Cilliers Cornelius, Nessler Ian, Christodolu Nikolas, Thurber Greg M
Department of Chemical Engineering and ‡Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Mol Pharm. 2017 May 1;14(5):1623-1633. doi: 10.1021/acs.molpharmaceut.6b01091. Epub 2017 Mar 31.
Monoclonal antibodies labeled with near-infrared (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab-800CW and -AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.
用近红外（NIR）荧光团标记的单克隆抗体在疾病检测、术中成像以及临床前和临床环境中治疗性抗体的药代动力学表征方面具有潜在应用。最近的研究表明，在足够高的标记度（DoL）下，将NIR荧光团与抗体缀合可能会显著改变抗体的处置情况；然而，其他报告显示用NIR荧光团标记后影响极小。在本研究中，我们用NIR染料IRDye 800CW（800CW）或Alexa Fluor 680（AF680）以1.2和0.3个染料/抗体的比例标记两种临床批准的抗体，赫赛汀（曲妥珠单抗）和阿瓦斯汀（贝伐单抗），并研究荧光团缀合对抗体血浆清除率和组织分布的影响。在0.3的DoL下，AF680缀合物在17天内表现出与未标记抗体相似的清除率，而800CW缀合物在4天后出现差异，这表明AF680更适合长期药代动力学研究。在1.2的DoL下，800CW缀合物在数小时后比未标记抗体清除得更快，这与其他已发表的报告一致。贝伐单抗 - 800CW和 - AF680缀合物的组织生物分布与使用放射性标记物报道的文献生物分布非常吻合。然而，在680 nm处更高的组织自发荧光导致在低剂量（约2 mg/kg）和AF680的0.3 DoL下，背景以上的检测受限，这表明800CW更适合短期生物分布测量和术中成像。总体而言，我们的研究表明，对于非位点特异性标记的抗体（具有泊松分布），0.3或更低的DoL对于限制NIR荧光团对抗体药代动力学的影响是理想的。
