Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

Zabetakis, Dan; Anderson, George P.; Bayya, Nikhil; Goldman, Ellen R.

doi:10.1371/journal.pone.0077678

Cited by 35 publications

(36 citation statements)

References 24 publications

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“…This suggests that antibody libraries designed with sequence variation only in heavy chain CDR3 have a reduced risk for displaying affinity/stability trade-offs. More generally, these and other findings [18–20] suggest that affinity-enhancing mutations have an increased risk of destabilization and that compensatory mutations are frequently required to maintain antibody thermodynamic stability.…”

Section: Antibody Affinity/stability Trade-offsmentioning

confidence: 59%

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

Rabia

Desai

Jhajj

et al. 2018

Biochemical Engineering Journal

113

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The widespread use of monoclonal antibodies for therapeutic applications has led to intense interest in optimizing several of their natural properties (affinity, specificity, stability, solubility and effector functions) as well as introducing non-natural activities (bispecificity and cytotoxicity mediated by conjugated drugs). A common challenge during antibody optimization is that improvements in one property (e.g., affinity) can lead to deficits in other properties (e.g., stability). Here we review recent advances in understanding trade-offs between different antibody properties, including affinity, specificity, stability and solubility. We also review new approaches for co-optimizing multiple antibody properties and discuss how these methods can be used to rapidly and systematically generate antibodies for a wide range of applications.

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Section: Antibody Affinity/stability Trade-offsmentioning

confidence: 59%

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

Rabia

Desai

Jhajj

et al. 2018

Biochemical Engineering Journal

113

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“…The cloning of sdAb A3, an anti-SEB Ab with a high T m (T m = 85°C), 58,39 was previously described and similar methods were used to construct the other sdAb clones. To construct the engineered A3C8 sdAb variant, the CDRs of the anti-RTA sdAb C8 were transferred into the framework regions of sdAb A3 to produce the thermostabilized A3C8 sdAb 59 . Two pet22 constructs of A3C8 were prepared for periplasmic expression─ a C-terminal His-tagged plasmid and a tag-free construct (referred to as A3C8stop).…”

Section: Methodsmentioning

confidence: 99%

Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

Legler

Compton

Hale

et al. 2016

mAbs

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Ricin is an A-B ribosome inactivating protein (RIP) toxin composed of an A-chain subunit (RTA) that contains a catalytic N-glycosidase and a B-chain (RTB) lectin domain that binds cell surface glycans. Ricin exploits retrograde transport to enter into the Golgi and the endoplasmic reticulum, and then dislocates into the cytoplasm where it can reach its substrate, the rRNA. A subset of isolated antibodies (Abs) raised against the RTA subunit protect against ricin intoxication, and RTA-based vaccine immunogens have been shown to provide long-lasting protective immunity against the holotoxin. Anti-RTA Abs are unlikely to cross a membrane and reach the cytoplasm to inhibit the enzymatic activity of the A-chain. Moreover, there is not a strict correlation between the apparent binding affinity (Ka) of anti-RTA Abs and their ability to successfully neutralize ricin toxicity. Some anti-RTA antibodies are toxin-neutralizing, whereas others are not. We hypothesize that neutralizing anti-RTA Abs may interfere selectively with conformational change(s) or partial unfolding required for toxin internalization. To test this hypothesis, we measured the melting temperatures (Tm) of neutralizing single-domain Ab (sdAb)-antigen (Ag) complexes relative to the Tm of the free antigen (Tm-shift = Tmcomplex – TmAg), and observed increases in the Tmcomplex of 9–20 degrees. In contrast, non-neutralizing sdAb-Ag complexes shifted the TmComplex by only 6–7 degrees. A strong linear correlation (r2 = 0.992) was observed between the magnitude of the Tm-shift and the viability of living cells treated with the sdAb and ricin holotoxin. The Tm-shift of the sdAb-Ag complex provided a quantitative biophysical parameter that could be used to predict and rank-order the toxin-neutralizing activities of Abs. We determined the first structure of an sdAb-RTA1-33/44-198 complex, and examined other sdAb-RTA complexes. We found that neutralizing sdAb bound to regions involved in the early stages of unfolding. These Abs likely interfere with steps preceding or following endocytosis that require conformational changes. This method may have utility for the characterization or rapid screening of other Ab that act to prevent conformational changes or unfolding as part of their mechanism of action.

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“…It contains the conserved disulfide bond between C22 and C99. Previous work has shown that CDR2 plays a critical role in both the affinity and the high thermal stability of sdAb A3 [22] . Structural and mutational studies have been employed to both understand the high melting temperature of sdAb A3 and to engineer additional stability into the protein [8] , [23] , [24] .…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

et al. 2014

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Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.

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Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

Cited by 35 publications

References 24 publications

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility

Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

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