Format of bispecific antibodies (BsAbs)-Controlled Fab arm exchange


Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs). This involves the recombination of antigen-binding arms (heavy chain–light chain (H-L) pairs) between individually expressed antibody molecules and is termed Fab-arm exchange, can be modified to make the routine generation of bsAbs feasible. The production of DuoBodies involves (i) separate expression and purification of two parental IgG1s containing two matching point mutations, one in either IgG1, at the CH3:CH3 interface, (ii) mixing of the parental IgGs; (iii) controlled reduction of interchain disulfide bonds in vitro, allowing the recombination of H-L pairs (driven by the matching mutations); and (iv) removal of reducing agent, allowing the reoxidation of interchain disulfide bonds to occur. Fragment-based formats are compatible with well-characterized and cost-effective prokaryotic and lower eukaryotic expression systems, offering the potential advantages of high yields.

Fig. 1. Graphical representation of the Controlled Fab arm exchange (cFAE) bispecific antibody production. (Adopted from: Steinhardt, J., Wu, Y., Fleming, R., Ruddle, B.T., Patel, P., Wu, H., Gao, C., Dimasi, N. (2020) Fab-arm exchange combined with selective protein a purification results in a platform for rapid preparation of monovalent bispecific antibodies directly from culture media. Pharmaceutics. 12, 3.)

Formats of bispecific antibodies (BsAbs)

Many formats have been developed for BsAb generation as listed in the following table.

FormatSchematic structureDescriptionExample BsAbTrademark Company
tandem VHHTandem VHH fragment-based BsAbN/A
tandem scFvPicture loading failed.Tandem ScFv fragment-based BsAbAMG330BiTETMAmgen
Dual-affinity re-targeting antibodyPicture loading failed.Tandem domain-exchanged Fv (can also be used to fuse with Fc domain to create whole Abs)FlotetuzumabDARTTMMacrogenics
DiabodyPicture loading failed.dimer of single-chain Fv (scFv) fragmentvixtimotamabReSTORETMAmphivena Therapeutics
(scFv)2-FabPicture loading failed.a Fab domain and two scFv domains bindA-337ITabTMGeneron/EVIVE Biotech
Rat–mouse hybrid IgGPicture loading failed.Full-size IgG-like half antibodies from two different speciesCatumaxomabTriomabTMTrion Pharma
Hetero heavy chain, Common light chainPicture loading failed.Hetero heavy chain, Common light chainEmicizumabART-IgTMGenentech/ Chugai/Roche
Controlled Fab arm exchangePicture loading failed.Recombin the parental half antibodies JNJ-64007957DuobodyTMGenmab/ Janssen
Hetero H, forced HL IgG1Picture loading failed.KIH technology for heterodimerization of 2 distinct H chains, replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond to enhance the cognate of H and L paringMEDI5752DuetMabTMMedImmune/ AstraZeneca
cH IgG1Picture loading failed.Identical heavy chains; 2 different light chains: one kappa (κ) and one lambda (λ)NI-1701κλ bodyTMNovimmune SA
Hetero H, CrossMabPicture loading failed.KIH technology; domain crossover of immunoglobulin domains in the Fab regionVanucizumabCrossMabTMRoche
scFv-Fab IgGPicture loading failed.Fab-Fc; ScFv-FcVibecotamab;
XmabTM (the engineered Fc to enhance the generation of heterodimeric Fc);
Xencor/Amgen; YZYBio
VH1-VH2-CH1-Fc1(G1) x VL2-VL1-CL-Fc2(G1)Picture loading failed.2 binding motif in one half antibodySAR440234CODV-IgTMSanofi
VL1-CL1-VH2-CH2-Fc x VH1-CH1 x VL2-CL2Picture loading failed.2 binding motif in one half antibodyEMB-01FIT-IgTMEPIMAB BIOTHERAPEUTICS
VH-1-TCR Cα x VL-1-TCR Cβ; VH-2-CH-2-Fc x VL-2-CL-2Picture loading failed.KIH technology; TCR Cα/Cβ is used to substitute the CH1 and CL domain in one armWuXibodyTMWuXi Biologics
C-terminal linker of FcPicture loading failed.Link the other molecules at the C-terminal of FcAPVO442ADAPTIR-FLEXTMAptevo Therapeutics
Fc antigen binding sitePicture loading failed.2 natural binding sites; 2 additional binding sites in the Fc loopFS118mAb2F-star Therapeutics