Format of bispecific antibodies (BsAbs)-VL1-CL1-VH2-CH2-Fc x VH1-CH1 x VL2-CL2


Tetravalent Fabs-In-Tandem immunoglobulins (FIT-Ig™) technology combines Fab fragments of any 2 parental mAbs create a tetravalent, dual-targeting single molecular entity, where the FabA is structurally fused to FabB in tandem at its N-terminus (Fig. 1a). A unique crisscross orientation of 2 sets of VH-CH1 and VL-CL evades any mispairing problem between two short chains and long chain. FIT-Ig have a complete Fc domain which is required for the formation of a disulfide-linked full IgG-like molecule. There is no peptide linker between two Fab moieties, no amino acid mutation in any area, and no altered Fab domain, which potentially reduce the immunogenicity risk. FIT-Ig design also provides enough flexibility allowing independent target engagement by the two antigen binding domains, as the two Fabs are connected only in one chain, instead of in both chains, therefore providing enough space for the lower domain to engage even a large antigen without any steric hindrance. FIT-Ig molecule is symmetrical and composed of long chains, 2 short chains during expression (Fig. 1b). The proper assembly is influenced by molar ratio of 3 polypeptide chains. It is known Fab domain usually have equivalent antigen specificity with mAb. FIT-Ig that utilizes 2 intact Fab domains tend to retains target binding of both parental mAbs. FIT-Ig molecule therefore can be designed based on the properties of the 2 parental mAbs.

Figure 1. A schematic illustration of the generation of a FIT-Ig molecule. (Adopted from: Gong, S., Wu, C. (2019) Generation of Fabs-in-tandem immunoglobulin molecules for dual-specific targeting. Methods, 154: 87-92.)

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