Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California
Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California.
J Nucl Med. 2019 Oct;60(10):1467-1473. doi: 10.2967/jnumed.118.223560. Epub 2019 Mar 15.
Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with F-labeled -cyclooctene (F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. We successfully synthesized a DML for conjugation of a fluorescent dye and F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.
基于抗体的双模（PET/荧光）成像可实现非侵入性全身评估的术前抗原特异性免疫 PET，以及用于可视化浅层组织层的术中荧光，以实现图像引导手术。我们开发了一种通用的双模连接子（DML），可促进与带有半胱氨酸残基的抗体片段进行位点特异性缀合，引入市售的荧光染料（通过胺反应性假体基团），并通过点击化学与 F 标记的环辛烯（F-TCO）快速有效地进行放射性标记。为了生成基于双模态抗体片段的成像剂，将 DML 用远红染料磺酸盐菁 5（sCy5）标记，位点特异性缀合到抗前列腺干细胞抗原（PSCA）cys-二抗体 A2 的 C 末端半胱氨酸上，然后通过点击化学与 F-TCO 进行放射性标记。在人 PSCA 阳性前列腺癌异种移植模型中，通过顺序免疫 PET 和光学成像评估了新的成像探针。通过体外生物分布证实了靶组织的摄取。我们成功合成了用于连接荧光染料和 F 的 DML。抗 PSCA cys-二抗体 A2 通过 DML 或 sCy5 进行位点特异性缀合，并通过点击化学与 F-TCO 进行放射性标记。免疫 PET 成像证实，在注射后 1 小时内即可对前列腺癌异种移植进行体内抗原特异性靶向。50 kDa 抗体片段的快速肾脏清除可实现当天成像。光学成像显示 PSCA 阳性异种移植中存在抗原特异性荧光信号，与周围组织和 PSCA 阴性异种移植形成高对比。DML 可实现抗体片段的抗原结合位点的位点特异性缀合，具有可控的连接子与蛋白质的比例，并将 2 种成像系统的信号分子组合到 1 个分子中。双模成像可以提供非侵入性的全身成像，具有器官水平的生物分布，并在手术期间通过荧光成像引导肿瘤边界的识别。
