Format of bispecific antibodies (BsAbs)-(scFv)2-Fab


Tribodies are multifunctional recombinant antibody derivatives. The Fab fragment serves as a specific heterodimerization signal, and the two scFv fragments are each fused to a different Fab chain. In this way we obtain a molecule of intermediate molecular weight (100 kDa) which allows incorporating three different antibody fragments (Fig. 1). This manifold, tribody, can be used to create trivalent molecules as well as bispecific molecules with bivalent binding to only one target, as trispecific molecules. These heterodimers are stable, with each of the binders retaining their specific affinities, with the bivalent tribody having higher affinity, and higher activation of T-cell proliferation and cytotoxicity in vivo. The molecule also has more favorable properties for toxic payload delivery. It can be argued that delivering a T-cell activating activity can be regarded as a toxic function. Antibodies with such a function remaining in the body for a longer time have the potency to accumulate aspecifically or form deposits in healthy tissue.

Fig. 1. A) Tribodies are genetically constructed by fusing entities (i.e. to the Fd and L chain of a Fab molecule. As a Fab is a natural disulphide stabilized heterodimer, the position of each function is exactly determined and a single homogeneous product is produced. C) Models of the molecule predict no sterical hindrance between the subunits. D) Even as the scFv are N-terminally linked, their binding site is facing outside. E) Tribodies can lead to 3x1 (trispecific), 2+1 (bispecific) or trivalent molecules, as well as immunotoxins, immunocytokines, enzyme fusions. F) The model easily allows trispecific, bispecific and trivalent antibody derivatives to be produced. (Adopted from: Mertens, N. (2011) Tribodies: Fab–scFv Fusion Proteins as a Platform to Create Multifunctional Pharmaceuticals. In: Kontermann, R. (eds) Bispecific Antibodies. Springer, Berlin, Heidelberg.)

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