Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas

Background
A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models.
Methods
An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments.
Results
89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models.
Conclusion
A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM.

背景
多形性胶质母细胞瘤（Glioblastoma Multiforme, GBM）诊疗领域的一项核心挑战，在于以无创方式实现肿瘤的精准诊断与进展评估。我们已鉴定出膜型1基质金属蛋白酶（Membrane-type 1 matrix metalloproteinase, MT1-MMP）可作为GBM成像的理想生物标志物：该蛋白积极参与肿瘤生长与进展，与肿瘤分级密切相关，且与GBM患者的不良预后显著关联。本研究报道了一款可在GBM模型中有效检测MT1-MMP的免疫PET（immunoPET）示踪剂的开发过程。
方法
将抗人MT1-MMP单克隆抗体（monoclonal antibody, mAb）LEM2/15与对异硫氰酸苄基去铁胺（p-isothiocyanatobenzyl-desferrioxamine, DFO-NCS）偶联，用于89Zr标记。我们在接种表达MT1-MMP的人GBM细胞（U251）以及作为阴性对照的不表达MT1-MMP的乳腺癌细胞（MCF-7）的异种移植小鼠中开展了生物分布与PET成像研究。此外，我们还采用了两种具有不同血脑屏障（blood-brain barrier, BBB）破坏程度的原位脑GBM模型——患者来源神经球（TS543）模型与U251细胞模型，用于PET成像实验。
结果
DFO-LEM2/15的89Zr标记实现了高标记产率（>90%）与高比活度（78.5 MBq/mg）。生物分布实验结果表明，89Zr-DFO-LEM2/15作为放射性示踪剂，在检测MT1-MMP阳性GBM肿瘤方面具有优异潜力。PET成像结果同样显示，与MT1-MMP阴性的MCF-7乳腺肿瘤相比，MT1-MMP阳性的U251 GBM肿瘤中出现了特异性且显著的89Zr-DFO-LEM2/15摄取。原位脑GBM模型的实验结果表明，示踪剂向肿瘤内的渗透高度依赖于被破坏的血脑屏障：与BBB通透性仅轻度升高的U251原位模型相比，89Zr-DFO-LEM2/15在BBB高度破坏的TS543肿瘤中展现出更高的聚集水平。组织学分析证实了该免疫偶联物在所有GBM模型中的检测特异性。
结论
本研究高效合成了一款新型抗MT1-MMP单克隆抗体示踪剂89Zr-DFO-LEM2/15。体内验证实验结果表明，该示踪剂可对MT1-MMP阳性GBM肿瘤实现高特异性对比度成像，同时为靶向MT1-MMP的免疫PET替代GBM非特异性成像提供了有力证据。