Relative Contribution of Framework and CDR Regions in Antibody Variable Domains to Multimerisation of Fv- and scFv-Containing Bispecific Antibodies

• Main Authors: Pallavi Bhatta, David P. Humphreys
• Format: Article
• Language: English
• Published: MDPI AG 2018-08-01
• Series: Antibodies
• Subjects: bispecific antibody; disulphide stabilised Fv; disulphide stabilised single chain Fv; monomer; thermal stability
• Online Access: http://www.mdpi.com/2073-4468/7/3/35

Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer levels during expression, and hence, undesirable therapeutic protein yields. We investigate the contribution of disulphide stabilised Fv and scFv to Fab-Fv and Fab-scFv multimerisation. We show that monomer levels of isolated Fv/scFv cannot always be used to predict monomer levels of Fab-linked Fv/scFv, and that Fab-scFv monomer levels are greater than the equivalent Fab-Fv. Through grafting bispecifics with framework/CDR-‘swapped’ Fv and scFv, we show that monomer levels of disulphide stabilised Fab-Fv and Fab-scFv can be improved by Fv framework ‘swapping’. The Fab-Fv and Fab-scFv can be considered representative of the significant number of bispecific antibody formats containing appended Fv/scFv, as we also used Fv framework ‘swapping’ to increase the monomer level of an IgG-scFv bispecific antibody. This research may, therefore, be useful for maximising the monomeric yield of numerous pharmaceutically-relevant bispecific formats in pre-clinical development.