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

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.

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…The pairing consistently allowed for >95% heterodimer formation when parental antibodies were combined with a reducing agent and exchanged. The rate constants have been determined for the mechanism of IgG1 BsAb formation [519].…”

biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies

“…The selection of the 30 mg/kg dose used in the studies described earlier was based on pilot studies showing evidence of efficacy at this dose (data not shown). As the biAb (in TBTI format) used in these studies demonstrated lower affinity (due to steric hindrance) to laminin-211 than the parental Ab ( Figure S2 B, left panel), we elected to engineer an improved version based on the Duobody format 29 , 30 (biAb-2) with higher affinities to both laminin-211 and βDG. This biAb-2 was used in a separate dose-response study in LARGE myd-3J mice.…”

A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody–Drug Conjugates

“…(1) the ease of parental antibody production applying standard IgG production procedures; (2) the opportunity to evaluate affinity, epitope bin, cellular binding, internalization, physico-chemical properties such as thermal stability or hydrophobicity, polyspecificity or self-interaction in IgG format with the same material in advance or in parallel to an HTP screening; (3) the high degree of heterodimerization; (4) the applicability for bsAb, but also biparatopic, ADC screening as reported herein. cFAE has been identified in human IgG4 antibodies [26,[41][42][43], extended to therapeutic bsAb production applying MEA as a reducing agent [25] and extensively studied, including the evaluation of MEA versus DTT or cFAE kinetics [44] and already applied in focused combinatorial screens [27], leading, among others, to the recently approved bispecific antibody amivantamab [4]. Although this is illustrative of an elegant, educated screening cascade based on deep biological knowledge, many novel modes of action necessitate complex and high-throughput screening setups targeting unprecedented biology without an a priori definition of optimal epitope combinations or their respective paratopes.…”