Negative tail fusions can improve ruggedness of single domain antibodies

Goldman, Ellen R.; Lee, P. Audrey Brozozog; Zabetakis, Dan; Turner, Kendrick B.; Walper, Scott A.; Liu, Jinny L.; Anderson, George P.

doi:10.1016/j.pep.2014.01.003

Cited by 23 publications

(29 citation statements)

References 43 publications

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“…Bacteria were grown and protein purified as described [9], [17]. Briefly, protein was purified through immobilized metal affinity chromatography and size exclusion.…”

Section: Methodsmentioning

confidence: 99%

“…However, when heated above their melting temperature at high concentration and for extended periods of time, many sdAb clones are prone to aggregation [9]. As aggregation is also a problem with scFvs, recombinant binding domains derived from conventional antibodies, several strategies have been reported that led to more soluble and stable recombinant antibody-binding domains (rAbs).…”

Section: Introductionmentioning

confidence: 99%

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Improving the biophysical properties of anti-ricin single-domain antibodies

Turner

Liu

Zabetakis

et al. 2015

Biotechnology Reports

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“…Bacteria were grown and protein purified as described [9], [17]. Briefly, protein was purified through immobilized metal affinity chromatography and size exclusion.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improving the biophysical properties of anti-ricin single-domain antibodies

Turner

Liu

Zabetakis

et al. 2015

Biotechnology Reports

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“…In other work, we looked at the effect of adding an α-synuclein tail to the C-terminus of a VHH as a general method for introducing negative charge to increase VHH stability (70). Addition of the negatively charged tail decreased aggregation, increased the ability of several VHHs to bind antigen after heating above their T m , and restored refolding ability in VHHs that lacked the canonical disulfide bond due to either cytoplasmic expression or mutation of the Cys residues that form the disulfide bond.…”

Section: Stabilizing Vhhsmentioning

confidence: 99%

“…Addition of the negatively charged tail decreased aggregation, increased the ability of several VHHs to bind antigen after heating above their T m , and restored refolding ability in VHHs that lacked the canonical disulfide bond due to either cytoplasmic expression or mutation of the Cys residues that form the disulfide bond. Impressively, a mutant which lacked the canonical disulfide bond and showed no ability to refold after heat denaturation, was able to regain almost 100% of its secondary structure after heating when expressed with the α-synuclein tail (70). Additionally, we observed that one of the cytoplasmically expressed VHHs with the α-synuclein tail melted at the low temperature associated with lack of the canonical disulfide bond formation, and refolded at the higher temperature observed with an intact canonical disulfide bond.…”

Section: Stabilizing Vhhsmentioning

confidence: 99%

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

et al. 2017

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Single domain antibodies (sdAbs) are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (Tms) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their Tm, making them stable until exposed to extreme temperatures. SdAbs derived from the variable heavy chains of camelid and shark heavy chain-only antibodies are termed VHH and VNAR, respectively, and generally exhibit some ability to refold and bind antigen after heat denaturation. This ability to refold varies from 0 to 100% and is a property dependent on both intrinsic factors of the sdAb and extrinsic conditions such as the sample buffer ionic strength, pH, and sdAb concentration. SdAbs have also been engineered to increase their solubility and refolding ability, which enable them to function even after exposure to temperatures that exceed their melting point. In addition, efforts to improve their stability at extreme pH and in the presence of chemical denaturants or proteases have been undertaken. Multiple routes have been employed to engineer sdAbs with these enhanced stabilities. The methods utilized to achieve these goals include grafting complementarity-determining regions onto stable frameworks, introduction of non-canonical disulfide bonds, random mutagenesis combined with stringent selection, point mutations such as inclusion of negative charges, and genetic fusions. Increases of up to 20°C have been realized, pushing the Tm of some sdAbs to over 90°C. Herein, we present an overview of the work done to stabilize sdAbs derived from camelids and sharks. Utilizing these various strategies sdAbs have been stabilized without significantly compromising their affinity, thereby providing superior reagents for detection, diagnostic, and therapeutic applications.

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“…Negatively charged residues, as observed in HEL4, have previously been shown to contribute favorably to protein solubility [96]. Aggregation resistance of antibody fragments can also be mediated by attaching negatively charged peptide sequences, as demonstrated by fusion of the acidic tail of α-synuclein [97], a penta glutamic acid tag [98] or a Glu-Ala dipeptide [99]. Similar solubilizing effects have been reported for the commonly used acidic FLAG-peptide [100, 101].…”

Section: Introductionmentioning

confidence: 99%

Engineered Autonomous Human Variable Domains

Nilvebrant

Tessier

Sidhu

2017

CPD

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The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

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Negative tail fusions can improve ruggedness of single domain antibodies

Cited by 23 publications

References 43 publications

Improving the biophysical properties of anti-ricin single-domain antibodies

Improving the biophysical properties of anti-ricin single-domain antibodies

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview

Engineered Autonomous Human Variable Domains

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