Domain Interactions in the Fab Fragment: A Comparative Evaluation of the Single-chain Fv and Fab Format Engineered with Variable Domains of Different Stability

Röthlisberger, Daniela; Honegger, Annemarie; Plückthun, Andreas

doi:10.1016/j.jmb.2005.01.053

Cited by 258 publications

(236 citation statements)

References 62 publications

(69 reference statements)

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“…37 In addition, pairing of the variable domains increases the stability of the variable LC domain due to interface contacts. 38 Ultimately, the folding of the multi-domain Fab is an interdependent process 39 and the above interpretation is consistent with the observations from the biophysical characterization of the antibody set we studied.…”

Section: Discussionsupporting

confidence: 86%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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(2015) Boosting antibody developability through rational sequence optimization , mAbs, 7:3, 505-515, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and longterm stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.

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Section: Discussionsupporting

confidence: 86%

Boosting antibody developability through rational sequence optimization

Seeliger

Schulz

Litzenburger

et al. 2015

mAbs

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Section: Discussionmentioning

confidence: 99%

“…26 This stabilizing effect of the constant domains has been attributed to the fixed distance and orientation of the domains rather than the formation of a tightly packed interface between variable and constant domains, which is loosely packed and rather small. 26 Consistent with this notion, the tethering of antibody variable domains by coiled-coil domains has been shown to result in increased thermal stability of Fv. 27 However, a recent study on the stability of chimeric Fab molecules has demonstrated that the positive effect on thermal stability was limited to the kappa constant domain and was not observed in Fabs comprising lambda or hybrid light chains.…”

Section: Discussionmentioning

confidence: 99%

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

Cooke

Arndt

Quan

et al. 2018

mAbs

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Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cϵ2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).

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“…The extent of recombination means that the vast majority of eukaryotic proteins contain more than one ''independently folding'' domain (1). Most protein folding studies consider these domains in isolation (2), but it may be important to consider these domains in their context; neighboring domains may be independent or there can be effects on both the stability and kinetic behavior in multidomain proteins (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). A complicating factor involves choice of domain boundaries (17).…”

mentioning

confidence: 99%

“…␤-Sandwich proteins are commonly found in multidomain proteins; a few have been studied in detail. Whereas the fnIII domains of fibronectin and the Ig domains of titin, with short linkers and few interdomain interactions, fold and unfold independently (12,13,15), similar domains with extensive interfaces (both Ig and ␥-crystallin domains) show increases in stability (8,16,19) and changes in folding kinetics. Only one study has used protein engineering to investigate the molecular basis for interdomain stabilizing effects in detail (20).…”

mentioning

confidence: 99%

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains

Batey

Clarke²

2006

Proc. Natl. Acad. Sci. U.S.A.

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Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor. alpha-helix ͉ protein folding ͉ spectrin ͉ m value ͉ equilibrium denaturation

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Domain Interactions in the Fab Fragment: A Comparative Evaluation of the Single-chain Fv and Fab Format Engineered with Variable Domains of Different Stability

Cited by 258 publications

References 62 publications

Boosting antibody developability through rational sequence optimization

Boosting antibody developability through rational sequence optimization

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains

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