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 (Gly(4)Ser) (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 (99m)Tc, 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 30kDa 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(1/2)alpha of 5.7 min, T(1/2)beta 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.

journal_name

World J Gastroenterol

authors

Fang J,Jin HB,Song JD

doi

10.3748/wjg.v9.i4.726

keywords:

subject

Has Abstract

pub_date

2003-04-01 00:00:00

pages

726-30

issue

4

eissn

1007-9327

issn

2219-2840

journal_volume

9

pub_type

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