Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19+CD20+CD27− IgM-naive B cells, >120,000 antibody clusters from CD19+CD20+CD27+ antigen–experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ–Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

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Signatures of selection in the human antibody repertoire: Selective sweeps, competing subclones, and neutral drift

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814213116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1261-1266 ◽

Cited By ~ 31

Author(s):

Felix Horns ◽

Christopher Vollmers ◽

Cornelia L. Dekker ◽

Stephen R. Quake

Keyword(s):

B Cell ◽

Binding Affinity ◽

Antibody Repertoire ◽

Human Antibody ◽

Selective Sweeps ◽

Human Immune System ◽

Evolutionary Processes ◽

Neutral Drift ◽

Cell Lineages ◽

Antibody Repertoires

Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the population genetic signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses in five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the relationship between B cell fitness and antibody binding affinity, we demonstrate the potential for using phylogenetic approaches to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.

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Specific In Vivo Deletion of B-Cell Subpopulations Expressing Human Immunoglobulins by the B-Cell Superantigen Protein L

Infection and Immunity ◽

10.1128/iai.72.6.3515-3523.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3515-3523 ◽

Cited By ~ 16

Author(s):

Muriel Viau ◽

Nancy S. Longo ◽

Peter E. Lipsky ◽

Lars Björck ◽

Moncef Zouali

Keyword(s):

B Cells ◽

B Cell ◽

Immune Defense ◽

Protein L ◽

Variable Regions ◽

Marginal Zone B Cells ◽

Human Immunoglobulins ◽

Cell Immunity ◽

B Cell Subsets

ABSTRACT Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.

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Regulatory Approved Monoclonal Antibodies Contain Framework Mutations Predicted From Human Antibody Repertoires

Frontiers in Immunology ◽

10.3389/fimmu.2021.728694 ◽

2021 ◽

Vol 12 ◽

Author(s):

Brian M. Petersen ◽

Sophia A. Ulmer ◽

Emily R. Rhodes ◽

Matias F. Gutierrez-Gonzalez ◽

Brandon J. Dekosky ◽

...

Keyword(s):

Monoclonal Antibodies ◽

High Frequency ◽

In Silico ◽

Sequence Data ◽

Antibody Repertoire ◽

Human Antibody ◽

Therapeutic Antibodies ◽

T Cell Epitopes ◽

Antibody Repertoires ◽

Dynamics Simulations

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

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Full-scale ‘naïve’ human antibody repertoires assembled from VH and VL variable regions

Human Antibodies ◽

10.3233/hab-1997-8106 ◽

1997 ◽

Vol 8 (1) ◽

pp. 37-42 ◽

Cited By ~ 3

Author(s):

Régis Sodoyer ◽

Isabelle Peubez ◽

Corinne Pion ◽

Joseline Dubayle ◽

Paul Jacquemot ◽

...

Keyword(s):

Full Scale ◽

Human Antibody ◽

Variable Regions ◽

Antibody Repertoires

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In Vivo Depletion of Cynomolgus Monkey B Cells Targeted by a Novel Monoclonal Antibody (Xi-20H5) Specific for a Shared Portion of the Human Immunoglobulin Variable Light Chain Lambda 1.

Blood ◽

10.1182/blood.v110.11.2354.2354 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2354-2354

Author(s):

Thierry Sornasse ◽

Keri Tate ◽

Kimberly Milner ◽

Thomas Theriault ◽

Dan W. Denney ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Light Chain ◽

Peripheral Blood ◽

Cynomolgus Monkey ◽

Patient Specific ◽

B Cell Malignancies ◽

Variable Regions ◽

Variable Light

Abstract Genitope is developing a novel, personalized treatment for surface immunoglobulin (Ig) positive B cell malignancies. This treatment is based on a panel of monoclonal antibodies (Mabs) directed against shared epitopes expressed on different subsets of the variable regions of human Ig. The concept of targeting surface tumor Ig was explored in a series of clinical trials performed by Dr. Ronald Levy and his colleagues at Stanford. In these studies, Mabs directed against patient specific (idiotypic) determinants of the surface tumor Ig produced significant clinical benefit in relapsed follicular non-Hodgkin’s lymphoma patients. A number of these patients have had long term remissions. In Genitope’s planned clinical use, each patient will receive a single Mab from the panel selected based on its reactivity with the patient’s tumor. The selected Mab will react with the patient’s tumor and a minority of normal B cells, leaving the majority of the normal B cell repertoire intact. The ability of the panel members to provide therapeutic effect requires binding to tumor surface Ig in the presence of serum containing soluble Ig molecules. Mab Xi-20H5, a member of this panel of antibodies, is specific for a shared determinant on the human Ig variable light chain lambda 1. It binds to 25 to 35% of normal human B cells from peripheral blood and to 20 to 30% of normal cynomolgus monkey B cells from peripheral blood. In this study, we sought to demonstrate that, despite the presence of serum Ig, Mab Xi-20H5 would bind to surface Ig expressed on monkey B cells in vivo, resulting in specific depletion of target B cells. Six naïve cynomolgus monkeys received 8 intravenous infusions of the Mab Xi-20H5 at a dose of 40 mg/kg on days 1, 2, 3, 4, 7, 10, 14 & 17. Two naïve control animals received 8 infusions of vehicle only following the same schedule. The frequencies of lymphocyte sub-populations and of target B cells were monitored by flow cytometry on plasma-depleted whole blood samples. Samples were collected 23 hours after each infusion. In addition, two baseline samples were collected prior to treatment. The frequencies of lymphocyte sub-populations and of target B cells were compared to the average of the two baseline measurements. Frequencies of target B cells bound by Mab Xi-20H5 decreased in all treated animals while no significant change was detectable in the control animals. The bulk of the reduction in target B cell frequencies was observed 23 hours after the first infusion (range: 22% – 62%, average 41%). Frequencies of target B cells continued to decrease moderately with additional daily infusions (days 2 – 4), resulting in maximum reduction in target B cell frequency at 23 h post infusion 4 (range: 39% – 78%, average 54%). The frequencies of total B and T lymphocytes did not significantly change during the treatment. In vivo administration of Mab Xi-20H5 results in depletion of target B cells in a manner consistent with the expectation of an immunotherapeutic Mab aimed at treating surface Ig expressing B cell malignancies.

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Highly sensitive and unbiased approach for elucidating antibody repertoires

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608649113 ◽

2016 ◽

Vol 113 (28) ◽

pp. 7846-7851 ◽

Cited By ~ 31

Author(s):

Sherry G. Lin ◽

Zhaoqing Ba ◽

Zhou Du ◽

Yu Zhang ◽

Jiazhi Hu ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germ Line ◽

Variable Region ◽

Cell Receptor ◽

Affinity Maturation ◽

Antigen Binding ◽

Variable Regions ◽

Antibody Repertoires ◽

B Lineage

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.

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The human antibody repertoire: Heavy and light chain variable region gene usage in six alloantibodies specific for human HLA class I and class II alloantigens

Molecular Immunology ◽

10.1016/0161-5890(95)00071-2 ◽

1995 ◽

Vol 32 (14-15) ◽

pp. 1105-1122 ◽

Cited By ~ 7

Author(s):

Jennifer S. Andris ◽

Sheena R. Abraham ◽

Virginia Pascual ◽

Maria P. Pistillo ◽

Stefano Mantero ◽

...

Keyword(s):

Light Chain ◽

Variable Region ◽

Hla Class I ◽

Class Ii ◽

Class I ◽

Antibody Repertoire ◽

Human Antibody ◽

Region Gene ◽

Variable Region Gene ◽

Gene Usage

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

10.1101/2020.12.31.424987 ◽

2021 ◽

Author(s):

Bailey B. Banach ◽

Gabriele Cerutti ◽

Ahmed S. Fahad ◽

Chen-Hsiang Shen ◽

Matheus Oliveira de Souza ◽

...

Keyword(s):

B Cells ◽

Light Chain ◽

Sequence Similarity ◽

Molecular Study ◽

Antibody Responses ◽

Human Antibody ◽

List Type ◽

Receptor Binding Site ◽

Healthy Human ◽

Antibody Class

SummaryUnderstanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2.HighlightsA molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires

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Regulatory approved monoclonal antibodies contain framework mutations predicted from human antibody repertoires

10.1101/2021.06.22.449488 ◽

2021 ◽

Author(s):

Brian M Petersen ◽

Sophia A Ulmer ◽

Emily R Rhodes ◽

Matias F Gutierrez Gonzalez ◽

Brandon J Dekosky ◽

...

Keyword(s):

Monoclonal Antibodies ◽

High Frequency ◽

In Silico ◽

Sequence Data ◽

Antibody Repertoire ◽

Human Antibody ◽

Therapeutic Antibodies ◽

T Cell Epitopes ◽

Antibody Repertoires ◽

Dynamics Simulations

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

Download Full-text