侵入性策略是 TNBC 免疫治疗的关键问题。

方法：我们开发了一种由吲哚菁绿（ICG）、钆二乙三胺五乙酸（Gd-DTPA）、人血清白蛋白（HSA）和阿特珠单抗组成的新型纳米颗粒-阿特珠单抗（NPs-Ate）。使用质谱验证了 Gd-DTPA 连接的效率，并使用纳米流式细胞术对 NPs-Ate 的粒径进行了表征。评估了合成的 NPs-Ate 的荧光稳定性、穿透深度和靶向特异性。使用 TNBC 细胞系和荷瘤小鼠模型来确定这种双模态近红外二区/磁共振成像（NIR-II/MRI）系统的可行性。此外，在 TNBC 荷瘤小鼠模型中，将 ICT 与化疗或放疗联合使用，以评估 PD-L1 的动态变化，并使用 NPs-Ate 预测治疗反应。

结果：阿特珠单抗，一种单克隆抗体，成功地用 ICG 和 Gd-DTPA 标记生成了 NPs-Ate。这在我们的近红外二区成像系统中显示出了很强的荧光信号，弛豫率（γ1）为 9.77 mM s。在荷瘤小鼠中，NIR-II 成像信号背景比（SBR）在 36 小时达到峰值 11.51，而 MRI 成像 SBR 在注射示踪剂 12 小时后达到最高值 1.95。NPs-Ate 特异性靶向表达 PD-L1 的细胞和肿瘤，能够在免疫治疗期间监测 PD-L1 状态。联合治疗导致肿瘤生长受到抑制，生存时间延长，PD-L1 表达增加，使用非侵入性的 NPs-Ate 成像系统可以有效地进行监测。

结论：NIR-II/MRI NPs-Ate 能有效反映免疫治疗过程中的 PD-L1 状态。在 NIR-II/MRI 成像引导下，对 TNBC 进行实时、非侵入性的免疫治疗及反应/预后监测是一种很有前途和创新的技术，具有广泛的临床应用前景。