Structure of a Therapeutic Full-Length Anti-NPRA IgG4 Antibody: Dissecting Conformational Diversity

Blech, Michaela; Hörer, Stefan; Kühn, Alexander; Kube, Sebastian; Göddeke, Hendrik; Kiefer, H.; Zang, Yuguo; Alber, Yannic; Westermann, Martin; Tully, Mark D.; Schäfer, Lars V.; Garidel, Patrick

doi:10.1016/j.bpj.2019.03.036

Cited by 18 publications

(29 citation statements)

References 67 publications

(61 reference statements)

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“…Traditionally, the structure of Abs has been illustrated by models as Y-shaped molecules (as shown in Figure 2). X-ray structures of Abs have mainly been limited to isolated Fab and Fc parts, even though a few structures of whole Abs have been published [60,61]. However, recent results using ion exchange-purified IgGs and chemical cross-linking in combination with MS have shown that native IgGs have a compact ("closed") structure [62].…”

Section: Discoverymentioning

confidence: 99%

Peptides, Antibodies, Peptide Antibodies and More

Trier

Hansen

Houen

2019

IJMS

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The applications of peptides and antibodies to multiple targets have emerged as powerful tools in research, diagnostics, vaccine development, and therapeutics. Antibodies are unique since they, in theory, can be directed to any desired target, which illustrates their versatile nature and broad spectrum of use as illustrated by numerous applications of peptide antibodies. In recent years, due to the inherent limitations such as size and physical properties of antibodies, it has been attempted to generate new molecular compounds with equally high specificity and affinity, albeit with relatively low success. Based on this, peptides, antibodies, and peptide antibodies have established their importance and remain crucial reagents in molecular biology.

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Section: Discoverymentioning

confidence: 99%

Peptides, Antibodies, Peptide Antibodies and More

Trier

Hansen

Houen

2019

IJMS

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“…With these structural rearrangements, the local environment also changes, which can affect surface properties. Recent studies highlight their inherent flexibility of antibodies and its impact on physicochemical properties such as binding promiscuity or aggregation propensity ( 10 , 48 , 49 , 50 , 51 , 52 , 53 ). Molecular dynamics simulations offer the unique opportunity to capture this flexibility at atomic resolution ( 54 ).…”

Section: Introductionmentioning

confidence: 99%

Conformational Ensembles of Antibodies Determine Their Hydrophobicity

Waibl

Fernández‐Quintero

Kamenik

et al. 2021

Biophysical Journal

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A major challenge in the development of antibody biotherapeutics is their tendency to aggregate. One root cause for aggregation is exposure of hydrophobic surface regions to the solvent. Many current techniques predict the relative aggregation propensity of antibodies via precalculated scales for the hydrophobicity or aggregation propensity of single amino acids. However, those scales cannot describe the nonadditive effects of a residue's surrounding on its hydrophobicity. Therefore, they are inherently limited in their ability to describe the impact of subtle differences in molecular structure on the overall hydrophobicity. Here, we introduce a physics-based approach to describe hydrophobicity in terms of the hydration free energy using grid inhomogeneous solvation theory (GIST). We apply this method to assess the effects of starting structures, conformational sampling, and protonation states on the hydrophobicity of antibodies. Our results reveal that high-quality starting structures, i.e., crystal structures, are crucial for the prediction of hydrophobicity and that conformational sampling can compensate errors introduced by the starting structure. On the other hand, sampling of protonation states only leads to good results when combined with high-quality structures, whereas it can even be detrimental otherwise. We conclude by pointing out that a single static homology model may not be adequate for predicting hydrophobicity.

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“…Fig. 1 displays the characteristic β -sheet architecture of antibodies ( 26 ). Midinfrared spectroscopic studies show that the secondary structure of antibodies is composed of ∼7–11% α -helix and 40–45% β -sheet ( 5 ).…”

Section: Introductionmentioning

confidence: 99%

Thermal and Chemical Unfolding of a Monoclonal IgG1 Antibody: Application of the Multistate Zimm-Bragg Theory

Garidel

Blech

et al. 2020

Biophysical Journal

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The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72 C with an unfolding enthalpy of DH cal $200-300 kcal/mol and a main transition at 85 C with an enthalpy of $900-1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter s $6 Â 10 À5 and a Gibbs free energy of unfolding of g nu $100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues n participating in the unfolding reaction. On average, n$220 5 50 amino acids are involved in the pretransition and n$850 5 30 in the main transition, accounting for $90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy DH cal and lowers the midpoint temperature T m. Both parameters depend linearly on the concentration of denaturant. The gua-nidineHCl concentrations needed to unfold mAb at 25 C are predicted to be 2-3 M for the pretransition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.

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Structure of a Therapeutic Full-Length Anti-NPRA IgG4 Antibody: Dissecting Conformational Diversity

Cited by 18 publications

References 67 publications

Peptides, Antibodies, Peptide Antibodies and More

Peptides, Antibodies, Peptide Antibodies and More

Conformational Ensembles of Antibodies Determine Their Hydrophobicity

Thermal and Chemical Unfolding of a Monoclonal IgG1 Antibody: Application of the Multistate Zimm-Bragg Theory

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