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Although antibody variable regions mediate antigen-specific binding, they can also mediate non-specific interactions with non-cognate antigens, impacting diverse immunological processes and the efficacy, safety, and half-life of antibody therapeutics. To understand the molecular basis of antibody non-specificity, we sorted two dissimilar human naïve antibody libraries against multiple reagents to enrich for variants with different levels of polyreactivity. Sequence analysis of >300,000 paired antibody variable regions revealed that the heavy chain primarily mediates human antibody polyreactivity, and this is due to the high positive charge, high hydrophobicity, and combinations thereof in the corresponding complementarity-determining regions, which can be predicted using a machine learning model developed in this work. Notably, a subset of the most important features governing antibody non-specific interactions, namely those that contain tyrosine, also govern specific antigen recognition. Our findings are broadly relevant for understanding fundamental aspects of antibody molecular recognition and the applied aspects of antibody-drug design., Competing Interests: Declaration of interests N.R., G.L., M.S.M., and S.K. are current or former employees of the company (Boehringer Ingelheim) that funded this research. P.M.T. is a member of the scientific advisory boards for Nabla Bio, Aureka Biotechnologies, and Dualitas Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 (V H ) 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 V H 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., (© 2024. The Author(s).)

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.

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., (© 2024. The Author(s).)

The long-term value of efficient antigen discovery includes gaining insights into the variety of potential cancer neoantigens, effective vaccines lacking adverse effects, and adaptive immune receptor (IR) targets for blocking adaptive IR-antigen interactions in autoimmunity. While the preceding goals have been partially addressed via big data approaches to HLA (human leukocyte antigen)-epitope binding, there has been little such progress in the big data setting for adaptive IR-epitope binding. This delay in progress for the latter is likely due to, among other things, the much more complicated adaptive IR repertoire in an individual compared to individual HLA alleles. Thus, results described here represent the application of an algorithm for efficient assessment of immunoglobulin heavy chain complementarity determining region-3 (IGH CDR3)-gliadin epitope interactions, with a focus on epitopes known to be associated with an immune response in celiac disease. The hydrophobic, chemical complementarity between celiac case IGH CDR3s and known celiac epitopes was found to be greater in comparison to the hydrophobic, chemical complementarity between the same celiac case IGH CDR3s and a series of control epitopes. Thus, the approaches indicated here likely offer guidance for the development of conveniently applied algorithms for antigen verification and discovery., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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.

Tetanus toxin (TeNT) is one of the most toxic proteins. Neutralizing antibodies against TeNT are effective in prevention and treatment. In this study, 14 anti-tetanus nanobodies were obtained from a phage display nanobody library by immunizing a camel with the C-terminal receptor-binding domain of TeNT (TeNT-Hc) as the antigen. After fusion with the human Fc fragment, 11 chimeric heavy-chain antibodies demonstrated nanomolar binding toward TeNT-Hc. The results of toxin neutralization experiments showed that T83-7, T83-8, and T83-13 completely protected mice against 20 × the median lethal dose (LD 50 ) at a low concentration. The neutralizing potency of T83-7, T83-8, and T83-13 against TeNT is 0.4 IU/mg, 0.4 IU/mg and 0.2 IU/mg, respectively. In the prophylactic setting, we found that 5 mg/kg of T83-13 provided the mice with full protection from tetanus, even when they were injected 14 days before exposure to 20 × LD 50 TeNT. T83-7 and T83-8 were less effective, being fully protective only when challenged 7 or 10 days before exposure, respectively. In the therapeutic setting, 12 h after exposure to TeNT, 1 ~ 5 mg/kg of T83-7, and T83-8 could provide complete protection for mice against 5 × LD 50 TeNT, while 1 mg/kg T83-13 could provide complete protection 24 h after exposure to 5 × LD 50 TeNT. Our results suggested that these antibodies represent prophylactic and therapeutic activities against TeNT in a mouse model. The T83-7, T83-8, and T83-13 could form the basis for the subsequent development of drugs to treat TeNT toxicity.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2 µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD 50 ) of BoNT/F in vitro. Injection of 5 mg/kg F20 at 1 h, 2 h, 3 h, and 4 h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 V H H, 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., (© 2024. The Author(s).)

Using an Ig H chain conferring specificity for N-acetyl-d-glucosamine (GlcNAc), we developed transgenic (VHHGAC39 TG) mice to study the role of self-antigens in GlcNAc-reactive B-1 B cell development. In VHHGAC39 TG mice, GlcNAc-reactive B-1 B cell development during ontogeny and in adult bone marrow was normal. However, adult TG mice exhibited a block at transitional-2 immature B cell stages, resulting in impaired allelic exclusion and accumulation of a B cell subset coexpressing endogenous Ig gene rearrangements. Similarly, VHHGAC39 B cell fitness was impeded compared with non-self-reactive VHJ558 B TG cells in competitive mixed bone marrow chimeras. Nonetheless, adult VHHGAC39 mice immunized with Streptococcus pyogenes produce anti-GlcNAc Abs. Peritoneal cavity B cells transferred from VHHGAC39 TG mice into RAG-/- mice also exhibited robust expansion and anti-GlcNAc Ab production. However, chronic treatment of young VHHGAC39 mice with GlcNAc-specific mAbs leads to lower GlcNAc-binding B cell frequencies while increasing the proportion of GlcNAc-binding B1-a cells, suggesting that Ag masking or clearance of GlcNAc Ags impedes maturation of newly formed GlcNAc-reactive B cells. Finally, BCR H chain editing promotes expression of endogenous nontransgenic BCR alleles, allowing potentially self-reactive TG B cells to escape anergy or deletion at the transitional stage of precursor B cell development. Collectively, these observations indicate that GlcNAc-reactive B cell development is sensitive to the access of autologous Ags., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies., (© 2024. The Author(s).)

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens., (Copyright © 2024 by The American Association of Immunologists, Inc.)

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Olia, Prabhakaran, Harris, Cheung, Gillespie, Gorman, Hoover, Morano, Ourahmane, Srikanth, Wang, Wu, Zhou, Andrews, Kanekiyo, Shapiro, McDermott and Kwong.)

CAR-T cell therapy is at the forefront of next-generation multiple myeloma (MM) management, with two B-cell maturation antigen (BCMA)-targeted products recently approved. However, these products are incapable of breaking the infamous pattern of patient relapse. Two contributing factors are the use of BCMA as a target molecule and the artificial scFv format that is responsible for antigen recognition. Tackling both points of improvement in the present study, we used previously characterized VHHs that specifically target the idiotype of murine 5T33 MM cells. This idiotype represents one of the most promising yet challenging MM target antigens, as it is highly cancer- but also patient-specific. These VHHs were incorporated into VHH-based CAR modules, the format of which has advantages compared to scFv-based CARs. This allowed a side-by-side comparison of the influence of the targeting domain on T cell activation. Surprisingly, VHHs previously selected as lead compounds for targeted MM radiotherapy are not the best (CAR-) T cell activators. Moreover, the majority of the evaluated VHHs are incapable of inducing any T cell activation. As such, we highlight the importance of specific VHH selection, depending on its intended use, and thereby raise an important shortcoming of current common CAR development approaches.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy.

Motivation: Antibody therapeutic candidates must exhibit not only tight binding to their target but also good developability properties, especially low risk of immunogenicity., Results: In this work, we fit a simple generative model, SAM, to sixty million human heavy and seventy million human light chains. We show that the probability of a sequence calculated by the model distinguishes human sequences from other species with the same or better accuracy on a variety of benchmark datasets containing >400 million sequences than any other model in the literature, outperforming large language models (LLMs) by large margins. SAM can humanize sequences, generate new sequences, and score sequences for humanness. It is both fast and fully interpretable. Our results highlight the importance of using simple models as baselines for protein engineering tasks. We additionally introduce a new tool for numbering antibody sequences which is orders of magnitude faster than existing tools in the literature., Availability and Implementation: All tools developed in this study are available at https://github.com/Wang-lab-UCSD/AntPack., (© The Author(s) 2024. Published by Oxford University Press.)

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics., Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated., Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo ., Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jiang, Wang, Guo, Cheng, Chen, Wang, Li, Du, Gao, Lu, Yu and Yang.)

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.

The development of bispecific antibodies that bind at least two different targets relies on bringing together multiple binding domains with different binding properties and biophysical characteristics to produce a drug-like therapeutic. These building blocks play an important role in the overall quality of the molecule and can influence many important aspects from potency and specificity to stability and half-life. Single-domain antibodies, particularly camelid-derived variable heavy domain of heavy chain (VHH) antibodies, are becoming an increasingly popular choice for bispecific construction due to their single-domain modularity, favorable biophysical properties, and potential to work in multiple antibody formats. Here, we review the use of VHH domains as building blocks in the construction of multispecific antibodies and the challenges in creating optimized molecules. In addition to exploring traditional approaches to VHH development, we review the integration of machine learning techniques at various stages of the process. Specifically, the utilization of machine learning for structural prediction, lead identification, lead optimization, and humanization of VHH antibodies.

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.

Quenchbodies (Q-bodies), a type of biosensor, are antibodies labeled with a fluorescent dye near the antigen recognition site. In the absence of an antigen, the dye is quenched by tryptophans in the antibody sequence; however, in its presence, the dye is displaced and therefore de-quenched. Although scFv and Fab are mainly used to create Q-bodies, this is the first report where a single-domain heavy chain V H from a semi-synthetic human antibody library formed the basis. To create a proof of concept "mini Q-body", a human anti-lysozyme single-domain V H antibody C3 was used. Mini Q-bodies were successfully developed using seven dyes. Different responses were observed depending on the dye and linker length; it was concluded that the optimal linker length for the TAMRA dye was C5, and rhodamine 6G was identified as the dye with the largest de-quenching response. Three single-domain antibodies with sequences similar to that of the C3 antibody were chosen, and the results confirmed the applicability of this method in developing mini Q-bodies. In summary, mini Q-bodies are an easy-to-use and time-saving method for detecting proteins.

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01 B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens 1 . 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., (© 2022. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates., Competing Interests: Declaration of interests Reported antibodies are in the process of being patented., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy- and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

In addition to conventional immunoglobulin, camelids and cartilaginous fish express a special class of antibody that consists only of heavy (H) chain (HCAbs). In the holocephalan elephantfish, there are two HCAb classes, one of which has evolved surprising features. The H-chain genes in cartilaginous fish are organized as 20-200 minigenes, or clusters, each consisting of VH, 1-3 DH, JH gene segments with one set of constant region exons. We report that HHC2 (holocephalan H-chain antibody 2) evolved from IgM H-chain clusters, but its DH gene segments have diverged considerably. The three DH in HHC2 clusters are A-rich, so that one to three potential reading frames for each DH encode lysine and arginine. All three are incorporated into the rearranged VDJ, ensuring that the ligand-binding site carries multiple basic residues, as cDNA sequences demonstrate. The electropositive character in HHC2 CDR3 is accompanied by a paucity of aromatic amino acids, the latter feature at variance to the established, interactive role of tyrosine not only in ligand-binding but generally at interfaces of protein complexes. The selection for these divergent HHC2 features challenges currently accepted ideas on what determines antibody reactivity and molecular recognition., (© 2021 Wiley-VCH GmbH.)

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ 1 -Cε 2-4 , each ∼53 kDa) and two constant κ L chains (C κ , each ∼12 kDa) and lacks a V domain. The presence of Cγ 1 instead of Cε 1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A β-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Young children and older adults suffer from enhanced susceptibility to infections with blood-borne pathogens. An essential step towards immunity is the establishment of a splenic marginal zone (sMZ), which is immature at below 2 years of age. At approximately 5 years of age, an adult level of protection is reached but wanes again in older adults. Although the infant sMZ is thought to contain mostly naïve B cells, memory B cells are recruited to and recirculate from the sMZ throughout life, and class-switched sMZ B cells dominate in older adults. For a better resolution of naïve versus memory B-cell subset accumulation in the sMZ, we performed a single cell-based gene expression analysis of (CD21 high IgM high ) sMZ B cells among five healthy donors (age 3 to 48 years) and validated the sMZ B-cell subset composition by flow cytometry of 147 spleen biopsies (age 0 to 82 years). We identified a major sMZ B-cell subpopulation, which is abundant at birth but decreases with age. These cells lack CD27 expression but carry a weak-to-intermediate memory B-cell signature. These CD27 neg sMZ B cells are either IGHV-unmutated or carry only a few IGHV mutations early in life but show average memory B-cell IGHV mutation frequencies (>3%) in adults. The activation and proliferation potential of CD27 neg sMZ B cells is significantly above that of non-sMZ B cells already in children. Our study suggests that the human sMZ B-cell pool changes with age, encompassing a major population of lowly Ig-mutated CD27neg but antigen-experienced B cells early in life., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kibler, Budeus, Küppers and Seifert.)

Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.

In 2013, we released the Structural Antibody Database (SAbDab), a publicly available repository of experimentally determined antibody structures. In the interim, the rapid increase in the number of antibody structure depositions to the Protein Data Bank, driven primarily by increased interest in antibodies as biotherapeutics, has led us to implement several improvements to the original database infrastructure. These include the development of SAbDab-nano, a sub-database that tracks nanobodies (heavy chain-only antibodies) which have seen a particular growth in attention from both the academic and pharmaceutical research communities over the past few years. Both SAbDab and SAbDab-nano are updated weekly, comprehensively annotated with the latest features described here, and are freely accessible at opig.stats.ox.ac.uk/webapps/newsabdab/., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Having a limited number of VH segments, cattle rely on uniquely long DH gene segments to generate CDRH3 length variation (3-70 aa) far greater than that in humans or mice. Bovine antibodies with ultralong CDRH3s (>50 aa) possess unusual structures and abilities to bind to special antigens. In this study, we replaced most murine endogenous DH segments with bovine DH genes, generating a mouse line termed B-DH. The use of bovine DH genes significantly increased the length variation of CDRH3 and consequently the Ig heavy chain repertoire in B-DH mice. However, no ultralong CDRH3 was observed in B-DH mice, suggesting that other factors, in addition to long DH genes, are also involved in the formation of ultralong CDRH3. The B-DH mice mounted a normal humoral immune response to various antigens, although the B-cell developmental paradigm was obviously altered compared with wild-type mice. Additionally, B-DH mice are not predisposed to the generation of autoantibodies despite the interspecies DH gene replacement. The B-DH mice reported in this study provide a unique model to answer basic questions regarding the synergistic evolution of DH and VH genes, VDJ recombination and BCR selection in B-cell development., (© 2021 John Wiley & Sons Ltd.)

The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro , and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (V H ) and light (V L ) chain domains (V H 3-9*01/V L 2-14*03) both altered ST3 gene expression in vitro ; however, C10 had fewer V L somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing V L swaps, C10V H /C27V L exhibited reduced ST3 binding and agglutination, but C27V H /C10V L binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the V L in their functional activity in vitro and in vivo . These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3. IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo , and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

A diverse immune repertoire is capable of recognizing the enormous universe of foreign antigens encountered over life. Aging has a profound impact on the immune repertoires. However, whether continuous age-related changes in the immune repertoires differ between sexes is unclear. In this study, the characteristics of immune repertoires stratified by sex during aging are deciphered by analyzing T-cell receptor β-chain (TRB) and immunoglobulin heavy chain (IGH) sequences in 361 healthy adults. A similar change was observed between males and females across their lifespan, whereas age-subgroup analysis revealed sex-specific signatures in TRB and IGH repertoires. In regard to TRB, in males, repertoire richness and evenness increases slightly before the age of 32 years and 45 years respectively, and decreases sharply thereafter. Intriguingly, in females, they decrease significantly until around the age 57 years old, and subsequently undergo a stable stage up to the age of 83 years. Although IGH repertoire evenness increases significantly with age in both sexes, richness decreases significantly with age in males but remains stable in females. Moreover, average length of IGH CDR3 increases with age. In conclusion, these findings provide fundamental insights into the mechanisms underlying sex differences in adaptive immunity., (Copyright © 2021 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Assembly of IgG-like asymmetric bispecific antibodies (bsAbs) requires heavy chain heterodimerization and cognate heavy-light chain pairings. Multiple strategies have been developed to solve these chain association issues. While these strategies greatly promote correct chain pairing, they normally cannot prevent low amount of chain mispaired byproducts from being generated. Besides, byproducts can also be generated as a result of discordant chain expression. The existence of various byproducts poses considerable challenges to downstream processing during the production of recombinant IgG-like bsAbs. In many cases, yield is greatly compromised for purity improvement. This mini review introduces eight IgG-like bsAb platforms, which share a common feature: they all contain built-in purification-facilitating elements in addition to chain pairing control designs. These platforms, by simultaneously providing solutions to the two issues associated with bsAb production (i.e., correct chain pairing and efficient purification), improve both efficiency and robustness of bsAb production., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Preterm labor (PTL) is the leading cause of neonatal morbidity and mortality worldwide. Whereas many studies have investigated the maternal immune responses that cause PTL, fetal immune cell activation has recently been raised as an important contributor to the pathogenesis of PTL. In this study, we analyzed lymphocyte receptor repertoires in maternal and cord blood from 14 term and 10 preterm deliveries, hypothesizing that the high prevalence of infection in patients with PTL may result in specific changes in the T cell and B cell repertoires. We analyzed TCR β-chain (TCR-β) and IgH diversity, CDR3 lengths, clonal sharing, and preferential usage of variable and joining gene segments. Both TCR-β and IgH repertoires had shorter CDR3s compared with those in maternal blood. In cord blood samples, we found that CDR3 lengths correlated with gestational age, with shorter CDR3s in preterm neonates suggesting a less developed repertoire. Preterm cord blood displayed preferential usage of a number of genes. In preterm pregnancies, we observed significantly higher prevalence of convergent clones between mother/baby pairs than in term pregnancies. Together, our results suggest the repertoire of preterm infants displays a combination of immature features and convergence with maternal TCR-β clones compared with that of term infants. The higher clonal convergence in PTL could represent mother and fetus both responding to a shared stimulus like an infection. These data provide a detailed analysis of the maternal-fetal immune repertoire in term and preterm patients and contribute to a better understanding of neonate immune repertoire development and potential changes associated with PTL., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dalloul, Laffleur, Dalloul, Wehbi, Jouan, Brauge, Derouault, Moreau, Kracker, Fischer, Durandy, Le Noir and Cogné.)

Human immunoglobulin G (IgG) is the primary component of the human serum antibody fraction, representing about 75% of the immunoglobulins and 10-20% of the total circulating plasma proteins. Generally, IgG sequences are highly conserved, yet the four subclasses, IgG1, IgG2, IgG3, and IgG4, differ in their physiological effector functions by binding to different IgG-Fc receptors (FcγR). Thus, despite a similarity of about 90% on the amino acid level, each subclass possesses a unique manner of antigen binding and immune complex formation. Triggering FcγR-expressing cells results in a wide range of responses, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and complement activation. Textbook knowledge implies that only B lymphocytes are capable of producing antibodies, which recognize specific antigenic structures derived from pathogens and infected endogenous or tumorigenic cells. Here, we review recent discoveries, including our own observations, about misplaced IgG expression in tumor cells. Various studies described the presence of IgG in tumor cells using immunohistology and established correlations between high antibody levels and promotion of cancer cell proliferation, invasion, and poor clinical prognosis for the respective tumor patients. Furthermore, blocking tumor-cell-derived IgG inhibited tumor cells. Tumor-cell-derived IgG might impede antigen-dependent cellular cytotoxicity by binding antigens while, at the same time, lacking the capacity for complement activation. These findings recommend tumor-cell-derived IgG as a potential therapeutic target. The observed uniqueness of Ig heavy chains expressed by tumor cells, using PCR with V(D)J rearrangement specific primers, suggests that this specific part of IgG may additionally play a role as a potential tumor marker and, thus, also qualify for the neoantigen category.

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., Competing Interests: Declaration of interests B.B.B., A.S.F., M.O., P.W., L.L., S.N.L.A., J.R.W., X.P., B.M., D.D.H., and B.J.D. declare competing financial interests in the form of a provisional patent application filed by the University of Kansas., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The thymus is a central lymphoid organ primarily responsible for the development of T cells. A small proportion of B cells, however, also reside in the thymus to assist negative selection of self-reactive T cells. Here we show that the thymus of human neonates contains a consistent contingent of CD138 + plasma cells, producing all classes and subclasses of immunoglobulins with the exception of IgD. These antibody-secreting cells are part of a larger subset of B cells that share the expression of signature genes defining mouse B1 cells, yet lack the expression of complement receptors CD21 and CD35. Data from single-cell transcriptomic, clonal correspondence and in vitro differentiation assays support the notion of intrathymic CD138 + plasma cell differentiation, alongside other B cell subsets with distinctive molecular phenotypes. Lastly, neonatal thymic plasma cells also include clones reactive to commensal and pathogenic bacteria that commonly infect children born with antibody deficiency. Thus, our findings point to the thymus as a source of innate humoral immunity in human neonates., (© 2021. The Author(s).)

Purpose: Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients' B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking., Methods: We performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults., Results: After gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency., Conclusion: Based on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Monoclonal antibodies (mAbs) are an important class of therapeutics used to treat cancer, inflammation, and infectious diseases. Identifying highly developable mAb sequences in silico could greatly reduce the time and cost required for therapeutic mAb development. Here, we present position-specific scoring matrices (PSSMs) for antibody framework mutations developed using baseline human antibody repertoire sequences. Our analysis shows that human antibody repertoire-based PSSMs are consistent across individuals and demonstrate high correlations between related germlines. We show that mutations in existing therapeutic antibodies can be accurately predicted solely from baseline human antibody sequence data. We find that mAbs developed using humanized mice had more human-like FR mutations than mAbs originally developed by hybridoma technology. A quantitative assessment of entire framework regions of therapeutic antibodies revealed that there may be potential for improving the properties of existing therapeutic antibodies by incorporating additional mutations of high frequency in baseline human antibody repertoires. In addition, high frequency mutations in baseline human antibody repertoires were predicted in silico to reduce immunogenicity in therapeutic mAbs due to the removal of T cell epitopes. Several therapeutic mAbs were identified to have common, universally high-scoring framework mutations, and molecular dynamics simulations revealed the mechanistic basis for the evolutionary selection of these mutations. Our results suggest that baseline human antibody repertoires may be useful as predictive tools to guide mAb development in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Petersen, Ulmer, Rhodes, Gutierrez-Gonzalez, Dekosky, Sprenger and Whitehead.)

This protocol is a comprehensive guide to phage display-based selection of virus neutralizing V H antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 10 11 ) 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 V H antibody domain, V H 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)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Immunoglobulin class switch recombination (CSR) plays a crucial role in adaptive immune responses through a change of the effector functions of antibodies and is triggered by T-cell-dependent as well as T-cell-independent antigens. Signals generated following encounter with each type of antigen direct CSR to different isotypes. At the genomic level, CSR occurs between highly repetitive switch sequences located upstream of the constant gene exons of the immunoglobulin heavy chain locus. Transcription of switch sequences is mandatory for CSR and is induced in a stimulation-dependent manner. Switch transcription takes place within dynamic chromatin domains and is regulated by long-range regulatory elements which promote alignment of partner switch regions in CSR centers. Here, we review recent work and models that account for the function of long-range transcriptional regulatory elements and the chromatin-based mechanisms involved in the control of CSR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dauba and Khamlichi.)

CD38 is the major NAD + -hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background., Competing Interests: FH and FKN receive a share of antibody sales via MediGate GmbH, a wholly owned subsidiary of the University Medical Center Hamburg-Eppendorf. PB and FKN are coinventors on a patent application on CD38-specific nanobodies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Baum, Eggers, Koenigsdorf, Menzel, Hambach, Staehler, Fliegert, Kulow, Adam, Haag, Bannas and Koch-Nolte.)