Balancing the extremes for antibody developability: hydrophobic and electrostatic germline framework signatures for CDR-loop compensation

In the past decade, the relevance of antibodies as therapeutics has increased substantially. Therefore, structural and functional characterization, in particular of the complementarity-determining regions (CDRs), is crucial to the design and engineering of antibodies with unique binding properties. Various studies have focused on classifying the CDR loops into a small set of main-chain conformations to facilitate antibody design by assuming that certain sequences can only adopt a limited number of conformations. Here, we present a kinetic classification of CDR loop structures as ensembles in solution. Using molecular dynamics simulations in combination with strong experimental structural information, we observe conformational transitions between canonical clusters and additional dominant solution structures in the micro-to-millisecond timescale for all CDR loops, independent of length and sequence composition. Besides identifying all relevant conformations in solution, our results revealed that various canonical cluster medians actually belong to the same kinetic minimum. Additionally, we reconstruct the kinetics and probabilities of the conformational transitions between canonical clusters, and thereby extend the model of static canonical structures to reveal a dynamic conformational ensemble in solution as a new paradigm in the field of antibody structure design. Abbreviations: CDR: Complementary-determining region; Fv: Antibody variable fragment; PCCA: Perron cluster analysis; tICA: Time-lagged independent component analysis; VH: Heavy chain variable region; VL: Light chain variable region

Roles of the respective loops at complementarity determining region on the antigen-antibody recognition

DMN Activity Pattern Under General Anesthesia Induced by Isoflurane is Dose-Dependent in Rats Brain

The combined approach of specific surface area (SSA), porosity, microprobe analysis (EMPA), transmission electron microscopy (TEM), scanning electron microscopy (SEM) equipped with EDX and infrared spectroscopy (FTIR) provided the mica mineral physico-chemical and morphological characterisation. The electrostatic surface properties were assessed through the determination of the Point of Zero Charge (pHPZC) by the drift method and the electrokinetic mica mineral features represented by the Isoelectric Point (pHIEP) which was carried out through zeta potential measurements. Adsorption tests were performed to correlate the surface charge behaviour of the mica mineral and its influence on the adsorption efficiency of two different dyes, namely: Safranin Orange (SO), as a cationic dye and Reactive Black 5 (RB5), as an anionic dye. The higher adsorption capacity SO dye was observed at pH 9 and achieved almost 83% of removal, while RB5 dye adsorption on mica surface had the highest result, about 45% of removal efficiency, on pH of 3. In both cases, the main mechanism identified that drove this results is the electrostatic force of attraction between the adsorbent edge surface charge (pH-dependent) and the ionic nature (anionic or cationic) of the pollutant dyes particles. The preliminary adsorption experiments demonstrated that the raw grounded mica mineral has a greater potential associated with its application on cationic dye removal in wastewater. The present study aimed to detail the main characteristics of the mica mineral in order to evaluate the potential use of such mineral residues in the removal efficiency of contaminated wastewater.

Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops.

Antigen binding by antibodies requires precise orientation of the complementarity- determining region (CDR) loops in the variable domain to establish the correct contact surface. Members of the family Camelidae have a modified form of immunoglobulin gamma (IgG) with only heavy chains, called Heavy Chain only Antibodies (HCAb). Antigen binding in HCAbs is mediated by only three CDR loops from the single variable domain (VHH) at the N-terminus of each heavy chain. This feature of the VHH, along with their other important features, e.g., easy expression, small size, thermo-stability and hydrophilicity, made them promising candidates for therapeutics and diagnostics. Thus, to design better VHH domains, it is important to thoroughly understand their sequence and structure characteristics and relationship. In this study, sequence characteristics of VHH domains have been analysed in depth, along with their structural features using innovative approaches, namely a structural alphabet. An elaborate summary of various studies proposing structural models of VHH domains showed diversity in the algorithms used. Finally, a case study to elucidate the differences in structural models from single and multiple templates is presented. In this case study, along with the above-mentioned aspects of VHH, an exciting view of various factors in structure prediction of VHH, like template framework selection, is also discussed.

Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (VH–VL). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71H (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71H, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71H on the paratope states and interface orientations has broad implications in the field of antibody engineering.

A computer-generated model of the single-chain variable V alpha V beta fragment of the RFL3.8 T-cell receptor (TCR) specific for fluorescein served as a starting point for mutagenesis aimed at identification of its antigen-contacting residues. Selected backbone segments of the model representing regions of prominent sequence similarity between antibodies and TCRs were least-squares superimposed onto the corresponding segments of the crystallographically resolved 4-4-20 antibody complexed with its antigen, fluorescein. The superimposition placed the antibody-bound fluorescein molecule close to a cavity on the surface of the TCR model formed by the complementarity-determining region (CDR) loops. Some of the TCR cavity forming loops displayed sequence motifs related to canonical CDR loops previously found in antibodies. Six putative amino acid contacts were identified and single-chain TCRs with mutations at each of these positions were expressed in Escherichia coli, purified, refolded, and assayed for fluorescein binding. Five of the six mutations resulted in a loss of detectable binding. These RFL3.8 antigen combining site residues are distributed among the beta 3, alpha 1, and alpha 2 CDR loops and show striking chemical similarity to the known fluorescein contact residues on 4-4-20. Thus, antibodies and TCRs are similar both in their overall architecture and in the chemical details of specific antigen recognition.

The adaptive immune system uses two main types of antigen receptors: T-cell receptors (TCRs) and antibodies. While both proteins share a globally similar β-sandwich architecture, TCRs are specialized to recognize peptide antigens in the binding groove of the major histocompatibility complex, while antibodies can bind an almost infinite range of molecules. For both proteins, the main determinants of target recognition are the complementarity-determining region (CDR) loops. Five of the six CDRs adopt a limited number of backbone conformations, known as the “canonical classes”; the remaining CDR (β3in TCRs and H3 in antibodies) is more structurally diverse. In this paper, we first update the definition of canonical forms in TCRs, build an auto-updating sequence-based prediction tool (available at http://opig.stats.ox.ac.uk/resources) and demonstrate its application on large scale sequencing studies. Given the global similarity of TCRs and antibodies, we then examine the structural similarity of their CDRs. We find that TCR and antibody CDRs tend to have different length distributions, and where they have similar lengths, they mostly occupy distinct structural spaces. In the rare cases where we found structural similarity, the underlying sequence patterns for the TCR and antibody version are different. Finally, where multiple structures have been solved for the same CDR sequence, the structural variability in TCR loops is higher than that in antibodies, suggesting TCR CDRs are more flexible. These structural differences between TCR and antibody CDRs may be important to their different biological functions.

Influenza viruses remain a persistent challenge to human health owing to their inherent ability to evade the immune response by antigenic drift. However, the discovery of broadly neutralizing antibodies (bnAbs) against divergent viruses has sparked renewed interest in a universal influenza vaccine and novel therapeutic opportunities. Here, a crystal structure at 1.70 Å resolution is presented of the Fab of the human antibody CH65, which has broad neutralizing activity against a range of seasonal H1 isolates. Previous studies proposed that affinity maturation of this antibody lineage pre-organizes the complementarity-determining region (CDR) loops into an energetically favorable HA-bound conformation. Indeed, from the structural comparisons of free and HA-bound CH65 presented here, the CDR loops, and in particular the heavy-chain CDR3, adopt the same conformations in the free and bound forms. Thus, these findings support the notion that affinity maturation of the CH65 lineage favorably preconfigures the CDR loops for high-affinity binding to influenza hemagglutinin.

αβ T cell receptors (TCRs) recognize peptides presented by major histocompatibility complex (MHC) proteins using multiple complementarity determining region (CDR) loops. TCRs display an array of poorly understood recognition properties, including specificity, cross-reactivity, and MHC restriction. Here we report a comprehensive thermodynamic deconstruction of the interaction between the A6 TCR and the Tax peptide presented by the class I MHC HLA-A*0201, uncovering the physical basis for the receptor's recognition properties. Broadly, our findings are in conflict with widely-held generalities regarding TCR recognition, such as the relative contributions of central and peripheral peptide residues and the roles of the hypervariable and germline CDR loops in engaging peptide and MHC. Instead we find that the recognition properties of the receptor emerge from the need to engage the composite peptide/MHC surface, with the receptor utilizing its CDR loops in a cooperative fashion such that specificity, cross-reactivity, and MHC restriction are inextricably linked.

Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed of 4 different kappa light chains paired with 4 different heavy chains. In this study, we systematically show that different heavy and light chain pairings strongly influence the paratope, interdomain interaction patterns and the relative VH-VL interface orientations. We observe changes in conformational diversity and substantial population shifts of the complementarity determining region (CDR) loops, resulting in distinct dominant solution structures and differently favored canonical structures. Additionally, we identify conformational changes in the structural diversity of the CDR-H3 loop upon different heavy and light chain pairings, as well as upon changes in sequence and structure of the neighboring CDR loops, despite having an identical CDR-H3 loop amino acid sequence. These results can also be transferred to all CDR loops and to the relative VH-VL orientation, as certain paratope states favor distinct interface angle distributions. Furthermore, we directly compare the timescales of sidechain rearrangements with the well-described transition kinetics of conformational changes in the backbone of the CDR loops. We show that sidechain flexibilities are strongly affected by distinct heavy and light chain pairings and decipher germline-specific structural features co-determining stability. These findings reveal that all CDR loops are strongly correlated and that distinct heavy and light chain pairings can result in different paratope states in solution, defined by a characteristic combination of CDR loop conformations and VH-VL interface orientations. Thus, these results have broad implications in the field of antibody engineering, as they clearly show the importance of considering paired heavy and light chains to understand the antibody binding site, which is one of the key aspects in the design of therapeutics.

A New Clustering of Antibody CDR Loop Conformations

ABSTRACTComputational modeling of antibody structures plays a critical role in therapeutic antibody design. Several antibody modeling pipelines exist, but no freely available methods currently model nanobodies, provide estimates of expected model accuracy, or highlight potential issues with the antibody's experimental development. Here, we describe our automated antibody modeling pipeline, ABodyBuilder, designed to overcome these issues. The algorithm itself follows the standard 4 steps of template selection, orientation prediction, complementarity-determining region (CDR) loop modeling, and side chain prediction. ABodyBuilder then annotates the ‘confidence’ of the model as a probability that a component of the antibody (e.g., CDRL3 loop) will be modeled within a root–mean square deviation threshold. It also flags structural motifs on the model that are known to cause issues during in vitro development. ABodyBuilder was tested on 4 separate datasets, including the 11 antibodies from the Antibody Modeling Assessment–II competition. ABodyBuilder builds models that are of similar quality to other methodologies, with sub–Angstrom predictions for the ‘canonical’ CDR loops. Its ability to model nanobodies, and rapidly generate models (∼30 seconds per model) widens its potential usage. ABodyBuilder can also help users in decision–making for the development of novel antibodies because it provides model confidence and potential sequence liabilities. ABodyBuilder is freely available at http://opig.stats.ox.ac.uk/webapps/abodybuilder.

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region