Framework Regions Research Articles

Significance of antibody numbering systems in the development of antibody engineering.

Immunotherapy has become increasingly popular in recent years for treating a variety of diseases including inflammatory, neurological, oncological, and auto-immune disorders. The significant interest in antibody development is due to the high binding affinity and specificity of an antibody against a specific antigen. Recent advances in antibody engineering have provided a different view on how to engineer antibodies in silico for therapeutic and diagnostic applications. In order to improve the clinical utility of therapeutic antibodies, it is of paramount importance to understand the various molecular properties which impact antigen targeting and its potency. In antibody engineering, antibody numbering (AbN) systems play an important role to identify the complementarity determining regions (CDRs) and the framework regions (FR). Hence, it is crucial to accurately define and understand the CDR, FR and the crucial residues of heavy and light chains that aid in the binding of the antibody to the antigenic site. Detailed understanding of amino acids positions are useful for modifying the binding affinity, specificity, physicochemical features, and half-life of an antibody. In this review, we have summarized the different antibody numbering systems that are widely used in antibody engineering and highlighted their significance. Here, we have systematically explored and mentioned the various tools and servers that harness different AbN systems.

IgMAT: immunoglobulin sequence multi-species annotation tool for any species including those with incomplete antibody annotation or unusual characteristics

BackgroundThe advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map dynamic changes in polyclonal responses precise framework and complementarity determining region annotation of rearranging genes is pivotal. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. For this reason, data agnostic tools able to learn from presented data can be very useful with new species or with novel datasets.ResultsWe have developed IgMAT, which utilises a reduced amino acid alphabet, that incorporates multiple HMM alignments into a single consensus to automatically annotate immunoglobulin sequences from most organisms. Additionally, the software allows the incorporation of user defined databases to better represent the species and/or antibody class of interest. To demonstrate the accuracy and utility of IgMAT, we present analysis of sequences extracted from structural data and immunoglobulin sequence datasets from several different species.ConclusionsIgMAT is fully open-sourced and freely available on GitHub (https://github.com/TPI-Immunogenetics/igmat) for download under GPLv3 license. It can be used as a CLI application or as a python module to be integrated in custom scripts.

The immunoglobulin A isotype of the Arabian camel (Camelus dromedarius) preserves the dualistic structure of unconventional single-domain and canonical heavy chains.

The evolution of adaptive immunity in Camelidae resulted in the concurrent expression of classic heterotetrameric and unconventional homodimeric heavy chain-only IgG antibodies. Heavy chain-only IgG bears a single variable domain and lacks the constant heavy (CH) γ1 domain required for pairing with the light chain. It has not been reported whether this distinctive feature of IgG is also observed in the IgA isotype. Gene-specific primers were used to generate an IgA heavy chain cDNA library derived from RNA extracted from the dromedary's third eyelid where isolated lymphoid follicles and plasma cells abound at inductive and effector sites, respectively. Majority of the cDNA clones revealed hallmarks of heavy chain-only antibodies, i.e. camelid-specific amino acid substitutions in framework region 1 and 2, broad length distribution of complementarity determining region 3, and the absence of the CHα1 domain. In a few clones, however, the cDNA of the canonical IgA heavy chain was amplified which included the CHα1 domain, analogous to CHγ1 domain in IgG1 subclass. Moreover, we noticed a short, proline-rich hinge, and, at the N-terminal end of the CHα3 domain, a unique, camelid-specific pentapeptide of undetermined function, designated as the inter-α region. Immunoblots using rabbit anti-camel IgA antibodies raised against CHα2 and CHα3 domains as well as the inter-α region revealed the expression of a ~52 kDa and a ~60 kDa IgA species, corresponding to unconventional and canonical IgA heavy chain, respectively, in the third eyelid, trachea, small and large intestine. In contrast, the leporine anti-CHα1 antibody detected canonical, but not unconventional IgA heavy chain, in all the examined tissues, milk, and serum, in addition to another hitherto unexplored species of ~45 kDa in milk and serum. Immunohistology using anti-CHα domain antibodies confirmed the expression of both variants of IgA heavy chains in plasma cells in the third eyelid's lacrimal gland, conjunctiva, tracheal and intestinal mucosa. We found that in the dromedary, the IgA isotype has expanded the immunoglobulin repertoire by co-expressing unconventional and canonical IgA heavy chains, comparable to the IgG class, thus underscoring the crucial role of heavy chain-only antibodies not only in circulation but also at the mucosal frontiers.

Matrixed CDR grafting: A neoclassical framework for antibody humanization and developability

Discovery and optimization of a biotherapeutic monoclonal antibody requires a careful balance of target engagement and physicochemical developability properties. To take full advantage of the sequence diversity provided by different antibody discovery platforms, a rapid and reliable process for humanization of antibodies from nonhuman sources is required. Canonically, maximizing homology of the human variable region (V-region) to the original germline was believed to result in preservation of binding, often without much consideration for inherent molecular properties. We expand on this approach by grafting the complementary determining regions (CDRs) of a mouse anti-LAG3 antibody into an extensive matrix of human variable heavy chain (VH) and variable light chain (VL) framework regions with substantially broader sequence homology to assess the impact on complementary determining region-framework compatibility through progressive evaluation of expression, affinity, biophysical developability, and function. Specific VH and VL framework sequences were associated with major expression and purification phenotypes. Greater VL sequence conservation was correlated with retained or improved affinity. Analysis of grafts that bound the target demonstrated that initial developability criteria were significantly impacted by VH, but not VL. In contrast, cell binding and functional characteristics were significantly impacted by VL, but not VH. Principal component analysis of all factors identified multiple grafts that exhibited more favorable antibody properties, notably with nonoptimal sequence conservation. Overall, this study demonstrates that modern throughput systems enable a more thorough, customizable, and systematic analysis of graft-framework combinations, resulting in humanized antibodies with improved global properties that may progress through development more quickly and with a greater probability of success.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Oligomannose-Type Glycans in the Antigen Binding Site Identify Origin and Prognosis of Diffuse Large B Cell Lymphoma

The acquisition of N-glycosylation sites (AGS) in the complementarity-determining region (CDR) of the tumor immunoglobulin (Ig) is an early clonal requirement of classic follicular lymphoma (FL). In FL, the AGS are occupied by oligomannose-type glycans, which mediate lymphoma Ig interaction with the microenvironmental lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This interaction induces a persistent low-level signal, favouring adhesion and ultimately survival and growth of the lymphoma cells in their protected environment . Oligomannose-type glycans can also occupy the CDR-located AGS of a subset of germinal-centre B cell-like (GCB) diffuse large B-cell lymphomas (DLBCL) but the consequences of AGS in DLBCL are less known. We determined the frequency, distribution, and composition of the glycan structures occupying the AGS in DLBCL using a discovery-validation approach with two large independent international public cohorts (BCC, dataset ID: EGAD00001003783; NCI accession phs001444.v1.p1), following our RNAseq Ig assembly pipeline (Ig-Seq-R) and high-resolution mass-spectrometry. Glycan types were compared to our FL cohort (12 samples) and correlated with DLBCL cell of origin (COO), genetic, clinical characteristics and outcome. The full IGHV-IGHD-IGHJ-IGHC transcript sequences were obtained from 251 DLBCL of the BCC, and 339 of the NCI cohort. Sample distribution by COO, clinical or molecular features, LymphGen subtype, progression-free survival (PFS) and overall survival (OS) were not different from the extended public cohorts. AGS were observed in 48-55% GCB-DLBCL, with 84-85% of them located in the CDR. Overall, frequency of CDR-located AGS contrasted dramatically between GCB-DLBCL (41-46%) and activated B-cell like (ABC)-DLBCL (7-10%). Also, within GCB-DLBCL, CDR-located AGS associated mostly with the EZB subtype (68-73%). However, only 66-61% EZB were AGS+ve, all of which were CDR+ve, while the remaining 34-39% had no AGS (AGS-ve EZB). The glycan structures of the synthetic F(ab)s from 35 AGS+ve DLBCL and 12 FL were analyzed by site-specific mass spectrometry. The DLBCL comprised 12 AGS+ve EZB and 23 AGS+ve non-EZB (14 CDR+ve, 9 located in the framework region). Glycan analysis revealed that all AGS+ve EZB and FL were invariably occupied by oligomannose-type glycans (Ig-Mann+ve DLBCL). In contrast, the other AGS+ve DLBCL were not occupied by oligomannose type glycans. They were either occupied by complex glycans (the remaining non-EZB GCB-DLBCL) or not occupied (preferentially the ABC-DLBCL), irrespective of AGS location. These data indicated that CDR+ve EZB Ig were universally and exclusively Ig-Mann+ve DLBCL, share the COO of FL, and have likely been influenced by an environmental driver distinct from the other GCB-DLBCL and ABC-DLBCL. We compared the clinical behavior of Ig-Mann+ve DLBCL versus other DLBCL subtypes including AGS-ve EZB and the other non-EZB GCB-DLBCL and ABC-DLBCL. In both the BCC and NCI cohorts, Ig-Mann+ve DLBCL status identified the subset with the worst PFS and OS of all GCB-DLBCL, not different from ABC-DLBCL (Figure 1A). This contrasted with the other GCB-DLBCL (AGS-ve/EZB and AGS+ve/non-EZB), which all shared an extremely good PFS and OS (Figure 1A). Univariate and multivariate analyses revealed that Ig-Mann+ve status was an independent prognostic factor for PFS and OS (Figure 1B). Gene expression profile analysis revealed that Ig-Mann+ve status associated with an enrichment of MYC, PI3K-Akt-mTORC1, pro-survival, and cell cycle pathways, while proinflammatory and apoptotic pathways were decreased in the Ig-Mann+ve DLBCL compared to AGS-ve/EZB and AGS+ve/non-EZB, independently of MYC translocations and double-hit signature. These data indicate identical COO of a EZB subset with FL cells, and point to a highly selective chronic environmental pressure on Ig-Mann+ve DLBCL negatively affecting patients survival. The combination of Ig gene analysis with the lymphGen classification can predict Ig glycan structure and provide a new fundamental approach to identifying the most aggressive GCB-DLBCL subtype, and the precise environmental tumour interaction (DC-SIGN:Ig-Mann) to intercept therapeutically early in the natural history of Ig-Mann+ve DLBCL.

Framework humanization optimizes potency of anti-CD72 nanobody CAR-T cells for B-cell malignancies

BackgroundApproximately 50% of patients who receive anti-CD19 CAR-T cells relapse, and new immunotherapeutic targets are urgently needed. We recently described CD72 as a promising target in B-cell malignancies and developed...

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Arginine cluster introduction on framework region in anti-lysozyme antibody improved association rate constant by changing conformational diversity of CDR loops.

Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an antibody to the antigen is critical for clinical efficacy of pharmaceuticals, many affinity maturation strategies have been developed. Although we previously reported an affinity maturation strategy in which the association rate of the antibody toward its antigen is improved by introducing a cluster of arginine residues into the framework region of the antibody, the detailed molecular mechanism responsible for this improvement has been unknown. In this study, we introduced five arginine residues into an anti-hen egg white lysozyme antibody (HyHEL10) Fab fragment to create the R5-mutant and comprehensively characterized the interaction between antibody and antigen using thermodynamic analysis, X-ray crystallography, and molecular dynamics (MD) simulations. Our results indicate that introduction of charged residues strongly enhanced the association rate, as previously reported, and the antibody-antigen complex structure was almost the same for the R5-mutant and wild-type Fabs. The MD simulations indicate that the mutation increased conformational diversity in complementarity-determining region loops and thereby enhanced the association rate. These observations provide the molecular basis of affinity maturation by R5 mutation.

VHH CDR-H3 conformation is determined by VH germline usage

VHHs or nanobodies are single antigen binding domains originating from camelid heavy-chain antibodies. They are used as diagnostic and research tools and in a variety of therapeutic molecules. Analyzing variable domain structures from llama and alpaca we found that VHHs can be classified into two large structural clusters based on their CDR-H3 conformation. Extended CDR-H3 loops protrude into the solvent, whereas kinked CDR-H3 loops fold back onto framework regions. Both major families have distinct properties in terms of their CDR-H3 secondary structure, how their CDR-H3 interacts with the framework region and how they bind to antigens. We show that the CDR-H3 conformation of VHHs correlates with the germline from which the antibodies are derived: IGHV3-3 derived antibodies almost exclusively adopt a kinked CDR-H3 conformation while the CDR-H3 adopts an extended structure in most IGHV3S53 derived antibodies. We do not observe any bias stemming from V(D)J recombination in llama immune repertoires, suggesting that the correlation is the result of selection processes during B-cell development. Our findings demonstrate a previously undescribed impact of germline usage on antigen interaction and contribute to a better understanding on how properties of the antibody framework shape the immune repertoire.

Late Jurassic paleogeography of the U.S. Cordillera from detrital zircon age and hafnium analysis of the Galice Formation, Klamath Mountains, Oregon and California, USA

The Upper Jurassic Galice Formation, a metasedimentary unit in the Western Klamath Mountains, formed within an intra-arc basin prior to and during the Nevadan orogeny. New detrital zircon U-Pb age analyses (N = 11; n = 2792) yield maximum depositional ages (MDA) ranging from ca. 160 Ma to 151 Ma, which span Oxfordian to Kimmeridgian time and overlap Nevadan contractional deformation that began by ca. 157 Ma. Zircon ages indicate a significant North American continental provenance component that is consistent with tectonic models placing the Western Klamath terrane on the continental margin in Late Jurassic time. Hf isotopic analysis of Mesozoic detrital zircon (n = 603) from Galice samples reveals wide-ranging εHf values for Jurassic and Triassic grains, many of which cannot be explained by a proximal source in the Klamath Mountains, thus indicating a complex provenance. New U-Pb ages and Hf data from Jurassic plutons within the Klamath Mountains match some of the Galice Formation detrital zircon, but these data cannot account for the most non-radiogenic Jurassic detrital grains. In fact, the in situ Cordilleran arc record does not provide a clear match for the wide-ranging isotopic signature of Triassic and Jurassic grains. When compiled, Galice samples indicate sources in the Sierra Nevada pre-batholithic framework and retroarc region, older Klamath terranes, and possibly overlap strata from the Blue Mountains and the Insular superterrane. Detrital zircon age spectra from strata of the Upper Jurassic Great Valley Group and Mariposa Formation contain similar age modes, which suggests shared sediment sources. Inferred Galice provenance within the Klamath Mountains and more distal sources suggest that the Galice basin received siliciclastic turbidites fed by rivers that traversed the Klamath-Sierran arc from headwaters in the retroarc region. Thus, the Galice Formation contains a record of active Jurassic magmatism in the continental arc, with significant detrital input from continental sediment sources within and east of the active arc. These westward-flowing river systems remained active throughout the shift in Cordilleran arc tectonics from a transtensional system to the Nevadan contractional system, which is characterized by sediment sourced in uplifts within and east of the arc and the thrusting of older Galice sediments beneath older Klamath terranes to the east.

Computational optimization of antibody humanness and stability by systematic energy-based ranking.

Conventional methods for humanizing animal-derived antibodies involve grafting their complementarity-determining regions onto homologous human framework regions. However, this process can substantially lower antibody stability and antigen-binding affinity, and requires iterative mutational fine-tuning to recover the original antibody properties. Here we report a computational method for the systematic grafting of animal complementarity-determining regions onto thousands of human frameworks. The method, which we named CUMAb (for computational human antibody design; available at http://CUMAb.weizmann.ac.il ), starts from an experimental or model antibody structure and uses Rosetta atomistic simulations to select designs by energy and structural integrity. CUMAb-designed humanized versions of five antibodies exhibited similar affinities to those of the parental animal antibodies, with some designs showing marked improvement in stability. We also show that (1) non-homologous frameworks are often preferred to highest-homology frameworks, and (2) several CUMAb designs that differ by dozens of mutations and that use different human frameworks are functionally equivalent.

A Chemiluminescence Enzyme Immunoassay Based on Biotinylated Nanobody and Streptavidin Amplification for Diazinon Sensitive Quantification

The advantages of genetic modification and preferable physicochemical qualities make nanobody (Nb) easy to develop a sensitive and stable immunosensor platform. Herein, an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA) based on biotinylated Nb was established for the quantification of diazinon (DAZ). The anti-DAZ Nb, named Nb-EQ1, with good sensitivity and specificity, was obtained from an immunized library via a phage display technique, where the molecular docking results indicated that the hydrogen bond and hydrophobic interactions between DAZ and complementarity-determining region 3 and framework region 2 in Nb-EQ1 played a critical role in the Nb-DAZ affinity processes. Subsequently, the Nb-EQ1 was further biotinylated to generate a bi-functional Nb-biotin, and then an ic-CLEIA was developed for DAZ determination via signal amplification of the biotin–streptavidin platform. The results showed that the proposed method based on Nb-biotin had a high specificity and sensitivity to DAZ, with a relative broader linear range of 0.12–25.96 ng/mL. After being 2-folds dilution of the vegetable samples matrix, the average recoveries were 85.7–113.9% with a coefficient of variation of 4.2–19.2%. Moreover, the results for the analysis of real samples by the developed ic-CLEIA correlated well with that obtained by reference method GC-MS (R2 ≥ 0.97). In summary, the ic-CLEIA based on biotinylated Nb-EQ1 and streptavidin recognition demonstrated itself to be a convenient tool for the quantification of DAZ in vegetables.

Selective targeting of pathogenic auto-reactive VH4–34 B cells with a rationally developed anti-VH4–34 antibody offers a new therapeutic approach for autoimmune disorders

Abstract B cells expressing B cell receptors (BCRs) specific for self-antigens are normally eliminated, or made anergic, through negative selection during B cell development. VH4–34 is an intrinsically autoreactive antibody (and BCR) heavy chain variable region that includes a germline encoded hydrophobic patch in framework region 1 (FR1). Autoantigens recognized by VH4–34 antibodies include N-acetyl lactosamine chains displayed on RBCs. In healthy individuals, <1% of B cells express VH4–34 encoded BCRs but during the loss of self-tolerance, characteristic of pathogenic disorders, the hydrophobic region of VH4–34 reacts to self-antigen, erroneously driving BCR activation and expansion. Expansion of VH4–34+ B cells and increased serum concentrations of VH4–34 antibodies has been implicated in systemic lupus erythematosus (50%) and virtually all cases of cold agglutinin disease. To date there have been no therapies against VH4–34 due to challenges of specifically targeting VH4–34 while avoiding other VH regions. We applied Hummingbird Bioscience’s Rational Antibody Discovery approach to develop anti-VH4–34 antibodies that bind to a computationally predicted and lineage conserved epitope in the hydrophobic FR1 region. The lead antibody, HMBD-011, demonstrates selective and high affinity binding to all tested VH4–34 antibodies, with no off-target binding. The binding epitope of HMBD-011 was confirmed by mutagenesis to be within the FR1 region of VH4–34. Further, HMBD-011 shows selective binding to cells that express VH4–34 BCRs. In vivo, HMBD-011 treatment resulted in rapid elimination of VH4–34+ antibodies and cells. In conclusion, HMBD-011 represents a promising precision therapy for VH4–34 driven autoimmune disorders.

Spatially resolved immune receptor repertoires from Visium transcriptomic libraries

Abstract Recent advances have enabled the integration of T cell receptor (TCR) sequencing into spatial transcriptomics. Current approaches yield critical information about in situ T cell responses directly from relevant tissues, yet are largely limited to alpha/beta TCRs, leaving the clonal dynamics of B cells and gamma/delta T cells underexplored. We therefore modified our previously reported ultra-efficient immune receptor repertoire sequencing (IRR-seq) method, Framework Region 3 AmplifiKation Sequencing (FR3AK-seq), for compatibility with the 10X Genomics Visium spatial transcriptomics platform. Here we report simultaneous acquisition of alpha/beta TCR, gamma/delta TCR, and immunoglobulin (B cell receptor, BCR) heavy/light chain sequences from a single multiplexed FR3AK-seq reaction using Visium transcriptomics-generated cDNA libraries. More specifically, we used the Visium spatial transcriptomics protocol to generate cDNA from serial sections of a lymph node with a metastatic lesion obtained from a patient with pancreatic ductal adenocarcinoma previously treated with immunotherapy. Notably, we detected sequences from all IRR chains as expected from the abundant immune infiltrates within these sections. Furthermore, we found that multiplexed FR3AK-seq yields technically reproducible repertoires, with the TCR beta chain repertoires also comparable to those obtained using a previously published method. We anticipate that this simple extension of the Visium technology will accelerate the simultaneous and comprehensive examination of in situ IRR dynamics beyond alpha/beta T cells.

The Plasma Cell Infiltrate Populating the Muscle Tissue of Patients with Inclusion Body Myositis Features Distinct B Cell Receptor Repertoire Properties.

Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.

Clonal Characterization and Somatic Hypermutation Assessment by Next-Generation Sequencing in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: A Detailed Description of the Technical Performance, Clinical Utility, and Platform Comparison

Somatic hypermutation status of the IGHV gene is essential for treating patients with chronic lymphocytic leukemia/small lymphocytic lymphoma. Unlike the conventional low-throughput method, assessment of somatic hypermutation by next-generation sequencing (NGS) has potential for uniformity and scalability. However, it lacks standardization or guidelines for routine clinical use. We critically assessed the performance of an amplicon-based NGS assay across 458 samples. Using a validation cohort (35 samples), the comparison of two platforms (Ion Torrent versus Illumina) and two primer sets [leader versus framework region 1 (FR1)] in their ability to identify clonotypic IGHV rearrangement(s) revealed 97% concordance. The mutation rates were identical by both platforms when using the same primer set (FR1), whereas a slight overestimation bias (+0.326%) was found when comparing FR1 with leader primers. However, for nearly all patients this did not affect the stratification into mutated or unmutated categories, suggesting that use of FR1 may provide comparable results if leader sequencing is not available and allowing for a simpler NGS laboratory workflow. In routine clinical practice (423 samples), the productive rearrangement was successfully detected by either primer set (leader, 97.7%; FR1, 94.7%), and a combination of both in problematic cases reduced the failure rate to 1.2%. Higher sensitivity of the NGS-based analysis also detected a higher frequency of double IGHV rearrangements (19.1%) compared with traditional approaches.

B cell M-CLL clones retain selection against replacement mutations in their immunoglobulin gene framework regions.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, accounting for 30-40% of all adult leukemias. The dynamics of B-lymphocyte CLL clones with mutated immunoglobulin heavy chain variable region (IgHV) genes in their tumor (M-CLL) can be studied using mutational lineage trees. Here, we used lineage tree-based analyses of somatic hypermutation (SHM) and selection in M-CLL clones, comparing the dominant (presumably malignant) clones of 15 CLL patients to their non-dominant (presumably normal) B cell clones, and to those of healthy control repertoires. This type of analysis, which was never previously published in CLL, yielded the following novel insights. CLL dominant clones undergo - or retain - more replacement mutations that alter amino acid properties such as charge or hydropathy. Although, as expected, CLL dominant clones undergo weaker selection for replacement mutations in the complementarity determining regions (CDRs) and against replacement mutations in the framework regions (FWRs) than non-dominant clones in the same patients or normal B cell clones in healthy controls, they surprisingly retain some of the latter selection in their FWRs. Finally, using machine learning, we show that even the non-dominant clones in CLL patients differ from healthy control clones in various features, most notably their expression of higher fractions of transition mutations. Overall, CLL seems to be characterized by significant loosening - but not a complete loss - of the selection forces operating on B cell clones, and possibly also by changes in SHM mechanisms.

Characterization of immunoglobulin-ε (IgE) to benefit therapeutic IgE engineering.

Several food proteins, e.g. in peanut, can trigger adversely immune responses that lead to severe, even fatal, clinical symptoms. Immunoglobulin-ε (IgE) is the main immunoglobulin isotype associated with such Type-I hypersensitivity. The allergic immune reactions are mediated by IgE and its interactions to the receptor FcεRIα located on mast cells. The cellular activation is triggered by the FcεRIα-bound IgE typically with multivalent antigen binding or occasionally without any presence of known antigens. Disrupting such interactions could intervene the allergic immune responses.Our previous study (doi: 10.1016/j.jaci.2019.03.028) suggested prolonged FcεRIα-IgE interactions led to emergence and over-presentation of the persistent IgE. Such effects were due to long-ranged communications between the IgE V-regions and the FcεRIα-binding site. Additionally, varying structural arrangements of the IgE complementarity-determining regions (CDRs) and framework regions (FWRs) of both the antibody heavy and light chains with influence from the CH-region could orient a single mutation in the VH-CDR2 to abrogate the non-specific binding of superantigen spA or could form inducible structural glutamine stretches that bound nickel Ni2+ without losing the antigen specificity (doi: 10.3389/fimmu.2021.676048). In fact, these work advocate analyzing IgE holistically as a whole, rather than a sum of its parts.In the current work riding on the long-ranged communications between the IgE antigen-binding and FcεRIα-binding regions, we further characterize the impact of the linking Cε2 on the IgE conformational changes upon antigen and/or FcεRIα binding, applied in peanut allergy. These characteristics elucidate the structural mechanisms for allosteric effects observed in the allergen and the FcεRIα binding. This will provide better understanding of initiating steps in the Type-I hypersensitivity as well as the crucial interdomain communications in the whole IgE structure, and hence further complement understanding the role of IgE in allergy pathogenesis.

Ab initio prediction and characterization of membrane binding of HIV-1 broadly neutralizing antibodies by multi-scale simulations.

Rare but potent broadly neutralizing antibodies (bnAbs) engage the HIV virion membrane and the membrane proximal exposed region (MPER) peptide during neutralization. A preliminary lipid mediated binding step is suggested to increase antigen avidity but it remains unclear how initial membrane association occurs and thus what features are essential to mature bnAbs against a lipid-antigen epitope. In this study, we derived computational methods to investigate biophysical features of MPER bnAbs driving membrane association. We performed microsecond atomistic simulations of Fabs docked in a lipid bilayer. Phospholipid binding in CDR loops is predicted in all 4 replicates of PGZL1, 4E10 and 10E8 where a phospholipid headgroup position closely matches the phosphate position in lipid-soaked x-ray crystallography structures (mean RMSD 0.6, 0.7, 2.7 Å respectively). Interaction profiles also highlight how framework regions FRH3 (PGZL1 and 4E10) or FRL3 (10E8) significantly associate with lipids. We then conducted fixed velocity pulling simulations for 4E10 and mutants with reduced affinity for lipid vesicles in vitro. Max force calculates differences in energy required to dissociate Fabs, establishing a bona fide tool to approximate membrane affinities. We expanded this platform to track evolution of binding affinity with inferred germline, intermediate, and mature PGZL1 variants that show increasing binding strengths. To reduce bias from pre-docked membrane conformations, we generated coarse grained models with a Fab initiated above a bilayer. Spontaneous membrane binding events (>1 µs) are detected in over half of the 18 independent 14 µs bnAb trajectories but only in 2 of 18 replicates for negative control BSA. Altogether we present a computational toolkit to biophysically evaluate residue-specific and surface level features of membrane binding antibodies and differentiate membrane affinities, facilitating future studies of complete MPER or other auto-reactive bnAb maturation pathways.