Improving solubility and refolding efficiency of human VHs by a novel mutational approach

Tanha, Jamshid; Nguyen, Thanh-Dung; Ng, Andy Cheuk-Him; Ryan, Shannon; Ni, Feng; MacKenzie, Ross

doi:10.1093/protein/gzl037

Cited by 36 publications

(26 citation statements)

References 2 publications

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“…The importance of FR4 residues in improving the aggregation resistance of immunoglobulin variable domains (V H Hs and V H s) has been suggested 69 , 70 . For example, V H Hs, which are known for their high aggregation resistance, have a highly conserved 105Q mutation compared with the aggregation prone V H s. Similarly, V H s with the 105Q mutation have been shown to have improved aggregation resistance compared with a corresponding wild-type V H .…”

Section: Discussionmentioning

confidence: 99%

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

Kim

Kandalaft

et al. 2013

mAbs

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We set out to gain deeper insight into the potential of antibody light chain variable domains (VLs) as immunotherapeutics. To this end, we generated a naïve human VL phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (VHs), we isolated a diversity of VL domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating VL repertoires are a rich source of non-aggregating domains. In addition, the VLs demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human VHs revealed that the VLs had similar overall profiles with respect to melting temperature (Tm), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of VLs did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their Tms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of VLs. Human VLs and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered VLs may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.

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Section: Discussionmentioning

confidence: 99%

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

Kim

Kandalaft

et al. 2013

mAbs

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“…Indeed, unlike the isolated human V H domains, nanobodies are highly soluble. In turn, solubility and folding properties of the human V H domains can be improved by introducing a few mutations at the contact surface with the V L domain based on the sequence comparison to the camelid VHH (8,9).…”

Section: Nanobodies Versus Fragments Of the Conventional Antibodies Amentioning

confidence: 99%

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Dmitriev

Lutsenko

Muyldermans

2016

Journal of Biological Chemistry

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Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy chain-only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

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“…Analogous mutational approaches to humanize V H Hs have also been attempted [84, 85]. However, these strategies generally only partially eliminate the tendency to aggregate and the modified V H domains may display poor expression levels, low solubility, low stability, increased stickiness on size-exclusion columns and/or impaired antigen binding [84–88]. Similar strategies have been applied to V-NARs, a process that is further complicated by the lower sequence homology and larger structural differences compared to human V H domains [89].…”

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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Improving solubility and refolding efficiency of human VHs by a novel mutational approach

Cited by 36 publications

References 2 publications

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

Engineered Autonomous Human Variable Domains

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