利用新型mCD3纤维连接蛋白支架对活体动物肿瘤浸润淋巴细胞的分子成像。

IF 3.9 2区 化学 Q1 BIOCHEMICAL RESEARCH METHODS
Bioconjugate Chemistry Pub Date : 2025-01-15 Epub Date: 2024-12-16 DOI:10.1021/acs.bioconjchem.4c00501
Char Wynter, Arutselvan Natarajan, Clyde John, Kaahini Jain, Ramasamy Paulmurugan
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引用次数: 0

摘要

肿瘤微环境(tumor microenvironment, TME)中癌细胞与免疫细胞的相互作用在决定肿瘤生长、转移和治疗反应中起着至关重要的作用。TME中的肿瘤浸润淋巴细胞(til)可能是各种治疗干预措施(包括化疗和免疫治疗)治疗反应的预测指标。因此,肿瘤免疫微环境成像对于选择最佳治疗策略具有重要意义。CD3蛋白是体内TILs诊断成像评估TME免疫状态的一个有希望的靶标。尽管许多抗cd3抗体已被探索用于此应用,但这些抗体的非特异性免疫激活限制了它们的应用。为了克服这一问题,我们利用酵母展示文库设计了一种新的纤维连接蛋白III结构域(FN3)蛋白结合物(mCD3-FN3;11.8 kDa)来对抗小鼠CD3抗原蛋白,以显示TILs在体内归巢到TME中。我们进行了体外和体内实验,以测试mCD3-FN3结合物的纯度以及在小鼠同基因肿瘤模型中的体内靶向性。我们使用与mCD3-FN3偶联的近红外800染料(IR800-mCD3-FN3)通过光学成像在体内跟踪TILs。我们使用三种不同的小鼠同基因肿瘤(C57BL/6小鼠的mCD3+ EL4肿瘤,BALB/c小鼠的mCD3- CT26结肠肿瘤和mCD3- 4T1乳腺肿瘤)在体内对TILs进行成像。将携带EL4肿瘤的C57BL/6小鼠分为阻断组[Blk]和非阻断组[Nblk];N = 3 /组),用于体内成像。阻断组在给予IR800-mCD3-FN3结合剂前2小时给予200 μg未标记的mCD3-FN3。每只小鼠注射25 μg的IR800-mCD3-FN3粘合剂,并使用IVIS光学成像系统跟踪一段时间。C57BL/6/EL4小鼠在注射IR800-mCD3-FN3结合剂后4和24 h成像,最终成像后24 h收集小鼠器官,用于离体组织学成像。在CT26和4T1肿瘤模型中,注射黏合剂后4、24和48 h对TME中的TILs进行成像。EL4肿瘤的近红外成像显示,IR800-mCD3-FN3在初始结合剂注射24 h后,能以高信本比检测到肿瘤和肿瘤细胞内的TILs,总辐射效率(平均TRE±SD)为6.5 × 1010±1.5 × 1010[光子/s]/[μW/cm2]。在IR800-mCD3-FN3结合剂给药前预先注射未标记的mCD3-FN3(Blk),与EL4-Nblk肿瘤相比,肿瘤中荧光信号明显减少(平均TRE±SD: 1.6 × 1010±4.1 × 109) (p = 0.006)。CT26和4T1肿瘤小鼠组,探针只能与肿瘤内的TILs结合,在48 h p.i.显示特异性成像信号(平均TRE±SD)分别为1.1 × 1011±5.2 × 1010和9.5 × 1010±4.6 × 1010。对于这些组,CT26和4T1肿瘤的离体肿瘤与肌肉的比例分别为20倍和27倍。这些结果清楚地证明了mCD3- fn3结合物与TME中T细胞表达的mCD3标记物的体内结合能力。肿瘤和EL4肿瘤动物器官的离体组织学分析以及CT26和4T1肿瘤的TILs成像(48 p.i)证实IR800-mCD3-FN3探针能够特异性结合T细胞表达的CD3标记物。综上所述,体外和体内数据表明,本研究所构建的mCD3- fn3结合物是一种很有前景的配体,可用于体内肿瘤的诊断成像,用于评估TME中表达TILs的mCD3。这可以作为评估肿瘤对治疗干预反应的预后标记物,以及成像肿瘤对免疫检查点阻断癌症治疗反应的诊断标记物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Molecular Imaging of Tumor-Infiltrating Lymphocytes in Living Animals Using a Novel mCD3 Fibronectin Scaffold.

The interaction between cancer cells and immune cells in the tumor microenvironment (TME) plays a crucial role in determining tumor growth, metastasis, and response to treatment. Tumor-infiltrating lymphocytes (TILs) in TME could be a predictive marker for treatment response in various therapeutic interventions, including chemotherapy and immunotherapy. Thus, imaging the tumor immune microenvironment is important for selecting the optimal treatment strategies in cancer therapy. The CD3 protein represents a promising target for diagnostic imaging of TILs in vivo to assess the immune state of the TME. Although many anti-CD3 antibodies have been explored for this application, the nonspecific immune activation by these antibodies limits their applications. To overcome this issue, we engineered a novel fibronectin III domain (FN3) protein binder (mCD3-FN3;11.8 kDa) against mouse CD3 antigen protein using a yeast display library to image TILs homing in vivo into the TME. We performed in vitro and in vivo assays to test the mCD3-FN3 binder purity as well as in vivo targetability in mouse models of syngeneic tumors. We used near-infrared 800 dye conjugated with mCD3-FN3 (IR800-mCD3-FN3) for in vivo tracking of TILs via optical imaging. We used three different syngeneic tumors in mice (mCD3+ EL4 tumor in C57BL/6 mice, mCD3- CT26 colon tumor, and mCD3- 4T1 breast tumor in BALB/c mice) for imaging TILs in vivo. C57BL/6 mice bearing EL4 tumors were separated into two groups (blocking [Blk] and nonblocking [Nblk]; n = 3 per group) and used for in vivo imaging. Blocking groups received 200 μg of unlabeled mCD3-FN3 2 h prior to the administration of IR800-mCD3-FN3 binder. Each mouse was administered with 25 μg of the IR800-mCD3-FN3 binder and tracked using an IVIS optical imaging system over time. C57BL/6/EL4 mice were imaged at 4 and 24 h post injection of the IR800-mCD3-FN3 binder, and mouse organs were collected at 24 h after final imaging and used for ex vivo histological imaging. In CT26 and 4T1 tumor models, TILs in TME were imaged 4, 24, and 48 h after binder injection. The NIR imaging of EL4 tumors showed that IR800-mCD3-FN3 can detect both TILs within the tumor and the tumor cells with a high signal-to-background ratio 24 h after initial binder injection with a total radiant efficiency (mean TRE ± SD) of 6.5 × 1010 ± 1.5 × 1010 [photons/s]/[μW/cm2]. The animals received preinjection of unlabeled mCD3-FN3(Blk) prior to IR800-mCD3-FN3 binder administration and showed a significant level of fluorescence signal reduction (mean TRE ± SD: 1.6 × 1010 ± 4.1 × 109) in the tumor when compared to the EL4-Nblk tumors (p = 0.006). The mouse group with CT26 and 4T1 tumors where the probe can only bind to TILs within the tumor showed a specific imaging signal (mean TRE ± SD) of 1.1 × 1011 ± 5.2 × 1010 and 9.5 × 1010 ± 4.6 × 1010, respectively, at 48 h p.i. For these groups, the ex vivo tumor-to-muscle ratios were 20- and 27-fold for CT26 and 4T1 tumors, respectively. These results clearly demonstrate the in vivo binding ability of the mCD3-FN3 binder to mCD3 marker expressed by T cells in the TME. The ex vivo histological analysis of tumors, and the organs of animals with EL4 tumors, and TILs imaging of CT26, and 4T1 tumors (at 48 p.i.) confirmed that the IR800-mCD3-FN3 probe was able to specifically bind to CD3 markers expressed by the T cells. In summary, both in vitro and in vivo data indicated that the engineered mCD3-FN3 binder by this study is a promising ligand for diagnostic imaging of tumors in vivo for the assessment of mCD3 expressing TILs in the TME. This can be used as a prognostic marker in evaluating tumor response to therapeutic intervention as well as a diagnostic marker in imaging tumor response to immune checkpoint blockade cancer therapies.

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来源期刊
Bioconjugate Chemistry
Bioconjugate Chemistry 生物-化学综合
CiteScore
9.00
自引率
2.10%
发文量
236
审稿时长
1.4 months
期刊介绍: Bioconjugate Chemistry invites original contributions on all research at the interface between man-made and biological materials. The mission of the journal is to communicate to advances in fields including therapeutic delivery, imaging, bionanotechnology, and synthetic biology. Bioconjugate Chemistry is intended to provide a forum for presentation of research relevant to all aspects of bioconjugates, including the preparation, properties and applications of biomolecular conjugates.
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