In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display.

Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-C_H3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated.

Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-C_H3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days.

Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

Keywords: antibody fragments, near-infrared fluorescence imaging, HER3, ErbB3, IRDye800CW

体内成像受成像探针的半衰期，组织渗透，生物分布和亲和力的影响。免疫球蛋白G（IgG）由具有已知功能的离散域组成，可为具有所需成像特性的工程抗体片段提供模板。在这里，我们设计了由抗体域的不同组合组成的基于抗体的成像探针，并用近红外荧光染料IRDye800CW对其进行了标记，并评估了它们的体内成像特性。基于抗体的成像探针基于使用噬菌体展示分离的抗HER3抗原结合片段（Fab）。

方法：我们构建了六种基于抗HER3抗体的成像探针：单链可变片段（scFv），Fab，双抗体，scFv-C _H 3，scFv-Fc和IgG。将IRDye800CW标记的，基于抗体的探针注入带有FaDu异种移植物的裸鼠中，并评估它们在异种移植物，肝脏和肾脏中的分布。

结果：这些成像探针与重组HER3和HER3阳性细胞系FaDu结合。分子量小于60 kDa的小抗体片段（scFv，双抗体和Fab）在异种移植物中迅速积累（注射后2-4小时（hpi）之间最大积累）并主要通过肾脏清除。scFv-C _H 3（80 kDa）具有快速清除能力，并在2-3 hpi之间在异种移植物中达到峰值，并以与Fab和双抗体相当的速率从异种移植物中清除。IgG和scFv-Fc在异种移植物中持续长达72 hpi，并主要分布于异种移植物和肝脏。用IgG和scFv-Fc成像探针观察到最高的异种移植荧光信号，并持续2-3天。

结论：这些结果突出了使用抗体片段优化清除率，肿瘤标记和生物分布特性的实用性，以开发用于图像引导手术或PET成像的抗HER3探针。

关键词：抗体片段，近红外荧光成像，HER3，ErbB3，IRDye800CW