Formats of bispecific antibodies and scaffolds described in the literature. (A) Bispecific quadroma generated by somatic fusion of two hybridomas, (B–J) bispecific formats developed by using knobs-in-holes (KiH) Fc heterodimerization strategy, (K) bispecific IgG1 developed by controlled Fab-arm exchange, (L) bispecific Fc-fusion constructs developed by electrostatic optimization, (M–O) bispecific formats developed by strand exchange, insertion of cleavage motif and expressing two light chains with single heavy chain, and (P–W) novel bispecifc or multi-specific scaffolds. Abbreviations: OAscFab-IgG, one-arm single-chain Fab-immunoglobulin gamma (IgG); dsFv-IgG, disulfide stabilized Fv-IgG; cFAE-IgG1, controlled Fab-arm exchanged-IgG1; scFv-Fc, charged pair single-chain Fv-Fc fusion; SEEDbody, strand-exchange engineered domain body; LUZ-Y, two-arm leucine zipper heterodimeric monoclonal antibodies; (κλ-body, kappa lambda body; BiTEs, bispecific T-cell engagers; BiKEs/TriKEs, bispecific/trispecific killer cell engagers; DART, dual-affinity retargeting molecules; mFc, monomeric Fc; Fcab, Fc antigen binding. Color codes are as follows: heterodimeric CH domains are shown in black and white; cognate light chains are shown in green and orange; the antigen-binding domains [VH (black/white), VL (green/orange), and VH–VL (paired white–orange/black–green)] are shown as indented bubble; additional specificity in trispecific molecules is shown by red/blue scFv or VH–VL pair (G,I,U); light chains of κλ-body (O) are shown in light and dark green; red bulge at CH3–CH3 interface depicts knobs-in-holes motif; curved arrows indicate cross-matched domains; interchain linkers are shown as red lines; interchain disulphide bonds are shown as black lines; engineered disulfide bonds are shown as yellow lines; red stripes depict exchanged sequences; black asterisks indicate site of mutations favoring heterodimerization; orange asterisks indicate protein A ablation mutation (J). +/− represents charged pair mutations; lightning bolt indicates cleavable peptide linkers.