Search

Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralizing public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-naive individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron-specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes a potential immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidence to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity., (© 2024. The Author(s).)

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.)

Catalytic antibodies possess a dual function that enables both antigen recognition and degradation. However, their time-consuming preparation is a significant drawback. This study developed a new method for quickly converting mice monoclonal antibodies into catalytic antibodies using site-directed mutagenesis. Three mice type monoclonal antibodies targeting hemagglutinin molecule of influenza A virus could be transformed into the catalytic antibodies by deleting Pro95 in CDR-3 of the light chain. No catalytic activity was observed for monoclonal antibodies and light chains. In contrast, the Pro95-deleted light chains exhibited a catalytic activity to cleave the antigenic peptide including the portion of conserved region of hemagglutinin molecule. The affinity of the Pro95-deleted light chains to the antigen increased approximately 100-fold compared to the wild-type light chains. In the mutants, three residues (Asp1, Ser92, and His93) come closer to the appropriate position to create the catalytic site and contributing to the enhancement of both catalytic function and immunoreactivity. Notably, the Pro95-deleted catalytic light chains could suppress influenza virus infection in vitro assay, whereas the parent antibody and the light chain did not. This strategy offers a rapid and efficient way to create catalytic antibodies from existing antibodies, accelerating the development for various applications in diagnostic and therapeutic applications., (© 2024. The Author(s).)

Abs are vital to human immune responses and are composed of genetically variable H and L chains. These structures are initially expressed as BCRs. BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated H and L chains, but advancements in single-cell sequencing now pair H and L chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired H and L chain sequences to build phylogenetic trees. We found that incorporating L chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree-building methods and persisted even when mixing bulk and single-cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some L chains were missing, such as when mixing single-cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for H and L chain gene partitions. Thus, we recommend using maximum likelihood methods with separate H and L chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io., (Copyright © 2024 by The American Association of Immunologists, Inc.)

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.)

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

According to classical immunology theory, immunoglobulin (Ig) is exclusively produced by differentiated B lymphocytes, which exhibit a typical tetrapeptide chain structure and are predominantly present on the surface of B cells and in bodily fluids. B-Ig is one of the critical effector molecules for humoral immune responses specifically recognising antigens and eliminating them. However, mounting evidence has demonstrated that Ig is widely expressed in non B lineage cells, especially malignant ones (referred to as non B-Ig). Interestingly, non B-Ig mainly resides in the cytoplasm and secretion, but to some extent on the cell surface. Furthermore non B-Ig not only displays a tetrapeptide chain structure but also shows free heavy chains and free light chains (FLCs). Additionally, Ig derived from non B cancer cell typically displays unique glycosylation modifications. Functionally, non B-Ig demonstrated diversity and versatility, showing antibody activity and cellular biological activity, such as promoting cell proliferation and survival, and it is implicated in cancer progression and some immune-related diseases, such as renal diseases., (© 2024. Springer Nature Singapore Pte Ltd.)

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 V L domain and stabilise an unexpected partially open LC dimer in which the two V L domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms., 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 © 2023 Elsevier Ltd. All rights reserved.)

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.

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).)

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.)

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications., Competing Interests: Authors SZ, LP, SK, PA, and BV are employees of Merck Healthcare KGaA. Author DK was taking part in an internship at Merck Healthcare KGaA at the time of this study. The remaining author declares 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 Klewinghaus, Pekar, Arras, Krah, Valldorf, Kolmar and Zielonka.)

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.)

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.

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.)

We studied the contribution of the light chain to functions of human monoclonal antibodies (mAbs) by measuring the relationships between the rate of mutations and cross-reactivity, binding affinity and neutralization activity. We analyzed 12 mAbs of two clonal families specific to the V2 region of HIV-1 derived from two chronically HIV-1 infected individuals. The clonal mAbs exhibited a range of reactivities, and the clones with superior properties were associated with the rate of mutations and the presence of particular mutated residues in the light chains, but not in the heavy chains. Our observations suggest that for some antibodies, the light chains play a vital role in antibody evolution toward more efficient ones and also suggest the importance of optimal residues rather than the rate of mutations in the variable fragment of the antibody., 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., (Published by Elsevier Inc.)

Purpose: CD19-redirected chimeric antigen receptor (CAR.CD19) T cells promote clinical responses in patients with relapsed/refractory B-cell non-Hodgkin lymphomas and chronic lymphocytic leukemia (CLL). However, patients showing sustained clinical responses after CAR.CD19-T treatment show increased infection risk due to compromised B-lymphocyte recovery. Mature B cell-derived malignancies express monoclonal immunoglobulins bearing either κ- or λ-light chains. We initially constructed CAR-T targeting the κ-light-chain (CAR.κ) and established a clinical study with it. After optimizing the CAR molecule, cells developed CAR-T targeting the λ-light chain (CAR.λ) and we explored their antitumor activity., Experimental Design: Using Igλ + lymphoma cell lines and patient-derived Igλ + CLL cells, we evaluated the in vitro tumor cytotoxicity and cytokine profiles of CAR.λ. We also assessed the in vivo efficacy of CAR.λ in xenograft Igλ + lymphoma models including a patient-derived xenograft (PDX) of mantle cell lymphoma, and the effects of λ- or κ-light chain-specific CAR-T on normal B lymphocytes in a humanized murine model., Results: CAR.λ demonstrated antitumor effects against Igλ + lymphoma cells and patient-derived CLL cells in vitro , and in vivo in xenograft and PDX Igλ + lymphoma murine models. Antitumor activity of CAR.λ was superimposable to CAR.CD19. Furthermore, we demonstrated in the humanized murine model that λ- or κ-light chain-specific CAR-T cells only depleted the corresponding targeted light chain-expressing normal B cells, while sparing the reciprocal light chain carrying B cells., Conclusions: Adoptive transfer of CAR.λ and CAR.κ-T cells represents a useful and alternative modality to CAR.CD19-T cells in treating mature B-cell malignancies with minimal impact on humoral immunity. See related commentary by Jain and Locke, p. 5736 ., (©2021 American Association for Cancer Research.)

Objectives: Follicular hyperplasias (FHs) with light chain-restricted (LCR) plasmacytoid/plasma cells (PCs) within germinal centers (GCs) based on immunohistochemistry (IHC)/in situ hybridization (ISH) can potentially lead to diagnostic error. This study aims to better characterize such cases, including their clinical implications., Methods: LC expression by IHC/ISH was quantitatively assessed in GCs of 17 FHs with LCRGCs. BCL2, CD10, BCL6, BCL2, immunoglobulin (Ig) heavy chains, IgG4, and Epstein-Barr encoding region stains were performed. In total, 8 cases had polymerase chain reaction (PCR)-based clonality studies., Results: All cases showed FH, including 4 with progressively transformed GCs (PTGCs); 0.8% to 52% (median, 21%) of the GCs were LCR; 13 of 17 had both κ- and λ-LCRGCs, and 4 of 17 had only κ-LCRGCs; 7 of 16 had prominent intrafollicular IgG4-positive cells. One case demonstrated BCL2-positive cells in focal LCRGCs but lacked BCL2 rearrangement. B-cell monoclonality was demonstrated in 3 of 8 cases (only after microdissection). Seven patients had autoimmune disorders, and 1 had had a transplant. Three patients had a history of lymphoma, 1 developed lymphoma, and 1 developed lymphomatoid granulomatosis subsequently., Conclusions: FHs with LCRGC by IHC/ISH are typically not associated with the development of lymphoma, even though they can express BCL2 and show monoclonality by PCR. They may be associated with increased intrafollicular IgG4-positive cells, PTGC, and autoimmunity., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)

Lenalidomide and dexamethasone (RD) is a standard treatment in relapsed/refractory immunoglobulin light chain (AL) amyloidosis (RRAL). We retrospectively investigated toxicity, efficacy and prognostic markers in 260 patients with RRAL. Patients received a median of two prior treatment lines (68% had been bortezomib-refractory; 33% had received high-dose melphalan). The median treatment duration was four cycles. The 3-month haematological response rate was 31% [very good haematological response (VGHR) in 18%]. The median follow-up was 56·5 months and the median overall survival (OS) and haematological event-free survival (haemEFS) were 32 and 9 months. The 2-year dialysis rate was 15%. VGHR resulted in better OS (62 vs. 26 months, P < 0·001). Cardiac progression predicted worse survival (22 vs. 40 months, P = 0·027), although N-terminal prohormone of brain natriuretic peptide (NT-proBNP) increase was frequently observed. Multivariable analysis identified these prognostic factors: NT-proBNP for OS [hazard ratio (HR) 1·71; P < 0·001]; gain 1q21 for haemEFS (HR 1·68, P = 0·014), with a trend for OS (HR 1·47, P = 0·084); difference between involved and uninvolved free light chains (dFLC) and light chain isotype for OS (HR 2·22, P < 0·001; HR 1·62, P = 0·016) and haemEFS (HR 1·88, P < 0·001; HR 1·59, P = 0·008). Estimated glomerular filtration rate (HR 0·71, P = 0·004) and 24-h proteinuria (HR 1·10, P = 0·004) were prognostic for renal survival. In conclusion, clonal and organ biomarkers at baseline identify patients with favourable outcome, while VGHR and cardiac progression define prognosis during RD treatment., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

We previously reported an antibody (clone ID: FR9, IgM-κ) that recognizes the sialyl oligosaccharide Neu5Acα2,6Galβ1,4GlcNAc as an epitope on glycoproteins and glycolipids. In the present study, we developed an antibody (clone ID: AFR45, IgM-κ) that recognizes Neu5Acα2,3Galβ1,4GlcNAc/Glc as an epitope on glycoproteins and glycolipids and compared the nucleotide and amino acid sequences of the immunoglobulin gene variable regions with those of FR9. The heavy chain variable (V H ) regions of FR9 and AFR45 were encoded by different V H gene segments, each of which was composed of a characteristic D gene segment. The major differences between V H genes encoding various antibodies deposited in public databases and FR9 and AFR45 were identified in the D gene segment, indicating that D genes play a critical role in determining the epitope specificity of these antibodies. Surprisingly, although FR9 and AFR45 were obtained independently from different mice immunized with different immunogens, the light chain variable (V L ) region nucleotide sequences were identical. The V L gene consisted of Igkv4-57 and Igkj4 gene segments (Igkv4-57j4), the sequences of which were identical to V L genes for a number of antibodies against meningococcal group C capsular polysaccharide deposited in public databases. As this polysaccharide is a sialic acid homopolymer, these results indicate that Igkv4-57j4 encodes a V L common to immunoglobulins that recognize sialylated glycans., Competing Interests: Declaration of competing interest The National Institute of Advanced Industrial Science and Technology holds patents (JP6172687, US9796785B2, and EP2993184B1) that relate to the materials reported in this article. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings., Competing Interests: The authors have declared that no competing interests exist.

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19., Materials and Methods: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them., Results: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients' odds ratio of 3.0401 was estimated., Conclusion: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.

The light chain (AL) amyloidosis is caused by the aggregation of light chain of antibodies into amyloid fibrils. There are plenty of computational resources available for the prediction of short aggregation-prone regions within proteins. However, it is still a challenging task to predict the amyloidogenic nature of the whole protein using sequence/structure information. In the case of antibody light chains, common architecture and known binding sites can provide vital information for the prediction of amyloidogenicity at physiological conditions. Here, in this work, we have compared classical sequence-based, aggregation-related features (such as hydrophobicity, presence of gatekeeper residues, disorderness, β-propensity, etc.) calculated for the CDR, FR or V L regions of amyloidogenic and non-amyloidogenic antibody light chains and implemented the insights gained in a machine learning-based webserver called "V L AmY-Pred" ( https://web.iitm.ac.in/bioinfo2/vlamy-pred/ ). The model shows prediction accuracy of 79.7% (sensitivity: 78.7% and specificity: 79.9%) with a ROC value of 0.88 on a dataset of 1828 variable region sequences of the antibody light chains. This model will be helpful towards improved prognosis for patients that may likely suffer from diseases caused by light chain amyloidosis, understanding origins of aggregation in antibody-based biotherapeutics, large-scale in-silico analysis of antibody sequences generated by next generation sequencing, and finally towards rational engineering of aggregation resistant antibodies.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope ( 2 KKTEETTLL 10 ) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two β-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

The kidney is one of the main organs affected by the autoimmune disease systemic lupus erythematosus. Lupus nephritis (LN) concerns 30-60% of adult SLE patients and it is significantly associated with an increase in the morbidity and mortality. The definitive diagnosis of LN can only be achieved by histological analysis of renal biopsies, but the invasiveness of this technique is an obstacle for early diagnosis of renal involvement and a proper follow-up of LN patients under treatment. The use of urine for the discovery of non-invasive biomarkers for renal disease in SLE patients is an attractive alternative to repeated renal biopsies, as several studies have described surrogate urinary cells or analytes reflecting the inflammatory state of the kidney, and/or the severity of the disease. Herein, we review the main findings in the field of urine immune-related biomarkers for LN patients, and discuss their prognostic and diagnostic value. This manuscript is focused on the complement system, antibodies and autoantibodies, chemokines, cytokines, and leukocytes, as they are the main effectors of LN pathogenesis.

Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations., Competing Interests: JG and BH were employed by the company Ferring Pharmaceuticals. JB, SC, and DF are employed by TU Darmstadt in frame of a collaboration project with Ferring Pharmaceuticals. 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 Bogen, Carrara, Fiebig, Grzeschik, Hock and Kolmar.)

Objective: Monoclonal protein (MP) exists in various diseases, and capillary electrophoresis (CE) has been widely used to detect MP. However, there is not much research on the application value of MP in the differential diagnosis of monoclonal gammopathies. This study aimed to explore MP's cutoff value for the differential diagnosis of multiple myeloma (MM) and other monoclonal gammopathies (MGs)., Methods: A retrospective analysis of 8167 cases was conducted. Serum MP was detected by CE, and the patients' clinical information was collected from the clinical database of our hospital., Results: 985 cases had MP with high peaks, and 91.1% were diagnosed with malignant diseases. The MP showed small peaks in 471 cases, and only 24.4% were diagnosed with malignant diseases. Among the MPs, the IgG-κ type was the most common type, followed by the IgG-λ, IgA-κ, IgA-λ, free λ light chain, IgM-κ, free κ light chain, double clone, and IgM-λ types. Differences in the MP of the IgG, IgA, IgM, and FLC types between the MM group and MGUS group were statistically different ( P <0.01). The MP of the IgG, IgA, and FLC types showed clear specificity and sensitivity in discriminating MM from other monoclonal gammopathies in ROC curve analysis. Serum IgM had statistical significance in the differential diagnosis between WM and other MGs ( P <0.01). However, there was no statistical significance in the differential diagnosis between MM and other MGs ( P =0.140). The cutoff values of the MP of the IgG, IgA, and FLC types were >18.67g/L, >13.86g/L, and >10.15g/L, respectively, for the differential diagnosis of MM and other MGs. The cutoff value of the MP of IgM for the WM diagnosis was >37.75 g/L., Conclusion: CE has good clinical application value in the diagnosis of monoclonal gammopathies, and MP can be used in the differential diagnosis of MM and other monoclonal gammopathies., (© 2021 by the Association of Clinical Scientists, Inc.)

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modified the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting antitumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies., Competing Interests: Competing interest statement: M.T., H.-L.C., and F.W.A. have filed a patent application on the new anti-PD1 antibodies described in the manuscript, and F.W.A. is a cofounder of Otoro Biopharmaceuticals.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Bispecific antibodies are an important and growing segment in antibody therapeutics, particularly in the immuno-oncology space. Manufacturing of a bispecific antibody with two different heavy chains is greatly simplified if the light chains can be the same for both arms of the antibody. Here, we introduce a strain of common light chain chickens, called OmniClic®, that produces antibody repertoires largely devoid of light chain diversity. The antibody repertoire in these chickens is composed of diverse human heavy chain variable regions capable of high-affinity antigen-specific binding and broad epitope diversity when paired with the germline human kappa light chain. OmniClic birds can be used in immunization campaigns for discovery of human heavy chains to different targets. Subsequent pairing of the heavy chain with a germline human kappa light chain serves to facilitate bispecific antibody production by increasing the efficiency of correct pairing. Abbreviations : AID: activation-induced cytidine deaminase; bsAb: bispecific antibody; CDR: complementarity-determining region; CL: light chain constant region; CmLC: common light chain; D: diversity region; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment crystallizable; FcRn: neonatal Fc receptor; FR: framework region; GEM: gel-encapsulated microenvironment; Ig: immunoglobulin; IMGT: the international ImMunoGeneTics information system®; J: joining region; KO: knockout; mAb: monoclonal antibody; NGS: next-generation sequencing; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PGC: primordial germ cell; PGRN: progranulin; TCR: T cell receptor; V: variable region; VK: kappa light chain variable region; VL: light chain variable region; VH: heavy chain variable region.

Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.

Bispecific (BsAb) and biparatopic (BpAb) antibodies emerged as promising formats for therapeutic biologics exhibiting tailor-made functional properties. Over recent years, chicken-derived antibodies have gained traction for diagnostic and therapeutic applications due to their broad epitope coverage and convenience of library generation. Here we report the first generation of a biparatopic common light chain (cLC) chicken-derived antibody by an epitope binning-based screening approach using yeast surface display. The resulting monospecific antibodies target conformational epitopes on domain II or III of the epidermal growth factor receptor (EGFR) with lower double- or single-digit nanomolar affinities, respectively. Furthermore, the domain III targeting variant was shown to interfere with epidermal growth factor (EGF) binding. Utilizing the Knob-into-Hole technology (KiH), a biparatopic antibody with subnanomolar affinity was generated that facilitates clustering of soluble and cell-bound EGFR and displayed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parental antibodies. This strategy for generating cLC-based biparatopic antibodies from immunized chickens may pave the way for their further development in therapeutic settings., Competing Interests: JG and BH were employed by the company Ferring Pharmaceuticals. JB, SC, and DF are employed by TU Darmstadt in frame of a collaboration project with Ferring Pharmaceuticals. 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 © 2020 Bogen, Carrara, Fiebig, Grzeschik, Hock and Kolmar.)

Immunoglobulin light-chain (AL) amyloidosis is a clonal plasma cell disorder characterised by production and deposition of misfolded monoclonal light chains in vital organs with potential to cause irreversible organ damage. The treatment of AL amyloidosis has evolved along the lines of multiple myeloma (MM) owing to clonal plasma cells being at the root of both disease processes. Treatment with melphalan and autologous haematopoietic cell transplantation, as well as proteasome inhibitors and immunomodulatory agents, are the standard of care for AL amyloidosis. While these treatment modalities are highly effective against the neoplastic plasma cells, patients often relapse and those with advanced disease may be unable to tolerate these treatments due to side-effects. Immunotherapy with monoclonal antibodies, bispecific antibodies, antibody-drug conjugates and chimeric antigen receptor T cells have revolutionised the treatment armamentarium for MM. These novel immunotherapy agents are in the early phases of evaluation and clinical development for patients with AL amyloidosis. The present review aims to discuss the role of novel immunotherapies currently in development and their potential for use in the treatment of AL amyloidosis., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (V L s) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant V L s and LCs differ in fibril formation. We addressed this question studying the effects of the V L mutations S20N and R61A within the isolated V L domain and in the full-length LC scaffold. Both V L variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to V L R61A. Our analyses revealed that the partially unfolded state of the V L R61A domain destabilizes the C L domain by non-native interactions, in turn leading to a further unfolding of the V L domain. In contrast, the folded mutant V L S20N and V L wt form native interactions with C L . These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the V L fold can have opposing effects on LC domain interactions, stability and amyloidogenicity., 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 © 2020 Elsevier Ltd. All rights reserved.)

In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as complementarity determining regions (CDRs) shaping the antigen-binding site, the paratope. Whereas it was assumed that certain sequences can only adopt a limited set of backbone conformations, in this study we present a kinetic classification of several paratope states in solution. Using molecular dynamics simulations in combination with experimental structural information we capture the involved conformational transitions between different canonical clusters and additional dominant solution structures occurring in the micro-to-millisecond timescale. Furthermore, we observe a strong correlation of CDR loop movements. Another important aspect when characterizing different paratope states is the relative V H /V L orientation and the influence of the distinct CDR loop states on the V H /V L interface. Conformational rearrangements of the CDR loops do not only have an effect on the relative V H /V L orientations, but also influence in some cases the elbow-angle dynamics and shift the respective distributions. Thus, our results show that antibodies exist as several interconverting paratope states, each contributing to the antibody's properties.

We previously discovered suppressor T cell-derived, antigen (Ag)-specific exosomes inhibiting mouse hapten-induced contact sensitivity effector T cells by targeting antigen-presenting cells (APCs). These suppressive exosomes acted Ag-specifically due to a coating of antibody free light chains (FLC) from Ag-activated B1a cells. Current studies are aimed at determining if similar immune tolerance could be induced in cutaneous delayed-type hypersensitivity (DTH) to the protein Ag (ovalbumin, OVA). Intravenous administration of a high dose of OVA-coupled, syngeneic erythrocytes similarly induced CD3 + CD8 + suppressor T cells producing suppressive, miRNA-150-carrying exosomes, also coated with B1a cell-derived, OVA-specific FLC. Simultaneously, OVA-immunized B1a cells produced an exosome subpopulation, originally coated with Ag-specific FLC, that could be rendered suppressive by in vitro association with miRNA-150. Importantly, miRNA-150-carrying exosomes from both suppressor T cells and B1a cells efficiently induced prolonged DTH suppression after single systemic administration into actively immunized mice, with the strongest effect observed after oral treatment. Current studies also showed that OVA-specific FLC on suppressive exosomes bind OVA peptides suggesting that exosome-coating FLC target APCs by binding to peptide-Ag-major histocompatibility complexes. This renders APCs capable of inhibiting DTH effector T cells. Thus, our studies describe a novel immune tolerance mechanism mediated by FLC-coated, Ag-specific, miRNA-150-carrying exosomes that act on the APC and are particularly effective after oral administration.

It is generally not well appreciated that Immunoglobulin A (IgA) can present as apparent biclonal monoclonal protein (M-protein) comprising monomers and polymers, despite the fact that Kyle pointed this out in 1999. In this clinical communication, we report on 3 patients with Ig-A multiple myeloma (MM) who displayed this phenomenon. Of these 3 patients, 2 had identical kappa light chains suggesting biclonality, while the other had 2 lambda light chains. Because the Ig-A M-proteins are clustered in the beta-area, accurate densitometric quantification is made difficult. Hence, we suggest assaying Ig-A levels and free light chains in concert with the M-protein levels to monitor these patients on therapy. In conclusion, when encountering biclonal Ig-A M-proteins with identical light chains, the laboratorian should add the 2 values and present one composite number to the clinician., (© 2020 by the Association of Clinical Scientists, Inc.)

Camelids are characterized by their unique adaptive immune system that exhibits the generation of homodimeric heavy-chain immunoglobulins, somatic hypermutation of T-cell receptors, and low genetic diversity of major histocompatibility complex (MHC) genes. However, short-read assemblies are typically highly fragmented in these gene loci owing to their repetitive and polymorphic nature. Here, we constructed a chromosome-level assembly of wild Bactrian camel genome based on high-coverage long-read sequencing and chromatin interaction mapping. The assembly with a contig N50 of 5.37 Mb and a scaffold N50 of 76.03 Mb, represents the most contiguous camelid genome to date. The genomic organization of immunoglobulin heavy-chain locus was similar between the wild Bactrian camel and alpaca, and genes encoding for conventional and heavy-chain antibodies were intermixed. The organizations of two immunoglobulin light-chain loci and four T cell receptor loci were also fully deciphered using the new assembly. Additionally, the complete classical MHC region was resolved into a single contig. The high-quality assembly presented here provides an essential reference for future investigations examining the camelid immune system., (© 2020 John Wiley & Sons Ltd.)

Light chain (AL) amyloidosis is a progressive, degenerative disease characterized by the misfolding and amyloid deposition of immunoglobulin light chain (LC). The amyloid deposits lead to organ failure and death. Our laboratory is specifically interested in cardiac involvement of AL amyloidosis. We have previously shown that the fibrillar aggregates of LC proteins can be cytotoxic and arrest the growth of human RFP-AC16 cardiomyocytes in vitro. We showed that adipose-derived mesenchymal stromal cells (AMSC) can rescue the cardiomyocytes from the fibril-induced growth arrest through contact-dependent mechanisms. In this study, we examined the transcriptome changes of human cardiomyocytes and AMSC in the presence of AL amyloid fibrils. The presence of fibrils causes a 'priming' immune response in AMSC associated with interferon associated genes. Exposure to AL fibrils induced changes in the pathways associated with immune response and extracellular matrix components in cardiomyocytes. We also observed upregulation of innate immune-associated transcripts (chemokines, cytokines, and complement), suggesting that amyloid fibrils initiate an innate immune response on these cells, possibly due to phenotypic transformation. This study corroborates and expands our previous studies and identifies potential new immunologic mechanisms of action for fibril toxicity on human cardiomyocytes and AMSC rescue effect on cardiomyocytes.

Serum κ and λ free light chain levels are markers of plasma cell proliferation, and their measurements have been included in recent guidelines by the International Myeloma Working Group for the management of patients with plasma cellular dyscrasias. Five in vitro diagnostic methods for the immunochemical quantification of serum free light chains (FLC) are available, three based on polyclonal antibodies (Freelite ® , The Binding Site; FLC ELISA κ and λ, Sebia; human κ and λ FLC, Diazyme Laboratories) and two on monoclonal antibodies (N Latex FLC, Siemens Healthineers; Seralite ® , Sebia). Several studies have shown that these methods cannot be used interchangeably for the follow-up of patients because measured κ and λ FLC concentrations may differ significantly, especially at high levels. Because no international reference material for the measurement of FLC is available, it is not possible to establish which method is the most accurate. For this reason, knowledge about the analytical and diagnostic performances of the assays used is important. The aim of this review is to describe the main analytical features of the κ and λ FLC assays and how they may influence the clinical use of these parameters.

Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs., (Copyright © 2020 Liao, Li, Yu, Yue, Su, Zheng, Jiang and Zhang.)

The measurement of serum-free light chains (FLC) is standard of care in the diagnosis and management of multiple myeloma (MM). The revised international myeloma working group (IMWG) implemented the involved FLC/noninvolved FLC (iFLC/niFLC) ratio as a biomarker for MM requiring treatment. Recently, a new definition of high-risk smoldering MM (SMM) including iFLC/niFLC ratio was published. These recommendations were solely based on a single assay method (Freelite assay). Today, two additional assays, N Latex FLC and ELISA-based Sebia FLC, are available. Here, we report on a single-center-study comparing results of all three different assays for FLC correlation and its potential implications for diagnostic and clinical use. In total, 187 samples from 47 MM patients were examined, and determination of FLC was performed. Comparison analyses showed similar FLC results for Sebia FLC and N Latex FLC assay with markedly lower absolute values for κ/λ ratio compared with Freelite. Values of λ FLC exhibited high variability. The ratio of iFLC/niFLC showed significant discrepancies among these assays. Our data demonstrate that the three available assays may result in markedly discrepant results, and should not be used interchangeably to monitor patients. Furthermore, modifications of the assay-specific diagnostic (iFLC/niFLC) thresholds for SMM and MM are recommended.

Antibodies from B-cell clonal lineages share sequence and structural properties as well as epitope specificity. Clonally unrelated antibodies can similarly share sequence and specificity properties and are said to be convergent. Convergent antibody responses against several antigens have been described in humans and mice and include different classes of shared sequence features. In particular, some antigens and epitopes can induce convergent responses of clonally unrelated antibodies with restricted heavy (V H ) and light (V L ) chain variable region germline segment usage without similarity in the heavy chain third complementarity-determining region (CDR H3), a critical specificity determinant. Whether these V germline segment-restricted responses reflect a general epitope specificity restriction of antibodies with shared V H /V L pairing is not known. Here, we investigated this question by determining patterns of antigen binding competition between clonally unrelated antigen-specific rat antibodies from paired-chain deep sequencing datasets selected based solely on V H /V L pairing. We found that antibodies with shared V H /V L germline segment pairings but divergent CDR H3 sequences almost invariably have restricted epitope specificity indicated by shared binding competition patterns. This epitope restriction included 82 of 85 clonally unrelated antibodies with 13 different V H /V L pairings binding in 8 epitope groups in 2 antigens. The corollary that antibodies with shared V H /V L pairing and epitope-restricted binding can accommodate widely divergent CDR H3 sequences was confirmed by in vitro selection of variants of anti-human epidermal growth factor receptor 2 antibodies known to mediate critical antigen interactions through CDR H3. Our results show that restricted epitope specificity determined by V H /V L germline segment pairing is a general property of rodent antigen-specific antibodies.

Background: At present, the role of autologous cells as antigen carriers inducing immune tolerance is appreciated. Accordingly, intravenous administration of haptenated syngeneic mouse red blood cells (sMRBC) leads to hapten-specific suppression of contact hypersensitivity (CHS) in mice, mediated by light chain-coated extracellular vesicles (EVs). Subsequent studies suggested that mice intravenously administered with sMRBC alone may also generate regulatory EVs, revealing the possible self-tolerogenic potential of autologous erythrocytes., Objectives: The current study investigated the immune effects induced by mere intravenous administration of a high dose of sMRBC in mice., Methods: The self-tolerogenic potential of EVs was determined in a newly developed mouse model of delayed-type hypersensitivity (DTH) to sMRBC. The effects of EV's action on DTH effector cells were evaluated cytometrically. The suppressive activity of EVs, after coating with anti-hapten antibody light chains, was assessed in hapten-induced CHS in wild-type or miRNA-150 -/- mice., Results: Intravenous administration of sMRBC led to the generation of CD9 + CD81+ EVs that suppressed sMRBC-induced DTH in a miRNA-150-dependent manner. Furthermore, the treatment of DTH effector cells with sMRBC-induced EVs decreased the activation of T cells but enhanced their apoptosis. Finally, EVs coated with antibody light chains inhibited hapten-induced CHS., Conclusions and Clinical Relevance: The current study describes a newly discovered mechanism of self-tolerance induced by the intravenous delivery of a high dose of sMRBC that is mediated by EVs in a miRNA-150-dependent manner. This mechanism implies the concept of naturally occurring immune tolerance, presumably activated by overloading of the organism with altered self-antigens., (© 2019 John Wiley & Sons Ltd.)

B cell clonal expansion is vital for adaptive immunity. High-throughput BCR sequencing enables investigating this process but requires computational inference to identify clonal relationships. This inference usually relies on only the BCR H chain, as most current protocols do not preserve H:L chain pairing. The extent to which paired L chains aids inference is unknown. Using human single-cell paired BCR datasets, we assessed the ability of H chain-based clonal clustering to identify clones. Of the expanded clones identified, <20% grouped cells expressing inconsistent L chains. H chains from these misclustered clones contained more distant junction sequences and shared fewer V segment mutations than the accurate clones. This suggests that additional H chain information could be leveraged to refine clonal relationships. Conversely, L chains were insufficient to refine H chain-based clonal clusters. Overall, the BCR H chain alone is sufficient to identify clonal relationships with confidence., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Homogeneous antibody-drug conjugates (ADCs) that use a highly reactive buried lysine (Lys) residue embedded in a dual variable domain (DVD)-IgG1 format can be assembled with high precision and efficiency under mild conditions. Here we show that replacing the Lys with an arginine (Arg) residue affords an orthogonal ADC assembly that is site-selective and stable. X-ray crystallography confirmed the location of the reactive Arg residue at the bottom of a deep pocket. As the Lys-to-Arg mutation is confined to a single residue in the heavy chain of the DVD-IgG1, heterodimeric assemblies that combine a buried Lys in one arm, a buried Arg in the other arm, and identical light chains, are readily assembled. Furthermore, the orthogonal conjugation chemistry enables the loading of heterodimeric DVD-IgG1s with two different cargos in a one-pot reaction and thus affords a convenient platform for dual-warhead ADCs and other multifaceted antibody conjugates., (Copyright © 2019 Elsevier Ltd. All rights reserved.)