Epitope-dependent thermodynamic signature of single-domain antibodies against hen egg lysozyme.

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.

An integrated computational pipeline for designing high-affinity nanobodies with expanded genetic codes.

Protein engineering and design principles employing the 20 standard amino acids have been extensively used to achieve stable protein scaffolds and deliver their specific activities. Although this confers some advantages, it often restricts the sequence, chemical space, and ultimately the functional diversity of proteins. Moreover, although site-specific incorporation of non-natural amino acids (nnAAs) has been proven to be a valuable strategy in protein engineering and therapeutics development, its utility in the affinity-maturation of nanobodies is not fully explored. Besides, current experimental methods do not routinely employ nnAAs due to their enormous library size and infinite combinations. To address this, we have developed an integrated computational pipeline employing structure-based protein design methodologies, molecular dynamics simulations and free energy calculations, for the binding affinity prediction of an nnAA-incorporated nanobody toward its target and selection of potent binders. We show that by incorporating halogenated tyrosines, the affinity of 9G8 nanobody can be improved toward epidermal growth factor receptor (EGFR), a crucial cancer target. Surface plasmon resonance (SPR) assays showed that the binding of several 3-chloro-l-tyrosine (3MY)-incorporated nanobodies were improved up to 6-fold into a picomolar range, and the computationally estimated binding affinities shared a Pearson's r of 0.87 with SPR results. The improved affinity was found to be due to enhanced van der Waals interactions of key 3MY-proximate nanobody residues with EGFR, and an overall increase in the nanobody's structural stability. In conclusion, we show that our method can facilitate screening large libraries and predict potent site-specific nnAA-incorporated nanobody binders against crucial disease-targets.

Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody.

Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.

Computer-guided library generation applied to the optimization of single-domain antibodies.

Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody-antigen interaction surface. Based on this characterization, we hypothesized that in-silico mutagenesis could be employed to guide the development of a small library for phage display with the goal of improving the affinity of an antibody for its antigen. Optimized antibody mutants were identified after three rounds of selection, in which an alanine residue at the core of the antibody-antigen interface was substituted by residues with large side-chains, generating diverse kinetic responses, and resulting in greater affinity (>10-fold) for the antigen.

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Evaluation of carotid arterial intima-media thickness (IMT) and its relation to clinical parameters in Japanese children.

The aim of this study was to evaluate the carotid arterial intima-media thickness (IMT) and its relation to clinical parameters in Japanese children. Fifty-two healthy children (39 boys and 13 girls), aged 6-14 years, were enrolled in this cross-sectional investigation study. IMT of the common carotid artery was determined using ultrasonography. We also investigated anthropometric parameters, blood pressure (BP), lifestyles and blood examinations. The mean value of IMT was 0.4 ± 0.1mm, which was lower than the normal value (1.0mm) in adults. IMT was positively correlated with age (r=0.340) and height (r=0.346) in boys, while it was positively correlated with body mass index (BMI) (r=0.584) and diastolic BP (DBP) (r=0.563) in girls. In addition, IMT was associated with sleeping hours and hours of watching television (TV) by using stepwise regression analysis. In conclusion, IMT increased with aging, and it was linked to some clinical parameters of atherosclerosis and lifestyles in children. Therefore, this reference data will be helpful for future assessment of age-related change in Japanese children in clinical practice, and IMT might be a good predictor of atherosclerosis in Japanese children.