Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH’s) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Atomically accurate de novo design of single-domain antibodies

We introduce AbDiffuser, an equivariant and physics-informed diffusion model for the joint generation of antibody 3D structures and sequences. AbDiffuser is built on top of a new representation of protein structure, relies on a novel architecture for aligned proteins, and utilizes strong diffusion priors to improve the denoising process. Our approach improves protein diffusion by taking advantage of domain knowledge and physics-based constraints; handles sequence-length changes; and reduces memory complexity by an order of magnitude, enabling backbone and side chain generation. We validate AbDiffuser in silico and in vitro. Numerical experiments showcase the ability of AbDiffuser to generate antibodies that closely track the sequence and structural properties of a reference set. Laboratory experiments confirm that all 16 HER2 antibodies discovered were expressed at high levels and that 57.1% of the selected designs were tight binders.

AbDiffuser: Full-Atom Generation of In-Vitro Functioning Antibodies

Abstract Antibodies are key proteins of the adaptive immune system, and there exists a large body of academic literature and patents dedicated to their study and concomitant conversion into therapeutics, diagnostics, or reagents. These documents often contain extensive functional characterisations of the sets of antibodies they describe. However, leveraging these heterogeneous reports, for example to offer insights into the properties of query antibodies of interest, is currently challenging as there is no central repository through which this wide corpus can be mined by sequence or structure. Here, we present PLAbDab (the Patent and Literature Antibody Database), a self-updating repository containing over 150,000 paired antibody sequences and 3D structural models, of which over 65 000 are unique. We describe the methods used to extract, filter, pair, and model the antibodies in PLAbDab, and showcase how PLAbDab can be searched by sequence, structure, or keyword. PLAbDab uses include annotating query antibodies with potential antigen information from similar entries, analysing structural models of existing antibodies to identify modifications that could improve their properties, and facilitating the compilation of bespoke datasets of antibody sequences/structures that bind to a specific antigen. PLAbDab is freely available via Github (https://github.com/oxpig/PLAbDab) and as a searchable webserver (https://opig.stats.ox.ac.uk/webapps/plabdab/). 

The Patent and Literature Antibody Database (PLAbDab): an evolving reference set of functionally diverse, literature-annotated antibody sequences and structures

Abstract Immunofocusing is a strategy to create immunogens that redirect humoral immune responses towards a targeted epitope and away from non-desirable epitopes. Immunofocusing methods often aim to develop “universal” vaccines that provide broad protection against highly variant viruses such as influenza virus, human immunodeficiency virus (HIV-1), and most recently, severe acute respiratory syndrome coronavirus (SARS-CoV-2). We use existing examples to illustrate five main immunofocusing strategies—cross-strain boosting, mosaic display, protein dissection, epitope scaffolding, and epitope masking. We also discuss obstacles for immunofocusing like immune imprinting. A thorough understanding, advancement, and application of the methods we outline here will enable the design of high-resolution vaccines that protect against future viral outbreaks. 

Bringing immunofocusing into focus

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries. 

Structural trends in antibody-antigen binding interfaces: a computational analysis of 1833 experimentally determined 3D structures

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant “public epitopes” at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid–binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system’s architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens. Description Antibodies GRAB their targets Different individuals will reproducibly generate antibodies against the same epitopes after exposure to a pathogen. However, the mechanisms driving the production of these “public antibodies” continue to be poorly understood. Shrock et al. used a phage display platform called VirScan to glean additional insights into this process. They found that many human V gene segments contained germline-encoded amino acid–binding (GRAB) motifs that bind particular amino acids and are essential for antibody recognition. Moreover, a comparison of mouse and human GRAB motifs uncovered only a partial overlap, which may explain the distinct public epitopes targeted by different species. —STS Antibody genes have GRAB motifs that bind specific amino acids and drive reproducible responses to immunodominant epitopes. INTRODUCTION Antibodies are generated by a DNA recombination mechanism occurring in the immunoglobulin heavy and light chain genes in which modular VDJ (for heavy) and VJ (for light) gene segments are combinatorially assembled. The vast complexity of the antibody repertoire allows many species to generate antibodies against virtually any protein. However, when different individuals are exposed to a given pathogen they often mount antibody responses to the same precise protein regions—or epitopes—from the pathogen. The mechanisms underlying these recurrent antibody responses to immunodominant “public epitopes” are not well understood. RATIONALE We set out to identify a collection of immunodominant public epitopes that would allow us to study mechanisms underlying recurrent antibody responses. We employed VirScan—a phage display platform programmed to display peptides covering the entire human virome—to identify the epitopes of antiviral antibodies from a large cohort of individuals in a high-throughput manner. Additionally, we isolated B cell receptors from different individuals that bound to model public epitopes in order to investigate their determinants of specificity. Finally, we performed a systematic analysis of antibody–antigen structures from the Protein Data Bank (PDB) to search for recurrent modes of antigen recognition. RESULTS We mapped 376 immunodominant public epitopes from 51 viral species to single-amino-acid resolution. Antibodies from different individuals that recognized the same public epitope often (i) shared light chain isotype (kappa or lambda) and (ii) bound the same precise critical residues in the epitope. Public epitopes showed biased amino acid composition, including a striking enrichment of lysine at the borders of public epitopes recognized by antibodies with lambda light chains. We examined 50 B cell receptors recognizing three model public epitopes in detail and observed conserved V gene segment usage but almost no conservation of heavy chain CDR3 sequences, indicating that key specificity determinants lay within the V gene segments themselves. Structural analysis of antibody–antigen complexes in the PDB uncovered 18 human V gene segments that harbor germline-encoded amino acid–binding (GRAB) motifs that specifically bind to particular amino acids. Among these were a family of six closely related lambda V gene segments with similar GRAB motifs specific for border lysines. We confirmed that the GRAB motifs we identified were critical for antibody recognition of two model public epitopes. Analysis of murine antibody–antigen structures revealed 21 V gene segment–encoded GRAB motifs that only partially overlapped with the human GRAB motifs, which may explain why there is little overlap between the public epitopes recognized across species. Thus, there appears to be a structural basis underlying the notable convergence in humoral immune responses to immunodominant public epitopes across humans and the differing public epitope selection across species. CONCLUSION Recurrent antibody responses to immunodominant public epitopes are a general feature of humoral immunity. We propose that they are driven by GRAB motifs, a germline-encoded component of the architecture of the antibody repertoire that predisposes antibodies to recognize particular structures and thus influences epitope selection and composition. Public epitopes likely arise in part because they are best aligned for recognition by GRAB motifs and can thus be bound by a relatively large precursor pool of B cells. GRAB motifs may have evolved to ensure efficient antibody responses to pathogens; the recurrent responses they engender across populations likely exert selective pressure on pathogens and influence host–pathogen coevolution. Uncovering origins of public antibody responses. VirScan reveals shared antibody responses to certain regions of viral proteins (“public epitopes”) across human populations. Heatmap color represents the strength of the antibody response in each individual (columns) to each peptide of a viral protein (rows). An analysis of antibody–antigen structures reveals that antibody genes harbor germline-encoded motifs that specifically bind certain amino acids and drive recognition of public epitopes.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by first engaging its cellular receptor angiotensin converting enzyme 2 (ACE2) to induce conformational changes in the virus-encoded spike protein and fusion between the viral and target cell membranes. We report here that certain monoclonal neutralizing antibodies against distinct epitopic regions of the receptor-binding domain of the spike can replace ACE2 to serve as a receptor and efficiently support membrane fusion and viral infectivity. These receptor-like antibodies can function in the form of a complex of their soluble immunoglobulin G with Fc-gamma receptor I, a chimera of their antigen-binding fragment with the transmembrane domain of ACE2 or a membrane-bound B cell receptor, indicating that ACE2 and its specific interactions with the spike protein are dispensable for SARS-CoV-2 entry. These results suggest that antibody responses against SARS-CoV-2 may expand the viral tropism to otherwise nonpermissive cell types; they have important implications for viral transmission and pathogenesis.

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Antibody-mediated cell entry of SARS-CoV-2

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Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses

Antibody-mediated cell entry of SARS-CoV-2