Production and characterization of bispecific single-chain antibody fragments.
We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli. The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells. The two domains are joined by a flexible peptide linker. Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions. After refolding of the bispecific protein, it was affinity-purified. As demonstrated by flow cytometry, both binding sites are retained in the refolded protein. Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv. Thus, these bispecific molecules show a potential anti-tumor activity.
De Jonge
J
J
Laboratory of Physiology, Medical School, Vrije Universiteit, Brussels, Belgium.
Brissinck
J
J
Heirman
C
C
Demanet
C
C
Leo
O
O
Moser
M
M
Thielemans
K
K
D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry; D006825:Hybridomas / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins