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

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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