Dual-affinity re-targeting proteins

Dual-affinity re-targeting proteins (DARTs) encompasses of two Fv fragments, containing two single antigen-binding sites formed when two Fv fragments heterodimerize. The Fv1 contains of a VH from antibody A and a VL from antibody B, whereas Fv2 contains VH from antibody B and VL from antibody A in the order of VL (1)-VH (2) and VL (2)-VH (1) (Fig. 1). This amalgamation permits DART to mimic natural interaction within an IgG molecule. Short linker sequences between the VL and VH segments encourage a posttranslational “diabody”-type association. The peptide linkers and the covalent linkage between the two DART chains limits the freedom of the antigen binding domains, resulting in a stable association between target and effector cells. DART can be synthesized in the mammalian expression systems. DART are more stable and potent. DART does not contain Fc region and, therefore it has a short serum half-life. Several DART antibodies are under development for T-cell redirection, modulation of receptor signaling and neutralization of viruses. DART molecules were found to be more consistent than BiTE molecules in targeting and killing B-cell lymphoma.

Fig. 1. The structure of dual-affinity re-targeting proteins (DARTs). The linker between heavy and light chains is as short as about five amino acids. Because of the short linker between the two domains of scFv, the two domains of the same scFv cannot pair and are forced to homodimerize with its homologous partner in another scFv. The adding of another cysteine residue at the end of VHA and VHB is helpful for the stability of this kind of bispecific antibody by forming a disulfide linkage (Adopted from: Asim A, Ejaj A, Qamar Z, Ahmar RM Mohammad O, Ghulam A. (2017) Recent advances in the development of novel protein scaffolds based therapeutics. International Journal of Biological Macromolecules. 102: 630-641). 

Formats of bispecific antibodies (BsAbs)

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