Tandemly repeated Fc domain augments binding avidities of antibodies for Fcgamma receptors, resulting in enhanced antibody-dependent cellular cytotoxicity.
Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antibodies exert anti-tumor effects. Here, we show that Fc multimerization augments the binding avidities for all of the low-affinity Fcgamma receptors, increasing ADCC activity very much. A chimeric antibody, designated M-Ab, was constructed with the V regions from mouse anti-CD20 mAb 1F5 and the C regions from human IgG1 and kappa chain. Two or three Fc domains were tandemly repeated downstream of the C-terminus of the M-Ab to give D0-Ab (Fc dimer Ab without a linker), T0-Ab (Fc trimer Ab without a linker), and T3-Ab (Fc trimer Ab with a (GGGGS)(3) linker in front of the second and third hinge regions). HPLC and SDS-PAGE analyses of the purified antibodies indicated that the H and L chains were appropriately linked with interchain disulfide bonds and that the Ab preparations did not contain aggregated molecules. Although flow cytometry indicated that Fc multimerization decreased the binding activity for CD20-bearing cells to 1/3 approximately 1/4, the binding avidities for the extracellular domains of low-affinity Fcgamma receptors were greatly augmented. The avidities were in the order of T3-Ab, T0-Ab, D0-Ab and M-Ab, with T3-Ab showing about 100 times greater avidity than M-Ab not only for FcgammaRIIIA, but also for FcgammaRIIA and FcgammaRIIB. The rank order of ADCC activity with human PBMC was the same, and T3-Ab induced ADCC at a 50-100 times less concentration, compared to M-Ab. These Fc tandem repeat antibodies are promising candidates for anti-tumor therapeutics, and should also be useful as tools to elucidate the biological roles of FcgammaRIIA, FcgammaRIIB, and FcgammaRIIIA.
Nagashima
Hiroaki
H
Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda, Chiba, Japan.
Tezuka
Tomoya
T
Tsuchida
Wakako
W
Maeda
Hiroaki
H
Kohroki
Junya
J
Masuho
Yasuhiko
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D018951:Antigens, CD20 / Q000276:immunology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000737:chemistry* / Q000276:immunology*; D020449:Repetitive Sequences, Amino Acid
D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D018076:DNA, Complementary; D017452:Receptors, IgG