Retargeting of CTL by an efficiently refolded bispecific single-chain Fv dimer produced in bacteria
Kurucz, I.; Titus, J.A.; Jost, C.R.; Jacobus, C.M.; Segal, D.M.
Journal of Immunology 154(9): 4576-4582
1995
ISSN/ISBN: 0022-1767 PMID: 7536774 Document Number: 450311
A single-chain bispecific Fv dimer (bs(sFV)-2) having specificity for mouse CD3-epsilon chain and human transferrin receptor was produced in bacterial inclusion bodies. To overcome difficulties associated with in vitro protein folding, we used a novel renaturation approach to obtain active bs(sFV)-2. The protein was dissolved in the weak ionic detergent sodium lauroylsarcosine, and disulfides were formed by oxidation in air. After oxidation, the bs(sFV)-2 exhibited very little covalent aggregation and migrated as a single species in nonreducing SDS-PAGE, suggesting that disulfides were correctly paired. The detergent was removed using an ion exchange resin and the protein fractionated by size exclusion chromatography. The recovered 65-kDa protein was monomeric in nondenaturing solvent, homogeneous by SDS-PAGE, and comprised 15 to 20% of material applied to the gel filtration column. This protein bound specifically to both mouse CD3-epsilon chain and human transferrin receptor with affinities indistinguishable from those of the parental Fabs or single-chain Fvs. The bs(sFv)-2 specifically redirected mouse cytotoxic T cells to lyse target cells expressing human transferrin receptor at picomolar concentrations. Bacterially produced and detergent oxidized bs(sFV)-2 molecules may therefore provide the abundant amounts of homogeneous active material required to redirect cytotoxic cells against tumors and other unwanted cells in animal models and in patients.