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


CONCLUSIONS
ND-1scFv shows significant immunoactivity, and better pharmacokinetic and biodistribution characteristics compared with intact mAbs, demonstrating the possibility as a carrier for tumor-imaging.
Fang
Jin
J
Key Lab of Cell Biology, Ministry of Public Health of China, China Medical University, 92 Beier Road, Heping District, Shenyang 110001, Liaoning Province, China.
Jin
Hong-Bin
HB
Song
Jin-Dan
JD
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000628:therapy*; D006207:Half-Life; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000627:therapeutic use*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000627:therapeutic use*; D008657:Metabolic Clearance Rate; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D057127:Single-Chain Antibodies
D000970:Antineoplastic Agents; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C475886:Nd-1scFv; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C055966:immunoglobulin Fv