A potential antibody repertoire diversification mechanism through tyrosine sulfation for biotherapeutics engineering and production.

The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody's binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed.

Avidity engineering of human heavy-chain-only antibodies mitigates neutralization resistance of SARS-CoV-2 variants.

Emerging SARS-CoV-2 variants have accrued mutations within the spike protein rendering most therapeutic monoclonal antibodies against COVID-19 ineffective. Hence there is an unmet need for broad-spectrum mAb treatments for COVID-19 that are more resistant to antigenically drifted SARS-CoV-2 variants. Here we describe the design of a biparatopic heavy-chain-only antibody consisting of six antigen binding sites recognizing two distinct epitopes in the spike protein NTD and RBD. The hexavalent antibody showed potent neutralizing activity against SARS-CoV-2 and variants of concern, including the Omicron sub-lineages BA.1, BA.2, BA.4 and BA.5, whereas the parental components had lost Omicron neutralization potency. We demonstrate that the tethered design mitigates the substantial decrease in spike trimer affinity seen for escape mutations for the hexamer components. The hexavalent antibody protected against SARS-CoV-2 infection in a hamster model. This work provides a framework for designing therapeutic antibodies to overcome antibody neutralization escape of emerging SARS-CoV-2 variants.

Evolutive emergence and divergence of an Ig regulatory node: An environmental sensor getting cues from the aryl hydrocarbon receptor?

One gene, the immunoglobulin heavy chain (IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3' end of the IgH gene locus (3'RR). Animal models have demonstrated an essential role of the 3'RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3'RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3'RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3'RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3'RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2.

General Trends of the Camelidae Antibody VHHs Domain Dynamics.

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen-antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design.

Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens¹. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens^2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes^23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

EXPRESSION OF IMMUNOGLOBULIN SEQUENCES HOMOLOGOUS TO ANTI-SARS-CoV-2 ANTIBODIES AND HIV IN CHRONIC LYMPHOCYTIC LEUKEMIA CASES.

BACKGROUND: Identification of epitopes recognized by leukemic B cells could provide insights into the molecular mechanisms of B cell transformation in chronic lymphocytic leukemia (CLL). The aim of this paper was to compare nucleotide sequences of immunoglobulin heavy chain variable region (IGHV) genes in CLL with known sequences directed against antigens of different origins available in public databases. MATERIALS AND METHODS: Analysis was performed in the groups of 412 unselected CLL patients with productive IGHV gene using polymerase chain reaction followed by direct sequencing. RESULTS: Homology between CLL Ig sequences and antibodies directed against autoantigens was found in 12 patients (2.9%), homology between CLL Ig sequences and antiviral antibodies - in 35 patients (8.5%). Most of these sequences belonged to stereotypical clusters. Among the sequences that have homology to antiviral antibodies, the most prevalent were cases homologous with antibodies against HIV (14 cases, 3.4%) and SARS-CoV-2 antigens (10 cases, 2.4%). None of the patients in our cohort was HIV-infected and the study was conducted before the emergence of SARS-CoV-2 virus. CONCLUSIONS: Suggestions could be made about the possible impact of past infection of SARS-CoV-2 virus on the pathogenesis of CLL. In particular, an increase in the proportion of CLL cases with the expression of some stereotyped BCR and/or an increase of CLL risk in the long-term period after SARS-CoV-2 virus infection is not excluded. This assumption needs to be verified by epidemiological data.

ImmunoTyper-SR: A computational approach for genotyping immunoglobulin heavy chain variable genes using short-read data.

Human immunoglobulin heavy chain (IGH) locus on chromosome 14 includes more than 40 functional copies of the variable gene (IGHV), which are critical for the structure of antibodies that identify and neutralize pathogenic invaders as a part of the adaptive immune system. Because of its highly repetitive sequence composition, the IGH locus has been particularly difficult to assemble or genotype when using standard short-read sequencing technologies. Here, we introduce ImmunoTyper-SR, an algorithmic tool for the genotyping and CNV analysis of the germline IGHV genes on Illumina whole-genome sequencing (WGS) data using a combinatorial optimization formulation that resolves ambiguous read mappings. We have validated ImmunoTyper-SR on 12 individuals, whose IGHV allele composition had been independently validated, as well as concordance between WGS replicates from nine individuals. We then applied ImmunoTyper-SR on 585 COVID patients to investigate the associations between IGHV alleles and anti-type I IFN autoantibodies, which were previously associated with COVID-19 severity.

A megadiverse naïve library derived from numerous camelids for efficient and rapid development of VHH antibodies.

The field of antibody development is under pressure to meet rising demands for speed, cost-effectiveness, efficacy, reliability, and large-scale production. It is costly and time-consuming to immunize animals and build a single-domain antibody (sdAb) library for each target. Using the variable domain (VHH) of heavy-chain only antibodies (HcAbs) derived from blood samples of 75 non-immunized camelid animals (51 alpacas, 13 llamas, 11 Bactrian camels), and spleens from two Bactrian camels, a naïve sdAb library with extensive megadiversity and reusability was constructed. The library was evaluated using next-generation DNA sequencing (NGS) and was found to contain hundreds of billions of unique clones. To confirm the availability of target-specific VHHs, a naive library was screened for a variety of targets. At least two VHH candidates were extracted for each target using a 20-day selection pipeline. Some binders had ultrahigh potencies, with binding affinities in the nanomolar range. This naïve library, in particular, offers the possibility of acquiring unique antibodies targeting antigens of interest with low feasible dissociation constant (kD) without the time, effort, and price associated in producing antibodies in animals via antigen injection. Overall, the study shows that the megadiverse naïve library provides a rapid, adaptable, and easy platform for antibody creation, emphasizing its therapeutic and diagnostic implications.

Global patterns of antigen receptor repertoire disruption across adaptive immune compartments in COVID-19.

Whereas pathogen-specific T and B cells are a primary focus of interest during infectious disease, we have used COVID-19 to ask whether their emergence comes at a cost of broader B cell and T cell repertoire disruption. We applied a genomic DNA-based approach to concurrently study the immunoglobulin-heavy (IGH) and T cell receptor (TCR) ß and δ chain loci of 95 individuals. Our approach detected anticipated repertoire focusing for the IGH repertoire, including expansions of clusters of related sequences temporally aligned with SARS-CoV-2-specific seroconversion, and enrichment of some shared SARS-CoV-2-associated sequences. No significant age-related or disease severity-related deficiencies were noted for the IGH repertoire. By contrast, whereas focusing occurred at the TCRß and TCRδ loci, including some TCRß sequence-sharing, disruptive repertoire narrowing was almost entirely limited to many patients aged older than 50 y. By temporarily reducing T cell diversity and by risking expansions of nonbeneficial T cells, these traits may constitute an age-related risk factor for COVID-19, including a vulnerability to new variants for which T cells may provide key protection.

Construction of a Large Size Human Immunoglobulin Heavy Chain Variable (VH) Domain Library, Isolation and Characterization of Novel Human Antibody VH Domains Targeting PD-L1 and CD22.

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

VHH Structural Modelling Approaches: A Critical Review.

VHH, i.e., VH domains of camelid single-chain antibodies, are very promising therapeutic agents due to their significant physicochemical advantages compared to classical mammalian antibodies. The number of experimentally solved VHH structures has significantly improved recently, which is of great help, because it offers the ability to directly work on 3D structures to humanise or improve them. Unfortunately, most VHHs do not have 3D structures. Thus, it is essential to find alternative ways to get structural information. The methods of structure prediction from the primary amino acid sequence appear essential to bypass this limitation. This review presents the most extensive overview of structure prediction methods applied for the 3D modelling of a given VHH sequence (a total of 21). Besides the historical overview, it aims at showing how model software programs have been shaping the structural predictions of VHHs. A brief explanation of each methodology is supplied, and pertinent examples of their usage are provided. Finally, we present a structure prediction case study of a recently solved VHH structure. According to some recent studies and the present analysis, AlphaFold 2 and NanoNet appear to be the best tools to predict a structural model of VHH from its sequence.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Observed Antibody Space: A diverse database of cleaned, annotated, and translated unpaired and paired antibody sequences.

The antibody repertoires of individuals and groups have been used to explore disease states, understand vaccine responses, and drive therapeutic development. The arrival of B-cell receptor repertoire sequencing has enabled researchers to get a snapshot of these antibody repertoires, and as more data are generated, increasingly in-depth studies are possible. However, most publicly available data only exist as raw FASTQ files, making the data hard to access, process, and compare. The Observed Antibody Space (OAS) database was created in 2018 to offer clean, annotated, and translated repertoire data. In this paper, we describe an update to OAS that has been driven by the increasing volume of data and the appearance of paired (VH/VL) sequence data. OAS is now accessible via a new web server, with standardized search parameters and a new sequence-based search option. The new database provides both nucleotides and amino acids for every sequence, with additional sequence annotations to make the data Minimal Information about Adaptive Immune Receptor Repertoire compliant, and comments on potential problems with the sequence. OAS now contains 25 new studies, including severe acute respiratory syndrome coronavirus 2 data and paired sequencing data. The new database is accessible at http://opig.stats.ox.ac.uk/webapps/oas/, and all data are freely available for download.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion, and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones 2-3 weeks after the onset of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34, and IGHV4-39 in COVID-19 patients were more abundant than those of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

Characterizing the BCR repertoire during lymphocyte reduction and recovery mediated by cyclophosphamide and granulocyte-macrophage colony-stimulating factor.

Leukopenia is a common manifestation of many diseases, including global outbreak SAS-CoV-2 infection. Granulocyte-macrophage colony-stimulating factor (GM -CSF) has been proved to be effective in promoting lymphocyte regeneration, but adverse immunological effects have also emerged. This study aim to investigate the effect of GM -CSF on BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration. Cyclophosphamide (CTX) and GM -CSF were used to inhibit and stimulate bone marrow hematopoiesis, respectively. High throughput sequencing was applied to detect the characteristics of BCR CDR3 repertoire in controls, CTX group and GM -CSF group. The white blood cells (WBCs) were quickly reduced (P < 0.05) with lymphocytes decreasing causing by CTX, and the WBCs and lymphocytes returned to the level of controls after GM -CSF treatment. The diversity of BCR heavy chain CDR3 repertoire was also significantly decreased in CTX group. Although there is still a big gap from the controls, the diversity was picked up after GM -CSF treatment. The expression of IGHD01-01, IGHD02-14 and IGHJ04-01 with high-frequency usage regularly and significantly changed in three groups, and many genes with low-frequency usage lost in CTX group and did not reappear in GM -CSF group. Moreover, two shared sequences and accounted for the highest proportion in GM -CSF group have been detected in animal model of chronic lymphocytic leukemia. These results revealed that GM -CSF can partially restore changes in the BCR heavy chain CDR3 repertoire while promoting lymphocyte regeneration, but it may also lead to rearrangement, proliferation and activation of abnormal B cells, which can provide a basis for further study on the adverse immunological effects and mechanism of GM -CSF treatment.

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

Protocol for constructing large size human antibody heavy chain variable domain (VH) library and selection of SARS-CoV-2 neutralizing antibody domains.

This protocol is a comprehensive guide to phage display-based selection of virus neutralizing VH antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 1011) naïve human antibody heavy chain domain library, (2) SARS-CoV-2 antigen expression and stable cell line construction, and (3) library panning for selection of SARS-CoV-2-specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

An Alternative Binding Mode of IGHV3-53 Antibodies to the SARS-CoV-2 Receptor Binding Domain.

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 because of structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization.