DEMELOPMENT OF RAKIOLABELED SMALLER ANTI-TENASCIN-CANTIBODY FOR CLINICAL USE

制备小鼠单克隆抗tenascin- c （TNC）抗体杂杂瘤细胞、抗TNC抗体Fab片段和精制TNC，制备新的放射性标记小抗体。对小鼠抗tnc抗体（4F1OTT和4C8MS）重链和轻链可变结构域（分别为V_H和V_L）的DNA片段进行克隆和组合，得到编码5‘-VH-linker-VL-FLAG-Cys-3’序列的scFv基因片段。从分泌小鼠抗TNC抗体的杂交瘤细胞中提取总RNA，逆转录得到编码V_H和V_L的第一链cDNA。凝胶纯化V_H和VL DNA片段，用重叠延伸PCR进行拼接。该基因在大肠杆菌细胞的全质表达提供了可溶性的scFv蛋白，其对TNC的亲和力与祖抗体Fab片段相似。合成了双功能螯合剂（EMCS-Bz-EDTA），并与半胱氨酸残基在scFv的C-te - More末端诱导反应。111大鼠心肌梗死后静脉注射in -anti- tnc - scfv （Ml）。测量各器官的生物分布。经放射自显像观察，急性心肌梗死大鼠坏死区周围肉芽组织中可见高放射性的^<111> in -anti-TNC- scfv，与免疫组化检测的TNC分子定位相对应。为了验证其临床应用价值，我们将^<111>In-anti-TNC-Fab静脉注射至心肌梗死大鼠。双同位素单光子发射计算机断层扫描成像（SPECT）。两种mIn-anti-TNC-Fab在坏死区域的累积值均高于非特异性fab抗体^<111>。超声心动图显示心肌梗死后心肌增大。此外，将^<111>In-anti- tnc - fab注射到实验性胶质瘤小鼠体内，揭示了肿瘤体内成像的可行性。该示踪剂可用于评价各种疾病。注射^<111>In-anti-TNC-Fab后的长期安全性也得到了认证。这项研究正计划申请专利。少

Hybridoma cells of mouse monoclonal anti-tenascin-C (TNC) antibody, Fab fragments of anti-TNC antibody, and refined TNC were prepared to make new radiolabeled smaller antibody against TNC. The DNA fragments encoding variable domains of heavy and light chains (V_H and V_L, respectively) in a mouse anti-TNC antibody (4F1OTT and 4C8MS) were cloned and combined to give the scFv gene fragment encoding the sequence of 5'-VH-linker-VL-FLAG-Cys-3'. The total RNA was extracted from hybridoma cells secreting mouse anti TNC antibody and reverse-transcribed to produce the first-strand cDNA encoding the V_H and V_L,. The V_H and VL DNA fragments were gel-purified and were spliced by overlap extension PCR. Peri plasmic expression of this gene in E. coli cells provided soluble scFv proteins that showed similar affinity to TNC to that of the Fab fragment of progenitor antibody.The bifunctional chelating agent (EMCS-Bz-EDTA) was synthesized and reacted with a cysteine residue, which was induced at C-te … More rminal of the scFv. 111In-anti-TNC-scFv was injected intravenously in rats after producing myocardial infarction (Ml). Biodistribution of each organ was measured. By autoradiography high radioactivity of ^<111>In-anti-TNC-scFv was observed in the granulation tissue around the necrotic area in the acute MI rat, which corresponds to the localization of TNC molecule detected by immunohistochemistry.To validate clinical usefulness, ^<111>In-anti-TNC-Fab was injected intravenously to MI rat. Dual-isotope single-photon emission computed tomography imaging (SPECT) was performed. Two kinds of mIn-anti-TNC-Fab accumulated higher than ^<111>In-non-specific-Fab antibody at necrotic area. A rat with larger accumulation of win-anti-INC-Fab at heart showed myocardial enlargement after MI by echocardiography. In addition, ^<111>In-anti-TNC-Fab was injected into experimental glioma mice and revealed the feasibility of in vivo imaging for tumor. This tracer was useful for evaluating various diseases. Long term safety after injection of ^<111>In-anti-TNC-Fab was also certified.This research is planning to apply a patent. Less