一种 3E8.scFv.Cys-IR800 缀合物，在原位结直肠癌模型中靶向 TAG-72。
A 3E8.scFv.Cys-IR800 Conjugate Targeting TAG-72 in an Orthotopic Colorectal Cancer Model.
机构信息
Department of Radiology, The Wright Center for Innovation in Biomedical Imaging, The Ohio State University, Columbus, OH, USA.
Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH, USA.
出版信息
Mol Imaging Biol. 2018 Feb;20(1):47-54. doi: 10.1007/s11307-017-1096-4.
PURPOSE
Optical surgical navigation (OSN) will be a potent tool to help surgeons more accurately and efficiently remove tumors. The purpose of this study was to evaluate a novel humanized 3E8 antibody (3E8 MAb) fragment site-specifically conjugated with IR800, 3E8.scFv.Cys-IR800, as a potential OSN agent to target colorectal adenocarcinoma.
PROCEDURES
An engineered single-chain variable fragment of 3E8 MAb (targeted to TAG-72), appending a C-terminal cysteine residue (3E8.scFv.Cys), was created and reacted with IRDye800-maleimide. 3E8.scFv.Cys-IR800 identity and purity were verified by MALDI-TOF mass spectra and 800 nm detected size exclusion column HPLC. In vitro human colon adenocarcinoma LS-174 T cells binding and competition assay validated biological functionality. We further evaluated the imaging ability and receptor-specific binding of 3E8.scFv.Cys-IR800 in an orthotopic LS-174 T mouse model.
RESULTS
A 1:1 dye to protein conjugate was achieved at greater than 90 % HPLC purity. A 1 nmol dose of 3E8.scFv.Cys-IR800 via intraperitoneal injection administration was sufficient to produce high tumor to background fluorescence contrast. Blocking competition studies both in vitro and in vivo using a different blocking protein, 3E8ΔCH2, demonstrated 3E8.scFv.Cys-IR800 binding specificity for TAG-72 antigen.
CONCLUSIONS
3E8.scFv.Cys-IR800 shows properties useful in a clinically viable OSN agent for colorectal cancer.
目的
光学手术导航（OSN）将成为帮助外科医生更准确、更有效地切除肿瘤的有力工具。本研究旨在评估一种新型的 3E8 抗体（3E8 MAb）片段，该片段特异性地与人源化 3E8 单链抗体（scFv）融合，并通过半胱氨酸连接到 IR800 上（3E8.scFv.Cys-IR800），作为一种潜在的用于靶向结直肠腺癌的 OSN 示踪剂。
方法
构建了靶向 TAG-72 的 3E8 MAb 的工程化单链可变片段（3E8.scFv.Cys），并在 C 末端添加一个半胱氨酸残基，然后与 IRDye800-马来酰亚胺反应。通过 MALDI-TOF 质谱和 800nm 检测的尺寸排阻柱 HPLC 验证 3E8.scFv.Cys-IR800 的身份和纯度。通过体外人结肠腺癌细胞 LS-174T 细胞结合和竞争实验验证了其生物学功能。我们进一步评估了 3E8.scFv.Cys-IR800 在 LS-174T 原位移植瘤小鼠模型中的成像能力和受体特异性结合。
结果
实现了大于 90%HPLC 纯度的 1:1 染料与蛋白偶联物。腹腔注射 1nmol 的 3E8.scFv.Cys-IR800 足以产生高肿瘤与背景的荧光对比度。体内和体外使用不同的阻断蛋白（3E8ΔCH2）进行阻断竞争研究，证明了 3E8.scFv.Cys-IR800 对 TAG-72 抗原的特异性结合。
结论
3E8.scFv.Cys-IR800 具有成为结直肠癌临床可行的 OSN 示踪剂的特性。
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