Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

Dennis R Goulet,Steven J Orcutt,Adam Zwolak,Theo Rispens,Aran F Labrijn,Rob N De Jong,William M Atkins,Mark L Chiu

doi:10.1074/jbc.ra117.000303

Abstract

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.

Highlights

Bispecific antibodies combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action
The heavy chain pairing problem has been addressed by incorporating complementary mutations into the CH3 region using technologies such as controlled Fab-arm exchange, knobs-into-holes, and electrostatic steering [17,18,19]. cFAE uses a minimal set of mutations and avoids the light chain pairing issue, as exchange of half-Abs can be performed without perturbing the correct heavy chain–light chain interaction
An understanding of the cFAE reaction is critically important for optimization of in vitro manufacturing, the precise chemical mechanism of bsAb formation using engineered IgG1 variants has not been established

Summary

Introduction

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Bispecific antibodies (bsAbs) combine the antigen specificities of two parental Abs to create multifunctional molecules capable of simultaneously binding two distinct targets. The small size of these Abs relative to intact immunoglobulin G (IgG) may allow for increased penetration to solid tumors, the absence of the Fc region abrogates binding to Fc receptors responsible for mediating immune effector functions and long serum half-life [15, 16]. Bispecific molecules based on the structure of full-length IgG retain Fc receptor binding but must be engineered to drive formation of the heavy chain heterodimer while retaining the correct pairing of heavy and light chains. In IgG4, these hinge cysteines are contained within the motif “CPSC,” whereas other isotypes contain “CPPC.” The presence of serine at position 228 confers the hinge with extra flexibility that allows for the for-

Methods

Results

Conclusion