Retargeting of CTL by an efficiently refolded bispecific single-chain Fv 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 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 non-denaturing 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.
Kurucz
I
I
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Titus
J A
JA
Jost
C R
CR
Jacobus
C M
CM
Segal
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011990:Receptors, Transferrin / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D000939:Epitopes; D007135:Immunoglobulin Variable Region; D011948:Receptors, Antigen, T-Cell; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; C061836:antigen T cell receptor, epsilon chain