Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains

Arbabi‐Ghahroudi, Mehdi; MacKenzie, Ross; Tanha, Jamshid

doi:10.1007/978-1-60761-652-8_22

Cited by 14 publications

(3 citation statements)

References 48 publications

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“…By comparing human scFvs selected from the CDR-modified Tomlinson repertoire (Holt et al, 2000), we additionally demonstrate a striking influence of CDR content on intrabody solubility and aggregation propensity. This observation is substantiated by studies of in vitro thermostability among CDR-modified human V H repertoires and camelid V HH s (Bond et al, 2003;Jespers et al, 2004;Arbabi-Ghahroudi et al, 2009b;Dudgeon et al, 2009), as well as by the findings of CDR loop grafting experiments, prompting some to conclude that nature has evolved a multitude of antibody frameworks as a consequence of selective pressures posed by hypervariable CDR residues on overall antibody structure and function (Honegger et al, 2009). An important implication of this study is that codons should be selected carefully when creating CDR-only repertoires so as to limit the frequency of positively charged amino-acid residues that potentially increase the risk for ionic aggregation at physiologic pH.…”

Section: Discussionmentioning

confidence: 75%

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm

Kvam

Sierks

Shoemaker

et al. 2010

Protein Engineering, Design and Selection

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Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and weak hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of principle, we demonstrate that the soluble expression of an aggregation-prone positively charged intrabody is modestly enhanced via cis or trans acidification using highly charged peptide tags (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use.

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Section: Discussionmentioning

confidence: 75%

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm

Kvam

Sierks

Shoemaker

et al. 2010

Protein Engineering, Design and Selection

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“…The ability of the human V L s to refold following thermal denaturation was assessed by determining their thermal refolding efficiencies (TREs), which is calculated as the ratio of the K D s for the binding of the native V L s to protein L ( K D n) to the K D s for the binding of the heat-denatured/cooled (refolded) V L s to protein L ( K D ref) 48 , 54 Fig.…”

Section: Resultsmentioning

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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“…The nomenclature used throughout this work to distinguish between wild-type and mutant V H Hs is exemplified as follows: “A4.2” denotes a wild-type V H H, “A4.2m” denotes a mutant V H H. To construct mutant V H Hs with a second disulfide bond, splice-overlap extension-polymerase chain reaction (SOE-PCR) [52] was performed using 4 primers for each V H H (Table S1) and two rounds of PCR essentially as described [53]. Ala or Gly and Ile codons at positions 54 and 78 (IMGT numbering system; http://imgt.cines.fr/), respectively, were changed to Cys codons through primer-forced mutation.…”

Section: Methodsmentioning

confidence: 99%

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

et al. 2011

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The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.

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Site-Directed Mutagenesis for Improving Biophysical Properties of VH Domains

Cited by 14 publications

References 48 publications

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm

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

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

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