Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies.
Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.
Kipriyanov
Sergey M
SM
Recombinant Antibody Research Group, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. s.kipriyanov@affimed.com
Moldenhauer
Gerhard
G
Braunagel
Michael
M
Reusch
Uwe
U
Cochlovius
Björn
B
Le Gall
Fabrice
F
Kouprianova
Olga A
OA
Von der Lieth
Claus Wilhelm
CW
Little
Melvyn
M
D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000941:Antigens / Q000378:metabolism; D018941:Antigens, CD19 / Q000235:genetics; D017252:CD3 Complex / Q000235:genetics; D019281:Dimerization; D004354:Drug Screening Assays, Antitumor; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D018033:Antibodies, Bispecific; D000941:Antigens; D018941:Antigens, CD19; D017252:CD3 Complex; D007106:Immune Sera; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv