Published online Apr 15, 2003. doi: 10.3748/wjg.v9.i4.726
Revised: December 25, 2002
Accepted: January 2, 2003
Published online: April 15, 2003
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: VH and VL 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 (T1/2α of 5.7 min, T1/2β 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.