Schematic representation of the different constructions of 56-65 combination. V1 is the initial version of the scDb-Fc 56-65. V2 is an optimized version based on 56-65 V1, where the variable region of the heavy chain (VH)-variable region of the light chain (VL) rigid linker is replaced with a flexible G4S linker. V3 is the further modification of 56-65 V2, involving the swapped positions of 56 and 65 on the scDb arm. The canonical bispecific IgG employs the knob-into-hole construction technique. For the 65 heavy chain, modifications include the introduction of S354C and T366W mutations to create the heavy chain knob. For the 56 heavy chain, modifications include Y349C, T366S, L368A, and Y407V, thus forming the heavy chain hole (435R and 436F prevent binding to protein A). This includes an 11 aa linker with the amino acid sequence RADSAAAGGPG, a 26 aa linker with the sequence RADAAAAGGGGSGGGGSGGGGSGGGG, an Up hinge with the sequence DKTHTCPPC, and a G4S hinge with the sequence GGGGSDKTHTCPPC.
