Fig. 1.
TsAb3v1 and TsAb2v2 antibody generation. (A) Schematic representation of the two TsAb constructs. cMet and HER3 VH-(G4S)3-VL scFvs were fused to the C-terminus of ‘knob-into-hole’ heavy chains of an IgG1 antibody backbone by a (G4S)2 connector. N-terminal scFabs targeting HER1 and IGF1R were designed as VL-CL-(G4S)6GG-VH-CH1 in the TsAB2v2 construct. TsAb3v1 consists of an IGF1R targeting one arm scFab and a disulfide stabilized Fab binding to HER1. (B) SDS-PAGE analysis of purified TsAb3v1 and TsAb2v2. (C) Analytical SEC of purified TsAb3v1 and TsAb2v2. (D) SPR analysis of simultaneous binding of TsAb3v1 and TsAb2v2 to HER1, HER3, IGF1R and cMet. A color code of the injection order of the extracellular receptor domains of IGF1R, HER3 and cMet on the tetraspecific antibodies bound to a HER1-coated CM5 chip is depicted below the graphs.