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

Pallavi Bhatta,David P Humphreys

doi:10.3390/antib7030035

Abstract

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.

Highlights

Through simultaneous binding to two antigens, bispecific antibodies can invoke synergistic or novel biology and may offer enhanced clinical efficacy via improved drug targeting
We expressed the scFv in the HL orientation as we have previously observed higher monomer levels for these variable domain sequences in the HL orientation compared to the LH orientation
This is an important consideration in this study since we wanted to witness the importance of the variable domain sequences in the multimerisation process rather than those driven by any linker constraints

Summary

Introduction

Through simultaneous binding to two antigens, bispecific antibodies can invoke synergistic or novel biology and may offer enhanced clinical efficacy via improved drug targeting. Fv and scFv are discrete antigen binding domains that have been fused to Fab or IgG scaffolds to confer multispecificity in a wide variety of formats (reviewed in [1]). Conversion of Fv to scFv through introduction of a polypeptide linker between the vH and vL was originally carried out to stabilise the relatively weak vH-vL interface, but dynamic domain exchange (‘breathing’) between proximal scFv monomers can result in variable levels of monomer, dimer and higher-order multimers [2]. The introduction of a disulphide (ds) bond in isolated scFv between the vH and vL prevents variable domain ‘breathing’. Between proximal scFv monomers (in isolated scFv or scFv-containing bispecific antibodies), is a spectrum of dimer, trimer and higher order species. In isolated disulphide stabilised scFv (dsscFv), the presence of a linker enables stable dimer/multimer formation (Figure 1B)

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Results

Conclusion