Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions

Perchiacca, Joseph M.; Bhattacharya, Moumita; Tessier, Peter M.

doi:10.1002/prot.23085

Cited by 93 publications

(108 citation statements)

References 62 publications

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“…The DDD and EEE variants showed largely reversible unfolding and refolding behavior (Fig. 5), consistent with our previous findings (39,40,50). Surprisingly, the NNN variant also displayed unfolding behavior that was largely reversible, whereas the QQQ and SSS variants unfolded irreversibly.…”

Section: Polar Cdr3 Mutations Strongly Impact Expression Levels Of A␤supporting

confidence: 91%

“…The SSS and QQQ variants displayed increased binding signals for disaggregated A␤ relative to fibrils. The IC 50 values for binding of the NNN, QQQ, and SSS variants to disaggregated A␤ were similar (200 -300 nM; Table 2). …”

Section: Charged and Non-charged Polar Mutations Differentiallymentioning

confidence: 86%

“…7A), we evaluated whether such multimers may influence the observed conformational specificity or IC 50 values of FIGURE 5. Impact of mutations at the edges of CDR3 on the apparent folding stability and reversibility of unfolding of the A␤17-42 V H domains.…”

Section: Charged and Non-charged Polar Mutations Differentiallymentioning

confidence: 99%

See 2 more Smart Citations

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Lee

Julian

Tiller

et al. 2016

Journal of Biological Chemistry

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Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (A␤). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V H ) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic A␤42 peptide segment (A␤ residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding V H domains. We find that V H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, A␤ V H domains with negatively charged CDR3 mutations show significant preference for recognizing A␤ fibrils relative to A␤ monomers, whereas the same V H domains with other polar CDR3 mutations recognize both A␤ conformers. We observe similar behavior for a V H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8 -37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.The misfolding and assembly of peptides and proteins into prefibrillar oligomers and amyloid fibrils is linked to several neurodegenerative diseases (1-4). To understand the contributions of polypeptide aggregation to such disorders, it is important to characterize the biochemical properties of aggregates. However, pre-amyloid and amyloid aggregates are difficult to characterize using many traditional biochemical methods that are routinely used for soluble proteins. High-resolution structural analysis of such aggregates is particularly challenging and must be performed using specialized methods such as solid state NMR (5-13) or x-ray crystallography of small peptide fragments (14 -16). These powerful structural methods generally lack the time resolution to probe pre-amyloid intermediates and oligomers unless they can be kinetically trapped.Given these challenges, antibodies with conformational specificity for prefibrillar oligomers and amyloid fibrils have proven valuable for biochemical characterization (17-28). An obvious strength of such antibodies is their ability to detect specific types of aggregates formed both in vivo and in vitro. The ability of conformational antibodies to bridge in vivo and in vitro studies is important for understanding the biochemical mechanisms that contribute to protein misfolding disorders.Several approaches have been used to generate conformational antibodies. The most widely used one is immuni...

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Section: Polar Cdr3 Mutations Strongly Impact Expression Levels Of A␤supporting

confidence: 91%

Section: Charged and Non-charged Polar Mutations Differentiallymentioning

confidence: 86%

See 1 more Smart Citation

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Lee

Julian

Tiller

et al. 2016

Journal of Biological Chemistry

Self Cite

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show abstract

“…These studies indicate that the controlling factor is the protein net charge. However other work, based on phage display [27] and rational mutagenesis [25,65,47], indicates that the spatial location of charged mutations controls the protein stability. However, care must be taken when engineering in charged mutations.…”

Section: Introductionmentioning

confidence: 99%

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Austerberry

Dajani

Panova

et al. 2017

European Journal of Pharmaceutics and Biopharmaceutics

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a b s t r a c tThe aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.

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“…A recent study compared and mutated the sequences of two related antibody domains -one aggregationprone and the other was its aggregation-resistant counterpart -to identify "aggregation hotspots" [255,342]. By swapping and mutagenesis analysis of CDR loops, an aggregation-prone human V H segment was converted into an aggregation-resistant one.…”

Section: Engineering To Improve Solubility and Viscositymentioning

confidence: 99%

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

Klein

Templeton

Schwenk

2014

Pure and Applied Chemistry

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This report discusses the history and mechanisms of vaccination of humans as well as the engineering of therapeutic antibodies. Deeper understanding of the molecular interactions involved in both acquired and innate immunity is allowing sophistication in design of modified and even synthetic vaccines. Recombinant DNA technologies are facilitating development of DNA-based vaccines, for example, with the recognition that unmethylated CpG sequences in plasmid DNA will target Toll-like receptors on antigen-presenting cells. Formulations of DNA vaccines with increased immunogenicity include engineering into plasmids with "genetic adjuvant" capability, incorporation into polymeric or magnetic nanoparticles, and formulation with cationic polymers and other polymeric and non-polymeric coatings. Newer methods of delivery, such as particle bombardment, DNA tattooing, electroporation, and magnetic delivery, are also improving the effectiveness of DNA vaccines. RNA-based vaccines and reverse vaccinology based on gene sequencing and bioinformatic approaches are also considered. Structural vaccinology is an approach in which the detailed molecular structure of viral epitopes is used to design synthetic antigenic peptides. Virus-like particles are being designed for vaccine deliveries that are based on structures of viral capsid proteins and other synthetic lipopeptide building blocks. A new generation of adjuvants is being developed to further enhance immunogenicity, based on squalene and other oil-water emulsions, saponins, muramyl dipeptide, immunostimulatory oligonucleotides, Toll-like receptor ligands, and lymphotoxins. Finally, current trends in engineering of therapeutic antibodies including improvements of antigen-binding properties, pharmacokinetic and pharmaceutical properties, and reduction of immunogenicity are discussed. Taken together, understanding the chemistry of vaccine design, delivery and immunostimulation, and knowledge of the techniques of antibody design are allowing targeted development for the treatment of chronic disorders characterized by continuing activation of the immune system, such as autoimmune disorders, cancer, or allergies that have long been refractory to conventional approaches.

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Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions

Cited by 93 publications

References 62 publications

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

Design and Optimization of Anti-amyloid Domain Antibodies Specific for β-Amyloid and Islet Amyloid Polypeptide

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

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