Sequence variants influencing the regulation of serum IgG subclass levels.

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4), encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here, we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years, we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma, autoimmune diseases, and blood traits. Seven variants map to the IGH locus, three to the Fcγ receptor (FCGR) locus, and two to the human leukocyte antigen (HLA) region, affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f), G2m(n) and G3m(b*) allotypes, and IgG1, IgG2 and IgG3, respectively. Additionally, we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3, ZPBP2, GSDMB, ORMDL3). Interestingly, the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining.

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Discovery of potent allosteric antibodies inhibiting EGFR.

In this work, we report the discovery of potent anti-epidermal growth factor receptor (EGFR) allosteric heavy-chain antibodies by combining camelid immunization and fluorescence-activated cell sorting (FACS). After immunization and yeast surface display library construction, allosteric clones were obtained by introducing the labeled EGF Fc fusion protein as an additional criterion for FACS. This sorting method enabled the identification of 11 heavy-chain antibodies that did not compete with the orthosteric ligand EGF for the binding to EGFR. These antibodies bind to a triple-negative breast cancer cell line expressing EGFR with affinities in the picomolar to nanomolar range. Those camelid-derived antibodies also exhibit interesting properties by modulating EGFR affinity for EGF. Moreover, they are also able to inhibit EGF-induced downstream signaling pathways. In particular, we identified one clone that is more potent than the approved blocking antibody cetuximab in inhibiting both PI3K/AKT and MAPK/ERK pathways. Our results suggest that allosteric antibodies may be potential new modalities for therapeutics.

Functional Divergence in the Affinity and Stability of Non-Canonical Cysteines and Non-Canonical Disulfide Bonds: Insights from a VHH and VNAR Study.

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

An IGHG1 variant exhibits polarized prevalence and confers enhanced IgG1 antibody responses against life-threatening organisms.

Evolutionary pressures sculpt population genetics, whereas immune adaptation fortifies humans against life-threatening organisms. How the evolution of selective genetic variation in adaptive immune receptors orchestrates the adaptation of human populations to contextual perturbations remains elusive. Here, we show that the G396R coding variant within the human immunoglobulin G1 (IgG1) heavy chain presents a concentrated prevalence in Southeast Asian populations. We uncovered a 190-kb genomic linkage disequilibrium block peaked in close proximity to this variant, suggestive of potential Darwinian selection. This variant confers heightened immune resilience against various pathogens and viper toxins in mice. Mechanistic studies involving severe acute respiratory syndrome coronavirus 2 infection and vaccinated individuals reveal that this variant enhances pathogen-specific IgG1+ memory B cell activation and antibody production. This G396R variant may have arisen on a Neanderthal haplotype background. These findings underscore the importance of an IGHG1 variant in reinforcing IgG1 antibody responses against life-threatening organisms, unraveling the intricate interplay between human evolution and immune adaptation.

SARS-CoV-2 JN.1 variant evasion of IGHV3-53/3-66 B cell germlines.

The severe acute respiratory syndrome coronavirus 2 variant JN.1 recently emerged as the dominant variant despite having only one amino acid change on the spike (S) protein receptor binding domain (RBD) compared with the ancestral BA.2.86, which never represented more than 5% of global variants. To define at the molecular level the JN.1 ability to spread globally, we interrogated a panel of 899 neutralizing human monoclonal antibodies. Our data show that the single leucine-455-to-serine mutation in the JN.1 spike protein RBD unleashed the global spread of JN.1, likely occurring by elimination of more than 70% of the neutralizing antibodies mediated by IGHV3-53/3-66 germlines. However, the resilience of class 3 antibodies with low neutralization potency but strong Fc functions may explain the absence of JN.1 severe disease.

Antibody-SGM, a Score-Based Generative Model for Antibody Heavy-Chain Design.

Traditional computational methods for antibody design involved random mutagenesis followed by energy function assessment for candidate selection. Recently, diffusion models have garnered considerable attention as cutting-edge generative models, lauded for their remarkable performance. However, these methods often focus solely on the backbone or sequence, resulting in the incomplete depiction of the overall structure and necessitating additional techniques to predict the missing component. This study presents Antibody-SGM, an innovative joint structure-sequence diffusion model that addresses the limitations of existing protein backbone generation models. Unlike previous models, Antibody-SGM successfully integrates sequence-specific attributes and functional properties into the generation process. Our methodology generates full-atom native-like antibody heavy chains by refining the generation to create valid pairs of sequences and structures, starting with random sequences and structural properties. The versatility of our method is demonstrated through various applications, including the design of full-atom antibodies, antigen-specific CDR design, antibody heavy chains optimization, validation with Alphafold3, and the identification of crucial antibody sequences and structural features. Antibody-SGM also optimizes protein function through active inpainting learning, allowing simultaneous sequence and structure optimization. These improvements demonstrate the promise of our strategy for protein engineering and significantly increase the power of protein design models.

Development of novel humanized VHH synthetic libraries based on physicochemical analyses.

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.

Adaptive Evolution of Two Distinct Adaptive Haplotypes of Neanderthal Origin at the Immunoglobulin Heavy-chain Locus in East Asian and European Populations.

Immunoglobulins (Igs) have a crucial role in humoral immunity. Two recent studies have reported a high-frequency Neanderthal-introgressed haplotype throughout Eurasia and a high-frequency Neanderthal-introgressed haplotype specific to southern East Asia at the immunoglobulin heavy-chain (IGH) gene locus on chromosome 14q32.33. Surprisingly, we found the previously reported high-frequency Neanderthal-introgressed haplotype does not exist throughout Eurasia. Instead, our study identified two distinct high-frequency haplotypes of putative Neanderthal origin in East Asia and Europe, although they shared introgressed alleles. Notably, the alleles of putative Neanderthal origin reduced the expression of IGHG1 and increased the expression of IGHG2 and IGHG3 in various tissues. These putatively introgressed alleles also affected the production of IgG1 upon antigen stimulation and increased the risk of systemic lupus erythematosus. Additionally, the greatest genetic differentiation across the whole genome between southern and northern East Asians was observed for the East Asian haplotype of putative Neanderthal origin. The frequency decreased from southern to northern East Asia and correlated positively with the genome-wide proportion of southern East Asian ancestry, indicating that this putative positive selection likely occurred in the common ancestor of southern East Asian populations before the admixture with northern East Asian populations.

Addressing Technical Pitfalls in Pursuit of Molecular Factors That Mediate Immunoglobulin Gene Regulation.

The expressed Ab repertoire is a critical determinant of immune-related phenotypes. Ab-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human Abs are composed of two identical H and L chain polypeptides derived from genes in IGH locus and one of two L chain loci. The combinatorial diversity that results from Ab gene rearrangement and the pairing of different H and L chains contributes to the immense diversity of the baseline Ab repertoire. During rearrangement, Ab gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the Ab repertoire associates with germline variation in IGH, implicating polymorphism in Ab gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. In this study, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype resolution. Using simulated and chromatin immunoprecipitation sequencing reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of the DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed Ab repertoires.

Analysis of immunoglobulin organization and complexity in mink (Neovison vison).

Mink are susceptible to viruses such as SARS-CoV-2, H1N1 and H9N2, so they are considered a potential animal model for studying human viral infections. Therefore, it is important to study the immune system of mink. Immunoglobulin (Ig) is an important component of humoral immunity and plays an important role in the body's immune defense. In this study, we described the gene loci structure of mink Ig germline by genome comparison, and analysed the mechanism of expression diversity of mink antibody library by 5'RACE and next-generation sequencing (NGS). The results were as follows: the IgH, Igκ and Igλ loci of mink were located on chromosome 13, chromosome 8 and chromosome 3, respectively, and they had 25, 36 and 7 V genes, 3, 5 and 7 J genes and 10 DH genes, respectively. Mink Ig heavy chain preferred the IGHV1, IGHD2 and IGHJ4 subgroups, κ chain mainly use the IGKV1, IGKJ1 and IGHL4 subgroups, and λ chain mainly use the IGLV3 and IGLJ3 subgroups. Linkage diversity analysis revealed that N nucleotide insertion was the main factor affecting the linkage diversity of mink Igs. On the mutation types of mink Ig Somatic Hypermutation (SHM), the high mutation types of heavy chain were mainly G > A, C > T, T > C, A > G, C > A, G > T, A > C, and T > G; the high mutation types of κ chain were G > A and T > C; and the high mutation types of λ chain were G > A and A > G. The objective of this study was to analyse the loci structure and expression diversity of Ig in mink. The results contribute to our comprehension of Ig expression patterns in mink and were valuable for advancing knowledge in mink immunogenetics, exploring the evolution of adaptive immune systems across different species, and conducting comparative genomics research.

Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.

RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

YY1-mediated enhancer-promoter communication in the immunoglobulin µ locus is regulated by MSL/MOF recruitment.

The rearrangement and expression of the immunoglobulin µ heavy chain (Igh) gene require communication of the intragenic Eµ and 3' regulatory region (RR) enhancers with the variable (VH) gene promoter. Eµ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eµ wild-type enhancer and that of Eµ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces µ gene expression and chromatin looping of Eµ to the 3' RR enhancer and VH promoter. Moreover, Mof heterozygosity in mice impaired µ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication.

UPF1 plays critical roles in early B cell development.

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.

A single-domain antibody library based on a stability-engineered human VH3 scaffold.

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua).

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Šresolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

IGH Complementarity Determining Region-3-Cytomegalovirus Protein Chemical Complementarity Linked to Better Overall Survival Probabilities for Glioblastoma.

Cytomegalovirus (CMV) has long been thought to have an association with glioblastoma multiforme (GBM), although the exact role of CMV and any subsequent implications for treatment have yet to be fully understood. This study addressed whether IGH complementarity determining region-3 (CDR3)-CMV protein chemical complementarity, with IGH CDR3s representing both tumor resident and blood-sourced IGH recombinations, was associated with overall survival (OS) distinctions. IGH recombination sequencing reads were obtained from (a) the Clinical Proteomic Tumor Analysis Consortium, tumor RNAseq files; and (b) the cancer genome atlas, blood exome-derived files. The Adaptive Match web tool was used to calculate chemical complementarity scores (CSs) based on hydrophobic interactions, and those scores were used to group GBM cases and assess survival probabilities. We found a higher OS probability for cases whose hydrophobic IGH CDR3-CMV protein chemical complementarity scores (Hydro CSs) were in the upper 50th percentile for several CMV proteins, including UL99 and UL123, as well as for CSs based on known B cell epitopes representing these proteins. We also identified multiple immune signature genes, including CD79A and TNFRSF17, for which higher RNA expression was associated with higher Hydro CSs. Results were consistent with the idea that stronger immunoglobulin responses to CMV are associated with better OS probabilities for GBM.

De Novo Synthesis and Structural Elucidation of CDR-H3 Loop Mimics.

The binding affinity of antibodies to specific antigens stems from a remarkably broad repertoire of hypervariable loops known as complementarity-determining regions (CDRs). While recognizing the pivotal role of the heavy-chain 3 CDRs (CDR-H3s) in maximizing antibody-antigen affinity and specificity, the key structural determinants responsible for their adaptability to diverse loop sequences, lengths, and noncanonical structures are hitherto unknown. To address this question, we achieved a de novo synthesis of bulged CDR-H3 mimics excised from their full antibody context. CD and NMR data revealed that these stable standalone ß-hairpin scaffolds are well-folded and retain many of the native bulge CDR-H3 features in water. In particular, the tryptophan residue, highly conserved across CDR-H3 sequences, was found to extend the kinked base of these ß-bulges through a combination of stabilizing intramolecular hydrogen bond and CH/π interaction. The structural ensemble consistent with our NMR observations exposed the dynamic nature of residues at the base of the loop, suggesting that ß-bulges act as molecular hinges connecting the rigid stem to the more flexible loops of CDR-H3s. We anticipate that this deeper structural understanding of CDR-H3s will lay the foundation to inform the design of antibody drugs broadly and engineer novel CDR-H3 peptide scaffolds as therapeutics.