CDR-H3 Loop Research Articles

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution.

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.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind onlyto up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

A Designer Quest for the Achilles’ Heel of Influenza

Monoclonal antibodies are attractive but, in certain applications, limited therapeutic modalities due to their large size and high specificity. In this issue of Structure, Sevy at al. describe a computationally designed cyclic peptide mimicking the CDRH3 loop of the C05 antibody against influenza showing the potential utility of designer biologics.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus invitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

Prediction of immunogenicity for humanized and full human therapeutic antibodies.

Immunogenicity is an important concern for therapeutic antibodies during drug development. By analyzing co-crystal structures of idiotypic antibodies and their antibodies, we found that anti-idiotypic antibodies usually bind the Complementarity Determining Regions (CDR) of idiotypic antibodies. Sequence and structural features were identified for distinguishing immunogenic antibodies from non-immunogenic antibodies. For example, non-immunogenic antibodies have a significantly larger cavity volume at the CDR region and a more hydrophobic CDR-H3 loop than immunogenic antibodies. Antibodies containing no Gly at the turn of CDR-H2 loop are often immunogenic. We integrated these features together with a machine learning platform to Predict Immunogenicity for humanized and full human THerapeutic Antibodies (PITHA). This method achieved an accuracy of 83% in leave-one-out experiment for 29 therapeutic antibodies with available crystal structures. The accuracy decreased to 65% for 23 test antibodies with modeled structures, because their crystal structures were not available, and the prediction was made with modeled structures. The server of this method is accessible at http://mabmedicine.com/PITHA.

Local and Global Rigidification Upon Antibody Affinity Maturation.

During the affinity maturation process the immune system produces antibodies with higher specificity and activity through various rounds of somatic hypermutations in response to an antigen. Elucidating the affinity maturation process is fundamental in understanding immunity and in the development of biotherapeutics. Therefore, we analyzed 10 pairs of antibody fragments differing in their specificity and in distinct stages of affinity maturation using metadynamics in combination with molecular dynamics (MD) simulations. We investigated differences in flexibility of the CDR-H3 loop and global changes in plasticity upon affinity maturation. Among all antibody pairs we observed a substantial rigidification in flexibility and plasticity reflected in a substantial decrease of conformational diversity. To visualize and characterize these findings we used Markov-states models to reconstruct the kinetics of CDR-H3 loop dynamics and for the first time provide a method to define and localize surface plasticity upon affinity maturation.

Geometric potentials from deep learning improve prediction of CDR H3 loop structures.

MotivationAntibody structure is largely conserved, except for a complementarity-determining region featuring six variable loops. Five of these loops adopt canonical folds which can typically be predicted with existing methods, while the remaining loop (CDR H3) remains a challenge due to its highly diverse set of observed conformations. In recent years, deep neural networks have proven to be effective at capturing the complex patterns of protein structure. This work proposes DeepH3, a deep residual neural network that learns to predict inter-residue distances and orientations from antibody heavy and light chain sequence. The output of DeepH3 is a set of probability distributions over distances and orientation angles between pairs of residues. These distributions are converted to geometric potentials and used to discriminate between decoy structures produced by RosettaAntibody and predict new CDR H3 loop structures de novo.ResultsWhen evaluated on the Rosetta antibody benchmark dataset of 49 targets, DeepH3-predicted potentials identified better, same and worse structures [measured by root-mean-squared distance (RMSD) from the experimental CDR H3 loop structure] than the standard Rosetta energy function for 33, 6 and 10 targets, respectively, and improved the average RMSD of predictions by 32.1% (1.4 Å). Analysis of individual geometric potentials revealed that inter-residue orientations were more effective than inter-residue distances for discriminating near-native CDR H3 loops. When applied to de novo prediction of CDR H3 loop structures, DeepH3 achieves an average RMSD of 2.2 ± 1.1 Å on the Rosetta antibody benchmark.Availability and ImplementationDeepH3 source code and pre-trained model parameters are freely available at https://github.com/Graylab/deepH3-distances-orientations.Supplementary information Supplementary data are available at Bioinformatics online.

Protection Against Influenza B Viruses by Human Monoclonal Antibodies that Target the Neuraminidase Active Site

Influenza B virus infections can cause severe disease in pediatric patients and in the elderly. The risk of such disease is exacerbated by the lower clinical effectiveness of some antiviral drugs against influenza B compared with influenza A virus infections especially in children. Recent efforts have advanced neuraminidase (NA) as a reemerging candidate for novel broadly protective influenza virus vaccines. Such efforts are assisted by the identification of new broadly neutralizing and protective antibodies that target the NA active site. Here, we describe seven human anti-NA monoclonal antibodies (mAbs) isolated from an influenza B virus-infected patient. All antibodies block NA activity, neutralize virus, mediate effector functions and are broadly protective in vivo. We show that the two most potent mAbs inhibit NA activity by blocking the active site using long CDR-H3 loops that engage highly conserved residues amongst influenza B viruses. The comprehensive functional and structural characterization of these antibodies provides a blueprint for the development of improved vaccines and therapeutics against influenza B viruses.

Antibody humanization-the Influence of the antibody framework on the CDR-H3 loop ensemble in solution.

Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.

Structural Diversity of Ultralong CDRH3s in Seven Bovine Antibody Heavy Chains.

Antigen recognition by mammalian antibodies represents the most diverse setting for protein-protein interactions, because antibody variable regions contain exceptionally diverse variable gene repertoires of DNA sequences containing combinatorial, non-templated junctional mutational diversity. Some animals use additional strategies to achieve structural complexity in the antibody combining site, and one of the most interesting of these is the formation of ultralong heavy chain complementarity determining region 3 loops in cattle. Repertoire sequencing studies of bovine antibody heavy chain variable sequences revealed that bovine antibodies can contain heavy chain complementarity determining region 3 (CDRH3) loops with 60 or more amino acids, with complex structures stabilized by multiple disulfide bonds. It is clear that bovine antibodies can achieve long, peculiarly structured CDR3s, but the range of diversity and complexity of those structures is poorly understood. We determined the atomic resolution structure of seven ultralong bovine CDRH3 loops. The studies, combined with five previous structures, reveal a large diversity of cysteine pairing variations, and highly diverse globular domains.

Structure of the Anti-C60 Fullerene Antibody Fab Fragment: Structural Determinants of Fullerene Binding

The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by - stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.

Thermal Stability of Single-Domain-Antibodies Estimated by MD Simulations

Antibodies bind to antigens with high specificity and affinity and have complementarity-determining region (CDR) loops, which play an important role in antigen binding. We previously predicted the structure of the CDR-H3 loop by using MD simulations [1] and used MD for the refinement and the ranking of decoys generated by rigid-body antibody-antigen docking [2]. Single-domain-antibodies, sdAbs, function like regular antibodies, however consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets. For applications such as antibody drugs and biosensors, an sdAb with a high thermal stability is required. In this work, we chose seven sdAbs, which have a wide range of Tm-values and have known structures. We then applied MD simulations to estimate their relative stability and compare them with the experimental data. Recently, we executed MD to validate the stability of docking configurations [3] and substrate binding structures [4]. Here, high temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the Tm-values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q-values of hydrophilic residues exhibited an even better correlation. Measuring the Q-value on a per-residue level enabled us to identify residues that contribute significantly to the instability and demonstrate how our analysis can be used in a mutant case-study. [1] H. Nishigami et al. Protein Eng. Des. Sel. 29, 477-484 (2016). [2] N. Shimba et al. J. Chem. Inf. Model. 56, 2005-2012 (2016). [3] G.-J. Bekker et al. J. Chem. Theory Comput. 13, 2389-2399 (2017). [4] N. Numoto et al. Biochemistry 57, 5289-5300 (2018).

Multistate design of influenza antibodies improves affinity and breadth against seasonal viruses

Influenza is a yearly threat to global public health. Rapid changes in influenza surface proteins resulting from antigenic drift and shift events make it difficult to readily identify antibodies with broadly neutralizing activity against different influenza subtypes with high frequency, specifically antibodies targeting the receptor binding domain (RBD) on influenza HA protein. We developed an optimized computational design method that is able to optimize an antibody for recognition of large panels of antigens. To demonstrate the utility of this multistate design method, we used it to redesign an antiinfluenza antibody against a large panel of more than 500 seasonal HA antigens of the H1 subtype. As a proof of concept, we tested this method on a variety of known antiinfluenza antibodies and identified those that could be improved computationally. We generated redesigned variants of antibody C05 to the HA RBD and experimentally characterized variants that exhibited improved breadth and affinity against our panel. C05 mutants exhibited improved affinity for three of the subtypes used in design by stabilizing the CDRH3 loop and creating favorable electrostatic interactions with the antigen. These mutants possess increased breadth and affinity of binding while maintaining high-affinity binding to existing targets, surpassing a major limitation up to this point.

Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties.

We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies.

Next-generation sequencing reveals new insights about gene usage and CDR-H3 composition in the horse antibody repertoire

Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.

Crystal structure of an L chain optimised 14F7 anti-ganglioside Fv suggests a unique tumour-specificity through an unusual H-chain CDR3 architecture

Targeted cancer immunotherapy offers increased efficacy concomitantly with reduced side effects. One antibody with promising clinical potential is 14F7, which specifically recognises the NeuGc GM3 ganglioside. This antigen is found in the plasma membrane of a range of tumours, but is essentially absent from healthy human cells. 14F7 can discriminate NeuGc GM3 from the very similar NeuAc GM3, a common component of cell membranes. The molecular basis for this unique specificity is poorly understood. Here we designed and expressed 14F7-derived single-chain Fvs (scFvs), which retained the specificity of the parent antibody. Detailed expression and purification protocols are described as well as the synthesis of the NeuGc GM3 trisaccharide. The most successful scFv construct, which comprises an alternative variable light chain (VLA), allowed structure determination to 2.2 Å resolution. The structure gives insights into the conformation of the important CDR H3 loop and the suspected antigen binding site. Furthermore, the presence of VLA instead of the original VL elucidates how this subdomain indirectly stabilises the CDR H3 loop. The current work may serve as a guideline for the efficient production of scFvs for structure determination.

Co-evolution of HIV Envelope and Apex-Targeting Neutralizing Antibody Lineage Provides Benchmarks for Vaccine Design

SummaryBroadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.

Lessons learned from merging wet lab experiments with molecular simulation to improve mAb humanization.

Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called ‘tyrosine cage’. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5.

Repertoire Analysis of Antibody CDR-H3 Loops Suggests Affinity Maturation Does Not Typically Result in Rigidification.

Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody’s antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR) loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity.

Single Virion Super-Resolution Microscopy Unveils Mechanistic Details of Env Glycoprotein Recognition by the Broadly Neutralizing HIV-1 Antibodies 4E10 and 10E8

Anti-HIV broadly neutralizing antibodies (bnAb-s) 4E10 and 10E8 bind their epitopes on the membrane proximal external region (MPER) of the Env surface glycoprotein. The exact mechanisms of recognition and neutralization of the native Env remain obscure. Both antibodies present an unusually long CDR-H3 loop that is essential for function and shown to interact with lipid bilayers. Here, using fluorescence correlation spectroscopy we characterize first the nature of antibody-lipid interactions and demonstrate that both, membrane molecular order and electrostatic surface charge, determine the behavior and diffusivity of anti-MPER bnAb-s. Super-resolution single virus experiments using a collection of engineered antibodies and different virus strains further demonstrate that, although 10E8 and 4E10 do not bind directly to the viral lipid envelope, antibody-membrane electrostatic and hydrophobic interactions are essential for Env recognition. Moreover, we find a strong correlation between neutralization potency and extent of binding to Env. These results shed light on the role of the hydrophobic CDR-H3 loop and delineate the molecular features that are essential for the recognition and blocking of the native Env.