Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

Abstract

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…One of the desired therapeutic properties for new therapeutics is multispecificity that will: 1) recruit effector cells to enhance killing; 2) prevent crosstalk between parallel/redundant signaling pathways to overcome resistance; 3) differentiate diseased cells from normal cells for improved safety and efficacy; and 4) potentially provoke synergetic effects. [1][2][3][4] Bispecific antibodies, which comprise a single molecule capable of recognizing 2 targets, have recently drawn attention as an emerging group of therapeutics to fulfill such unmet medical needs. Such characteristics are not only appreciated for the aforementioned functions, but also greatly favored by pharmaceutical companies from a cost-of-goods perspective.…”

“…5,6 It is produced from a mouse/rat quadroma (hybrid hybridoma) cell, in theory resulting in 12.5% of the total products possessing the desired dual specificity, though preferential pairing between intra-species HC and LC would most likely give rise to a higher amount of bispecific IgG with correct assembly. 3 Such a prodigal process can be largely improved by expression and purification of individual antibodies followed by chemical coupling of 2 IgGs (IgG 2 ) or Fab fragments (F(ab') 2 ). 7 This chemical method has also been utilized to conjugate a pharmacophore peptide heterodimer to the catalytic center of a scaffold IgG, resulting in the CovX-Body.…”

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

“…A number of alternative approaches for the same purpose have been developed recently. 9,10,11,12,13,14 Upon application of one of these methods alone, however, there still remains a mixture of 4 compounds except for cases where the chain association issue is circumvented by a common LC approach. 15 The LC mispairing problem is more difficult to address because a total of 4 possible pairings of heavy and light chains have to be considered.…”

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…[6][7][8][9][10][11][12] Several solutions have been described for the generation of BsAb with an IgG chain-like sequence and structure (IgG-Bs). 13 For example, IgG-Bs have been produced by expressing the IgGs separately and subsequently mixing them, 14,15 but these methods require both site-specific mutations and reduction and oxidation, which complicate downstream development. Moreover, these approaches require the production of 2 separate cell lines expressing the 2 antibody pairs.…”

“…The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs, [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods.…”