Construction, expression and tumor targeting of a single-chain Fv against human colorectal carcinoma

Abstract

AIM: A single-chain antibody fragment, ND-1scFv, against human colorectal carcinoma was constructed and expressed in E.coli, and its biodistribution and pharmacokinetic properties were studied in mice bearing tumor. METHODS: V_H and V_L genes were amplified from hybridoma cell IC-2, secreting monoclonal antibody ND-1, by RT-PCR, and connected by linker (Gly₄Ser)₃ to form scFv gene, which was cloned into expression vector pET 28a(+) and finally expressed in E.coli. The expressed product ND-1scFv was purified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDS-PAGE and ELISA. ND-1scFv was labeled with ^99mTc, and then injected into mice bearing colorectal carcinoma xenograft for phamacokinetic study in vivo. RESULTS: SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 30 kDa with purity of 94%. ELIAS assay revealed that ND-1scFv retained the immunoactivity of parent mAb, being capable of binding specifically to human colorectal carcinoma cell line expressing associated antigen. Radiolabeled ND-1scFv exhibited rapid tumor targeting, with specific distribution in mice bearing colorectal carcinoma xenograft observed as early as 1 h following injection. In vivo pharmacokinetic studies also demonstrated that ND-1scFv had very rapid plasma clearance (T_1/2α of 5.7 min, T_1/2β of 2.6 h). CONCLUSION: ND-1scFv shows significant immunoactivity, and better pharmacokinetic and biodistribution characteristics compared with intact mAbs, demonstrating the possibility as a carrier for tumor-imaging.