A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

Abstract

<jats:p>We previously reported efficient heavy-chain assembly of heterodimeric bispecific antibodies by exchanging the interdomain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α and β domains of the human T-cell receptor, a technology known as bispecific engagement by antibodies based on the T-cell receptor (BEAT). Efficient heterodimerization in mammalian cell transient transfections was observed, but levels were influenced by the nature of the binding arms, particularly in the Fab-scFv-Fc format. In this study, we report a single amino acid change that significantly and consistently improved the heterodimerization rate of this format (≥95%) by inducing partial disorder in one homodimer species without affecting the heterodimer. Correct folding and assembly of the heterodimer were confirmed by the high-resolution (1.88–1.98 Å) crystal structure presented here. Thermal stability and 1-anilinonaphthalene-8-sulfonic acid–binding experiments, comparing original BEAT, mutated BEAT, and “knobs-into-holes” interfaces, suggested a cooperative assembly process of heavy chains in heterodimers. The observed gain in stability of the interfaces could be classified in the following rank order: mutated BEAT &gt; original BEAT &gt; knobs-into-holes. We therefore propose that the superior cooperativity found in BEAT interfaces is the key driver of their greater performance. Furthermore, we show how the mutated BEAT interface can be exploited for the routine preparation of drug candidates, with minimal risk of homodimer contamination using a single Protein A chromatography step.</jats:p>

Mutational biases promote neutral increases in the complexity of protein interaction networks following gene duplication

“…Furthermore, although mutational effects on binding for homomers and heteromers are highly correlated, our FoldX predictions suggest that there is a small percentage of mutations that could directly stabilize one complex while destabilizing the other (Figure 3A, Figure S3A). Our model shows that changes in the proportion of the three complexes could therefore arise through a few mutations, matching previous reports of homomer evolution (Ashenberg et al, 2011;Garcia-Seisdedos et al, 2017;Hochberg et al, 2018) as well as heteromer evolution (Stutz & Blein, 2020;Emlaw et al, 2021).…”

“…This is supported by empirical observations. Studies show that homomeric specificity can be achieved with just 1-3 substitutions (Ashenberg et al , 2011; Garcia-Seisdedos et al , 2017; Stutz & Blein, 2020; Emlaw et al , 2021) and marginal differences in binding affinity (Hochberg et al , 2018). On the other hand, there are regions of the solution space that are much less sensitive to these mutational effects, for example, when the difference in ΔG bind between the homodimers and the heterodimer is already very large.…”

Mega-scale experimental analysis of protein folding stability in biology and protein design

“…Protein sequences vary by more than ten orders of magnitude in thermodynamic folding stability (the ratio of unfolded to folded molecules at equilibrium) (1,2). Even single point mutations that alter stability can have profound effects on health and disease (3)(4)(5), pharmaceutical development (6)(7)(8), and protein evolution (9)(10)(11)(12)(13). Thousands of point mutants have been individually studied over decades to quantify the determinants of stability (14,15), but these studies highlight a challenge: similar mutations can have widely varying effects in different protein contexts, and these subtleties remain difficult to predict despite substantial effort (16,17).…”

“…Protein sequences vary by more than ten orders of magnitude in thermodynamic folding stability (the ratio of unfolded to folded molecules at equilibrium) (Dill, 1990; Goldenzweig and Fleishman, 2018). Even single point mutations that alter stability can have profound effects on health and disease (Stein et al, 2019; Wang and Moult, 2001; Yue et al, 2005), pharmaceutical development (Rodríguez-Rodríguez et al, 2012; Stutz and Blein, 2020; Wang et al, 2021), and protein evolution (Agozzino and Dill, 2018; Bloom et al, 2006; Drummond and Wilke, 2008; Gong et al, 2013). Thousands of point mutants have been individually studied over decades to quantify the determinants of stability (Nikam et al, 2021; Xavier et al, 2021), but these studies highlight a challenge: similar mutations can have widely varying effects in different protein contexts, and these subtleties remain difficult to predict despite substantial effort (Laimer et al, 2015; Schymkowitz et al, 2005).…”

Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins

“… 16 , 47 , 48 More recently, multiple alternative approaches to enable correct heavy-chain association have been described, such as relying on charge interactions. 7–11 , 14 , 29 , 49 …”