The monoclonal antibody (mAb) AF-20 binds to a cell-surface glycoprotein expressed uniformly and selectively on all primary hepatocellular carcinoma cells (HCC) and metastases examined thus far. The AF-20 mAb has also been showed to bind to other human tumor cells such as breast and colon thus suggesting that antigen expression may not be limited to HCC. In this study, we have cloned the variable region (V) gene of the AF-20 IgG from hybridoma mRNA and constructed an AF-20 single-chain Fv (scFv) which was expressed in soluble form in yeast. The AF-20 scFv was shown to bind specifically to the same epitope as mAb AF-20 with a binding affinity of 4nM. The AF-20 scFv was also internalized into tumor cells in a manner identical to that of the original mAb AF-20. The scFv was employed for cellular internalization of virus-sized fluorescent quantum dots. In addition, to demonstrate the versatility of the antibody, we constructed an immunotoxin composed of AF-20 scFv fused to the highly cytotoxic recombinant toxin gelonin (rGel). The AF20scFv/rGel fusion toxin was expressed in bacteria, purified and then evaluated for its efficacy against three different tumor cell lines in vitro. The IC50 of the AF-20 scFv/rGel was 100 nm, 35 nM and 3.5 nM respectively for log-phase FOCUS (HCC), L3.6pl (pancreatic) and PC3(prostate) tumor cells compared to the IC50 for rGel itself(2,000nM, 1,000nM and 100nM). The AF20 scFv/rGel construct was therefore between 20 and 30 fold more active against target cells than the free rGel toxin. This data further demonstrates a potentially broader capability of the AF-20 scFv to serve as a targeting module for tumors other than just hepatocellular carcinoma. The AF-20 scFv is a potential internalization vector for toxins, enzymes, radionuclides and virus particles for targeted therapy of HCC and other tumors. Supported by NSF biotechnology process engineering center (BPEC). Research conducted, in part, by the Clayton Foundation for Research.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]