Pietersz G A, Patrick M R, Chester K A
Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.
J Nucl Med. 1998 Jan;39(1):47-56.
We describe the engineering of a novel single-chain fragment (scFv) metallothionein (MET) containing anti-carcinoembryonic antigen (CEA) antibody (referred to as MET-scFv) for use as a diagnostic imaging agent in colorectal cancer.
Site-directed cloning of annealed oligonucleotides, containing both the MET and a c-myc tag sequence, into a pUC19-based expression vector enabled soluble secreted protein expression from Escherichia coli. Affinity purification was used to purify the protein using an anti-c-myc affinity column. The specificity of both the unlabeled and labeled MET-scFv for CEA was demonstrated by solid-phase enzyme-linked immunosorbent assay and radioimmunoassay and by fluorescence-activated cell sorting analysis on CEA-expressing human colorectal LS-174T cells. Technetium-99m labeling was achieved using a Zn2+ transchelation step, enabling direct 99mTc transfer without separate reduction of MET. In vitro stability was demonstrated by fast protein liquid chromatography analysis of labeled MET-scFv, incubated with bovine serum albumin (BSA), transferrin and mouse serum. Last, in vivo pharmacokinetics, biodistribution and imaging were performed.
Yields of 6 mg/liter induced culture purified protein were achieved. Successful site-specific labeling was demonstrated using a Zn2+ transchelation modification of a pretinning method, which also enabled lower amounts of the reducing agent to be used. The specificity for CEA was retained after labeling. Despite a rapid serum clearance (t(1/2alpha) = 2.8 min), adequate localization to tumor of 5.37% injected dose/g at 4 hr was demonstrated. Moreover, the short-lived t(1/2alpha) of scFv, its early tumor targeting and rapid blood-pool clearance gave tumor-to-blood ratios of 2.07 by 4 hr, enabling early gamma camera imaging. Successful and specific imaging was achieved using LS-174T xenografts in nude mice by 3-6 hr.
A recombinant MET containing scFv was successfully expressed, purified and labeled with 99Tc. The stable site-specific labeling of 99Tc, combined with the rapid plasma clearance of the scFv, led to successful early in vivo imaging of xenografted mice.
我们描述了一种新型单链片段（scFv）金属硫蛋白（MET）的构建，该蛋白含有抗癌胚抗原（CEA）抗体（称为MET-scFv），用作结直肠癌的诊断成像剂。
通过将含有MET和c-myc标签序列的退火寡核苷酸定向克隆到基于pUC19的表达载体中，使大肠杆菌能够表达可溶性分泌蛋白。使用抗c-myc亲和柱通过亲和纯化来纯化蛋白质。通过固相酶联免疫吸附测定和放射免疫测定以及对表达CEA的人结肠LS-174T细胞进行荧光激活细胞分选分析，证明了未标记和标记的MET-scFv对CEA的特异性。使用锌离子转螯合步骤实现了锝-99m标记，无需单独还原MET即可直接进行99mTc转移。通过对与牛血清白蛋白（BSA）、转铁蛋白和小鼠血清一起孵育的标记MET-scFv进行快速蛋白质液相色谱分析，证明了其体外稳定性。最后，进行了体内药代动力学、生物分布和成像研究。
诱导培养的纯化蛋白产量达到6 mg/升。使用预锡化方法的锌离子转螯合修饰证明了成功的位点特异性标记，这也使得还原剂的用量减少。标记后对CEA的特异性得以保留。尽管血清清除迅速（t(1/2α)=2.8分钟），但在4小时时仍显示出对肿瘤的充分定位，为注射剂量的5.37%/克。此外，scFv的半衰期短（t(1/2α)）、早期肿瘤靶向和快速血池清除使得4小时时肿瘤与血液的比率为2.07，从而能够进行早期γ相机成像。在3至6小时内，使用裸鼠中的LS-174T异种移植瘤成功实现了特异性成像。
成功表达、纯化并使用99Tc标记了含有scFv的重组MET。99Tc的稳定位点特异性标记，结合scFv的快速血浆清除，导致异种移植小鼠体内早期成像成功。
