Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies

Julian, Mark C.; Lee, Christine C.; Tiller, Kathryn E.; Rabia, Lilia A.; Day, Evan K.; Schick, Arthur J.; Tessier, Peter M.

doi:10.1093/protein/gzv050

Cited by 37 publications

(46 citation statements)

References 75 publications

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“…The antibody (V H ) domains that we isolated by co-selecting for affinity and stability17 are summarized in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently observed strong affinity/stability trade-offs during the directed evolution of a human antibody (V H ) domain specific for the Alzheimer’s Aβ42 peptide17. We generated mutant libraries of a stable V H antibody scaffold (B1a18) that was initially grafted with Aβ residues 33–42 in CDR3, displayed the mutants on the surface of yeast, and selected for V H domains with improved binding affinity.…”

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confidence: 99%

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Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

Julian

Garde

et al. 2017

Sci Rep

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The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability.

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“…The antibody (V H ) domains that we isolated by co-selecting for affinity and stability17 are summarized in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

Julian

Garde

et al. 2017

Sci Rep

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“…bility, we first analyzed the relative affinity of the A␤17-42 domains for Protein A. This analysis is motivated by the fact that folded V H 3 domains (such as the A␤17-42 domains) have a Protein A binding site on their scaffold, and the relative binding of Protein A to V H 3 domains is correlated with V H stability (45,48,49). Using an ELISA assay to measure V H binding to immobilized Protein A, we found that the NNN, QQQ, and SSS variants had similar IC 50 values of ϳ1-2 nM, whereas the DDD and EEE variants had higher IC 50 values (ϳ5-10 nM; Fig.…”

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

confidence: 99%

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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“…However, it is more challenging to select sets of rare mutations that improve binding but are not destabilizing. The importance of simultaneously optimizing affinity and stability was recently demonstrated in selections of yeast-displayed gammabodies where simultaneous selection for antigen binding and Protein A binding was employed [121]. …”

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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Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies

Cited by 37 publications

References 75 publications

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

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

Engineered Autonomous Human Variable Domains

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