scFv-Fab IgG/ XmAb

The XmAb enables alterations with desirable effects to the Fc domain of the antibodies. The modification increases affinity to the neonatal Fc receptor which prevents the antibody from degradation. Hence, this interaction extends the antibody’s halflife of this therapeutic drug. In order to construct the XmAb format an antibody heavy and light chain and a scFv Fcfusion were subcloned into vectors. The scFv and Fc region were connected with a GS-linker. The Fc region was altered with substitutions in order to increase the differences in pI between the two heavy chains. This would increase the pI differences between homodimers and heterodimers, which would then facilitate the purification of heterodimers. For the production of the proteins, plasmids encoding all chains were co-transfected into HEK cells. The antibody was further purified using protein A chromatography and ion exchange chromatography. Due to alterations in the Fc domain, pI differences can be used in the purification. The risk of immunogenicity was also minimized by the XmAb.

Fig. 1. A schematic illustration of XmAb format (Adopted from: Svahn, C.F., Khan. A., Wahlsten. A., Larsson, T., Koivula, T., Andersson, T. (2019) Drugs of the Future - Bispecific Antibodies : An investigaion of future development needs. In proceedings: Svahn 2019 Drugs OT.)

Formats of bispecific antibodies (BsAbs)

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