(scFv)2 Fab/ Tribodies

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.