Mahalingam Sakkarapalayam M, Dudkin Vadim Y, Goldberg Shalom, Klein Donna, Yi Fang, Singhal Sunil, O'Neil Karyn T, Low Philip S
Janssen Research & Development , 1400 McKean Road, Springhouse, Pennsylvania 19477, United States.
Department of Surgery, University of Pennsylvania Perelman School of Medicine , Philadelphia, Pennsylvania 19104, United States.
Bioconjug Chem. 2017 Nov 15;28(11):2865-2873. doi: 10.1021/acs.bioconjchem.7b00566. Epub 2017 Oct 17.
Tumor-targeted near-infrared fluorescent dyes have the potential to improve cancer surgery by enabling surgeons to locate and resect more malignant lesions where good visualization tools are required to ensure complete removal of malignant tissue. Although the tumor-targeted fluorescent dyes used in humans to date have been either small organic molecules or high molecular weight antibodies, low molecular weight protein scaffolds have attracted significant attention because they penetrate solid tumors almost as efficiently as small molecules, but can be infinitely mutated to bind almost any antigen. Here we describe the use of a 10 kDa protein scaffold, a Centyrin, to target a near-infrared fluorescent dye to tumors that overexpress the epidermal growth factor receptor (EGFR) for fluorescence-guided surgery (FGS). We have developed and optimized the dose and time required for imaging small tumor burdens with minimal background fluorescence in real-time fluorescence-guided surgery of EGFR-expressing tumor xenografts in murine models. We demonstrate that the Centyrin-near-infrared dye conjugate (CNDC) binds selectively to human EGFR cancer cells with an EC of 2 nM, localizes to EGFR tumor xenografts in athymic nude mice and that uptake of the dye in xenografts is significantly reduced when EGFR are blocked by preinjection of excess unlabeled Centyrin. Taken together, these data suggest that CNDCs can be used for intraoperative identification and surgical removal of EGFR-expressing lesions and that Centyrins targeted to other tumor-specific antigens should prove similarly useful in fluorescence guided surgery of cancer. In addition, we demonstrate that the CNDC is detected in the NIR region of the spectrum and can be utilized for fluorescence-guided surgery (FGS). In addition, we propose that with its eventual complete clearance from EGFR-negative tissues and its quantitative retention in the tumor mass for >24 h, a Centyrin-targeted NIR dye should provide excellent tumor contrast when injected at least 6-8 h before initiation of cancer surgery in human patients.
肿瘤靶向近红外荧光染料有潜力改善癌症手术，使外科医生能够定位并切除更多恶性病变，因为在确保完全切除恶性组织时需要良好的可视化工具。尽管迄今为止用于人类的肿瘤靶向荧光染料要么是小分子有机化合物，要么是高分子量抗体，但低分子量蛋白质支架已引起了极大关注，因为它们穿透实体瘤的效率几乎与小分子一样高，但可以进行无限突变以结合几乎任何抗原。在此，我们描述了使用一种10 kDa的蛋白质支架（一种Centyrin），将近红外荧光染料靶向到过表达表皮生长因子受体（EGFR）的肿瘤，用于荧光引导手术（FGS）。我们已经开发并优化了在小鼠模型中对表达EGFR的肿瘤异种移植进行实时荧光引导手术时，以最小背景荧光成像小肿瘤负荷所需的剂量和时间。我们证明，Centyrin-近红外染料偶联物（CNDC）以2 nM的半数有效浓度（EC）选择性地与人EGFR癌细胞结合，定位于无胸腺裸鼠的EGFR肿瘤异种移植中，并且当通过预先注射过量未标记的Centyrin阻断EGFR时，染料在异种移植中的摄取显著减少。综上所述，这些数据表明CNDC可用于术中识别和手术切除表达EGFR的病变，并且靶向其他肿瘤特异性抗原的Centyrin在癌症的荧光引导手术中应同样有用。此外，我们证明CNDC在光谱的近红外区域被检测到，可用于荧光引导手术（FGS）。此外，我们提出，由于其最终从EGFR阴性组织中完全清除，并在肿瘤块中定量保留超过24小时，当在人类患者开始癌症手术前至少6 - 8小时注射时，Centyrin靶向的近红外染料应能提供出色的肿瘤对比度。
