RETARGETING OF CTL BY AN EFFICIENTLY REFOLDED BISPECIFIC SINGLE-CHAINFV DIMER PRODUCED IN BACTERIA

A single-chain bispecific Fv dimer (bs(sFv)(2)) having specificity for mouse CD3 epsilon chain and human transferrin receptor was produced i n bacterial inclusion bodies. To overcome difficulties associated with in vitro protein folding, we used a novel renaturation approach to ob tain active bs(sFv)(2). The protein was dissolved in the weak ionic de tergent sodium lauroylsarcosine, and disulfides were formed by oxidati on in air. After oxidation, the bs(sFv)(2) exhibited very little coval ent aggregation and migrated as a single species in nonreducing SDS-PA CE, suggesting that disulfides were correctly paired. The detergent wa s removed using an ion exchange resin and the protein fractionated by size exclusion chromatography. The recovered 65-kDa protein was monome ric in nondenaturing solvent, homogeneous by SDS-PAGE, and comprised 1 5 to 20% of material applied to the gel filtration column. This protei n bound specifically to both mouse CD3 epsilon chain and human transfe rrin receptor with affinities indistinguishable from those of the pare ntal Fabs or single-chain Fvs. The bs(sFv)(2) specifically redirected mouse cytotoxic T cells to lyse target cells expressing human transfer rin receptor at picomolar concentrations. Bacterially produced and det ergent oxidized bs(sFv)(2) molecules may therefore provide the abundan t amounts of homogeneous active material required to redirect cytotoxi c cells against tumors and other unwanted cells in animal models and i n patients.