Antibody fragments as inhibitors of Japanese radish acid phosphatase

Vh (heavy-chain variable region) and Vl (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and Mab-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar Vh and identical Vl domains. Initially, the Vh and Vl genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named Mab-11 and Mab-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified Vh and Vl genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18